32 human secreted proteins
18 claims: 2 independent, 16 dependent
- 1Polinucleótido seleccionado do grupo consistindo de:nucleotidica REIVINDICAÇÕES integral (b) um integral polinucleótido tendo da SEQ ID N°: 23;polinucleótido tendo daquela sequência de sequência sequência ADNc incluída nucleotidica no Depósito ATCC N° 209075, que é hibridável sob condições restringentes a SEQ ID N°: 23;150 (d) 150 um fragmento polinucleotidico nucleótidos contíguos da SEQ ID N°: um fragmento polinucleotidico que 23;que nucleótidos da sequência de ADNc incluída ATCC N° 209075 que é hibridável sob compreende compreende no Depósito condições restringentes a SEQ ID N°: 23;(e) um polinucleótido codificando um fragmento polipeptidico, em que o referido fragmento polipeptidico compreende 40 ou 50 resíduos de aminoácidos contíguos de comprimento da SEQ ID N°: 60;(f) um polinucleótido codificando um fragmento polipeptidico codificado pela sequência de ADNc incluída no Depósito ATCC N° 209075, que é hibridável sob condições restringentes a SEQ ID N°: 23, em que o referido fragmento polipeptidico compreende 40 ou 50 resíduos de aminoácidos contíguos da SEQ ID N°: 60;(g) um polinucleótido codificando um fragmento polipeptidico da SEQ ID N°: 60, ou um seu fragmento polipeptidico, codificado pela sequência de ADNc incluída no Depósito ATCC N° 209075, em que o referido fragmento polipeptídico compreende 40 ou 50 resíduos de aminoácidos contíguos de comprimento e tem actividade biológica;(h) um polinucleótido codificando um polipéptido de comprimento completo da SEQ ID N°: 60 ou sendo codificado pela sequência de ADNc incluída no Depósito ATCC N° 209075, que é hibridável sob condições restringentes a SEQ ID N°: 23;(i) um polinucleótido que é, pelo menos, 95% idêntico a, pelo menos, 150 nucleótidos contíguos de um polinucleótido como definido em qualquer uma de (a) a (h) ;e (j) um polinucleótido codificando um polipéptido tendo uma sequência de aminoácidos, pelo menos, 95% idêntica à sequência de aminoácidos de um polipéptido codificado por um polinucleótido de qualquer uma de (a) a (i);ou a cadeia complementar de polinucleótido (a) a (j).
- 2Polinucleótido da reivindicação 1 que é ADN ou ARN.
- 3Gene correspondendo à sequência de ADNc da SEQ ID N°:23 ou codificando o polipéptido tendo a sequência de aminoácidos da SEQ ID N°: 60 .
- 4Vector compreendendo o polinucleótido da reivindicação 1 ou 2.
- 5Método de preparação de uma célula hospedeira recombinante, compreendendo a introdução do polinucleótido da reivindicação 1 ou 2, ou do vector da reivindicação 4.
- 6Célula hospedeira recombinante, contendo o polinucleótido da reivindicação 1 ou 2, ou o vector da reivindicação 4 ou produzida pelo método da reivindicação 5.
- 7Célula hospedeira recombinante da reivindicação 6 que expressa um polipéptido codificado pelo polinucleótido da reivindicação 1 ou 2.
- 8Método de preparação de um polipéptido codificado pelo polinucleótido da reivindicação 1 ou 2, compreendendo;(a) cultivar a célula hospedeira recombinante da reivindicação 7 sob condições, o referido polipéptido seja expresso;e (b) recuperar o referido polipéptido.
- 9Polipéptido codificado pelo polinucleótido da reivindicação 1 ou 2 ou obtenível pelo método da reivindicação 8.
- 10Anticorpo que se liga especificamente ao polipéptido da reivindicação 9.
- 11Antagonista compreendendo um polinucleótido anti-sentido em relação ao polinucleótido da reivindicação 1 ou 2, capaz de se ligar ao referido polinucleótido e bloquear a tradução no polipéptido codificado.
- 12Método para identificação de antagonistas ou agonistas do polipéptido da reivindicação 9, compreendendo:(a) produzir células que expressam o polipéptido da reivindicação 9;(b) colocar em contacto o polipéptido produzido na etapa (a) com uma amostra de teste potencialmente contendo um antagonista ou agonista;e (c) identificar um antagonista ou agonista, através da observação da ligação e inibição ou estimulação de actividade do referido polipéptido.
- 13Método de diagnóstico in vitro de um estado patológico, ou uma susceptibilidade a um estado patológico, num indivíduo, compreendendo:(a) determinar a presença ou ausência de uma mutação no
- 14Método de diagnóstico in vitro de um estado patológico, ou uma susceptibilidade a um estado patológico, num indivíduo, compreendendo:(a) determinar a presença ou quantidade de expressão do polipéptido da reivindicação 9 numa amostra biológica;e (b) diagnosticar um estado patológico, ou uma susceptibilidade a um estado patológico, com base na presença ou quantidade de expressão do polipétido.
- 15Método para identificação de um parceiro de ligação ao polipéptido da reivindicação 9, compreendendo:(a) colocar em contacto o polipéptido da reivindicação 9 com um parceiro de ligação;e (b) determinar se o parceiro de ligação influencia uma actividade do polipéptido.
- 16Composição farmacêutica compreendendo o polinucleótido da reivindicação 1 ou 2, o polipéptido da reivindicação 9, um ADN codificando e capaz de expressar o referido polipéptido in vivo, o anticorpo da reivindicação 10 ou o antagonista da reivindicação 11 e, opcionalmente, um veiculo farmaceuticamente aceitável.
- 17Composição de diagnóstico compreendendo o polinucleótido da reivindicação 1 ou 2, ou o anticorpo da reivindicação 10.
- 18Método para a produção de uma composição farmacêutica, compreendendo as etapas do método da reivindicação 12 e (d) formular o composto identificado na etapa (c) numa forma farmaceuticamente aceitável.
Independent claims18
2,613 paragraphs in 465 sections, as filed
DESCRIPTION
HUMAN PROTEINS SEGREGATED
Field of the Invention
This invention relates to newly identified polynucleotides and polypeptides encoded by these polynucleotides, uses of such polynucleotides and polypeptides, and their production.
Background of the Invention
Unlike bacteria, which exist as a single compartment surrounded by a membrane, human and other eukaryotic cells are subdivided by membranes into many functionally distinct compartments. Each membrane compartment, or organelle, contains different proteins essential for organelle function. The cell uses screening signals, which are amino acid motifs located on the protein, to target proteins to particular cell organelles.
A type of screening signal, called a signal sequence, a signal peptide, or a conductive sequence, directs a class of proteins to an organelle called an endoplasmic reticulum (ER). ER separates membrane proteins from all other types of proteins. Once located in the ER, both protein groups can be further directed to another organelle called the Golgi apparatus. Here, Golgi distributes proteins to vesicles, including secretory vesicles, the cell membrane, lysosomes, and other organelles.
Proteins directed to the ER by a signal sequence may be released into the extracellular space as a secreted protein. For example, secreted protein-containing vesicles may fuse with the cell membrane and release their contents into extracellular space - a process called exocytosis. Exocytosis may occur constitutively or upon receipt of a triggering signal. In the latter case, proteins are stored in secretory vesicles (or secretory granules) until exocytosis is triggered. Similarly, proteins residing at the cell membrane can also be secreted into the extracellular space by proteolytic cleavage of a protein-binding ligand to the membrane.
Notwithstanding the great progress made in recent years, only a small number of genes encoding secreted human proteins have been identified. These secreted proteins include the commercially valuable human insulin, interferon, Factor VIII, human growth hormone, tissue plasminogen activator and erythropoietin. Thus, in light of the omnipresent role of secreted proteins in human physiology, there is a need for the identification and characterization of novel secreted human proteins and the genes encoding them. This knowledge will enable anyone to detect, treat and prevent medical disorders through the use of secreted proteins or the genes that encode them.
Summary of the Invention
The present invention relates to novel polynucleotides and encoded polypeptides. Furthermore, the present invention relates to recombinant vectors, host cells, antibodies and methods for producing polypeptides and polynucleotides. Diagnostic methods for detecting polypeptide-related disorders and pharmaceutical compositions are also provided. The invention further relates to screening methods for identifying polypeptide binding agents.
Specifically, the present invention relates to a polynucleotide selected from the group consisting of:
(a) a polynucleotide having the full nucleotide sequence of SEQ ID NO: 23;
(b) a polynucleotide having the full nucleotide sequence of that cDNA sequence included in ATCC Depot No. 209075 which is hybridizable under conditions restricting SEQ ID NO: 23;
(c) a polynucleotide fragment comprising
150 contiguous nucleotides of SEQ ID NO: 23;
(d) a polynucleotide fragment comprising
150 nucleotides of the cDNA sequence included in ATCC Depot No. 209075 which is hybridizable under stringent conditions to SEQ ID NO: 23;
(e) a polynucleotide encoding a polypeptide fragment, wherein said polypeptide fragment comprises 40 or 50 contiguous amino acid residues in length of SEQ ID NO: 60;
(f) a polynucleotide encoding a polypeptide fragment encoded by the cDNA sequence included in ATCC Depot No. 209075, which is hybridizable under conditions stringent to SEQ ID NO: 23, wherein said polypeptide fragment comprises 40 or 50 contiguous amino acid residues of SEQ ID NO: 60;
(g) a polynucleotide encoding a polypeptide fragment of SEQ ID NO: 60, or a polypeptide fragment thereof, encoded by the cDNA sequence included in ATCC Depot No. 209075, wherein said polypeptide fragment comprises 40 or 50 contiguous amino acid residues in length and has biological activity;
(h) a polynucleotide encoding a full length polypeptide of SEQ ID NO: 60 or being encoded by the cDNA sequence included in ATCC Depot No. 209075, which is hybridizable under conditions restricting SEQ ID NO: 23;
(i) a polynucleotide that is at least 95% identical to at least 150 contiguous nucleotides of a polynucleotide as defined in any one of (a) to (h);
(j) a polynucleotide encoding a polypeptide having an amino acid sequence at least 95% identical to the amino acid sequence of a polypeptide encoded by a polynucleotide of any of (a) to (i); and or the polynucleotide complementary strand (a) to (j).
The present invention also relates to the gene corresponding to the cDNA sequence of SEQ ID NO: 23 or encoding the polypeptide having the amino acid sequence of SEQ ID NO: 60. In addition, the present invention relates to a vector comprising the polynucleotide of the invention.
The present invention also relates to a method of preparing a recombinant host cell comprising introducing the polynucleotide or vector of the invention.
Further, the present invention relates to a recombinant host cell containing the polynucleotide or vector of the invention or produced by the aforementioned method of preparing a recombinant host cell.
The present invention also relates to a method of preparing a polypeptide encoded by the polynucleotide of the invention comprising;
(a) culturing the aforementioned recombinant host cell expressing said polypeptide, under conditions that said polypeptide is expressed; and (b) recovering said polypeptide.
Further, the present invention relates to a polypeptide encoded by the polynucleotide of the invention or obtainable by the aforementioned method of preparing a polypeptide.
The present invention also relates to an antibody that specifically binds to the polypeptide of the invention.
The present invention further relates to an antagonist comprising an antisense polynucleotide to the polynucleotide of the invention capable of binding said polynucleotide and blocking translation into the encoded polypeptide.
The present invention also relates to a method for identifying antagonists or agonists of the polypeptide of the invention, comprising:
(a) producing cells expressing said polypeptide;
(b) contacting the polypeptide produced in step (a) with a test sample potentially containing an antagonist or agonist; and (c) identifying an antagonist or agonist by observing binding and inhibiting or stimulating activity of said polypeptide.
Further, the present invention relates to an in vitro diagnostic method of a pathological condition, or a susceptibility to a pathological condition, in an individual comprising:
(a) determining the presence or absence of a mutation in the polynucleotide or gene of the invention; and (b) diagnosing a pathological condition, or a susceptibility to a pathological condition, based on the presence or absence of said mutation.
The present invention also relates to an in vitro diagnostic method of a pathological condition, or a susceptibility to a pathological condition, in an individual comprising:
(a) determining the presence or amount of expression of the polypeptide of the invention in a biological sample; and (b) diagnosing a pathological condition, or a susceptibility to a pathological state, based on the presence or amount of expression of the polypeptide.
Further, the present invention relates to a method for identifying a binding partner for the polypeptide of the invention, comprising:
(a) contacting said polypeptide with a binding partner; and (b) determining whether the binding partner influences a polypeptide activity.
Additionally, the present invention relates to a pharmaceutical composition comprising the polynucleotide of the invention, the polypeptide of the invention, a DNA encoding and capable of expressing said polypeptide in vivo, the antibody of the invention, the antagonist of the invention and, optionally, a pharmaceutically acceptable carrier.
The present invention also relates to a diagnostic composition comprising the polynucleotide or antibody of the invention.
Finally, the present invention also relates to a method for producing a pharmaceutical composition comprising the steps of the aforementioned method for identifying antagonists or agonists and (d) formulating the compound identified in step (c) in a pharmaceutically acceptable form.
Detailed Description
<td rowspan="2">The following understanding of d description.</td><td rowspan="2">Definitions Undetermined</td><td colspan="2">are provided to facilitate the</td>
<td>terms used in</td><td>all this</td>
<td>In the present</td><td>invention,</td><td>isolated refers</td><td>the material</td>
removed from its original environment (eg, the natural environment if it is naturally occurring) and thus is altered by man's hand from its natural state. For example, an isolated polynucleotide could be part of a vector or a composition of matter or could be contained within a cell and be further isolated because that particular vector, composition of matter or cell is not the original environment of the subject. polynucleotide.
In the present invention, a secreted protein refers to those proteins capable of being directed to the ER, secretory vesicles or extracellular space as a result of a signal sequence, as well as to those proteins released within the extracellular space, without necessarily containing a sequence. signal. If the secreted protein is released into the extracellular space, the secreted protein may undergo extracellular processing to produce a mature protein. Release within the extracellular space can occur by many mechanisms, including exocytosis and proteolytic cleavage.
As used herein, a polynucleotide refers to a molecule having a nucleic acid sequence contained within the
SEQ ID NO: 23 or the cDNA contained within the clone deposited with ATCC Deposit No. 209075 or variants thereof as defined above in (a) to (j). For example, the polynucleotide may contain the nucleotide sequence of the full length cDNA sequence, including the 5 'and 3' untranslated sequences, the coding region, with or without the signal sequence, the secreted protein coding region, as well as fragments nucleic acid sequence epitopes, domains and variants as defined above in (a) to (j) - In addition, as used herein, A polypeptide refers to a molecule having the translated amino acid sequence produced from the polynucleotide as defined above in general.
In the present description, the full length sequence, identified as SEQ ID NO: X, was often produced by overlapping sequences contained in multiple clones (contig analysis). A representative clone containing all or most of the sequence for SEQ ID NO: X was filed with the American Type Culture Collection (ATCC). As shown in Table 1, each clone is identified by a cDNA Clone ID and ATCC Deposit Number. ATCC is located at 10801 University Boulevard, Manassas, Virginia 20110-2209, USA. The ATCC filing was made in accordance with the terms of the Budapest Treaty, in the international recognition of the filing of microorganisms for patenting purposes.
A polynucleotide of the present invention also includes those polynucleotides capable of hybridizing under stringent hybridization conditions to the sequences contained in SEQ ID NO: 23, its complement, or cDNA within the clone deposited with ATCC Depot No. 209075, provided that the hybridization sequence has at least 95% sequence identity with at least 150 contiguous nucleotides of a defined polynucleotide in any one of claims 1 (a) to (h) or encode a polypeptide having at least one amino acid sequence 95% identical to the amino acid sequence of a polypeptide encoded by a polynucleotide of any one of claims 1 (a) to (i). Stringent hybridization conditions refer to an overnight incubation at 42 ° C in a solution comprising 50% formamide, 5x SSC (750 mM NaCl, 75 mM sodium citrate), 50 mM (pH 7.6), 5x Denhardt's solution, 10% dextran sulfate and cut denatured salmon sperm DNA at 20 pg / ml, followed by washing the filters in 0.1x SSC at about 100 ° C. 65 ° C.
Nucleic acid molecules that hybridize to the polynucleotides of the present invention are also described under stringent low hybridization conditions. Changes in hybridization stringency and signal detection are mainly accomplished by manipulating formamide concentration (lower percentages of formamide result in decreased stringency); saline conditions or temperature. For example, low stringency conditions include overnight incubation at 37 ° C in a solution comprising 6X SSPE (20X SSPE = 3 M NaCl; NaH<sub>2</sub>POWDER<sub>4</sub> at 0.2 M; 0.02 M EDTA, pH 7.4), 0.5% SDS, 30% formamide, 100 pg / ml salmon sperm blocking DNA; followed by washes at 50 ° C with 1XSSPE, 0.1% SDS. Additionally, in order to achieve even lower stringency, washes performed after stringent hybridization can be performed at higher salt concentrations (eg, 5X SSC).
It is to be noted that variations in the above conditions may be realized by the inclusion and / or substitution of alternative blocking reagents used to suppress the background in hybridization experiments. Typical blocking reagents include Denhardt's reagent, BLOTTO, heparin, denatured salmon sperm DNA, and commercially available proprietary formulations. Inclusion of specific blocking reagents may require modification of the hybridization conditions described above due to compatibility issues.
Of course, a polynucleotide that hybridizes only to polyA + sequences (such as any terminal polyA + 3 'apparatus of a cDNA shown in the sequence listing), or to a complementary segment of T (or U) residues, would not be included in the definition of polynucleotide. as such a polynucleotide would hybridize to any nucleic acid molecule containing a poly (A) segment or to its complement (eg, in practical terms, any double stranded cDNA clone).
The polynucleotide of the present invention may be composed of any polyribonucleotide or polydeoxyribonucleotide which may be unmodified RNA or DNA or modified RNA or DNA. For example, polynucleotides may be composed of single stranded and double stranded DNA, DNA which is a mixture of single stranded and double stranded regions, single stranded and double stranded RNA, and RNA which is a mixture of single stranded and single stranded regions. double stranded, hybrid molecules comprising DNA and RNA which may be single stranded or more typically double stranded or a mixture of single stranded and double stranded regions. Additionally, the polynucleotide may be composed of triple stranded regions, comprising RNA or DNA, or RNA and
DNA. A polynucleotide may also contain one or more modified bases or modified DNA or RNA structures for stability or for other reasons. Modified bases include, for example, tritylated bases and unusual bases such as inosine. A variety of modifications may be made to DNA and RNA; thus polynucleotide encompasses chemically, enzymatically or metabolically modified forms.
The polypeptide of the present invention may be composed of amino acids linked together by peptide bonds or modified peptide bonds, ie, peptide isoesters and may contain amino acids other than 20 amino acids encoded by gene. Polypeptides may be modified by natural processes, such as post-translational processing, or by chemical modification techniques that are well known in the art. Such modifications are well described in basic texts and more detailed monographs, as well as in a voluminous research literature. Modifications may occur anywhere in a polypeptide, including the peptide structure, amino acid side chains, and amino or carboxyl ends. It will be understood that in a given polypeptide, the same type of modification may be present at the same or to varying degrees at various sites. Also, a particular polypeptide may contain many types of modifications. Polypeptides may be branched, for example, as a result of ubiquitination and they may be cyclic, with or without branching. The cyclic, branched and cyclic branched polypeptides may result from natural post-translational processes or may be prepared by synthetic methods. Modifications include acetylation, acylation, ADP ribosylation, amidation, covalent conjugation of flavin, covalent conjugation of a heme moiety, covalent conjugation of a nucleotide or nucleotide derivative, covalent conjugation of a lipid or lipid derivative, covalent conjugation of phosphotidylinositol, crosslinking, cyclization, disulfide bond formation, demethylation, covalent crosslinking, cysteine formation, pyroglutamate formation, formylation, gamma carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristylation, oxidation, pegylation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer mediated addition
Amino acid RNA for proteins such as arginylation and ubiquitination (See, for example, PROTEINS - STRUCTURE AND MOLECULAR PROPERTIES,
2<sup>The</sup>
Ed.,
TE Creighton, WH Freeman and Company, New York
POSTTRANSLATIONAL COVALENT MODIFICATION OF PROTEINS,
B.
Ç.
Johnson, Ed., Academic Press, New York, p. 1-12 (1983);
Seifter et al. Meth Enzymol 182: 626-646 (1990); Rattan et al.,
Ann NY Acad Sci 663: 48-62 (1992)).
SEQ ID NO: X refers to a polynucleotide sequence, while SEQ ID NO: Y refers to a polypeptide sequence, both sequences identified by an integer specified in Table 1.
A polypeptide having biological activity refers to polypeptides exhibiting activity similar to, but not necessarily identical to, an activity of a polypeptide of the present invention, including mature forms, as measured in a particular biological assay, with or without dose dependence. Where dose dependence exists, it need not be identical to that of the polypeptide but rather substantially similar to dose dependence in a given activity compared to the polypeptide of the present invention (1). e., the candidate polypeptide will exhibit greater activity or should not exceed about 25 times less and preferably should not exceed about ten times less activity and most preferably should not exceed about three times less. less activity, relative to the polypeptide of the present invention).
Polynucleotides and Polypeptides of the Invention or Disclosed herein
FEATURES OF NS GENE-CODED PROTEIN 1
This gene maps to chromosome 3 and therefore the polynucleotides disclosed herein can be used in binding analysis as a marker for chromosome 3.
This gene is expressed in a variety of fetal tissues, including brain, liver and lung, and to a lesser extent in adult tissues, particularly skin.
Accordingly, the disclosed Polynucleotides and polypeptides are useful as reagents for differential identification of tissue (s) or cell type (s) present in a biological sample and for diagnosing diseases and conditions that include, but are not limited to, a variety of cancers, particularly from the brain, liver and lung. Similarly, polypeptides and antibodies directed to these polypeptides are useful in providing immunological probes for differential identification of tissue (s) or cell type (s). For a number of prior tissue or cell disorders, particularly the central nervous system, hepatic system, and hepatic system, expression of this gene at significantly higher or lower levels may be routinely detected in certain tissues (e.g. brain, and other tissue of the nervous system, liver, lung and skin, and cancerous and injured tissues) or body fluids (eg, serum, plasma, urine, synovial fluid, or spinal fluid) or other tissue or cell specimen taken from a subject having such a disorder, relative to the level of standard gene expression, 1. e., the level of expression in healthy body fluid or tissue of an individual not having the disorder.
Tissue distribution indicates that polynucleotides and polypeptides corresponding to this gene are useful as a target for a variety of blocking agents, as they are likely to be involved in the promotion of a variety of cancers.
GENE N2-CODED PROTEIN CHARACTERISTICS
The polypeptides disclosed herein comprise the sequence:
MSVPAFIDISEE DQAAE LRAYLKSKGAEISEENSE GGLHVDLAQIIEAC
DVCLEEDDKDVESVMNSWSLLLILEPDKQEAUESLCEKLVKFREGERPSLRLQ
LLSNLFHGMDKNTPVRYTVYCSLIKVAASCGAIQYIPTELDQVRKWISDWNLTT
EKKHTLLRLLYEALVDCKKSDAASKVMVELLGSYTEDNASQARVDAHRCIVRA
LKDPNAFLFDHLLTLKPVKFLEGELLIDLLTIFVSAKLASYVKFYQNNKDFIDSL
GLLHEQNMAKMRLLTFMGMAVENKEISFDTMQQELQIGADDVEAFVIDAVRTK
MVYCKIDQTQRKVWSHSTHRTFGKQQWQQLYDTLNAWKQNLNKVKNSLLS
LSDT (SEQ ID NO: 85), MSVPAFIDISEED (SEQ ID NO: 86),
QAAELRAYLKSKG AE (SEQ ID NO: 87),
ISEENSEGGLHVDLAQI (SEQ ID NO: 88),
IEACDVCLKED DKDVESV (SEQ ID NO:),
VARPSSLFRSAWSCEW (SEQ ID NO: 90), LRLQLLS NLFHG (SEQ ID NO: 91),
KDVESVMNSWSLLUL (SEQ ID NO: 92), DAASKVMV ELLGSYTEDNASQARVDA (SEQ ID NO: 93) and / or VEAFVIDAVR (SEQ ID NO: 94). Polynucleotides encoding these polypeptides are also disclosed.
This gene is expressed in bone and, to a lesser extent, in the brain, lung, T cells, muscle, skin, testis, spleen and macrophages.
Accordingly, the disclosed polypeptide polynucleotides are useful as identification reagents present in a biological sample and for diagnosing diseases and conditions that include, but are not limited to, bone cancer, osteoarthritis and autoimmune diseases.
Similarly, polypeptides and antibodies directed to these polypeptides are useful in providing immunological probes for differential identification of a number of prior tissue or cell disorders, particularly the immune system and skeletal system, the expression of this gene at significantly higher levels. or lower can be routinely detected in certain tissues and cell types (e.g. brain and other nervous system tissue, T cells and other immune system cells and tissue, lung, muscle, skin and injured tissues) or body fluids (eg, serum, plasma, urine, synovial fluid or spinal fluid) or another tissue or cell sample taken from an individual having such a disorder, relative to the standard gene expression level, ie, the level of healthy tissue or body fluid expression of an individual not having the disorder. Preferred epitopes include those comprising a sequence shown in SEQ ID NO: 49 as residues: Arg-31 to Ser-37, Met-50 to Val-56, Glu-80 to Trp-87, Thr-94 to His-99,
Tyr-129 to Ser-135, Tyr-193 to Phe-199, Ser-274 to Gln-285 and / or
Ala-293 to Lys-302.
GENE N2-CODED PROTEIN CHARACTERISTICS
The translation product of this gene shares sequence homology with various kinases. The closest homolog is mouse TIF1 which is a mouse nuclear protein. TIF1 improves RXR and RAR AF-2 in yeast and interacts, in a ligand-dependent manner, with various nuclear receptors in yeast and mammalian cells, as well as in vitro. Notably, these interactions require the amino acids constituting the AF-2 activation domain conserved in all active NRs. Furthermore, estrogen receptor (ER) antagonist hydroxy tamoxifen cannot promote the interaction of ER-TIF1. TIF1, which contains several conserved domains found in transcriptional regulatory proteins, is proposed to be a ligand-dependent AF-2 mediator. Interestingly, in mouse T18 oncoprotein, the N-terminal fraction of TIF1 is fused to B-raf.
This gene is expressed primarily in activated T cells and, to a lesser extent, in various other tissues, including testes and brain.
Therefore, disclosed are tissue differential (s) polynucleotides and polypeptides useful as reagents for identification or cell type (s) present in a biological sample and for diagnosing diseases and conditions that include, but are not limited to, autoimmune diseases, AIDS, leukemias and various other cancers. Similarly, polypeptides and antibodies directed to these polypeptides are useful in providing immunological probes for differential identification of tissue (s) or cell type (s). For a number of prior tissue or cell disorders, particularly the immune system, expression of this gene at significantly higher or lower levels may be routinely detected in certain tissues and cell types (e.g. T cells and other cells and tissue of the immune system and brain and other nervous system tissue and cancerous and injured tissues) or body fluids (eg, serum, plasma, urine, synovial fluid or spinal fluid) or other tissue sample or cell taken from an individual having such a disorder, relative to the level of standard gene expression, ie, the level of expression in healthy body tissue or fluid of an individual not having the disorder. Preferred epitopes include those comprising a sequence shown in SEQ ID NO: 50 as residues: Ala-31 to Glu-36.
Tissue distribution and TIF homology indicate that polynucleotides and polypeptides corresponding to this gene are useful for nuclear receptor modulation and ligand interaction in various immune disorders.
GENE NODE 4 PROTEIN CHARACTERISTICS
This gene maps to chromosome 11. Accordingly, the polynucleotides disclosed herein may be used in ligation analysis as a marker for chromosome 11. In specific embodiments, the disclosed polypeptides comprise the sequence:
: °:
95)
LVAPRF QRKFKAKQLTPRILEAHQNVAQLSLAEAQLRFIQAWQSL
96) ,
VGD WKTWRFSNMRQWNVNWDIR
EEIDCTEEEMMVFAALQYH INKLSQS (SEQ
EEIDCTEEEMMVFAALQYHINKLSQS polynucleotides encoding these
No.
ID
<td>ISSEQ</td><td>ID</td><td>No.</td><td> 97),</td>
<td>ID</td><td>No.</td><td> 98)</td><td>and / or</td>
<td>ID</td><td>No.</td><td> 99) .</td><td>The</td>
<td colspan="2">ilipeptides</td><td>also</td><td>are</td>
disclosed.
This gene is expressed primarily in various types of leukocytes, including monocytes, T cells and neutrophils, and to a lesser extent in a limited number of other tissues, including umbilical vein and liver.
Accordingly, the disclosed polynucleotides and polypeptides are useful as reagents for differential identification of tissue (s) or cell type (s) present in a biological sample and for diagnosing diseases and conditions that include, but are not limited to , various diseases of the immune system, including AIDS, immunodeficiency diseases, and autoimmune disorders. Similarly, polypeptides and antibodies directed to these polypeptides are useful in providing immunological probes for differential identification of tissue (s) or cell type (s). For a number of prior tissue or cell disorders, particularly the immune system, expression of this gene at significantly higher or lower levels may be routinely detected in certain tissues and cell types (e.g. blood cells, liver and vascular tissue other cancerous and injured tissues) or body fluids (eg, serum, plasma, urine, synovial fluid or spinal fluid) or other tissue or cell sample taken from an individual having such a disorder, relative to the standard level of gene expression, ie, the level of expression in healthy body fluid or tissue of an individual not having the disorder. Preferred epitopes include those comprising a sequence shown in SEQ ID NO: 51 as residues: Ser-3 to Pro-9, Leu-17 to Leu-29, Asp-64 to Pro-69, He-105 to Gln-110, The-183 to Gln-200, Cys-239 to Arg-247, Ser-256 to Met-261, Gln-280 to Ala-296, Arg-310 to Thr-321, Lys-363 to Asp-368, Ser- 395 to Trp-400 and / or Thr-443 to Asp-453.
Tissue distribution indicates that polynucleotides and polypeptides corresponding to this gene are useful for replacement therapy in a variety of immune system disorders.
NS 5 GENE-CODED PROTEIN CHARACTERISTICS
This gene is mainly expressed in the brain and little or nothing in any other tissue.
Accordingly, the polynucleotides and polypeptides disclosed herein are useful as reagents for differential identification of tissue (s) or cell type (s) present in a biological sample and for diagnosing diseases and conditions that include, but are not limited to. a, mood disorders, bipolar disorder and unipolar depression. Similarly, polypeptides and antibodies directed to these polypeptides are useful in providing immunological probes for differential identification of tissue (s) or cell type (s). For a number of prior tissue or cell disorders, particularly the central nervous system, expression of this gene at significantly higher or lower levels may be routinely detected in certain tissues (e.g. brain and other nervous system tissue and cancerous and injured tissues) or body fluids (eg, serum, plasma, urine, synovial fluid or spinal fluid) or other tissue or cell sample taken from an individual having such a disorder, relative to the standard level of gene expression, ie, the level of expression in healthy body fluid or tissue of an individual not having the disorder. Preferred epitopes include those comprising a sequence shown in SEQ ID NO: 52 as residues: Met-1 to Gly-8, Pro-10 to Arg-17, Pro-45 to Ser-55 and / or Gly-63 to Tyr- 74
Tissue distribution of this gene, mainly in the brain, indicates that polynucleotides and polypeptides corresponding to this gene are useful for detecting / treating neurodegenerative disease states and behavioral disorders such as Alzheimer's disease, Parkinson's disease, Huntington's, schizophrenia, mania, dementia, paranoia, obsessive compulsive disorder and panic disorder. Also, given the brain specific expression of this gene, the promoter region of this gene contains a brain specific element that could be used for targeting expression of brain vector systems in gene replacement therapy.
GENE N-CODED PROTEIN CHARACTERISTICS<sup>and</sup> 6
This gene maps to chromosome 1 and therefore the polynucleotides disclosed herein can be used in binding analysis as a marker for chromosome 1.
This gene is abundantly expressed in rhabdomyosarcoma, is expressed at a high level and in different regions of the brain and pituitary gland, and to a lesser extent in a variety of other tissues.
Accordingly, the disclosed polynucleotides and polypeptides are useful as identification reagents present in a biological sample and for diagnosing diseases and conditions that include, but are not limited to, neurological disorders and muscle disorders.
Similarly, polypeptides and antibodies directed to these polypeptides are useful in providing immunological probes for differential identification.
For a number of prior tissue or cell disorders, particularly brain disorders, expression of this gene at significantly higher or lower levels may be routinely detected in certain tissues (eg, smooth muscle, brain and other tissue of the nervous and pituitary system and cancerous and injured tissues) or body fluids (e.g. serum, plasma, urine, synovial fluid or spinal fluid) or other tissue or cell sample taken from an individual having such a disorder relative to the standard gene expression level, ie, the expression level in body tissue or fluid healthy condition of an individual not having the disorder.
Abundant expression of this gene in rhabdomyosarcoma indicates a role for the protein product in the detection and / or treatment of skeletal muscle disorders, including muscle degeneration, muscle wasting and rhabdomyolysis. In addition, expression in the brain indicates a role for the protein product of this gene in detecting / treating neurodegenerative disease states and behavioral disorders such as Alzheimer's disease, Parkinson's disease, Huntington's disease, schizophrenia, mania, dementia, paranoia, obsessive compulsive disorder and panic disorder.
GENE CODIFIED PROTEIN CHARACTERISTICS # 27 This translation product of this gene shares sequence homology with the TDAG51 gene thought to be important in mediating apoptosis and cell death by binding TCR stimulation to Fas expression. In specific embodiments, the polypeptides of the invention comprise the sequence:
<td>KELSFARIKAVECVESTGR HIYFTLV (SEQ GWNAQITLGLVKFKNQQ (SEQ ID NO: 101).</td><td>ID</td><td>No.</td><td>100) and / or</td>
<td>This gene is expressed in several</td><td>fabrics</td><td colspan="2">including</td>
<td>macrophages.</td><td></td><td></td><td></td>
Accordingly, disclosed are tissue differential (s) polynucleotides and polypeptides useful as reagents for identifying or cell type (s) present in a biological sample and for diagnosing diseases and conditions that include, but are not limited to, immune disorders. Similarly, polypeptides and antibodies directed to these polypeptides are useful in providing immunological probes for differential identification of tissue (s) or cell type (s). For a number of prior tissue or cell disorders, particularly the immune system, expression of this gene at significantly higher or lower levels may be routinely detected in certain tissues and cell types (eg, macrophages and other blood cells and cancerous tissues). and injured) or body fluids (e.g. serum, plasma, urine, synovial fluid or spinal fluid) or other tissue or cell sample taken from an individual having such a disorder relative to the standard gene expression level, ie, the level of expression in body tissue or fluid. healthy condition of an individual not having the disorder. Preferred epitopes include those comprising a sequence shown in SEQ ID NO: 54 as residues: Met-1 to
Pro-9, Gln-43 to Glu-49 and / or Phe-95 to Arg-102.
Tissue distribution and homology to the TDAG51 gene indicate that polynucleotides and polypeptides corresponding to this gene are useful for diagnosis and intervention of immune disorders such as immunodeficiency, allergy, infection, inflammation, tissue / organ transplantation.
GENE N2-CODED PROTEIN CHARACTERISTICS 8
This gene is expressed in breast tissue and amniotic cells and, to a lesser extent, in smooth muscle, T cells, and infant brain.
Accordingly, the polynucleotides and polypeptides disclosed herein are useful as reagents for differential identification of tissue (s) or cell type (s) present in a biological sample and for diagnosing diseases and conditions that include, but are not limited to , fetal insufficiency syndrome and embryonic wasting. Similarly, polypeptides and antibodies directed to these polypeptides are useful in providing immunological probes for differential identification of tissue (s) or cell type (s). For a number of prior tissue or cell disorders, particularly the female reproductive system, expression of this gene at significantly higher or lower levels may be routinely detected in certain tissues and cell types (e.g. breast tissue, amniotic cells, smooth muscle, brain and other nervous system tissue and T cells and other immune system cells and tissue, and cancerous and injured tissues) or body fluids (eg, serum, plasma, urine, synovial fluid or spinal fluid) or other tissue or cell sample taken from an individual having such a disorder, relative to the standard level of gene expression, i. e., the level of expression in healthy body tissue or fluid of an individual not having the disorder.
FEATURES OF NS 9 GENE-ENCODED PROTEIN
In specific embodiments, the polypeptides disclosed herein comprise the sequence:
LVLGLSXLNNSYNFSF (SEQ ID NO: 102), HWIGSQAEEGQYSLNF (SEQ ID NO: 103), HNCNNSVPGKEHPFDITVM (SEQ ID NO: 104), FIKYVLSD KEKKVFGIV (SEQ ID NO: 105), IPMQVQ (106) , IPMQVL ANVAYII (SEQ ID NO: 107), DGKVAVNLAKLKLFR (SEQ ID NO: 108) and / or IREKNPDGFLSAA (SEQ ID NO: 109). Polynucleotides encoding these polypeptides are also disclosed.
This gene is mainly expressed in the fetal liver, spleen and pituitary gland and, to a lesser extent, in multiple tissues.
Accordingly, the disclosed polynucleotides and polypeptides are useful as reagents for differential identification of tissue (s) or cell type (s) present in a biological sample and for diagnosing diseases and conditions that include, but are not limited to , immune disorders and cancer. Similarly, polypeptides and antibodies directed to these polypeptides are useful in providing immunological probes for differential identification of tissue (s) or cell type (s). For a number of prior tissue or cell disorders, particularly the hepatic, immune and hematopoietic systems, expression of this gene at significantly higher or lower levels can be routinely detected in certain tissues (e.g. liver, spleen and pituitary gland and cancerous and injured tissues) or body fluids (eg, serum, plasma, urine, synovial fluid or spinal fluid) or other tissue or cell specimen taken from an individual having such a disorder, with respect to at the standard gene expression level, ie, the level of expression in healthy body fluid or tissue of an individual not having the disorder. Preferred epitopes include those comprising a sequence shown in SEQ ID NO: 56 as residues: Ser-62 to Cys-71, Thr-78 to Leu-86, Ser-104 to
Lys-109, Ser-130 to Ala-135 and / or Gln-168 to Asp-174.
Tissue distribution indicates that polynucleotides and polypeptides corresponding to this gene are useful for diagnosis and treatment of liver disorders and disorders of the immune and hematopoietic systems such as liver failure, hepatitis, alcoholic liver disease, portal hypertension, toxic liver injury. , liver transplantation and liver neoplasm. Expression in the fetal liver and spleen also indicates its function in hematopoiesis and therefore the gene may be useful in hematopoietic disorders including anemia, leukemia or cancer radiotherapy / chemotherapy. Expression in the pituitary gland may indicate its use in endocrine disorders with systemic or specific manifestations.
GENE N2-CODED PROTEIN CHARACTERISTICS 10
The translation product of this gene shares sequence homology with a chicken DNA binding protein thought to be important in transcriptional regulation of gene expression. Polypeptides disclosed herein comprise the sequence: MMFGGYETI (SEQ ID NO: 110), YRDESSSELSVDSEVEFQLYSQIH (SEQ ID NO: 111), YAQDLDDVIREEEHEEKNSGNSESSSSKPNQKKLIVLSDSEVI
QLSDGSEVITLSDEDSIYRCKGKNVRVQAQENAHGLSSSLQSNELVDKKKSK EKPKSEERSGVIREVMIIEVSSSEEEESTISEGDNVESW (SEQ ID NO: 112), MLLG
CEVDDKDDDILLNLVGCENSVTEGEDGINWSIS (SEQ ID NO: 113), DKDIEAQI
ANNRTPGRWT (SEQ ID NO: 114), QRYYSANKNIICRNCDKRGHLSKNCPLP RKV (SEQ ID NO: 115) and / or RRCFLCSRRGHLLYSCPAPLCEYCPVPKMLDHS
CLFRHSWDKQCDRCHMLGHYTDACTEDNRQYHLTTKPGPPKKPKTPSRPSAL AYCYHCAQKGHYGHECPEREVYDPSPVSPFICYYXDKYEIQEREKRLKQKIKV XKKNGVIPEPSKLPYIKQANNWDQNNNHDDKNKNW Polynucleotides encoding these polypeptides are also disclosed.
This gene is expressed in the tonsils and bone marrow.
Accordingly, the disclosed polynucleotides and polypeptides are useful as reagents for differential identification of tissue (s) or cell type (s) present in a biological sample and for diagnosing diseases and conditions that include, but are not limited to, disorders of the immune, hematopoietic and lymphatic systems. Similarly, polypeptides and antibodies are useful in providing differential identification of those polypeptide targeting immunological probes for tissue (s) or cell type (s).
For a number of prior tissue or cell disorders, particularly the immune, hematopoietic and lymphatic systems, expression of this gene at significantly higher or lower levels can be routinely detected in
tonsils and bone marrow and cancerous tissues or body fluids (e.g.
serum, plasma, urine, synovial fluid or spinal fluid) or other tissue or cell sample taken from an individual having such a disorder, relative to the standard level of gene expression, i.
e., the level of expression in healthy body tissue or fluid of an individual not having the disorder.
Tissue distribution and DNA binding protein homology indicates that polynucleotides and polypeptides corresponding to this gene are useful for the treatment and diagnosis of disorders in the immune, hematopoietic and lymphatic systems.
FEATURES OF NS 11 GENE-ENCODED PROTEIN
This gene is expressed in dendritic and T cells.
Accordingly, the disclosed polynucleotides and polypeptides are useful as reagents for differential identification of the tissue (s) or cell type (s) present in a biological sample and for diagnosing diseases and conditions which include, but are not limited to. limited to, immune system disorders. Similarly, polypeptides and antibodies directed to these polypeptides are useful in providing immunological probes for differential identification of tissue (s) or cell type (s). For a number of prior tissue or cell disorders, particularly the immune system, expression of this gene at significantly higher or lower levels may be routinely detected in certain tissues and cell types (e.g. dendritic cells and T cells and other cells and tissue of the immune system and cancerous and injured tissues) or body fluids (eg, serum, plasma, urine, synovial fluid or spinal fluid) or other tissue or cell sample taken from a individual having such a disorder, relative to the level of standard gene expression, ie<sub>r</sub> the level of expression in healthy body tissue or fluid of an individual not having the disorder.
Tissue distribution indicates that the protein products of this gene are useful for the treatment and diagnosis of immune system disorders, particularly those involving dendritic or T cells, such as inflammation.
NS 12 GENE-CODED PROTEIN CHARACTERISTICS
This gene is expressed in activated neutrophils, endothelial cells, T cells and, to a lesser extent, in the brain and liver.
Accordingly, the polynucleotides and polypeptides disclosed herein are useful as reagents for differential identification of tissue (s) or cell type (s) present in a biological sample and for diagnosing diseases and conditions that include, but are not limited to AIDS, immune disorders and susceptibility to infectious disease. Similarly, polypeptides and antibodies directed to these polypeptides are useful in providing immunological probes for differential identification of tissue (s) or cell type (s). For a number of prior tissue or cell disorders, particularly the immune system and skin, expression of this gene at significantly higher or lower levels can be routinely detected in certain tissues and cell types (e.g. neutrophils and other blood cells, endothelial cells, T cells and other cells and immune system tissue, brain and other nervous and liver tissue and cancerous and injured tissues) or body fluids (eg, serum, plasma, urine, synovial fluid or spinal fluid) or other tissue or cell sample taken from an individual having such a disorder, relative to the standard level of gene expression, i. e., the level of expression in healthy body tissue or fluid of an individual not having the disorder. Preferred epitopes include those comprising a sequence shown in SEQ ID NO: 59 as residues: Glu-41 to Val-46.
This gene product is useful for the diagnosis and / or treatment of a variety of disorders, hematopoietic disorders, neurological disorders, including liver disease disorders and disorders involving angiogenesis.
GENE N-CODED PROTEIN CHARACTERISTICS<sup>s</sup> 13
This gene is expressed in keratinocytes and, to a lesser extent, in endothelial cells and placenta.
Accordingly, the polynucleotides and polypeptides of the invention are useful as reagents for differential identification of tissue (s) or cell type (s) present in a biological sample and for diagnosing diseases and conditions that include, but are not limited to , impaired healing. Similarly, polypeptides and antibodies directed to these polypeptides are useful in providing immunological probes for differential identification of tissue (s) or cell type (s). For a number of prior tissue or cell disorders, particularly skin disorders, expression of this gene at significantly higher or lower levels can be routinely detected in certain tissues and cell types (eg, keratinocytes and other skin cells, endothelial cells). and placenta and cancerous and injured tissues) or body fluids (e.g. serum, plasma, urine, synovial fluid or spinal fluid) or other tissue or cell sample taken from an individual having such a disorder relative to the standard level of gene expression, ie, the level of expression in body tissue or fluid. healthy condition of an individual not having the disorder. Preferred epitopes include those comprising a sequence shown in SEQ ID NO: 60 as residues: Pro-35 to Trp-42, Ala-53 to Asp-62 and / or Arg-103 to Pro-113.
Tissue distribution indicates that the protein products of this gene are useful for the treatment of scarring deficiency and skin disorders.
GENE N-CODED PROTEIN CHARACTERISTICS<sup>s</sup> 14
This gene is expressed in the kidney and to a lesser extent in embryonic tissues.
Accordingly, the polynucleotides and polypeptides disclosed herein are useful as reagents for differential identification of tissue (s) or cell type (s) present in a biological sample and for diagnosing diseases and conditions that include, but are not limited to , renal insufficiency. Similarly, polypeptides and antibodies directed to these polypeptides are useful in providing immunological probes for differential identification of tissue (s) or cell type (s). For a number of prior tissue or cell disorders, particularly the kidney, expression of this gene at significantly higher or lower levels can be routinely detected in certain tissues (eg, kidney, embryonic tissue and other rapidly developing tissue (eg, in division) and cancerous and injured tissues) or body fluids (e.g. serum, plasma, urine, synovial fluid or spinal fluid) or other tissue or cell sample taken from an individual having such a disorder relative to the standard gene expression level, ie, the expression level in body tissue or fluid healthy condition of an individual not having the disorder.
GENE N2-ENCODED PROTEIN CHARACTERISTICS 15
This gene is expressed primarily in the brain and, to a lesser extent, in the liver.
Accordingly, the polynucleotides and polypeptides disclosed herein are useful as reagents for differential identification of tissue (s) or cell type (s) present in a biological sample and for diagnosing diseases and conditions that include, but are not limited to , depression, manic depression and other mental disorders. Similarly, polypeptides and antibodies directed to these polypeptides are useful in providing immunological probes for differential identification of tissue (s) or cell type (s).
For a number of prior tissue or cell disorders, particularly the central nervous system, expression of this gene at significantly higher or lower levels may be routinely detected in certain tissues (eg, brain and other nervous system tissue and liver and tissues). cancerous and injured) or body fluids (e.g. serum, plasma, urine, synovial fluid or spinal fluid) or other tissue or cell sample taken from an individual having such a disorder relative to the standard gene expression level, ie, the expression level in body tissue or fluid healthy condition of an individual not having the disorder.
Tissue distribution indicates that the protein products of this gene are useful for treating central nervous system disorders such as depression and other mental illnesses.
GENE NODE 16 CODED PROTEIN CHARACTERISTICS
This gene is expressed in fetal brain and, to a lesser extent, in placenta, endothelial cells, fetal lung and T cells.
Accordingly, the polynucleotides and polypeptides disclosed herein are useful as reagents for differential identification of tissue (s) or cell type (s) present in a biological sample and for diagnosing diseases and conditions that include, but are not limited to , restenosis, birth defects and immune disorders. Similarly, polypeptides and antibodies directed to these polypeptides are useful in providing immunological probes for differential identification of tissue (s) or cell type (s). For a number of prior tissue or cell disorders, particularly the cardiovascular system and developmental process, expression of this gene at significantly higher or lower levels can be routinely detected in certain tissues and cell types (eg, placenta, endothelial cells). , lung and T cells and other cells and tissue of the immune system and cancerous and injured tissues) or body fluids (e.g. serum, plasma, urine, synovial fluid or spinal fluid) or other tissue or cell sample taken from an individual having such a disorder relative to the standard gene expression level, ie, the level of expression in body tissue or fluid. healthy condition of an individual not having the disorder. Preferred epitopes include those comprising a sequence shown in SEQ ID NO: 63 as residues: Gln-36 to Lys-42 and / or Glu-89 to
Arg-104.
Tissue distribution indicates that the protein products of this gene are useful for the development of agonists and / or antagonists for treatment of nervous system disorders and fetal development.
FEATURES OF NS 17 GENE-ENCODED PROTEIN
This gene is expressed in hemangiopericytoma and, to a lesser extent, in fetal tissue.
Accordingly, the polynucleotides and polypeptides disclosed herein are useful as reagents for differential identification of tissue (s) or cell type (s) present in a biological sample and for diagnosing diseases and conditions that include, but are not limited to , hemangiopericytomas and other cancers, as well as developmental disorders. Similarly, polypeptides and antibodies directed to these polypeptides are useful in providing immunological probes for differential identification of tissue (s) or cell type (s). For a number of prior tissue or cell disorders, expression of this gene at significantly higher or lower levels can be routinely detected in certain tissues (e.g. vascular tissue, pericytic tissue and developing tissue and cancerous and injured tissues) or body fluids (eg, serum, plasma, urine, synovial fluid or spinal fluid) or other tissue or cell sample taken from an individual having such disorder, relative to the level of standard gene expression, ie, the level of expression in healthy body tissue or fluid of an individual not having the disorder. Preferred epitopes include those comprising a sequence shown in SEQ ID NO: 64 as residues: Glu-43 to Pro-51, Gly-71 to Arg-82, Pro-96 to Arg-103, and / or Thr-130 to Gly- 140
Polynucleotides and polypeptides related to this gene are considered to be useful for the treatment and diagnosis of tumors, particularly hemangiopericytomas, and for the treatment of developmental disorders.
Characteristics of NS 18 GENE-CODED PROTEIN
This gene is expressed in fetal liver and to a lesser extent in brain and T cells.
Accordingly, the polynucleotides and polypeptides disclosed herein are useful as reagents for differential identification of tissue (s) or cell type (s) present in a biological sample and for diagnosing diseases and conditions that include, but are not limited to , fetal disorders, fetal development and immune disorders. Similarly, polypeptides and antibodies directed to these polypeptides are useful in providing immunological probes for differential identification of tissue (s) or cell type (s). For a number of prior tissue or cell disorders, particularly the hepatic system, nervous system and immune system, expression of this gene at significantly higher or lower levels can be routinely detected in certain tissues and cell types (eg, liver, brain and other nervous system tissue and T cells and other immune system cells and tissue and cancerous and injured tissues) or body fluids (e.g. serum, plasma, urine, synovial fluid or spinal fluid) or other tissue or cell sample taken from an individual having such a disorder relative to the standard gene expression level, ie, the level of expression in body tissue or fluid. healthy condition of an individual not having the disorder.
Tissue distribution indicates that the protein products of this gene are useful for identifying agonists and / or antagonists for treating mental illnesses such as schizophrenia and depression. The gene product may also be useful for monitoring fetal development during pregnancy.
GENE NODE 19 CODIFIED PROTEIN CHARACTERISTICS
This gene is expressed in T cells and, to a lesser extent, in the brain.
Accordingly, the polynucleotides and polypeptides disclosed herein are useful as reagents for differential identification of tissue (s) or cell type (s) present in a biological sample and for diagnosing diseases and conditions that include, but are not limited to , central nervous system disorders and immune disorders. Similarly, polypeptides and antibodies directed to these polypeptides are useful in providing immunological probes for differential identification of a number of prior tissue or cell disorders, particularly the central nervous system and immune system, expression of this gene at significantly higher levels. Upper or lower can be routinely detected in certain tissues and cell types (e.g. T cells and other cells and tissue of the immune system and brain and other nervous system tissue and cancerous and injured tissues) or body fluids (eg, serum, plasma, urine, synovial fluid or spinal fluid) or other tissue sample or cell taken from an individual having such a disorder, relative to the standard level of gene expression, i.
e., the level of expression in healthy body tissue or fluid of an individual not having the disorder. Preferred epitopes include those comprising a sequence shown in
SEQ ID NO: 66 as residues: Lys-69 to Leu-74, Ser-92 to Phe-97, Asp-109 to Leu-117, Leu-142 to Ser-159, Thr-166 to Glu-183, Ala -191 to Glu-205 and / or Pro-213 to Glu-220.
Tissue distribution indicates that the protein products of this gene are useful for the development of drugs for treating disorders affecting the central nervous system and the immune system.
GENE N2 ENCODED PROTEIN CHARACTERISTICS 20
The translation product of this gene shares sequence homology with a C. elegans ORF that appears to be a transmembrane protein. (See Genbank Access No. 790406). This contig has two probable phase shifts between the +2 and +3 phases, based on homology to the C. elegans gene. This phase shift can be easily resolved by sequencing the deposited clone. Furthermore, this gene maps to chromosome 8 and therefore can be used as a marker in binding analysis for chromosome 8.
This gene is ubiquitously expressed, including T cells and amygdala.
Accordingly, the polynucleotides and polypeptides disclosed herein are useful as reagents for differential identification of tissue (s) or cell type (s) present in a biological sample and for diagnosing diseases and conditions that include, but are not limited to , cancer. Similarly, polypeptides and antibodies directed to these polypeptides are useful in providing immunological probes for differential identification of tissue (s) or cell type (s). For a number of prior tissue or cell disorders, particularly the immune system and endocrine system, expression of this gene at significantly higher or lower levels can be routinely detected in certain tissues and cell types (eg, T cells and other cells). and immune system tissue, amygdala and cancerous and injured tissues) or body fluids (e.g. serum, plasma, urine, synovial fluid or spinal fluid) or other tissue or cell sample taken from an individual having such a disorder relative to the standard level of gene expression, ie, the level of expression in body tissue or fluid. healthy condition of an individual not having the disorder.
Tissue distribution ubiquitates C. transmembrane-like protein.
and homology to an elegans, indicates that the protein product of this gene in both vertebrates as diagnosis or treatment plays an important role in invertebrates and is useful for disorders related to this gene.
NS 21 GENE-ENCODED PROTEIN CHARACTERISTICS
This gene is expressed primarily in embryonic cells and testes and to a lesser extent in ovarian, hepatoma, kidney, endothelium and smooth muscle cells.
Accordingly, the polynucleotides and polypeptides disclosed herein are useful as reagents for differential identification of tissue (s) or cell type (s) present in a biological sample and for diagnosing diseases and conditions that include, but are not limited to , metabolic disorder, abnormal embryonic development and tumor. Similarly, polypeptides and antibodies directed to these polypeptides are useful in providing immunological probes for differential identification of tissue (s) or cell type (s). For a number of prior tissue or cell disorders, particularly embryonic or vascular tissues, expression of this gene at significantly higher or lower levels can be routinely detected in certain tissues and cell types (e.g. ovarian tissue and other reproductive tissue, kidney, endothelial cells and smooth muscle cells and cancerous and injured tissues) or body fluids (eg, serum, plasma, urine, synovial fluid or spinal fluid) or other tissue sample or cell taken from an individual having such a disorder, relative to the level of standard gene expression, ie, the level of expression in healthy body fluid or tissue of an individual not having the disorder.
Tissue distribution and homology to NADH dehydrogenase indicates that polynucleotides and polypeptides corresponding to this gene are useful for diagnosis and / or treatment of metabolic disorders, particularly involving embryonic and vascular tissues.
GENE CODIFIED PROTEIN CHARACTERISTICS NS 22 The translation product of this gene shares sequence homology with the alpha-adrenergic receptor thought to be important in neuronal signal transmission.
This gene is expressed primarily in the breast lymph node and, to a lesser extent, in uterine cancer and testicular tumor.
Accordingly, the polynucleotides and polypeptides disclosed herein are useful as reagents for differential identification of tissue (s) or cell type (s) present in a biological sample and for diagnosing diseases and conditions that include, but are not limited to , neurological disorders. Similarly, polypeptides and antibodies directed to these polypeptides are useful in providing immunological probes for differential identification of tissue (s) or cell type (s). For a number of prior tissue or cell disorders, particularly neurological, breast lymph node, uterine and testicular cancer, expression of this gene at significantly higher or lower levels may be routinely detected in certain tissues (eg, breast tissue, lymphoid tissue, uterine and testicular tissue and other reproductive tissue and cancerous and injured tissues) or body fluids (e.g. serum, plasma, urine, synovial fluid, or spinal fluid) or other tissue or cell sample taken from an individual having such a disorder, relative to the standard level of gene expression, e.g. e., the level of expression in healthy body tissue or fluid of an individual not having the disorder.
Tissue distribution and homology to the alpha 1C adrenergic receptor indicate that polynucleotides and polypeptides corresponding to this gene are useful for transmitting signals to neurons.
GENE N-CODED PROTEIN CHARACTERISTICS<sup>s</sup> 23
The translation product of this gene shares homology of the G protein-linked receptor sequence, which is thought to be important in mediating a wide variety of physiological functions and belongs to a superfamily of limb genes, ranging from guimiocin receptor to receptor. of bradykinin.
This gene was also recently cloned by another group, designating the platelet activating receptor homologue gene.
(See Genbank Access N °
2580588). Preferred polypeptide fragments comprise the following amino acids: LSIIFLAFVSIDRCLQL (SEQ ID NO:
117) and
GSCFAIWAFIQENTNHRCVSIY
LINLLTADFLLTLALPVKIWDLGVAPWKLKIFHCQVTACLIYIN (SEQ ID NO: 118).
Polynucleotide fragments encoding these polypeptide fragments are also preferred.
This gene is mainly expressed in immune cells, particularly lymphocytes.
Accordingly, the polynucleotides and polypeptides disclosed herein are useful as reagents for differential identification of tissue (s) or cell type (s) present in a biological sample and for diagnosing diseases and conditions which include, but are not limited to , lymphocyte disorders and other immune cells. Similarly, polypeptides and antibodies directed to these polypeptides are useful in providing immunological probes for differential identification of tissue (s) or cell type (s). For a number of prior tissue or cell disorders, particularly the immune system, expression of this gene at significantly higher or lower levels may be routinely detected in certain tissues and cell types (e.g. lymphocytes and other cells and tissue of the immune system and cancerous and injured tissues) or body fluids (eg, serum, plasma, urine, synovial fluid or spinal fluid) or other tissue or cell sample taken from an individual having such disorder, relative to the level of standard gene expression, ie, the level of expression in healthy body tissue or fluid of an individual not having the disorder. Preferred epitopes include those comprising a sequence shown in SEQ ID NO: 70 as residues: Asp-59 to
Asn-65, Lys-72 to Trp-79, Tyr-110 to Val-121 and / or Ala-204 to
Asn-215.
Tissue distribution and G protein-linked receptor homology indicate that the polynucleotides and polypeptides corresponding to this gene are useful as a chemokine receptor in lymphocytes that regulate immune response.
NS 24 GENE-CODED PROTEIN CHARACTERISTICS
The translation product of this gene shares sequence homology with the protein disulfide isomerase, which is thought to be important in protein folding and protein-protein interaction. This gene also shares homology with genes having thioredoxin domains. (See Access No. 1943817). This gene also maps to chromosome 9 and therefore may be useful in binding analysis as a marker for chromosome 9.
This gene is expressed primarily in tumor tissues and to a lesser extent in a wide variety of normal tissues.
Accordingly, the polynucleotides and polypeptides disclosed herein are useful as reagents for differential identification of tissue (s) or cell type (s) present in a biological sample and for diagnosing diseases and conditions that include, but are not limited to , disorders due to inappropriate protein folding and protein-protein interaction. Similarly, polypeptides and antibodies directed to these polypeptides are useful in providing immunological probes for differential identification of tissue (s) or cell type (s). For a number of prior tissue or cell disorders, particularly the tumorigenic process, expression of this gene at significantly higher or lower levels may be routinely detected in certain tissues (e.g. cancerous and injured tissues) or body fluids (eg, serum, plasma, urine, synovial fluid or spinal fluid) or other tissue or cell sample taken from an individual having such a disorder, relative to the standard level of gene expression, ie, the level of expression in healthy body fluid or tissue of an individual not having the disorder. Preferred epitopes include those comprising a sequence shown
<td>at SEQ</td><td>ID No: 71</td><td colspan="3">as waste: Glu-</td><td> 78</td><td>the Asn-83,</td><td>Asp-91</td><td>The</td>
<td>Gln-100,</td><td>Glu-122</td><td>The</td><td>Ser-128,</td><td>Arg-137</td><td>The</td><td>Pro-143,</td><td>Asp-15 7</td><td>The</td>
<td>Asn-162,</td><td>Glu-168</td><td>The</td><td>Asn-174,</td><td>Ser-199</td><td>The</td><td>Gly-206,</td><td>Pro-213</td><td>The</td>
<td>Wing 218,</td><td>Glu-251</td><td>The</td><td>Thr-257,</td><td>Ser-353</td><td>The</td><td>His-361,</td><td>Gly-363</td><td>The</td>
<td>Wing 375,</td><td>Pro-382 a</td><td>Phe</td><td>-387 and / or</td><td>Arg-401 a</td><td colspan="2">Leu-406.</td><td></td><td></td>
Tissue distribution and homology to the protein disulfide isomerase indicate that the polynucleotides and polypeptides corresponding to this gene are useful for protein fold regulation and protein-protein interaction in tumor tissues.
GENE N2-CODED PROTEIN CHARACTERISTICS 25
This gene is expressed primarily in leukocytes involved in immune defense, including T cells, macrophages, neutrophils and, to a lesser extent, synovial membrane, adrenal gland tumor, adipose and placenta.
Accordingly, the polynucleotides and polypeptides disclosed herein are useful as reagents for differential identification of tissue (s) or cell type (s) present in a biological sample and for diagnosing diseases and conditions that include, but are not limited to , defects or disorders in leukocytes. Similarly, polypeptides and antibodies directed to these polypeptides are useful in providing immunological probes for differential identification of tissue (s) or cell type (s). For a number of prior tissue or cell disorders, particularly the immune and defense systems, expression of this gene at significantly higher or lower levels can be routinely detected in certain tissues and cell types (e.g. leukocytes and other cells and tissues of the immune system, synovial membrane, adrenal gland, adipose and placenta, and cancerous and injured tissues) or body fluids (eg, serum, plasma, urine, synovial fluid or spinal fluid) or other tissue or cell sample taken from an individual having such a disorder, relative to the standard gene expression level, ie, the level of healthy tissue or body fluid expression of an individual not having the disorder.
Tissue distribution indicates that polynucleotides and polypeptides corresponding to this gene are useful for regulating leukocyte function and may be used for diagnosis and treatment of disorders in immune and defense systems.
GENE N2-CODED PROTEIN CHARACTERISTICS 26
This gene is expressed in a variety of tissues and cell types, including colon cancer, breast cancer, neutrophils, T cells, spinal cord, fibroblasts and vascular endothelial cells.
Accordingly, the polynucleotides and polypeptides disclosed herein are useful as reagents for differential identification of tissue (s) or cell type (s) present in a biological sample and for diagnosing diseases and conditions that include, but are not limited to. a, cancer, disorder and abnormalities in leukocytes and other tissues. Similarly, polypeptides and antibodies directed to these polypeptides are useful in providing immunological probes for differential identification of tissue (s) or cell type (s). For a number of prior tissue or cell disorders, particularly those involved in tumorigenesis and immune defense systems, expression of this gene at significantly higher or lower levels can be routinely detected in certain tissues and cell types (eg, colon, breast tissue, neutrophils, T cells and
6th other blood cells, spinal cord and other nervous system tissue, endothelial cells, vascular and fibroblast tissues and cancerous and injured tissues) or body fluids (eg serum, plasma, urine, synovial fluid or spinal fluid) or other tissue sample or cell taken from an individual having such a disorder, relative to the standard gene expression level, ie, the level of expression in healthy body fluid or tissue of an individual not having the disorder.
Tissue distribution indicates that polynucleotides and polypeptides corresponding to this gene are useful for diagnosis and treatment of cancer or immune system disorders.
GENE N-CODED PROTEIN CHARACTERISTICS<sup>s</sup> The translation product of this gene shares sequence homology with a mouse pancreatic polypeptide. (See Genbank Access No. 200464). Thus, this gene is likely to have activity similar to that of mouse pancreatic polypeptide.
Preferred polypeptide fragments comprise the amino acid sequence:
APLETMQNKPRAPQKRALPFPEL
ELRDYASVLTRYSLGLRNKEPSLGHRWGTQKLGRSPC (SEQ ID NO: 119). Also preferred are polynucleotide fragments encoding this polypeptide fragment.
This gene is expressed primarily in neutrophils and, to a lesser extent, in induced endothelial cells.
Accordingly, the polynucleotides and polypeptides disclosed herein are useful as reagents for differential identification of tissue (s) or cell type (s) present in a biological sample and for diagnosing diseases and conditions that include, but are not limited to , neutrophil or leukocyte adhesion disorders. Similarly, polypeptides and antibodies directed to these polypeptides are useful in providing immunological probes for differential identification of tissue (s) or cell type (s). For a number of prior tissue or cell disorders, particularly the immune system, expression of this gene at significantly higher or lower levels may be routinely detected in certain tissues and cell types (e.g. neutrophils and other blood cells and endothelial cells and cancerous and injured tissues) or body fluids (eg, serum, plasma, urine, synovial fluid or spinal fluid) or other tissue or cell sample taken from an individual having such a disorder relative to the standard level of gene expression, ie, the level of expression in healthy body fluid or tissue of an individual not having the disorder.
Tissue distribution indicates that polynucleotides and polypeptides corresponding to this gene are useful for regulating neutrophil or leukocyte adhesion to endothelial cells. It can be used to diagnose or treat disorders associated with neutrophils and vascular endothelial cells.
FEATURES OF NS 28 GENE-ENCODED PROTEIN
This gene is mainly expressed in prostate BPH.
Accordingly, the polynucleotides and polypeptides disclosed herein are useful as reagents for differential identification of tissue (s) or cell type (s) present in a biological sample and for diagnosing diseases and conditions that include, but are not limited to , benign prostate hypertrophy. Similarly, polypeptides and antibodies directed to these polypeptides are useful in providing immunological probes for differential identification of tissue (s) or cell type (s). For a number of prior tissue or cell disorders, particularly the male urinary system, expression of this gene at significantly higher or lower levels can be routinely detected in certain tissues (e.g. prostate and cancerous and injured tissues) or body fluids (eg, serum, plasma, urine, synovial fluid or spinal fluid) or other tissue or cell sample taken from an individual having such a disorder with respect to the level of gene expression pattern, ie the level of expression in healthy body fluid or tissue of an individual not having the disorder.
Tissue distribution indicates that polynucleotides and polypeptides corresponding to this gene are useful for diagnosis and treatment of benign prostate hypertrophy or prostate cancer.
GENE N-CODED PROTEIN CHARACTERISTICS<sup>2</sup> 29
The translation product of this gene shares sequence homology with C16C10.7, a C. elegans gene similar to zinc finger protein, a protein involved in DNA ligation. Thus, this protein is expected to share certain biological activities with C16C10.7, including DNA binding activity.
This gene is expressed primarily in activated T cells and, to a lesser extent, in fetal brain, TNF-induced amniotic cells and epididymis.
Accordingly, the polynucleotides and polypeptides disclosed herein are useful as reagents for differential identification of tissue (s) or cell type (s) present in a biological sample and for diagnosing diseases and conditions that include, but are not limited to , immune or neurodegenerative disorders. Similarly, polypeptides and antibodies directed to these polypeptides are useful in providing immunological probes for differential identification of tissue (s) or cell type (s). For a number of prior tissue or cell disorders, particularly the central nervous and immune systems, expression of this gene at significantly higher or lower levels may be routinely detected in certain tissues and cell types (e.g. T cells and other immune cells and tissue, brain and other nervous system tissue, amniotic and epididymal cells and other reproductive tissue and cancerous and injured tissues) or body fluids (eg, serum, plasma, urine, synovial fluid or spinal fluid) or other tissue or cell sample taken from an individual having such a disorder, relative to the standard level of gene expression, i. e., the level of expression in healthy body tissue or fluid of an individual not having the disorder.
Tissue distribution indicates that the protein products of this gene are useful for the diagnosis and treatment of immune and / or neurodegenerative disorders and promotion of neuron survival and differentiation.
GENE N2 30 CODED PROTEIN CHARACTERISTICS
This gene is expressed primarily in T cells and, to a lesser extent, in the bone marrow.
Accordingly, the polynucleotides and polypeptides disclosed herein are useful as reagents for identification present in a biological sample and for diagnosing diseases and conditions that include, but are not limited to, immune disorders, including autoimmune disease. Similarly, polypeptides and antibodies directed to these polypeptides are useful in providing immunological probes for differential identification of tissue (s) or cell type (s). For a number of prior tissue or cell disorders, particularly the immune system, expression of this gene at significantly higher or lower levels can be routinely detected in certain tissues and cell types (eg, τ cells and other cells and tissue of the immune system and bone marrow and cancerous and injured tissues) or body fluids (e.g. serum, plasma, urine, synovial fluid or spinal fluid) or other tissue or cell sample taken from an individual having such a disorder relative to the standard gene expression level, ie, the level of expression in body tissue or fluid. healthy condition of an individual not having the disorder. This gene is thought to map to chromosome 4: Transcript map: WI-11395, Ch.4, D4S395D4S414; White point map: WI-11395, Ch.4, 498.0 cR; dbSTS accesses: G21269.
Tissue distribution indicates that the protein products of this gene are useful for diagnosis and treatment of immunologically mediated disorders, as they are thought to play a role in the proliferation, survival, differentiation and / or activation of a variety of hematopoietic cells, including early progenitors. or hematopoietic stem cells.
GENE N2 ENCODED PROTEIN CHARACTERISTICS 31
This gene is mainly expressed in human skin.
Accordingly, the polynucleotides and polypeptides disclosed herein are useful as reagents for differential identification of tissue (s) or cell type (s) present in a biological sample and for diagnosing diseases and conditions that include, but are not limited to , scarring and skin cancers. Similarly, polypeptides and antibodies directed to these polypeptides are useful in providing immunological probes for differential identification of tissue (s) or cell type (s). For a number of prior tissue or cell disorders, particularly the integumentary system, expression of this gene at significantly higher or lower levels can be routinely detected in certain tissues (e.g. cancerous and injured skin and tissues) or body fluids (eg, serum, plasma, urine, synovial fluid or spinal fluid) or other tissue or cell sample taken from an individual having such a disorder with respect to the level of gene expression pattern, ie, the level of expression in healthy body tissue or fluid of an individual not having the disorder.
Tissue distribution indicates that the protein products of this gene are useful for diagnosis and treatment of skin cancers and scarring.
N2 GENE-CODE PROTEIN CHARACTERISTICS 32 translation product of this gene shares sequence homology with Human Tear Albumin (Genbank Accession No. gil307518) and rat Odor Binding Protein (Genbank Accession No. gil207551), which thinks are both important in molecular binding and transport.
This gene is mainly expressed in endometrial tumor.
Accordingly, the disclosed acute polynucleotides and polypeptides are useful as reagents for differential identification of the tissue (s) or cell type (s) present in a biological sample and for diagnosing diseases and conditions that include, but are not limited to , cancers of the endometrium, skin and hematopoietic system. Similarly, polypeptides and antibodies directed to these polypeptides are useful in providing immunological probes for differential identification of tissue (s) or cell type (s). For a number of prior tissue or cell disorders, particularly the hematopoietic system, expression of this gene at significantly higher or lower levels may be routinely detected in certain tissues (e.g. cells and tissue of the immune and endometrium system and other reproductive system tissue and cancerous and injured tissues) or body fluids (eg, serum, plasma, urine, synovial fluid or spinal fluid) or other tissue or cell specimen taken from an individual having such a disorder, relative to the standard level of gene expression, ie, the level of expression in healthy body fluid or tissue of an individual not having the disorder.
Tissue distribution and homology to the molecular binding and transport gene family indicate that the protein products of this gene are useful for the diagnosis and treatment of endometrial and hematopoietic cancers, as well as for the treatment of autoimmune disorders such as inflammation.
5
<td>Gene No.</td><td>ID of Clone cDNA</td><td>No. and Date of Deposit ATCC</td><td>Vector</td><td>NT SEQ ID No. X</td><td>Seq. NT Total</td><td>5 'NT of Seq. in Clone</td><td>3 'NT of Seq. in Clone</td><td>5 'NT of Cotton Initiation</td><td>5 'NT of First AA from Pep. Signal</td><td>AA SEQ ID No. Y</td><td>Pep's first. Signal</td><td>Pep's last AA. Signal</td><td>First AA from Portion Segregated</td><td>Last AA from ORF</td>
<td> 1</td><td>HSVBZ80</td><td> 209075 05/22/97</td><td>Uni-ZAP XR</td><td> 11</td><td> 1169</td><td> 64</td><td> 1060</td><td> 162</td><td> 162</td><td> 48</td><td> 1</td><td> 38</td><td> 39</td><td> 145</td>
<td> 2</td><td>HTAAU21</td><td> 209075 05/22/97</td><td>Uni-ZAP XR</td><td> 12</td><td> 1310</td><td> 1</td><td> 1310</td><td> 283</td><td> 283</td><td> 49</td><td> 1</td><td> 18</td><td> 19</td><td> 311</td>
<td> 3</td><td>HTLEK16</td><td> 209075 05/22/97</td><td>Uni-ZAP XR</td><td> 13</td><td> 1139</td><td> 19</td><td> 1111</td><td></td><td> 251</td><td> 50</td><td> 1</td><td> 21</td><td> 22</td><td> 45</td>
<td> 4</td><td>HUSIR91</td><td> 209075 05/22/97</td><td>pSportl</td><td> 14</td><td> 2271</td><td> 743</td><td> 2271</td><td> 59</td><td> 59</td><td> 51</td><td> 1</td><td> 23</td><td> 24</td><td> 466</td>
<td> 4</td><td>HUSIR91</td><td> 209075 05/22/97</td><td>pSportl</td><td> 43</td><td> 2581</td><td> 1035</td><td> 2154</td><td> 1148</td><td> 1148</td><td> 80</td><td> 1</td><td> 27</td><td> 28</td><td> 207</td>
<td> 5</td><td>HADMC21</td><td> 209075 05/22/97</td><td>pBluescript</td><td> 15</td><td> 626</td><td> 60</td><td> 479</td><td> 91</td><td> 91</td><td> 52</td><td> 1</td><td> 51</td><td> 52</td><td> 82</td>
<td> 6</td><td>HAGFM45</td><td> 209075 05/22/97</td><td>Uni-ZAP XR</td><td> 16</td><td> 2118</td><td> 1170</td><td> 2058</td><td> 1248</td><td> 1248</td><td> 53</td><td> 1</td><td> 16</td><td> 17</td><td> 62</td>
<td> 7</td><td>HAIBE65</td><td> 209075 05/22/97</td><td>Uni-ZAP XR</td><td> 17</td><td> 1076</td><td> 396</td><td> 993</td><td> 528</td><td> 528</td><td> 54</td><td> 1</td><td> 31</td><td> 32</td><td> 123</td>
<td> 8</td><td>HAQBH57</td><td> 209075 05/22/97</td><td>Uni-ZAP XR</td><td> 18</td><td> 1379</td><td> 420</td><td> 1306</td><td> 618</td><td> 618</td><td> 55</td><td> 1</td><td> 25</td><td> 26</td><td> 179</td>
9th (continuation)
<td>Gene No.</td><td>ID of Clone cDNA</td><td>No. and Date of Deposit ATCC</td><td>Vector</td><td>NT SEQ ID No. X</td><td>Seq. NT Total</td><td>5 'NT of Seq. in Clone</td><td>3 'NT of Seq. in Clone</td><td>5 'NT of Cotton Initiation</td><td>5 'NT First AA from Pep. Signal</td><td>AA SEQ ID No. Y</td><td>Pep's first. Signal</td><td>Pep's last AA. Signal</td><td>First AA from Portion Segregated</td><td>Last AA from ORF</td>
<td> 9</td><td>HATCX80</td><td> 209075 05/22/97</td><td>Uni-ZAP XR</td><td> 19</td><td> 1337</td><td> 47</td><td> 1337</td><td> 199</td><td> 199</td><td> 55</td><td> 1</td><td> 18</td><td> 19</td><td> 286</td>
<td> 10</td><td>HCFLQ84</td><td> 209075 05/22/97</td><td>pSportl</td><td> 20</td><td> 1390</td><td> 237</td><td> 1390</td><td> 410</td><td> 410</td><td> 57</td><td> 1</td><td> 20</td><td> 21</td><td> 33</td>
<td> 11</td><td>HCFLS78</td><td> 209075 05/22/97</td><td>pSportl</td><td> 21</td><td> 1431</td><td> 178</td><td> 981</td><td> 420</td><td> 420</td><td> 58</td><td> 1</td><td> 21</td><td> 22</td><td> 23</td>
<td> 12</td><td>HTADI12</td><td> 209075 05/22/97</td><td>Uni-ZAP XR</td><td> 22</td><td> 2539</td><td> 69</td><td> 2539</td><td> 104</td><td> 104</td><td> 59</td><td> 1</td><td> 27</td><td> 28</td><td> 45</td>
<td> 13</td><td>HEMCM42</td><td> 209075 05/22/97</td><td>Uni-ZAP XR</td><td> 23</td><td> 1041</td><td> 48</td><td> 1007</td><td> 58</td><td> 58</td><td> 60</td><td> 1</td><td> 29</td><td> 30</td><td> 113</td>
<td> 14</td><td>HEONP72</td><td> 209075 05/22/97</td><td>pSportl</td><td> 24</td><td> 1952</td><td> 1</td><td> 1947</td><td> 181</td><td> 181</td><td> 61</td><td> 1</td><td> 19</td><td> 20</td><td> 31</td>
<td> 15</td><td>HFCDW34</td><td> 209075 05/22/97</td><td>Uni-ZAP XR</td><td> 25</td><td> 1228</td><td> 321</td><td> 1228</td><td> 525</td><td> 525</td><td> 62</td><td> 1</td><td> 24</td><td> 25</td><td> 80</td>
<td> 16</td><td>HTTEU91</td><td> 209075 05/22/97</td><td>Uni-ZAP XR</td><td> 26</td><td> 1340</td><td> 325</td><td> 1340</td><td> 15</td><td> 15</td><td> 53</td><td> 1</td><td> 18</td><td> 19</td><td> 103</td>
<td> 17</td><td>HHGBF89</td><td> 209075 05/22/97</td><td>Lambda ZAP II</td><td> 27</td><td> 806</td><td> 31</td><td> 806</td><td> 77</td><td> 77</td><td> 54</td><td> 1</td><td> 19</td><td> 20</td><td> 145</td>
<td> 17</td><td>HHGBF89</td><td> 209075</td><td>LambdaZAP</td><td> 45</td><td> 796</td><td> 31</td><td> 796</td><td> 77</td><td> 77</td><td> 82</td><td> 1</td><td> 25</td><td> 25</td><td> 145</td>
(continuation)
<td>Gene No.</td><td>ID of Clone cDNA</td><td>No. and Date of Deposit ATCC</td><td>Vector</td><td>NT SEQ ID No. X</td><td>Seq. NT Total</td><td>5 'NT of Seq. in Clone</td><td>3 'NT of Seq. in Clone</td><td>5 'NT of Cotton Initiation</td><td>5 'NT First AA from Pep. Signal</td><td>AA SEQ ID No. Y</td><td>Pep's first. Signal</td><td>Pep's last AA. Signal</td><td>First AA from Portion Segregated</td><td>Last AA from ORF</td>
<td></td><td></td><td> 05/22/97</td><td>II</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>
<td> 18</td><td>HKIYQ55</td><td> 209075 05/22/97</td><td>pBluescript</td><td> 28</td><td> 696</td><td> 1</td><td> 584</td><td> 98</td><td> 98</td><td> 55</td><td> 1</td><td> 17</td><td> 18</td><td> 30</td>
<td> 19</td><td>HKMLN27</td><td> 209075 05/22/97</td><td>pBluescript</td><td> 29</td><td> 1007</td><td> 71</td><td> 963</td><td> 129</td><td> 129</td><td> 66</td><td> 1</td><td> 23</td><td> 24</td><td> 259</td>
<td> 20</td><td>HKIAC30</td><td> 209022 05/08/97</td><td>Uni-ZAP XR</td><td> 30</td><td> 2017</td><td> 125</td><td> 2007</td><td> 161</td><td> 161</td><td> 67</td><td> 1</td><td></td><td></td><td> 22</td>
<td> 21</td><td>HKIXB95</td><td> 209022 05/08/97</td><td>pBluescript</td><td> 31</td><td> 699</td><td> 196</td><td> 699</td><td> 230</td><td> 230</td><td> 68</td><td> 1</td><td> 22</td><td> 23</td><td> 26</td>
<td> 22</td><td>HLMIY86</td><td> 209022 05/08/97</td><td>Lambda ZAP II</td><td> 32</td><td> 1254</td><td> 1</td><td> 1264</td><td></td><td> 342</td><td> 69</td><td> 1</td><td> 16</td><td> 17</td><td> 28</td>
<td> 23</td><td>HLYAZ51</td><td> 209022 05/08/97</td><td>pSportl</td><td> 33</td><td> 997</td><td> 74</td><td> 997</td><td> 205</td><td> 205</td><td> 70</td><td> 1</td><td> 20</td><td> 21</td><td> 215</td>
<td> 24</td><td>HMQDT36</td><td> 209022 05/08/97</td><td>Uni-ZAP XR</td><td> 34</td><td> 1914</td><td> 37</td><td> 1897</td><td> 192</td><td> 192</td><td> 71</td><td> 1</td><td> 32</td><td> 33</td><td> 406</td>
<td> 25</td><td>HNEDF25</td><td> 209022 05/08/97</td><td>Uni-ZAP XR</td><td> 35</td><td> 1020</td><td> 11</td><td> 1020</td><td></td><td> 211</td><td> 72</td><td> 1</td><td></td><td></td><td> 8</td>
<td> 26</td><td>HNFET17</td><td> 209022 05/08/97</td><td>Uni-ZAP XR</td><td> 36</td><td> 781</td><td> 31</td><td> 781</td><td> 100</td><td> 100</td><td> 73</td><td> 1</td><td></td><td></td><td> 33</td>
8th (continuation)
<td>Gene No.</td><td>ID of Clone cDNA</td><td>No. and Date of Deposit ATCC</td><td>Vector</td><td>NT SEQ ID No. X</td><td>Seq. NT Total</td><td>5 'NT of Seq. in Clone</td><td>3 'NT of Seq. in Clone</td><td>5 'NT of Cotton Initiation</td><td>5 'NT First AA from Pep. Signal</td><td>AA SEQ ID No. Y</td><td>Pep's first. Signal</td><td>Pep's last AA. Signal</td><td>First AA from Portion Segregated</td><td>Last AA from ORF</td>
<td> 27</td><td>HNHCR46</td><td> 209022 05/08/97</td><td>Uni-ZAP XR</td><td> 37</td><td> 966</td><td> 507</td><td> 948</td><td></td><td> 575</td><td> 74</td><td> 1</td><td> 29</td><td> 30</td><td> 56</td>
<td> 28</td><td>HPWAS91</td><td> 209022 05/08/97</td><td>Uni-ZAP XR</td><td> 38</td><td> 416</td><td> 1</td><td> 416</td><td> 95</td><td> 95</td><td> 75</td><td> 1</td><td> 24</td><td> 25</td><td> 25</td>
<td> 29</td><td>HWTAW41</td><td> 209022 05/08/97</td><td>Uni-ZAP XR</td><td> 39</td><td> 1114</td><td> 804</td><td> 1114</td><td> 843</td><td> 843</td><td> 76</td><td> 1</td><td></td><td></td><td> 14</td>
<td> 30</td><td>HBMUT52</td><td> 209022 05/08/97</td><td>Uni-ZAP XR</td><td> 40</td><td> 602</td><td> 142</td><td> 602</td><td> 204</td><td> 204</td><td> 77</td><td> 1</td><td> 26</td><td> 27</td><td> 32</td>
<td> 31</td><td>HERAG83</td><td> 209022 05/08/97</td><td>Uni-ZAP XR</td><td> 41</td><td> 970</td><td> 1</td><td> 970</td><td> 110</td><td> 110</td><td> 78</td><td> 1</td><td> 22</td><td> 23</td><td> 22</td>
<td> 32</td><td>HETFI51</td><td> 209022 05/08/97</td><td>Uni-ZAP XR</td><td> 42</td><td> 1002</td><td> 1</td><td> 1002</td><td> 43</td><td> 43</td><td> 79</td><td> 1</td><td> 21</td><td> 22</td><td> 172</td>
<td> 32</td><td>HETFI51</td><td> 209022 05/08/97</td><td>Uni-ZAP XR</td><td> 47</td><td> 981</td><td> 1</td><td> 981</td><td> 23</td><td> 23</td><td> 84</td><td> 1</td><td> 17</td><td> 18</td><td> 30</td>
Table 1 summarizes the information corresponding to each Gene No. previously described. The nucleotide sequence identified as NT SEQ ID NO: X was assembled from partially homologous (overlapping) sequences obtained from the clone ID cDNA identified in Table 1 and, in some cases, from additional related DNA clones. Overlapping sequences were grouped into a single contiguous high redundancy sequence (usually three to five overlapping sequences at each nucleotide position), resulting in a final sequence identified as SEQ ID NO: X.
CDNA Clone ID was deposited on the date and given the corresponding deposit number listed under ATCC Deposit No. Z and Date. Some of the deposits contain multiple different clones corresponding to the same gene. Vector refers to the type of vector contained in the cDNA Clone ID.
Seq. Total NT refers to the total number of nucleotides in the contig identified by Gene No. The deposited clone may contain all or most of these sequences, reflected by the nucleotide position indicated as 5 'NT of Seq. Clone and 3 'NT Seq. of SEQ ID NO: X Clone. The nucleotide position of SEQ ID NO: X of the putative initiation codon (methionine) is identified as 5 'NT Initiation Cotton. Similarly, the nucleotide position of SEQ ID NO: X of the predicted signal sequence is identified as 5 'NT of First Pep's AA. Signal.
The translated amino acid sequence starting with methionine is identified as AA SEQ ID NO: Y, although other reading steps can also be easily translated using known molecular biology techniques.
The first and last amino acid positions of SEQ ID NO: Y of the predicted signal peptide are identified as Pep's First AA. Pep's Sign and Last AA. Signal. The predicted first amino acid position of SEQ ID NO: Y of the secreted moiety is identified as Predicted First Segregated Portion AA. Finally, the amino acid position of SEQ ID NO: Y of the last amino acid in the open reading frame is identified as Last AA of ORF.
SEQ ID to SEQ
ID
N: Y translated are sufficiently accurate and otherwise suitable for a variety of uses well known in the art further described below. For example, SEQ ID NO: X is useful for designing nucleic acid hybridization probes that will detect nucleic acid sequences contained in SEQ ID NO: X or the cDNA contained in the deposited clone. These probes will also hybridize to nucleic acid molecules in biological samples, thereby enabling a variety of forensic and diagnostic methods of the invention. Similarly, the identified polypeptides of SEQ ID NO: Y may be used to produce antibodies that specifically bind to secreted proteins encoded by the cDNA clones identified in Table 1.
However, DNA sequences produced by sequencing reactions may contain sequencing errors. The mistakes
<td>exist</td><td>how</td><td>nucleotides</td><td>wrong</td><td>identified</td><td>or</td><td>how</td>
<td>inserts</td><td>or</td><td>deletions of</td><td>nucleotides</td><td>in sequence</td><td>in</td><td>DNA</td>
<td>produced</td><td>. The</td><td colspan="5">nucleotides wrongly inserted or deleted</td>
cause phase shifts in the reading phases of the predicted amino acid sequence. In these cases, the predicted amino acid sequence differs from the actual amino acid sequence, although the DNA sequence produced may be more than 99.9% identical to the actual DNA sequence (for example, insertion or deletion of a base in a reading frame). 1000 bases).
Accordingly, for those applications requiring precision in nucleotide sequence or amino acid sequence, the present invention provides not only the nucleotide sequence produced, identified as
SEQ ID NO: 23 and the predicted translated amino acid sequence, identified as SEQ ID NO:
60, but also a plasmid DNA sample containing a human cDNA of the invention deposited with the ATCC as shown in Table 1. The nucleotide sequence of each deposited clone can be readily determined by sequencing the deposited clone according to known methods. . The predicted amino acid sequence can then be verified from such deposits. In addition, the amino acid sequence of the protein encoded by a particular clone can also be directly determined by peptide sequencing or by expression of the protein in a suitable host cell containing the deposited human cDNA, protein collection and sequence determination.
The present invention also relates to genes corresponding to SEQ ID NO: 23, SEQ ID NO: 60 or the deposited clone. The corresponding gene may be isolated according to known methods using the sequence information disclosed herein. Such methods include preparing probes or primers from the disclosed sequence and identifying or amplifying the corresponding gene from appropriate sources of genomic material.
Species homologs are also provided in the present invention as long as they are encompassed by claim 1. Species homologs may be isolated and identified by preparing suitable probes or primers from the sequences provided herein and by screening for a suitable source of nucleic acid to the desired homologue.
The polypeptides of the invention may be prepared in any suitable manner. Such polypeptides include isolated naturally occurring polypeptides, recombinantly produced polypeptides, synthetically produced polypeptides, or polypeptides produced by a combination of these methods. Means for preparing such polypeptides are well understood in the art.
The polypeptides may be in the form of a secreted protein, including the mature form or may be a part of a larger protein, such as a fusion protein (see below).
It is often advantageous to include an additional amino acid sequence that contains secretory or conductive sequences, pro-sequences, sequences that aid in purification, such as multiple histidine residues or an additional sequence for stability during recombinant production.
The polypeptides of the present invention are preferably provided in an isolated form and preferably are substantially purified. A recombinantly produced version of a polypeptide, including the secreted polypeptide, can be substantially purified by the one-step method described in Smith and Johnson, Gene 67: 31-40 (1988). Polypeptides of the invention may also be purified from natural or recombinant sources using antibodies of the invention derived against the secreted protein in methods which are well known in the art.
Signal Sequences
Methods for predicting whether a protein has a signal sequence, as well as the cleavage point for that sequence, are available. For example, McGeoch's method, Virus Res. 3: 271-286 (1985), uses information from a short region.
N-terminally charged and a subsequent uncharged region of the complete (uncleaved) protein. The von Heinje method, Nucleic Acids Res. 14: 4683-4690 (1986) utilizes the information of the residues surrounding the cleavage site, typically residues -13 to +2, where +1 indicates the amino terminus of the secreted protein. The prediction accuracy of known mammalian secretory protein cleavage points for each of these methods is in the range of 75-80%. (von Heinje, supra). However, both methods do not always produce the same predicted cleavage point (s) for a given protein.
In the present case, the deduced amino acid sequence of the secreted polypeptide was analyzed by a computer program called
Nielsen et al., Protein
Cellular engineering of a protein based on amino acid sequence.
As part of this computational location prediction, McGeoch von Heinje's methods can be used. Analysis of the amino acid sequences of the secreted proteins described herein by this program provided the results shown in Table 1.
However, as one skilled in the art would understand, cleavage sites sometimes vary from organism to organism and cannot be predicted with absolute certainty. Accordingly, the present invention provides secreted polypeptides having a sequence shown in SEQ ID NO: 60 which has an N-terminus starting within 5 residues (ie, + or - 5 residues) of the predicted cleavage point. Similarly, it is also recognized that in some cases the signal sequence cleavage of a secreted protein is not entirely uniform, resulting in more than one secreted species. These polypeptides and polynucleotides encoding such polypeptides are contemplated by the present invention.
Moreover, the signal sequence identified by the above analysis may not necessarily predict the naturally occurring signal sequence. For example, the naturally occurring signal sequence may be further upstream from the predicted signal sequence. However, it is likely that the predicted signal sequence will be able to direct the secreted protein into the ER. These polypeptides and polynucleotides encoding such polypeptides are contemplated by the present invention.
Polynucleotide and Polypeptide Variants
Variant refers to a polynucleotide or polypeptide differing from the polynucleotide or polypeptide of the present invention but retaining its essential properties. In general, variants are generally closely similar and in many regions identical to the polynucleotide or polypeptide of the present invention.
4
By a polynucleotide having a nucleotide sequence at least, for example, 95% identical to a reference nucleotide sequence of the present invention, means that the nucleotide sequence of the polynucleotide is identical to the reference sequence, except that the polynucleotide sequence may be include up to five point mutations per 100 nucleotides of the reference nucleotide sequence encoding the polypeptide. In other words, in order to obtain a polynucleotide having a nucleotide sequence at least 95% identical to a reference nucleotide sequence, up to 5% of the nucleotides in the reference sequence may be deleted or substituted with another nucleotide, or a nucleotide. Nucleotide numbers up to 5% of the total nucleotides in the reference sequence can be inserted into the reference sequence. The search sequence may be an integral sequence shown in Table 1, the ORF (open reading frame) or any specified fragment as described herein.
In practice, if any particular nucleic acid or polypeptide molecule is at least 90%, 95%, 96%, 97%, 98% or 99% identical to a nucleotide sequence of the present invention, it can be determined in conventional manner. using known computer programs. A preferred method for determining the best overall match between a search sequence (a sequence of the present invention) and a submitted sequence, also referred to as a global sequence alignment, can be determined using the FASTDB computer program based on the search algorithm. Brutlag et al. {Comp. App. Biosci. (1990) 6: 237-245). In a sequence alignment, the search and subject sequences are both DNA sequences. An RNA sequence can be compared by converting U to T. The result of said overall sequence alignment is in percent identity. Preferred parameters used in a FASTDB alignment of DNA sequences to calculate percent identity are: Matrix = Unitary, k-tuple = 4, Pairing Error Penalty = 1, Join Penalty = 30, Randomization Group Length = 0 , Exclusion Score = 1, Gap Penalty = 5, Gap Size Penalty = 0.05, Window Size = 500, or the length of the submitted nucleotide sequence, whichever is smaller.
If the submitted sequence is shorter than the search sequence by virtue of 5 'or 3' deletions, not because of internal deletions, a manual correction of the results should be performed. This is because the FASTDB program does not take into account 5 'and 3' truncations of the submitted sequence when calculating percent identity. For 5 'or 3' end truncated subject sequences relative to the search sequence, percent identity is correlated by calculating the base number of the search sequence that is 5 'and 3' from the submitted sequence, which is not matching / aligned as a percentage of the total bases of the search string. Whether a nucleotide is matched / aligned is determined by results of FASTDB sequence alignment. This percentage is then subtracted from the percent identity calculated by the previous FASTDB program using the specified parameters to arrive at a final percent identity score. This corrected score is that used for the purposes of the present invention. Only bases outside the 5 'and 3' bases of the submitted sequence, as displayed by the FASTDB alignment, which do not match / align with the search sequence, are calculated for the purpose of manually adjusting the percent identity score.
For example, a 90 base subject sequence is aligned to a 100 base search sequence to determine percent identity. Deletions occur at the 5 'end of the submitted sequence and therefore the FASTDB alignment does not show a match / alignment of the first 10 bases at the 5' end. The 10 unpaired bases represent 10% of the sequence (number of bases at the 5 'and 3' ends not matching / total number of bases in the search sequence), so 10% is subtracted from the percent identity score calculated by the FASTDB program. If the remaining 90 bases were perfectly matched, the final percent identity would be 90%. In another example, a 90 base subject sequence is compared to a 100 base search sequence. This time, the deletions are internal deletions, so there are no 5 'or 3' bases of the submitted sequence that do not match / align with the search. In this case, the percent identity calculated by FASTDB is not manually corrected. Again, only 5 'and 3' bases of the submitted sequence that do not match / align with the search sequence are manually corrected. For the purposes of the present invention, no further manual corrections should be made.
By a polypeptide having an amino acid sequence at least, for example, 95% identical to a search amino acid sequence of the present invention, means that the amino acid sequence of the subject polypeptide is identical to the search sequence, except that the subject polypeptide sequence may include up to five amino acid changes per 100 amino acids of the search amino acid sequence. In other words, in order to obtain a polypeptide having an amino acid sequence at least 95% identical to a search amino acid sequence, up to 5% of the amino acid residues in the submitted sequence may be inserted, deleted. ) or substituted with other amino acids. These reference sequence changes may occur at the terminal amino or carboxyl positions of the reference amino acid sequence or anywhere between those terminal positions, individually dispersed between residues in the reference sequence or at one or more contiguous groups within the reference sequence.
In practice, if any particular polypeptide is at least 90%, 95%, 96%, 97%, 98% or 99% identical, for example, to the amino acid sequence shown in Table 1 or the DNA-encoded amino acid sequence. deposited, the clone can be determined in conventional manner using known computer programs. A preferred method for determining the best overall match between a search sequence (a sequence of the present invention) and a submitted sequence, also referred to as a global sequence alignment, can be determined using the FASTDB computer program based on the search algorithm. Brutlag et al. (Comp. App. Biosci. (1990) 6: 237-245). In a sequence alignment, the search and subject sequences are either nucleotide sequences or both amino acid sequences. The result of said overall sequence alignment is in percent identity. Preferred parameters used in an FASTDB amino acid alignment are: Matrix = MAP, k-tuple = 2, Pairing Error Penalty = 1, Join Penalty = 20, Randomization Group Length = 0, Exclusion Score = 1, Size of
Window = Sequence Length, Gap Penalty = 5, Gap Size Penalty = 0.05 Window Size = 500 or the length of the submitted amino acid sequence, whichever is shorter.
If the submitted sequence is shorter than the search sequence due to N-terminal or C-terminal deletions, not due to internal deletions, a manual correction to the results should be performed. This is because the FASTDB program does not take into account N-terminal or C-terminal truncations of the submitted sequence when calculating percent global identity. For N and C-terminated truncated subject sequences relative to the search sequence, percent identity is correlated by calculating the number of N-terminal and C-terminal search sequence residues of the submitted sequence that do not match / aligned with a corresponding submitted residue as a percentage of the total bases of the search sequence. Whether a residue is matched / aligned is determined by FASTDB sequence alignment results. This percentage is then subtracted from the percent identity calculated by the previous FASTDB program using the specified parameters to arrive at a final percent identity score. This final percent identity score is what is used for the purposes of the present invention. Only residues for the N and C ends of the submitted sequence that do not match / align with the search sequence are considered for the purpose of manually adjusting the percent identity score. That is, only the search residue positions outside the N-terminal and C-terminal residues farthest from the submitted sequence.
For example, a submitted sequence of 90 amino acid residues is aligned to a 100 residue search sequence to determine percent identity. Deletion occurs at the N-terminus of the submitted sequence and therefore FASTDB alignment does not show a match / alignment of the first 10 residues at the N-terminus. The 10 unpaired residues represent 10% of the sequence (number of residues at unmatched N and C ends / total number of residues in the search sequence), so 10% is subtracted from the percent identity score calculated by the FASTDB program. If the remaining 90 residues were perfectly matched, the final identity percentage would be 90%. In another example, a 90 residue submitted sequence is compared to a 100 residue search sequence. This time, the deletions are internal deletions, so that there are no residues at the N or C end of the submitted sequence that do not match / align with the search. In this case, the percent identity calculated by FASTDB is not manually corrected. Again, only the residue positions outside the N-terminal and C-terminal ends of the submitted sequence, as shown in FASTDB alignment, which do not match / align with the search sequence, are manually corrected. For the purposes of the present invention, no further manual corrections should be made.
Variants may contain changes in coding regions, non-coding regions or both. Especially preferred are polynucleotide variants containing alterations that produce silent substitutions, additions or deletions, but do not alter the properties or activities of the encoded polypeptide. Nucleotide variants produced by silent substitutions due to degeneracy of the genetic code are preferred. Moreover, variants in which 5-10, 1-5 or 1-2 amino acids are substituted, deleted or added in any combination are also preferred. Polynucleotide variants can be produced for a variety of reasons, eg, optimizing codon expression for a particular host (changing codons in human mRNA to those preferred by a bacterial host, such as E. coli).
Naturally occurring variants are called allelic variants and refer to one of several alternative forms of a gene occupying a particular locus on a chromosome of an organism. (Genes II, Lewin, B., ed., John Wiley & Sons, New York (1985)). These allelic variants may vary at the polynucleotide and / or polypeptide level. Alternatively, non-naturally occurring variants may be produced by mutagenesis techniques or by direct synthesis.
Using known methods of protein engineering and recombinant DNA technology, variants may be generated to enhance or alter the characteristics of the polypeptides of the present invention. For example, one or more amino acids from the N-terminus or C-terminus of the secreted protein may be deleted without substantial loss of biological function. The authors of Ron et al., J. Biol. Chem. 268: 2984-2988 (1993) reported variant KGF proteins having heparin binding activity even after deletion of 3, 8, or 27 amino-terminal amino acid residues. Similarly, interferon gamma exhibited up to ten-fold activity after deletion of 8-10 amino acid residues from the carboxyl end of this protein. (Dobeli et al., J. Biotechnology 7: 199-216 (1988)).
Moreover, ample evidence shows that variants often retain a biological activity similar to that of the naturally occurring protein. For example, Gayle and colleagues (J. Biol. Chem 268: 22105-22111 (1993)) conducted extensive mutational analysis of human cytokine IL-1a. They used random mutagenesis to produce over 3500 individual IL-1a mutants that averaged 2.5 amino acid changes per variant over the entire length of the molecule. Multiple mutations were examined at each possible amino acid position. The researchers found that most of the molecule could be altered with little effect on either [binding or biological activity]. (See, Summary). In fact, only 23 unique amino acid sequences from more than 3,500 nucleotide sequences examined produced a protein that differed significantly in wild-type activity.
Further, even if deletion of one or more amino acids from the N-terminus or C-terminus of a polypeptide results in modification or loss of one or more biological functions, other biological activities may still be retained. For example, the ability of a deletion variant to induce and / or bind to antibodies that recognize the secreted form is likely to be retained when less than most residues of the secreted form are removed from the N-terminus or C-terminus. Whether a particular polypeptide devoid of N-terminal or C-terminal residues of a protein retains such immunogenic activities can be readily determined by routine methods described herein and otherwise known in the art.
Thus, the invention further includes polypeptide variants within the scope of the claims showing substantial biological activity. Such variants include deletions, insertions, inversions, repeats and substitutions, selected according to general rules known in the art, to have little effect on activity. For example, guidance regarding how to prepare phenotypically silent amino acid substitutions is provided in Bowie, JU. et al., Science 247: 1306-1310 (1990), in which the authors indicate that there are two main strategies for studying the tolerance to alteration of an amino acid sequence.
The first strategy explores the tolerance of amino acid substitutions by natural selection during the evolution process. By comparing amino acid sequences in different species, conserved amino acids can be identified. These conserved amino acids are likely to be important for protein function. In contrast, amino acid positions where substitutions were tolerated by natural selection indicate that these positions are not critical for protein function. Thus, positions tolerating amino acid substitution could be modified while still maintaining the biological activity of the protein.
The second strategy uses genetic engineering to introduce amino acid changes at specific positions of a gene, cloned to identify regions critical for protein function. For example, point-directed mutagenesis or alanine screening mutagenesis (introduction of unique alanine mutations at each residue in the molecule) may be used. (Cunningham and Wells, Science 244: 1081-1085 (1989)). The resulting mutant molecules can then be tested for biological activity.
As the authors state, these two strategies have revealed that proteins are surprisingly tolerant of amino acid substitutions. The authors further indicate that amino acid changes are likely to be permissive to certain amino acid positions in the protein. For example, deeper amino acid residues (within the tertiary structure of the protein) require nonpolar side chains, while few surface side chain characteristics are generally conserved. Furthermore, conservative tolerated amino acid substitutions involve substitution of aliphatic or hydrophobic amino acids Ala, Val, Leu and Ile; replacement of Ser and Thr hydroxyl residues; substitution of acidic residues Asp and Glu; replacement of Asn and Gin amide residues, replacement of Lys, Arg and His basic residues; replacement of the aromatic residues Phe, Tyr and Trp and replacement of the small amino acids Ala, Ser, Thr, Met and Gly. In addition to conservative amino acid substitution, variants of the present invention as defined in the claims include (i) substitutions with one or more of the non-conserved amino acid residues, where the substituted amino acid residues may or may not be one encoded by the genetic code, or (ii) substitution with one or more amino acid residues having a substituent group; or (iii) fusion of the mature polypeptide with another compound, such as a compound to increase the stability and / or solubility of the polypeptide (e.g. polyethylene glycol) or (iv) fusion of the polypeptide with additional amino acids, such as an IgG Fc fusion peptide region, or secretory or conductive sequence or facilitating sequence purification. Such variant polypeptides are believed to be within the knowledge of those skilled in the art from the present teachings.
For example, polypeptide variants containing amino acid substitutions of amino acids loaded with other charged or neutral amino acids may produce proteins with improved characteristics, such as less aggregation.
Aggregation of pharmaceutical formulations reduces activity and increases clearance due to the immunogenic activity of the aggregate.
(Pinckard et al., Clin. Exp. Immunol.
2: 331-340 (1967); Robbins et al., Diabetes 36: 838-845 (1987);
Cleland et al., Crit. Rev. Therapeutic Drug Carrier Systems
10:307-377 (1993)).
Polynucleotide and Polypeptide Fragments
In the present description, a polynucleotide fragment refers to a short polynucleotide having a nucleic acid sequence contained in the clone deposited or shown in SEQ ID NO: 23. Short nucleotide fragments have at least
<td>least about</td><td>l from</td><td> 15</td><td>nt at least about</td><td>in</td><td> 20</td><td>nt,</td><td>, at least,</td>
<td>about 30</td><td>nt</td><td>or,</td><td>at least about</td><td> 40</td><td>nt</td><td>in</td><td>length.</td>
<td>It is intended</td><td>what</td><td>one</td><td>fragment at least</td><td> 20</td><td>nt</td><td>in</td><td>length,</td>
for example, include 20 or more contiguous bases of the cDNA sequence contained in the deposited clone or nucleotide sequence shown in SEQ ID NO: 23. These nucleotide fragments are useful as probes and diagnostic primers as discussed herein. Larger fragments (eg, 150, 500, 600 nucleotides) are part of the invention.
Furthermore, representative examples of polynucleotide fragments of the description include, for example, fragments having a sequence of about nucleotide number 1-50, 51-100, 101-150, 151-200, 201-250, 251-300, 301 -350, 351-400, 401-450, 451-500, 501-550, 551-600, 651-700, 701-750, 751-800, 800-850, 851-900, 901-950 or 951-1000 SEQ ID NO: 23 or cDNA contained in the deposited clone. In this context about includes the particularly large or smaller enumerated ranges in several (5, 4, 3, 2 or 1) nucleotides, at one end or both ends. Preferably, these fragments encode a polypeptide that has biological activity. More preferably, these polynucleotides may be used as probes or primers as discussed herein.
In the present description, a polypeptide fragment refers to a short amino acid sequence contained in SEQ ID NO: 60 or encoded by the cDNA contained in the deposited clone. Protein fragments may be independent or comprised within a larger polypeptide, of which the fragment forms a part or region, most preferably as a single contiguous region. Representative examples of polypeptide fragments of the description include, for example, fragments of about amino acid number 1-20, 21-40, 41-60, 61-80 or 81-100. Moreover, the polypeptide fragments of the invention may be about 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140 or 150 amino acids in length. In this context about includes the particularly large or minor ranges listed in several (5, 4, 3, 2 or 1) amino acids at one or both ends.
Preferred polypeptide fragments include the secreted protein as well as the mature form. Additional preferred polypeptide fragments include the secreted protein or mature form, having a continuous series of deleted amino or carboxy-terminal residues, or both. For example, any number of amino acids, ranging from 1-60, may be deleted from the amino terminus, either from the secreted polypeptide or from the mature form. Similarly, any number of amino acids, ranging from 1-30, may be deleted from the carboxy end of the secreted protein or mature form. Furthermore, any combination of the above amino and carboxyl end deletions is preferred. Similarly, polynucleotide fragments encoding these polypeptide fragments are also preferred.
Also preferred are polypeptide and polynucleotide fragments, characterized by structural or functional domains, such as fragments comprising alpha helix and alpha helix, beta sheet formation regions and beta sheet, fold and regions of folding, spiral and spiral forming regions, hydrophilic regions, hydrophobic regions, alpha amphiphilic regions, beta amphiphilic regions, flexible regions, surface forming regions, substrate binding regions and regions of high antigenic index. Polypeptide fragments of SEQ ID NO: 60 within conserved domains are specifically contemplated by the present invention. In addition, polynucleotide fragments encoding these domains are also contemplated.
Other preferred fragments are biologically active fragments. Biologically active fragments are those exhibiting activity similar to, but not necessarily identical to, an activity of the polypeptide of the present invention.
The biological activity of the fragments may include improved desired activity or decreased undesirable activity.
Epitopes & Antibodies
In the present description, epitopes refer to polypeptide fragments having antigenic or immunogenic activity in an animal, especially a human. A preferred embodiment of a polypeptide fragment of the present invention relates to fragments comprising an epitope. Accordingly, polynucleotides of the present invention encoding such fragments are also preferred. A region of a protein molecule to which an antibody can bind is defined as an antigenic epitope. In contrast, an immunogenic epitope is defined as a part of a protein that elicits an antibody response. (See, for example, Geysen et al., Proc. Natl. Acad. Sci. USA 81: 3998-4002 (1983)).
Fragments that function as epitopes can be produced by any conventional means. (See, eg, Houghten, RA, Proc. Natl. Acad. Sci. USA 82: 5131-5135 (1985), further described in US Patent No. 4,631,211).
In the present description, antigenic epitopes preferably contain a sequence of at least seven, more preferably at least nine and most preferably between about 15 to about 30 amino acids. Antigenic epitopes are useful for deducing antibodies, including monoclonal antibodies that specifically bind to the epitope.
(See, for example, Wilson et al., Cell 37: 767-778 (1984);
Sutcliffe, JG et al., Science 219: 660-666 (1983)).
Similarly, immunogenic epitopes may be used to induce antibodies according to methods well known in the art. (See, for example, Sutcliffe et al., Supra; Wilson et al., Supra; Chow, M. et al., Proc. Natl. Acad. Sci. USA 82: 910-914; and Bittle, FJ et al. J. Gen. Virol 66: 2347-2354 (1985)). A preferred immunogenic epitope includes the secreted protein. Immunogenic epitopes may be presented together with a carrier protein, such as an albumin, to an animal system (such as rabbit or mouse) or, if sufficiently long (at least about 25 amino acids), without a carrier. However, it has been shown that immunogenic epitopes comprising only 8 to 10 amino acids are sufficient to deduce antibodies capable of binding at least linear epitopes on a denatured polypeptide (e.g. in a Western blot).
As used herein, the term antibody (Ab) or monoclonal antibody (Mab) is intended to include intact molecules as well as antibody fragments (such as, for example, Fab and F (ab ') 2 fragments) that are capable of binding. specifically the protein. Fab and F (ab ') 2 fragments lack the intact antibody Fc fragment, are cleared faster from circulation and may have less nonspecific tissue binding than an intact antibody. (Wahl et al., J. Nucl. Med. 24: 316-325 (1983)). Thus, these fragments are preferred, as are products from a FAB or other immunoglobulin expression library. Furthermore, the antibodies of the present invention include chimeric, single chain and humanized antibodies.
Fusion Proteins
Any polypeptide of the present invention may be used to produce fusion proteins. For example, the polypeptide of the present invention, when fused to a second protein, may be used as an antigenic tail. Antibodies raised against the polypeptide of the present invention may be used to indirectly detect the second protein by binding to the polypeptide. Furthermore, because the secreted proteins target cellular locations based on traffic signals, the polypeptides of the present invention may be used as targeting molecules once fused to other proteins.
Examples of domains that may be fused to polypeptides of the present invention include not only heterologous signal sequences, but also other heterologous functional regions. The fusion does not necessarily need to be direct but can occur via ligand sequences.
In addition, fusion proteins may also be engineered to improve characteristics of the polypeptide of the present invention. For example, a region of additional amino acids, particularly charged amino acids, may be added to the N-terminus of a polypeptide to improve stability and persistence during host cell purification or subsequent manipulation and storage. Also, peptide moieties may be added to the polypeptide to facilitate purification. Such regions may be removed prior to final preparation of the polypeptide. The addition of peptide moieties to facilitate manipulation of polypeptides are familiar and routine techniques in the art.
In addition, the polypeptides of the present invention, including fragments and specifically epitopes, may be combined with portions of the immunoglobulin constant domain (IgG), resulting in chimeric polypeptides. These fusion proteins facilitate purification and show an increased half life in vivo. A cited example describes chimeric proteins consisting of the first two domains of the human CD4 polypeptide and several domains of the mammalian immunoglobulin heavy or light chain constant regions. (EP A 394827; Traunecker et al., Nature 331: 84-86 (1988)). Fusion proteins having disulfide-linked dimeric structures (by virtue of IgG) may also be more efficient in binding and neutralizing other molecules than the secreted monomeric protein or protein fragment alone. (Fountoulakis et al., J. Biochem. 270: 3958-3964 (1995)).
Similarly, EP-A-0464533 (Canadian equivalent document 2045869) discloses fusion proteins comprising several constant region portions of immunoglobulin molecules together with another human protein or part thereof. In many cases, the Fc part in a fusion protein is beneficial in therapy and diagnosis and thus may result, for example, in improved pharmacokinetic properties (EP-A 0232262). Alternatively, deletion of the Fc moiety would be desired after the fusion protein has been expressed, detected and purified. For example, the Fc moiety may prevent therapy and diagnosis if the fusion protein is used as an antigen for immunizations. In drug discovery, for example, human proteins, such as hIL-5, were fused to Fc moieties for the purpose of high throughput screening assays to identify hIL-5 antagonists.
(See, D. Bennett et al., J. Molecular Recognition 8: 52-58 (1995); K. Johanson et al., J. Biol. Chem. 270: 9459-9471 (1995)).
Furthermore, the polypeptides of the present invention may be fused to marker sequences, such as a peptide that facilitates purification of the fused polypeptide. In preferred embodiments, the marker amino acid sequence is a hexahistidine peptide, such as the tag tail provided in a pQE vector (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, CA, 91311), among others, many of which are commercially available. As described in Gentz et al., Proc. Natl. Acad. Know. USA 86: 821-824 (1989), for example, hexahistidine provides convenient purification of the fusion protein. Another useful peptide tail for purification, the HA tail, corresponds to an epitope derived from the influenza hemagglutinin protein. Brooks et al., Cell, 37: 767 (1984).
Thus, any of these foregoing fusions may be engineered using the polynucleotides or polypeptides of the present invention.
Production of Vectors, Host Cells and Protein
The present invention also relates to vectors containing the polynucleotide of the present invention, host cells and the production of polypeptides by recombinant techniques. The vector may be, for example, a phage, plasmid, viral or retroviral vector. Retroviral vectors may be replication competent or replication defective. In the latter case, viral propagation will generally occur only in complementing host cells.
Polynucleotides may be linked to a vector containing a selectable marker for propagation in a host. In general, a plasmid vector is introduced into a precipitate, such as a calcium phosphate precipitate or into a complex with a charged lipid. If the vector is a virus, it may be packaged in vitro using an appropriate packaging cell line and then transduced into host cells.
The polynucleotide insert should be operably linked to an appropriate promoter, such as the phage lambda PL promoter, E. coli lac, trp, phoA and tac promoters, early and late SV40 promoters, and retroviral LTR promoters, to name a few. . Other suitable promoters will be known to the person skilled in the art. Expression constructs will additionally contain sites for transcription initiation, termination and, in the transcribed region, a ribosome binding site for translation. The coding portion of the transcripts expressed by the constructs will preferably include an early translation initiation codon and a termination codon (UAA, UGA or UAG) appropriately positioned at the end of the polypeptide for translation.
As indicated, expression vectors will preferably include at least one selectable marker. Such markers include resistance of dihydrofolate reductase, G418 or neomycin, to eukaryotic cell culture and tetracycline, kanamycin or ampicillin resistance genes for cultivation in E. coli and other bacteria. Representative examples of suitable hosts include, but are not limited to, bacterial cells, such as E. cells. coli, Streptomyces and Salmonella typhimurium; fungal cells, such as yeast cells; insect cells such as Drosophila S2 and Spodoptera Sf9 cells; animal cells, such as CHO, COS, 293 and Bowes melanoma cells; and plant cells. Appropriate culture media and conditions for the host cells described above are known in the art.
Preferred vectors for use in bacteria include pQE70, pQE60 and pQE-9, available from QIAGEN, Inc .; pBluescript vectors, Phagescript vectors, pNH8A, pNH16a, pNH18A, pNH46A available from Stratagene Cloning Systems, Inc .; and ptrc99a, pKK223-3, pKK233-3, pDR540, pR1T5 available from Pharmacy Biotech, Inc. Preferred eukaryotic vectors include pWLNEO, pSV2CAT, pOG44, pXT1 and pSG, available from Stratagene; and pSVK3, pBPV, pMSG and pSVL, available from Pharmacia. Other suitable vectors will be readily apparent to one skilled in the art.
The introduction of the construct into the host cell may be affected by calcium phosphate transfection, DEAE-dextran mediated transfection, cationic lipid mediated transfection, electroporation, transduction, infection or other methods. Such methods are described in many standard laboratory manuals, such as Davis et al., Methods In Molecular Biology (1986). It is specifically contemplated that the polypeptides of the present invention may in fact be expressed by a host cell devoid of a recombinant vector.
A polypeptide of this invention may be recovered and purified from recombinant cell cultures by well known methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity, hydroxylapatite chromatography and lectin chromatography. Most preferably, high performance liquid chromatography (HPLC) is employed for purification.
The polypeptides of the present invention, and preferably the secreted form, may also be recovered from: products purified from natural sources, including body fluids, tissues and cells, either directly isolated or cultured; chemical synthetic process products; and products produced by recombinant techniques from a prokaryotic or eukaryotic host including, for example, bacterial, yeast, higher plant, insect and mammalian cells. Depending on the host employed in a recombinant production process, the polypeptides of the present invention may be glycosylated or may be non-glycosylated. Additionally, the polypeptides of the invention may also include an initial modified methionine residue, in some cases as a result of host mediated processes. Thus, it is known in the art that the N-terminal methionine encoded by the translation initiation codon is generally removed with high efficiency from any protein after translation into all eukaryotic cells. Although N-terminal methionine in most proteins is also efficiently removed in most prokaryotes, for some proteins, this prokaryotic removal process is inefficient, depending on the nature of the amino acid to which N-terminal methionine is covalently attached.
Uses of Polynucleotides
<td>Each</td><td>From</td><td>polynucleotides</td><td>on here</td><td>identified</td><td>Can be</td>
<td>used</td><td>in</td><td>numerous forms</td><td>how</td><td>reagents.</td><td>The following</td>
<td>description</td><td>must</td><td colspan="3">be considered exemplary</td><td>and use</td>
known techniques.
The polynucleotides of the present invention are useful for chromosomal identification. There is a continuing need to identify new chromosomal markers, as few chromosome labeling reagents based on actual sequence data (repeat polymorphisms) are presently available. Each polynucleotide of the present invention may be used as a chromosomal marker.
Briefly, sequences can be mapped to chromosomes by preparing PCR primers (preferably 15-25 bp) from the sequence shown in SEQ ID NO: 23. Primers can be selected using computer analysis, from primers do not encompass more than one predicted exon in genomic DNA. These primers are then used for PCR screening of somatic cell hybrids containing individual human chromosomes. Only those hybrids containing the human gene corresponding to SEQ ID NO: 23 will produce an amplified fragment.
Similarly, somatic hybrids provide a rapid method of PCR mapping from polynucleotides to particular chromosomes. Three or more clones may be assigned per day using a single thermal cycler. Furthermore, sub-localization of polynucleotides can be achieved with panels of specific chromosomal fragments. Other gene mapping strategies that may be used include in situ hybridization, pre-screening with labeled chromosomes separated by flow cytometry, and pre-selection by hybridization to specific chromosome-building DNA libraries.
Precise chromosomal localization of polynucleotides can also be achieved using fluorescence in situ hybridization (FISH) of a metaphase chromosomal propagation. This technique utilizes polynucleotides as short as 500 or 600 bases; however, 2000-4000 bp polynucleotides are preferred. For a review of this technique, see Verma et al., Human Chromosomes: A Manual of Basic Techniques, Pergamon Press, New York (1988).
For chromosome mapping, polynucleotides may be used individually (to label a single chromosome or a single site on that chromosome) or in panels (for labeling multiple sites and / or multiple chromosomes). Preferred polynucleotides correspond to non-coding regions of cDNA, because coding sequences are most likely conserved within gene families, thereby increasing the likelihood of cross hybridization during chromosomal mapping.
Once a polynucleotide has been mapped to an accurate chromosomal location, the physical position of the polynucleotide can be used in binding analysis. Binding analysis establishes co-inheritance between a chromosomal location and presentation of a particular disease. (Disease mapping data are found, for example, in V. McKusick, Mendelian Inheritance in Man (available on the Internet from the Johns Hopkins University Welch Medical Library ')). Assuming 1 megabase mapping resolution and one gene per 20 kb, a precisely located cDNA for a disease-associated chromosomal region could be 50-500 potential causative genes.
Thus, after co-inheritance has been established, differences in the polynucleotide and corresponding gene between affected and unaffected individuals can be examined. First, visible structural changes in chromosomes, such as deletions or translocations, are examined in chromosomal propagation or by PCR. If there are no structural changes, the presence of point mutations is ascertained. Mutations observed in some or all affected individuals, but not normal individuals, indicate that the mutation may cause the disease. However, complete sequencing of the polypeptide and corresponding gene from several normal individuals is required to distinguish mutation from a polymorphism. If a new polymorphism is identified, this polymorphic polypeptide may be used for further binding analysis.
In addition, increased or decreased gene expression in affected individuals as compared to unaffected individuals can be evaluated using polynucleotides of the present invention. Any of these changes (altered expression, chromosomal rearrangement or mutation) can be used as a diagnostic or prognostic marker.
In addition to the foregoing, a polynucleotide may be used to control gene expression through triple helix formation or antisense DNA or RNA. Both methods are based on polynucleotide binding to DNA or RNA. For these techniques, preferred polynucleotides are usually 20 to 40 bases in length and complementary to the region of the gene involved in transcription (triple helix - see Lee et al., Nucl. Acids Res. 6: 3073 (1979); Cooney et al., Science 241: 456 (1988); and Dervan et al., Science 251: 1360 (1991)) or to the mRNA itself (antisense - Okano, J. Neurochem. 56: 560 (1991); Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRC Press, Boca Raton, FL (1988)). Triple helix formation optimally results in a cut off of RNA transcription from DNA, while antisense RNA hybridization blocks the translation of an mRNA molecule into polypeptide. Both techniques are effective in model systems and the information disclosed herein may be used to design antisense or triple helix polynucleotides in an effort to treat disease.
The polynucleotides of the present invention are also useful in gene therapy. One goal of gene therapy is to insert a normal gene into an organism having a defective gene in an effort to correct the genetic defect. The polynucleotides disclosed in the present invention offer a means of targeting such genetic defects in a highly accurate manner. Another objective is to insert into a new gene that was not present in the host genome thereby producing a new trait in the host cell.
Polynucleotides are also useful for identifying individuals from minute biological samples. The US military, for example, is considering the use of restriction fragment length polymorphism (RFLP) to identify its personnel. In this technique, an individual's genomic DNA is digested with one or more restriction enzymes and probed in a Southern blot to produce unique bands for personnel identification. This method does not suffer from the current limitations of ID Cards that can be lost, exchanged or stolen, making positive identification difficult. The polynucleotides of the present invention may be used as additional DNA markers for RFLP.
Polynucleotides of the present invention may also be used as an alternative to RFLP by determining the actual base-by-base DNA sequence of selected portions of an individual's genome. These sequences can be used to prepare PCR primers for amplification and isolation of that selected DNA which can then be sequenced. Using this technique, individuals can be identified by the fact that each individual will have a unique set of DNA sequences. After establishing a unique ID database for an individual, positive identification of that individual, alive or dead, can be performed from extremely small tissue samples.
Forensic biology also benefits from the use of DNA-based identification techniques as disclosed herein. DNA sequences taken from very small biological samples, such as tissues, eg<sub>r</sub> Hair or skin, or body fluids, eg, blood, saliva, semen, etc., can be amplified using PCR. In a prior art technique, gene sequences amplified from polymorphic loci, such as HLA class II DQa gene, are used in forensic biology to identify individuals. (Erlich, H., PCR Technology, Freeman and Co. (1992)). After these specific polymorphic loci are amplified, they are digested with one or more restriction enzymes, producing a DNA-labeled Southern blot band corresponding to the HLA class II DQa gene. Similarly, the polynucleotides of the present invention may be used as polymorphic markers for forensic purposes.
There is also a need for reagents capable of identifying the source of a particular tissue. Such a need arises, for example, in a forensic scenario when confronted with tissue of unknown origin. Suitable reagents may comprise, for example, particular tissue specific DNA probes or primers prepared from the sequences of the present invention. Panels of such reagents may identify tissue by species and / or organ type. In a similar manner, these reagents may be used to screen tissue cultures for contamination.
At a minimum, the polynucleotides of the present invention may be used as molecular weight markers in Southern gels, as diagnostic probes for the presence of a specific mRNA in a particular cell type, as a probe for subtracting known sequences in the discovery process. new polynucleotides for the selection and preparation of oligomers for conjugation to a gene chip or other support to deduce anti-DNA antibodies using DNA immunization techniques and as an antigen to elicit an immune response.
Uses of Polypeptides
Each of the polypeptides identified herein may be used in numerous ways. The following description is to be considered exemplary and uses known techniques.
A polypeptide of the present invention may be used to assay protein levels in a biological sample using antibody based techniques. For example, protein expression in tissues can be studied with classical immunohistological methods. (Jalkanen, M., et al.<sub>r</sub> J. Cell. Biol. 101: 976-985 (1985); Jalkanen, M., et al., J. Cell. Biol.
105:3087-3096 (1987)).
Other antibody-based methods useful for detecting protein gene expression include immunoassays, such as enzyme-linked immunosorbent assay (ELISA) and radioimmunoassay (RIA).
Suitable antibody assay markers are known in the art and include enzyme enzymes such as glucose oxidase and radioisotopes such as iodine (1251,
1211), carbon (140), sulfur (35S), tritium (3H), indium (112ln) and technetium (99mTc) and fluorescent markers such as fluorescein and rhodamine and biotin.
In addition to assaying secreted protein levels in a biological sample, proteins can also be detected in vivo by imaging. Antibody labels or markers for in vivo protein imaging include those detectable by X-ray, NMR or ESR. For X-ray, suitable markings include radioisotopes, such as barium or cesium, which emit detectable radiation but are not manifestly harmful to the individual. Suitable NMR and ESR markers include those with a detectable characteristic spin, such as deuterium that can be incorporated into the antibody by labeling nutrients for the relevant hybridoma.
A protein-specific antibody or antibody fragment that has been labeled with an appropriate detectable imaging moiety, such as a radioisotope (e.g., 1311, 112ln, 99mTc), a radiopaque substance, or a magnetic resonance detectable material nuclear, is introduced (e.g. parenterally, subcutaneously or intraperitoneally) into the mammal. It will be understood in the art that the size of the subject and the imaging system employed will determine the amount of imaging fraction required to produce diagnostic images.
In the case of a radioisotope fraction, for a human individual, the amount of radioactivity injected will normally range from about 5 to 20 millicuries of
99mTc.
The labeled antibody or antibody fragment will then preferably accumulate at the site of cells containing the specific protein. In vitro tumor imaging described in SW
Burchiel et “Immunopharmacokinetics of Radiolabeled
Antibodies and
Their fragments. (Chapter 13 in Tumor Imaging:
The Radiochemical Detection of Cancer, SW
Burchiel and BA
Rhodes, eds., Masson Publishing Inc. (1982)).
Accordingly, the invention provides a method of diagnosing a disorder which involves (a) assaying the expression of a polypeptide of the present invention in an individual's cells or body fluid; (b) comparing the level of gene expression with a standard level of gene expression, whereby an increase or decrease in the level of polypeptide gene expression tested compared to the standard level of expression is indicative of a disorder.
In addition, the polypeptides of the present invention may be used to treat disease. For example, a polypeptide of the present invention may be administered to patients in an effort to restore missing or decreased levels of the polypeptide (eg, insulin) to supplement missing or decreased levels of a different polypeptide (eg, hemoglobin S by hemoglobin B). , to inhibit the activity of a polypeptide (eg, an oncogene), to activate the activity of a polypeptide (e.g. by binding to a receptor) to reduce the activity of a membrane-bound receptor by competing with free ligand (eg, soluble TNF receptors used to reduce inflammation) or to lead to a desired response. (eg, blood vessel growth).
Similarly, antibodies directed to a polypeptide of the present invention may also be used to treat disease. For example, administration of an antibody directed to a polypeptide of the present invention may bind and reduce overproduction of the polypeptide. Similarly, administration of an antibody may activate the polypeptide, such as by binding to a membrane bound (receptor) polypeptide.
At a minimum, the polypeptides of the present invention may be used as molecular weight markers on SDS-PAGE gels or molecular sieve gel filtration columns using methods well known to those skilled in the art. Polypeptides may also be used to elicit antibodies, which in turn are used to measure protein expression from a recombinant cell as a means of assessing host cell transformation. In addition, the polypeptides of the present invention may be used to test the following biological activities.
Biological Activities
The polynucleotides and polypeptides of the present invention may be used in assays to test for one or more biological activities. If these polynucleotides and polypeptides exhibit activity in a particular assay, it is likely that these molecules may be involved in diseases associated with biological activity. Thus, polynucleotides and polypeptides could be used to treat the associated disease.
Immune Activity
A polypeptide or polynucleotide of the present disclosure may be useful in treating immune system deficiencies or disorders by activating or inhibiting proliferation, differentiation or mobilization (chemotaxis) of immune cells. Immune cells develop through a process called hematopoiesis, producing myeloid cells (platelets, red blood cells, neutrophils and macrophages) and lymphoid cells (B and T lymphocytes) from pluripotent stem cells. The etiology of these immune deficiencies or disorders may be genetic, somatic, such as cancer or some autoimmune disorders, acquired (eg, by chemotherapy or toxins) or infectious. In addition, a polynucleotide or polypeptide of the present disclosure may be used as a marker or detector for a particular disease or immune system disorder.
A polynucleotide or polypeptide of the present disclosure may be useful in treating or detecting hematopoietic cell deficiencies or disorders. The polypeptide or polynucleotide could be used to enhance differentiation and proliferation of hematopoietic cells, including pluripotent stem cells, in an effort to treat those disorders associated with a decrease in certain (or many) hematopoietic cell types. Examples of immune deficiency syndromes include but are not limited to: blood protein disorders (eg agammaglobulinemia, dysgamaglobulinemia), ataxia telangiectasia, common variable immunodeficiency, Digeorge syndrome, HIV infection, HTLV-BLV infection, adhesion deficiency syndrome leukocyte count, lymphopenia, phagocytic bactericidal dysfunction, severe combined immunodeficiency (SCIDs), Wiskott-Aldrich Disorder, anemia, thrombocytopenia or hemoglobinuria.
Furthermore, a polypeptide or polynucleotide of the present disclosure could also be used to modulate hemostatic (bleeding arrest) or thrombolytic (clot formation) activity. For example, by increasing hemostatic or thrombolytic activity, the polynucleotide or polypeptide could be used to treat blood clotting disorders (eg, fibrinogenemia, factor deficiencies), blood platelet disorders (eg thrombocytopenia) or wounds resulting from trauma, surgery or other causes. Alternatively, a polynucleotide or polypeptide of the present disclosure which may decrease hemostatic or thrombolytic activity could be used to inhibit or dissolve coagulation. These molecules could be important in treating heart attacks (stroke), stroke or scarring.
A polynucleotide or polypeptide of the present disclosure may also be useful in treating or detecting autoimmune disorders. Many autoimmune disorders result from improper recognition of self as foreign material by immune cells. This inappropriate recognition results in an immune response leading to destruction of host tissue. Therefore, administration of a polypeptide or polynucleotide of the present disclosure that inhibits an immune response, particularly T cell proliferation, differentiation or chemotaxis, may be an effective therapy in preventing autoimmune disorders.
Examples of autoimmune disorders that can be treated or detected include, but are not limited to: Addison's disease, Haemolytic anemia, Antiphospholipid syndrome, Rheumatoid arthritis, Dermatitis, Allergic encephalomyelitis, Glomerulonephritis, Goodpasture's syndrome, Severe disease, Severe myasthenia, Nevitis, Ophthalmia, Pemphigoid bullous disease, Penhypholithiasis , Stiff-Man Syndrome, Autoimmune Thyroiditis, Systemic Lupus Erythematosus, Autoimmune Pulmonary Inflammation, Guillain-Barre Syndrome, insulin-dependent diabetes mellitus and inflammatory autoimmune eye disease.
Similarly, allergic reactions and conditions such as asthma (particularly allergic asthma) or other respiratory problems may also be treated by a polypeptide or polynucleotide of the present disclosure. Furthermore, these molecules may be used to treat anaphylaxis, hypersensitivity to an antigenic molecule, or blood group incompatibility.
A polynucleotide or polypeptide of the present disclosure may also be used to treat and / or prevent organ rejection or graft versus host disease (GVHD). Organ rejection occurs by destruction of host immune cell from transplanted tissue through an immune response.
Similarly, an immune response is also involved in
GVHD but in this case the foreign transplanted immune cells destroy the host tissues. Administration of a polypeptide or polynucleotide of the present disclosure that inhibits an immune response, particularly T cell proliferation, differentiation or chemotaxis, may be an effective therapy in preventing organ rejection or GVHD.
Similarly, a polypeptide or polynucleotide of the present disclosure may also be used to modulate inflammation. For example, the polypeptide or polynucleotide may inhibit proliferation and differentiation of cells involved in an inflammatory response. These molecules may be used to treat inflammatory, chronic and acute conditions, including infection-associated inflammation (e.g. septic shock, sepsis or systemic inflammatory response syndrome (SIRS)), ischemia-reperfusion injury, endotoxin lethality, arthritis, complement-mediated hyperacute rejection, nephritis, cytokine or chemokine-induced lung injury, inflammatory bowel disease, Crohn's disease or resulting from overproduction of cytokines (eg, TNF or IL-1).
Hyperproliferative Disorders
A polypeptide or polynucleotide of the present disclosure may be used to treat or detect hyperproliferative disorders, including neoplasms. The polypeptide or polynucleotide may inhibit the proliferation of the disorder through direct or indirect interactions. Alternatively, the polypeptide or polynucleotide may proliferate other cells that may inhibit hyperproliferative disorder.
For example, by enhancing an immune response, particularly by enhancing antigenic qualities of the hyperproliferative disorder or by proliferating, differentiating or mobilizing T cells, hyperproliferative disorders may be treated. This immune response may be enhanced by enhancing an existing immune response or by initiating a new immune response. Alternatively, diminishing an immune response may also be a method of treating hyperproliferative disorders, such as a chemotherapeutic agent.
Examples of hyperproliferative disorders that can be treated or detected by a polynucleotide or polypeptide of the present invention include, but are not limited to, neoplasms located in the abdomen, bone, breast, digestive system, liver, pancreas, peritoneum, endocrine glands (supra- renal, parathyroid, pituitary, testis, ovary, thymus, thyroid), eye, head and neck, nervous (central and peripheral), lymphatic, pelvic system, skin, soft tissue, spleen, thoracic and urogenital.
Similarly, other hyperproliferative disorders may also be treated or detected by a polynucleotide or polypeptide of the present disclosure. Examples of such hyperproliferative disorders include, but are not limited to: hypergammaglobulinemia, lymphoproliferative disorders, paraproteinemia, purpura, sarcoidosis, Sezary's syndrome, Waldenstron's disease, Gaucher's disease, histiocytosis, and any other hyperproliferative system, in addition to a neoplasia previously listed organic.
Infectious disease
A polypeptide or polynucleotide of the present disclosure may be used to treat or detect infectious agents. For example, by enhancing the immune response, particularly by increasing B and / or T cell proliferation and differentiation, infectious diseases may be treated. The immune response may be enhanced by ameliorating an existing immune response or by initiating a new immune response. Alternatively, the polypeptide or polynucleotide may also directly inhibit the infectious agent without necessarily eliciting an immune response.
Viruses are an example of an infectious agent that can cause disease or symptoms that can be treated or detected by a polynucleotide or polypeptide of the present disclosure. Examples of viruses include, but are not limited to the following viral DNA and RNA families:
Arbovirus,
Adenoviridae,
Arenaviridae,
Arterivirus,
Birnaviridae,
Bunyaviridae,
Caliciviridae,
Circoviridae,
Coronaviridae,
Flavi viridae,
100
Hepadnaviridae (Hepatite), Herpesviridae (such as Cytomegalovirus, Herpes Simplex, Herpes Zoster), Mononegavirus (eg, Paramyxoviridae, Morbilivirus, Rhabdoviridae), Orthomyxoviridae (eg, Influenza), Papovaviridae, Parvoviridae (Picomaviridae), or Vaccinia , Reoviridae (eg, Rotavirus), Retroviridae (HTLV-I, HTLV-II, Lentivirus) and Togaviridae (eg, Rubivirus). Viruses within these families can cause a variety of diseases or symptoms, including but not limited to: arthritis, bronchiolitis, encephalitis, eye infections (eg, conjunctivitis, keratitis), chronic fatigue syndrome, hepatitis (A, B, C, E , Chronic Active, Delta), meningitis, opportunistic infections (e.g. (AIDS), pneumonia, Burkitt's lymphoma, chickenpox, haemorrhagic fever, Measles, Mumps, Parainfluenza, Rabies, common cold, Polio, leukemia, Rubella, sexually transmitted diseases, skin diseases (eg, Kaposi, warts) and viremia. A polypeptide or polynucleotide of the present disclosure may be used to treat or detect any of these symptoms or diseases.
Similarly, bacterial or fungal agents which may cause disease or symptoms and which may be treated or detected by a polynucleotide or polypeptide of the present disclosure, include but are not limited to the following bacterial families.
Gram-negative and Gram-positive fungi:
Actinomycetales
g.,
Corinebacterium,
Mycobacterium,
Norcardia),
Aspergillosis,
anthrax,
Borrelia,
Bacteroidaceae,
Blastomycosis,
Bordetella,
Brucellosis,
Candidiasis,
Campylobacter,
Coccidioidomycosis,
Cryptococcosis,
Dermatocycoses,
Enterobacteriaceae (Klebsiella, Salmonella, Serratia, Yersinia),
Erysipelothrix, Helicobacter, Legionellosis, Leptospirosis,
101
Listeria, Mycoplasmatales, Neisseriaceae (eg, Acinetobacter, Gonorrhea, Meningococcus), Pasteurellacea Infections (eg, Actinobacillus, Heamophilus, Pasteurella), Pseudomonas, Rickettsiaceae, Chlamydiaceae, Syphilis and Staphylococcus. These bacterial or fungal families may cause the following diseases or symptoms, including but not limited to: bacteremia, endocarditis, eye infections (conjunctivitis, tuberculosis, uveitis), gingivitis, opportunistic infections (e.g. AIDS-related infections), paronychia, prosthetic-related infections, Reiter's disease, respiratory tract infections such as whooping cough or empyema, sepsis, Lyme disease, cat-scratch disease, dysentery, paratyphoid fever, intoxication Food, Typhoid, Pneumonia, Gonorrhea, Meningitis, Chlamydia, Syphilis, Diphtheria, Leprosy, Paratuberculosis, Tuberculosis, Lupus, Botulism, Gangrene, Tetanus, Impetigo, Rheumatic Fever, Scarlet fever, sexually transmitted diseases, skin diseases (eg, cellulite, dermatocycosis), toxemia, urinary tract infections, wound infections. A polypeptide or polynucleotide of the present disclosure may be used to treat or detect any of these symptoms or diseases.
Furthermore, parasitic agents causing disease or symptoms that may be treated or detected by a polynucleotide or polypeptide of the present description include, but are not limited to, the following families: Amebiasis, Babesiosis,
Coccidiosis,
Cryptosporidiosis,
Dientamoebiasis,
Dourine,
Ectoparasitic,
Giardiasis,
Helminthiasis,
Leishmaniasis,
Theileriasis,
Toxoplasmosis,
Trypanosomiasis and Trichomonas.
These parasites can cause a variety of diseases or symptoms, including but not limited to: Scabies, Thrombiculiasis, Eye Infections, Intestinal Disease (eg, Dysentery,
102 giardiasis), liver disease, lung disease, opportunistic (eg, AIDS-related) infections, malaria, pregnancy complications and toxoplasmosis. A polypeptide or polynucleotide of the present disclosure may be used to treat or detect any of these symptoms or diseases.
Preferably, treatment using a polypeptide or polynucleotide of the present disclosure could be either by administering an effective amount of the polypeptide to the patient or by removing cells from the patient by providing the cells with the polynucleotide and returning the engineered cells to the patient. ill (ex vivo therapy). In addition, the polypeptide or polynucleotide may be used as an antigen in a vaccine to elicit an immune response against infectious disease.
Regeneration
A polynucleotide or polypeptide of the present disclosure may be used to differentiate, proliferate and attract cells, leading to tissue regeneration. (See, Science 276: 59-87 (1997)). Tissue regeneration could be used for tissue damaged by burn defects, incisions or ulcers), osteoporosis, osteochondritis, liver disease), surgery, including fibrosis, reperfusion injury or repair, replacement or protection of birth defects, trauma (wounds, age, disease). (eg
periodontal, cosmetic plastic surgery insufficiency systemic cytokine damage.
103
Tissues that could be regenerated using the present invention include organs (eg, pancreas, liver, intestine, kidney, skin, endothelium), muscle (smooth, skeletal or cardiac), vascular (including vascular endothelium), nervous, hematopoietic and skeletal tissue ( bone, cartilage, tendon and ligament). Preferably, regeneration occurs with or without scar reduction. Regeneration may also include angiogenesis.
Further description, a polynucleotide or polypeptide of the present may enhance the regeneration of difficult to cure tissues. For example, increased tendon / ligament regeneration could accelerate recovery time after injury. The polynucleotide or polypeptide could also be used prophylactically in an effort to prevent damage. Specific diseases that could be treated include tendonitis, carpal tunnel syndrome and other tendon or ligament defects. An additional example of tissue healing of non-healing wounds includes pressure ulcers, vascular-associated ulcers, surgical and traumatic wounds.
Similarly, nerve and brain tissue could also be regenerated by using a polynucleotide or polypeptide of the present disclosure to proliferate and differentiate nerve cells. Diseases that could be treated using this method include central and peripheral nervous system disorders, neuropathies, or mechanical and traumatic disorders (eg, spinal cord disorders, head trauma, cerebrovascular disease, and stroke). Specifically, diseases associated with peripheral nerve damage, peripheral neuropathy (eg, resulting from chemotherapy or other medical therapies), neuropathies
104 Central nervous system disorders (eg, Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, and Shy-Drager syndrome) could all be treated using the polynucleotide or polypeptide of the present disclosure.
Chemotaxis
A polynucleotide or polypeptide of the present disclosure may have chemotaxis activity. A chemotactic molecule attracts or mobilizes cells (eg, monocytes, fibroblasts, neutrophils, T cells, mast cells, eosinophils, epithelial and / or endothelial cells) to a particular body site, such as inflammation, infection or hyperproliferation site. The mobilized cells may then fight and / or cure the particular trauma or abnormality.
A polynucleotide or polypeptide of the present disclosure may enhance the chemotaxis activity of particular cells. These chemotactic molecules can then be used to treat inflammation, infection, hyperproliferative disorders or any immune system disorder by increasing the number of cells targeted to a particular location in the body. For example, chemotactic molecules may be used to treat wounds and other trauma to tissues by attracting immune cells to the injured site. The chemotactic molecules of the present disclosure may also attract fibroblasts that may be used to treat wounds.
105
It is also contemplated that a polynucleotide or polypeptide of the present disclosure may inhibit chemotactic activity. These molecules could also be used to treat disorders. Thus, the polynucleotide or polypeptide could be used as a chemotaxis inhibitor.
Liaison Activity
A polypeptide of the present disclosure may be used to screen for molecules that bind to the polypeptide or for molecules to which the polypeptide binds. Binding of the polypeptide and molecule may activate (agonist), increase, inhibit (antagonist) or decrease the activity of the polypeptide or linked molecule. Examples of such molecules include antibodies, oligonucleotides, proteins (eg, receptors) or small molecules.
Preferably, the molecule is closely related to the natural ligand of the polypeptide, eg, a ligand fragment, or a natural substrate, a ligand, a structural or functional mimetic. (See, Coligan et al., Current Protocols in Immunology 1 (2): Chapter 5 (1991)). Similarly, the molecule may be closely related to the natural receptor to which the polypeptide binds or at least one receptor fragment capable of being bound by the polypeptide (e.g. g., active site). In either case, the molecule may be rationally designed using known techniques.
Preferably, screening for these molecules involves the production of appropriate cells expressing the
106 polypeptides as a secreted protein or on the cell membrane. Preferred cells include mammalian cells, yeast, Drosophila or
AND.
coli.
Cells expressing the polypeptide (or cell membrane containing the polypeptide contact molecule) are then preferably placed on with a test compound, potentially containing observing binding, stimulation or inhibition of activity of the polypeptide or molecule.
The assay may simply test for binding of a candidate compound to the polypeptide, where binding is detected by a label, or in an assay involving competition with a labeled competitor. Additionally, the assay can test whether the candidate compound results in a signal produced by binding to the polypeptide.
Alternatively, the assay may be performed using cell free preparations, polypeptide / molecule attached to a solid support, chemical libraries or mixtures of natural products. The assay may also simply comprise the steps of mixing a candidate compound with a solution containing a polypeptide, measuring the activity or binding of the polypeptide / molecule and comparing the activity or binding of the polypeptide / molecule to a standard.
Preferably, an ELISA may measure the level or activity of polypeptide in a sample (eg, biological sample) using a monoclonal or polyclonal antibody. The antibody may measure the level or activity of polypeptide by binding directly or indirectly to the polypeptide or by competing with the polypeptide for a substrate.
107
All of these prior assays may be used as diagnostic or prognostic markers. The molecules identified using these assays may be used to treat disease or to lead to a particular outcome in a patient (eg, blood vessel growth) through activation or inhibition of the polypeptide / molecule. In addition, assays can identify agents that can inhibit or enhance polypeptide production from properly engineered cells or tissues.
Accordingly, the invention includes a method of identifying compounds that bind to a polypeptide of the invention, comprising the steps of: (a) incubating a candidate binding compound with a polypeptide of the invention; and (b) determine if binding has occurred. Further, the description includes a method of identifying agonists / antagonists, comprising the steps of: (a) incubating a candidate compound with a polypeptide of the invention, (b) assaying a biological activity and (b) determining whether a biological activity of the polypeptide has been altered.
Other activities
A polypeptide or polynucleotide of the present invention may also increase or decrease the differentiation or proliferation of embryonic stem cells in addition to, as discussed above, the hematopoietic lineage.
A polypeptide or polynucleotide of the present invention may also be used to modulate mammalian characteristics such as body height, weight, hair color, color of
108 eyes, skin, percentage of fat, pigmentation, size and shape (eg, cosmetic surgery). Similarly, a polypeptide or polynucleotide of the present invention may be used to modulate mammalian metabolism affecting energy catabolism, anabolism, processing, utilization and storage.
A polypeptide or polynucleotide of the present invention may be used to alter a mammal's mental state or physical state through the influence of biorhythms, heart rhythms, depression (including depressive disorders), tendency to violence, pain tolerance, reproductive abilities ( preferably by Activin or Inhibin-like activity), hormone or endocrine levels, appetite, libido, memory, stress or other cognitive qualities.
A polypeptide or polynucleotide of the present invention may also be used as a food additive or preservative in order to increase or decrease storage capacities, fat content, lipid, protein, carbohydrate, vitamins, minerals, cofactors or other nutritional components.
Other Items
An isolated nucleic acid molecule comprising a nucleotide sequence that is at least 95% identical to a sequence of at least about 50 contiguous nucleotides in the nucleotide sequence of SEQ ID NO: 23 as defined in Table is described. 1.
109
Also described is a nucleic acid molecule, wherein said contiguous nucleotide sequence is included in the nucleotide sequence of SEQ ID NO: 23, in the position range beginning with the nucleotide around the position of the 5 'Nucleotide of the Clone Sequence. and ending with the nucleotide around the position of the 3 'Nucleotide of the Clone Sequence, as defined for SEQ ID NO: 23 in Table 1.
Also described is a nucleic acid molecule, wherein said contiguous nucleotide sequence is included in the nucleotide sequence of SEQ ID NO: 23, in the position range starting with the nucleotide around the position of the 5 'Primer Nucleotide and ending with the nucleotide around the position of the 3 'Nucleotide of the Clone Sequence, as defined for SEQ ID NO: 23 in Table 1.
Similarly, a nucleic acid molecule is described, wherein said contiguous nucleotide sequence is included in the nucleotide sequence of SEQ ID NO: 23, in the position range starting with the nucleotide around the position of the 5 'Nucleotide Signal Peptide First Amino Acid and ending with the nucleotide around the position of the 3 'Nucleotide of the Clone Sequence, as defined for SEQ ID NO: 23 in Table 1.
A preferred embodiment of the present invention is an isolated nucleic acid molecule, comprising a nucleotide sequence that is at least 95% identical to a sequence of at least about 150 contiguous nucleotides in the nucleotide sequence of SEQ ID NO: : 23.
110
Further isolated is an isolated nucleic acid molecule comprising a nucleotide sequence that is at least 95% identical to a sequence of at least about 500 contiguous nucleotides in the nucleotide sequence of SEQ ID NO: 23.
Further described is a nucleic acid molecule, comprising a nucleotide sequence that is at least 95% identical to the nucleotide sequence of SEQ ID NO: 23, starting with the nucleotide around the position of the 5 'Nucleotide of the First Amino Acid of the Peptide Signal and terminating with the nucleotide around the position of the 3 'Nucleotide of the Clone Sequence, as defined for SEQ ID NO: 23 in Table 1.
Further described is a nucleic acid molecule.
<td colspan="5">isolated, comprising a nucleotide sequence which is at least</td>
<td>at least 95% identical SEQ ID NO: 23.</td><td>the sequence</td><td>nucleotide</td><td colspan="2">complete</td>
<td>It is also described</td><td>a molecule</td><td colspan="2">of nucleic acid</td><td>isolated</td>
hybridizing under stringent hybridization conditions to a nucleic acid molecule, wherein said hybridizing nucleic acid molecule does not hybridize under stringent hybridization to a nucleic acid molecule having a nucleotide sequence consisting of only in waste A or only in waste T.
Also preferred is a DNA molecule comprising a human cDNA clone, identified as ATCC Deposit No. 209075, a DNA molecule that is contained in material deposited with the American Type Culture Collection and assigned the Number of
111
ATCC deposit shown in Table 1 for said cDNA Clone Identifier.
Also described is an isolated nucleic acid molecule comprising a nucleotide sequence that is at least 95% identical to a sequence of at least 50 contiguous nucleotides in the nucleotide sequence of a human cDNA clone, a DNA molecule that is contained within. deposit assigned ATCC Deposit Number 209075.
An isolated nucleic acid molecule is also described, wherein said sequence of at least 50 contiguous nucleotides is included in the nucleotide sequence of the complete open reading frame sequence encoded by said human cDNA clone.
Also preferred is an isolated nucleic acid molecule comprising a nucleotide sequence that is at least 95% identical to the sequence of at least 150 contiguous nucleotides in the nucleotide sequence encoded by said human cDNA clone.
Further isolated is an isolated nucleic acid molecule comprising a nucleotide sequence that is at least 95% identical to the sequence of at least 500 contiguous nucleotides in the nucleotide sequence encoded by said human cDNA clone.
Further isolated is an isolated nucleic acid molecule comprising a nucleotide sequence that is at least 95% identical to the complete nucleotide sequence encoded by said human cDNA clone.
112
Further described is a method for detecting in a biological sample a nucleic acid molecule, comprising a nucleotide sequence that is at least 95% identical to a sequence of at least 50 contiguous nucleotides in a sequence selected from the group consisting of: a nucleotide sequence of SEQ ID NO: 23 as defined in Table 1; and a nucleotide sequence encoded by a human cDNA clone, identified by a cDNA Clone Identifier in Table 1 and contained in deposit number ATCC Deposit 209075; A method comprising a step of comparing a nucleotide sequence of at least one nucleic acid molecule in said sample to a sequence selected from said group and determining whether the sequence of said nucleic acid molecule in said sample is at least 95%. % identical to that selected sequence.
Also described is the above method, wherein said sequence comparison step comprises determining the extent of nucleic acid hybridization between nucleic acid molecules in said sample and a nucleic acid molecule comprising said sequence selected from said group. Similarly, the above method is also described, wherein said sequence comparison step is performed by comparing the nucleotide sequence determined from a nucleic acid molecule in said sample to said sequence selected from said group . Nucleic acid molecules may comprise DNA molecules or RNA molecules.
Further described is a method for identifying the species, tissue or cell type of a biological sample, a method comprising a step of detecting molecules of
113 nucleic acids in said sample, if any, comprising a nucleotide sequence that is at least 95% identical to a sequence of at least 50 contiguous nucleotides in a sequence selected from the group consisting of: a nucleotide sequence of SEQ ID NO: : 23 as defined in Table 1; and a nucleotide sequence encoded by a human cDNA clone, identified by a cDNA Clone Identifier in Table 1 and contained in the deposit with ATCC Deposit Number 209075.
The method for identifying the species, tissue or cell type of a biological sample may comprise a step of detecting nucleic acid molecules comprising a nucleotide sequence in a panel of at least two nucleotide sequences, wherein at least one The sequence in said panel is at least 95% identical to a sequence of at least 50 contiguous nucleotides in a sequence selected from said group.
Also preferred is a method for diagnosing in a subject a condition associated with abnormal gene structure or expression encoding a secreted protein identified in Table 1, a method comprising a detection step in a biological sample obtained from said subject, nucleic acid molecules, if any, comprising a nucleotide sequence that is at least 95% identical to a sequence of at least 50 contiguous nucleotides, in a sequence selected from the group consisting of: a nucleotide sequence of SEQ ID NO: 23; and a nucleotide sequence encoded by a human cDNA clone, identified by a cDNA Clone Identifier in Table 1 and contained in the deposit with ATCC Deposit Number 209075.
114 A method for diagnosing a condition may comprise a step of detecting nucleic acid molecules comprising a nucleotide sequence in a panel of at least two nucleotide sequences, wherein at least one sequence in said panel is at least one. 95% identical to a sequence of at least 50 contiguous nucleotides in a sequence selected from said group.
Also described is a composition of matter comprising isolated nucleic acid molecules, wherein the nucleotide sequences of said nucleic acid molecules comprise a panel of at least two nucleotide sequences, wherein at least one sequence in said panel is, at least,
95% identical to a sequence of at least contiguous nucleotides in a sequence selected from the group consisting of:
a nucleotide sequence of the
SEQ ID
23, as defined in Table 1;
and a nucleotide sequence encoded by a human cDNA clone, identified by a cDNA Clone Identifier in Table 1 and contained in the deposition with ATCC Deposit Number 209075. Nucleic acid molecules may comprise DNA molecules or RNA molecules.
An isolated polypeptide comprising at least one amino acid sequence is also described.
90% identical to a sequence of at least about 10 contiguous amino acids in the amino acid sequence of SEQ ID NO:
60 as defined in
Table 1
115
Also described is a polypeptide, wherein said contiguous amino acid sequence is included in the amino acid sequence of SEQ ID NO: 60, in the position range starting with the residue around the position of the First Amino Acid of the Segmentation and ending with the amino acid sequence. residue around the Last Amino Acid of the Open Reading Phase, as shown for
SEQ ID NO: 60, in Table 1.
An isolated polypeptide comprising at least 90% amino acid sequence identical to a sequence of at least about 30 contiguous amino acids in the amino acid sequence of SEQ ID NO: 60 is also described.
An isolated polypeptide comprising at least 90% amino acid sequence identical to a sequence of at least about 100 contiguous amino acids in the amino acid sequence of SEQ ID NO: 60 is also described.
An isolated polypeptide comprising at least 95% amino acid sequence identical to the complete amino acid sequence of SEQ ID NO: 60 is also described.
An isolated polypeptide comprising at least 90% amino acid sequence identical to a sequence of at least about 10 contiguous amino acids in the complete amino acid sequence of a secreted protein encoded by a human cDNA clone is further described. identified by a cDNA Clone Identifier in Table 1 and contained in the deposit with ATCC Deposit Number 209075.
Further described is a polypeptide, wherein said contiguous amino acid sequence is included in the sequence of
116 amino acids of a secreted portion of the secreted protein, encoded by a human cDNA clone, identified by a cDNA Clone Identifier in Table 1 and contained in the deposit with ATCC Deposit Number 209075.
An isolated polypeptide comprising at least 95% amino acid sequence identical to a sequence of at least about 30 contiguous amino acids in the amino acid sequence of the secreted portion of the protein encoded by a human cDNA clone is also described. identified by a cDNA Clone Identifier in Table 1 and contained in the deposit with ATCC Deposit Number 209075.
An isolated polypeptide comprising at least 95% amino acid sequence identical to a sequence of at least about 100 contiguous amino acids in the amino acid sequence of the secreted portion of the protein encoded by a human cDNA clone is also described. identified by a cDNA Clone Identifier in Table 1 and contained in the deposit with ATCC Deposit Number 209075.
An isolated polypeptide comprising at least 95% amino acid sequence identical to the amino acid sequence of the secreted portion of the protein encoded by the human cDNA clone contained in the deposit number ATCC 209075 is preferred.
Further disclosed is an isolated antibody that specifically binds to a polypeptide, comprising an amino acid sequence that is at least 90% identical to a sequence of at least 10 contiguous amino acids in a sequence selected from the group consisting of: a sequence of amino acids of SEQ ID
117
: °: 60 as defined in Table 1; and a complete amino acid sequence of a protein encoded by a human cDNA clone, identified by a cDNA Clone Identifier in Table 1 and contained in deposit number ATCC 209075.
Further described is a method for detecting in a biological sample a polypeptide, comprising an amino acid sequence that is at least 90% identical to a sequence of at least 10 contiguous amino acids in a sequence selected from the group consisting of: a sequence of amino acids of SEQ ID NO: 60 as defined in Table 1; and a complete amino acid sequence of a protein encoded by a human cDNA clone, identified by a cDNA Clone Identifier in Table 1 and contained in deposit number ATCC 209075; A method comprising a step of comparing an amino acid sequence of at least one polypeptide molecule in said sample with a sequence selected from said group and determining whether the sequence of said polypeptide molecule in said sample is at least 90% identical to that in said sequence of at least 10 contiguous amino acids.
Also described is the above method, wherein said step of comparing an amino acid sequence of at least one polypeptide molecule in said sample with a sequence selected from said group comprises determining the specific binding extent of polypeptides in said sample. to an antibody that specifically binds to a polypeptide, comprising an amino acid sequence that is at least 90% identical to a sequence of at least 10 contiguous amino acids in a sequence selected from the group
118 consisting of: an amino acid sequence of SEQ ID NO: 60 as defined in Table 1; and a complete amino acid sequence of a protein encoded by a human cDNA clone, identified by a cDNA Clone Identifier in Table 1 and contained in deposit number ATCC 209075.
Also described is the above method, wherein said sequence comparison step is performed by comparing the amino acid sequence determined from a polypeptide molecule in said sample with said sequence selected from said group.
Also described is a method for identifying the species, tissue or cell type of a biological sample, a method comprising a step of detecting polypeptide molecules in said sample, if any, comprising an amino acid sequence that is at least 90% identical to a sequence of at least 10 contiguous amino acids in a sequence selected from the group consisting of: an amino acid sequence of SEQ ID NO: 60 as defined in Table 1; and a complete amino acid sequence of a secreted protein encoded by a human cDNA clone, identified by a cDNA Clone Identifier in Table 1 and contained in deposit number ATCC Deposit 209075.
Also described is the above method for identifying the species, tissue or type of method comprising a polypeptide comprising a panel of at least two at least one sequence in the cell of a biological sample, step of detecting molecules an amino acid sequence in a amino acid sequences, wherein said panel is at least 90%
119 identical to a sequence of at least 10 contiguous amino acids in a sequence selected from the group above.
Also preferred is a method for diagnosing, in an individual, a condition associated with the abnormal structure or expression of a gene encoding a secreted protein identified in Table 1, a method comprising a detection step in a biological sample obtained from the polypeptide molecules of the gene. said subject comprising an amino acid sequence in a panel of at least two amino acid sequences, wherein at least one sequence in said panel is at least 90% identical to a sequence of at least 10 contiguous amino acids in a sequence selected from the group consisting of: an amino acid sequence of SEQ ID NO: 60, as defined in Table 1; and a complete amino acid sequence of a secreted protein encoded by a human cDNA clone, identified by a cDNA Clone Identifier in Table 1 and contained in deposit number ATCC Deposit 209075.
In either of these methods, the step of detecting said polypeptide molecules includes the use of an antibody.
Also described is an isolated nucleic acid molecule, comprising a nucleotide sequence that is at least 95% identical to a nucleotide sequence encoding a polypeptide, wherein said polypeptide comprises an amino acid sequence that is at least 90% identical. to a sequence of at least 10 contiguous amino acids in a sequence selected from the group consisting of: an amino acid sequence of SEQ ID NO: 60; and an amino acid sequence
120 of a secreted protein encoded by a human cDNA clone, identified by a cDNA Clone Identifier in Table 1 and contained in the deposit with ATCC Deposit Number 209075.
An isolated nucleic acid molecule is also described, wherein said nucleotide sequence encoding a polypeptide has been optimized for expression of said polypeptide in a prokaryotic host.
Also isolated is an isolated nucleic acid molecule wherein said polypeptide comprises an amino acid sequence selected from the group consisting of: an amino acid sequence of SEQ ID NO: 60 as defined in Table 1; and a complete amino acid sequence of a secreted protein encoded by a human cDNA clone, identified by a cDNA Clone Identifier in Table 1 and contained in deposit number ATCC Deposit 209075.
Further described is a method of preparing a recombinant vector comprising inserting any of the above isolated nucleic acid molecules into a vector. Also preferred is the recombinant vector produced by this method. Also preferred is a method of preparing a recombinant host cell comprising introducing the vector into a host cell as well as the recombinant host cell produced by this method.
Also preferred is a method of preparing an isolated polypeptide comprising culturing this recombinant host cell under conditions such that said polypeptide is expressed and recovering said polypeptide.
121 polypeptide. Also preferred is this method of preparing an isolated polypeptide, wherein said recombinant host cell is a eukaryotic cell and said polypeptide is a secreted portion of a secreted human protein, comprising an amino acid sequence selected from the group consisting of: a sequence of amino acids of SEQ ID NO: 60, beginning with the residue at the position of the First Amino Acid of the Segregated Portion of SEQ ID NO: 60 shown in Table 1 and said position of the First Amino Acid Segregated Portion of SEQ ID NO: 60 is defined in Table 1; and an amino acid sequence of a secreted portion of a protein encoded by a human cDNA clone, identified by a cDNA Clone Identifier in Table 1 and contained in the deposition with ATCC Deposit Number 209075. The isolated polypeptide produced by this method It is also preferred.
Also described is a method of treating an individual in need of an increased level of secreted protein activity, which method comprises administering to such an individual a pharmaceutical composition comprising an amount of an effective isolated polypeptide, polynucleotide or antibody of the claimed invention. to increase the level of said protein activity in said individual.
Having the invention generally described, it will be more readily understood by reference to the following examples which are provided by way of illustration and should not be construed as limiting.
122
Examples
Example 1: Isolation of a Selected cDNA Clone from
From Deposited Sample
Each cDNA clone in a cited ATCC deposit is contained in a plasmid vector. Table 1 identifies the vectors used to construct the cDNA library from which each clone was isolated. In many cases, the vector used to construct the library is a phage vector from which a plasmid has been excised. The Table immediately below correlates the related plasmid for each phage vector used in constructing the cDNA library. For example, if a particular clone is identified in Table 1 as being isolated on the deposited Lambda vector it is in pBluescript.
Vector Used for
Build the library
Lambda Zap
Uni-Zap XR
Zap Express lafmid BA pSportl pCMVSport 2.0 pCMVSport 3.0 pCR®2.1
Zap, the corresponding clone
Deposited Plasmid
Corresponding pBluescript (pBS) pBluescript (pBS) pBK plasmid BA pSportl pCMVSport 2.0 pCMVSport 3.0 pCR®2.1
123
Lambda Zap Vectors (US Patents N<sup>the</sup> 5,128,256 and 5,286,636), Uni-Zap XR (US Pat.<sup>the</sup> 5,128,256 and 5,286,636), Zap Express (US Pat.<sup>the</sup> 5,128,256 and 5,286,636), pBluescript (pBS) (Short, UM et al., Nucleic Acids Res. 16: 7583-7600 (1988);
Alting Mees, Μ. A. and Short, JM, Nucleic Acids Res. 17: 9494 (1989)) and pBK (Alting-Mees, A. A. et al., Strategies 5: 58-61 (1992)), are commercially available from Stratagene Cloning Systems, Inc., 11011 N. Torrey Pines Road, La Uolla, CA, 92037. pBS contains an ampicillin resistance gene and pBK contains a neomycin resistance gene. Both can be transformed into the E. coli XL-1 Blue strain, also available from Stratagene. PBS comes in 4 forms SK +, SK-, KS + and KS. S and K refer to the orientation of the polylinker with respect to T7 and T3 primers flanking the polyligantine region (S is for Saci and K is for Kpnl which are the first sites at each respective end of the ligand). + or refers to the orientation of the origin of replication fl (ori), such that in one orientation, single stranded rescue initiated from the ori orientation generates sense stranded DNA and in the other antisense.
The pSportl, pCMVSport 2.0 and pCMVSport 3.0 vectors were obtained from Life Technologies, Inc., PO Box 6009, Gaithersburg, MD 20897. All Sport vectors contain an ampicillin resistance gene and can be transformed into the E. coli strain. DH10B, also available from Life Technologies. (See, for example, Gruber, CE, et al., Focus 15:59 (1993)). The lafmid BA vector (Bento Soares, Columbia
University, NI) contains an ampicillin resistance gene and can be transformed into the E. coli XL-1 Blue strain. The pCR®2.1 vector, which is available from Invitrogen, 1600 Faraday Avenue, Carlsbad, CA 92008, contains a gene from
124 ampicillin resistance and can be transformed into the E. coli DH10B strain, available from Life Technologies. (See, for example, Clark, JM, Nuc. Acids Res. 16: 9677-9686 (1988) and Mead, D. et al., Bio / Technology 9: (1991)). Preferably, a polynucleotide of the present invention does not comprise the phage vector sequences identified for the particular clone in Table 1, as well as the corresponding plasmid vector sequences designated above.
The material deposited in the sample was assigned the ATCC Deposit Number cited in Table 1, as any given clone may also contain one or more additional plasmids, each comprising a different cDNA clone than that given clone. Thus, deposits sharing the same ATCC Deposit Number contain at least one plasmid for each cDNA clone identified in Table 1. Typically, each ATCC depot sample cited in Table 1 comprises a mixture of approximately equal amounts (by weight) of about 50 plasmid DNAs, each containing a different cDNA clone; but such a sample deposit may include plasmids for about 50 cDNA clones, up to about 500 cDNA clones.
Two approaches can be used to isolate a particular clone from the plasmid DNA sample deposit cited for that clone in Table 1. First, a plasmid is directly isolated by screening clones using a polynucleotide probe corresponding to SEQ ID NO: : X.
125
In particular, a specific 30-40 nucleotide polynucleotide is synthesized using an Applied Biosystems DNA synthesizer according to the sequence described. The oligonucleotide is labeled, for example, with<sup>32</sup>Ρ-γ-ΑΤΡ using
T4 polynucleotide kinase is purified according to routine methods.
(eg, Maniatis et al., Molecular Cloning: A
Laboratory
Manual,
Cold spring harbor
Press, Cold Spring, NY (1982)). The plasmid mixture is transformed into a suitable host as indicated above (as described above).
XL-1 Blue (Stratagene)), using techniques known to those skilled in the art, such as those provided by the vector supplier or in related publications or patents cited above. Transformants are plated on 1.5% agar plates (containing the appropriate selection agent, eg, ampicillin) to a density of about 150 transformants (colonies) per plate. These plates are screened using Nylon membranes according to routine methods for bacterial colony screening (eg, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2<sup>The</sup> Edition, (1989), Cold Spring Harbor Laboratory Press, pages 1.93 to 1.104) or other techniques known to those skilled in the art.
Alternatively, two 17-20 nucleotide primers derived from both ends of SEQ ID NO: X (ie, within the region of SEQ ID NO: X linked by the 5 'NT and the 3' NT of the clone defined in Table 1 ) are synthesized and used to amplify the desired cDNA using the deposited cDNA plasmid as a template. The polymerization chain reaction is carried out under routine conditions, for example in 25 µl reaction mixture with 0.5 µg of the above cDNA template. A convenient reaction mixture is MgCl<sub>2</sub> 1.5-5 mM, 0.01% (w / v) gelatin, 20 pM of each dATP, dCTP, dGTP, dTTP, 25 pmol of
126 each primer and 0.25 Taq polymerase Units. Thirty-five PCR cycles (denaturation at 94 ° C for 1 min; pairing at 55 ° C for 1 min; elongation at 72 ° C for 1 min) are performed with a Perkin-Elmer Cetus automated thermocycler . The amplified product is analyzed by agarose gel electrophoresis and the expected molecular weight DNA band is excised and purified. The PCR product is found to be the sequence selected by subcloning and sequencing the DNA product.
Various methods are available for identifying non-coding 5 'or 3' portions of a gene that may not be present in the deposited clone. These methods include, but are not limited to, filter probing, clone enrichment using specific probes and similar or identical protocols for 5 'and 3' RACE protocols that are well known in the art. For example, a method similar to RACE 5 'is available to produce the missing 5' end of a desired full length transcript. (Fromont-Racine et al., Nucleic Acids Res. 21 (7): 1683-1684 (1993)).
Briefly, an RNA-specific oligonucleotide is ligated to the 5 'ends of an RNA population, presumably containing full length gene RNA transcripts. A primer set containing a primer specific for the ligated RNA oligonucleotide and a primer specific for a known sequence of the gene of interest is used to PCR amplify the 5 'portion of the desired full length gene. This amplified product can then be sequenced and used to produce the full length gene.
127
This prior method begins with total RNA isolated from the desired source, although poly-A + RNA may be used. The RNA preparation can then be treated, if necessary, with phosphatase to eliminate 5 'phosphate groups in degraded or damaged RNA that may interfere with the later RNA ligase step. Phosphatase should then be inactivated and RNA treated with acid tobacco pyrophosphatase to remove the cap structure present at the 5 'ends of messenger RNA.
This reaction leaves the 5 'phosphate group at the 5' end of cleaved capped RNA which can then be ligated to an RNA oligonucleotide using T4 RNA ligase.
This modified RNA preparation is used as a template for first strand cDNA synthesis using a gene specific oligonucleotide. The first strand synthesis reaction is used as a template for PCR amplification of the desired 5 'end using a primer specific for the ligated RNA oligonucleotide and a primer specific for the known gene sequence of interest. 0 The resulting product is then sequenced and analyzed to confirm that the 5 'end sequence belongs to a desired gene.
Example 2: Isolation of Genomic Clones Matching a Polynucleotide
A human genomic P1 library (Genomic Systems, Inc.) is PCR screened using primers selected for the cDNA sequence corresponding to SEQ ID NO: X according to the method described in Example 1. (See also Sambrook) .
128
Example 3: Tissue Distribution of Polypeptide
The tissue distribution of polynucleotide mRNA expression of the present invention is determined using protocols for Northern blot analysis, described, among others, by Sambrook et al. For example, a cDNA probe produced by the method described in Example 1 and labeled with P using the rediprime ™ DNA labeling system (Amersham Life Science) according to the manufacturer's instructions. After labeling, the probe is purified using CHROMA SPIN-100 ™ column (Clontech Laboratories, Inc.) according to manufacturer protocol number PT1200-1. The purified labeled probe is then used to examine various human tissues for mRNA expression.
Transfers
Northern of
Multiple Tissues (MTN), containing various human tissues or human immune system (TM) tissues (Clontech), are examined with the labeled probe using hybridization solution.
ExpressHyb ™ (Clontech) according to manufacturer protocol number PT1190-1. After hybridization and washing, blots are mounted and exposed to film at -70 ° C overnight and films are developed according to standard procedures.
Example 4: Chromosome Mapping of Polynucleotides
An oligonucleotide primer set is designed according to the sequence at the terminus
5 'from SEQ
ID #:
X. The primer preferably comprises about
100 nucleotides. This set of primers is then used in a polymerization chain reaction under
129 following set of conditions: 30 seconds, 95 ° C; minute, ° C; minute, 70 ° C. This cycle is repeated 32 times, followed by a 5 minute cycle at 70 ° C. Human, mouse, and hamster DNA is used as a template in addition to a somatic cell hybrid panel containing chromosomes or individual chromosome fragments (Bios, Inc). Reactions are analyzed on either 8% polyacrylamide gels or 3.5% agarose gels. Chromosomal mapping is determined by the presence of an approximately 100 bp PCR fragment in the particular somatic cell hybrid.
Example 5: Bacterial Expression of a Polypeptide
A polynucleotide encoding a polypeptide of the present invention is amplified using PCR oligonucleotide primers corresponding to the 5 'and 3' ends of the DNA sequence as outlined in Example 1 to synthesize insert fragments. Primers used to amplify the cDNA insert should preferably contain restriction sites, such as BamHI and XbaI, at the 5 'end of the primers, to clone the amplified product within the expression vector. For example, BamHI and Xbal correspond to the enzyme restriction sites in the pQE-9 bacterial expression vector. (Qiagen, Inc., Chatsworth, CA). This plasmid vector encodes antibiotic resistance (Amp<sup>r</sup>), a bacterial origin of replication (ori), an IPTG-adjustable promoter / operator (P / O), a ribosomal binding site (RBS), a 6-histidine tail (6-His), and restriction enzyme cloning sites .
The pQE-9 vector is digested with BamHI and XbaI and the amplified fragment is ligated into the pQE-9 vector, maintaining the phase of
130 reading started on bacterial RBS. The ligation mixture is then used to transform E. coli M 15 / rep4 strain (Qiagen, Inc.) which contains multiple copies of plasmid pREP4, which expresses the lacl repressor and also confers kanamycin resistance (Kan<sup>r</sup>). Transformants are identified by their ability to grow on LB plates and ampicillin / kanamycin resistant colonies are selected. Plasmid DNA is isolated and confirmed by restriction analysis.
Clones containing the desired constructs are grown overnight (0 / N) in liquid culture in LB media supplemented with either Amp (100 pg / mL) or Kan (25 pg / mL). The O / N culture is used to inoculate a large culture at a ratio of 1: 100 to 1: 250. Cells are grown to an optical density 600 (OD<sup>600</sup>) between 0.4 and 0.6. IPTG (Isopropyl-BD-thiogalate pyranoside) is then added to a final concentration of 1 mM. IPTG induces, by inactivating the lacl repressor, clearing P / O, leading to increased gene expression.
The cells are cultured for an extra 3 to 4 hours. The cells are then harvested by centrifugation (20 min. At 6000 X g). The cell pellet is solubilized in the chaotropic agent, 6 Molar Guanidine HCl, by stirring for 3-4 hours at 4 ° C. Cell debris is removed by centrifugation and the supernatant containing the polypeptide is loaded onto a nickel nitrile tri-acetic acid (Ni-NTA) affinity resin column (available from QIAGEN, Inc., supra). Proteins with a 6 x His tail bind to high affinity Ni-NTA resin and can be purified
131 in a simple one-step process (for details see: The
QIAexpressionist (1995) QIAGEN, Ine., Supra).
Briefly, the supernatant is loaded onto the column in 6 M guanidine-HCl, pH 8, the column is first washed with 10 volumes of 6 M guanidine-HCl, pH 8, then washed with 10 volumes of 6 guanidine-HCl M, pH 6 and finally the polypeptide is eluted with 6 M guanidine-HCl, pH 5.
The purified protein is then renatured by its dialysis against phosphate buffered saline (PBS) or 50 mM Na-acetate buffer, pH 6, plus 200 mM NaCl. Alternatively, the protein may be successfully refolded while immobilized on the Ni-NTA column. The recommended conditions are as follows: renature using a linear gradient of 6 Ml urea in 500 mM NaCl, 20% glycerol, 20 mM Tris / HCl, pH 7.4, containing protease inhibitors. Renaturation should be performed over a period of 1.5 hours or more. After renaturation, proteins are eluted by the addition of 250 mM immidazole. Imidazole is removed by a final dialysis step against PBS buffer or 50 mM sodium acetate, pH 6, plus 200 mM NaCl. Purified protein is stored at 4<sup>O</sup> C or frozen at -80 ° C.
In addition to the above expression vector, the present invention further includes an expression vector comprising phage operator and promoter elements operably linked to a polynucleotide of the present invention, called pHE4a. (ATCC Access Number 209645, filed February 25, 1998). This vector contains: 1) a neomycin phosphotransferase gene as a selection marker, 2) an E. coli origin of replication, 3) a promoter sequence
132 phage T5, 4) two lac operator sequences, 5) one Shine-Delgarno sequence and 6) the lactose operon repressor gene (laclq). The origin of replication (oriC) is derived from pUC19 (LTI, Gaithersburg, MD). Promoter and operator sequences are synthetically prepared.
DNA can be inserted into pHEa by restriction of the vector with Ndel and XbaI, BamHI, XhoI or Asp718, running the restricted product on a gel and isolation of the larger fragment (the filler fragment should be about 310 base pairs). The DNA insert is generated according to the PCR protocol described in Example 1 using PCR primers having restriction sites for Ndel (5 'primer) and Xbal, BamHI, Xhol or Asp718 (3' primer). The PCR insert is gel purified and restricted with compatible enzymes. The insert and vector are ligated according to standard protocols.
The engineered vector could easily be substituted in the previous protocol to express protein in a bacterial system.
Example 6: Purification of a Polypeptide from a
Inclusion Body
The following alternative method may be used to purify an E. coli expressed polypeptide when present as inclusion bodies. Unless otherwise specified, all of the following steps are conducted at 4-10 ° C.
133
Upon completion of the E. coli fermentation production phase, the cell culture is cooled to 4-10 ° C and cells are harvested by continuous centrifugation at 15000 rpm (Heraeus Sepatech). Based on the expected protein yield per unit weight of cell paste and the amount of purified protein required, an appropriate amount of cell paste by weight is suspended in a buffer solution containing 100 mM Tris, 50 mM EDTA, pH 7, 4 The cells are dispersed to a homogeneous suspension using a high shear mixer.
The cells are then lysed by passing the solution through a microfluidizer (Microfuidics, Corp. or APV Gaulin, Inc.) twice at 4000-6000 psi. The homogenate is then mixed with NaCl solution to a final concentration of 0.5 M NaCl, followed by centrifugation at 7000 xg for 15 min. The resulting pellet is washed again using 0.5 M NaCl, 100 mM Tris, 50 mM EDTA, pH 7.4.
The resulting washed inclusion bodies are solubilized with 1.5 M guanidine hydrochloride (GuHCl) for 2-4 hours. After centrifugation at 7000 xg for 15 min., The pellet is discarded and the supernatant-containing polypeptide is incubated at 4 ° C overnight to allow for additional GuHCl extraction.
Following high speed centrifugation (30000 xg) to remove insoluble particles, the GuHCl-solubilized protein is refolded by rapidly mixing the GuHCl extract with 20 volumes of 50 mM sodium pH 4.5 NaCl buffer. at 150 mM, 2 mM EDTA by vigorous stirring. The diluted refolded protein solution is kept at 4 ° C without
134 mixing for 12 hours before further purification steps.
In order to clarify the refolded polypeptide solution, a previously prepared tangential filtration unit equipped with a 0.16 µm membrane filter with appropriate surface area (eg, Filtron) balanced with 40 mM sodium acetate is employed. pH 6.0. The filtered sample is loaded onto a cation exchange resin (eg, Poros HS-50, Perseptive Biosystems). The column is washed with 40 mM sodium acetate, pH 6.0 and eluted with 250 mM, 500 mM, 1000 mM and 1500 mM NaCl in the same buffer in a stepwise mode. The absorbance at 280 nm of the effluent is continuously monitored. Fractions are collected and further analyzed by SDS-PAGE.
Fractions containing the polypeptide are then pooled and mixed with 4 volumes of water. The diluted sample is then loaded onto a previously prepared serial column assembly of strong anion exchange resins (Poros HQ-50, Perseptive Biosystems) and weak anionic resins (Poros CM-20, Perseptive Biosystems). The columns are equilibrated with 40 mM sodium acetate, pH 6.0. Both columns are washed with 40 mM sodium acetate, pH 6.0, 200 mM NaCl. The CM-20 column is then eluted using a 10 column volume linear gradient, ranging from 0.2 M NaCl, 50 mM sodium acetate, pH 6.0 and NaCl a
1.0 M, 50 mM sodium acetate, pH 6.5.
Fractions are collected under monitoring A<sub>2</sub>constant flow of effluent.
Fractions containing the polypeptide (determined, for example, by
16% SDS-PAGE) are then aggregated.
Following the previous refolding and purification steps, the resulting polypeptide should exhibit greater than 95% purity. No
135 Important contaminant bands should be observed from Commassie blue stained 16% SDS-PAGE gel when 5 µg of purified protein is loaded. Purified protein can also be tested for endotoxin / LPS contamination and typically the LPS content is less than 0.1 ng / mL according to LAL assays.
Example 7: Cloning and Expression of a Polypeptide in a
Baculovirus
Expression System
In this example, the plasmid shuttle vector pA2 is used to insert a polynucleotide into a baculovirus to express a polypeptide. This expression vector contains the strong Authedpha californica nuclear polyhedrosis virus (AcMNPV) polyhedrin promoter, followed by convenient restriction sites such as BamHI, Xba I and Asp718. The simian virus 40 (SV40) polyadenylation site is used for efficient polyadenylation. For easy selection of recombinant virus, the plasmid contains the E. coli beta-galactosidase gene, under the control of a weak Drosophila promoter in the same orientation, followed by the polyhedrin gene signal. The inserted genes are flanked on both sides by viral sequences for homologous recombination of cellular mediation with wild-type viral DNA to produce a viable virus expressing the cloned polynucleotide.
Many other baculovirus vectors may be used in place of the above vector, such as pAc373, pVL941 and pAcIM1, as one skilled in the art would readily understand, provided
136 the construct provides appropriately localized signals for transcription, translation, secretion and the like, including a signal peptide and an in-phase AUG as required. Such vectors are described, for example, in Luckow et al., Virology 170: 31-39 (1989).
Specifically, the cDNA sequence contained in the deposited clone, including the AUG initiation codon and the naturally associated leader sequence identified in Table 1, is amplified using the PCR protocol described in Example 1. If the naturally occurring signal sequence is used To produce the secreted protein, the pA2 vector does not require a second signal peptide. Alternatively, the vector may be modified (pA2 GP) to include a baculovirus leader sequence using the standard method described in Summers et al., A Manual of Methods for Baculovirus Vectors and Insect Cell Culture Procedures, Texas Agricultural Experimental Station Bulletin No. 1555 (1987).
The amplified fragment is isolated from a 1% agarose gel using a commercially available kit (Geneclean, BIO 101 Inc., La Jolla, Ca.). The fragment is then digested with appropriate restriction enzymes and further purified on a 1% agarose gel.
The plasmid is digested with the corresponding restriction enzymes and optionally can be dephosphorylated using calf intestinal phosphatase using routine procedures known in the art. DNA is then isolated from a 1% agarose gel using a commercially available kit (Geneclean BIO 101 Inc., La Jolla, Ca.).
137 The fragment and the dephosphorylated plasmid are linked together with T4 DNA ligase. E. coli HB101 or other suitable E. coli hosts, such as XL-1 Blue cells (Stratagene Cloning Systems, La Jolla, CA) are transformed with the ligation mixture and propagated in culture plates. Bacteria containing the plasmid are identified by DNA digestion of individual colonies and analysis of the digestion product by gel electrophoresis. The sequence of the cloned fragment is confirmed by DNA sequencing.
Five pg of a polynucleotide-containing plasmid is co-transfected with 1.0 pg of commercially available linearized baculovirus DNA (BaculoGold ™ baculovirus DNA, Pharmingen, San Diego, CA) using the lipofection method described by Felgner et al. ., Proc. Natl. Acad. Know. USA 84: 7413-7417 (1987). One µg of BaculoGold ™ virus DNA and 5 µg of the plasmid are mixed in a sterile well of a microtiter plate containing 50 µl Grace serum free medium (Life Technologies Inc., Gaithersburg, MD). Subsequently, 10 µl Lipofectin plus 90 µl Grace medium is added, mixed and incubated for 15 minutes at room temperature. Then, the transfection mixture is added dropwise to Sf9 insect cells (ATCC CRL 1711), inoculated into a 35 mm tissue culture dish with 1 ml serum free Grace medium. The plate is then incubated for 5 hours at 27 ° C. The transfection solution is then removed from the plate and 1 ml Grace insect medium supplemented with 10% fetal calf serum is added. Cultivation is then continued at 27 ° C for four days.
After four days, the supernatant is collected and a plaque assay is performed as described by Summers and Smith, supra. IS
138 A Blue Gal agarose gel (Life Technologies Inc., Gaithersburg) is used to allow easy identification and isolation of blue expressing clones producing blue stained plaques. (A detailed description of such a plaque assay can also be found in the user's guide for insect cell culture and baculovirology, distributed by Life Technologies Inc., Gaithersburg, page 9-10). After appropriate incubation, the blue stained plates are collected with a micropipettor tip (eg, Eppendorf). The agar containing the recombinant viruses is then resuspended in a microcentrifuge tube containing 200 µl Grace medium and the suspension containing the recombinant baculovirus is used to infect inoculated Sf9 cells in 35 mm dishes. Four days later, the supernatants from these culture plates are collected and then stored at 4 ° C.
In order to verify polypeptide expression, Sf9 cells are cultured in Grace medium supplemented with FBS at
10% heat inactivated.
Cells are infected with the polynucleotide-containing recombinant baculovirus at a multiplicity of infection (MOI) of about 2.
If radiolabelled proteins are desired, 6 hours later the medium is removed and replaced with SF900 II medium minus methionine and cysteine (available from Life Technologies Inc., Rockville, MD). After 42 hours, 5 pCi of<sup>35</sup>S-methionine and 5 pCi of <sup>35</sup>S-cysteine (available from Amersham). The cells are further incubated for 16 hours and then harvested by centrifugation. Proteins in the supernatant as well as intracellular proteins are analyzed by SDS-PAGE, followed by autoradiography (radiolabeled).
139
Microsequencing of the amino acid amino acid sequence of purified protein can be used to determine the amino-terminal sequence of the protein produced.
Example 8: Expression of a Mammalian Cell Polypeptide The polypeptide of the present invention may be expressed in a mammalian cell. A typical mammalian expression vector contains a promoter element that mediates mRNA transcription initiation, a protein coding sequence, and signals required for transcription termination and transcription polyadenylation. Additional elements include enhancers, Kozak sequences, and intermediate sequences flanked by donor and acceptor sites for RNA excision. Highly efficient transcription is achieved with SV40 early and late promoters, Retrovirus long terminal repeats (LTRS), eg, RSV, HTLVI, HIVI and cytomegalovirus early promoter (CMV). However, cellular elements (eg the human actin promoter) may also be used.
Expression vectors suitable for use in the practice of the present invention include, for example, vectors such as pSVL and pMSG (Pharmacia, Uppsala, Sweden), pRSVcat (ATCC 37152), pSV2dhfr (ATCC 37146), pBC12MI (ATCC 67109), pCMVSport 2.0 and pCMVSport 3.0. Mammalian host cells that could be used include human Hela, 293, H9 and Jurkat cells, NIH3T3 and C127 mouse cells, Cos 1, Cos 7 and CV1 cells, QC1-3 quail cells, mouse L cells and ovarian cells. of Chinese hamster (CHO).
140
Alternatively, the polypeptide may be expressed in stable cell lines containing the integrated polynucleotide within a chromosome. Co-transfection with a selectable marker such as dhfr, gpt, neomycin, hygromycin allows the identification and isolation of transfected cells.
The transfected gene can also be amplified to express large amounts of the encoded protein. The DHFR (dihydrofolate reductase) marker is useful in the development of cell lines carrying several hundred or even several thousand copies of the gene of interest. (See, eg, Alt, FW, et al., J. Biol. Chem. 253: 1357-1370 (1978); Hamlin, JL and Ma, C., Biochem. Et Biophys. Acta, 1097: 107-143 ( 1990); Page, MJ and Sydenham, A., Biotechnology 9: 64-68 (1991)). Another useful selection marker is the glutamine synthase (GS) enzyme (Murphy et al., Biochem J. 227: 277-279 (1991); Bebbington et al., Bio / Technology 10: 169-175 (1992)). Using these markers, mammalian cells are cultured in selective medium and cells with the highest resistance are selected. These cell lines contain the amplified gene (s) integrated within a chromosome. Chinese hamster ovary (CHO) and NSO cells are often used for protein production.
Derived from plasmid pSV2-dhfr (ATCC Accession No. 37146), the expression vectors pC4 (ATCC Accession No. 209646) and pC6 (ATCC Accession No. 209647) contain the strong Rous Sarcoma Virus (LTR) promoter (Cullen). et al., Molecular and Cellular Biology, 438-447 (March 1985)) plus a fragment of the CMV enhancer (Boshart et al., Cell 41: 521-530 (1985)). Multiple cloning sites, eg, with the BamHI, Xbal and Asp718 enzyme restriction cleavage sites, facilitate cloning of the
141 interest. The vectors also contain the 3 'intron, the polyadenylation signal and termination of the rat preproinsulin gene, and the mouse DHFR gene under control of the SV40 early promoter.
Specifically, plasmid pC6, for example, is digested with appropriate restriction enzymes and then dephosphorylated using calf intestinal phosphatase by methods known in the art. The vector is then isolated from a 1% agarose gel.
A polynucleotide of the present invention is amplified according to the protocol outlined in Example 1. If the naturally occurring signal sequence is used to produce the secreted protein, the vector does not require a second signal peptide. Alternatively, if the naturally occurring signal sequence is not used, the vector may be modified to include a heterologous signal sequence. (See, e.g. WO 96/34891.) Amplified fragment is isolated from a 1% agarose gel using a commercially available kit (Geneclean, BIO 101 Inc., La Jolla, Ca.). The fragment is then digested with appropriate restriction enzymes and further purified on a 1% agarose gel.
The amplified fragment is then digested with the same restriction enzymes and purified on a 1% agarose gel. The isolated fragment and the dephosphorylated vector are then ligated with T4 DNA ligase. E. coli HB 101 or XL-1 Blue cells are then transformed and the bacteria containing the fragment
142 inserted into plasmid pC6 are identified using, for example, enzyme restriction analysis.
Chinese hamster ovary cells lacking an active DHFR gene are used for transfection.
Five pg of the pC6 expression plasmid are co-transfected with
0.5 pg of plasmid pSVneo using lipofectin (Felgner et al., Supra). Plasmid pSV2-neo contains a dominant selectable marker, the Tn5 neo gene encoding an enzyme that confers resistance to a group of antibiotics, including G418. Cells are inoculated into MEM minus alpha supplemented with 1 mg / mL G418.
After 2 days, the cells are trypsinized and inoculated into hybridoma cloning plates (Greiner,
Germany) in MEM minus alpha, supplemented with 10, 25 or 50 ng / mL methotrexate plus 1 mg / mL G418. After about 10-14 days, single clones are trypsinized and then seeded in
6-well petri or mL glass flasks using nM, 100 nM, 200 nM,
400 nM, 800 nM). Clones growing at the highest concentrations of methotrexate are then transferred to new 6-well plates containing even higher concentrations of mM, 2 pM, 5 pM, mM, 20 mM). The same process is repeated until clones growing at a concentration of 100
200 pM Expression of the desired gene product is analyzed, for example, by SDS-PAGE and Western blotting or by reverse phase HPLC analysis.
Example 9: Protein Fusion
The polypeptides of the present invention are preferably fused to other proteins. These fusion proteins
143 can be used for a variety of applications. For example, the fusion of the present His tail, HA tail, protein A, IgG domains and maltose binding protein polypeptides facilitates purification. (See Example 5; see also EP A 394827; Traunecker, et al., Nature 331: 84-86 (1988)). Similarly, fusion to IgG-β, IgG-3 and albumin increases the half-life in vivo. Nuclear localization signals fused to the polypeptides of the present invention may direct the protein to a specific subcellular location, while the covalent heterodimer or homodimers may increase or decrease the activity of a fusion protein. Fusion proteins can also create chimeric molecules having more than one function. Finally, fusion proteins may increase the solubility and / or stability of the fused protein compared to unfused protein. All of the types of fusion proteins described above may be prepared by modifying the following protocol, which outlines the fusion of a polypeptide to an IgG molecule or the protocol described in Example 5.
Briefly, the human Fc portion of the IgG molecule can be PCR amplified using primers spanning the 5 'and 3' ends of the sequence described below. These primers should also have convenient enzyme restriction sites that will facilitate cloning within an expression vector, preferably a mammalian expression vector.
For example, if pC4 (Accession No. 209646) is used, the human Fc moiety may be ligated within the BamHI cloning site. It should be noted that the BamHI 3 'site should not be destroyed.
144
Then, the vector containing the human Fc moiety is again restricted with BamHI, linearizing the vector, and a polynucleotide of the present invention, isolated by the PCR protocol described in Example 1, is ligated into this BamHI site. Note that the polynucleotide is cloned without a stop codon, otherwise a fusion protein will not be produced.
If the naturally occurring signal sequence is used to produce the secreted protein, pC4 does not require a second signal peptide. Alternatively, if the naturally occurring signal sequence is not used, the vector may be modified to include a heterologous signal sequence. (See, eg, WO 96/34891).
Human IgG Fc Region:
GGGATCCGGAGCCCAAATCTTCTGACAAAACTCACACATGCCCACCGTGCC CAGCACCTGAATTCGAGGGTGCACCGTCAGTCTTCCTCTTCCCCCCAAAACC CAAGGACACCCTCATGATCTCCCGGACTCCTGAGGTCACATGCGTGGTGGT GGACGTAAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACG GCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAAC AGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTG AATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAACCCCC ATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGT GTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCT GACCTGCCTGGTCAAAGGCTTCTATCCAAGCGACATCGCCGTGGAGTGGGA GAGCAATGGGCAGCCGGAGAACAACTACAAGCACGCCTCCCGTGCTGG ACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCAGCAAGG
GGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGC ACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGAGTGC GACGGCCGCGACTCTAGAGGAT (SEQ ID N ^: 1)
145
<img file="PT1015477E_D0001.tif" />
<img file="PT1015477E_D0002.tif" />
Antibodies of the present invention may be prepared by a variety of methods. (See, Current Protocols, Chapter 2). For example, cells expressing a polypeptide of the present invention are administered to an animal to induce the production of polyclonal antibody-containing sera. In a preferred method, a secreted protein preparation is prepared and purified to make it substantially free of natural contaminants. Such a preparation is then introduced into an animal to produce higher specific activity polyclonal antisera.
In the most preferred method, the antibodies of the present invention are monoclonal antibodies (or protein binding fragments thereof). Such monoclonal antibodies may be prepared using hybridoma technology. (Kohler et al., Nature 256: 495 (1975); Kohler et al., Eur. J. Immunol. 6: 511 (1976); Kohler et al., Eur. J. Immunol. 6: 292 (1976); Hammerling et al., In: Monoclonal Antibodies and T-Cell Hybridomas, Elsevier, NY, pp. 563-681 (1981)). In general, such processes involve immunization of an animal (preferably a mouse) with polypeptide or, more preferably, with a secreted polypeptide expressing cell. Such cells may be cultured in any suitable tissue culture medium; however, it is preferred to grow cells in Earle's modified Eagle's medium, supplemented with 10% fetal bovine serum (inactivated at about 56 ° C) and supplemented with about 10 g / l non-essential amino acids, about 1000 U / kg. ml of penicillin and about 100 pg / ml streptomycin.
146
Splenocytes from such mice are extracted and fused to a suitable myeloma cell line. Any suitable myeloma cell line may be employed in accordance with the present invention; however, it is preferred to employ the parent myeloma cell line (SP2O) available from the ATCC. Following fusion, hybridoma cells are selectively maintained in HAT medium and then cloned by limiting dilution as described by Wands et al. (Gastroenterology 80: 225-232 (1981)). Hybridoma cells obtained by such selection are then assayed to identify antibody secreting clones capable of binding to the polypeptide.
Alternatively, additional antibodies capable of binding to the polypeptide can be produced in a two step process using anti-idiotypic antibodies. Such a method makes use of the fact that the antibodies themselves are antigens and therefore an antibody that binds to a second antibody can be obtained. According to this method, protein specific antibodies are used to immunize an animal, preferably a mouse. Splenocytes from such an animal are then used to produce hybridoma cells and hybridoma cells are screened to identify clones that produce an antibody whose ability to bind to protein-specific antibody can be blocked by the polypeptide. Such antibodies comprise anti-idiotypic antibodies to the protein specific antibody and may be used to immunize an animal to induce formation of additional protein specific antibodies.
It will be understood that Fab and F (ab ') 2 and other antibody fragments of the present invention may be used in accordance with
147 with the methods disclosed herein. Such fragments are typically produced by proteolytic cleavage using enzymes such as papain (to produce Fab fragments) or pepsin (to produce F (ab ') 2 fragments). Alternatively, secreted protein binding fragments may be produced by the application of recombinant DNA technology or by synthetic chemistry.
For in vivo use of antibodies in humans, it may be preferred to use humanized chimeric monoclonal antibodies. Such antibodies may be made using hybrid constructs derived from hybridoma cells producing the monoclonal antibodies described above. Methods for producing chimeric antibodies are known in the art. (See, for review, Morrison, Science 229: 1202 (1985); Oi et al., BioTechniques 4: 214 (1986); Cabilly et al., US Patent No. 4816567; Taniguchi et al., EP 171496; Morrison et al., EP 173494; Neuberger et al., WO 8601533; Robinson et al., WO 8702671; Boulianne et al., Nature 312: 643 (1984); Neuberger et al., Nature 314: 268 (1985)).
Example 11: Production of Segregated Protein for High Capacity Screening Assays
The following protocol produces a supernatant containing a polypeptide to be tested. The supernatant may then be used in the Screening Assays described in Examples 13-20.
First, dilute the Poly-D-Lysine stock solution (644587 Boehringer-Mannheim) (1 mg / mL in PBS) 1:20 in PBS (without
148 17-516F Biowhittaker) to a 50 pg / mL working solution. Add 200 µl of this solution to each well (24-well plates) and incubate at rt for 20 minutes. Ensure solution distribution over each well (note: a 12-channel two-channel tipped pipettor can be used). Aspirate out the Poly-D-Lysine solution and rinse with 1 mL PBS (Phosphate Buffered Saline). PBS should remain in the well until just prior to plating the cells and the plates may be coated with polylysine in advance for up to two weeks.
Plaque 293T cells (not transporting cells beyond P + 20) at 2 x 10<sup>5</sup> cells / well in 5 ml DMEM (Dulbecco's Modified Eagle's Medium) (with 4.5 G / L glucose and L-glutamine (12-604F Biowhittaker)) / 10% heat inactivated FBS (14-503F Biowhittaker ) / lx Penstrep (17-602E Biowhittaker). Let the cells grow overnight.
The next day, mix each other in a sterile solution container: 300 µl Lipofectamine (18324-012 Gibco / BRL) and 5 mL Optimem I (31985070 Gibco / BRL) / 96-well plate. With a small volume multichannel pipettor, approximately 2 µg aliquot from an expression vector containing a polynucleotide insert produced by the methods described in Examples 8 or 9 into an appropriately labeled 96-well round bottom plate. Using a multichannel pipettor, add 50 µL of the Lipofectamine / Optimem I mixture to each well. Pipette up and down gently to mix. Incubate at rt 15-45 minutes. After about 20 minutes, use a multichannel pipettor to add 150 µL of Optimem I to each well. As a control, a vector DNA plate
149 without an insert should be transfected with each set of transfections.
Preferably, transfection should be performed through teamwork of the following tasks. Through teamwork, the time spent washing is cut in half and the cells do not spend too much time in PBS. First, person aspirates out the media from four 24-well cell plates and then person B each well with 0.5-1 mL of PBS. Person A then aspirates out the rinsed PBS and person B using a two-channel two-tipped 12-channel pipettor adds 200 µl DNA / Lipofectamine / Optimem I complex to the odd wells first, then to the eleven wells, each row in the 24-well plates. Incubate at 37 ° C for 6 hours.
While cells are incubating, prepare appropriate media, 1% BSA in DMEM with 1x penstrep or CHO-5 media (116.6 mg / l CaCl<sub>2</sub> (anid.); 0.00130 mg / L CuSO<sub>4</sub>-5H<sub>2</sub>O; 0.050 mg / l Fe (NO<sub>3</sub>) <sub>3</sub>-9H<sub>2</sub>O; 0.417 mg / l FeSO<sub>4</sub>-7H<sub>2</sub>O; 311.80 mg / l KCl; 28.64 mg / L MgCl<sub>2</sub>; 48.84 mg / l MgSO<sub>4</sub>; 6995.50 mg / l NaCl; 2400.0 mg / L NaHCO<sub>3</sub>; 62.50 mg / l NaH<sub>2</sub>POWDER<sub>4</sub>-H<sub>2</sub>0; 71.02 mg / L Na<sub>2</sub>HPO<sub>4</sub>; 0.4320 mg / L ZnSO<sub>4</sub>-7H<sub>2</sub>O; 0.002 mg / L Arachidonic Acid; 1.022 mg / L Cholesterol; 0.070 mg / L DL-alphaTocopherol Acetate; 0.0520 mg / L Linoleic Acid; 0.010 mg / L Linolenic Acid; 0.010 mg / L Myristic Acid; 0.010 mg / L Oleic Acid; 0.010 mg / L Palmitic Acid; 0.010 mg / L Palmitic Acid; 100 mg / L Pluronic F-68; 0.010 mg / L Stearic Acid; 2.20 mg / l Tween 80; 4551 mg / l D-Glucose; 130.85 mg / ml L-Alanine; 147.50 mg / ml L-Arginine-HCL; 7.50 mg / mL L-Asparagine-H<sub>2</sub>O; 6.65 mg / ml L-Aspartic Acid; 29.56 mg / mL L-Cystine-2HCl-H<sub>2</sub>O; 31.29 mg / mL L-Cystine-2HCl; 7.35 mg / mL
150 L-Glutamic Acid; 365.0 mg / ml L-Glutamine; 18.75 mg / mL of
<td>Glycine; 52.48</td><td>mg / mL</td><td colspan="3">of L-Histidine-HCl-H<sub>2</sub>O;</td><td> 106,97</td><td>mg / mL</td><td>in</td>
<td>L-Isoleucine;</td><td> 111,45</td><td>mg / mL</td><td>in</td><td>L-Leucine;</td><td> 163,75</td><td>mg / mL</td><td>in</td>
<td>L-Lysine HCl;</td><td> 32, 34</td><td>mg / mL</td><td>in</td><td>L-Methionine;</td><td> 68,48</td><td>mg / mL</td><td>in</td>
<td>L-Phenylalainine</td><td> ; 40,0</td><td>mg / mL</td><td>in</td><td>L-Proline;</td><td> 26,25</td><td>mg / mL</td><td>in</td>
<td>L-Serine; 101</td><td colspan="2">0.05 mg / mL of</td><td>L</td><td>Treonine;</td><td> 19,22</td><td>mg / mL</td><td>in</td>
L-Tryptophan; 91.79 mg / mL L-Tyrosine-2Na-2H<sub>2</sub>0; 99.65 mg / ml L-Valine; 0.0035 mg / L Biotin; 3.24 mg / l D-Ca Pantothenate; 11.78 mg / l Choline Chloride; 4.65 mg / L Folic Acid; 15.60 mg / l i-lnositol; 3.02 mg / l Niacinamide; 3.00 mg / l Pyridoxal HCl; 0.031 mg / L Pyridoxine HCl; 0.319 mg / L Riboflavin; 3.17 mg / L Thiamine HCl; 0.365 mg / L Thymidine; and 0.680 mg / L Vitamin B<sub>12</sub>; 25 mM HEPES Buffer; 2.39 mg / L Na Hypoxanthine; 0.105 mg / L Lipoic Acid; 0.081 mg / L Putrescine-2HCl Sodium; 55.0 mg / L Sodium Pyruvate; 0.0067 mg / L Sodium Selenite; 20 μΜ Ethanolamine; 0.122 mg / L Ferric Citrate; 41.70 mg / l Methyl-B-Cyclodextrin complexed with Linoleic Acid; 33.33 mg / L Methyl-BCcyclodextrin complexed with Oleic Acid; and 10 mg / L Retinal-complexed Methyl-BCcyclodextrin) with 2 mM Glutamine and 1x penstrep. (BSA (81-068-3 Bayer) 100 g, dissolved in 1 L of DMEM to a 10% BSA stock solution). Filter media and collect 50 µL for endotoxin assay in 15 mL conical polystyrene.
The transfection reaction is preferably terminated by teamwork at the end of the incubation period. Person A aspirates removal of transfection media, while person B adds 1.5 mL of appropriate media to each well. Incubate at 37 ° C for 45 or 72 hours, depending on the media used: 1% BSA for 45 hours or CHO-5 for 72 hours.
151
On day four, using a 300 µl multichannel pipettor, aliquot 600 µl into a 1 mL depth plate and the remaining supernatant into a 2 mL depth well. Supernatants from each well can then be used in the assays described in Examples 13-20.
It is specifically understood that when activity is obtained in any of the assays described below using a supernatant, the activity originates either from the polypeptide directly (eg, as a secreted protein) or by the polypeptide inducing expression of other proteins which are then segregated into the supernatant. Thus, the invention further provides a method of identifying protein in the supernatant, characterized by an activity in a particular assay.
Example 12: Construction of GAS Reporter Construction
One signal transduction pathway involved in cell differentiation and proliferation is called the Jaks-STAT pathway. Jaks-STAT pathway-activated proteins bind to elements of the GAS gamma activation site or interferon-responsive element (ISRE), located on the promoter in many genes. Binding of a protein to these elements alters the expression of the associated gene.
The GAS and ISRE elements are recognized by a class of transcription factors called Signal Transducers and Transcription Activators, or STAT. There are six members of the STAT family. Statl and Stat3 are present in many types of
152 cells, as is Stat2 (as a response to IFN-alpha is widespread). Stat4 is more restricted and not in many cell types, although it has been found in class I helper T cells after treatment with IL-12. Stat5 was originally called breast growth factor, but was found at higher concentrations in other cells, including myeloid cells. It can be activated in tissue culture cells by many cytokines.
STATs are activated to translocate from the cytoplasm to the nucleus after tyrosine phosphorylation by a set of kinases, known as the Janus Kinase (Jaks) family. Jaks represent a distinct family of soluble tyrosine kinases and include Tyk2, Jakl, Jak2 and Jak3. These kinases exhibit significant sequence similarity and are generally catalytically inactive in resting cells.
Jaks are activated by a wide range of receptors, summarized in the Table below. (Adapted from review by Schidler and Darnell, Ann. Rev. Biochem. 64: 621-51 (1995)). A family of cytokine receptors capable of activating Jaks is divided into two groups: (a) Class 1 includes receptors for IL-2, IL-3, IL-4, IL-6, IL-7, IL-1. 9, IL-11, IL-12, IL-15, Epo, PRL, GH, G-CSF, GM-CSF, LIF, CNTF and thrombopoietin; and (b) Class 2 includes IFN-Î ±, IFN-g and IL-10. Class 1 receptors share a conserved cysteine motif (a set of four conserved cysteines and a tryptophan) and a WSXWS motif (a proximal membrane region encoding Trp-Ser-Xxx-Trp-Ser (SEQ ID NO: 2) ).
Thus, on binding of a ligand to a receptor, Jaks are activated which in turn activate STAT which then
153 translocate and bind to GAS elements. The integral process is encompassed by the Jaks-STAT signal transduction pathway.
Therefore, activation of the Jaks-STAT pathway, reflected by binding of the GAS element or the ISRE element, can be used to indicate proteins involved in cell proliferation and differentiation. For example, growth factors and cytokines are known to activate the Jaks-STAT pathway. (See Table below). Thus, by using GAS elements linked to reporter molecules, activators of the Jaks-STAT pathway can be identified.
<td>Calling</td><td colspan="2">tyk2 JAKs</td><td>Jak2</td><td>Jak3</td><td>ST AT</td><td>GAS (elements) or</td>
<td></td><td></td><td>Jakl</td><td></td><td></td><td></td><td>ISRE</td>
<td>IFN Family</td><td></td><td></td><td></td><td></td><td></td><td></td>
<td>IFN-a / B</td><td> +</td><td> +</td><td> -</td><td> -</td><td> 1,2,3</td><td>ISRE</td>
<td>IFN-g</td><td></td><td> +</td><td> +</td><td> -</td><td> 1</td><td>GAS (IRFI> Lys6> IFP)</td>
<td> 11-10</td><td> +</td><td> 7</td><td> 7</td><td> -</td><td> 1,3</td><td></td>
<td>Gpl30 family</td><td></td><td></td><td></td><td></td><td></td><td></td>
<td>IL-6</td><td> +</td><td> +</td><td> +</td><td> 7</td><td> 1,3</td><td>GAS (IRFl> Lys6> IFP)</td>
<td>(Pleiotropic)</td><td></td><td></td><td></td><td></td><td></td><td></td>
<td>II-</td><td> 7</td><td> +</td><td> 7</td><td> 7</td><td> 1,3</td><td></td>
<td>11 (Pleiotropic)</td><td></td><td></td><td></td><td></td><td></td><td></td>
<td>OnM (Pleiotropic)</td><td> 7</td><td> +</td><td> +</td><td> 7</td><td> 1,3</td><td></td>
<td>LIF (Pleiotropic)</td><td></td><td> +</td><td> +</td><td> 7</td><td> 1,3</td><td></td>
<td>CNTF</td><td> -/ +</td><td> +</td><td> +</td><td> 7</td><td> 1,3</td><td></td>
154 (continuation)
<td>Calling</td><td colspan="2">tyk2 JAKs</td><td>Jak2</td><td>Jak3</td><td>ST AT</td><td>GAS (elements) or</td>
<td></td><td></td><td>Jakl</td><td></td><td></td><td></td><td>ISRE</td>
<td>(Pleiotropic)</td><td></td><td></td><td></td><td></td><td></td><td></td>
<td>G-CSF</td><td> 7</td><td> +</td><td> 7</td><td> 7</td><td> 1,3</td><td></td>
<td>(Pleiotropic)</td><td></td><td></td><td></td><td></td><td></td><td></td>
<td>IL-12</td><td> +</td><td> -</td><td> +</td><td> +</td><td> 1,3</td><td></td>
<td>(Pleiotropic)</td><td></td><td></td><td></td><td></td><td></td><td></td>
<td>GC Family</td><td></td><td></td><td></td><td></td><td></td><td></td>
<td>IL-2 (lymphocytes)</td><td> -</td><td> +</td><td> -</td><td> +</td><td> 1,3,5</td><td>GAS</td>
<td>IL-4</td><td> -</td><td> +</td><td> -</td><td> +</td><td> 6</td><td>GAS (IRFI =</td>
<td>(lymph / myeloid)</td><td></td><td></td><td></td><td></td><td></td><td>IFP >> Ly6) (IgH)</td>
<td>IL-7 (lymphocytes)</td><td> -</td><td> +</td><td> -</td><td> +</td><td> 5</td><td>GAS</td>
<td>IL-9 (lymphocytes)</td><td> -</td><td> +</td><td> -</td><td> +</td><td> 5</td><td>GAS</td>
<td>IL-13 (lymphocyte)</td><td> -</td><td> +</td><td> 7</td><td> 7</td><td> 6</td><td>GAS</td>
<td>IL-15</td><td> 7</td><td> +</td><td> 7</td><td> +</td><td> 5</td><td>GAS</td>
<td>Gpl40 family</td><td></td><td></td><td></td><td></td><td></td><td></td>
<td>IL-3 (myeloid)</td><td> -</td><td> -</td><td> +</td><td> -</td><td> 5</td><td>GAS (IRFI> IFP »Ly6)</td>
<td>IL-5 (myeloid)</td><td> -</td><td> -</td><td> +</td><td> -</td><td> 5</td><td>GAS</td>
<td>GM-CSF (myeloid)</td><td> -</td><td> -</td><td> +</td><td> -</td><td> 5</td><td>GAS</td>
<td>Hormone family</td><td></td><td></td><td></td><td></td><td></td><td></td>
<td>of growth</td><td></td><td></td><td></td><td></td><td></td><td></td>
<td>GH</td><td> 7</td><td> -</td><td> +</td><td> -</td><td> 5</td><td></td>
<td>PRL</td><td> ?</td><td> + /-</td><td> +</td><td> -</td><td> 1,3,5</td><td></td>
<td>EPO</td><td> 7</td><td></td><td> +</td><td></td><td> 5</td><td>GAS (B-</td>
155 (continuation)
Binding tyk2 JAKs Jak2 Jak3 STAT GAS (elements) or
Jakl ISRE
CAS> IRFI = IFP >> Ly6)
Receiver of
Tyrosine kinases
<td>EGF</td><td> 9</td><td> +</td><td> +</td><td colspan="2"> 1,3</td><td>GAS</td><td>(IRF1)</td>
<td>PDGF</td><td> 7</td><td> +</td><td> +</td><td> -</td><td> 1,3</td><td></td><td></td>
<td>CSF-1</td><td> 7</td><td> +</td><td> +</td><td> -</td><td> 1,3</td><td>GAS</td><td>(not IRF1)</td>
In order to construct a synthetic GAS-containing promoter element that is used in the Biological Assays described in Examples 13-14, a PCR based strategy is employed to produce a GAS-SV40 promoter sequence. 0 The 5 'primer contains four serial copies of the IRF1 promoter-verified GAS binding site previously shown to bind STAT following induction with a range of cytokines (Rothman et al., Immunlty 1: 457-468 (1994)). ), although other GAS or ISRE elements may be used as an alternative. The 5 'primer also contains 18 bp of the sequence complementary to the SV40 early promoter sequence and is flanked with an XhoI site. The 5 'primer sequence is:
5 ': GCGCCTCGAGATTTCCCCGAAATCTAGATTTCCCCGAAATGATTTCCCCG AAATGATTTCCCCGAAATATCTGCCATCTCAATTAG: 3' (SEQ ID NA 3) Complementary to SV40 promoter is flanked with a Hind IIIG: TCA IDG: 3G
156
PCR amplification is performed using the SV40 promoter template present on plasmid B-gal: promoter, obtained from Clontech. The resulting PCR fragment is digested with XhoI / Hind III and subcloned into BLSK2-. (Stratagene). Sequencing with forward and reverse primers confirms that the insert contains the following sequence:
5 ': CTCGAGATTrCCCCGAAATCTAGATTTCCCCGAAATGATTTCCCCGAAATG ATTTCCCCGAAATATCTGCCATCTCAATTAGTCAGCAACCATAGTCCCGCCC CTAACTCCGCCCATCCCGCCCCTAACTCCGCCCAGTTCCGCCCATTCTCCGC CCCATGGCTGACTAATTTTTTTTATTTATGCAGAGGCCGAGGCCGCCTCGGC CTCTGAGCTATTCCAGAAGTAGTGAGGAGGCTTTTTTGGAGGCCTAGGCTTT TGCAAAAAGCTT: 3 * (* SEQ ID NO: 5)
With this GAS promoter element linked to the SV40 promoter, a GAS: SEAP2 reporter construct is then manipulated. Here, the reporter molecule is a secreted alkaline phosphatase or SEAP. Clearly, however, it may be any reporter molecule instead of SEAP in this or any of the other Examples. Well-known reporter molecules that may be used instead of SEAP include chloramphenicol acetyltransferase (CAT), luciferase, alkaline phosphatase, β-galactosidase, green protein
<td>fluorescent (GFP) antibody.</td><td>or any</td><td>protein</td><td>detectable</td><td>for one</td>
<td>The sequence</td><td colspan="2">previous confirmed</td><td>that the</td><td>element</td>
<td>synthetic promoter</td><td>GAS-SV40 is</td><td>subcloned</td><td>inside</td><td>vector</td>
<td colspan="2">pSEAP-Promoter, obtained from</td><td>Clontech,</td><td>using</td><td>HindIII</td>
and Xhol, effectively replacing the SV40 promoter with the amplified promoter element GAS: SV40 to create the GAS-SEAP vector. However, this vector does not contain a resistance gene.
157 neomycin and therefore not preferred for mammalian expression systems.
Thus, to produce stable mammalian cell lines expressing the GAS-SEAP reporter, the GAS-SEAP cassette is removed from the GAS-SEAP vector using Sall and Notl and inserted into a vector frame containing the neomycin resistance gene, such as as pGFP-1 (Clontech) using these restriction sites at the multiple cloning site to create the GAS-SEAP / Neo vector. After this vector is transfected into mammalian cells, this vector can then be used as a reporter molecule for GAS binding as described in Examples 13-14.
Using the above description other constructs may be prepared and substituting GAS with a different promoter sequence. For example, the construction of reporter molecules containing NFK-B and EGR promoter sequences is described in Examples 15 and 16. However, many other promoters may be substituted using the protocols described in these Examples. For example, the SRE, IL-2, NFAT or Osteocalcin promoters may be substituted alone or in combination (e.g. GAS / NF-KB / EGR, GAS / NF-KB, IJ-2 / NFAT, or NF-KB / GAS). Similarly, other cell lines may be used to test reporter construct activity, such as HELA (epithelial), HUVEC (endothelial), Reh (cell B), Saos-2 (osteoblast), HUVAC (aortic) or Cardiomyocyte.
158
Example 13: High Capacity Trace Assay for
T cell activity.
The following protocol is used to evaluate T cell activity by identifying factors, such as growth factors and cytokines, that may proliferate or differentiate into T cells. T cell activity is assessed using the GAS / SEAP / Neo construct , produced in Example 12. Thus, factors that increase SEAP activity indicate the ability to activate the Jaks-STAT signal transduction pathway. The T cell used in this assay is Jurkat T cells (ATCC Accession No. TIB-152), although Molt-3 cells (ATCC Accession No. CRL-1552) and Molt-4 cells (ATCC Accession No. CRL- 1582).
Jurkat T cells are lymphoblastic CD4 + Th1 helper cells. To produce stable cell lines, approximately 2 million Jurkat cells are transfected with the GAS-SEAP / neo vector using DMRIE-C (Life Technologies) (transfection procedure described below). Transfected cells are inoculated to a density of approximately 20,000 cells per well and transfectants resistant to 1 mg / ml genticin are selected. Resistant colonies are expanded and then tested for their response to increasing interferon gamma concentrations. The dose response of a selected clone is demonstrated.
Specifically, the following protocol will produce enough cells for 75 wells containing 200 µl of cells. Thus, it is either scaled up or performed in multiple to produce enough cells for multiple 96-well plates. Jurkat cells are maintained in RPMI + 10% serum with
159
1% Pen-Strep. Combine 2.5 mL OPTI-MEM (Life Technologies) with 10 pg plasmid DNA in a T25 flask. Add 2.5 mL OPTI-MEM containing 50 µl DMRIE-C and incubate at room temperature for 15-45 min.
During the incubation period, count the cell concentration, centrifuge the required number of cells (10<sup>7</sup> transfection) and resuspend in OPTI-MEM to a final concentration of 10<sup>7</sup> cells / mL. Then add 1 mL of 1 x 10<sup>7</sup> cells in OPTI-MEM to a T25 flask and incubate at 37 ° C for 6 h. After incubation, add 10 mL RPMI + 15% serum.
Jurkat RPMI reporter lines + 10% serum, 1 mg / ml These cells are treated polypeptide as produced Example 11.
: Stable GAS-SEAP is maintained in Genticin and 1% Pen-Strep.
with supernatants containing one by the protocol described in
On the day of treatment with the supernatant, cells should be washed and resuspended in fresh RPMI + 10% serum to a density of 500000 cells per mL. The exact number of cells required will depend on the number of supernatants being screened.
For a well plate approximately 10 million cells are required (for plates,
100 millions of cells).
Transfer the cells to a triangular reservoir boat to dose the cells into a well plate using a 12-channel pipette. Using a 12-channel pipette, transfer 200 µL of cells into
160 each well (thus adding 100000 cells per well).
After all plates have been inoculated, 50 µl of the supernatants are transferred directly from the 96-well plate containing the supernatant into each well using a 12-channel pipette. Additionally, a dose of exogenous interferon gamma (0.1, 1.0, 10 ng) is added to wells H9, H10 and H11 to serve as additional positive controls for the assay.
96-well plates containing supernatant-treated Jurkat cells are placed in an incubator for 48 h. (Note: This time is variable between 48-72 h.). 35 µL samples from each well are then transferred to an opaque 96-well plate using a 12-channel pipette. Opaque plates should be covered (using cellophane covers) and stored at -20 ° C until SEAP assays according to Example 17 are performed. Plates containing the remaining treated cells are placed at 4 ° C and serve as a source of material for repeating the assay in a specific well if desired.
As a positive control, 100 Units / ml interferon gamma known to activate Jurkat T cells can be used. Over 30-fold induction is typically observed in the positive control wells.
161
<img file="PT1015477E_D0003.tif" />
High Capacity Screening Test
Identifying Myeloid Activity The following protocol is used to evaluate myeloid activity by identifying factors such as growth factors and cytokines that may proliferate or differentiate into myeloid cells. Myeloid activity is assessed using the GAS / SEAP / Neo construct produced in Example 12. Thus, factors that increase SEAP activity indicate the ability to activate the Jaks-STAT signal transduction pathway. The myeloid cell used in this assay is U937, a pre-monocytic cell line, although TF-1, HL60 or KG1 may be used.
In order to transiently transfect U937 cells with the GAS / SEAP / Neo construct produced in Example 12, a DEAE-Dextran method is used (Kharbanda et al., 1994, Cell Growth & Differentiation, 5: 259-265). . First, collect 2xl0e<sup>7</sup> U937 cells and wash with PBS. U937 cells are usually cultured in RPMI 1640 medium containing 10% heat-inactivated fetal bovine serum (FBS) supplemented with 100 units / mL penicillin and 100 mg / mL streptomycin.
The cells were then suspended in 1 ml 20 mM Tris-HCl buffer (pH 7.4) containing 0.5 mg / ml DEAE-Dextran, 8 pg GAS-SEAP2 plasmid DNA, 140 mM NaCl, 5 mM KCl, Na<sub>2</sub>HPO<sub>4</sub>.7H<sub>2</sub>O at 375 μΜ, MgCl<sub>2</sub> at 1 mM and CaCl<sub>2</sub> at 675 μΜ. Incubate at 37 ° C for 45 min.
Wash the cells with RPMI 1640 medium containing 10% FBS and then resuspend in 10 mL complete medium and incubate at 37 ° C for 36 h.
162
Stable GAS-SEAP / U937 cells are obtained by growing cells in 400 pg / ml G418. G418 free medium is used for routine growth, but every month or every two months, cells should be re-cultured in 400 pg / mL G418 for two subcultures.
These cells are tested by collecting 1x10<sup>8</sup> cells (are sufficient for assaying ten 96-well plates) and washed with PBS. Suspend the cells in 200 mL of the growth medium described above, with a final density of 5x10<sup>5</sup> cells / mL. Plate 200 µl cells per well into the 96-well plate (or 1x10<sup>5</sup> cells / well).
Add 50 µL of the supernatant prepared by the protocol described in Example 11. Incubate at 37 ° C for 48 to 72 h. As a positive control, 100 Units / ml interferon gamma known to activate U937 cells can be used. Over 30-fold induction is typically observed in the positive control wells. SEAP the supernatant according to the protocol described in Example 17.
Example 15: High Capacity Screening Assay Identifying Neuronal Activity.
When cells undergo differentiation and proliferation, a group of genes is activated through many different signal transduction pathways. One such gene, EGR1 (early growth response gene 1), is induced in various tissues and cell types upon activation. The EGR1 promoter is responsible for such induction. Using the EGR1 promoter linked to reporter molecules, cell activation can be assessed.
163
In particular, the following protocol is used to evaluate neuronal activity in PC12 cell lines. PC12 cells (rat phenochromocytoma cells) are known to differentiate and / or proliferate by activation with a number of mitogens such as TPA (tetradecanoyl phorbol acetate), NGF (nerve growth factor) and EGF ( epidermal growth). EGR1 gene expression is activated during this treatment. Thus, by stably transfecting PC12 cells with a construct containing an EGR promoter linked to the SEAP reporter, activation of PC12 cells can be assessed.
The EGR / SEAP reporter construct can be grouped by the following protocol. The EGR-1 (-633 to +1) promoter sequence (Sakamoto K et al., Oncogene 6: 867-871 (1991)) can be PCR amplified from human genomic DNA using the following primers:
5 'GCGCTCGAGGGATGACAGCGATAGAACCCCGG -3' (SEQ ID NO: 6)
5 'GCGAAGCTTCGCGACTCCCCGGATCCGCCTC-3' (SEQ ID NO: 7)
Using the GAS: SEAP / Neo vector produced in Example 12, the amplified EGR1 product can then be inserted into this vector. Linearize the GAS: SEAP / Neo vector using Xhol / HindIII restriction enzymes by removing the GAS / SV40 filler. Restrict the amplified EGR1 product with these same enzymes. Bind vector and EGR1 promoter.
In order to prepare 96-well cell culture plates, two mL of a coating solution (1:30 dilution of type I collagen [Upstate Biotech Inc. Cat No. 08-115) in 30% ethanol (filter sterilized) ) are added by a
164 10 cm plate, or 50 mL per well of the 96-well plate and allowed to air dry for 2 h.
PC12 cells are routinely cultured in RPMI-1640 medium (Bio Whittaker) containing 10% horse serum (JRH BIOSCIENCES, Cat. No. 12449-78P), 5% heat-inactivated fetal bovine serum (FBS) supplemented with 100 units / ml penicillin and 100 pg / ml streptomycin in a pre-coated 10 cm tissue culture dish. Every three or four days, one to four subcultures are performed. Cells are scraped off the plates and resuspended by pipetting up and down for more than 15 times.
Transfect the EGR / SEAP / Neo construct to PC12 using the Lipofectamine protocol described in Example 11. Stable EGR-SEAP / PC12 cells are obtained by growing cells in 300 pg / ml G418. G418 free medium is used
<td>for routine growth,</td><td>but every</td><td>month or</td><td>two</td><td>in</td><td>two</td>
<td>months, the cells should be</td><td colspan="2">re-cultivated in 300</td><td>pg / mL</td><td>in</td><td>G418</td>
<td>for two subcultures.</td><td></td><td></td><td></td><td></td><td></td>
<td>In order to rehearse for</td><td>activity</td><td>neuronal,</td><td>an</td><td colspan="2">board of</td>
cm with cells around 70 to 80% confluent is traced by removing the old medium. Wash cells once with PBS (Phosphate Buffered Saline). Then subject the cells to starvation in serum-poor medium (RPMI-1640 containing 1% horse serum and 0.5% FBS with antibiotics) overnight.
The next morning, remove the medium and wash the cells with PBS. Scrape the cells from the plate, suspend the well cells in 2 mL of serum-poor medium. Count the number of cells and
165 add more serum-poor media to reach a final cell density of 5x10<sup>5</sup> cells / mL.
Add 200 μL of the cell suspension to each well of the 96-well plate (1x10 equivalent).<sup>5</sup> cells / well). Add 50 µL of supernatant produced by Example 11, 37 ° C, for 48 to 72 h. As a positive control, a growth factor known to activate PC12 cells through EGR, such as 50 ng / pL neuronal growth factor (NGF), can be used. Over 50-fold SEAP induction is typically found in the positive control wells. SEAP the supernatant according to Example 17.
Example 16: High Capacity T Cell Activity Screening Assay
NF-κΒ (Nuclear Factor kB) is a transcription factor activated by a wide variety of agents, including inflammatory cytokines IL-1 and TNF, CD30 and CD40, lymphotoxin alfa and lymphotoxin beta, by exposure to LPS or thrombin and by expression of certain viral gene products. As a transcription factor, NF-κΒ regulates the expression of genes involved in immune cell activation, apoptosis control (NF-κΒ appears to shield apoptosis cells), B and T cell development, antiviral and antimicrobial responses and to multiple stress.
Under unstimulated conditions, NF-κΒ is retained in the cytoplasm with I-κΒ (kB inhibitor). However, upon stimulation, Ι-kB is phosphorylated and degraded, causing displacement of NF-kB to the nucleus, thereby activating gene transcription.
166 target. NF-κΒ-activated target genes include IL-2, IL-6, GM-CSF, ICAM-1 and MHC class 1.
Because of their central role and ability to respond to a range of stimuli, reporter constructs using the NF-κΒ promoter element are used to screen for supernatants produced in Example 11. NF-kB activators or inhibitors would be useful in treating disease. . For example, NF-κΒ inhibitors could be used to treat diseases related to acute or chronic activation of NF-κB, such as rheumatoid arthritis.
In order to construct a vector containing the NF-κΒ promoter element, a PCR based strategy is employed. The upstream primer contains four serial copies of the NF-κΒ binding site (GGGGACTTTCCC) (SEQ ID NO: 8), 18 bp complementary to the 5 'end of the SV40 early promoter sequence and is flanked with a site. Xhol:
5 ': GCGGCCTCGAGGGGACTTTCCCGGGGACTTTCCGGGGACTTTCCGGGAC TTTCCATCCTGCCATCTCAATTAG: 3' (SEQ ID N<sup>s</sup>: 9) Downstream primer is complementary to the 3 'end of the SV40 promoter and is flanked with a Hind III site:
5 ': GCGGCAAGCTTTTTGCAAAGCCTAGGC: 3' (SEQ ID NO: 4)
PCR amplification is performed using the SV40 promoter template present on plasmid pB-gal: promoter, obtained from Clontech. The resulting PCR fragment is digested with XhoI and HindIII and subcloned into BLSK2-. (Stratagene) Sequencing with primers T7 and T3 confirms that the insert contains the following sequence:
167 ': CTCGAGGGGACTTTCCCGGGGACTTTCCGGGGACTTTCCGGGACTTTCC ATCTGCCATCTCAATTAGTCAGCAACCATAGTCCCGCCCCTAACTCCGCCCA TCCCGCCCCTAACTCCGCCCAGTTCCGCCCATTCTCCGCCCCATGGCTGACT AATTTTTTTTATTrATGCAGAGGCCGAGGCCGCCTCGGCCTCTGAGCTATTC CAGAAGTAGTGAGGAGGCTTTTTTGGAGGCCTAGGCTTTTGCAAAAAGCTT: 3' (SEQ ID NO:<sup>s</sup>: 10)
Then replace the minimal SV40 promoter element present in plasmid SEAP2-promoter (Clontech) with this NF-KB / SV40 fragment using XhoI and HindIII. However, this vector does not contain a neomycin resistance gene and therefore is not preferred for mammalian expression systems.
To produce stable mammalian cell lines, the NF-KB / SV40 / SEAP cassette is removed from the above NF-kB / SEAP vector using the restriction enzymes Sall and Notl and inserted into a vector containing neomycin resistance. Particularly, the NF-KB / SV40 / SEAP cassette was inserted into pGFP-1 (Clontech), replacing the GFP gene after restriction of pGFP-1 with SalI and Notl.
After the NF-KB / SV40 / SEAP / Neo vector is created, stable Jurkat T cells are created and maintained according to the protocol described in Example 13. Similarly, the method for assaying supernatants with these stable Jurkat T cells is also. is described in Example 13. As a positive control, exogenous TNF alpha (0.1, 1, 10 ng) is added to wells H9, H10 and H11, with 5-10 fold activation typically observed.
168
Example 17: Assay for SEAP Activity
As a reporter molecule for the assays described in Examples 13-16, SEAP activity is assayed using the Tropix Phospho-light kit (Cat. BP-400) according to the following general procedure. The Tropix Phospho-light kit provides the Dilution, Assay, and Reaction Buffs used below.
Start a dispenser with the 2.5x Dilution Buffer and dispense 15 pL of 2.5x dilution buffer into Optiplate plates containing 35 pL of a supernatant. Seal the plates with a plastic sealant and incubate at 65 ° C for 30 min. Separate Optiplate plates to avoid uneven heating.
Cool the samples to room temperature for 15 minutes. Empty the dispenser and start with the Test Buffer. Add 50 µL Assay Buffer and incubate at room temperature 5 min. Empty the doser and start with the Reaction Buffer (see table below). Add 50 µL of Reaction Buffer and incubate at room temperature for 20 minutes. Since the chemiluminescent signal strength is time dependent and takes about 10 minutes to read 5 plates on the luminometer, 5 plates should be treated at a time and the second set started 10 minutes later.
Read the relative light unit on the luminometer. Mark H12 as white and print the results. An increase in chemiluminescence indicates reporter activity.
169
Reaction Buffer Formulation:
<td>No. of Plates</td><td>Rxn Buffer Diluent (mL)</td><td>CSPD (mL)</td>
<td> 10</td><td> 60</td><td> 3</td>
<td> 11</td><td> 65</td><td> 3,25</td>
<td> 12</td><td> 70</td><td> 3,5</td>
<td> 13</td><td> 75</td><td> 3, 75</td>
<td> 14</td><td> 80</td><td> 4</td>
<td> 15</td><td> 85</td><td> 4,25</td>
<td> 16</td><td> 90</td><td> 4,5</td>
<td> 17</td><td> 95</td><td> 4, 75</td>
<td> 18</td><td> 100</td><td> 5</td>
<td> 19</td><td> 105</td><td> 5,25</td>
<td> 20</td><td> 110</td><td> 5,5</td>
<td> 21</td><td> 115</td><td> 5, 75</td>
<td> 22</td><td> 120</td><td> 6</td>
<td> 23</td><td> 125</td><td> 6,25</td>
<td> 24</td><td> 130</td><td> 6,5</td>
<td> 25</td><td> 135</td><td> 6,75</td>
<td> 26</td><td> 140</td><td> 7</td>
<td> 27</td><td> 145</td><td> 7,25</td>
<td> 28</td><td> 150</td><td> 7,5</td>
<td> 29</td><td> 155</td><td> 7, 75</td>
<td> 30</td><td> 160</td><td> 8</td>
<td> 31</td><td> 165</td><td> 8,25</td>
<td> 32</td><td> 170</td><td> 8,5</td>
<td> 33</td><td> 175</td><td> 8, 75</td>
170
<td>No. of Plates</td><td>Rxn Buffer Diluent (mL)</td><td>CSPD (mL)</td>
<td> 34</td><td> 180</td><td> 9</td>
<td> 35</td><td> 185</td><td> 9,25</td>
<td> 36</td><td> 190</td><td> 9,5</td>
<td> 37</td><td> 195</td><td> 9, 75</td>
<td> 38</td><td> 200</td><td> 10</td>
<td> 39</td><td> 205</td><td> 10, 25</td>
<td> 40</td><td> 210</td><td> 10, 5</td>
<td> 41</td><td> 215</td><td> 10, 75</td>
<td> 42</td><td> 220</td><td> 11</td>
<td> 43</td><td> 225</td><td> 11, 25</td>
<td> 44</td><td> 230</td><td> 11, 5</td>
<td> 45</td><td> 235</td><td> 11, 75</td>
<td> 4 6</td><td> 240</td><td> 12</td>
<td> 47</td><td> 245</td><td> 12, 25</td>
<td> 48</td><td> 250</td><td> 12, 5</td>
<td> 49</td><td> 255</td><td> 12, 75</td>
<td> 50</td><td> 260</td><td> 13</td>
<td>Example</td><td>18: Rehearsal</td><td>Tracking</td><td>High Capacity</td>
<td>Identifying</td><td>Changes in</td><td>Concentration</td><td>of Small Molecule and</td>
<td>Permeability</td><td>Membrane</td><td></td><td></td>
Binding of a ligand to a receptor is known to alter intracellular levels of small molecules, such as calcium, potassium, sodium and pH, as well as to alter membrane potential.
171
These changes can be measured in an assay to identify supernatants that bind to receptors of a particular cell. While the following protocol describes a calcium assay, this protocol can be easily modified to detect changes in potassium, sodium, pH, membrane potential or any other small molecule that can be detected by a fluorescent probe.
The following assay uses Fluorometric Imaging Plate Reader (FLIPR) to measure changes in fluorescent molecules (Molecular Probes) that bind to small molecules. Clearly, any fluorescent molecule detecting a small molecule may be used in place of the fluorescent calcium fluo-3 molecule used herein.
For adherent cells, seed the cells at 10000-20000 cells / well in a shallow bottom black Co-star 96-well plate. The plate is incubated in a CO incubator.<sub>2</sub> for 20 hours. The adherent cells are washed twice in a Biotek washer with 200 µl HBSS (Hank Balanced Saline) leaving 100 µl buffer after the final wash.
A stock solution of 1 mg / ml fluo-3 is prepared in 10% pluronic acid DMSO. To load cells with fluo-3, 50 pL of 12 pg / ml fluo-3 is added to each well. The plate is incubated at 37 ° C in a CO incubator.<sub>2</sub>for 60 min. The plate is washed four times in the Biotek HBSS washer, leaving 100 µl of buffer.
For nonadherent cells, the cells are separated from the culture media by concentration at the bottom of the tube. Cells are resuspended to 2-5x10<sup>6</sup> cells / mL with HBSS in a tube
172 50 ml conical flask. 4 µl of 1 mg / mL fluo-3 solution in 10% pluronic acid DMSO is added to each mL of cell suspension. The tube is then placed in a water bath at 37 ° C for 30-60 min. Cells are washed twice with HBSS, resuspended to 1x10<sup>6</sup> cells / ml and dispensed into a microplate, 100 pL / well. The plate is centrifuged at 1000 rpm for 5 min. The plate is then washed once in 200 µl Denley CellWash, followed by an aspiration step to 100 µl final volume.
For a non-cell based assay, each well contains a fluorescent molecule, such as fluo-3. Supernatant is added to the well and a change in fluorescence is detected.
In order to measure intracellular calcium fluorescence,
FLIPR is set to the following parameters: (1) System gain is 300-800 mW; (2) Exposure time is 0.4 seconds;
(3) F / chamber diaphragm is F / 2; (4) Excitation is 488 nm; (5)
Emission is 530 nm; and (6) Sample addition is 50 pL. Increased emission at 530 nm indicates an extracellular signaling event that resulted in an increase in Ca concentration.<sup>++</sup> intracellular.
Example 19:
High Capacity Screening Test
Identifying Tyrosine Kinase Activity
Protein Tyrosine Kinases (PTK) represent a diverse group of transmembrane and cytoplasmic kinases. Within the Protein Kinase Receptors (RPTK) group are receptors for a range of mitogenic and metabolic growth factors including PDGF, FGF, EGF, NGF, HGF, and Insulin receptor subfamilies. Additionally, there is
173 A large family of RPTKs relative to which the corresponding ligand is unknown. RPTK ligands mainly include small secreted proteins, but also membrane and extracellular matrix proteins.
Activation of RPTK by ligands involves ligand-mediated receptor dimerization, resulting in transphosphorylation of receptor subunits and activation of cytoplasmic tyrosine kinases. Cytoplasmic tyrosine kinases include receptor-associated tyrosine kinases from the src family (e.g. src, yes, lck, lyn, fyn) and non-receptor and cytosolic protein tyrosine kinases, such as the Jak family, whose members mediate signal transduction triggered by the cytokine receptor superfamily (eg, Interleukins , Interferons, GM-CSF and Leptin).
Due to the wide range of known factors capable of stimulating tyrosine kinase activity, it is of interest to identify novel secreted human proteins capable of activating tyrosine kinase signal transduction pathways. Accordingly, the following protocol is designed to identify those novel secreted human proteins capable of activating tyrosine kinase signal transduction pathways.
Seed target cells (eg, primary gueratinocytes) at a density of approximately 25,000 cells per well in 96-well Loprodyne Silent Screen Plates drawn to Nalge Nunc (Naperville, IL). The plates are sterilized with two 30-minute rinses with 100% ethanol, rinsed with water and dried overnight. Some plates are coated for 2 h with 100 ml cell culture grade type I collagen (50 mg / ml), gelatin (2%) or polylysine
174 (50 mg / mL), all of which can be purchased from Sigma Chemicals (St. Louis, MO), or 10% Matrigel, purchased from Becton Dickinson (Bedford, MA), or calf serum, rinsed with PBS and stored at 4 ° C. ° C. Cell growth in these plates is assayed by seeding 5000 cells / well in growth medium and indirect cell number quantification using alamarBlue as described by the manufacturer Alamar Biosciences, Inc. (Sacramento, CA). After 48 h., Becton Dickinson Falcon No. 3071 plate covers (Bedford, MA) are used to cover the Loprodyne Silent Screen Plates. Falcon Microtest III cell culture plates may also be used in some proliferation experiments.
In order to prepare extracts, A431 cells are inoculated onto the nylon membranes of Loprodyne plates (20000/200 mL / well) and grown overnight in complete medium. The cells are quiesced by incubation in serum free basal medium for 24 h. After 5-20 minutes of EGF treatment (60 ng / mL) or 50 µl of the supernatant produced in Example 11, the medium was removed and 100 mL of extraction buffer ((20 mM HEPES, pH 7.5, NaCl a 0.15 M, 1% Triton X-100, 0.1% SDS, Na<sub>3</sub>POWDER<sub>4</sub> at 2 mM, Na<sub>4</sub>P<sub>2</sub>2 mM O7 and a protease inhibitor cocktail (No. 1836170) obtained from Boeheringer Mannheim (Indianapolis, IN) are added to each well and the plate is shaken on a rotary shaker for 5 minutes at 4 ° C. The plate is then placed in a vacuum transfer manifold and the extract filtered through the 0.45 mm membrane bottoms of each well using household vacuum. The extracts are collected in a 96 well capture / plate at the bottom of the vacuum manifold and immediately placed on ice. In order to obtain extracts
175 clarified by centrifugation, the contents of each well after detergent solubilization for 5 minutes are removed and centrifuged for 15 minutes at 4 ° C at 16000 x g.
Test the filtered extracts for levels of tyrosine kinase activity. While many methods of detecting tyrosine kinase activity are known, a method is described herein.
In general, the tyrosine kinase activity of a supernatant is assessed by determining its ability to phosphorylate a tyrosine residue on a specific substrate (a biotinylated peptide). Biotinylated peptides that may be used for this purpose include PSK1 (corresponding to amino acids 6-20 of cdc2-p34 cell division kinase) and PSK2 (corresponding to amino acids 1-17 of gastrin). Both peptides are substrates for a range of tyrosine kinases and are available from Boehringer Mannheim.
The tyrosine kinase reaction is implemented by adding the following components in order. First add 10 pL of 5 pM Biotinylated Peptide, then 10 pl ATP / Mg<sup>2+ </sup>(5 mM ATP / MgCl<sub>2</sub> 10 µl of 5x Assay Buffer (40 mM imidazole hydrochloride, pH 7.3, 40 mM beta-glycerophosphate, 1 mM EGTA, MgCl<sub>2</sub> at 100 mM, MnCl<sub>2</sub> 5 mM, 0.5 mg / ml BSA), then 5 µl Sodium Vanadate (1 mM) and then 5 µl water. Mix components gently and preincubate the reaction mixture at 30 ° C for 2 min. Start the reaction by adding 10 µl of the control enzyme or the filtered supernatant.
176
The tyrosine kinase assay reaction is then terminated by adding 10 µl of 120 mM EDTA and placing the reactions on ice.
Tyrosine kinase activity is determined by transferring 50 µl aliquot of reaction mixture to a microtiter plate module (MTP) and incubating at 37 ° C for 20 min. This allows the streptavadin-coated 96-well plate to associate with the biotinylated peptide. Wash the
<td>300 MTP module</td><td>pL / well of</td><td>PBS</td><td>four times.</td><td>Then,</td>
<td>add 75 pL of</td><td>antibody</td><td>anti</td><td>-phosphotyrosine</td><td>in conjunction with</td>
<td>horseradish peroxidase</td><td>(anti-P-Tyr-</td><td>-POD</td><td>(0.5 pg / mL)) to</td><td>each well and</td>
<td>incubate at 37 ° C</td><td colspan="2">during a</td><td>hour. Wash the</td><td>well as</td>
<td>previously.</td><td></td><td></td><td></td><td></td>
Then add 100 µL peroxidase substrate solution (Boehringer Mannheim) and incubate at room temperature for at least min. (up to 30 min.).
Measure the absorbance of the sample at 405 nm using an ELISA reader. The level of bound peroxidase activity is quantified using an ELISA reader and reflects the level of tyrosine kinase activity.
Example 20:
High Capacity Screening Test
Identifying Phosphorylation Activity
As a potential alternative and / or complement to the protein tyrosine kinase activity assay described in Example 19, an assay that detects activation (phosphorylation) of important intracellular signal transduction intermediates may also be used. For example, as described below, a
177 Particular assay can detect tyrosine kinases phosphorylation Erk-1 and Erk-2. However, phosphorylation of other molecules such as Raf, JNK, p38 MAP, Map kinase kinase (MEK), MEK kinase, Src, Muscle Specific Kinase (MuSK), IRAK, Tec and Janus, as well as any other phosphoserine molecule phosphotyrosine or phosphotreonine can be detected by replacing these molecules with Erk-1 or Erk-2 in the following assay.
Specifically, assay plates are prepared by coating the wells of a 96-well ELISA plate with 0.1 ml protein G (1 pg / ml) for 2 h at temp. environment (ok). The plates are then rinsed with PBS and blocked with 3% BSA / PBS for 1 h at rt. Protein G plates are then treated with 2 commercial monoclonal antibodies (100 ng / well) against Erk- 1 and Erk-2 (1 hr at rt) (Santa Cruz Biotechnology). (In order to detect other molecules, this step can be easily modified by replacing a monoclonal antibody detecting any of the molecules described above). After 3-5 rinses with PBS, the plates are stored at 4 ° C until use.
A431 cells are inoculated at 20,000 / well in a 96-well Loprodyne filter plate and grown overnight in growth medium. The cells are then starved for 48 h in basal medium (DMEM) and then treated with EGF (6 ng / well) or 50 µl of the supernatants obtained in Example 11 for 5-20 minutes. The cells are then solubilized and the extracts filtered directly into the assay plate.
After incubation with the extract for 1 h at rt, the wells are rinsed again. As a positive control, it is
178 A commercial MAP kinase preparation (10 ng / well) is used instead of the A431 extract. Plates are then treated with a commercial polyclonal (rabbit) antibody (1 pg / ml) that specifically recognizes the phosphorylated epitope of Erk-1 and Erk-2 kinases (1 h at RT). This antibody is biotinylated by standard procedures. Bound polyclonal antibody is then quantified by successive incubations with fluorescence stimulation reagent, Europopepteptavidin and Europium, on the Wallac DELFIA (temporal resolution fluorescence) instrument. An increased fluorescent signal above the background indicates phosphorylation.
Example 21: Method for Determining Gene Changes Corresponding to a Polynucleotide
RNA isolated from whole families or diseased individuals presenting with a phenotype of interest (such as a disease) should be isolated. CDNA is then produced from these RNA samples using protocols known in the art. (See, Sambrook). The cDNA is then used as a template for PCR employing primers surrounding regions of interest in SEQ ID NO: X. Suggested PCR conditions consist of 35 cycles at 95 ° C for 30 seconds; 60-120 seconds at 52-58 ° C; and 60-120 seconds at 70 ° C using buffer solutions described in Sidransky, D., et al., Science 252: 706 (1991).
PCR products are then sequenced using primers labeled at any 5 'end with T4 polynucleotide kinase employing SequiTherm Polymerase. (Epicentre Technologies). The intron-exon edges of selected exons are also determined and the PCR products
179 analyzed in order to confirm the results. PCR products harboring suspected mutations are then cloned and sequenced to validate the results of direct sequencing.
PCR products are cloned into T-tailed vectors as described in Holton, TA and Graham, MW, Nucleic Acids Research, 19: 1156 (1991) and sequenced with T7 polymerase (United States Biochemical). Affected individuals are identified by mutations not present in unaffected individuals.
Genomic rearrangements are also observed as a method of determining changes in a gene corresponding to a polynucleotide. Genomic clones, isolated according to Example 2, are Nick-translated with digoxigenindesoxyuridine 5'-triphosphate (Boehringer Manheim), and FISH, performed as described in Johnson, Cg. et al., Methods Cell Biol. 35: 73-99 (1991). Hybridization with the labeled probe is performed using a large excess of human cot-1 DNA for specific hybridization to the corresponding genomic locus.
Chromosomes are contrasted with 4,6-diamino-2phenylidole and propidium iodide, producing a combination of C and R bands. Aligned images for precise mapping are obtained using a triple band filter set (Chroma Technology, Brattleboro, VT) , in combination with a chamber with cooled charge coupling device (Photometrics, Tucson, AZ) and variable excitation wavelength filters. (Johnson, Cv. Et al., Genet. Anal. Tech. Appl., 8:75 (1991)). Imaging, analysis and chromosomal fractional length measurements are performed using ISee
180
Graphical Program System. (Inovision Corporation, Durham, NC). Chromosomal changes in the probe-hybridized genomic region are identified as insertions, deletions and translocations. These changes are used as a diagnostic marker for an associated disease.
Example 22: Method for Detecting Abnormal Levels of a Polypeptide in a Biological Sample
A polypeptide of the present invention may be detected in a biological sample and, if an increased or decreased level of the polypeptide is detected, this polypeptide is a marker for a particular phenotype. Detection methods are numerous, and thus it is understood that one skilled in the art can modify the following assay to suit his particular needs.
For example, antibody sandwich ELISAs are used to detect polypeptides in a sample, preferably a biological sample. Wells of a microtiter plate are coated with specific antibodies at a final concentration of 0.2 to 10 pg / mL. Antibodies are monoclonal or polyclonal and are produced by the method described in Example 10. The wells are blocked so that non-specific binding of the polypeptide to the wells is reduced.
The coated wells are then incubated for> 2 hours at rt with a sample containing the polypeptide. Preferably, serial dilutions of the sample should be used to validate the results.
181
The plates are then washed three times with deionized or distilled water to remove unbound polypeptide.
Then 50 µl of antibody-specific alkaline phosphatase conjugate at a concentration of 25-400 ng is added and incubated for 2 hours at room temperature. The plates are again washed three times with water.
<td colspan="2">deionized or on.</td><td>distilled from</td><td>way to</td><td>remove conjugate no</td>
<td colspan="2">Add</td><td>75 pL of solution</td><td colspan="2">of 4-methylumbelliferyl substrate</td>
<td>phosphate</td><td>(MUP)</td><td>or p-nitrophenyl</td><td>phosphate</td><td>(NPP) to each well and</td>
<td>incubate,</td><td>1 hour</td><td>, the temperature</td><td>environment.</td><td>Measure the reaction with a</td>
microtiter plate reader. Prepare a standard curve using serial dilutions of a control sample and plot the X-axis polypeptide concentration (log scale) and Y-axis fluorescence or absorbance (linear scale). Interpolate the concentration of polypeptide in the sample using the standard curve.
Example 23: Formulating a Polypeptide
The secreted polypeptide composition will be formulated and dosed in a manner consistent with good medical practice, taking into account the clinical condition of the diseased individual (especially the side effects of treatment with the secreted polypeptide alone), the site of distribution, the method of administration, the administration program and other factors known to doctors. The amount effective for the present purposes is thus determined by such considerations.
182
As a general proposition, the total pharmaceutically effective amount of secreted polypeptide administered parenterally per dose will be in the range of about 1 pg / kg / day to 10 mg / kg / day of patient body weight although, as noted above, will be subject to therapeutic discretion. More preferably, this dose is at least 0.01 mg / kg / day and most preferably for humans between about 0.01 and 1 mg / kg / day for the hormone. If provided continuously, the secreted polypeptide is typically administered at a dose rate of from about 1 pg / kg / hour to about 50 pg / kg / hour, by 1-4 injections per day or by continuous subcutaneous infusions, e.g. using a mini pump. An intravenous solution bag may also be employed. The duration of treatment required to observe changes and the interval after treatment for responses to appear to vary, depending on the desired effect.
Pharmaceutical compositions containing the secreted protein of the invention are administered orally, rectally, parenterally, intracystically, intravaginally, intraperitoneally, topically (as powders, ointments, gels, drops or transdermal patch), orally or as an oral or nasal spray. Pharmaceutically acceptable carrier refers to a thickener, diluent, non-toxic, solid, semi-solid or liquid encapsulating material or auxiliary formulation of any kind. The term parenteral as used herein refers to modes of administration which include intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous and intraarticular injection and infusion.
The secreted polypeptide is also suitably administered by sustained release systems. Suitable examples of
183 prolonged release compositions include semipermeable polymeric matrices in the form of shaped pieces, eg films, or microcapsules. Extended release matrices include polylacids (US Pat. No. 3773919, EP 58481), L-glutamic acid and gamma-ethylglutamate copolymers (Sidman, U. et al., Biopolymers 22: 547-556 (1983)) , poly (2-hydroxyethyl methacrylate) (R. Langer et al., J. Biomed. Mater. Res. 15: 167-277 (1981) and R. Langer, Chem. Tech. 12: 98-105 (1982)), ethylene vinyl acetate (R. Langer et al.) Or poly-D- (-) -3-hydroxybutyric acid (EP 133988). Prolonged release compositions also include liposome entrapped polypeptides. Liposomes containing the secreted polypeptide are prepared by methods known per se: DE 3218121; Epstein et al., Proc. Natl. Acad. Know. USA 82: 3688-3692 (1985); Hwang et al., Proc. Natl. Acad. Know. USA 77: 4030-4034 (1980); EP 52322; EP 36676; EP 88046; EP 143949; EP 142641; Japanese Patent Application 83-118008; Pat. US N<sup>the</sup> 4,485,045 and 4,544,545; and EP 102324. Usually, liposomes are of the small unilamellar type (about 200-800 Angstroms), wherein the lipid content is greater than about 30 mol. percentage of cholesterol, the proportion being
<td>selected segregated.</td><td>adjusted</td><td>for therapy</td><td>great</td><td>in</td><td>polypeptide</td>
<td colspan="2">For administration</td><td colspan="2">parenteral, in a form</td><td>in</td><td>achievement the</td>
<td>polypeptide</td><td>segregated</td><td>is formulated in</td><td>general,</td><td colspan="2">through your</td>
mixing to a desired purity in an injectable dosage form (solution, suspension or emulsion, with a pharmaceutically acceptable carrier, ie one which is non-toxic to the receptors, the dosages and concentrations employed, and is compatible with other ingredients of the For example, the formulation preferably does not include oxidizing agents.
184 and other compounds that are known to be deleterious to polypeptides.
In general, formulations are prepared by uniformly and intimately contacting the polypeptide with liquid carriers or finely divided solid carriers or both. Then, if necessary, the product is formed into the desired formulation. Preferably, the carrier is a parenteral carrier, more preferably a solution that is isotonic with the recipient's blood. Examples of such carrier vehicles include water, saline, Ringer's solution and dextrose solution. Non-aqueous vehicles, such as fixed oils and ethyl oleate, are also useful herein, as are liposomes.
The vehicle suitably contains minor amounts of additives, such as substances that improve isotonicity and chemical stability. Such materials are non-toxic to receptors at the dosages and concentrations employed and include buffers such as phosphate, citrate, succinate, acetic acid and other organic acids or their salts; antioxidants, such as ascorbic acid; low molecular weight polypeptides (less than about ten residues), eg polyarginine or tripeptides;
proteins such as serum albumin, gelatin or immunoglobulins;
hydrophilic polymers, such as polyvinylpyrrolidone; amino acids, such as glycine, glutamic acid, aspartic acid or arginine;
monosaccharides, disaccharides and other carbohydrates, including cellulose or derivatives thereof, glucose, mannose or dextrins; chelating agents, such as EDTA; sugar alcohols, such as mannitol or sorbitol; homologous ions such as sodium; and / or nonionic surfactants such as polysorbates, poloxamers or PEGs.
185 Secreted polypeptide is typically formulated in such carriers at a concentration of about 0.1 mg / mL to 100 mg / mL, preferably 1-10 mg / mL, at a pH of about 3 to
8 It will be understood that the use of certain of the above excipients, vehicles or stabilizers will result in the formation of polypeptide salts.
Any polypeptide to be used for therapeutic administration may be sterile. Sterility is easily achieved by filtration through filtration membranes.
, membranes of
0.2 micron). Therapeutic polypeptide compositions are generally placed within a container having a sterile access port, for example an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle.
Typically, polypeptides will be stored in unit or multi-dose containers, for example, sealed ampoules or vials, as an aqueous solution or as a lyophilized formulation for reconstitution. As an example of a lyophilized formulation, 10 mL vials are filled with 5 mL of 1% (w / v) filtration sterile aqueous polypeptide solution and the resulting mixture is lyophilized. The infusion solution is prepared by reconstituting the lyophilized polypeptide using bacteriostatic Water for Injection.
The invention also provides a pharmaceutical package or kit comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions of the invention. Associated with such container (s) may be a warning as prescribed by a government agency
186 regulating the preparation, use or sale of medicinal or biological products, a warning reflecting the agency's approval of the preparation, human. Additionally, therapeutics may be employed.
use or sale of the polypeptides of together with for administration of the present invention other compounds
Example 24:
Method of
Treatment of Decreased Levels of
<img file="PT1015477E_D0004.tif" />
It will be appreciated that conditions caused by a decrease in the standard or normal expression level of a secreted protein in an individual may be treated by administering the polypeptide of the present invention, preferably in secreted form. Thus, the invention also provides a method of treating an individual in need of an increased level of the polypeptide comprising administering to such an individual a pharmaceutical composition comprising an amount of the polypeptide in order to increase the level of activity of the polypeptide in such a subject. individual.
For example, a patient with decreased polypeptide levels receives a daily dose of 0.1-100 pg / kg of the polypeptide for six consecutive days. Preferably, the polypeptide is in secreted form. Exact details of the dosing schedule, based on administration and formulation, are provided in Example 23.
187
Example 25: Method of Treatment of Increased Levels of
Antisense technology is used to inhibit the production of a polypeptide of the present invention. This technology is an example of a method of lowering the levels of a polypeptide, preferably a secreted form, by virtue of a variety of etiologies, such as cancer.
For example, to a patient diagnosed with abnormally increased levels of a polypeptide, antisense polynucleotides are administered intravenously at 0.5, 1.0, 1.5, 2.0 and 3.0 mg / kg / day for 21 days. This treatment is repeated after a rest period of 7 days if the treatment was well tolerated. Antisense polynucleotide formulation is provided in Example 23.
Example 26: Treatment Method Using Gene Therapy
One method of gene therapy transplants fibroblasts that are capable of expressing a polypeptide to a patient. In general, fibroblasts are obtained from an individual by skin biopsy. The resulting tissue is placed in tissue culture medium and separated into small pieces. Small portions of the tissue are placed on a damp surface of a tissue culture flask, approximately ten pieces are placed in each flask. The bottle is turned upside down, tightly sealed and left at room temperature overnight. After 24 hours at room temperature, the flask is inverted and the tissue portions remain attached to the bottom of the flask and fresh media (eg, Ham F12 media,
188 10% FBS, penicillin and streptomycin). The flasks are then incubated at 37 ° C for approximately one week.
At this time, new media is added and subsequently changed every several days. After two more weeks in culture, a fibroblast monolayer emerges. The monolayer is trypsinized and weighed into larger vials.
pMV-7 (Kirschmeier, PT et al., DNA, 7: 219-25 (1988)), flanked by long terminal repeats of Moloney murine sarcoma virus, is digested with EcoRI and HindIII and subsequently treated with calf intestinal phosphatase. . The linear vector is fractionated on agarose gel and purified using glass beads.
The cDNA encoding a polypeptide of the present invention can be amplified using PCR primers that correspond to the 5 'and 3' end sequences as shown in Example 1. Preferably, the 5 'primer contains an EcoRI site and the primer 3 'includes a HindIII site. Equal amounts of the linear structure of the Moloney murine sarcoma virus and the amplified EcoRI and HindIII fragment are added together in the presence of T4 DNA ligase. The resulting mixture is maintained under appropriate conditions for ligation of the two fragments. The ligation mixture is then used to transform HB101 bacteria which are then plated onto kanamycin-containing agar to confirm that the vector has the gene of interest properly inserted.
Amphotropic packaging cells pA317 or GP + aml2 are cultured in tissue culture to confluent density,
189 in Dulbecco's Modified Eagle's Medium (DMEM) with 10% calf serum (CS), penicillin and streptomycin. The MSV vector containing the gene is then added to the media and the packaging cells transduced with the vector. Packaging cells now produce infectious viral particles
<td>containing</td><td>the gene (the</td><td>cells of</td><td colspan="2">packing</td><td>are now,</td>
<td>referred to</td><td>like cells</td><td>producers).</td><td></td><td></td><td></td>
<td>Are</td><td>added</td><td>new media</td><td>at</td><td>cells</td><td>producers</td>
transduced, and subsequently the media is collected from a 10 cm plate of confluent producer cells. The spent media containing the infectious viral particles is filtered through a millipore filter to remove detached producer cells and these media are then used to infect fibroblast cells. Media is removed from a subconfluent fibroblast plate and rapidly replaced with media from the producing cells. These media are removed and replaced with new media. If the virus titer is high then virtually all fibroblasts will be infected and no selection is required. If the titer is too low then a retroviral vector must be used that has a selectable marker such as neo or his. After fibroblasts have been efficiently infected, fibroblasts are analyzed to determine if protein is produced.
The manipulated fibroblasts are then transplanted to the host, either alone or after being cultured to confluence in cytodex microvessel beads 3.
190
LIST OF SEQUENCES (1) GENERAL INFORMATION:
(i) APPLICANT: Human Genome Sciences, Inc., et al.
(ii) TITLE OF INVENTION: 32 Segregated Human Proteins (iii) SEQUENCE NUMBER: 120 (iv) ADDRESS FOR CORRESPONDENCE:
(A) ADDRESSEE: Human Genome Sciences, Inc.
(B) STREET: 9410 Key West Avenue (C) CITY: Rockville (D) STATE: Maryland (E) COUNTRY: USA (F) POST CODE: 20850 (v) COMPUTER READING FORM:
(A) MEDIA TYPE: Floppy Disk, 3.50 inch, 1.4 Mb storage (B) COMPUTER: HP Vectra 486/33 (C) OPERATING SYSTEM: MSDOS, Version 6.2 (D) SOFTWARE: ASCII Text (vi) DATA FROM THIS REQUEST:
(A) APPLICATION NUMBER:
(B) DATE OF PRESENTATION: 27 May 1998 (C) CLASSIFICATION:
191 (vii) PREVIOUS REQUEST DATA:
(A) APPLICATION NUMBER:
(B) DATE OF PRESENTATION:
(viii) MANDATORY / AGENT INFORMATION:
(A) NAME: A. Anders Brookes (B) REGISTRATION NUMBER: 36373 (C) REFERENCE / REGISTRATION NUMBER: PZ006PCT (vi) TELECOMMUNICATIONS INFORMATION:
(A) PHONE: (301) 309-8504 (B) TELEFAX: (301) 309-8439 (2) INFORMATION FOR SEQ ID NO: 1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 733 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: double (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:
192
GGGATCCGGA GCCCAAATCT TCTGACAAAA CTCACACATG CCCACCGTGC CCAGCACCTG60
AATTCGAGGG TGCACCGTCA GTCTTCCTCT TCCCCCCAAA ACCCAAGGAC ACCCTCATGA120
TCTCCCGGAC TCCTGAGGTC ACATGCGTGG TGGTGGACGT AAGCCACGAA GACCCTGAGG180
TCAAGTTCAA CTGGTACGTG GACGGCGTGG AGGTGCATAA TGCCAAGACA AAGCCGCGGG240
AGGAGCAGTA CAACAGCACG TACCGTGTGG TCAGCGTCCT CACCGTCCTG CACCAGGACT300
GGCTGAATGG CAAGGAGTAC AAGTGCAAGG TCTCCAACAA AGCCCTCCCA ACCCCCATCG360
AGAAAACCAT CTCCAAAGCC AAAGGGCAGC CCCGAGAACC ACAGGTGTAC ACCCTGCCCC420
CATCCCGGGA TGAGCTGACC AAGAACCAGG TCAGCCTGAC CTGCCTGGTC AAAGGCTTCT480
ATCCAAGCGA CATCGCCGTG GAGTGGGAGA GCAATGGGCA GCCGGAGAAC AACTACAAGA540
CCACGCCTCC CGTGCTGGAC TCCGACGGCT CCTTCTTCCT CTACAGCAAG CTCACCGTGG600
ACAAGAGCAG GTGGCAGCAG GGGAACGTCT TCTCATGCTC CGTGATGCAT GAGGCTCTGC660
ACAACCACTA CACGCAGAAG AGCCTCTCCC TGTCTCCGGG TAAATGAGTG CGACGGCCGC720
GACTCTAGAG GAT733 (2) INFORMATION FOR SEQ ID NO: 2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 5 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:
Trp Ser Xaa Trp Ser
193 (2) INFORMATION FOR SEQ ID NO: 3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 86 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: double (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:
GCGCCTCGAG ATITCCCCGA AATCTAGATT TCCCCGAAAT GATTTCCCCG AAATGATTTC 60
CCCGAAATAT CTGCCATCTC AATTAG 86 (2) INFORMATION FOR SEQ ID NO: 4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 27 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: double (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:
GCGGCAAGCT TTTTGCAAAG CCTAGGC 27
194 (2) INFORMATION FOR SEQ ID NO: 5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 271 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: double (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:
CTCGAGATTT TRANSGAAATC TAGATTTCCC CGAAATGATT TCCCCGAAAT GATTTCCCCG60
AAATATCTGC CATCTCAATT AGTCAGCAAC CATAGTCCCG TRANSTAACTC CGCCCATCCC120
GCCCCTAACT CCGCCCAGTT CCGCCGATTC TCCGCCCCAT GGCTGACTAA ΤΤΠΤΓΓΓΑΤ180
TTATGCAGAG GCCGAGGCCG CCTCGGCCTC TGAGCTATTC CAGAAGTAGT GAGGAGGCTT240
TTTTGGAGGC CTAGGCTTTT GCAAAAAGCT T271 (2) INFORMATION FOR SEQ ID NO: 6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 32 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: double (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:
GCGCTCGAGG GATGACAGCG ATAGAACCCC GG 32
195 (2) INFORMATION FOR SEQ ID NO: 7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 31 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: double (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7:
GCGAAGCTTC GCGACTCCCC GGATCCGCCT C 31 (2) INFORMATION FOR SEQ ID NO: 8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 12 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: double (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8:
GGGGACTTTC CC 12 (2) INFORMATION FOR SEQ ID NO: 9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 73 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: double (D) TOPOLOGY: linear
196 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9:
GCGGCCTCGA GGGGACTTTC CCGGGGACTT TCCGGGGACT TTCCGGGACT TTCCATCCTG 60
CCATCTCAAT TAG (2) INFORMATION FOR SEQ ID NO: 10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 256 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: double (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10:
CTCGAGGGGA CTTTCCCGGG GACTTTCCGG GGACTTTCCG GGACTTTCCA TCTGCCATCT60
CAATTAGTCA GCAACCATAG TCCCGCCCCT AACTCCGCCC ATCCCGCCCC TAACTCCGCC120
CAGTTCCGCC CATTCTCCGC CCCATGGCTG ACTAATTTTT ΊΤΓΑΤΤΓΑΤΌ CAGAGGCCGA180
GGCCGCCTCG GCCTCTGAGC TATTCCAGAA GTAGTGAGGA GGCTTTTTTG GAGGCCTAGG240
CTTTTGCAAA AAGCTT256 (2) INFORMATION FOR SEQ ID NO: 11:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1169 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: double (D) TOPOLOGY: linear
197 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11:
GGGGCGCAAA TAGGGTCAGT GGGCCGCTTG GCGKTGTTCG TTGCGGTACC AGGTCCGCGT60
GAGGGGTTCG GGGGTTCTGG GCAGGCACAA TQGCGTCTCG AGCAGGCCCG CGAGCGGCCG120
RCACCGACGC AGCGAGCTTT CAGCACCGGG AGCGCGTCGC CATGCACTAC CAGATGAGTG180
TGACCCTCAA GTATGAAATC AAGAAGCTGA TCTACGTACA TCTGGTCATA TGGCTGCTGC240
TGGTTGCTAA GATGAGCGTG GGACACCTGA GGCTCTTGTC ACATGATCAG GTGGCCATGC300
CCTATCAGTG GGAATACCCG TATTTGCTGA GCATTTTGCC CTCTCTCTTG GGCCTTCTCT360
CCTTTCCCCG CAACAACATT AGCTACCTGG TGCTCTCCAT GATCAGCATG GGACTCTTTT420
CCATCGCTCC ACTCATTTAT GGCAGCATGG AGATGTTCCC TGCTGCACAG CCTTCTACCG480
CCATGGCAAG GCCTACCGTT TCCTCTTTGG TTTTTCTGCC GTTTCCATCA TGTACCTGGT540
GTTGGTGTTG GCAGTGCAAG TGCATGCCTG GCAGTTGTAC TACAGCAAGA AGCTCCTAGA600
CTCTTGGTTC ACCAGCACAC AGGAGAAGAA GCATAAATGA AGCCTCTTTG GGGTGAAGCC660
TGGACATCCC ATCGAATGAA AGGACACTAG TACAGCGGTT CCAAAATCCC TTCTGGTGAT720
TTTAGCAGCT GTGATGTTGG TACCTGGTGC AGACCCAGGC CAAAGTTCTG GAAAGCTCCT780
TTTGCCATCT GCTGAGGTGG CAAAACTATA ATTTATTCCT GGTTGGCTAG AACTGGGTGA840
CCAACAGCTA TGAAACAAAT TTCAGCTGTT TGAAGTTGAA CTTTGAGGTT TTTCTTTAAG900
AATGAGCTTC GTCCTTGCCT CTACTCGGTC ATTCTCCCCA TTTCCATCCA TTACCCCTTA960
GCCA1TGAGA CTAAAGGAAA TAGGGAATAA ATCAAATTAC TTCATCTCTA GGTCACGGGT1020
CAGGAAACAT TTGGGCAGCT GCTCCCTTGG CAGCTGTGGT CTCCTCTGCA AAGCATTTTA1080
ATTAAAAACC TCAATAAAGA TGCCCTGCCC ACAAAAAAAA AAAAAAAAAA AATTCGGGGG1140
GGGGCCCGGG NAACCAATTN GCCCCTANA1169 (2) INFORMATION FOR SEQ ID NO: 12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1310 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: double (D) TOPOLOGY: linear
198 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12:
<td>AATTCGGCAC GAGGCAGCGT CGCGCGGCCC AGTTCCCTTT TCCGGTCGGC GTGGTCTTGC</td><td> 60</td>
<td>GAGTGGAGTG TCCGCTGTGC CCGGGCCTGC ACCATGAGCG TCCCGGCCTT CATCGACATC</td><td> 120</td>
<td>AGTGAAGAAG ATCAGGCTGC TGAGCTTCGT GCTTATCTGA AATCTAAAGG AGCTGAGATT</td><td> 180</td>
<td>TCAGAAGAGA ACTCGGAAGG TGGACTTCAT GTTGATTTAG CTCAAATTAT TGAAGCCTGT</td><td> 240</td>
<td>GATGTGTGTC TGAAGGAGGA TGATAAAGAT GTTGAAAGTG TGATGAACAG TGTGGTATCC</td><td> 300</td>
<td>CTACTCTTGA TCCTGGAACC AGACAAGCAA GAAGCTTTGA TTGAAAGCCT ATGTGAAAAG</td><td> 360</td>
<td>CTGGTCAAAT TTCGCGAAGG TGAACGCCCG TCTCTGAGAC TGCAGTTGTT AAGCAACCTT</td><td> 420</td>
<td>TTCCACGGGA TGGATAAGAA TACTCCTGTA AGATACACAG TGTATTGCAG CCTTATTAAA</td><td> 480</td>
<td>GTGGCAGCAT CTTGTGGGGC CATCCAGTAC ATCCCAACTG AGCTGGATCA AGTTAGAAAA</td><td> 540</td>
<td>TGGATTTCTG ACTGGAATCT CACCACTGAA AAAAAGCACA CCCTTTTAAG ACTACTTTAT</td><td> 600</td>
<td>GAGGCACTTG TGGATTGTAA GAAGAGTGAT GCTGCTTCAA AAGTCATGGT GGAATTGCTC</td><td> 660</td>
<td>GGAAGTTACA CAGAGGACAA TGCTTCCCAG GCTCGAGTTG ATGCCCACAG GTGTATTGTA</td><td> 720</td>
<td>CGAGCATTGA AAGATCCAAA TGCATTTCTT TTTGACCACC TTCTTACTTT AAAACCAGTC</td><td> 780</td>
<td>AAGTTTTTGG AAGGCGAGCT TATTCATGAT CTTTTAACCA TTTTTGTGAG TGCTAAATTG</td><td> 840</td>
<td>GCATCATATG TCAAGTTTTA TCAGAATAAT AAAGACTTCA TTGATTCACT TGGCCTGTTA</td><td> 900</td>
<td>CATGAACAGA ATATGGCAAA AATGAGACTA CTTACTTTTA TGGGAATGGC AGTAGAAAAT</td><td> 960</td>
<td>AAGGAAATTT CTTTTGACAC AATGCAGCAA GAACTTCAGA TTGGAGCTGA TGATGTTGAA</td><td> 1020</td>
<td>GCATTTGTTA TTGACGCCGT AAGAACTAAA ATGGTCTACT GCAAAATTGA TCAGACCCAG</td><td> 1080</td>
<td>AGAAAAGTAG TTGTCAGTCA TAGCACACAT CGGACATTTG GAAAACAGCA GTGGCAACAA</td><td> 1140</td>
<td>CTGTATGACA CACTTAATGC CTGGAAACAA AATCTGAACA AAGTGAAAAA CAGCCTTTTG</td><td> 1200</td>
<td>AGTCTTTCTG ATACCTGAGT TTTTATGCTT ATAATTTTTG TTCITTGAAA AAAAAGCCCT</td><td> 1260</td>
<td>AAATCATAGT AAAACATTAT AAACTAAAAA AAAAAAAAAA AAAAAAAAA</td><td> 1310</td>
199 (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1139 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: double (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13:
AGGGCANACT TACAGAGATA TCATATGAGA CCCTCGAGCG GTGCCGGAGC GCASCCAGGT OATTGCCTGG GANGAAGTCT GTCAACAAAG ATGATGAGTT TCTTCTGCTT TGTGATGGGT GTCCCAAGAT GGAGGCTGTC CCAGAAGGAG AGGTGGAGGG AGAATTCACT CAGAAGCCTG OTGGTTATTC GCTGAACTTC TCAGAGGGTG GCCGAGAAAG CCCAGCAGCA GGGCCTCGGT GGCGGCGACT CTCTATGCGG AACCACCACA TGGAGATGGA GTCCCATGAT GCAGCCTGGC TGAGTGGGTA CCGGCGCATC ATCAAAAATC TGCTCAGGGG AGGGTACACC AGCTCAGAGG ACAACTGCCA GACTTTCAAC GAGGATGACT GCCGCTTCTT CGAGAGCCGC TGGGAGGAGT GCAAGGGAGG TGGGGAGTCA CCTTGTGGCA CATTTTCACC TGCTGATGCT GCCCTGGGTC TGACCTTNCC CTTGGCAGTG TRANSACATCC TCCTCTTGCT CCTCAAGTAA GAGGTGCAGA AAAGAAAGAA AAAAAWAAAÀ AAAAAAAAA
TCACCCCTCG CATTCGTGTC TGGCGCCAGA GTGCTTGTGC CTGGGCCAGC TGGAGAGGTC TGACATGTCT AGTCTGCCGG AAGGGTGACA GTRACCGTGG CTGCCACATT TACTGCCATC ATTGGTTCTG TACTGTCTGT TTGGCTCAGC GTTTCCCAAA GCGTGGCCAG AAGCGGAAAA ATGGCCGCCG ACGCCGGGTA CTGTTGAGGG ACTCGGAAGA AGGGCTCTCC CCCTCCAAGC GTGATCTCAC ATTTTGCGAG ATTATCCTGA CTTTCCTAGA GCCTGTGAAC CCACGTTTGG CTATGGATTT TTCCACCATG CGGGAGCGGC AGTTTGCGGC TGATGCCCTC CTGGTATTTG CTGAAGTAGG CAAGGCTGGG CACATCATGC TTTATCAGGG AAAACAGGCC AATCTGTGAG TCTCCCCCCA CCTTCCAAAC AAAAACCTGC CAGACTCAAG TCAGATACAA CCCTGATTTT TCTTATTCCT ACATCCCTTT CTCCCTTCCC GATGAGGTCC TTCTGGACTA AAAGCCAAAA AAAAAAAAAA AAAAAAAA AAAAAAAAN
120
180
240
300
360
420
480
540
600
660
720
780
840
900
960
1020
1080
1139
200 (2) INFORMATION FOR SEQ ID NO: 14:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2271 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: double (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14:
GTTCCGGGGG ATGCCAGCTC ACTTCTCGGA CAGCGCCCAG ACTGAGGCCT GCTACCACAT60
GCTGAGCCGG ACCEAGCCGC CACCCGACCC CCTCCTGCTC CAGCGTCTGC CACGGCCCAG120
CTCCCTGTCA GACAAGACCC AGCTCCACAG CAGGTGGCTG GACTCGTCGC GGTGTCTCAT180
GCAGCAGGGC ATCAAGGCCG GGGACGCACT CTGGCTGCGC TTCAAGTACT ACAGCTTCTT240
CGATTTGGAT CCCAAGACAG ACCCCGTGCG GCTGACACAG CTGTATGAGC AGGCCCGGTG300 ggacctgctg ctggaggaga ttgactgcac cgaggaggag ATGATGGTGT ttgccgccct gcagtaggggagggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggcggcgcgzczczc aret it's up?
360
420
201
<td>CCCAGGGCTG</td><td>GACGACCTGG</td><td>ATGTGGCCCT</td><td>GAGCAACCTG</td><td>GAGGTGAAGC</td><td>TGGAGGGGTC</td><td> 480</td>
<td>GGCGCCCACA</td><td>GATGTGCTGG</td><td>ACAGCCTCAC</td><td>CACCATCCCA</td><td>GAGCTCAAGG</td><td>ACCATCTCCG</td><td> 540</td>
<td>AATCTTTCGG</td><td>ACCGAGAAGC</td><td>TGACCCTGAA</td><td>GGGCTACCGC</td><td>CAACACTGGG</td><td>TGGTGTTCAA</td><td> 600</td>
<td>GGAGACCACA</td><td>CTGTCCTACT</td><td>ACAAGAGCCA</td><td>GGACGAGGCC</td><td>CCTGGGGACC</td><td>CCATTCAGCA</td><td> 660</td>
<td>GCTCAACCTC</td><td>AAGGGCTGTG</td><td>AGGTGGTTCC</td><td>CGATGTTAAC</td><td>GTCTCCGGCC</td><td>AGAAGTTCTG</td><td> 720</td>
<td>CATTAAACTC</td><td>CTAGTGCCCT</td><td>TRANSTGAGGG</td><td>CATGAGTGAG</td><td>ATCTACCTGC</td><td>GGTGCCAGGA</td><td> 780</td>
<td>TGAGCAGCAG</td><td>TATGCCCGCT</td><td>GGATGGCTGG</td><td>CTGCCGCCTG</td><td>GCCTCCAAAG</td><td>GCCGCACCAT</td><td> 840</td>
<td>GGCCGACAGC</td><td>AGCTACACCA</td><td>GCGAGGTGCA</td><td>GGCCATCCTG</td><td>GCCTTCCTCA</td><td>GCCTGCAGCG</td><td> 900</td>
<td>CACGGGCAGT</td><td>GGGGGCCCGG</td><td>GCAACCACCC</td><td>CCACGGCCCT</td><td>GATGCCTCTG</td><td>CCGAGGGCCT</td><td> 960</td>
<td>CAACCCCTAC</td><td>GGCCTCGTTG</td><td>CCCCCCGTTT</td><td>CCAGCGAAAG</td><td>TTCAAGGCCA</td><td>AGCAGCTCAC</td><td> 1020</td>
<td>CCCACGGATC</td><td>CTGGAAGCCC</td><td>ACCAGAATGT</td><td>GGCCCAGTTG</td><td>TCGCTGGCAG</td><td>AGGCCCAGCT</td><td> 1080</td>
<td>GCGCTTCATC</td><td>CAGGCCTGGC</td><td>AGTCCCTGCC</td><td>CGACTTCGGC</td><td>ATCTCCTATG</td><td>TCATGGTCAG</td><td> 1140</td>
<td>GTTCAAGGGC</td><td>AGCAGGAAAG</td><td>ACGAGATCCT</td><td>GGGCATCGCC</td><td>AACAACCGAC</td><td>TGATCCGCAT</td><td> 1200</td>
<td>CGACTTGGCC</td><td>GTGGGCGACG</td><td>TGGTCAAGAC</td><td>CTGGCGTTTC</td><td>AGCAACATGC</td><td>GCCAGTGGAA</td><td> 1260</td>
<td>TGTCAACTGG</td><td>GACATCCGGC</td><td>AGGTGGCCAT</td><td>CGAGTTTGAT</td><td>GAACACATCA</td><td>ATGTGGCCTT</td><td> 1320</td>
<td>CAGCTGCGTG</td><td>TCTGCCAGCT</td><td>GCCGAATTGT</td><td>ACACGAGTAT</td><td>ATCGGGGGCT</td><td>ACATTTTCCT</td><td> 1380</td>
<td>GTCGACGCGG</td><td>GAGCGGGCCC</td><td>GTGGGGAGGA</td><td>GCTGGATGAA</td><td>GACCTCTTCC</td><td>TGCAGCTCAC</td><td> 1440</td>
<td>CGGGGGCCAT</td><td>gaggccttct</td><td>GAGGGCTGTC</td><td>TGATTGCCCC</td><td>TGCCCTGCTC</td><td>ACCACCCTGT</td><td> 1500</td>
<td>CACAGCCACT</td><td>CCCAAGCCCA</td><td>CACCCACAGG</td><td>GGCTCACTGC</td><td>CCCACACCCG</td><td>CTCCAGGCAG</td><td> 1560</td>
<td>GCACCCAGCT</td><td>GGGCATTTCA</td><td>CCTGCTGTCA</td><td>CTGACTTTGT</td><td>GCAGGCCAAG</td><td>GACCTGGCAG</td><td> 1620</td>
<td>GGCCAGACGC</td><td>TGTACCATCA</td><td>CCCAGGCCAG</td><td>GGATGGGGGT</td><td>GGGGGTCCCT</td><td>GAGCTCATGT</td><td> 1680</td>
<td>GGTGCCCCCT</td><td>TTCCTTGTCT</td><td>GAGTGGCTGA</td><td>GGCTGATACC</td><td>CCTGACCTAT</td><td>CTGCAGTCCC</td><td> 1740</td>
<td>CCAGCACACA</td><td>AGGAAGACCA</td><td>GATGTAGCTA</td><td>CAGGATGATG</td><td>AAACATGGTT</td><td>TCAAACGAGT</td><td> 1800</td>
<td>TCTTTCTTGT</td><td>TACTTTTTAA</td><td>AATTTCTTTT</td><td>TTATAAATTA</td><td>ΑΤΑΤΤΊΤΑΤΤ</td><td>GTTGGATCCT</td><td> 1860</td>
<td>CCTCCTTTCT</td><td>CTGGAGCTGT</td><td>GCTTGGGGCT</td><td>ACTCTGACAC</td><td>TCTGTCTCTT</td><td>CATCACCAGC</td><td> 1920</td>
<td>CAAGGAAAGG</td><td>GGCTTTCCTG</td><td>ATAAAGACAA</td><td>GAGTTGGTTA</td><td>GAGAAAGGGA</td><td>CACCTAAGTC</td><td> 1980</td>
<td>AGTCTAGGGT</td><td>TGGAAGCTAG</td><td>GAGAGAGGTG</td><td>AGGGCAGAAG</td><td>GGCACAGCTT</td><td>TCAGGAACAA</td><td> 2040</td>
<td>GGAATAGGGG</td><td>CTGGGGTKGT</td><td>KGTTCTCACG</td><td>GGTAGGCGTA</td><td>CCTGCAGGGC</td><td>CTCCTTGAAG</td><td> 2100</td>
202
TACTTGGGAA GGAGGAAGCC ATCAGTATTC CCTGGAGTCA GAATCACCCC ATTGGCAGAG
2160
CGGAAGAAGG GTATTCCATC TGCTGACAGA GCCAGAGATG
TGACTCATGC CCTCCCCGAA
2220
GGCAAAGTCA GCTCCTGCTT TGTCCAGACT CACCTGCCAG AGCCAGGGGT C
2271 (2) INFORMATION FOR SEQ ID NO: 15:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 626 base pairs (B) TYPE: nucleic acid (C) chain type: double (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15:
ACAACAAACA TCGAAAATCG ANTATGTGCC CCGAAAAGTC GGAACGCAGG CÁATCAGTCC60
GCACGMGCGC AAGTTCAACA TGAAGATGAT ATGAGGCCGG GGCGGGGGGC AGGGACCCCC120
GGGCGGCCGG GCAGGGGAAG GGGCCTGGCC GCCACCTGCT CACTCTCCAG TCCTTCCCAC180
CTCCTCCCTA CCCTTCTACA CACGTTCTCT TTCTCCCTCC CGCCTCCGTC CCCTGCTGCC240
ACCOUNT CCCCACCACC TGCCCTCCTT CTACCAGGAC CTCAGAAGCC CAGACCTGGG300
GACCCCACCT ACACAGGGGC ATTGACAGAC TGGAGTTCAA AGCCGACGAA CCGACACGCG360
GCAGAGTCAA TAATTCAATA AAAAAGTTAC GAACTTTCTC TGTAACTTGG GTTTCAATAA420
TTATGGATTT TTATGAAAAC TTGAAATAAT AAAAAGAGAA AAAAACTATT TCCTATAGCT480
AGTCGGAATG CAAACTTTTG ACGTCCTGAT TGCTCCAGGG CCCTCTTTCC AACTCAGTTT540
CTTGTTTTTC CTCTTCCTCC TCCTCCTCTT CTTCCTCCTT TCTTTCTCTT NCCCCATGGG600
GGAGGGGTTC ATTCAGGGAA AACAGG626
203 (2) INFORMATION FOR SEQ ID NO: 16:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2118 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: double (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16:
TTTTCCAGCC ATGTCACTAA TTGTGAATTC CTACCAACTA TTGACAGAAT ACAGAGTTGA60
TTTTTTAATA AAAAGTTATA TATAATTATC CCTTTAATTA AAGGGAGCAA AGGGGCGTTC120
CACATGGACA GAGGCTTGGA CCGAGGCCTG GTCACAGCAG CGAGCATCCA GGGTTTGCAG180
GGACGATGTT ACAGACTCTG TTTTCTGCCT GGCGTTTCAC TTGTGTCTGC TCCTAGCCTG240
TGCTCTGCCA GCAGCACAGA CATCTGCTCC ATCAGACCTC TTCCATTTTG CACAGGGAGT300
GCAGGAGGTG AATGTTCACT TTCTGTTCTC CAGTGTCACT GTTCTGTTTC CACGGGATGG360
AAAGCGCATG GGCCTGTGTC CATTGTAGAT TTCCTTCTAG ATTTCTGTGT ACACACACTT420
GATTGTTCTG GATGAATGTC TTTTTTAATA CTCCGAAAAT TTCATCATCT AAGAAAATGA480
TTCCATACAA ATAACTCAGC ACACAAGTGA CCCAGGACAT ATGCCTGCCA AAGGGATGTG540
TTAGAAGGCT GCCTTCTCAT GCGCATTGTC ACTTGGATCT TGTGGTGAGG ACGGCCCCAT600
CTTTCTTGCC ACAGATTGAG GCCACTTTTG AGCAAGGGAG ATCCTGGAGT TAAGACAGGT660
GTTGGGGGCA GCCTGTATTT TACCCTAGGG GCAGGTCTGC ATGGTGACCC CACATYGCAC720
TGGTAAACCA TTTGAGTCCC ACTCTTCATC CTGGAAGTGG GAACTGGAGT CCCACCCACA780
GTGCATTCAG AAAGCATGCT GTGTGGGGGC TGCTTCTCAG GAGGCCAGGC CCTTCTGAGC840
GGAACCGTCC TGGAGAGAGC CTGCCCTCGT TTCCAGGCTG CAGCCGTAAC GCACTTTCTC900
CCAGGCTGAG GGCGGGTGTT CTGGGGTGTC TGCCCTCTGT CGGCCCTGCT TCCTGCCAGG960
ACGTGGCCTC TTCCGATCCT TTTCTCTCAG ACACTGGAGG TCTCTTCTGC CATTGTGCTG1020
GTCCCATCCC AAGAATTGTA GGACAGAGAC CACACTGGGT CGGCGGACAC AAAGTCCATC1080
CAGGACCCAG GCCGCAGAGG GAGCAGGAAG AGATGCTGAT AGTTTGATCT AGAAACCAGC1140
AGCTACTGGC TCAAATTCAG GTTCTGGCGT CAAATAGCGA CATTTCCAGT TTCTCTTAAA1200
AACCGTGTTT GGTTTCAGTT GGGATAGGCT TGTTTTGTCT GTTGAAAATG TTTCTAGTTT1260
204
<td>TTTTTCTTTC</td><td>ATTTTTCTCT</td><td>CATTCCATTT</td><td>CTGCCTTAAC</td><td>TTTAGTTTGT</td><td>TCACAGGGAG</td><td> 1320</td>
<td>GCAAAGCTGA</td><td>CATGAACCTT</td><td>TTGTCGTGGG</td><td>ACTTCAGGCC</td><td>ACATTGGCTT</td><td>GAAGGCATTC</td><td> 1380</td>
<td>GTTTCCTTCT</td><td>GGGGTGGGGA</td><td>CAGGCCCTCA</td><td>TGGCAGGCTT</td><td>GTTCCCGTGG</td><td>CTCTGAGCGA</td><td> 1440</td>
<td>GGCCTCTTCC</td><td>TGCTGGGCTC</td><td>CCAGACTCCT</td><td>GCATCCAGGC</td><td>ACCACCTTC</td><td>TCGGCTTCTG</td><td> 1500</td>
<td>GTTTTTCTTT</td><td>CTTTTTGGTA</td><td>GAACACAACA</td><td>TCTACCATTC</td><td>AGTTAAACCT</td><td>TCTTTATCTC</td><td> 1560</td>
<td>CTCCTYTGGC</td><td>ATCCATTTTT</td><td>CCAAAGAAGA</td><td>GTCGAGTCCT</td><td>CTGAGGTCTG</td><td>TGCTTGAAAR</td><td> 1620</td>
<td>CCGTCCGAAC</td><td>GCATTCTTGT</td><td>TAGCTTTGCT</td><td>TTTCTCCCCA</td><td>TATCCCAAGG</td><td>CGAAGCGCTG</td><td> 1680</td>
<td>agattcttcc</td><td>ATCTAAAAAA</td><td>CCCTCGACCC</td><td>GAAACCCTCA</td><td>CCAGATAAAC</td><td>TACAGTTTGT</td><td> 1740</td>
<td>TTAGGAGGCC</td><td>CTGACCTTCA</td><td>TGGTGTCTTT</td><td>GAAGCCCAAC</td><td>CACTCGGTTT</td><td>CCTTCGGATT</td><td> 1800</td>
<td>TTCCTCCCTT</td><td>TGTTCGGGGT</td><td>TTGGTTTGGC</td><td>TCCTCTGTGT</td><td>GTGTCCGTAT</td><td>CTTGTTCGGT</td><td> 1860</td>
<td>GTCCTCGAGG</td><td>TTGAGCTTCA</td><td>CTCCACTGCG</td><td>GCAGAGGCAG</td><td>CGTGCACACT</td><td>CGGATTTGCT</td><td> 1920</td>
<td>ACGTTTCTAT</td><td>ATATCTTGAA</td><td>GCTAAATGTA</td><td colspan="2">TATATGAGTA GTTTGCCATG</td><td>AGATAACACA</td><td> 1980</td>
<td>GTGTAAACAG</td><td>TAGACACCCA</td><td>GAAATCGTGA</td><td>CTTCTGTGTT</td><td>CTCTCCATTT</td><td>GAGTATTTTG</td><td> 2040</td>
<td>ΤΑΑΊΤΓΓΤΓΓ</td><td>GAAATATTTG</td><td>TGGACATAAA</td><td>TAAAACCAAG</td><td>CTACACTACA</td><td>AAAAAAAAAA</td><td> 2100</td>
<td>AAAAAAACTG</td><td>GAGACTAG</td><td></td><td></td><td></td><td></td><td> 2118</td>
(A) LENGTH: 1076 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: double (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17:
GCCCAAGGAG CTCAGCTTCG CCCGCATCAA GGCCGTTGAG
TGCGTGGAGA GCACCGGGCG
CCACATCTAC TTCACGCTGG TGACCGAAGG GWGCGGCGAG ATCGACTTCC
GCTGCCCCCT
120
GGAAGATCCC GGCTGGAACG CCCAGATCAC
CCTAGGCCTG GTCAAGTTCA AGAACCAGCA
180
205
GGCCATCCAG ACAGTGCGGG CACCGGGCGC ACCATCTTTC CTTCGTTGTT CCCTCTGGCT CATGGACGTG TGGAGGAGGC GAAGGCTACG CAGGGCTGAG CATGCCCAGC TCAGGGGCTT GGCATAGGAG CCCCCTCCCT CCCTGGCTTT GTGCCAGGCC TCAGGAATCA GGAGGCCAGC CACTCCAGAA TGGCCTCTGG CTCCTGGGGG TCTCCTGCTG
TTTGCACACT TCTTCCCCCA GGTGGTGGGG GTGAGGGGGG TGCCCTGCAA AGGGCAGTNA CCGTTAACCA TTTTGGCCTN
CCGCGCAGAG CCTCGGGACC CTTCATGCTA CCCACCACCT TGTGGGGGCA CGGCTGTSYT GCTGGAGCTG
CCTCTAYGCA TCTTCCCCCC GTTTGGCCAT TAGCATTTCA ACCACAAAAA AAAAAAAAAA ATAGGGGGGN GGTTTTTAAA
GGGACCCTCG TGTCCTAAAC CAGTGCTGAG GTCAAGGCAG CCATGTGGCA AGGTGGAAGG CGAGCCCTGG GAGGAGGGCA ATGGTCCCAG ACTCTCAGGA ATATGGCATG AGGGGGAGCT CCTGCAGACT GCTCTGCCGCCGCCGCCGCCGCCGCCGCCGCCGCCG
ACTGCGGTGT TCGGCCTGAA TGTCTTTCCC CAAATGAAGA AAAAACNTGG GGGGGGGGCC AATTAATTGG GCCCGG
240
300
360
420
480
540
600
660
720
780
840
900
960
1020
1076 (2) INFORMATION FOR SEQ ID NO: 18:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1379 base pairs (B) TYPE: nucleic acid (C) chain type: double (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 18:
GGCACGAGCA CCCTCCCACA CCTCCCTGAA CTTCCATCTG ATCGACTTCA ACTTGCTGAT
GGTGACCACC ATCGTTCTGG GCCGCCGCTT CATTGGGTCC ATCGTGAAGG AGGCCTCTCA
120
206
GAGGGGGAAG GTCTCCCTCT TTCGCTCCAT CCTGCTGTTC CTCACTCGCT TCACCGTTCT 180 CACGGCAACA GGCTGGAGTC TGTGCCGATC CCTCATCCAC CTCTTCAGGA CCTACTCCTT 240 CCTGAACCTC CTGTTCCTCT GCTATCCGTT TGGGATGTAC ATTCCGTTCC TGCARCTGAA 300 TTKCGAMCTY CGSAAGAGAA GCCTCTTCAA CCACATGGCC TCCATGGGGC CCCGGGAGGC 360 GGTCAGTGGC CTGGCAAAGA GCCGGGACTA CCTCCTGACA CTGCGGGAGA CGTGGAAGCA 420 GCACASAAGA CAGCTGTATG GCCCGGACGC CATGCCCACC CATGCCTGCT GCCTCTCGCC 480 CAGCCTCATC CGCAGTGAGG TGGAGTTCCT CAAGATGGAC TTCAACTGGC 540 GCATGAAGGA AGTGCTCGTS AGCTCCATGC TGAGCGCCTA CTATGTGGCC TTTGTGCCTG TYTGGTTCGT 600 GAAGAACACA CATTACTATG ACAAGCGCTG GTCCTGTGNA ACTCTTCCTG CTGGTGTCCA 660 TCAGCACCTC CGTGATCCTC ATGCAGCACC TGCTGCNTGC CAGCTACTGT GACCTGCTGC 720 ACAAGGCCGC CGCCCATCTG GGCTGTTGGC AGAAGGTGGA CCCAGCGCTG TGCTCCAACG 780 TGCTGCAGCA CCCGTGGACT GAAGAATGCA TGTGGCCGCA GGGCGTGCTG GTGAAGCACA 840 GCAAGAACGT CTACAAAGCC GTAGGCCAMW ACAAMGTGGC TATCCCCTCT GACGTCTCCC 900 ACTTCCGCTT CCAKTTCTTT TTCAGCAAAC CCCTGCGGAT CCTCAACATC CTCCTGCTGC 960 TGGAGGGCGC TGTCATTGTC TATCAGCTGT ACTCCCTAAT GTCCTCTGAA 1020 AGACCATCTC GCTGGCCCTC ATCCTCTTCA
ACCACCGTTT CTCCTGAGCC CTGGGGTCAC CTCAGGGACA GCGTCCAGGC TTCAGCAAGG 1200 GCTCCCTGGC AAGGGGCTGT TGGGTAGAAG TGGTGGTGGG GGGGACAAAA GACAAAAAAA 1260 TCCACCAGAG CTTTGTATTT TTGTTACGTA CTGTTTCTTT GATAATTGAT GTGATAAGGA 1320 AAAAAAAAAA AAAAAGTCCT ATTTTTATAC TCCCAANMAA NAAAAAGCGG CCGAAAGCT 1379 (2) INFORMATION FOR SEQ ID NO: 19:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1337 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: double
207 (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 19:
<td>CTGGTGTTGG GCCTGAGCCN CCTCAACAAC TCCTACAACT TCAGTTTCCA CGTGGTGATC</td><td> 60</td>
<td>GGCTCTCAGG CGGAAGAAGG CCAGTACAGC CTGAACTTCC ACAACTGCAA CAATTCAGTG</td><td> 120</td>
<td>CCAGGAAAGG AGCATCCATT CGACATCACG GTGATGATCC GGGAGAAGAA TRANSGATGGC</td><td> 180</td>
<td>TTCCTGTCGG CAGCGGAGAT GCCCCTTTTC AAGCTCTACA TGGTCATGTC CGCCTGCTTC</td><td> 240</td>
<td>CTGGCCGCTG GCATCTTCTG GGTGTCCATC CTCTGCAGGA ACACGTACAG CGTCTTCAAG</td><td> 300</td>
<td>ATCCACTGGC TCATGGCGGC CTTGGCCTTC ACCAAGAGCA TCTCTCTCCT CTTCCACAGC</td><td> 360</td>
<td>ATCAACTACT ACTTCATCAA CAGCCAGGGG CCACCCCATC GAAGGCCTTG CCGKCATGTA</td><td> 420</td>
<td>CTACATCGCA CACCTGCTGA AGGGCGCCCT CCTCTTCATC ACCATCGCCC TGATTGGCTC</td><td> 480</td>
<td>AGGCTGGGCT TCATCAAGTA CGTCCTGTCG GATAAGGAGA AGAAGGTCTT TGGGATCGTG</td><td> 540</td>
<td>ATCCCCATGC AGGTCCTGGC CAACGTGGCC TACATCATCA TCGAGTCCCG CGAGGAAGGC</td><td> 600</td>
<td>GCCACGAACT ACGTGCTGTG GAAGGAGATT TTGTTCCTGG TGGACCTCAT CTGCTGTGGT</td><td> 660</td>
<td>GCCATCCTGT TCCCCGTAGT CTGGTCCATC CGGCATCTCC AGGATGCGTC TGGCACAGAC</td><td> 720</td>
<td>GGGAAGGTGG CAGTGAACCT GGCCAAGCTG AAGCTGTTCC GGCATTACTA TGTCATGGTC</td><td> 780</td>
<td>ATCTGCTACG TCTACTTCAC CCGCATCATC GCCATCCTGC TGCAGGTGGC TGTGCCCTTT</td><td> 840</td>
<td>CAGTCGCAGT GGCTGTACMA GCTCTTGGTG GARGGCTCCA CCCTGGCCTT CTTCGTGCTC</td><td> 900</td>
<td>ACGGGCTACA AGTTCCAGCC CACAGGGAAC AACCCGTACC TGCAGCTGCC CCAGGAGGAC</td><td> 960</td>
<td>GAGGAGGATG TTCAGATGGA GCAAGTAATG ACGGACTCTG GGTTCCGGGA AGGCCTCTCC</td><td> 1020</td>
<td>AAAGTCAACA AAACAGCCAG CGGGCGGGAA CTGTTATGAT CACCTCCACA TCTCAGACCA</td><td> 1080</td>
<td>AAGGGTCGTC CTCCCCCAGC ATTTCTCACT CCTGCCCTTC TTCCACAGCG TATGTGGGGA</td><td> 1140</td>
<td>GGTGGAGGGG TCCATGTGGA CCAGGCGCCC AGCTCCCGGG ACSCCGGTTC ccggacaagc</td><td> 1200</td>
<td>CCATTTGGAA GAAGAGTCCC TTCCTCCCCC CAAATATTGG GCAGCCCTGT CCTTACCCCG</td><td> 1260</td>
<td>GGACCACCCC TCCCTTCCAG CTATGTGTAC AATAATGACC AATCTGTTTG GCTAAAAAAA</td><td> 1320</td>
<td>AAAAAAAAAA AACTCGA</td><td> 1337</td>
208 (2) INFORMATION FOR SEQ ID NO: 20:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1390 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: double (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 20:
<td>GCCGTTTTGG</td><td>TTCCCGGTTG</td><td>GTGCTTCCTG</td><td>TTCGCAGCTG</td><td>CGGCACTTCA</td><td>AGGTTACTGA</td><td> 60</td>
<td>CTTTTTATGA</td><td>TGTTTGGTGG</td><td>CTATGAGACT</td><td>ATAGAWGCRT</td><td>RSGRRGATGA</td><td>TYTTTATCGA</td><td> 120</td>
<td>GATGAGTCAT</td><td>CTAGTGAACT</td><td>GAGTGTTGAT</td><td>AGTGAGGTGG</td><td>AATTTCAACT</td><td>CTATAGCCAA</td><td> 180</td>
<td>ATTCATTATG</td><td>CCCAAGATCT</td><td>TGATGATGTC</td><td>ATCAGGGAGG</td><td>AAGAGCATGA</td><td>AGAAAAGAAC</td><td> 240</td>
<td>TCTGGGAATT</td><td>CGGAATCTTC</td><td>GAGTAGTAAA</td><td>CCAAATCAGA</td><td>AGAAGCTAAT</td><td>CGTCCTTTCA</td><td> 300</td>
<td>GATAGTGAGG</td><td>TCATCCAGCT</td><td>GTCAGATGGG</td><td>TCAGAGGTCA</td><td>TCACTTTGTC</td><td>TGATGAAGAC</td><td> 360</td>
<td>AGTATTTATA</td><td>GATGTAAAGG</td><td>AAAGAATGTT</td><td>AGAGTTCAAG</td><td>CACAAGAAAA</td><td>TGCCCATGGT</td><td> 420</td>
<td>CTTTCTTCTT</td><td>CTCTTCAATC</td><td>TAATGAGCTG</td><td colspan="2">GTTGATAAGA AATGCAAGAG</td><td>TGATATTGAG</td><td> 480</td>
<td>AAGCCTAAAT</td><td>CTGAAGAGAG</td><td>ATCAGGTGTA</td><td>ATCCGAGAGG</td><td>TCATGATTAT</td><td>AGAGGTCAGT</td><td> 540</td>
<td>TCAAGTGAAG</td><td>AGGAAGAGAG</td><td>CACCATTTCA</td><td>GAAGGTGATA</td><td>ATGTGGAAAG</td><td>CTGGATGCTA</td><td> 600</td>
<td>CTGGGATGTG</td><td>AAGTAGATGA</td><td>TAAAGATGAT</td><td>GATATCCTTC</td><td>TCAACCTTGT</td><td>GGGATGTGAA</td><td> 660</td>
<td>AACTCTGTTA</td><td colspan="2">CTGAAGGAGA AGATGGTATA</td><td>AACTGGTCCA</td><td>TCAGTGACAA</td><td>AGACATTGAG</td><td> 720</td>
<td>GCCCAGATAG</td><td>CTAATAACCG</td><td>AACACCTGGA</td><td>AGATGGACCC</td><td>AGCGGTACTA</td><td>TTCAGCCAAC</td><td> 780</td>
<td>AAAAACATTA</td><td>TCTGTAGAAA</td><td>TTGTGACAAA</td><td>CGTGGTCATT</td><td>TATCAAAAAA</td><td>CTGCCCCTTA</td><td> 840</td>
<td>CCACGAAAAG</td><td>TTCGTCGCTG</td><td>CTTCCTGTGC</td><td>TCCAGGAGAG</td><td>GACATCTCCT</td><td>GTATTCCTGT</td><td> 900</td>
<td>CCAGCCCCCC</td><td>TTTGCGAATA</td><td>CTGTCCTGTG</td><td>CCTAAGATGT</td><td>TGGACCACTC</td><td>ATGTCTITTC</td><td> 960</td>
<td>AGACATTCCT</td><td>GGGATAAACA</td><td>GTGTGACCGA</td><td>TGTCATATGC</td><td>TAGGCCACTA</td><td>TACAGATGCT</td><td> 1020</td>
209
TGCACAGAAA TCTGGAGGCA GTATCACCTA ACGACCAAAC CTGGACCACC CAAAAAGCCG
AAGACCCCTT CAAGACCATC AGCCTTAGCA TATTGCTATC ACTGCGCGCA AAAAGGCCAT
TATGGACACG AATGTCCAGA AAGAGAAGTG TATGACCCGT CTCCAGTATC TCCATTCATC
TGCTACTATG RTGACAAATA TGAAATTCAG GAGAGAGAAA AGAGACTAAA ACAAAAATA
AAAGTANTCA AGAAAAATGG GGTTATCCCA GAGCCATCCA AGCTACCTTA TATAAAAGCA
GCAAATGAGA ACCCCCACCA TGATATAAGG AAGGGCCGTG CCTCATGGAA AAGCAACAGG TGGCCTCAAG (2) INFORMATION FOR SEQ ID NO: 21:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1431 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: double (D) TOPOLOGY: linear
1080
1140
1200
1260
1320
1380
1390 (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 21:
GCCTGCAGTC GACACTAGTG GATCCAAAGA CTCAGTCTCC CTGGCGAGCG ACGGGCAGAA TGGATCCCAG AAGGTCGCGA AGGCAGTACC AATGTCTTTT CCACCTCATT TGAATCGCCC GATCCCACCC CCGCAGTTTC CAGGATTTCC TCCTGTACCA ATGAGCATTA TGGCTCCTGC GGTTGGAAAG CATTTGGGCG CAAGAAAGGA TGAAAATTGT GGTCCTACTA CCACTGTTTT CATGCTTATA AGACAACTCT TAGCTAAATG AGGTGCTTCC GGAAAGCTTC AAGCCTTCGG
ATTCGGCCTG TGCGAGTAGG CGCTTGGGCA ATCTCGAACC AGTGGAGCGC ACTCGTAACC GTTTCCTCAG CGGCGGACTG CTGCAGTAAG TCCCATGGGA ATCCCAGCAC TCCCACCAGG TCCACCTGTA CCTCCAGGGA CCCCAATGAT TCCAACTGTC TTAGTACCCA CTGTGTCTAT TCATCCAGGC TTAAAGGCTA AAGAAAATGA TGTTGGCAAC ATTTCCGAGA AAGCTTCAGA TGGTTTGGTT TTGAGCTGGA AGAGAGTACA ATTCTGTGAG TÀCAAGGAGC CAGAATCTAC
120
180
240
300
360
420
480
540
600
210
CCTCCGTGCA CTCAGATTAT TACATGACCT GCAAATTGGA GAGAAAAAGC TACTCGTTAA660
AGTTGATGCA AAGACAAAGG CACAGCTGGA TGAATGGAAA GCAAAGAAGA AAGCTTCTAA720
TGGGAATGCA AGGCCAGAAA CTGTCACTAA TGACGATGAA GAAGCCTTGG ATGAAGAAAC780
AAAGAGGAGA GATCAGATGA TTAAAGGGGC TATTGAAGTT TTAATTCGTG AATACTCCAG840
TGAGCTAAAT GCCCCCTCAC AGGAATCTGA TTCTCACCCC AGGAAGAAGA AGAAGGAAAA900
GAAGGAGGAC ATTTTCCGCA GATTTCCAGT GGCCCCACTG ATCCCTTATC CACTCATCAC960
TAAGGAGGAT ATAAATGCTA TAGAAATGGA AGAAGACAAA AGAGACCTGA TATCTCGAGA1020
GATCAGCAAA TTCAGAGACA CACATAAGAA ACTGGAAGAA GAGAAAGGCA AAAAGGAAAA1080
AGAAAGACAG GAAATTGAGA AAGAACGGAG AGAAAGAGAG AGGGAGCGTC AAAGGGAACG1140
AGAAAGGCGA GAACGGGAAC GAGAAAGGGA AAGAGAACGT GAACGAGAAA AGGAGAAAGA1200
ACGGGAGCGG GAACGAGAAC GGGATAGGGA CCGTGACCGG ACAAAAGAGA GAGACCGAGA1260
TCGGGATCGA GAGAGAGATC GTGACCGGGA TAGAGAAAGG AGCTCAGATC GTAATAAGGA1320
TCGCATTCGA TCAAGAGAAA AAAGCAGAGA TCGTGAAAGG GAACGAGAGC GGGAAAGAGA1380
GAGAGAGAGA GAACGAGAGC GAGAACGAGA ACGGGAGCGA GAGAGAGAAG C1431 (2) INFORMATION FOR SEQ ID NO: 22:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2539 bp (B) TYPE: nucleic acid (C) CHAIN TYPE: double (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 22:
GGGTGCAGGA GTGCCACCCC CAGGGCCCTG TCAACCTCTC TTTTCTCCTC CATGGCTGTC60
TGCCTGCGTA TCTGTCTCTG AGAATCCTCG GGGCGGTCAG GGGATGTCAG GAGGGGÀAGG120
AGCCGCCCTC CCTATCTTGC TGCTCCTCTT GGCACTCAGG GGCACCTTCC ATGGAGCCAG180
211
<td>ACCGGGTGGA</td><td>GGGGCTTCTG</td><td>GGATTTGGTG</td><td>TCTGCTGCTG</td><td>CCAGAGCAGG</td><td>AACCCCCAGT</td><td> 240</td>
<td>CTAGGACTTG</td><td>GGCATTTTAA</td><td>CAGGGAGAAA</td><td>GTAGTGGCTT</td><td>CCCTTTTCTC</td><td>TCTCTCCTCC</td><td> 300</td>
<td>TTTTTCCCTT</td><td>TAAGCCCACA</td><td>GATTCAGGTC</td><td>ATGCCAAAAG</td><td>CTCTCTGGTT</td><td>GTAACCTGGA</td><td> 360</td>
<td>GACATGTGGA</td><td>GGGGAATGGC</td><td>GATGGGATTA</td><td>TAGGACTCTC</td><td>CCCATCTCGG</td><td>GCCCTGACCC</td><td> 420</td>
<td>TGACCCTTGC</td><td>CACCAACCCA</td><td>AAGACAGCTG</td><td>GTGGGTTTCC</td><td colspan="2">CCTTGGAGAM AATCCTGCGT</td><td> 480</td>
<td>TTGCCTGGGC</td><td>CGGCCCTGGC</td><td>TGCCCTCAGC</td><td>TTTCGCTGAT</td><td>CTGCCCGGCC</td><td>TGGAGCCTCC</td><td> 540</td>
<td>CATCACCCCG</td><td>CTTCTTGTTG</td><td>GGCCTCAGGC</td><td>ACTGGTTACC</td><td>AGAAGGGGGT</td><td>CTGGGTCTGC</td><td> 600</td>
<td>TCAGGAATCA</td><td>TGTTTTGTAG</td><td>CACCTCCTGT</td><td>TGGAGGGGTG</td><td>GAGGGATGTT</td><td>ACCAGGCCCC</td><td> 660</td>
<td>AGGCTGAGAC</td><td>TAGAACCCCA</td><td>TCTTCCCTGA</td><td>GCCAGGCTGA</td><td>GACTAGAACC</td><td>CCATCTTCCC</td><td> 720</td>
<td>CACCACGCCA</td><td>TRANSTGTGST</td><td>KGCTACAGGA</td><td>GCACAGTAGT</td><td>GAAGGCCTGA</td><td>GCTCCAGGTT</td><td> 780</td>
<td>TGAAAGACCC</td><td>AACTGGAGCG</td><td>TGGGGCGGGC</td><td>AGGCAGGGGT</td><td>TAGTGAAAGG</td><td>ACACTTCCAG</td><td> 840</td>
<td>GGTTAGGACA</td><td>GAGCAITTAG</td><td>CCTTCTGGAA</td><td>GAACCCCTGC</td><td>CTGGGGTGGG</td><td>ACTGTGCAGG</td><td> 900</td>
<td>CCAGAGAAGG</td><td>TGGCATGGGC</td><td>CTGAACCCAC</td><td>CTGGACTGAC</td><td>TTCTGCACTG</td><td>AAGCCACAGA</td><td> 960</td>
<td>TGGAGGGTAG</td><td>GCTGGTGGGT</td><td>GGGGGTGGTT</td><td>CGTTCTCTAG</td><td>CCGGGGCAGA</td><td>CACCCAGCTG</td><td> 1020</td>
<td>GCTGGGTCCT</td><td>TCCTCAGCCT</td><td>TGCCTCCTCC</td><td>TGTCCCCAAC</td><td>CCTTTCCTTT</td><td>CCTCCTGCTT</td><td> 1080</td>
<td>GCGGACTGCT</td><td>GGTCCCCTCT</td><td>CCTTCCCTCC</td><td>TTCCAGCTGT</td><td>TTCTAGTTAC</td><td>CACCTÀCCCC</td><td> 1140</td>
<td>TGGGCCGTGG</td><td>ACTGATCAGA</td><td>CCAGCATTCA</td><td>AAATAAAAGT</td><td>TTGTTCCAAG</td><td>TTGACAGTGT</td><td> 1200</td>
<td>GGTGCTCCCT</td><td>GCCCAGCCCC</td><td>TCCAGGTGGA</td><td>GGTGCTGCCA</td><td>CGGGAACGCA</td><td>GTTGCTCTGC</td><td> 1260</td>
<td>CTGCCCTGGG</td><td>TRANSTGGCGA</td><td>CANTGGGAGC</td><td>AGGGCAGTGC</td><td>TGTGAGGAGC</td><td>CCAGCTTTCC</td><td> 1320</td>
<td>CAGTCAGGCA</td><td>GGCATGGCW</td><td>CCGTGTTCAG</td><td>GCTCCCTCAC</td><td>CAGCTGGTGA</td><td>CACGGGACAA</td><td> 1380</td>
<td>GCTTACAAAC</td><td>CTTCTCTGAA</td><td>CCTCAGTTTT</td><td>CTCATTTACA</td><td>AGAGGCAAAG</td><td>CATCCATCAC</td><td> 1440</td>
<td>clTGTGlOGA</td><td>TTCARAGAAT</td><td>GTRAGGCCCT</td><td>GGGGTGTCCT</td><td>ACACAAuGGA</td><td>AAGGCTTGCT</td><td> 1500</td>
<td>CAGTGAGCGG</td><td>TCTGCACACC</td><td>GTTAGCCACC</td><td>CTGCCACCTC</td><td>TGTGCCCTGG</td><td>GCAGGCTCCA</td><td> 1560</td>
<td>AAGGAAAGCT</td><td>CTGGCTGGGA</td><td>CTGCCRGGAG</td><td>TCTCACACGC</td><td>TCCTGTTGAC</td><td>ATTCCCAGCA</td><td> 1620</td>
<td>GCYGCCCCTG</td><td>AGGTCGATGT</td><td>TTGTTCTGTT</td><td>TTTCTTTTTC</td><td>TTTTTTGAGA</td><td>CGGAGTCTCG</td><td> 1680</td>
<td>CTGTGTTGCC</td><td>AGGCTGGAGT</td><td>GCAGTGGTGT</td><td>GATCTCTGCT</td><td>CACTGCAACC</td><td>TCCGCCTGCC</td><td> 1740</td>
<td>AGTTTCAAGT</td><td>GATTCTCTGC</td><td>CTCAGCCTTC</td><td>TGAGTAGCTG</td><td>GGACTACAGG</td><td>TGCACGCCAC</td><td> 1800</td>
212
<td>CACGCCCAGC TAACTTTTTG TATTTWAGTA GAGACAGGGT TTCGCCATGT CGGCCAGGGT</td><td> 1860</td>
<td>GGTCTTGATC TCCTGACCTC ATGATCCACC CGCCTCAGCC TCCCAAAGTG CTGGGATTAC</td><td> 1920</td>
<td>AGGTATGAGC CACCGCACCG GGCCTGTTCT ATTTTTCTAG TTAAGGGAAC TGAAGCTCAG</td><td> 1980</td>
<td>ARAGGTGTCA CCAGCARGTG TTCATTCCCA TGCCAGCCTT GCCCCCCGGC TTTTCCCAGG</td><td> 2040</td>
<td>CAGGCTCCTG CGTGCCCACT GGCTCCAGCC TGGTCCTCTG TCTCTTGGCT GCTTCACTCC</td><td> 2100</td>
<td>TGCTCTTTGT CCCGACTCTG GCCCTGCTTA CAGGGGCCAC TACCTGCTGG TGCCTCCATA</td><td> 2160</td>
<td>ACAAGCGTCT GGCGTTGAGA TRANSTGGCAT GGCAGGGGCT TTGGGGTCTG GTTTCCACAA</td><td> 2220</td>
<td>GGCTTAGCCA TGGCAGAACC TCGTTTTATT TTAACTCTTT GCCCCTACAA ACAAACAGCA</td><td> 2280</td>
<td>GTACTTGCCA GAACCATTCT TGGGATTCAG GAGCTCGGGC GACTGCCTTG GCCTCTGGCC</td><td> 2340</td>
<td>GCACCCAGGA GGGTGGGGTT GGATCTGTGT AGTTGCCAGG CCCACACCTG CCAGCAGGGG</td><td> 2400</td>
<td>GCTGACTGGA TCCATGCTTT ACTGTGTTTA ATGGGGGTAA CAGGGGTCCC TACAGCCCTC</td><td> 2460</td>
<td>CCAGYTAAAM ATTTGGAACA AAACACCAGC CCTTTTGTAG TGGATGCAGA ATAAAATTGT</td><td> 2520</td>
<td>TAATCCAATC AAAAAAAAA</td><td> 2539</td>
(2) INFORMATION FOR SEQ ID NO: 23:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1041 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: double (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 23:
TCGACCCACG CGTCCGCCCA CGCGTCCGCC CACGCGTCCG GGCGCAGGAC GTGCACTATG60
GCTCGGGGCT CGCTGCGCCG GTTGCTGCGG CTCCTCGTGC TGGGGCTCTG GCTGGCGTTG120
CTGCGCTCCG TGGCCGGGGA GCAAGCGCCA GGCACCGCCC CCTGCTCCCG CGGCAGCTCC180
TGGAGCGCGG ACCTGGACAA GTGCATGGAC TGCGCGTCTT GCAGGGCGCG ACCGCACAGC240
213
GACTTCTGCC TGGGCTGCGC TGCAGCACCT CCTGCCCCCT TCCGGCTGCT TTGGCCCATC 300 CTTGGGGGCG CTCTGAGCCT GACCTTCGTG CTGGGGCTGC TTTCTGGCTT TTTGGTCTGG 360 AGACGATGCC GCAGAGAGAG AAGTTCACCA CCCCCATAGA GGAGACCGGC GGAGAGGGCT 420 GCCCAGCTGT GGCGCTGATC CAGTGACAAT GTGCCCCCTG CCAGCCGGGG CTCGCCCACT 480 CATCATTCAT TCATCCATTC TAGAGCCAGT CTCTGCCTCC CAGACGCGGC GGGAGCAAGC 540 TCCTCCAACC ACAAGGGGGG TGGGGGGCGG TGAATCACCT CYGAGGCCTG GGCCCAGGGT 600 TCAGGGGAAC TTCCAAGGTG TCTGGTTGCC CTGCCTCTGG CTCCAGAACA 660 GAAAGGGAGC CTCACGCTGG CTCACACAAA ACAGCTGACA CTGACTAAGG AACTGCAGCA TTTGCACAGG 720 GGAGGGGGGT GCCCTCCTTC CTAGAGGCCC TGGGGGCCAG GCTGACTTGG GGGGCAGACT 780 TGACACTAGG CCCCACTCAC TCAGATGTCC TGAAATTCCA CCACGGGGGT CACCCTGGGG 840 GGTTAGGGAC CTATTTTTAA CACTAGGGGG CTGGCCCACT AGGAGGGCTG GCCCTAAGAT 900 ACAGACCCCC CCAACTCCCC AAAGCGGGGA GGAGATATTT ATTTTGGGGA GAGTTTGGAG 960 GGGAGGGAGA ATTTATTAAT AAAAGAATCT TTAACTTTAA AAAAAAAAAA AAAAAAGGGC 1020 GGCCGCTCTA GAGGATCCCT C 1041 (2) INFORMATION FOR SEQ ID NO: 24:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1962 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: double (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 24:
ACCCACGCGT CCGGTACAAA ACACAGTTTT CATTAGATTT AGGGTTGCAT ATTAAAAACT CACTCAGGAT ATAACACACT ATAATAGAAA ATGGTTTGTA TACTGGTTCT GACACTTGTT
ATTCTATGAA AATTTTGAGA TTATTAGAAA60
ATATCCATTT TGCCTTATTA TTTAGTGTCT120
ATGTAGACTT CAGAATCAGG TATATTTGAG180
AGCTATTCAT CTTTGGTAAA TTCCCCATTA240
214
<td>CCCTTTGTKC ACCTAIWIGT GGGGATCAGT GCATAGTGTG TGTWAAGCAT TTAATACCTG</td><td> 300</td>
<td>GCAAGTGTTC AGCAAATTTT TTGTTCTATA ΤΑΤΤΓΑΤΤΑΤ TTGATTATTG GCCCTGAGGA</td><td> 360</td>
<td>GTAGGTGTTT GTTTGTTTGT TTGTTTGTTT AGTTTTATTT CTCATCTCCT CAGGAACACA</td><td> 420</td>
<td>AATGAAACTT GGATAITOTT ATGGTCCTTT TNATAATATA ΤΤΤΑΤΓΑΤΤΤ TCAGCAATTN</td><td> 480</td>
<td>ATTCTTGTTA AAACAATTTC TTATGACAAG TTACTCATCT TCAATGGTGA GAAGAAATCT</td><td> 540</td>
<td>AGCTCAGAAT AATATATTTT TAGTGTTTGT ATCTCTGGAT ACTCATTTTG CTCATTGCCA</td><td> 600</td>
<td>CGTAAAGTAA AAAAATACAT AAATTAGCTT ATTCCAATGT AATATCTTCA GGATAGTCAT</td><td> 660</td>
<td>GGGCAAGGAA TTAATCACAT TAAGAGATAA CTGCAACTÀA GCACTATTTG AGGTGACTTC</td><td> 720</td>
<td>TGTGGAAAAA AAATTAATYC TTTACCATTG CAGCGTTCTG CCCTAGGTCC AAATGTTACC</td><td> 780</td>
<td>AAAATCACTC TAGAATCTTT TCTTGCCTGG AAGAAAAGGA AAAGACAAGA AAAGATTGAT</td><td> 840</td>
<td>AAACTTGAAC AAGATATGGA AAGAAGGAAA GCTGACTTCA AAGCAGGGAA AGCACTAGTG</td><td> 900</td>
<td>ATCAGTGGTC GTGAAGTGTT TGAATTTCGT CCTGAACTGG TCAATGATGA TGATGAGGAA</td><td> 960</td>
<td>GCAGATGATA CCCGCTACAC CCAGGGAACA GGTGGTGATG ÀGGTTGATGA TTCAGTGAGT</td><td> 1020</td>
<td>GTAAATGACA TAGATTTAAG CCTGTACATC CCAAGAGATG TAGATGAAAC AGGTATTACT</td><td> 1080</td>
<td>GTAGCCAGTC TTGAAAGATT CAGCACATAT ACTTCAGATA AAGATGAAAA CAAATTAAGT</td><td> 1140</td>
<td>GAAGCTTCTG GAGGTAGGGC TGAAAATGGT GAAAGAAGTG ACTTGGAAGA GGACAACGAG</td><td> 1200</td>
<td>AGGGAGGGAA CGGAAAATGG AGCCATTGAT GCTGTTCCTG TTGATGAAAA TCTTTTCACT</td><td> 1260</td>
<td>GGAGAGGATT TGGATGAACT AGAAGAAGAA TTAAATACAC TTGATTTAGA AGAATGACAC</td><td> 1320</td>
<td>CAAACACATC GCTGAAAAAA TTAAGTCAGC TCAGCACGAG TTGAAATTGA CTACATTAAT</td><td> 1380</td>
<td>TTCTTTCCAC CTAGAATCAA CAGGATGTTT ATTTCCTATG CTGATTCTGG AGGAGTTAAC</td><td> 1440</td>
<td>CTCCTGCAAA AAAGGCATCT TGTCCCTACA TCTTCTCTTC TGACTTTGGC TACATCTCAT</td><td> 1500</td>
<td>AGTAAGTTCA GAGTAGTTCA TGATAAATTG AAAATATAAT GGTCATTGCA GAAAATGA1T</td><td> 1560</td>
<td>GATGTTGTAA CTGTCCACCC AAGTAAGAAG TGTATCTGCC TTTCCATCTT TTGGTTTCA</td><td> 1620</td>
<td>TTTGGGCATG TGCTATTACC AGAAACAACA AACTTATATT TAAAATACCC TTCATTTGAC</td><td> 1680</td>
<td>ACAGTTTTTA ATGAGTGATT TAATTTCCTC TGTATTTGTA TCTTTAGAAG ACTGCCTAAA</td><td> 1740</td>
<td>ACATGAGCAC TGTACTTCAT AAAGGAAACG CGTATGCAGA TTCAGTATTG TGTATCTTTG</td><td> 1800</td>
<td>GACAATTAGA TGGACATTTA AAATGGAACT TCTTTTATCT GACAGGATCA GCTACAATGC</td><td> 1860</td>
<td>CCTGTG1TAA ATTGTTTAAA AGTTTCCCTT TTCTTTTTTG CCAATAAAGT TGTAAATAAA</td><td> 1920</td>
<td>GACCATCATA CATTAAAATC CAAAAAAAAA AAAAAAAAA AA</td><td> 1962</td>
215 (2) INFORMATION FOR SEQ ID NO: 25:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1228 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: double (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 25:
GGCTGCCCAG GCCCCGCACT GGAAGAGCCT GAGCCCCAGC AGCCACTCCC CAGAGGGGAG GTGTAAGGCA GCTGCATCTG CACCGAGCTC ACCCTCTGTC CGCACCGCTG TTGCCCTGAC TGACATCATC CAACAGGAAG CGTCACCCTG ACAGTGGTAT AGCAGCCACT CCAGCCTCTG GGAGAGGCCT GTCCCACGGA GCCCAGAGGC GGCCTCCAGA CCTCGCCCAT GACGGGAGGA CGGCTGCCCT ATCCATGTTC CATGTCTCCA TGAGCTGCAT CTTGGGCTTG GTGCTGCCCC GGTGGCGCTG GGGCCTGCCA NTGCCTGCAG CTTCGGGGGC TGGCAGGGGG CCGAGTCCTG CTAGCAGGGA TCCTGGCCCG GGTGCTGAAT TCTGTGGCCT CCCCAGAGGA CGTCCAGGCC CAGTGGAAGA TGTTGCAGTG CCATCCTCAC CATTTACACC GCAGAAATGA CACCGCACGC AGCCCACGGC TCCCTCGACT CTGGGGCACG
CCAGCAGCAA GATGTGACCG YTGTGCCGAT GCCTCCACCT CTGCTGCCTG GGGGTCCAGT CCTCCTGGAC CAGCCGTGCC TCTGCCCCGC AACGCCGGAT ATCACATTGG TTCTGCCCCC AGGGAGGAGA CAGAAGCCTG GGCCAGGTGA CTGCAGCAGC CACCCTGGAT GTGGCTGTTC TGCTGTGCGT GGCCCTGGGA CAGCTGGACC GTCTGTGGCT GAACATCAGG GGCAAGGAGG CGCCACTGCC AGTGATGACC GGTGGTTTCC TGGCCTATGN TTCCAGCCTG ACCTGGTGCT GGCCCCCACG CTGCACTCCT GGCTGCAATG GCCCTCCTGG AGGAGAACTC CACACCCCAG GGAGAGGCAC CTCCTAGCCT AGGCCCTTCC CTGATGTACC TGAGAGGGCA GCTGGAGCCT CTGGTGGCTT GAAATCGGCC AAGGTGGGAG CAGCGCCCCG CGGCCGCGAT CCGGACCCCA GAACCCCGCC CACTCCCAAT CarregGCGCCC
120
180
240
300
360
420
480
540
600
660
720
780
840
900
960
1020
216
CGCCCTCTCC
CACCCGTGCT
TCCCCCGCTC
CACCCCTCAC
CTCACCTCGC
CCCSGCCCCA
1080
CCCATCGCGC
CCCGGCCCGT
CCCATCGAGG
CCCATGCAAC
CCACGCTCGG
TYCCGTTCCG
1140
GCCCCTGCGC
TCKCGCTKNS
TTCGCTCCCC
GCCCTTGCGC
CGTTAGTAAA
CATCGCTCAA
1200
ACGAAAAAAA AAAAAAAAAA
AAACTCGA
1228 (2) INFORMATION FOR SEQ ID NO: 26:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1340 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: double (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 26:
AATTCGGCAG AGAGATGGCC GCCCCCGTGG TTCAAGCCAA AGTTATTGAC ACTCAACAGA AGCTAAACAG AACGAAAAAG CATGCACATC ATGAGACTAA CATGTATGAA GGTGTAGGAA TTCACAGTCA GCTGTTAGAG AAGCAGAAAA AGAAAAAGTC CTACCTGGAG CGACGTTAAA GATGGCACGA AGGGCCCAGT AGGGAGCCTC TTCCTGGTGG GGGCAGAGGA GTGTCTGCAG GCTTTATTTG GATGGCCTGG CAACATCACA TCCCAGCCCT GGAGTTTTTA CCCGGCTTTG CTCGGGATGT GTGATGAACT CCCAGGAGAG CTCTGCCTGA GCTTTCCACT AGGCACGCCA CTGCTAAGCC TGGTTTTCTT GAGCAGAGGG
ATCTAGAGCT GAAGAAGGCC TTCACAGAGC60
AGGTGAAGCT CGCAGACATA CAGATTGAAC120
TTACAGATAC AGAGATCATG ACTTTGGTAG180
GAATGTTTAT TCTTCAGTCC AAGGAAGCAA240
TAGCAGAAGA AAAAATTAAA GAACTAGAAC300
GGAAGCTGAG GACAACATCC GGGAGATGCT360
TCTGGGAAGC TCTTCCTCCT GCCCCTCCCA420
GGAAACAGCT TCTCCTCTGC CCCGATGGAT480
TTTTCTGCAT CACCCTGAGC CCCATTTGCT540
CTGCCACCTC TGCCCAGGAC ACKCTTCCCT600
GGAAGATGGG AGCCAGGGCA AGATAGGAAG660
GCCAGACCAA TAAAAAGCGT CTGTCCCACT720
ATGGAACAGA GGGTGAGAGA GGCAGTGGCC780
217
GTCTCCACCT CAGCTCCTGC TCCCTCTGCA TCAGAGCCCT TCCTTTCTTG GGGGATGGGC 840 CTTGCCNTCT TCTCTTTTCC CTTCCTGTAC CTTTGACTAA CGCTCAGCTT CCGGGCCTGC 900 ATGCAGTAGA CAGAAGAGGA AGAAAGAACA GATGTTCACA GCTGAATCTC AGTGAACAGA 960 ATAGCAGTCC CTGGATGGCA GTCTGCCTAA AGATTCCTTT CCCTGCCTTC TCCCATACAT 1020 TCCAAAAGGA AGTTCAACAG TAAGCAGCAC CTCCAAGACT GTCTCCTTTY GGCCARTATC 1080 ATAAGATGGA CGCCATAATC CTGAGGCCTC CTAGAGGCTG AGGGGGCAAC GGTGTGATCC 1140 AGCTGGCTCA TCCCAGCCAG GTGGGCCAAT TATTCAATTT TCAAGAATTT 1200 TGTTGCAAGC CAGTTGTCAA ACACAGCCAT TATAATTATG TAAATTTGCA AATTATGTTA AAAACAAGGA 1260 CAATAAATAT TCAAAATGCA TCCCTAAWWA AAAAAAAAAA AANGGGNGGC CGCNCTAGGG 1320 GATCCACG ID: 1340 GACCCC ID: 40
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 806 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: double (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 27:
ACCTTCTTCC ATGTTTAGTC CCTTGGGCTC TGCTACCCTC CTGCTGGAGG TGAGAGCATC 60 CTGTGTCCAA CCAGAGATGC CCTCTGGCTT TCAGACCTGC CTCCTTTTCA CCCTCAGCCC 120 TTTCTCACTC AGCAAAATTG TGGGGGTCCC TAGTCAGCAG CTCCCTGGGC AGCTCTCTGA 180 GCAAGGTGGT CTCTGTGGTC ATGAAGGAGA GCCGGCTAGG ACAGTGCCGG AAACTCAGCT 240 GCCTCTCCCC TTCAACTCAG CTGGCCCCCC GCACCTGAAG TGCACAGGAG CCGGGAAGAG 300 AGTCTGGAGC CCACCCCGGA GGGCAGCACA GGAGGTGTCT 360 CTGCAGCTGG TGTCCTGCCA CCCCTGCAGG CAGCACACGT CCCGGGCATT CTCCTTAGCC ACAGACAGAA 420 CAGCCAGTGC
218
CAGAGTCTGC TGTCGTTCCC CTTTAAGCAC ACTCATTCAC CACACCCGAG GAGGCCAGAG 480
GTGCAGGGAG CATGGGCTGT CGCTTCCCCT TTAAGCACAC TCATTCACCA CACCCGAGGA 540 GGCCAGAAGT GCAGGGAGCA TGGGCTGGGT GCACCTCCGC AGGAGAGAAG GCTGAGCCAC 600 CGCCGTCCCG GGAGCCCGGC TCCCAGGCCT CTCGTTTTCC CCTACCTCCC TAAGACTTTT 660 CTGTCACTCT CTGGCCATTG AAAGGCTTCT GTTCCTTAAA GTGCTGTTAC ACTCTCCTTT 720 CCCAGGATGC AGCAAGCCAA AACAGTACCA CTGCACGTCA GCCTGGGTGA CAGAGTGAGA 780 CCCTATCTTA AAAAAAAAAA ΑΑΑΆΑΑ 806 (2) INFORMATION FOR SEQ ID NO: 28 :
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 696 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: double (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 28:
GAGTTCCCNA CGCGGTGGCG NCCGTTTTAG AAATTAGTGG
ATCCCCCCGG GCTGGCAGGG
AATTCGGCAC GAGCACAGAG GAAAGCGGGT GCCCGGCATG
GCCATCCTGA TGTTGCTGGC
120
GGGATCCCCA TGCACCTTGT CCTTCTCCAC TGATACTGGC AGCTCGGCTC CTGGACCCAA
180
GATCCCTTGA GTGGAATTCT GCAGTGCAAG AGCCCTTCGT GGGAGCTGTC CCATCTTTCC
240
ATGGTCCCCA GTCTCCCCTC CACTTGGTGG GGTCACCAAC
TACTCACCAG AAGGGGGCTT
300
ACCAAGAAAG CCCTAAAAAG CTGTTGACTT ATCTGCGCTT GTTCCAACTC
TTATGCCCCC
360
AACCTGCCCT ACCACCACCA CGCGCTCAGC
CTGATGTGTT TACATGGTAC TGTATGTATG
420
GGAGAGCAGA CTGCACCCTC CAGCAACAAC AGATGAAAGC
CAGTGAGCCT ACTAACCGTG
480
CCATCTTGCA AACTACACTT TAAAAAAAAC
TCATTGCTTT GTATTCTAGT AACCAATATG
540
TGCAGTATAC GTTGAATGTA TATGAACATA CTTTCCTATT TCTGTTCTTT
GAAAATGTCA
600
219
GAAATATTTT TTTCTTTCTC ATTTTATGTT GAACTAAAAA GGA1TAAAAA AAAAATCTCC
660
AGAMAAAAAA AAAAAAAAAA AAATTACTGC ggtccg
696 (2) INFORMATION FOR SEQ ID NO: 29:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1007 base pairs (B) TYPE: nucleic acid (C) chain type: double (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 29:
AATTCGGCAC GAGGAAAAAA TACCATTTGT GTATGATACC CAATTTGGAT CTCAATTTGG 60 ATAGAGATTT GGTGCTTCCA GATGTRAGTT ATCAGGTGGA ATCCAGTGAG GAGGATCAGT 120 CTCAGACTAT GGATCCTCAA GGACAAACTC TGCTGCTTTT TCTCTTTGTG GATTTCCACA 180 GTGCATTTCC AGTCCAGCAA ATGGAAATCT GGGGAGTCTA TACTTTGCTC ACAACTCATC 240 TCAATGCCAT CCTTGTGGAG AGCCACAGTG TAGTGCAAGG TTCCATCCAA TTCACTGTGG 300 ACAAGGTCTT GGAGCAACAT CACCAGGCTG CCAAGGCTCA GCAGAAACTA CAGGCCTCAC 360 TCTCAGTGGC TGTGAACTCC ATCATGAGTA TTCTGACTGG AAGCACTAGG AGCAGCTTCC 420 GAAAGATGTG TCTCCAGACC CTTCAAGCAG CTGACACACA AGAGTTCAGG ACCAAACTGC 480 ACAAAGTATT TCGTGAGATC ACCCAACACC AATTTCTTCA CCACTGCTCA TGTGAGGTGA 540 AGCAGCTAAC CCTAGAAAAA AAGGACTCAG CCCAGGGCAC TGAGGACGCA CCTGATAACA 600 GCAGCCTGGA GCTCCTAGCA GATACCAGCG GGCAAGCAGA AAACAAGAGG CTCAAGAGGG 660 GCAGCCCCCG CATAGAGGAG ATGCGAGCTC TGCGCTCTGC CAGGGCCCCG AGCCCGTCAG 720 AGGCCGCCCC GCGCCGCCCG GAAGCCACCG CGGCCCCCCT CACTCCTAGA GGAAGGGAGC 780 ACCGCGAGGC TCACGGCAGG GCCCTGGCGC CGGGCAGGGC GAGCCTCGGA 840 AGCCGCCTGG
220
AGGACGTCCT GTGGCTGCAG GAGGTCTCCA ACCTGTCAGA GTGGCTGAGT CCCAGCCCTG
GGCCCTGAGC CGGGTCCCCT TNCGCÀAGCG CCCACCGATC CGGARGCTGC GGGCAGCCGT
TATCCCGTGG TTTAATAAAG TGCCGCGCGC TCACCAAAAA AAAAAAA (2) INFORMATION FOR SEQ ID NO: 30:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2017 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: double (D) TOPOLOGY: linear
900
960
1007 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 30:
AATTCGGCAC GAGCGGATCC GTTGCGGCTG CCGCCAGGGG TAGCGGTGTA GCTGCGCACG CGTAGCGTCT GGCAGCCCGG CGCCATCTTC GGGAGCGGCG GGTACTGCTT GCTCCTCGGC GCCTGGGCTG GAACGACCCT GACAGAATGT ACTATGACCG CTATACCACC TCCCGCAGCT AGGCACAGCT GGTTGTGATT CTTATACCCC GGATGGGTAT GATGTACAGT GGGAATGTAA AAAAACTGTG GTGAGCTGTG AAGGCTATGA TTCTTGTGGC TTGGAGTATA ATTTAGATTA GTCTGGAAAG CAGCACGGCT TTGCCTCTTT GGATTCCTGT AACATGAGTG GATTGATTAC AGTCTATAAG CTGTTCCTGA GTGACGGGCA
CAGCTCTGCA GTCGGGCCGT TCCTTCGCCG TCGCGCGCGC TACCGCACCC AGGTTCGGCC ATCGAGCGCC ATGGCCGCAG CCTGCGGGCC TTGCATTTGT TTCTGCTGAC CGCGGGCCCT TGCTGCGGGA TGTAAAAGCT CTTACCCTCC GGATCCCATC CCACAGTTGA AATGTGTTGG AAAAGTCATA CAGTGTCAGA ACAAAGGCTG GACGGACTTA GATATTGCAT ACAAATTTGG GTCCTCTGAA GACCAGTATG TACTAAGAGG TACAGAACTT GGCCTGCAGA AACTGAAGGA CTCTGATTAT TATTATAAGT GGTCCTCGGC CATCGTGGTA CTCCTTGGGA TCGCCTTTGT GTATTCTCCT CCACCGTACT CTGAGTATCC
120
180
240
300
360
420
480
540
600
660
720
780
221
TCCATTTTCC CACCGTTACC AGAGATTCAC CAACTCAGCA GGACCTCCTCLOADAGGCTT840
TAAGTCTGAG TTCACAGGAC CACAGAATAC TGGCCATGGT GCAACTTCTG GTTTTGGCAG900
TGCTTTTACA GGACAACAAG GATATGAAAA TTCAGGACCA GGGTTCTGGA CAGGCTTGGG960
AACTGGTGGA ATACTAGGAT ATTTGTTTGG CAGCAATAGA GCGGCAACAC CCTTCTCAGA1020
CTCGTGGTAC TACCCGTCCT ATCCTCCCTC CTACCCTGGC ACGTGGAATA GGGCTTACTC1080
ACCCCTTCAT GGAGGCTCGG GCAGCTATTC GGTATGTTCA AACTCAGACA CGAAAACCAG1140
AACTGCATCA GGATATGGTG GTACCAGGAG ACGATAAAGT AGAAAGTTGG AGTCAAACAC1200
TGGATGCAGA AATTTTGGAT TTTTCATCAC TTTCTCTTTA GAAAAAAAGT ACTACCTGTT1260
AACAATTGGG AAAAGGGGAT ATTCAAAAOT TCTGTGGTGT TATGTCCAGT GTAGCITTIT1320
GTATTCTATT ATTTGAGGCT AAAAGTTGAT GTGTGACAAA ATACTTATGT GTTGTATGTC1380
AGTGTAACAT GCAGATGTAT ATTGCAGTTT TTGAAAGTGA TCATTACTGT GGAATGCTAA1440
AAATACATTA ATTTCTAAAA CCTGTGATGC CCTAAGAAGC ATTAAGAATG AAGGTGTTGT1500
ACTAATAGAA ACTAAGTACA GAAAATTTCA GTTTTAGGTG GTTGTAGCTG ATGAGTTATT1560
ACCTCATAGA GACTATAATA TTCTATTTGG TATTATATTA TTTGATGTTT GCTGTTCTTC1620
AAACATTTAA ATCAAGCTTT GGACTAATTA TGCTAATTTG TGAGTTCTGA TCACTTTTGA1680
GCTCTGAAGC TTTGAATCAT TCAGTGGTGG AGATGGCCTT CTGGTAACTG AATATTACCT1740
TCTGTAGGAA AAGGTGGAAA ATAAGCATCT AGAAGGTTGT TGTGAATGAC TCTGTGCTGG1800
CAAAAATGCT TGAAACCTCT ATATTTCTTT CGTTCATAAG AGGTAAAGGT CAAATTTTTC1860
AACAAAAGTC TTTTAATAAC AAAAGCATGC AGTTCTCTGT GAAATCTCAA ATATTGTTGT1920
AATAGTCTGT TTCAATCTTA AAAAGAATCA ATAAAAACAA ACAAGGGAAA AAAAAAAAA1980
AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAA2017 (2) INFORMATION FOR SEQ ID NO: 31:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 699 bp (B) TYPE: nucleic acid (C) CHAIN TYPE: double
222 (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 31:
GNGTTTTTTC CAGCCAGGAA GTGACCGNTA CTGCAGCACG AGANAGATTG GTTGGGTTGG60
TTGRAAATGA CYCTGAACAT TTATTTCCAT TGCAATTTCT GTGGCTGAGG AGACTTAAAC120
TTTACAAGTA TTATCCTTTT AAGATCATTT TAATTTTAGT TGAGTGCAGA GGGCTTTTAT180
AACAAACGTG CAGAAATTTT GGAGGGCTGT GATTTTTCCA GTATTAAACA TGCATGCATT240
AATCTTGCAG TTTATTTTCT CATTGTGTAT GTATATATCG CTTTTCTCTG CAGCACGATT300
TCTCTTTTGA TAAWKCCCTT TAGGGCACAA CTAGTTATCA GTAACTGAAT GTATCTTAAT360
CATTATGGCT GCTTCTGTTT TTTCATTAAC AAAGGTTATT CATATGTTAG CATATAGTTT420
CTTTGCACCC ACTATTTATG TCTGAATCAT TTGTCACAAG AGAGTGTGTG CTGATGAGAT480
TGTAAGTTTG TGTGTTTAAA CTTTrTTTTG AGCGAGGGAA GAAAAAGCTG TATGCATTTC540
ATTGCTGTCT ACAGGTTTCT TTCAGATTAT GTTCATGGGT TTGTGTGTAT ACAATATGAA600
GAATGATCTG AAGTAATTGT GCTGTATTTA TGTTTATTCA CCAGTCTTTG ATTAAATAAA660
AAGGAAAACC AGAAAAAAAA AAAAAAAAA AAAAAAAAA699 (2) INFORMATION FOR SEQ ID NO: 32:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1264 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: double (D) TOPOLOGY: linear
223 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 32:
<td>GGCACGAGGG CACTGTTTCC TCAGTCCATG GCTGAGTACA TCACCGGTGT TTTCTCTCTT</td><td> 60</td>
<td>A1TCCTCCCA TCAAGCCTAA AAGGAATCTC TA1TGGAGAT ACTGCCATTA GTGTTCCTTT</td><td> 120</td>
<td>TATAGGTGAG GAACTGAGGC ATAKAGGGTT TRANSAGTTGA ACCAACTGAT AAATAGTAGA</td><td> 180</td>
<td>ACTTGGATTT TAATTCAGTC TTGATGCCAG GGATAAGGCT CTTACTTTCT ACCTTAGGCT</td><td> 240</td>
<td>AITTCTAGGA AACGCAGGAG AGTGTTGAAG GGGCAGAGAA AGGGATCCAG TTCCTTTCTG</td><td> 300</td>
<td>TCCCGCATCC TAGTCCCTGA GAAGCAAAGA ARAATGTGTG GCTTCTTTTG CTTTGCTTTT</td><td> 360</td>
<td>GTTGTCATCC CACACATCTC CAGGGGAMCT GGGCTCTTGA TCTTGGSCTC TTCCCCTTTA</td><td> 420</td>
<td>ACTGTTAAGT GGGAGCARGT AAGGGGGTAC AGTAGGGCTG GCCTGGAGTT AGAGGCTTGG</td><td> 480</td>
<td>ATGCCTTAGC TCCTCTGTCT GCACTCCAGA ACTGCCTGAC TTCATTTCGT ATGTTGTCCT</td><td> 540</td>
<td>TTGTTTTGAC AATTGATCCA TGTCCCAGTC CGTCTCTTCT TCCTTCTTGA TACTTACACT</td><td> 600</td>
<td>GCTTCTTTCT GTTGGTTTCC AGTGTTTAAC ACTGTATACA ACAGTGACGA CAACGTGTTT</td><td> 660</td>
<td>GTGGGGGCCC CCACGGGCAG CGGGAAGACT ATTTGTGCAG AGTTTGCCAT CCTGCGAATG</td><td> 720</td>
<td>CTGCTGCAGA GCTCGGAGGG GCGCTGTGYS WCWTCACCM CCATGGAGGC CCTGGCCAGA</td><td> 780</td>
<td>RCAGGTATGA CGTGGCGCTG TGTCATGTGA ATTTCCCAAG AAGCAT1TCA TCTGTGATTC</td><td> 840</td>
<td>CGTATGAAGG CTTTCTAAGC CCTGAAATTT GCAGGGTCAT TTCCTCAGTT TGTGTATTAA</td><td> 900</td>
<td>AGAAAAGCTG ACCEAGCCAA GCGTGGTGGC TCACGCCTGT AATCCCAGCA CTTTGGGAGG</td><td> 960</td>
<td>CCGAGGCGGG CAGATCTCCG GAGATCAGGA GTTCGAGACC AGCCTGGCCA ACATGGTGRA</td><td> 1020</td>
<td>ACCCTGTCTC TACTAAAAWT ACAGAAATTA GCTGGGNGTG GTGGTGTGCG CCTGTAATCC</td><td> 1080</td>
<td>CAGCTACTTG GAAGGCTGAG GCAGGAGAAT CGCTTGAACC CGGGAGGCGG AGGTTGCAGT</td><td> 1140</td>
<td>GAGCCAAGTT CGCACCACTG CACTCCAGCC TGGGCAACAA GAGCGAGACT TCATCTCAAA</td><td> 1200</td>
<td>AAAAAAAAAA AAAAACTCGA GGGGGGGCCC GGTACCCAAT TCGCCCTATA GTGATCGTAT</td><td> 1260</td>
TACA 1264 (2) INFORMATION FOR SEQ ID NO: 33:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 997 base pairs
224 (B) TYPE: nucleic acid (C) CHAIN TYPE: double (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 33:
<td>attggaagtt</td><td>GTTTTGCAAC</td><td>CTGGGCTTTT</td><td>ATACAGAAGA</td><td>ATACGAATCA</td><td>CAGGTGTGTG</td><td> 60</td>
<td>agcatctact</td><td>ΤΑΑΤΓΑΑΤΤΤ</td><td>GCTTACAGCC</td><td>GATTTCCTGC</td><td>TTACTCTGGC</td><td>ATTACCAGTG</td><td> 120</td>
<td>aaaattgttg</td><td>TTGACTTGGG</td><td>TGTGGCACCT</td><td>TGGAAGCTGA</td><td>AGATATTCCA</td><td>CTGCCAAGTA</td><td> 180</td>
<td>ACAGCCTGCC</td><td>TCATCTATAT</td><td>CAATATGTAT</td><td>TTATCAATTA</td><td>TCTTCTTAGC</td><td>ATTTGTCAGC</td><td> 240</td>
<td>attgaccgct</td><td>GTCTTCAGCT</td><td>GACACACAGC</td><td>TGCAAGATCT</td><td>ACCGAATACA</td><td>AGAACCCGGA</td><td> 300</td>
<td>TTTGCCAAAA</td><td>TGATATCAAC</td><td>CGTTGTGTGG</td><td>CTAATGGTCC</td><td>TTCTTATAAT</td><td>GGTGCCAAAT</td><td> 360</td>
<td>ATGATGATTC</td><td>CCATCAAAGA</td><td>CATCAAGGAA</td><td>AAGTCAAATG</td><td>TGGGTTGTAT</td><td>GGAGTTTAAA</td><td> 420</td>
<td>AAGGAATTTG</td><td>GAAGAAATTG</td><td>GCATTTCCTG</td><td>ACAAATTTCA</td><td>TATGTGTAGC</td><td>AATATTTTTA</td><td> 480</td>
<td>AATTTCTCAG</td><td>CCATCATTTT</td><td>AATATCCAAT</td><td>TGCCTTGTAA</td><td>TTCGACAGCT</td><td>CTACAGAAAC</td><td> 540</td>
<td>AAAGATAATG</td><td>AAAATTACCC</td><td>AAATGTGAAA</td><td>AAGGCTCTCA</td><td>TCAACATACT</td><td>TTTAGTGACC</td><td> 600</td>
<td>ACGGGCTACA</td><td>TCATATGCTT</td><td>TGTTCCTTAC</td><td>CACATTGTCC</td><td>GAATCCCGTA</td><td>TACCCTCAGC</td><td> 660</td>
<td>CAGACAGAAG</td><td>TCATAACTGA</td><td>TTGCTCAACC</td><td>AGGATTTCAC</td><td>TCTTCAAAGC</td><td>CAAAGAGGCT</td><td> 720</td>
<td>ACACTGCTCC</td><td>TGGCTGTGTC</td><td>GAACCTGTGC</td><td>TTTGATCCTA</td><td>TCCTGTACTA</td><td>TCACCTCTCA</td><td> 780</td>
<td>AAAGCATTCC</td><td>GCTCAAAGGT</td><td>CACTGAGACT</td><td>TTTGCCTCMC</td><td>CTAAAGAGAC</td><td>CAAGGTYAGA</td><td> 840</td>
<td>AAGAAAAATT</td><td>AAGANGTGGA</td><td>AATAATGGCT</td><td>AAAAGACAGG</td><td>NTTTTTGTGG</td><td>TACCAATTCT</td><td> 900</td>
<td>GGGCTTTATG</td><td>GGACCNTAAA</td><td>G1TATTATAG</td><td>CTTGGAAGGT</td><td>AAAAAAAAAA</td><td>AAAGGGNGGG</td><td> 960</td>
<td>CGCTCTAGAG</td><td>GTTCCCCGAG</td><td>GGGCCAGCTT</td><td>AGGGTGC</td><td></td><td></td><td> 997</td>
(2) INFORMATION FOR SEQ ID NO: 34:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1914 base pairs (B) TYPE: nucleic acid
225 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 34:
(C) CHAIN TYPE: double (D) TOPOLOGY: linear
GTGTGAGAGG CCTCTCTGGA AGTTGTCCCG GACGAGGTGC CGCTGCCTGG AGAATCCTCC TCCTAACCCA ACCCAACCTA GCCCAGTCCC CCAGCGTTAC CATGCATCCT GCCGTCTTCC TGCTCCTGGT AACTTGGGTT TTTACTCCTG agaatataga tgaaatttta AACAATGCTG GGTGTCGTTT CAGTCAGATG TTGCATCCAA AAGAATTTCC AAATGAAAAT CAAGTAGTGT ACATAGCCCA GAGATACAGG ATAAGCAAAT TGATGATGAA GAGAGAATAC AGGGGTCAGC GGCAACAAAA AAGTGACCCC ATTCAAGAAA ATCCCAGCAA AAGAAATATC ATTGGATATT TTTTTGAACG AGTAGCGAAT ATTTTGCATG ATGTTTCAAA ACCGGAAAGA TATAGTGGCG CTGCTCCGGA TATGGTGTAC TTGGGAGCTA TTCAAGATAA ATGTGTTCCT CTTGTCCGAG CAGAAGAAGG ACTGCCTTTT CTCATACTCT AAATATTCCA GAATGAAGTA GCTCGGCAAT TACATCCCGA TTGTGACAAA TTTAGACATC ATTGTCCTGT AATCGCTATTACATTACTACTACTACTACTACTACTATTACATTACTATTAC
GGTGTTCGCC GCTGGAGCCC GGGTCGAGAG GCTGCCGTCG GCTCCCGGAG CCCAGCCCTT AGCCGCCAGC GCCTGTCCCT GTCACGGACC TATCCTTACC CGACCTCAGA TGCTCCCTTC TAACAACTGA AATAACAAGT CTTGATACAG ATGTTGCTTT AGTAAATTTT TATGCTGACT TTTTTGAGGA AGCTTCCGAT GTCATTAAGG TTGCCAGAGT TGATTGTGAT CAGCACTCTG ACCCAACCCT CÀAATTGTTT CGTAATGGGA GATCAGTGAA AGCATTGGCA GATTACATCA TTCGGGACTT AGCAGAAATC ACCACTCTTG TTGAGCAAAA GGACTCGGAC AACTATAGAG ATGACTGTGC CTTTCTTTCT GCATTTGGGG ACAACATAAT CTACAAACCA CCAGGGCATT TGACAAATTT TGATGTGACT TACAATTGGA AAATAACATT TGAAAATGGA GAGGAATTGA TTCACATGAA AGAAGATACA GAAAGTTTAG TAATAAGTGA AAAAGGTACA ATAAACTTTT CTCTTCTGCA CATACAGAAA ACTCCAGCAG GGCATATGTA TGTGTTTGGA CTCTTAG CTTACTACTA CTTACTACTA CTTACTACTA
120
180
240
300
360
420
480
540
600
660
720
780
840
900
960 1020 1080 1140 1200 1260 1320
226
ATGTAGCAAG
CAGTCCACCT
GAGAGCTCCT
TCCAGAAACT AGCACCCAGT
ATACTCTATT
GAGGGATCGA
GATGAGCTTT
AAAAACTTGA AAAACAGTTT
CAACAGCAGC
ATCAACCTAC
GTGGTGGAAA
TAGTAAACCT
GTATTTTTAT
TTTGAATAAA
CAGAAAGAAA
TTTTGGGTTT
TCAAAATGCA
TTGTCATTTA
ATATAGTAGC
CTCTTAAAAA
TTAAAAATAA
AAATCAGAGG
CCTATCTCCA
CTTTAAATCT
ATCAAATGAA
AGGTGACATT
GCCAGAAACT
TACCATTAAC
GGACTTAGGG
ATGTTTCCTG
TGTCGTATGT
GCTTTTCTTT
TTGGTATTTT
CAGTATCTCA
TTTCTCAAAG
CTAAAGAGAT
AGGGGAATAA
ATTAAAGTTT
TCACACTGNA AAAAAAAAAA
ATATTTTCAT
ΤΤΑΑΤΓΤΤΤΤ
AAAAAAAAAC
GTCCTGTAAA
TTGCACTACT
CTTTCATATG
ATACATTCTG
AAAAAAAAAC
<td>GAATATAGGT</td><td> 1380</td>
<td>GTAAGCCTTT</td><td> 1440</td>
<td>AATTCTATGT</td><td> 1500</td>
<td>TCTCCCCGAC</td><td> 1560</td>
<td>CTGCTAGGAT</td><td> 1620</td>
<td>AGTTTTATAA</td><td> 1680</td>
<td>AGGGTAGGGA</td><td> 1740</td>
<td>ATCAATTCTG</td><td> 1800</td>
<td>GATACTTGGG</td><td> 1860</td>
<td>TCGA</td><td> 1914</td>
(2) INFORMATION FOR SEQ ID NO: 35:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1020 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: double (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 35:
CCOTTNNTTT TTTTTTTTTG CAAGACAAAA ΤΑΤΑΟΓΓΓΑΤ TGTGACAGCA AATGCACATA 60 GTGCTGTAGG TAAGGCATGC TACTAGGAAT CTGCATATAA TCAAAAGCCA GTATGGAAAT 120 GAATGGAAAT GAATGCTGTT GTTCTCAGAT TGAGTCCATG GTGGAGAAAG GATAGTTTGT 180 GTCCACTTAT TTCAAATGCA GTATCATACC TACTTAATCA GTTACCTATG CTTCTAACCA 240 ACAGCCCAGT GGCAAATAGG AGGAACTTAA CTGTACTCAG AAGTCACTTT TAATATCAAC 300 GACAGAAATA TTTCACTAAT TCAACTGAGG CAAATTTCCT TTCTAGACAA AGGACCTAGA 360 AATTGAGCAT GCAAAACATC CATCCATTCA TTCATTCAAA TAATTAGCCA ATTTTACCGT 420 CATTTAATTC CACCAGAAGC AAATACTAGA ATATCTAGAA GTAGTTTGGG TAAAGAAACA 480
227
TTTACATTTT TTGATCCCTT AGAGAAGAGC CTCTACAGAC AAAGCAGTAT AATGTTTTTT TGCGGGTTTC GTTTAAGAGA TTTATAAGGT
AATATTGTGT TGTATGTGAG TGAAAAAAAT TTGTCAAAAA TTTTAGACAA TGCATGAATG AACAGTATGC TGAAAAAAAA GTTTTTARGC
AATGTCATAA GGTACAAAGT GCTAAATAAG ATCAATGCAA GTTGCTTCAT CACATTGACA TTTCATTTAA CTTTACTACT AACACAGGAC
ATTTGGGGCT
ACAGTTTTCG AGATCTAGGC AACTGAGGGG 1TCCCCCTTT TTCTGTTCAA ACAAAGAATA ATGAAAATTG CGGTNGAACC
AAAATAACAC
TTTCAACAGC CTTTGATGGA GAAAGGCTGA TCTAAAACAG CTGTTTTCTA 1TATATGCAT CTTATCAAAT GANCAAATTT
CAGGTCAAAT TGAACTTCTG AACTATTAGG AATGCTTTGT ATGCAGATTA AATGCAACAC GGTCAATTTA ACTCTCCTCT ATAATTATAC
540
600
660
720
780
840
900
960
1020 (2) INFORMATION FOR SEQ ID NO: 36:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 781 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: double (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 36:
AACTCCTGAC CTCAAGTGCT CCACCTGCGT RAGCCACTGC GCCTGGCTGA TCCCAGCACT TGGCCTACTT TGAACAGCAA ACTTGTTGCT CCAGCTTCAA GCAGGGTGTG AATTGGCCAG TGTGGCTTTC AGCTGCGGGCCA GCGTGACGTGCCTGTCTGTC
TGGCTTCCCA AAGTGCTGGG ATACAGGAGT TTTMAAATGA TGCCGCTCAA AGCCGTGACT GCTGTTGTCA ACCTGAAGGC CTCTCAAATG TGTCAGATCT CAGGAGTCCT GTGTTGAGAG GGTGGGGAAA GCCAGGCGGTC TAG GACGACT TAGGAGGAGGAGGAGT
120
180
240
300
360
420
228
GGGTGGGGCC AGAGGGACCA
ATGGTATCTG CATATTAGCC AGCCCAAGAG CTGTCACTGC ATAGACTGAA GCCAGAACAG ATTTATGAGA CTTCTTAGTC TCCCAGCATT TTGGGAAGCC
GTGCCCTCCT CAGTGCTTAG CCTCTCCACC TTCTTTCTCC TTCTTTCTCC CTGGGAAGAA TGCCACACCC TCGCCTTAAT AAATATGAGG GAGGTTGGAT GAGGTGGGAG GATCCCITGA
GGGCAGAGCC ACCTGCAGCA CGCTGAATCA TTTCCCTCAA TGCGTGGACT CTGCCTGGTG TCCTTGCTAG GTGTTCTCAG GTGGTGGCTT GTGCCTGTAA AGCCAGGAGT TTGAGACAAG
480
540
600
660
720
780
781 (2) INFORMATION FOR SEQ ID NO: 37:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 966 bp (B) TYPE: nucleic acid (C) CHAIN TYPE: double (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 37:
GGCACGAGGA AGCAGCTGGG GGCTGATCAG TGCCTATTTG AGTGGCAGCT CCTCTTGAAA AAAGGGCACT GCCCTTCCCA GAACTTGAGC ACAGCTTGGG GCTGAGGAAC AAAGAGCCTT TGGGCAGG AG CCCCTGTTCT GAAGGGTCCC AGAACCACTC TAAAGAAGAA CAGAGAGACG GGCACACCTG GGCGTACAGG NGAGGGGCGC GTGGTGGGGA AAGGAGCCCC CCAAAGTGTC CGGGCCACTG CTGGGATCAG TGGTGCCATG GGAAGCGTGC CCAAATCTTT SCATCACCAT
GGGGAGCACG CAGCCCTCCG ATTGCAGGGC CAATGCAGAA CAAGCCCAGG GCCCCACAGA TCCGGGACTA CGCATCTGTT CTCACCAGAT CCCTGGGCCA CAGGTGGGGG ACCCAGAAGC AGGGCCACAC CACAGATGCT GCTGACGTGC CAGGAGCACA GAGGARGTGC GGCCAGGGGA AGGACACTTC GAGGCTGACA GGAGACCCAC AGAGCATGAA GCAGCAGGAA GGAGCTCCCT GAGCAAGCGA GGTTGTTTGG CCTGAAAGCC GTAGGCAGTC ACCTCGCTCC TCAGCACTCG
120
180
240
300
360
420
480
540
600
229
GGGCAGGACA GAAGCTTGCT GTCTGCTCAC CAGACATCCT GTGCTGCCCT ACAGACACCT660
TGCTCGCCAG CCATCCCCAC TCACTTCTGA CCGGGACCCA ATTCTCTGGC CAAACCCAGG720
CTCTAGCACC GTCTTGGTGT GCTTGAGAAA CATCTAGTTT AAGTCAAAAT CCAATGTCTT780
TTTAATATAT AGACTATATG TACCTATGGA CTAGAGGTGA ATATATATAC ATCATATCAA840
ATTCAAGTGA CCCAGTATTT CGGGAGAACC CACTATGTCC CCAGCCTGCA TGGGAAGCTG900
GGGATTCTGG CATGAACTGC ACCTTATCTT CCTCGAGGGG GGGCCGGTAC CAATTGCCNA960
TAGTGG966 (2) INFORMATION FOR SEQ ID NO: 38:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 416 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: double (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 38:
GAATTCGGCA CGAGGTAATA GGAGCCCTCG TACCTCTTGT GTTCCTTACA AACÀTTCTCA
TCAGTAGCTC TACGCGTTGA CTGGGTGGTT TCARATGGCT
GGTATACACA GGGCTTTCTT
120
GGTGTTCTGT CTCTGGGGCT TARCTTTGTG TGTGGTTGGA
GGGCCCTGGT GAGATTGGAA
180
GTACCAGAGA GTGCTGTGTC AGGGGCAGAG GGGCCTGTCG
CTGGAGCTGG AGGGTGCCTG
240
CCTTTGTGTC TGACTCARTC TCCTGTCTGC CTTGCCCCCT CAGGGTCTCG
CCAGCCCAGC
300
CTCTGTGGGA ATCTAAAAGG ARTGGATGTG GACGTKTGAC CAAGCACATC
TCAGCTTTTA
360
ATACCTGGGC TATTTATAGA CCTTTGGGGG GAATNGCTTG TGGAACAACA AGGGTT
416
230 (2) INFORMATION FOR SEQ ID NO: 39:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1114 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: double (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 39:
<td>TGTGTATTTG GGGGGACTGA AGGGTACGTG GGGCGAAACA AAACCGGCCA TGGCAGCAGC</td><td> 60</td>
<td>GGAGGAGGAG GACGGGGGCC CCGAAGCCAA AATCGCGAGC GGGGCGGGGC GGGCGCGACC</td><td> 120</td>
<td>TTCGAATGTA ATATATGTTT GGAGÀCTGCT CGGGAAGCTG TGGTCAGTGT GTGTGGCCAC</td><td> 180</td>
<td>CTGTACTGTT GGCCATGTCT TCATCAGTCG CTGGAGACAC GGCCAGAACG GCAAGAGTGT</td><td> 240</td>
<td>CCAGTATGTA AAGCTGGGAT CAGCAGAGAG AAGGTTGTCC CGCTTTATGG GCGAGGGAGC</td><td> 300</td>
<td>CAGAAGCCCC AGGATCCCAG ATTAAAAACT CCACCCCGCC CCCAGGGCCA GAGACCAGCT</td><td> 360</td>
<td>CCGGAGAGCA GAGGGGGATT CCAGCCATTT GGTGATACCG GGGGCTTCCA CTTCTCATTT</td><td> 420</td>
<td>ggtgttggtg cttttccctt tggctttttc accaccgtct tcaatgccca tgagcctttc</td><td> 480</td>
<td>CGCCGGGGTA CAGGTGTGGA TCTGGGACAG GGTCACCCAG CCTCCAGCTG GCAGGATTCC</td><td> 540</td>
<td>CTCTTCCTGT TTCTCGCCAT CTTCTTCTTT TTTTGGCTGC TCAGTATTTG AGCTATGTCT</td><td> 600</td>
<td>GCTTCCTGCC CACCTCCAGC CAGAGAAGAA TCAGTATTGA GGGTCCCTGC TGACCCITCC</td><td> 660</td>
<td>GTACTCCTGG ACCCCCTTGA ACCESSCCTATT TCTGHTGGCT AAGGCCAGCC CTGGACATTG</td><td> 720</td>
<td>TCCAGGAAGG CCTGGGGAGG AGGAGTGAAG TCTGTGCATA GATGGGAGAG CCTTCTGCTC</td><td> 780</td>
<td>AGAGGCTCAC TCAGTAACGT TGTTTAATTC TCTGCCCTGG GGAAGGAGGA TGGATTGAGA</td><td> 840</td>
<td>GAATGTCTTT CTCCTCTCCT AAGTCTTTGC TTTCCCTGAT TTCTTGATTT GATCTTCAAA</td><td> 900</td>
<td>GGTGGGCAAA GTTCCCTCTG ACTCTTCCCC CACTCCCCAT CTTACTGA1T ΤΑΑΤΓΤΑΑΤΤ</td><td> 960</td>
<td>TTTCACTCCC CAGAGTCTAA TATGGATTCT GACTCTTAAG TGCTTCCGCC CCCTCACTAC</td><td> 1020</td>
<td>CTCCTTTAAT ACAAATTCAA TAAAAAAGGT GAAATATAAA AAAAAAAAA AAAAAACYCG</td><td> 1080</td>
<td>GGGGGGGCCC CGGTCCCCAT TCCCTTTGGG GGGT</td><td> 1114</td>
231 (2) INFORMATION FOR SEQ ID NO: 40:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 602 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: double (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 40:
GGGTCGACCC ACGCGTCCGT CCCAGGCCAC AAGACATTTC CTGCTCGGAA CCTTGTTTAC60
TAATTGTCTC TGTGGCACAT TTTGTTTCCC GTGCCTTGGG TGTCAAGTTG CAGCTGATAT120
GAATGAATGC TGTCTGTGTG GAACAAGCGT CGCAATGAGG ACTCTCTACA GGACCCGATA180
TGGCATCCCT GGATCTATTT GTGATGACTA TATGGCAACT CTTTGCTGTC CTCATTGTAC240
TCTTTGCCAA ATCAAGAGAG ATATCAACAG AAGGAGAGCC ATGCGTACTT TCTAAAAACT300
GATGGTGAAA AGCTCTTACC GAAGCAACAA AATTCAGCAG ACACCTCTTC AGCTTGAGTT360
CTTCACCATC TTTTGCAACT GAAATATGAT GGATATGCTT AAGTACAACT GATGGCATGA420
AAAAAATCAA ATTTTTGATT TATTATAAAT GAATGTTGTC CCTGAACTTA GCTAAATGGT480
GCAACTTAGT TTCTCCTTGC TTTCATATTA TCGAATTTCC TGGCTTATAA ACTTTTTAAA540
TTACATTTGA AATATAAACC AAATGAAATA TTTTACTGAA AAAAAAAAA AAAAAANCCC600
CA602 (2) INFORMATION FOR SEQ ID NO: 41:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 970 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: double
232 (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 41:
GGCAGAGCTT AGGAGAACAG CTCCCTTTGG ATCCCTNTCA AAGGTGATAC CATTGGCTCC60
CAGCTTAGAG TAAGAAGCTC TGAGAAGTTG AATGAAGGGT GAGATAGAGA TGCTGAACCC120
ATTCTTSCAG CTTCTTCTAG TGTTGTTATT TCCAGAATGG CCAACACCCC TACATTGATA180
CATAAACACA TTCCAAGGCC TTGTGTAATA CAAAGTTCAC CGTCCTCCTG GAATAGGAGC240
CCTGGGTTCT AGTTCTCACT CTGCCACTGG GGGAAAATCC AATTAAAGTC TGGTTTAGTC300
AGCTTGGGTC ACCATAGACT GGGTGGCTTA AACAGCAGAC ATTTATTTCT GGTAGTTTCT360
GGAGGCTACA AATCTAAGAG CAAGGTGCCA GCATGGTCAC ATTCTGGTGA GGGSCCTCTT420
CCTGGCTTGT AGACGGCTGC YTTCTCACCG TGTGCTCACA TAGCCTTTCG TGTGTGTGTG480
TGTGTGTGTG TGCGTKCGTG CAAGCTTCCK GATGTCTCTT CTTAGAAGGA CACCAACCCC540
ATCATGAGAG CCCTACTCTC ATGACTTAGC CTAACCCTAA TTACCCTCCA AAGGCCCCAT600
CTCCAAATGC CATCACATTG GAGGGTAGAG CTTCAACATA GGGATTTTGG GGGACACAAA660
CATTCAGTCC ATAACAAAGG CTGTAGTCCT TARTTTCCTT GTCTGTGAAA TGAGAGTGTT720
GAGATTCTTT CTAGCCTTTA TCATTTATAA TTCTGTGAGA TGTAGATTTG CATTATTTTC780
GAGTTCGAGT TATATGAAAT GTTTCCCTCT ACATTTTCTT GGGCAACTGA GAACTGAATA840
GGGCTAGGTT TAAATAGAGT TAGGCAGTTA GGCTTATTCT TTTATTTAAT AAGCATTTTT900
GGAGCATCTA CGGTGTTCCA GGAACTGAAC TGTTGTAAAC ATTGGAGCTG TAACAGAGAA960
CAAAAGAGAC970 (2) INFORMATION FOR SEQ ID NO: 42:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1002 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: double (D) TOPOLOGY: linear
233 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 42:
<td>GAATTCGGCA</td><td>CGAGCCGAGG</td><td>TCGGCAGCAC</td><td>AGAGCTCTGG</td><td>AGATGAÀGAC</td><td>CCTGTTCCTG</td><td> 60</td>
<td>GGTGTCACGC</td><td>TCGGMCTGGC</td><td>CGCTGCCCTG</td><td>TCCTTMACCC</td><td>TGGRGGAGGA</td><td>GGATATCACA</td><td> 120</td>
<td>GGGACCTGGT</td><td>ACGTGAAGGC</td><td>CATGGTGGTC</td><td>GATAAGACTT</td><td>TCCGGAGACA</td><td>GGAGGCCCAG</td><td> 180</td>
<td>AAGGTGTCCC</td><td>CAGTGAAGGT</td><td>GACAGCCCTG</td><td>GGCGGTGGGA</td><td>AGTTGGAAGC</td><td>CACGTTCACC</td><td> 240</td>
<td>TTCATGAGGG</td><td>AGGATCGGTG</td><td>CATCCAGAAG</td><td>AAAATCCTGR</td><td>TGCGGAAGAC</td><td>GGAGGAGCCT</td><td> 300</td>
<td>GGCAAATACA</td><td>GCGCCTGTGA</td><td>GCCCCTCCCC</td><td>CAYTCCCACC</td><td>CCCACCYTCC</td><td>CCCACCGCCA</td><td> 360</td>
<td>ACCCCAGTGC</td><td>ACCAGCCTCC</td><td>ACAGGTAGAG</td><td>AGTGCCCAGG</td><td>crccccnTr</td><td>GCCAGGGCCC</td><td> 420</td>
<td>CAGCTCTGCC</td><td>CACCTCCAAG</td><td>GAGGGGCTGG</td><td>CCTCTCCTTC</td><td>CTGGGGGGCT</td><td>GGTGGCCCTG</td><td> 480</td>
<td>ACATCAGACA</td><td>CCGGGTGTGA</td><td>CAGGCTTGTC</td><td>CGGAGTCGAG</td><td>ATGGACCAGA</td><td>TCACGCCTGC</td><td> 540</td>
<td>CCTCTGGGAG</td><td>GCCCTAGCCA</td><td>TTGACACATT</td><td>GAGGAAGCTG</td><td>AGGATTGGGA</td><td>CAAGGAGGCC</td><td> 600</td>
<td>AAGGATTAGA</td><td>TGGGGGCAGG</td><td>AAGCTCATGT</td><td>ACCTGCAGGA</td><td>GCTGCCCAGG</td><td>AGGGACCAYT</td><td> 660</td>
<td>ACATCTTTTA</td><td>CTGCAAAGAC</td><td>CAGCACCATG</td><td>GGGGCSTGCT</td><td>CCACATGGGA</td><td>AAGCTTGTGG</td><td> 720</td>
<td>GTAGGAATTC</td><td>TGATACCAAC</td><td>CGGGAGGCCC</td><td>TGGAAGAATT</td><td>TAAGAAATTG</td><td>GTGCAGCGCA</td><td> 780</td>
<td>AGGGACTCTC</td><td>GGAGGAGGAC</td><td>ATTTTCACGC</td><td>CCCTGCAGAC</td><td>GGGAAGCTGC</td><td>GTTCCCGAAC</td><td> 840</td>
<td>ACTAGGCAGC</td><td>ACCUPGGTCT</td><td>GCACCTCCAG</td><td>AGCCCACCCT</td><td>ACCACCAGAC</td><td>ACAGAGCCCG</td><td> 900</td>
<td>GACCACCTGG</td><td>ACCTACCCTC</td><td>CAGCCATGAC</td><td>CCTTCCCTGC</td><td>TCCCACCCAC</td><td>CTGACTCCAA</td><td> 960</td>
<td>ATAAAGTCCT</td><td>TCTCCCCCAA</td><td>AAAAAAAAAA</td><td>AAAAAAACTC</td><td>GA</td><td></td><td> 1002</td>
(2) INFORMATION FOR SEQ ID NO: 43:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2581 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: double (D) TOPOLOGY: linear
234 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 43:
<td>TGCAAAACCA</td><td>CTGGACACTG</td><td>GACAAGTACG</td><td>GGATCCTGGS</td><td>CGACGCACGC</td><td>CTCTTCTTTG</td><td> 60</td>
<td>GGCCCCAGCA</td><td>CCGGSCCGTC</td><td>ATCCTTCGGT</td><td>TGTCCAACCG</td><td>CCGCGCACTG</td><td>CGCCTCCGTG</td><td> 120</td>
<td>CCAGCTTCTC</td><td>CCAGCCCCTC</td><td>TTCCAGGCTG</td><td>TGGSTGCCAT</td><td>CTGCCGCCTC</td><td>CTCAGCATCC</td><td> 180</td>
<td>GGCACCCCGA</td><td>GGAGCTGTCC</td><td>CTGCTCCGGG</td><td>CTCCTGAGAA</td><td>GAAGGAGAAG</td><td>AAGAAGAAAG</td><td> 240</td>
<td>AGAAGGAGCC</td><td>AGAGGAAGAG</td><td>CTCTATGACT</td><td>TGAGCAAGGT</td><td>TGTCTTGGCT</td><td>GGGGGCGTGG</td><td> 300</td>
<td>CACCTGCACT</td><td>GTTCCGGGGG</td><td>ATGCCAGCTC</td><td>ACTTCTCGGA</td><td>CAGCGCCCAG</td><td>ACTGAGGCCT</td><td> 360</td>
<td>GCTACCACAT</td><td>GCTGAGCCGG</td><td>ACCAGCCGC</td><td>CACCCGACCC</td><td>CCTCCTGCTC</td><td>CAGCGTCTGC</td><td> 420</td>
<td>CACGGCCCAG</td><td>CTCCCTGTCA</td><td>GACAAGACCC</td><td>AGCTCCACAG</td><td>CAGGTGGCTG</td><td>GACTCGTCGC</td><td> 480</td>
<td>ggtgtctcat</td><td>GCAGCAGGGC</td><td>ATCAAGGCCG</td><td>GGGACGCACT</td><td>CTGGCTGCGC</td><td>TTCAAGTACT</td><td> 540</td>
<td>ACAGCTTCTT</td><td>CGATTTGGAT</td><td>CCCAAGACAG</td><td>ACCCCGTGCG</td><td>GCTGACACAG</td><td>CTGTATGAGC</td><td> 600</td>
<td>AGGCCCGGTG</td><td>GGACCTGCTG</td><td>CTGGAGGAGA</td><td>TTGACTGCAC</td><td>CGAGGAGGAG</td><td>ATGATGGTGT</td><td> 660</td>
<td>TTGCCGCCCT</td><td>GCAGTACCAC</td><td>ATCAACAAGC</td><td>TGTCCCAGAG</td><td>CGGGGAGGTG</td><td>GGGGAGCCGG</td><td> 720</td>
<td>CTGGCACAGA</td><td>CCCAGGGCTG</td><td>GACGACCTGG</td><td>ATGTGGCCCT</td><td>GAGCAACCTG</td><td>GAGGTGAAGC</td><td> 780</td>
<td>TGGAGGGGTC</td><td>GGCGCCCACA</td><td>GATCTGCTGG</td><td>ACAGCCTCAC</td><td>CACCATCCCA</td><td>GAGCTCAAGG</td><td> 840</td>
<td>ACCATCTCCG</td><td>AATCTTTCGG</td><td>ACCGAGAAGC</td><td>TGACCCTGAA</td><td>GGGCTACCGC</td><td>CAACACTGGG</td><td> 900</td>
<td>TGGTGTTCAA</td><td>GGAGACCACA</td><td>CTGTCCTACT</td><td>ACAAGAGCCA</td><td>GGACGAGGCC</td><td>CCTGGGGACC</td><td> 960</td>
<td>CCATTCAGCA</td><td>GCTCAACCTC</td><td>AAGGGCTGTG</td><td>AGGTGGTTCC</td><td>CGATGTTAAC</td><td>GTCTCCGGCC</td><td> 1020</td>
<td>AGAAGTTCTG</td><td>CATTAAACTC</td><td>CTAGTGCCCT</td><td>TRANSTGAGGC</td><td>ATGAGTGAGA</td><td>TCTACCTGCG</td><td> 1080</td>
<td>GTGCCAGGAT</td><td>GAGCAGCAGT</td><td>ATGCCCGCTG</td><td>GATGGCTGGC</td><td>TGCCGCCTGG</td><td>CCTCCAAAGG</td><td> 1140</td>
<td>CCGCACCATG</td><td>GCCGACAGCA</td><td>GCTACACCAG</td><td>CGAGGTGCAG</td><td>GCCATCCTGG</td><td>CYTTCCTCAG</td><td> 1200</td>
<td>CCTGGAGCGC</td><td>ACGGGCAGTG</td><td>GGGGCCCGGG</td><td>CAACCACCCC</td><td>CACGGCCCTG</td><td>ATGCCTCTGC</td><td> 1260</td>
<td>CGAGGGCCTC</td><td>AACCCCTACG</td><td>GCCTCGTTGC</td><td>ACCGTTTC</td><td>CAGCGAAAGT</td><td>TCAAGGCCAA</td><td> 1320</td>
<td>GCAGCTCACC</td><td>CCACGGATCC</td><td>TGGAAGCCCA</td><td>CCAGAATGTG</td><td>GCCCAGTTGT</td><td>CGCTGGCAGA</td><td> 1380</td>
<td>GGCCCAGCTG</td><td>CGCTTCATCC</td><td>AGGCCTGGCA</td><td>GTCCCTGCCC</td><td>GACTTCGGCA</td><td>TCTCCTATGT</td><td> 1440</td>
<td colspan="2">CATGGTCAGG TTCAAGGGCA</td><td>GCAGGAAAGA</td><td>CGAGATCCTG</td><td>GGCATCGCGA</td><td>AGAACCGACT</td><td> 1500</td>
235
GATCCGCATC GACTTGGCCG TGGGCGACGT GGTCAAGACC TGGCGTTTCA GCAACATGCG1560
CCAGTGGAAT GTCAACTGGG ACATCCGGCA NGTGGCCATC GAGTTTGATG AACACATCAA1620
TGTGGCCTTC AGCTGCGTGT CTGCCAGCTG CCGAATTGTA CACGAGTATA TCGGGGGCTA1680
CATTTTCCTG TCGACGCGGG AGNGGGCCCG TGGGGAGGAG CTGGATGAAG ACCTCTTCCT1740
GCAGCTCACC GGGGGCCATG AGGCCTTCTG AGGGCTGTCT GATTGCCCCT GCCCTGCTCA1800
CCACCCTGTC ACAGCCACTC CCAAGCCCAC ACCCACAGGG GCTCACTGCC CCACACCCGC1860
TCCAGGCAGG CACCCAGCTG GGCATTTCAC CTGCTGTCAC TGACTTTGTG CAGGCCAAGG1920
ACCTGGCAGG GCCAGACGCT GTACCATCAC CCAGGCCAGG GATGGGGGTG GGGGTCCCTG1980
AGCTCATGTG GTGCCCCCTT TCCTTGTCTG AGTGGCTGAG GCTGATACCC CTGACCTATC2040
TGCAGTCCCC CAGCACÀCAA GGAAGACCAG ATGTAGCTAC AGGATGATGA AACATGGTTT2100
CAAACGAGTT CTTTCTTGTT ACTTTITAAA ATTTCTTTTT TATAAATTAA TATTTTATTG2160
TTGGATCCTC CTCCTTTCTC TGGAGCTGTG CTTGGGGCTA CTCTGACACT CTGTCTCTTC2220
ATCACCAGCC AAGGAAAGGG GCTTTCCTGA TAAAGACAAG AGTTGGTTAG AGAAAGGGAC2280
ACCTAAGTCA GTCTAGGGTT GGAAGCTAGG AGAGAGGTGA GGGCAGAAGG GCACAGCTTT2340
CAGGAACAAG GAATAGGGGC TGGGGTKGTK GTTCTCACGG GTAGGCGGTA CCTGCAGGGC2400
CTCCTTGAAG TACTTGGGAA GGAGGAAGCC ATCAGTATTC CCTGGAGTCA GAATCACCCC2460
ATTGGCAGAG CGGAAGAAGG GTATTCCATC TGCTGACAGA GCCAGAGATG TGACTCATGC2520
CCTCCCCGAA GGCAAAGTCA GCTCCTGCTT TGTCCAGACT CACCTGCCAG AGCCAGGGGT2580
C2581 (2) INFORMATION FOR SEQ ID NO: 44:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1764 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: double (D) TOPOLOGY: linear
236 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 44:
<td>GAATTCGGCA CGAGGATGAT ATTCCTACTA TTCCTCACCC CACTCTGGCT GCAAAAAGGA</td><td> 60</td>
<td>AGTGCAGGGA AAATGAGTGG GGAGTTCCTG TATGCCAGTC TGTTTCAATG GAACTATTTT</td><td> 120</td>
<td>TGGAGGAATA AAAAAGTATG CTAGATTATA TTGGTACGAT AGGCATTTTC TTACATTGCA</td><td> 180</td>
<td>TATAGTCTGC TTTGGCTTTT ACCTGTTGAG GGGAAGAATG AGGAGAGGAT AAAAATCATT</td><td> 240</td>
<td>GTÀTCCCCTA GAGAAGGAAT ATCAAAATCC ATTTAATAAA AAAACTCATA CTAAGAATAA</td><td> 300</td>
<td>AATTGCATAG tgttttattc tcctttgttc ataattaaac ACAAGATATT TTAAATTGTC</td><td> 360</td>
<td>AAATCAGTTT CTTTATGAAA AAATATGACC TGTATGCCTT TATTCTCTCC TTTCCTTCTT</td><td> 420</td>
<td>CCCACCCGTC GCTTCTTTTC TTCTCTTCCT TTTTTTCTTT CCTTGTCCTC TGACTAAATG</td><td> 480</td>
<td>AAGAACAAAC ATTTGATAAA AGCCACTGCC AATTCATGAT AAAAATTCAC AGCAAAGTTG</td><td> 540</td>
<td>GTACAGAAAA GAACTTTCTC TGCGTGTTAA AGGGTGCCTC TCCCATGCTC TCAGCAAATA</td><td> 600</td>
<td>TTTAATGATG AAATCTTATT AATAATCACT GTAGAACCAA GAATTAAACT AGTATACCCA</td><td> 660</td>
<td>CTGTCTTGGC TTGTAATCAA CAATATACAG GTGGTTCTAG CCAGTGCAAT AAGACAAGAG</td><td> 720</td>
<td>AAACAAAAAT GTTATAAGGC CTGGAAAAGA TGAAACAAAC TGTTATTCAC AAAATACTGT</td><td> 780</td>
<td>CTATACAGAA TGCTCAGTGT CTTTTTTTCT TTTCTTTTTT TTAAACTTTA GTGAGATACC</td><td> 840</td>
<td>CTTCTGCCCT ATCTTAAAAT CACGTGGTGG GGGGTGGTGT CTGCACTTGA AACAGGACAC</td><td> 900</td>
<td>TTGGTTCCTG GGTTTAGCAT TGACCTTGCC AGCTTGGTYT GGCAGCTGAG TTGTTGGACT</td><td> 960</td>
<td>AGGAAGCGTC CYTGCAGGTT GTGKTCTGKT ACCTCTCTGT AAAGCCTGAA AGCATCCTAC</td><td> 1020</td>
<td>SATTGCATTT GCTAGKTCTC AGTAGAGCTA TTTAACAAGA ATCTGGAAAC ATTTTYCCTG</td><td> 1080</td>
<td>AGGGCTCTCT TTAGACAGCA GTAAAATGTA GCTGGAGACA TATTGAGTAA ATGGAAAAGA</td><td rowspan="2"> 1140 1200</td>
<td>AAAATCTAAT GAGGCCAGGA ATTTTTTTAA TCTTCTATTC TCACAGAAGG CCTCAAGGAG</td>
<td>AACACCATAA TTCATATTTT ACTCAKGTGG GTTAGGCATA AAGCCTCCCC CATAGATCCA</td><td> 1260</td>
<td>ATAACCTGTA RGTGTYCTGG TTTTGAAATT GCACCTGCTT ACATKGCTGG ATCNTAGCAC</td><td> 1320</td>
<td>TAAWTCACAC RGCAACGGCT TCTGGTTCAA TKGTTCATTA CTTGGGAATG TCAGATTGCC</td><td> 1380</td>
<td>AGAGAGCAGC CTGATGTTTA CATCCAATCG GCAATGCCTT AGGAAATCAG TTTTAATTAC</td><td> 1440</td>
<td>AATCTCACGT AGCAGCACTG CACTCAACCT TCAGAGAGGC TGGGATTTGT GTTGAACCTA</td><td> 1500</td>
<td>CATCTTATAG CTGTGCAGAA AATGCCTGTC CGACTGGGTC ATGCAAAATG GACAGCAAAG</td><td> 1560</td>
<td>TCAGCAGAAC CTTAGAAAAG ATGACACAGC AAGTGGAACA CAGCTGGATC ATCCCCCGTC</td><td> 1620</td>
237
CTGTCAAGCG TGCAGTGCTC TCTGGCCCCT TTTTAAAACA AGGGAACCCA GTTGGCGTTT
GCCTTTCAGC TTCCCCATTC TGATATAAAA ATCTGTGACC CAGCAGCTTT AACCATAAAA
AAAAAAAAAA AAAAAAAAAC TCGA
1680
1740
1764 (2) INFORMATION FOR SEQ ID NO: 45:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 796 bp (B) TYPE: nucleic acid (C) CHAIN TYPE: double (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 45:
ACCTTCTTCC ATGTTTAGTC CCTTGGGCTC CTGTGTGCAA CCAGAGATGC CCTCTGGCTT TTTCTCACTC AGCAAAATTG TGGGGGTCCC GCAAGGTGGT CTCTGTGGTC ATGAAGGAGA GCCTCTCCCC TTCAACTCAG CTGGCCCCCC AGTCTGGAGC CCACCCCGGA GGGCAGCACA CCCCTGCAGG CAGMACACGT CCCGGGCATT CAGAGTCTGC TGTCGYTTCC CCTTTAAGCA GGTCCAGGGA GCATGGGCTG TCGTTCCCCT GGCCAGAAGT GCAGGGAGCA TGGGCTGGGT CGCCGTCCCG GGAGCCCGGC TCCCAGGCCT CTGTCACTCT CTGGCCATTG AAAGGCTTCT CCCAGGATGC AGCAAGCCAA AACAGTACCA CCCTATCTTA AAAAAA
TGCTACCCTC CTGCTGGAGG TGAGAGCATC TCAGACCTGC CTGCTTTTCA CCCTCAGCCC TAGTCAGCAG CTCCCTGGGC AGCTCTCTGA GCCGGCTAGG ACAGTGCCGG AAACTCAGCT GCACCTGAAG TGCACAGGAG CCGGGAAGAG GGAGGTGTCT YTGCAGCTGG TGTCCTGCMA CTCCTTAGCC ACAGACAGAA CAGCCAGTGC CACTCATTCA CCACACCCGA GGAGGCCAGA TTAAGCACAC TCATTCACCA CACCCGAGGA GCACCTCCGC AGGAGAGAAG GCTGAGCCAC CTCGTTTTCC CCTACCTCCC TAAGACTTTT GTTCCTTAAA GTGCTGTTAC ACTCTCCTTT CTGCACGTCA GCCTGGGTGA CAGAGTGAGA
120
180
240
300
360
420
480
540
600
660
720
780
796
238 (2) INFORMATION FOR SEQ ID NO: 46:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1705 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: double (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 46:
TGGCCATGGA CTGGAGACCC TCAAGGAGCC TCGAGATCTG ACAAGGCTGA CTGGCTATGC AGTGGAGACC GGATGACATC CCAGAAAGCG CACAAACAAT GTCCATGCAG GTTCTACCAC CACGGAAGAT GCTGCACAGC CTTGGCCACAGC CTTGGCCACA
AGCGCTAGAA CAAAGAAACA CAAAGCCGCC TGGTGACCAT GCGCGAGCCC TGCTGAGACC CAGCGCTGGG TACATGCCGT TTCCACAAGG GCCGTGGCGG TGCCTCGATA TTCCAGCGTT CCGTTCAGTG CTGCCCAAGG CTG CTTACAG CTG
GGTTTAGATT AAGATGCTAA GTGGAGATGT GGCTGGGTTG CTGCTGCTGT TACCTGAAGA ATGAGGCCTT ATGCTCAGTG CTGGGCGACA AGAGCAGGTT TAGCTCAAGA TGGCAGAGCT TCCATCGTCC ACACGGCCCCCCGCCTCCACGCCTCCACGCCTCCACGCCT
TTGAAACAGC
TCACCAAACA ACATCTCAGC ACATGTTGAT GCGCTACCTA TGGGTGACCT
TGCTTTGGGT GCTAGCAGAG
GAGAGAAGCG
TAATGATGCT TCCTGCCCAG GTACCATGGT TCAAACTCTT GGGCATCTCT CTACAGGCTG CCAAAAGTCC GGAGTTGGTG CAACGTCTGC
AAAGAAGGAT GGCTGACTGG AGGAGAGCAC CGACATCGCC CCTCAAGAAG CAAGTCCCTG GAGAAGCATC AACGATCGCT GTCCAGGTGC GCCTATTATT AAGGACACAA TACCATGCCA TTCAACATCT AAAGTGAAAA GCCGGCCATGCCCGCGCCTG
TTCCTTGGAT GCCAGAAATA GTCAAGGCCA CGCAAACTGG CTGGACAGCC GTGCAGCTGC CTGAGTTTAA TTGAGGAAGC TGGAGCAGCT ACTGGATGCT TGCTTGGCAA TCCATCGCCA CCAGGTTCCT TACTCTTCACCT CCTACT CCTACT CCTACT CCTACT CCTACTACT CCTACTACT CCTAGACTACT
120
180
240
300
360
420
480
540
600
660
720
780
840
900
960
1020
1080
239
<td>CATCTTCTCC</td><td>GCCTCTTCCT ACGACGTGCT ACACCTGGTT GAGTTCTACC TGGAGGAAGG</td><td> 1140</td>
<td>GATCACTGAT</td><td>GAAGAAGCCA TCTCCCTCAT CGACCTGGAG GTGCTGAGAC CCAAGCGGGA</td><td> 1200</td>
<td>TGACAGACAG</td><td>CTAGAGATTT GCAAACAACA GCTCCCAGAT TCTTGCGGCT AGTGGGAGAC</td><td> 1260</td>
<td>CAAGGGACTC</td><td>CATCGGAGAT NAGGACCCGT TCACAGCTAA GCTRAGCTTT GAGCAAGGTG</td><td> 1320</td>
<td>GCTCARAGTT</td><td>CGTGCCAGTG CTGGTGAGCC GGCTGGTGCT GCGCTCCATG AGCCGCCGGG</td><td> 1380</td>
<td>ATGTCCTCAT</td><td>CAAGCGATGG TRANSCACCCC TGAGGTGGCA ATACTTCCGC TCACTGCTGC</td><td> 1440</td>
<td>CTGACGCCTC</td><td>CATTACCATG TGCCCCTCCT GCTTCCAGAT GTTCCATTCT GAGGACTATG</td><td> 1500</td>
<td>AGTTGCTGGT</td><td>GCTTCAGCAT GGCTGCTGCC CCTACTGCCG CAGGTGCAAG GATGACCCTG</td><td> 1560</td>
<td>GCCCATGACC</td><td>AGCATCCTGG GGACGGCCTG CACCCTCTGC CCGCCTTGGG GTCTGCTGGG</td><td> 1620</td>
<td>CTGTGAAGGA</td><td>GAATAAAGAG TTAAACTGTC AAAAAAAAAA AAAAAAAAA AAAAAAAAAA</td><td> 1680</td>
<td>AAAAAAAAAA</td><td>AAAAAAAAAA AAANA</td><td> 1705</td>
(2) INFORMATION FOR SEQ ID NO: 47:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 981 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: double (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 47:
TCGGCAGCAC AGAGCTCTGG AGATGAAGAC CCTGTTCCTG GGTGTCACGC
TCGGCCTGGC
GCTGCCCTGT CCTTCACCCT GGRGGAGGAG GATATCACAG GGACCTGGTA CGTGAAGGCC
120
ATGGTGGTCG ATAAGACTTT CCGGAGACAG GAGGCCCAGA AGGTGTCCCC
AGTGAAGGTG
180
ACAGCCCTGG GCGGTGGGAA GTTGGAAGCC ACGTTCACCT TCATGAGGGA GGATCGGTGC
240
ATCCAGAAGA AAATÇCTGRT GCGGAAGACG GAGGAGCCTG GCAAATACAG CGCCTGTGAG
300
CCTCCCCC AYTCCCACCC CCACCYTCCC CCACCGCCAA
360
240
CAGGTAGAGA GTGCCCAGGC TGCCCTTTTG CCAGGGCCCC AGCTCTGCCC ACCTCCAAGG 420 AGGGGCTGGC CTCTCCTTCC TGGGGGGCTG GTGGCCCTGA CATCAGACAC CGGGTGTGAC 480 AGGCTTGTCC GCAGTCGAGA TGGACCAGAT CACGCCTGCC CTCTGGGAGG CCCTAGCCAT 540 TGACACATTG AGGAAGCTGA GGATTGGGAC AAGGAGGCCA AGGATTAGAT GGGGGCAGGA AGCTCATGTA CCTGCAGGAG 600 CTGCCCAGGA GGGACCAYTA CATCTTTTAC TGCAAAGACC 660 AGCACCATGG GGGCSTGCTC CACATGGGAA AGCTTGTGGG TAGGAATTCT GATACCAACC 720 GGGAGGCCCT GGAAGAATTT AAGAAATTGG TGCAGCGCAA GGGACTCTCG 780 GAGGAGGACA
TTTTCACGCC
CCTGCAGACG
GGAAGCTGCR
TTCCCGAACA
CTAGGCAGCC CCCGGGTCTG
840
CACCTCCAGA
GCCCACCCTA
CCACCAGACA
CAGAGCCCGG
ACCACCTGGA CCTACCCTCC
900 agccatgacc
CTTCCCTGCT
CCCACCCACC
TGACTCCAAA
TAAAGTCCTT CTCCCCCAAA
960
AAAAAAAAAA
AAAAAACTCG
981 (2) INFORMATION FOR SEQ ID NO: 48:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 146 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 48:
<td>Met</td><td>His</td><td>Tyr</td><td>Gin</td><td>Met</td><td>To be</td><td>Go</td><td>Thr</td><td>Read</td><td>Lys</td><td>Tyr</td><td>Glu</td><td>Ile</td><td>Lys</td><td>Lys</td><td>Read</td>
<td> 1</td><td></td><td></td><td></td><td> 5</td><td></td><td></td><td></td><td></td><td> 10</td><td></td><td></td><td></td><td></td><td> 15</td><td></td>
<td>Ile</td><td>Tyr</td><td>Go</td><td>His</td><td>Read</td><td>Go</td><td>Ile</td><td>Trp</td><td>Read</td><td>Read</td><td>Read</td><td>go</td><td>Allah</td><td>Lys</td><td>Met</td><td>To be</td>
<td></td><td></td><td></td><td> 20</td><td></td><td></td><td></td><td></td><td> 25</td><td></td><td></td><td></td><td></td><td> 30</td><td></td><td></td>
<td>Go</td><td>Gly</td><td>His</td><td>Read</td><td>Arg</td><td>Read</td><td>Read</td><td>To be</td><td>His</td><td>Asp</td><td>Gin</td><td>Go</td><td>Allah</td><td>Met</td><td>Pro</td><td>Tyr</td>
<td></td><td></td><td> 35</td><td></td><td></td><td></td><td></td><td> 40</td><td></td><td></td><td></td><td></td><td> 45</td><td></td><td></td><td></td>
<td>Gin</td><td>Trp</td><td>Glu</td><td>Tyr</td><td>Pro</td><td>Tyr</td><td>Read</td><td>Read</td><td>To be</td><td>Ile</td><td>Read</td><td>Pro</td><td>To be</td><td>Read</td><td>Read</td><td>Gly</td>
55 60
241
<td colspan="2">Leu Leu 65</td><td colspan="3">Be Phe Pro</td><td colspan="2">Arg Asn 70</td><td>Asn</td><td colspan="3">Ile Ser Tyr 75</td><td colspan="4">Leu Vai Leu Ser</td><td>Met 80</td>
<td>Ile</td><td>To be</td><td>Met</td><td>Gly</td><td>Read</td><td>Phe</td><td>To be</td><td>Ile</td><td>Allah</td><td>Pro</td><td>Read</td><td>Ile</td><td colspan="2">Tyr gly</td><td>To be</td><td>Met</td>
<td></td><td></td><td></td><td></td><td> 85</td><td></td><td></td><td></td><td></td><td> 90</td><td></td><td></td><td></td><td></td><td> 95</td><td></td>
<td>Glu</td><td>Met</td><td>Phe</td><td>Pro</td><td>Allah</td><td>Allah</td><td>Gin</td><td>Pro</td><td>To be</td><td>Thr</td><td>Allah</td><td>Met</td><td>Allah</td><td>Arg</td><td>Pro</td><td>Thr</td>
<td></td><td></td><td></td><td> 100</td><td></td><td></td><td></td><td></td><td> 105</td><td></td><td></td><td></td><td></td><td> 110</td><td></td><td></td>
<td>Go</td><td>To be</td><td>To be</td><td>Read</td><td>Go</td><td>Phe</td><td>Read</td><td>Pro</td><td>Phe</td><td>Pro</td><td>To be</td><td>Cys</td><td>Thr</td><td colspan="2">Trp cys</td><td>Trp</td>
<td></td><td></td><td> 115</td><td></td><td></td><td></td><td></td><td> 120</td><td></td><td></td><td></td><td></td><td> 125</td><td></td><td></td><td></td>
<td>Cys</td><td>Trp</td><td>Gin</td><td>Cys</td><td>Lys</td><td>Cys</td><td>Met</td><td>Pro</td><td>Gly</td><td>To be</td><td>Cys</td><td>Thr</td><td>Thr</td><td colspan="2">Arg Wing</td><td>To be</td>
<td></td><td> 130</td><td></td><td></td><td></td><td></td><td> 135</td><td></td><td></td><td></td><td></td><td> 140</td><td></td><td></td><td></td><td></td>
To be Xaa
145 (2) INFORMATION FOR SEQ ID NO: 49:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 312 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 49:
<td>Met</td><td>Asn</td><td>To be</td><td>Go</td><td>Go</td><td>To be</td><td>Read</td><td>Read</td><td>Read</td><td>Ile</td><td>Read</td><td>Glu</td><td colspan="2">Pro Asp Lys</td><td>Gin</td>
<td> 1</td><td></td><td></td><td></td><td> 5</td><td></td><td></td><td></td><td></td><td> 10</td><td></td><td></td><td></td><td> 15</td><td></td>
<td>Glu</td><td>Allah</td><td>Read</td><td colspan="2">Ile glu</td><td>To be</td><td>Read</td><td>Cys</td><td>Glu</td><td>Lys</td><td>Read</td><td>Go</td><td>Lys phe</td><td>Arg</td><td>Glu</td>
<td></td><td></td><td></td><td> 20</td><td></td><td></td><td></td><td></td><td> 25</td><td></td><td></td><td></td><td> 30</td><td></td><td></td>
<td>Gly</td><td>Glu</td><td>Arg</td><td>Pro</td><td>To be</td><td>Read</td><td>Arg</td><td>Read</td><td>Gin</td><td>Read</td><td>Read</td><td>To be</td><td>Asn leu</td><td>Phe</td><td>His</td>
<td></td><td></td><td> 35</td><td></td><td></td><td></td><td></td><td> 40</td><td></td><td></td><td></td><td></td><td> 45</td><td></td><td></td>
<td>Gly</td><td>Met</td><td>Asp</td><td>Lys</td><td>Asn</td><td>Thr</td><td>Pro</td><td>Go</td><td>Arg</td><td>Tyr</td><td>Thr</td><td>Go</td><td>Tyr cys</td><td>To be</td><td>Read</td>
<td></td><td> 50</td><td></td><td></td><td></td><td></td><td> 55</td><td></td><td></td><td></td><td></td><td> 60</td><td></td><td></td><td></td>
<td>Ile</td><td>Lys</td><td>Go</td><td>Allah</td><td>Allah</td><td>To be</td><td>Cys</td><td>Gly</td><td>Allah</td><td>Ile</td><td>Gin</td><td>Tyr</td><td>Ile pro</td><td>Thr</td><td>Glu</td>
<td> 65</td><td></td><td></td><td></td><td></td><td> 70</td><td></td><td></td><td></td><td></td><td> 75</td><td></td><td></td><td></td><td> 80</td>
<td>Read</td><td>Asp</td><td>Gin</td><td>Go</td><td>Arg</td><td>Lys</td><td>Trp</td><td>Ile</td><td>To be</td><td>Asp</td><td>Trp</td><td>Asn</td><td>Read thr</td><td>Thr</td><td>Glu</td>
90 95
242
<td colspan="16">Lys Lys His Thr Leu Leu Arg Leu Leu Tyr Glu Wing Leu Go Asp Cys</td>
<td colspan="2"></td><td colspan="3"> 100</td><td colspan="6"> 105</td><td colspan="5"> 110</td>
<td>Lys</td><td>Lys</td><td>To be</td><td>Asp</td><td>Allah</td><td>Allah</td><td>To be</td><td>Lys</td><td>Go</td><td>Met</td><td>Go</td><td>Glu</td><td>Read</td><td>Read</td><td>Gly</td><td>To be</td>
<td></td><td></td><td> 115</td><td></td><td></td><td></td><td></td><td> 120</td><td></td><td></td><td></td><td></td><td> 125</td><td></td><td></td><td></td>
<td>Tyr</td><td>Thr</td><td>Glu</td><td>Asp</td><td>Asn</td><td>Allah</td><td>To be</td><td>Gin</td><td>Allah</td><td>Arg</td><td>Go</td><td>Asp</td><td>Allah</td><td>His</td><td>Arg</td><td>Cys</td>
<td></td><td> 130</td><td></td><td></td><td></td><td></td><td> 135</td><td></td><td></td><td></td><td></td><td> 140</td><td></td><td></td><td></td><td></td>
<td>Ile</td><td>Go</td><td>Arg</td><td>Allah</td><td>Read</td><td colspan="2">Lys asp</td><td>Pro</td><td>Asn</td><td>Allah</td><td>Phe</td><td>Read</td><td>Phe</td><td>Asp</td><td>His</td><td>Read</td>
<td> 145</td><td></td><td></td><td></td><td></td><td> 150</td><td></td><td></td><td></td><td></td><td> 155</td><td></td><td></td><td></td><td></td><td> 160</td>
<td>Read</td><td>Thr</td><td>Read</td><td>Lys</td><td>Pro</td><td>Go</td><td>Lys</td><td>Phe</td><td>Read</td><td>Glu</td><td colspan="2">Gly Glu</td><td>Read</td><td>Ile</td><td>His</td><td>Asp</td>
<td></td><td></td><td></td><td></td><td> 165</td><td></td><td></td><td></td><td></td><td> 170</td><td></td><td></td><td></td><td></td><td> 175</td><td></td>
<td>Read</td><td>Read</td><td>Thr</td><td>Ile</td><td>Phe</td><td>Go</td><td>To be</td><td>Allah</td><td>Lys</td><td>Read</td><td>Allah</td><td>To be</td><td>Tyr</td><td>Go</td><td>Lys</td><td>Phe</td>
<td></td><td></td><td></td><td> 180</td><td></td><td></td><td></td><td></td><td> 185</td><td></td><td></td><td></td><td></td><td> 190</td><td></td><td></td>
<td>Tyr</td><td>Gin</td><td>Asn</td><td>Asn</td><td>Lys</td><td>Asp</td><td>Phe</td><td>Ile</td><td>Asp</td><td>To be</td><td>Read</td><td>Gly</td><td>Read</td><td>Read</td><td>His</td><td>Glu</td>
<td></td><td></td><td> 195</td><td></td><td></td><td></td><td></td><td> 200</td><td></td><td></td><td></td><td></td><td> 205</td><td></td><td></td><td></td>
<td>Gin</td><td>Asn</td><td>Met</td><td>Allah</td><td>Lys</td><td>Met</td><td>Arg</td><td>Read</td><td>Read</td><td>Thr</td><td>Phe</td><td>Met</td><td>Gly</td><td>Met</td><td>Allah</td><td>Val</td>
<td></td><td> 210</td><td></td><td></td><td></td><td></td><td> 215</td><td></td><td></td><td></td><td></td><td> 220</td><td></td><td></td><td></td><td></td>
<td>Glu</td><td>Asn</td><td>Lys</td><td>Glu</td><td>Ile</td><td>To be</td><td>Phe</td><td>Asp</td><td>Thr</td><td>Met</td><td>Gin</td><td>Gin</td><td>Glu</td><td>Read</td><td>Gin</td><td>Ile</td>
<td> 225</td><td></td><td></td><td></td><td></td><td> 230</td><td></td><td></td><td></td><td></td><td> 235</td><td></td><td></td><td></td><td></td><td> 240</td>
<td>Gly</td><td>Allah</td><td colspan="2">Asp Asp</td><td>Go</td><td>Glu</td><td>Allah</td><td>Phe</td><td>Go</td><td>Ile</td><td>Asp</td><td>Allah</td><td>Go</td><td>Arg</td><td>Thr</td><td>Lys</td>
<td></td><td></td><td></td><td></td><td> 245</td><td></td><td></td><td></td><td></td><td> 250</td><td></td><td></td><td></td><td></td><td> 255</td><td></td>
<td>Met</td><td>Go</td><td>Tyr</td><td>cys</td><td>Lys</td><td>Ile</td><td>Asp</td><td>Gin</td><td>Thr</td><td>Gin</td><td>Arg</td><td>Lys</td><td>Go</td><td>val</td><td>Val</td><td>To be</td>
<td></td><td></td><td></td><td> 260</td><td></td><td></td><td></td><td></td><td> 265</td><td></td><td></td><td></td><td></td><td> 270</td><td></td><td></td>
<td>His</td><td>To be</td><td>Thr</td><td>His</td><td>Arg</td><td>Thr</td><td>Phe</td><td>Gly</td><td>Lys</td><td>Gin</td><td>Gin</td><td>Trp</td><td>Gin</td><td>Gin</td><td>Read</td><td>Tyr</td>
<td></td><td></td><td> 275</td><td></td><td></td><td></td><td></td><td> 280</td><td></td><td></td><td></td><td></td><td> 285</td><td></td><td></td><td></td>
<td>Asp</td><td>Thr</td><td>Read</td><td>Asn</td><td>Allah</td><td>Trp</td><td>Lys</td><td>Gin</td><td>Asn</td><td>Read</td><td>Asn</td><td>Lys</td><td>Go</td><td>Lys</td><td>Asn</td><td>To be</td>
<td></td><td> 290</td><td></td><td></td><td></td><td></td><td> 295</td><td></td><td></td><td></td><td></td><td> 300</td><td></td><td></td><td></td><td></td>
<td>Read</td><td>Read</td><td>To be</td><td>Read</td><td>To be</td><td>Asp</td><td>Thr</td><td>Xaa</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>
305 310 (2) INFORMATION FOR SEQ ID NO: 50:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 47 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear
243 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 50:
<td>Gly</td><td>Gly</td><td>Cys</td><td>Pro</td><td>Arg</td><td>Arg</td><td>Arg</td><td>Read</td><td>Go</td><td>Read</td><td>Tyr</td><td>Cys</td><td>Read</td><td>Phe</td><td>Gly</td><td>To be</td>
<td> 1</td><td></td><td></td><td></td><td> 5</td><td></td><td></td><td></td><td></td><td> 10</td><td></td><td></td><td></td><td></td><td> 15</td><td></td>
<td>Allah</td><td>Gly</td><td>Gly</td><td>Gly</td><td>Arg</td><td>Ile</td><td>His</td><td>To be</td><td>Glu</td><td>Allah</td><td>Trp</td><td>Phe</td><td>Pro</td><td>Lys</td><td>Allah</td><td>Trp</td>
<td></td><td></td><td></td><td> 20</td><td></td><td></td><td></td><td></td><td> 25</td><td></td><td></td><td></td><td></td><td> 30</td><td></td><td></td>
<td>Pro</td><td>Glu</td><td>Allah</td><td>Glu</td><td>Lys</td><td>Trp</td><td>Read</td><td>Phe</td><td>Allah</td><td>Glu</td><td>Read</td><td>Read</td><td>Arg</td><td>Gly</td><td>Xaa</td><td></td>
<td></td><td></td><td> 35</td><td></td><td></td><td></td><td></td><td> 40</td><td></td><td></td><td></td><td></td><td> 45</td><td></td><td></td><td></td>
<td> (2)</td><td>IN</td><td>FOR</td><td colspan="2">.MATION</td><td>PAJ</td><td>FROG</td><td>SEQ</td><td>ID</td><td>No.</td><td>. AND</td><td> >1:</td><td></td><td></td><td></td><td></td>
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 467 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 51:
<td colspan="16">Met Leu Be Arg Pro Gin Pro Pro Asp Pro Read Leu Read Leu Gin Arg</td>
<td colspan="3"> 1</td><td colspan="2"> 5</td><td colspan="7"> 10</td><td colspan="4"> 15</td>
<td>Read</td><td>Pro</td><td>Arg</td><td>Pro</td><td>To be</td><td>To be</td><td>Read</td><td>To be</td><td>Asp</td><td>Lys</td><td>Thr</td><td>Gin</td><td>Read</td><td>His</td><td>To be</td><td>Arg</td>
<td></td><td></td><td></td><td> 20</td><td></td><td></td><td></td><td></td><td> 25</td><td></td><td></td><td></td><td></td><td> 30</td><td></td><td></td>
<td>Trp</td><td>Read</td><td>Asp</td><td>To be</td><td>To be</td><td>Arg</td><td>Cys</td><td>Read</td><td>Met</td><td>Gin</td><td>Gin</td><td>Gly</td><td>Ile</td><td>Lys</td><td>Allah</td><td>Gly</td>
<td></td><td></td><td> 35</td><td></td><td></td><td></td><td></td><td> 40</td><td></td><td></td><td></td><td></td><td> 45</td><td></td><td></td><td></td>
<td>Asp</td><td>Allah</td><td>Read</td><td>Trp</td><td>Read</td><td>Arg</td><td>Phe</td><td>Lys</td><td>Tyr</td><td>Tyr</td><td>To be</td><td>Phe</td><td>Phe</td><td>Asp</td><td>Read</td><td>Asp</td>
<td></td><td> 50</td><td></td><td></td><td></td><td></td><td> 55</td><td></td><td></td><td></td><td></td><td> 60</td><td></td><td></td><td></td><td></td>
<td>Pro</td><td>Lys</td><td>Thr</td><td>Asp</td><td>Pro</td><td>Go</td><td>Arg</td><td>Read</td><td>Thr</td><td>Gin</td><td>Read</td><td>Tyr</td><td>Glu</td><td>Gin</td><td>Allah</td><td>Arg</td>
<td> 65</td><td></td><td></td><td></td><td></td><td> 70</td><td></td><td></td><td></td><td></td><td> 75</td><td></td><td></td><td></td><td></td><td> 80</td>
<td>Trp</td><td>Asp</td><td>Read</td><td>Read</td><td>Read</td><td>Glu</td><td>Glu</td><td>Ile</td><td>Asp</td><td>Cys</td><td>Thr</td><td>Glu</td><td>Glu</td><td>Glu</td><td>Met</td><td>Met</td>
<td></td><td></td><td></td><td></td><td> 85</td><td></td><td></td><td></td><td></td><td> 90</td><td></td><td></td><td></td><td></td><td> 95</td><td></td>
<td>Go</td><td>Phe</td><td>Allah</td><td>Allah</td><td>Read</td><td>Gin</td><td>Tyr</td><td>His</td><td>Ile</td><td>Asn</td><td>Lys</td><td>Read</td><td>To be</td><td>Gin</td><td>To be</td><td>Gly</td>
<td></td><td></td><td></td><td> 100</td><td></td><td></td><td></td><td></td><td> 105</td><td></td><td></td><td></td><td></td><td> 110</td><td></td><td></td>
244
<td rowspan="2">Glu</td><td rowspan="2">Go</td><td rowspan="2">Gly 115</td><td rowspan="2">Glu</td><td colspan="3" rowspan="2">Pro Wing Gly</td><td colspan="9">Thr Asp Pro Gly Leu Asp Asp Leu Asp</td>
<td colspan="3"> 120</td><td colspan="6"> 125</td>
<td>Go</td><td>Allah</td><td>Read</td><td>To be</td><td>Asn</td><td>Read</td><td>Glu</td><td>Go</td><td>Lys</td><td>Read</td><td>Glu</td><td>Gly</td><td colspan="2">To be a wing</td><td>Pro</td><td>Thr</td>
<td></td><td> 130</td><td></td><td></td><td></td><td></td><td> 135</td><td></td><td></td><td></td><td></td><td> 140</td><td></td><td></td><td></td><td></td>
<td>Asp</td><td>Go</td><td>Read</td><td>Asp</td><td>To be</td><td>Read</td><td>Thr</td><td>Thr</td><td>Ile</td><td>Pro</td><td>Glu</td><td>Read</td><td colspan="2">Lys asp</td><td>His</td><td>Read</td>
<td> 145</td><td></td><td></td><td></td><td></td><td> 150</td><td></td><td></td><td></td><td></td><td> 155</td><td></td><td></td><td></td><td></td><td> 160</td>
<td>Arg</td><td>Ile</td><td>Phe</td><td>Arg</td><td>Pro</td><td>Arg</td><td>Lys</td><td>Read</td><td>Thr</td><td>Read</td><td>Lys</td><td colspan="3">Gly Tyr Arg</td><td>Gin</td><td>His</td>
<td></td><td></td><td></td><td></td><td> 165</td><td></td><td></td><td></td><td></td><td> 170</td><td></td><td></td><td></td><td></td><td> 175</td><td></td>
<td>Trp</td><td>Go</td><td>Go</td><td>Phe</td><td>Lys</td><td>Glu</td><td>Thr</td><td>Thr</td><td>Read</td><td>To be</td><td>Tyr</td><td>Tyr</td><td>Lys</td><td>To be</td><td>Gin</td><td>Asp</td>
<td></td><td></td><td></td><td> 180</td><td></td><td></td><td></td><td></td><td> 185</td><td></td><td></td><td></td><td></td><td> 190</td><td></td><td></td>
<td>Glu</td><td>Allah</td><td>Pro</td><td>Gly</td><td>Asp</td><td>Pro</td><td>Ile</td><td>Gin</td><td>Gin</td><td>Read</td><td>Asn</td><td>Read</td><td>Lys</td><td colspan="2">Gly cys</td><td>Glu</td>
<td></td><td></td><td> 195</td><td></td><td></td><td></td><td></td><td> 200</td><td></td><td></td><td></td><td></td><td> 205</td><td></td><td></td><td></td>
<td>Go</td><td>Go</td><td>Pro</td><td>Asp</td><td>Go</td><td>Asn</td><td>Go</td><td>To be</td><td>Gly</td><td>Gin</td><td>Lys</td><td>Phe</td><td>Cys</td><td>Ile</td><td>Lys</td><td>Read</td>
<td></td><td> 210</td><td></td><td></td><td></td><td></td><td> 215</td><td></td><td></td><td></td><td></td><td> 220</td><td></td><td></td><td></td><td></td>
<td>Read</td><td>Go</td><td>Pro</td><td>To be</td><td>Pro</td><td>Glu</td><td>Gly</td><td>Met</td><td>To be</td><td>Glu</td><td>Ile</td><td>Tyr</td><td>Read</td><td>Arg</td><td>Cys</td><td>Gin</td>
<td> 225</td><td></td><td></td><td></td><td></td><td> 230</td><td></td><td></td><td></td><td></td><td> 235</td><td></td><td></td><td></td><td></td><td> 240</td>
<td>Asp</td><td>Glu</td><td>Gin</td><td>Gin</td><td>Tyr</td><td>Allah</td><td>Arg</td><td>Trp</td><td>Met</td><td>Allah</td><td colspan="2">Gly cys</td><td>Arg</td><td>Read</td><td>Allah</td><td>To be</td>
<td></td><td></td><td></td><td></td><td> 245</td><td></td><td></td><td></td><td></td><td> 250</td><td></td><td></td><td></td><td></td><td> 255</td><td></td>
<td>Lys</td><td>Gly</td><td>Arg</td><td>Thr</td><td>Met</td><td>Allah</td><td>Asp</td><td>To be</td><td>To be</td><td>Tyr</td><td>Thr</td><td>To be</td><td>Glu</td><td>Go</td><td>Gin</td><td>Allah</td>
<td></td><td></td><td></td><td> 260</td><td></td><td></td><td></td><td></td><td> 265</td><td></td><td></td><td></td><td></td><td> 270</td><td></td><td></td>
<td>Ile</td><td>Read</td><td>Allah</td><td>Phe</td><td>Read</td><td>To be</td><td>Read</td><td>Gin</td><td>Arg</td><td>Thr</td><td>Gly</td><td>To be</td><td colspan="2">Gly Gly</td><td>Pro</td><td>Gly</td>
<td></td><td></td><td> 275</td><td></td><td></td><td></td><td></td><td> 280</td><td></td><td></td><td></td><td></td><td> 285</td><td></td><td></td><td></td>
<td>Asn</td><td>His</td><td>Pro</td><td>His</td><td>Gly</td><td>Pro</td><td>Asp</td><td>Allah</td><td>To be</td><td>Allah</td><td>Glu</td><td>Gly</td><td>Read</td><td>Asn</td><td>Pro</td><td>Tyr</td>
<td></td><td> 290</td><td></td><td></td><td></td><td></td><td> 295</td><td></td><td></td><td></td><td></td><td> 300</td><td></td><td></td><td></td><td></td>
<td>Gly</td><td>Read</td><td>Go</td><td>Allah</td><td>Pro</td><td>Arg</td><td>Phe</td><td>Gin</td><td>Arg</td><td>Lys</td><td>Phe</td><td>Lys</td><td>Allah</td><td>Lys</td><td>Gin</td><td>Read</td>
<td> 305</td><td></td><td></td><td></td><td></td><td> 310</td><td></td><td></td><td></td><td></td><td> 315</td><td></td><td></td><td></td><td></td><td> 320</td>
<td>Thr</td><td>Pro</td><td>Arg</td><td>Ile</td><td>Read</td><td>Glu</td><td>Allah</td><td>His</td><td>Gin</td><td>Asn</td><td>Go</td><td>Allah</td><td>Gin</td><td>Read</td><td>To be</td><td>Read</td>
<td></td><td></td><td></td><td></td><td> 325</td><td></td><td></td><td></td><td></td><td> 330</td><td></td><td></td><td></td><td></td><td> 335</td><td></td>
<td>Allah</td><td>Glu</td><td>Allah</td><td>Gin</td><td>Read</td><td>Arg</td><td>Phe</td><td>Ile</td><td>Gin</td><td>Allah</td><td>Trp</td><td>Gin</td><td>To be</td><td>Read</td><td>Pro</td><td>Asp</td>
<td></td><td></td><td></td><td> 340</td><td></td><td></td><td></td><td></td><td> 345</td><td></td><td></td><td></td><td></td><td> 350</td><td></td><td></td>
<td>Phe</td><td>Gly</td><td>Ile</td><td>To be</td><td>Tyr</td><td>Go</td><td>Met</td><td>Go</td><td>Arg</td><td>Phe</td><td>Lys</td><td>Gly</td><td>To be</td><td>Arg</td><td>Lys</td><td>Asp</td>
<td></td><td></td><td> 355</td><td></td><td></td><td></td><td></td><td> 360</td><td></td><td></td><td></td><td></td><td> 365</td><td></td><td></td><td></td>
245
<td>Glu</td><td>Ile</td><td>Read</td><td>Gly ile</td><td>Allah</td><td>Asn</td><td colspan="2">Asn arg</td><td>Read</td><td>Ile</td><td>Arg</td><td>Ile</td><td>Asp</td><td>Read</td><td>Allah</td>
<td></td><td> 370</td><td></td><td></td><td></td><td> 375</td><td></td><td></td><td></td><td></td><td> 380</td><td></td><td></td><td></td><td></td>
<td>Go</td><td colspan="2">Gly asp</td><td>Go Go</td><td>Lys</td><td>Thr</td><td colspan="2">Trp Arg</td><td>Phe</td><td>To be</td><td>Asn</td><td>Met</td><td>Arg</td><td>Gin</td><td>Trp</td>
<td> 385</td><td></td><td></td><td></td><td> 390</td><td></td><td></td><td></td><td></td><td> 395</td><td></td><td></td><td></td><td></td><td> 400</td>
<td>Asn</td><td>Go</td><td>Asn</td><td>Trp Asp</td><td>Ile</td><td>Arg</td><td>Gin</td><td>Go</td><td>Allah</td><td>Ile</td><td>Glu</td><td>Phe</td><td>Asp</td><td>Glu</td><td>His</td>
<td></td><td></td><td></td><td> 405</td><td></td><td></td><td></td><td></td><td> 410</td><td></td><td></td><td></td><td></td><td> 415</td><td></td>
<td>Ile</td><td>Asn</td><td>Go</td><td>Phe Wing</td><td>To be</td><td>Cys</td><td>Go</td><td>To be</td><td>Allah</td><td>To be</td><td>Cys</td><td>Arg</td><td>Ile</td><td>Go</td><td>His</td>
<td></td><td></td><td></td><td> 420</td><td></td><td></td><td></td><td> 425</td><td></td><td></td><td></td><td></td><td> 430</td><td></td><td></td>
<td>Glu</td><td>Tyr</td><td>Ile</td><td colspan="2">Gly Gly Tyr</td><td>Ile</td><td>Phe</td><td>Read</td><td>To be</td><td>Thr</td><td>Arg</td><td>Glu</td><td>Arg</td><td>Allah</td><td>Arg</td>
<td></td><td></td><td> 435</td><td></td><td></td><td></td><td> 440</td><td></td><td></td><td></td><td></td><td> 445</td><td></td><td></td><td></td>
<td colspan="2">Gly Glu</td><td>Glu</td><td colspan="3">Read Asp Glu Asp</td><td>Read</td><td>Phe</td><td>Read</td><td>Gin</td><td>Read</td><td>Thr</td><td colspan="2">Gly Gly</td><td>His</td>
<td></td><td> 450</td><td></td><td></td><td></td><td> 455</td><td></td><td></td><td></td><td></td><td> 460</td><td></td><td></td><td></td><td></td>
Glu Ala Phe
465 (2) INFORMATION FOR SEQ ID NO: 52:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 83 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 52:
<td rowspan="2">Met 1</td><td colspan="15">Arg Pro Gly Arg Gly Wing Gly Thr Pro Gly Arg Pro Gly Arg Gly</td>
<td colspan="5"> 5</td><td colspan="5"> 10</td><td colspan="5"> 15</td>
<td>Arg</td><td>Gly</td><td>Read</td><td>Allah</td><td>Allah</td><td>Thr</td><td>Cys</td><td>To be</td><td>Read</td><td>To be</td><td>To be</td><td>Pro</td><td>To be</td><td>His</td><td>Read</td><td>Read</td>
<td></td><td></td><td></td><td> 20</td><td></td><td></td><td></td><td></td><td> 25</td><td></td><td></td><td></td><td></td><td> 30</td><td></td><td></td>
<td>Pro</td><td>Thr</td><td>Read</td><td>Read</td><td>His</td><td>Thr</td><td>Phe</td><td>To be</td><td>Phe</td><td>To be</td><td>Read</td><td>Pro</td><td>Pro</td><td>Pro</td><td>To be</td><td>Pro</td>
<td></td><td></td><td> 35</td><td></td><td></td><td></td><td></td><td> 40</td><td></td><td></td><td></td><td></td><td> 45</td><td></td><td></td><td></td>
<td>Allah</td><td>Allah</td><td>Pro</td><td>Arg</td><td>Gin</td><td>Pro</td><td>To be</td><td>Pro</td><td>Pro</td><td>Allah</td><td>Read</td><td>Read</td><td>Read</td><td>Pro</td><td>Gly</td><td>Pro</td>
55 60
246
Gin Lys Pro Arg Pro Gly Asp Pro Tyr Thr Gly Wing Read Thr Asp
70 75 80
Trp Ser Xaa (2) INFORMATION FOR SEQ ID NO: 53:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 63 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 53:
<td>Met</td><td>Phe leu</td><td>Val</td><td>Phe</td><td>Phe</td><td>Read</td><td>To be</td><td>Phe</td><td>Phe</td><td>To be</td><td>His</td><td>To be</td><td>Ile</td><td>To be</td><td>Allah</td>
<td> 1</td><td></td><td></td><td> 5</td><td></td><td></td><td></td><td></td><td> 10</td><td></td><td></td><td></td><td></td><td> 15</td><td></td>
<td>Read</td><td>Thr leu</td><td>Val</td><td>Cys</td><td>To be</td><td>Gin</td><td colspan="3">Gly Gly Lys</td><td>Allah</td><td>Asp</td><td>Met</td><td>Asn</td><td>Read</td><td>Read</td>
<td></td><td></td><td> 20</td><td></td><td></td><td></td><td></td><td> 25</td><td></td><td></td><td></td><td></td><td> 30</td><td></td><td></td>
<td>To be</td><td>Trp Asp</td><td>Phe</td><td>Arg</td><td>Pro</td><td>His</td><td>Trp</td><td>Read</td><td>Glu</td><td>Gly</td><td>Ile</td><td>Arg</td><td>Phe</td><td>Read</td><td>Read</td>
<td></td><td> 35</td><td></td><td></td><td></td><td></td><td> 40</td><td></td><td></td><td></td><td></td><td> 45</td><td></td><td></td><td></td>
<td colspan="3">Gly Trp Gly Gin</td><td>Allah</td><td>Read</td><td>Met</td><td>Allah</td><td>Gly</td><td>Read</td><td>Phe</td><td>Pro</td><td>Trp</td><td>Read</td><td>Xaa</td><td></td>
<td></td><td> 50</td><td></td><td></td><td></td><td> 55</td><td></td><td></td><td></td><td></td><td> 60</td><td></td><td></td><td></td><td></td>
(2) INFORMATION FOR SEQ ID NO: 54:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 124 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear
247 (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 54:
<td>Met</td><td colspan="13">Arg Gly Be Trp His Arg Be Pro Read Pro Wing Go Go</td><td>Read</td><td>Pro</td>
<td> 1</td><td></td><td></td><td></td><td> 5</td><td></td><td></td><td></td><td></td><td> 10</td><td></td><td></td><td></td><td></td><td> 15</td><td></td>
<td>To be</td><td>Go</td><td>Read</td><td>Gin</td><td>Thr</td><td>Allah</td><td>Read</td><td>To be</td><td>Pro</td><td>Read</td><td>Allah</td><td>Read</td><td>Cys</td><td>Gin</td><td>Allah</td><td>Trp</td>
<td></td><td></td><td></td><td> 20</td><td></td><td></td><td></td><td></td><td> 25</td><td></td><td></td><td></td><td></td><td> 30</td><td></td><td></td>
<td>Arg</td><td>Arg</td><td>Allah</td><td>Go</td><td>Pro</td><td>His</td><td>Gly</td><td>Go</td><td>Pro</td><td>To be</td><td>Gin</td><td>Arg</td><td>Read</td><td>Arg</td><td>Asn</td><td>Gin</td>
<td></td><td></td><td> 35</td><td></td><td></td><td></td><td></td><td> 40</td><td></td><td></td><td></td><td></td><td> 45</td><td></td><td></td><td></td>
<td>Glu</td><td>Allah</td><td>To be</td><td>Read</td><td>Go</td><td>Pro</td><td>Lys</td><td colspan="2">Gly Vai</td><td>Pro</td><td>Arg</td><td>Allah</td><td>Trp</td><td>Tyr</td><td>Pro</td><td>Gly</td>
<td></td><td> 50</td><td></td><td></td><td></td><td></td><td> 55</td><td></td><td></td><td></td><td></td><td> 60</td><td></td><td></td><td></td><td></td>
<td>Pro</td><td>Read</td><td>Gin</td><td>Asn</td><td>Gly</td><td>Read</td><td>Trp</td><td>Thr</td><td>His</td><td>Read</td><td>Glu</td><td>Lys</td><td>Gly</td><td>Glu</td><td>Read</td><td>Read</td>
<td> 65</td><td></td><td></td><td></td><td></td><td> 70</td><td></td><td></td><td></td><td></td><td> 75</td><td></td><td></td><td></td><td></td><td> 80</td>
<td>Gly</td><td>Read</td><td>Lys</td><td>Pro</td><td>Thr</td><td>Pro</td><td colspan="2">Gly Gly</td><td>Read</td><td>Read</td><td>Read</td><td>Read</td><td>Arg</td><td>To be</td><td>Phe</td><td>Trp</td>
<td></td><td></td><td></td><td></td><td> 85</td><td></td><td></td><td></td><td></td><td> 90</td><td></td><td></td><td></td><td></td><td> 95</td><td></td>
<td>Asp</td><td>Pro</td><td>His</td><td>Pro</td><td>To be</td><td>Arg</td><td>Pro</td><td>Phe</td><td>Read</td><td>Cys</td><td>Thr</td><td>Read</td><td>Read</td><td>Pro</td><td>Pro</td><td>Pro</td>
<td></td><td></td><td></td><td> 100</td><td></td><td></td><td></td><td></td><td> 105</td><td></td><td></td><td></td><td></td><td> 110</td><td></td><td></td>
<td>Read</td><td>Xaa</td><td>Ile</td><td>Phe</td><td>Pro</td><td>Pro</td><td colspan="3">Read Arg Cys</td><td>To be</td><td>Allah</td><td>Xaa</td><td></td><td></td><td></td><td></td>
<td></td><td></td><td> 115</td><td></td><td></td><td></td><td></td><td> 120</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>
(2) INFORMATION FOR SEQ ID NO: 55:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 180 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 55:
Met Thr Be Ala Gly Pro Goes Xaa Leu Phe Leu Leu Will Be Lie Ser 15 10 15
Thr Be Go Ile Leu Met Gin His Leu Leu Xaa Wing Be Tyr Cys Asp 20 25 30
248
<td rowspan="2">Read</td><td colspan="4" rowspan="2">Read His Lys Wing Wing 35</td><td rowspan="2">Allah</td><td colspan="9">His Leu Gly Cys Trp Gin Lys Val Asp</td>
<td colspan="2"> 40</td><td colspan="7"> 45</td>
<td>Pro</td><td>Allah 50</td><td>Read</td><td>Cys</td><td>To be asn</td><td>Val 55</td><td>Read</td><td>Gin</td><td>His</td><td>Pro</td><td>Trp 60</td><td>Thr</td><td>Glu</td><td>Glu</td><td>Cys</td>
<td>Met 65</td><td>Trp</td><td>Pro</td><td>Gin</td><td>Gly val 70</td><td>Read</td><td>Val</td><td>Lys</td><td>His</td><td>To be 75</td><td>Lys</td><td>Asn</td><td>Val</td><td>Tyr</td><td>Lys 80</td>
<td>Allah</td><td>Val</td><td>Gly</td><td>Xaa</td><td>Xaa Xaa 85</td><td>Val</td><td>Allah</td><td>Ile</td><td>Pro 90</td><td>To be</td><td>Asp</td><td>Val</td><td>To be</td><td>His 95</td><td>Phe</td>
<td>Arg</td><td>Phe</td><td>Xaa</td><td>Phe 100</td><td>Phe Phe</td><td>To be</td><td>Lys</td><td>Pro 105</td><td>Read</td><td>Arg</td><td>Ile</td><td>Read</td><td>Asn 110</td><td>Ile</td><td>Read</td>
<td>Read</td><td>Read</td><td>Read 115</td><td>Glu</td><td>Gly Wing</td><td>Val</td><td>Ile 120</td><td>Val</td><td>Tyr</td><td>Gin</td><td>Read</td><td>Tyr 125</td><td>To be</td><td>Read</td><td>Met</td>
<td>To be</td><td>To be 130</td><td>Glu</td><td>Lys</td><td>Trp his</td><td>Gin 135</td><td>Thr</td><td>Ile</td><td>To be</td><td>Read</td><td>Allah 140</td><td>Read</td><td>Ile</td><td>Read</td><td>Phe</td>
<td>To be</td><td>Asn</td><td>Tyr</td><td>Tyr</td><td>Allah</td><td>Phe</td><td>Phe</td><td>Lys</td><td>Leu Leu</td><td>Arg</td><td>Asp</td><td>Arg</td><td>Read</td><td>Val</td><td>Read</td>
<td> 145</td><td></td><td></td><td></td><td></td><td> 150</td><td></td><td></td><td></td><td> 155</td><td></td><td></td><td></td><td></td><td> 160</td>
<td>Gly</td><td>Lys</td><td>Allah</td><td>Tyr</td><td>To be</td><td>Tyr</td><td>To be</td><td>Allah</td><td>To be pro</td><td>Gin</td><td>Arg</td><td>Asp</td><td>Read</td><td>Asp</td><td>His</td>
<td></td><td></td><td></td><td></td><td> 165</td><td></td><td></td><td></td><td> 170</td><td></td><td></td><td></td><td></td><td> 175</td><td></td>
<td>Arg</td><td>Phe</td><td>To be</td><td>xaa</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>
<td></td><td></td><td></td><td> 180</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>
(2) INFORMATION FOR SEQ ID NO: 56:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 287 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 56:
Met Pro Read Phe Lys Leu Tyr Met Val Met Ser Wing Cys Phe Leu Wing 15 10 15
249
<td rowspan="2">Allah</td><td colspan="2" rowspan="2">Gly ile</td><td colspan="2" rowspan="2">Phe Trp 20</td><td colspan="11">Val Ser Ile Read Cys Arg Asn Thr Tyr Ser Val</td>
<td colspan="4"> 25</td><td colspan="7"> 30</td>
<td>Phe</td><td>Lys</td><td>Ile</td><td>His</td><td>Trp</td><td>Read</td><td>Met</td><td>Allah</td><td>Allah</td><td>Read</td><td>Allah</td><td>Phe</td><td>Thr</td><td>Lys</td><td>To be</td><td>Ile</td>
<td></td><td></td><td> 35</td><td></td><td></td><td></td><td></td><td> 40</td><td></td><td></td><td></td><td></td><td> 45</td><td></td><td></td><td></td>
<td>To be</td><td>Read</td><td>Read</td><td>Phe</td><td>His</td><td>To be</td><td>Ile</td><td>Asn</td><td>Tyr</td><td>Tyr</td><td>Phe</td><td>Ile</td><td>Asn</td><td>To be</td><td>Gin</td><td>Gly</td>
<td></td><td> 50</td><td></td><td></td><td></td><td></td><td> 55</td><td></td><td></td><td></td><td></td><td> 60</td><td></td><td></td><td></td><td></td>
<td>Pro</td><td>Pro</td><td>His</td><td colspan="2">Arg Arg</td><td>Pro</td><td>Cys</td><td>Arg</td><td>His</td><td>Val</td><td>Read</td><td>His</td><td>Arg</td><td>Thr</td><td>Pro</td><td>Allah</td>
<td> 65</td><td></td><td></td><td></td><td></td><td> 70</td><td></td><td></td><td></td><td></td><td> 75</td><td></td><td></td><td></td><td></td><td> 80</td>
<td>Glu</td><td colspan="2">Gly arg</td><td>Pro</td><td>Pro</td><td>Read</td><td>His</td><td>His</td><td>His</td><td>Arg</td><td>Pro</td><td colspan="2">Asp Trp</td><td>Read</td><td>Arg</td><td>Read</td>
<td></td><td></td><td></td><td></td><td> 85</td><td></td><td></td><td></td><td></td><td> 90</td><td></td><td></td><td></td><td></td><td> 95</td><td></td>
<td>Gly</td><td>Phe</td><td>Ile</td><td>Lys</td><td>Tyr</td><td>Val</td><td>Read</td><td>To be</td><td>Asp</td><td>Lys</td><td>Glu</td><td>Lys</td><td>Lys</td><td>Val</td><td>Phe</td><td>Gly</td>
<td></td><td></td><td></td><td> 100</td><td></td><td></td><td></td><td></td><td> 105</td><td></td><td></td><td></td><td></td><td> 110</td><td></td><td></td>
<td>Ile</td><td>Val</td><td>Ile</td><td>Pro</td><td>Met</td><td>Gin</td><td>Val</td><td>Read</td><td>Allah</td><td>Asn</td><td>Val</td><td>Allah</td><td>Tyr</td><td>Ile</td><td>Ile</td><td>Ile</td>
<td></td><td></td><td> 115</td><td></td><td></td><td></td><td></td><td> 120</td><td></td><td></td><td></td><td></td><td> 125</td><td></td><td></td><td></td>
<td>Glu</td><td>To be</td><td>Arg</td><td>Glu</td><td>Glu</td><td>Gly</td><td>Allah</td><td>Thr</td><td>Asn</td><td>Tyr</td><td>Val</td><td>Read</td><td>Trp</td><td>Lys</td><td>Glu</td><td>Ile</td>
<td></td><td> 130</td><td></td><td></td><td></td><td></td><td> 135</td><td></td><td></td><td></td><td></td><td> 140</td><td></td><td></td><td></td><td></td>
<td>Read</td><td>Phe</td><td>Read</td><td>Val</td><td>Asp</td><td>Read</td><td>Ile</td><td>Cys</td><td>Cys</td><td>Gly</td><td>Allah</td><td>Ile</td><td>Read</td><td>Phe</td><td>Pro</td><td>Val</td>
<td> 145</td><td></td><td></td><td></td><td></td><td> 150</td><td></td><td></td><td></td><td></td><td> 155</td><td></td><td></td><td></td><td></td><td> 160</td>
<td>Val</td><td>Trp</td><td>To be</td><td>Ile</td><td>Arg</td><td>His</td><td>Read</td><td>Gin</td><td>Asp</td><td>Allah</td><td>To be</td><td>Gly</td><td>Thr</td><td colspan="3">Asp Gly Lys</td>
<td></td><td></td><td></td><td></td><td> 165</td><td></td><td></td><td></td><td></td><td> 170</td><td></td><td></td><td></td><td></td><td> 175</td><td></td>
<td>Val</td><td>Allah</td><td>Val</td><td>Asn</td><td>Read</td><td>Allah</td><td>Lys</td><td>Read</td><td>Lys</td><td>Read</td><td>Phe</td><td>Arg</td><td>His</td><td colspan="2">Tyr tyr</td><td>Val</td>
<td></td><td></td><td></td><td> 180</td><td></td><td></td><td></td><td></td><td> 185</td><td></td><td></td><td></td><td></td><td> 190</td><td></td><td></td>
<td>Met</td><td>Val</td><td>Ile</td><td colspan="2">Cys tyr</td><td>Val</td><td>Tyr</td><td>Phe</td><td>Thr</td><td>Arg</td><td>Ile</td><td colspan="2">Ile wing</td><td colspan="2">Ile leu</td><td>Read</td>
<td></td><td></td><td> 195</td><td></td><td></td><td></td><td></td><td> 200</td><td></td><td></td><td></td><td></td><td> 205</td><td></td><td></td><td></td>
<td>Gin</td><td>Val</td><td>Allah</td><td>Val</td><td>Pro</td><td>Phe</td><td>Gin</td><td>Trp</td><td>Gin</td><td>Trp</td><td>Read</td><td colspan="2">Tyr Xaa</td><td>Read</td><td>Read</td><td>Val</td>
<td></td><td> 210</td><td></td><td></td><td></td><td></td><td> 215</td><td></td><td></td><td></td><td></td><td> 220</td><td></td><td></td><td></td><td></td>
<td>Glu</td><td>Gly</td><td>To be</td><td>Thr</td><td>Read</td><td>Allah</td><td>Phe</td><td>Phe</td><td>Val</td><td>Read</td><td>Thr</td><td colspan="2">Gly tyr</td><td>Lys</td><td>Phe</td><td>Gin</td>
<td> 225</td><td></td><td></td><td></td><td></td><td> 230</td><td></td><td></td><td></td><td></td><td> 235</td><td></td><td></td><td></td><td></td><td> 240</td>
<td>Pro</td><td>Thr</td><td>Gly</td><td>Asn</td><td>Asn</td><td>Pro</td><td>Tyr</td><td>Read</td><td>Gin</td><td>Read</td><td>Pro</td><td>Gin</td><td>Glu</td><td>Asp</td><td>Glu</td><td>Glu</td>
<td></td><td></td><td></td><td></td><td> 245</td><td></td><td></td><td></td><td></td><td> 250</td><td></td><td></td><td></td><td></td><td> 255</td><td></td>
<td>Asp</td><td>Val</td><td>Gin</td><td>Met</td><td>Glu</td><td>Gin</td><td>Val</td><td>Met</td><td>Thr</td><td>Asp</td><td>To be</td><td>Gly</td><td>Phe</td><td>Arg</td><td>Glu</td><td>Gly</td>
<td></td><td></td><td></td><td> 260</td><td></td><td></td><td></td><td></td><td> 265</td><td></td><td></td><td></td><td></td><td> 270</td><td></td><td></td>
<td>Read</td><td>To be</td><td>Lys</td><td>Val</td><td>Asn</td><td>Lys</td><td>Thr</td><td>Allah</td><td>To be</td><td>Gly</td><td>Arg</td><td>Glu</td><td>Read</td><td>Read</td><td>Xaa</td><td></td>
<td></td><td></td><td> 275</td><td></td><td></td><td></td><td></td><td> 280</td><td></td><td></td><td></td><td></td><td> 285</td><td></td><td></td><td></td>
250 (2) INFORMATION FOR SEQ ID NO: 57:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 34 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 57:
Met Pro Met Go Phe Leu Leu Leu Phe Asn Leu Met Ser Trp Leu Ile 15 10 15
Arg Asn Ala Arg Go Ile Leu Arg Be Leu Asn Leu Lys Arg Asp Gin 20 25 30
Vai xaa (2) INFORMATION FOR SEQ ID NO: 58:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 58:
Met Lys Ile Go Go Read Leu Pro Leu Phe Leu Leu Wing Thr Phe Pro 15 10 15
Arg Lys Leu Gin Thr Cys Leu Xaa
251 (2) INFORMATION FOR SEQ ID NO: 59:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 47 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 59:
Met Ser Gly Gly Glu Gly Wing Ward Leu Pro Ile Leu Leu Leu Leu Leu
Wing Read Arg Gly Thr Phe His Gly Wing Arg Pro Gly Gly Gly Wing Ser 20 25 30
Gly He Trp Cys Leu Leu Leu Pro Glu Gin Glu Pro Pro Go Xaa
40 45 (2) INFORMATION FOR SEQ ID NO: 60:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 114 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 60:
Met Wing Arg Gly Ser Read Arg Arg 15
Leu Trp Leu Wing Leu Leu Arg Ser 20
Thr Wing Pro Cys Ser Arg Gly Ser 3540
Leu Leu Arg Leu Leu Go Leu Gly
1015
Go Wing Gly Glu Gin Wing Pro Gly
2530
Be Trp Be Asp Wing Read Asp Lys
252
<td>Cys</td><td colspan="2">Met Asp Cys</td><td>Allah</td><td>To be</td><td>Cys</td><td>Arg</td><td>Allah</td><td>Arg</td><td>Pro his</td><td>To be</td><td>Asp</td><td>Phe</td><td>Cys</td>
<td></td><td> 50</td><td></td><td></td><td></td><td> 55</td><td></td><td></td><td></td><td> 60</td><td></td><td></td><td></td><td></td>
<td>Read</td><td colspan="2">Gly Cys Wing</td><td>Allah</td><td>Allah</td><td>Pro</td><td>Pro</td><td>Allah</td><td>Pro</td><td>Phe arg</td><td>Read</td><td>Read</td><td>Trp</td><td>Pro</td>
<td> 65</td><td></td><td></td><td></td><td> 70</td><td></td><td></td><td></td><td></td><td> 75</td><td></td><td></td><td></td><td> 80</td>
<td>Ile</td><td>Read</td><td>Gly Gly</td><td>Allah</td><td>Read</td><td>To be</td><td>Read</td><td>Thr</td><td>Phe</td><td>Vai Leu</td><td>Gly</td><td>Read</td><td>Read</td><td>To be</td>
<td></td><td></td><td></td><td> 85</td><td></td><td></td><td></td><td></td><td> 90</td><td></td><td></td><td></td><td> 95</td><td></td>
<td>Gly</td><td>Phe</td><td>Leu Vai</td><td colspan="3">Trp Arg Arg</td><td>Cys</td><td>Arg</td><td>Arg</td><td>Glu Arg</td><td>To be</td><td>To be</td><td>Pro</td><td>Pro</td>
<td></td><td></td><td> 100</td><td></td><td></td><td></td><td></td><td> 105</td><td></td><td></td><td></td><td> 110</td><td></td><td></td>
Pro Xaa (2) INFORMATION FOR SEQ ID NO: 61:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 32 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 61:
Met Go Cys Ile Leu Go Leu Thr Leu Go Tyr Be Go Leu Go 15 10 15
Asn Ser Pro Leu Pro Phe Goes His Leu Xaa Goes Gly Ile Ser Ala Xaa 20 25 30 (2) SEQ ID: 62:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 81 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear
253 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 62:
<td>Met</td><td>Thr</td><td>Gly</td><td>Gly</td><td>Phe</td><td>Read</td><td>To be</td><td>Cys</td><td>Ile</td><td>Read</td><td>Gly</td><td>Read</td><td>Val</td><td>Read</td><td>Pro</td><td>Read</td>
<td> 1</td><td></td><td></td><td></td><td> 5</td><td></td><td></td><td></td><td></td><td> 10</td><td></td><td></td><td></td><td></td><td> 15</td><td></td>
<td>Allah</td><td>Tyr</td><td>Xaa</td><td>To be</td><td>To be</td><td>Read</td><td>Thr</td><td>Trp</td><td>Cys</td><td>Trp</td><td>Trp</td><td>Arg</td><td>Trp</td><td>Gly</td><td>Read</td><td>Pro</td>
<td></td><td></td><td></td><td> 20</td><td></td><td></td><td></td><td></td><td> 25</td><td></td><td></td><td></td><td></td><td> 30</td><td></td><td></td>
<td>Xaa</td><td>Pro</td><td>Allah</td><td>Gly</td><td>Pro</td><td>Pro</td><td>Arg</td><td>Cys</td><td>Thr</td><td>Pro</td><td>Gly</td><td>Cys</td><td>Asn</td><td>Allah</td><td>To be</td><td>Gly</td>
<td></td><td></td><td> 35</td><td></td><td></td><td></td><td></td><td> 40</td><td></td><td></td><td></td><td></td><td> 45</td><td></td><td></td><td></td>
<td>Allah</td><td>Gly</td><td>Arg</td><td>Gly</td><td>Pro</td><td>To be</td><td>Pro</td><td>Gly</td><td>Pro</td><td>Pro</td><td>Gly</td><td>Gly</td><td>Glu</td><td>Read</td><td>His</td><td>Thr</td>
<td></td><td> 50</td><td></td><td></td><td></td><td></td><td> 55</td><td></td><td></td><td></td><td></td><td> 60</td><td></td><td></td><td></td><td></td>
<td>Pro</td><td>Allah</td><td>To be</td><td>Arg</td><td>Asp</td><td>Pro</td><td>Gly</td><td>Pro</td><td>Gly</td><td>Allah</td><td>Glu</td><td>Trp</td><td>Arg</td><td>Gly</td><td>Thr</td><td>To be</td>
<td> 65</td><td></td><td></td><td></td><td></td><td> 70</td><td></td><td></td><td></td><td></td><td> 75</td><td></td><td></td><td></td><td></td><td> 80</td>
Xaa (2) INFORMATION FOR SEQ ID NO: 63:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 104 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 63:
<td rowspan="2">Met 1</td><td rowspan="2">Allah</td><td colspan="2" rowspan="2">Pro Val Wing 5th</td><td colspan="2" rowspan="2">Asp Leu</td><td colspan="8">Glu Leu Lys Lys Wing Phe Thr Glu Leu</td>
<td colspan="7"> 10</td><td> 15</td>
<td>Gin</td><td>Allah</td><td>Lys val 20</td><td>Ile</td><td>Asp</td><td>Thr</td><td>Gin</td><td>Gin 25</td><td>Lys</td><td>Val</td><td>Lys</td><td>Read</td><td>Allah 30</td><td>Asp Ile</td>
<td>Gin</td><td>Ile</td><td>Glu Gin 35</td><td>Read</td><td>Asn</td><td>Arg</td><td>Thr 40</td><td>Lys</td><td>Lys</td><td>His</td><td>Allah</td><td>His 45</td><td>Read</td><td>Thr asp</td>
<td>Thr</td><td>Glu 50</td><td>Ile met</td><td>Thr</td><td>Read</td><td>Val 55</td><td>Asp</td><td>Glu</td><td>Thr</td><td>Asn</td><td>Met 60</td><td>Tyr</td><td>Glu</td><td>Gly val</td>
<td colspan="2">Gly arg 65</td><td>Met phe</td><td>Ile</td><td>Read 70</td><td>Gin</td><td>To be</td><td>Lys</td><td>Glu</td><td>Allah 75</td><td>Ile</td><td>His</td><td>To be</td><td>Gin Leu 80</td>
254
Read Glu Lys Gin Lys Ile Wing Glu Glu Lys Ile Lys Glu
90 95
Lys Lys Ser Tyr Leu Glu Arg Arg
100 (2) INFORMATION FOR SEQ ID NO: 64:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 146 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 64:
<td colspan="2" rowspan="2">Met pro 1</td><td rowspan="2">To be</td><td colspan="7">Gly Phe Gin Thr Cys Leu Leu</td><td rowspan="2">Phe</td><td colspan="3" rowspan="2">Thr Leu Ser</td><td rowspan="2">Pro 15</td><td rowspan="2">Phe</td>
<td colspan="3"> 5</td><td colspan="4"> 10</td>
<td>To be</td><td>Read</td><td>To be</td><td>Lys</td><td colspan="2">Ile Vai</td><td>Gly</td><td>Go</td><td>Pro</td><td>To be</td><td>Gin</td><td>Gin</td><td>Read</td><td>Pro</td><td colspan="2">Gly gin</td>
<td></td><td></td><td></td><td> 20</td><td></td><td></td><td></td><td></td><td> 25</td><td></td><td></td><td></td><td></td><td> 30</td><td></td><td></td>
<td>Read</td><td>To be</td><td>Glu</td><td colspan="3">Gin Gly Gly</td><td>Read</td><td>Cys</td><td>Gly</td><td>His</td><td>Glu</td><td>Gly</td><td>Glu</td><td>Pro</td><td>Allah</td><td>Arg</td>
<td></td><td></td><td> 35</td><td></td><td></td><td></td><td></td><td> 40</td><td></td><td></td><td></td><td></td><td> 45</td><td></td><td></td><td></td>
<td>Thr</td><td>Go</td><td>Pro</td><td>Glu</td><td>Thr</td><td>Gin</td><td>Read</td><td>Pro</td><td>Read</td><td>Pro</td><td>Phe</td><td>Asn</td><td>To be</td><td>Allah</td><td>Gly</td><td>Pro</td>
<td></td><td> 50</td><td></td><td></td><td></td><td></td><td> 55</td><td></td><td></td><td></td><td></td><td> 60</td><td></td><td></td><td></td><td></td>
<td>Pro</td><td>His</td><td>Read</td><td>Lys</td><td>cys</td><td>Thr</td><td colspan="3">Gly Wing Gly</td><td>Lys</td><td>Arg</td><td>Go</td><td>Trp</td><td>To be</td><td>Pro</td><td>Pro</td>
<td> 65</td><td></td><td></td><td></td><td></td><td> 70</td><td></td><td></td><td></td><td></td><td> 75</td><td></td><td></td><td></td><td></td><td> 80</td>
<td>Arg</td><td>Arg</td><td>Allah</td><td>Allah</td><td>Gin</td><td>Glu</td><td>Go</td><td>To be</td><td>Read</td><td>Gin</td><td>Read</td><td>Go</td><td>To be</td><td>Cys</td><td>His</td><td>Pro</td>
<td></td><td></td><td></td><td></td><td> 85</td><td></td><td></td><td></td><td></td><td> 90</td><td></td><td></td><td></td><td></td><td> 95</td><td></td>
<td>Cys</td><td>Arg</td><td>Gin</td><td>His</td><td>Thr</td><td>To be</td><td>Arg</td><td>Allah</td><td>Phe</td><td>To be</td><td>Read</td><td>Allah</td><td>Thr</td><td colspan="2">Asp Arg</td><td>Thr</td>
<td></td><td></td><td></td><td> 100</td><td></td><td></td><td></td><td></td><td> 105</td><td></td><td></td><td></td><td></td><td> 110</td><td></td><td></td>
<td>Allah</td><td>To be</td><td>Allah</td><td>Arg</td><td>Go</td><td>Cys</td><td colspan="2">Cys arg</td><td>To be</td><td>Pro</td><td>Read</td><td>To be</td><td>Thr</td><td>Read</td><td>Ile</td><td>His</td>
<td></td><td></td><td> 115</td><td></td><td></td><td></td><td></td><td> 120</td><td></td><td></td><td></td><td></td><td> 125</td><td></td><td></td><td></td>
<td>His</td><td>Thr</td><td>Arg</td><td colspan="2">Gly Gly</td><td>Gin</td><td colspan="2">Arg cys</td><td>Arg</td><td>Glu</td><td>His</td><td>Gly</td><td>Read</td><td>To be</td><td>Read</td><td>Pro</td>
<td></td><td> 130</td><td></td><td></td><td></td><td></td><td> 135</td><td></td><td></td><td></td><td></td><td> 140</td><td></td><td></td><td></td><td></td>
Read Xaa
145
255 (2) INFORMATION FOR SEQ ID NO: 65:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 31 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 65:
<td>Met</td><td>Wing Ile Leu Met Leu Leu Wing</td><td>Gly</td><td>To be</td><td>Pro cys</td><td>Thr</td><td>Read</td><td>Be Phe</td>
<td> 1</td><td> 5</td><td></td><td> 10</td><td></td><td></td><td></td><td> 15</td>
<td>To be</td><td>Thr Asp Thr Gly Ser Ser Ala</td><td>Pro</td><td>Gly</td><td>Pro lys</td><td>Ile</td><td>Pro</td><td>Xaa</td>
<td></td><td> 20</td><td> 25</td><td></td><td></td><td></td><td> 30</td><td></td>
<td> 2)</td><td>SEQ INFORMATION</td><td>ID</td><td>No.</td><td> : 66:</td><td></td><td></td><td></td>
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 260 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 66:
<td>Met</td><td>Asp</td><td>Pro</td><td>Gin</td><td>Gly</td><td>Gin</td><td>Thr</td><td>Read</td><td>Read</td><td>Read</td><td>Phe</td><td>Read</td><td>Phe</td><td>Go</td><td>Asp</td><td>Phe</td>
<td> 1</td><td></td><td></td><td></td><td> 5</td><td></td><td></td><td></td><td></td><td> 10</td><td></td><td></td><td></td><td></td><td> 15</td><td></td>
<td>His</td><td>To be</td><td>Allah</td><td>Phe</td><td>Pro</td><td>Go</td><td>Gin</td><td>Gin</td><td>Met</td><td>Glu</td><td>Ile</td><td colspan="2">Trp gly</td><td>Go</td><td>Tyr</td><td>Thr</td>
<td></td><td></td><td></td><td> 20</td><td></td><td></td><td></td><td></td><td> 25</td><td></td><td></td><td></td><td></td><td> 30</td><td></td><td></td>
<td>Read</td><td>Read</td><td>Thr</td><td>Thr</td><td>His</td><td>Read</td><td>Asn</td><td>Allah</td><td>Ile</td><td>Read</td><td>Go</td><td>Glu</td><td>To be</td><td>His</td><td>To be</td><td>Go</td>
<td></td><td></td><td> 35</td><td></td><td></td><td></td><td></td><td> 40</td><td></td><td></td><td></td><td></td><td> 45</td><td></td><td></td><td></td>
<td>Go</td><td>Gin</td><td>Gly</td><td>To be</td><td>Ile</td><td>Gin</td><td>Phe</td><td>Thr</td><td>Go</td><td>Asp</td><td>Lys</td><td>Go</td><td>Read</td><td>Glu</td><td>Gin</td><td>His</td>
<td></td><td> 50</td><td></td><td></td><td></td><td></td><td> 55</td><td></td><td></td><td></td><td></td><td> 60</td><td></td><td></td><td></td><td></td>
256
<td rowspan="2">His 65</td><td colspan="4" rowspan="2">Gin Wing Wing Lys</td><td colspan="2" rowspan="2">Gin Wing 70</td><td colspan="9">Gin Lys Leu Gin Wing Be Read Be Go</td>
<td colspan="4"> 75</td><td colspan="5"> 80</td>
<td>Allah</td><td>Go</td><td>Asn</td><td>To be</td><td>Ile</td><td>Met</td><td>To be</td><td>Ile</td><td>Read</td><td>Thr</td><td>Gly</td><td>To be</td><td>Thr</td><td>Arg</td><td>To be</td><td>To be</td>
<td></td><td></td><td></td><td></td><td> 85</td><td></td><td></td><td></td><td></td><td> 90</td><td></td><td></td><td></td><td></td><td> 95</td><td></td>
<td>Phe</td><td>Arg</td><td>Lys</td><td>Met</td><td>Cys</td><td>Read</td><td>Gin</td><td>Thr</td><td>Read</td><td>Gin</td><td>Allah</td><td>Allah</td><td>Asp</td><td>Thr</td><td>Gin</td><td>Glu</td>
<td></td><td></td><td></td><td> 100</td><td></td><td></td><td></td><td></td><td> 105</td><td></td><td></td><td></td><td></td><td>Ί10</td><td></td><td></td>
<td>Phe</td><td>Arg</td><td>Thr</td><td>Lys</td><td>Read</td><td>His</td><td>Lys</td><td>Go</td><td>Phe</td><td>Arg</td><td>Glu</td><td>Ile</td><td>Thr</td><td>Gin</td><td>His</td><td>Gin</td>
<td></td><td></td><td> 115</td><td></td><td></td><td></td><td></td><td> 120</td><td></td><td></td><td></td><td></td><td> 125</td><td></td><td></td><td></td>
<td>Phe</td><td>Read</td><td>His</td><td>His</td><td>Cys</td><td>To be</td><td>Cys</td><td>Glu</td><td>Go</td><td>Lys</td><td>Gin</td><td>Read</td><td>Thr</td><td>Read</td><td>Glu</td><td>Lys</td>
<td></td><td> 130</td><td></td><td></td><td></td><td></td><td> 135</td><td></td><td></td><td></td><td></td><td> 140</td><td></td><td></td><td></td><td></td>
<td>Lys</td><td>Asp</td><td>To be</td><td>Allah</td><td>Gin</td><td>Gly</td><td>Thr</td><td>Glu</td><td>Asp</td><td>Allah</td><td>Pro</td><td>Asp</td><td>Asn</td><td>To be</td><td>To be</td><td>Read</td>
<td> 145</td><td></td><td></td><td></td><td></td><td> 150</td><td></td><td></td><td></td><td></td><td> 155</td><td></td><td></td><td></td><td></td><td> 160</td>
<td>Glu</td><td>Read</td><td>L me</td><td>Allah</td><td>Asp</td><td>Thr</td><td>To be</td><td>Gly</td><td>Gin</td><td>Allah</td><td>Glu</td><td>Asn</td><td>Lys</td><td>Arg</td><td>Read</td><td>Lys</td>
<td></td><td></td><td></td><td></td><td> 165</td><td></td><td></td><td></td><td></td><td> 170</td><td></td><td></td><td></td><td></td><td> 175</td><td></td>
<td colspan="2">Arg Gly</td><td>To be</td><td>Pro</td><td>Arg</td><td>Ile</td><td>Glu</td><td>Glu</td><td>Met</td><td>Arg</td><td>Allah</td><td>Read</td><td>Arg</td><td>To be</td><td>Allah</td><td>Arg</td>
<td></td><td></td><td></td><td> 180</td><td></td><td></td><td></td><td></td><td> 185</td><td></td><td></td><td></td><td></td><td> 190</td><td></td><td></td>
<td>Allah</td><td>Pro</td><td>To be</td><td>Pro</td><td>To be</td><td>Glu</td><td>Allah</td><td>Allah</td><td>Pro</td><td colspan="2">Arg Arg</td><td>Pro</td><td>Glu</td><td>Allah</td><td>Thr</td><td>Allah</td>
<td></td><td></td><td> 195</td><td></td><td></td><td></td><td></td><td> 200</td><td></td><td></td><td></td><td></td><td> 205</td><td></td><td></td><td></td>
<td>Allah</td><td>Pro</td><td>Read</td><td>Thr</td><td>Pro</td><td>Arg</td><td colspan="2">Gly arg</td><td>Glu</td><td>His</td><td>Arg</td><td>Glu</td><td>Allah</td><td>His</td><td colspan="2">Gly arg</td>
<td></td><td> 210</td><td></td><td></td><td></td><td></td><td> 215</td><td></td><td></td><td></td><td></td><td> 220</td><td></td><td></td><td></td><td></td>
<td>Allah</td><td>Read</td><td>Allah</td><td>Pro</td><td colspan="2">Gly arg</td><td>Allah</td><td>To be</td><td>Read</td><td>Gly</td><td>To be</td><td>Arg</td><td>Read</td><td>Glu</td><td>Asp</td><td>Go</td>
<td> 225</td><td></td><td></td><td></td><td></td><td> 230</td><td></td><td></td><td></td><td></td><td> 235</td><td></td><td></td><td></td><td></td><td> 240</td>
<td>Read</td><td>Trp</td><td>Read</td><td>Gin</td><td colspan="2">Glu Vai</td><td>To be</td><td>Asn</td><td>Read</td><td>To be</td><td>Glu</td><td>Trp</td><td>Read</td><td>To be</td><td>Pro</td><td>To be</td>
245 250 255
Pro Gly Pro Xaa
260 (2) INFORMATION FOR SEQ ID NO: 67:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 23 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear
257 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 67:
Met Wing Wing Cys Wing Gly Pro Gly Wing Wing Gly Wing Wing Cys Ser Ser 15 10 15
Cys Wing Ile Cys Phe Cys Xaa (2) INFORMATION FOR SEQ ID NO: 68:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 27 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 68:
Met His Wing Leu Ile Leu Gin Phe Ile Phe Ser Leu Cys Met Tyr Ile 15 10 15
Be Read Phe Be Ward Wing Arg Phe Read Phe Xaa
25 (2) INFORMATION FOR SEQ ID NO: 69:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear
258 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 69:
Leu Leu Leu Leu Cys Phe Cys Cys His Pro Thr His Leu Gin Gly Xaa 15 10 15
Trp Wing Leu Asp Leu Gly Leu Phe Pro Phe Asn Cys Xaa
25 (2) INFORMATION FOR SEQ ID NO: 70:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 216 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 70:
<td colspan="4" rowspan="2">Met Tyr Leu Ser 1</td><td colspan="12">Ile Ile Phe Read Ala Phe Will Be He Asp Arg Cys</td>
<td colspan="2"> 5</td><td colspan="2" rowspan="2">Cys lys</td><td colspan="6"> 10</td><td colspan="2" rowspan="2">15 Pro gly</td>
<td colspan="3">Leu Gin Leu</td><td>Thr 20</td><td>His</td><td>To be</td><td>He 25</td><td>Tyr</td><td>Arg</td><td>He</td><td>Gin</td><td>Glu 30</td>
<td>Phe</td><td>Allah</td><td>Lys</td><td>Met</td><td>Ile</td><td>To be</td><td>Thr</td><td>Go</td><td>Go</td><td>Trp</td><td>Read</td><td>Met</td><td>Go</td><td>Read</td><td>Read</td><td>Ile</td>
<td></td><td></td><td> 35</td><td></td><td></td><td></td><td></td><td> 40</td><td></td><td></td><td></td><td></td><td> 45</td><td></td><td></td><td></td>
<td>Met</td><td>Go</td><td>Pro</td><td>Asn</td><td>Met</td><td>Met</td><td>Ile</td><td>Pro</td><td>He</td><td>Lys</td><td>Asp</td><td>Ile</td><td>Lys</td><td>Glu</td><td>Lys</td><td>To be</td>
<td></td><td> 50</td><td></td><td></td><td></td><td></td><td> 55</td><td></td><td></td><td></td><td></td><td> 60</td><td></td><td></td><td></td><td></td>
<td>Asn</td><td>Go</td><td colspan="2">Gly cys</td><td>Met</td><td>Glu</td><td>Phe</td><td>Lys</td><td>Lys</td><td>Glu</td><td>Phe</td><td colspan="2">Gly arg</td><td>Asn</td><td>Trp</td><td>His</td>
<td> 65</td><td></td><td></td><td></td><td></td><td> 70</td><td></td><td></td><td></td><td></td><td> 75</td><td></td><td></td><td></td><td></td><td> 80</td>
<td>Read</td><td>Read</td><td>Thr</td><td>Asn</td><td>Phe</td><td>Ile</td><td>Cys</td><td>Go</td><td>Allah</td><td>Ile</td><td>Phe</td><td>Read</td><td>Asn</td><td>Phe</td><td>To be</td><td>Allah</td>
<td></td><td></td><td></td><td></td><td> 85</td><td></td><td></td><td></td><td></td><td> 90</td><td></td><td></td><td></td><td></td><td> 95</td><td></td>
<td>Ile</td><td>Ile</td><td>Read</td><td>Ile</td><td>To be</td><td>Asn</td><td>Cys</td><td>Read</td><td>Go</td><td>Ile</td><td>Arg</td><td>Gin</td><td>Read</td><td colspan="2">Tyr arg</td><td>Asn</td>
<td></td><td></td><td></td><td> 100</td><td></td><td></td><td></td><td></td><td> 105</td><td></td><td></td><td></td><td></td><td> 110</td><td></td><td></td>
<td>Lys</td><td>Asp</td><td>Asn</td><td>Glu</td><td>Asn</td><td>Tyr</td><td>Pro</td><td>Asn</td><td>Go</td><td>Lys</td><td>Lys</td><td>Allah</td><td>Read</td><td colspan="2">Ile asn</td><td>He</td>
<td></td><td></td><td> 115</td><td></td><td></td><td></td><td></td><td> 120</td><td></td><td></td><td></td><td></td><td> 125</td><td></td><td></td><td></td>
259
<td>Read</td><td>Read</td><td>Go</td><td>Thr</td><td>Thr</td><td>Gly</td><td>Tyr</td><td>Ile</td><td>Ile</td><td>Cys</td><td>Phe</td><td>Go</td><td>Pro</td><td>Tyr</td><td>His</td><td>Ile</td>
<td></td><td> 130</td><td></td><td></td><td></td><td></td><td> 135</td><td></td><td></td><td></td><td></td><td> 140</td><td></td><td></td><td></td><td></td>
<td>Go</td><td>Arg</td><td>Ile</td><td>Pro</td><td>Tyr</td><td>Thr</td><td>Read</td><td>To be</td><td>Gin</td><td>Thr</td><td>Glu</td><td>Go</td><td>Ile</td><td>Thr</td><td>Asp</td><td>Cys</td>
<td> 145</td><td></td><td></td><td></td><td></td><td> 150</td><td></td><td></td><td></td><td></td><td> 155</td><td></td><td></td><td></td><td></td><td> 160</td>
<td>To be</td><td>Thr</td><td>Arg</td><td>Ile</td><td>To be</td><td>Read</td><td>Phe</td><td>Lys</td><td>Allah</td><td>Lys</td><td>Glu</td><td>Allah</td><td>Thr</td><td>Read</td><td>Read</td><td>Read</td>
<td></td><td></td><td></td><td></td><td> 165</td><td></td><td></td><td></td><td></td><td> 170</td><td></td><td></td><td></td><td></td><td> 175</td><td></td>
<td>Allah</td><td>Go</td><td>To be</td><td>Asn</td><td>Read</td><td>Cys</td><td>Phe</td><td>Asp</td><td>Pro</td><td>Ile</td><td>Read</td><td colspan="2">Tyr tyr</td><td>His</td><td>Read</td><td>To be</td>
<td></td><td></td><td></td><td> 180</td><td></td><td></td><td></td><td></td><td> 185</td><td></td><td></td><td></td><td></td><td> 190</td><td></td><td></td>
<td>Lys</td><td>Allah</td><td>Phe</td><td>Arg</td><td>To be</td><td>Lys</td><td>Go</td><td>Thr</td><td>Glu</td><td>Thr</td><td>Phe</td><td>Allah</td><td>To be</td><td>Pro</td><td>Lys</td><td>Glu</td>
<td></td><td></td><td> 195</td><td></td><td></td><td></td><td></td><td> 200</td><td></td><td></td><td></td><td></td><td> 205</td><td></td><td></td><td></td>
<td>Thr</td><td>Lys</td><td>Go</td><td>Arg</td><td>Lys</td><td>Lys</td><td>Asn</td><td>Xaa</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>
<td></td><td> 210</td><td></td><td></td><td></td><td></td><td> 215</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>
<td> (2)</td><td colspan="4">INFORMATION</td><td colspan="3">FOR SEQ</td><td>ID</td><td>No.</td><td> 71</td><td></td><td></td><td></td><td></td><td></td>
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 407 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 71:
<td rowspan="2">Met 1</td><td rowspan="2">His</td><td colspan="14">Pro Wing Go Phe Leu Be Leu Pro Asp Leu Arg Cys Be Leu</td>
<td colspan="4"> 5</td><td colspan="7"> 10</td><td colspan="3"> 15</td>
<td>Read</td><td>Read</td><td colspan="2">Leu Vai</td><td>Thr</td><td>Trp</td><td>Go</td><td>Phe</td><td>Thr</td><td>Pro</td><td>Go</td><td>Thr</td><td>Thr</td><td>Glu</td><td>Ile</td><td>Thr</td>
<td></td><td></td><td></td><td> 20</td><td></td><td></td><td></td><td></td><td> 25</td><td></td><td></td><td></td><td></td><td> 30</td><td></td><td></td>
<td>To be</td><td>Read</td><td>Asp</td><td>Thr</td><td>Glu</td><td>Asn</td><td>Ile</td><td>Asp</td><td>Glu</td><td>Ile</td><td>Read</td><td>Asn</td><td>Asn</td><td>Allah</td><td>ASp</td><td>Go</td>
<td></td><td></td><td> 35</td><td></td><td></td><td></td><td></td><td> 40</td><td></td><td></td><td></td><td></td><td> 45</td><td></td><td></td><td></td>
<td>Allah</td><td>Read</td><td>Go</td><td>Asn</td><td>Phe</td><td>Tyr</td><td>Allah</td><td colspan="2">Asp Trp</td><td>Cys</td><td>Arg</td><td>Phe</td><td>To be</td><td>Gin</td><td>Met</td><td>Read</td>
<td></td><td> 50</td><td></td><td></td><td></td><td></td><td> 55</td><td></td><td></td><td></td><td></td><td> 60</td><td></td><td></td><td></td><td></td>
<td>His</td><td>Pro</td><td>Ile</td><td>Phe</td><td>Glu</td><td>Glu</td><td>Allah</td><td colspan="2">Be asp</td><td>Go</td><td>Ile</td><td>Lys</td><td>Glu</td><td>Glu</td><td>Phe</td><td>Pro</td>
70 75 80
260
<td colspan="4" rowspan="2">Asn Glu Asn Gin</td><td rowspan="2">Go 85</td><td rowspan="2">Go</td><td colspan="9">Phe Ala Arg Goes Asp Cys Asp Gin His Ser</td>
<td colspan="6"> 90</td><td></td><td colspan="2"> 95</td>
<td>Asp</td><td>Ile</td><td>Allah</td><td>Gin 100</td><td>Arg</td><td>Tyr</td><td>Arg</td><td>Ile</td><td>To be 105</td><td>Lys</td><td>Tyr</td><td>Pro</td><td>Thr leu 110</td><td>Lys</td><td>Read</td>
<td>Phe</td><td>Arg</td><td>Asn 115</td><td>Gly</td><td>Met</td><td>Met</td><td>Met</td><td>Lys 120</td><td>Arg</td><td>Glu</td><td>Tyr</td><td>Arg</td><td>Gly gin 125</td><td>Arg</td><td>To be</td>
<td>Go</td><td>Lys 130</td><td>Allah</td><td>Read</td><td>Allah</td><td>Asp</td><td>Tyr 135</td><td>Ile</td><td>Arg</td><td colspan="2">Gin Gin</td><td>Lys 140</td><td>Be asp</td><td>Pro</td><td>Ile</td>
<td>Gin 145</td><td>Glu</td><td>Ile</td><td colspan="2">Arg Asp</td><td>Read 150</td><td>Allah</td><td>Glu</td><td>Ile</td><td>Thr</td><td>Thr 155</td><td>Read</td><td>Asp Arg</td><td>To be</td><td>Lys 160</td>
<td>Arg</td><td>Asn</td><td>Ile</td><td>Ile</td><td colspan="2">Gly tyr 165</td><td>Phe</td><td>Glu</td><td>Gin</td><td>Lys 170</td><td>Asp</td><td>To be</td><td>Asp Asn</td><td colspan="2">Tyr arg 175</td>
<td>Go</td><td>Phe</td><td>Glu</td><td>Arg 180</td><td>Go</td><td>Allah</td><td>Asn</td><td>Ile</td><td>Read 185</td><td>His</td><td>Asp</td><td>Asp</td><td>Cys Wing 190</td><td>Phe</td><td>Read</td>
<td>To be</td><td>Allah</td><td>Phe 195</td><td colspan="2">Gly asp</td><td>Go</td><td>To be</td><td>Lys 200</td><td>Pro</td><td>Glu</td><td>Arg</td><td>Tyr</td><td colspan="2">To be Gly Asp 205</td><td>Asn</td>
<td>Ile</td><td>Ile 210</td><td>Tyr</td><td>Lys</td><td>Pro</td><td>Pro</td><td>Gly 215</td><td>HiS</td><td>To be</td><td>Allah</td><td>Pro</td><td>Asp 220</td><td>Met Vai</td><td>Tyr</td><td>Read</td>
<td>Gly 225</td><td>Allah</td><td>Met</td><td>Thr</td><td>Asn</td><td>Phe 230</td><td>Asp</td><td>Go</td><td>Thr</td><td>Tyr</td><td>Asn 235</td><td>Trp</td><td>Ile gin</td><td>Asp</td><td>Lys 240</td>
<td>Cys</td><td>Go</td><td>Pro</td><td>Read</td><td>Go 245</td><td>Arg</td><td>Glu</td><td>Ile</td><td>Thr</td><td>Phe 250</td><td>Glu</td><td>Asn</td><td>Gly Glu</td><td>Glu 255</td><td>Read</td>
<td>Thr</td><td>Glu</td><td>Glu</td><td>Gly 260</td><td>Read</td><td>Pro</td><td>Phe</td><td>Read</td><td>Ile 265</td><td>Read</td><td>Phe</td><td>His</td><td>Met lys 270</td><td>Glu</td><td>Asp</td>
<td>Thr</td><td>Glu</td><td>To be 275</td><td>Read</td><td>Glu</td><td>Ile</td><td>Phe</td><td>Gin 280</td><td>Asn</td><td>Glu</td><td>Go</td><td>Allah</td><td>Arg gin 285</td><td>Read</td><td>Ile</td>
<td>To be</td><td>Glu 290</td><td>Lys</td><td>Gly</td><td>Thr</td><td>Ile</td><td>Asn 295</td><td>Phe</td><td>Read</td><td>His</td><td>Allah</td><td colspan="2">Asp Cys Asp 300</td><td>Lys</td><td>Phe</td>
<td>Arg 305</td><td>His</td><td>Pro</td><td>Read</td><td>Read</td><td>His 310</td><td>Ile</td><td>Gin</td><td>Lys</td><td>Thr</td><td>Pro 315</td><td>Allah</td><td>Asp Cys</td><td>Pro</td><td>Go 320</td>
<td>Ile</td><td>Allah</td><td>lie</td><td>Asp</td><td>To be 325</td><td>Phe</td><td>Arg</td><td>His</td><td>Met</td><td>Tyr 330</td><td>Go</td><td>Phe</td><td>Gly asp</td><td>Phe 335</td><td>Lys</td>
<td>Asp</td><td>Go</td><td>Read</td><td>Ile 340</td><td>Pro</td><td>Gly</td><td>Lys</td><td>Read</td><td>Lys 345</td><td>Gin</td><td>Phe</td><td>Go</td><td>Phe asp 350</td><td>Read</td><td>His</td>
<td>To be</td><td>Gly</td><td>Lys 355</td><td>Read</td><td>His</td><td>Arg</td><td>Glu</td><td>Phe 360</td><td>His</td><td>His</td><td>Gly</td><td>Pro</td><td>Asp Pro 365</td><td>Thr</td><td>Asp</td>
<td>Thr</td><td>Allah 370</td><td>Pro</td><td>Gly</td><td>Glu</td><td>Gin</td><td colspan="2">Gin Wing 375</td><td>Asp</td><td>Go</td><td>Allah</td><td>To be 380</td><td>To be pro</td><td>Pro</td><td>Glu</td>
<td>To be 385</td><td>To be</td><td>Phe</td><td>Gin</td><td>Lys</td><td>Read 390</td><td>Allah</td><td>Pro</td><td>To be</td><td>Glu</td><td>Tyr 395</td><td>Arg</td><td>Tyr thr</td><td>Read</td><td>Read 400</td>
261
Arg Asp Arg Asp Glu Leu Xaa
405 (2) INFORMATION FOR SEQ ID NO: 72:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 72:
Tyr Leu Ile Ser Tyr Leu Cys Phe Xaa
5th (2) INFORMATION FOR SEQ ID NO: 73:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 34 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 73:
Met Pro Leu Lys Wing Val Thr Trp Pro Leu Thr Asn Ser Lys Leu Val 15 10 15
Wing Wing Val Val Asn Leu Lys Wing Be Gin Met Pro Wing Be Ser Arg 20 25 30
Val Xaa
262 (2) INFORMATION FOR SEQ ID NO: 74:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 57 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 74:
<td>Gin</td><td>To be</td><td>Pro</td><td>Arg</td><td>To be</td><td>To be</td><td>Allah</td><td>Read</td><td>Gly</td><td>Allah</td><td>Gly</td><td>Gin</td><td>Lys</td><td>Read</td><td>Allah</td><td>Go</td>
<td> 1</td><td></td><td></td><td></td><td> 5</td><td></td><td></td><td></td><td></td><td> 10</td><td></td><td></td><td></td><td></td><td> 15</td><td></td>
<td>Cys</td><td>To be</td><td>Pro</td><td>Asp</td><td>Ile</td><td>Read</td><td>Cys</td><td>Cys</td><td>Pro</td><td>Thr</td><td>Asp</td><td>Thr</td><td>Read</td><td>Read</td><td>Allah</td><td>To be</td>
<td></td><td></td><td></td><td> 20</td><td></td><td></td><td></td><td></td><td> 25</td><td></td><td></td><td></td><td></td><td> 30</td><td></td><td></td>
<td>His</td><td>Pro</td><td>His</td><td>To be</td><td>Read</td><td>Read</td><td>Thr</td><td>Gly</td><td>Thr</td><td>Gin</td><td>Phe</td><td>To be</td><td>Gly</td><td>Gin</td><td>Thr</td><td>Gin</td>
<td></td><td></td><td> 35</td><td></td><td></td><td></td><td></td><td> 40</td><td></td><td></td><td></td><td></td><td> 45</td><td></td><td></td><td></td>
<td>Allah</td><td>Read</td><td>Allah</td><td>Pro</td><td>To be</td><td>Trp</td><td>Cys</td><td>Allah</td><td>Xaa</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>
<td></td><td> 50</td><td></td><td></td><td></td><td></td><td> 55</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>
(2) INFORMATION FOR SEQ ID NO: 75:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 26 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 75:
Met Wing Gly Ile His Arg Wing Phe Leu Go Phe Cys Leu Trp Gly Leu 15 10 15
Xaa Read Cys Goes Gly Gly Pro Trp Xaa
25
263 (2) INFORMATION FOR SEQ ID NO: 76:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 76:
Met Ser Phe Ser Ser Pro Lys Ser Leu Leu Ser Leu Ile Ser Xaa 15 10 15 (2) SEQ ID: 77:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 33 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 77:
Met Thr Ile Trp Gin Leu Phe Ala Go Leu Ile Go Leu Phe Ala Lys 15 10 15 Be Arg Glu Ile Be Thr Glu Gly Glu Pro Cys Will Read Ser Lys Asn 20 25 30
Xaa (2) INFORMATION FOR SEQ ID NO: 78:
(i) SEQUENCE CHARACTERISTICS:
264 (A) LENGTH: 23 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 78:
Met Leu Asn Pro Phe Xaa Gin Leu Leu Leu Goes Leu Leu Phe Pro Glu 15 10 15
Trp Pro Thr Pro Leu His Xaa (2) INFORMATION FOR SEQ ID NO: 79:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 173 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 79:
<td rowspan="2">Met 1</td><td colspan="3" rowspan="2">Lys Thr Leu</td><td colspan="11">Phe Leu Gly Go Thr Leu Gly Leu Wing Ala Wing Leu</td>
<td> 5</td><td colspan="5"> 10</td><td colspan="5"> 15</td>
<td>To be</td><td>Xaa</td><td>Thr</td><td>Read</td><td>Xaa</td><td>Glu</td><td>Glu</td><td>Asp</td><td>Ile</td><td>Thr</td><td>Gly thr</td><td>Trp</td><td>Tyr</td><td>Go</td><td>Lys</td>
<td></td><td></td><td></td><td> 20</td><td></td><td></td><td></td><td></td><td> 25</td><td></td><td></td><td></td><td> 30</td><td></td><td></td>
<td>Allah</td><td>Met</td><td>Go</td><td>Go</td><td>Asp</td><td>Lys</td><td>Thr</td><td>Phe</td><td>Arg</td><td>Arg</td><td>Gin Glu</td><td>Allah</td><td>Gin</td><td>Lys</td><td>Go</td>
<td></td><td></td><td> 35</td><td></td><td></td><td></td><td></td><td> 40</td><td></td><td></td><td></td><td> 45</td><td></td><td></td><td></td>
<td>To be</td><td>Pro</td><td>Go</td><td>Lys</td><td>Go</td><td>Thr</td><td>Allah</td><td>Read</td><td colspan="3">Gly Gly Gly Lys</td><td>Read</td><td>Glu</td><td>Allah</td><td>Thr</td>
<td></td><td> 50</td><td></td><td></td><td></td><td></td><td> 55</td><td></td><td></td><td></td><td> 60</td><td></td><td></td><td></td><td></td>
<td>Phe</td><td>Thr</td><td>Phe</td><td>Met</td><td>Arg</td><td>Glu</td><td colspan="2">Asp Arg</td><td>Cys</td><td>Ile</td><td>Gin lys</td><td>Lys</td><td>Ile</td><td>Read</td><td>Xaa</td>
<td> 65</td><td></td><td></td><td></td><td></td><td> 70</td><td></td><td></td><td></td><td></td><td> 75</td><td></td><td></td><td></td><td> 80</td>
<td>Arg</td><td>Lys</td><td>Thr</td><td>Glu</td><td>Glu</td><td>Pro</td><td colspan="2">Gly lys</td><td>Tyr</td><td>To be</td><td>Cys Wing</td><td>Glu</td><td>Pro</td><td>Read</td><td>Pro</td>
90 95
265
<td>His</td><td colspan="2">To be his</td><td>Pro</td><td>His Xaa</td><td>Pro pro</td><td>Pro</td><td>Pro</td><td>Thr</td><td>Pro</td><td>Val his</td><td>Gin</td><td>Pro</td>
<td></td><td></td><td></td><td> 100</td><td></td><td></td><td> 105</td><td></td><td></td><td></td><td> 110</td><td></td><td></td>
<td>Pro</td><td colspan="2">Gin Val</td><td>Glu</td><td>To be a wing</td><td>Gin Wing</td><td>Allah</td><td>Read</td><td>Read</td><td>Pro</td><td>Gly pro</td><td>Gin</td><td>Read</td>
<td></td><td></td><td> 115</td><td></td><td></td><td> 120</td><td></td><td></td><td></td><td></td><td> 125</td><td></td><td></td>
<td>Cys</td><td>Pro</td><td>Pro</td><td>Pro</td><td colspan="2">Arg Arg Gly Trp</td><td>Pro</td><td>Read</td><td>Read</td><td>Pro</td><td>Gly Gly</td><td>Read</td><td>Val</td>
<td></td><td> 130</td><td></td><td></td><td></td><td> 135</td><td></td><td></td><td></td><td> 140</td><td></td><td></td><td></td>
<td>Allah</td><td>Read</td><td>Thr</td><td>To be</td><td>Asp Thr</td><td>Gly cys</td><td>Asp</td><td>Arg</td><td>Read</td><td>Val</td><td>Arg Ser</td><td>Arg</td><td>Asp</td>
<td> 145</td><td></td><td></td><td></td><td> 150</td><td></td><td></td><td></td><td> 155</td><td></td><td></td><td></td><td> 160</td>
<td>Gly</td><td>Pro</td><td>Asp</td><td>His</td><td>Cys Wing</td><td>Pro leu</td><td colspan="2">Gly Gly</td><td>Pro</td><td>To be</td><td>His</td><td></td><td></td>
165 170 (2) INFORMATION FOR SEQ ID NO: 80:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 208 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 80:
Met Wing Asp Ser Ser Tyr Thr Ser Glu Val 15 10
Gin Wing Ile L> I Wing Phe
Read Be Read Read Gin Arg Thr Gly Gly Gly Pro Gly Asn His Pro His 20 25 30
<td rowspan="2">Gly</td><td rowspan="2">Pro</td><td colspan="4" rowspan="2">Asp Wing Be Wing 35</td><td rowspan="2">Glu</td><td colspan="9">Gly Leu Asn Pro Tyr Gly Leu Val Wing</td>
<td colspan="2"> 40</td><td colspan="7"> 45</td>
<td>Pro</td><td>Arg</td><td>Phe</td><td>Gin</td><td>Arg</td><td>Lys</td><td>Phe</td><td>Lys</td><td>Allah</td><td>Lys</td><td>Gin</td><td>Read</td><td>Thr</td><td>Pro</td><td>Arg</td><td>Ile</td>
<td></td><td> 50</td><td></td><td></td><td></td><td></td><td> 55</td><td></td><td></td><td></td><td></td><td> 60</td><td></td><td></td><td></td><td></td>
<td>Read</td><td>Glu</td><td>Allah</td><td>His</td><td>Gin</td><td>Asn</td><td>Val</td><td>Allah</td><td>Gin</td><td>Read</td><td>To be</td><td>Read</td><td>Allah</td><td>Glu</td><td>Allah</td><td>Gin</td>
<td> 65</td><td></td><td></td><td></td><td></td><td> 70</td><td></td><td></td><td></td><td></td><td> 75</td><td></td><td></td><td></td><td></td><td> 80</td>
<td>Read</td><td>Arg</td><td>Phe</td><td>Ile</td><td>Gin</td><td>Allah</td><td>Trp</td><td>Gin</td><td>To be</td><td>Read</td><td>Pro</td><td>Asp</td><td>Phe</td><td>Gly</td><td>Ile</td><td>To be</td>
<td></td><td></td><td></td><td></td><td> 85</td><td></td><td></td><td></td><td></td><td> 90</td><td></td><td></td><td></td><td></td><td> 95</td><td></td>
<td>Tyr</td><td>Val</td><td>Met</td><td>Val</td><td>Arg</td><td>Phe</td><td>Lys</td><td>Gly</td><td>To be</td><td>Arg</td><td>Lys</td><td>Asp</td><td>Glu</td><td>Ile</td><td>Read</td><td>Gly</td>
<td></td><td></td><td></td><td> 100</td><td></td><td></td><td></td><td></td><td> 105</td><td></td><td></td><td></td><td></td><td> 110</td><td></td><td></td>
266
<td>Ile</td><td>Allah</td><td>Asn</td><td colspan="2">Asn arg</td><td>Read</td><td>Ile</td><td>Arg</td><td>Ile</td><td>Asp</td><td>Read</td><td colspan="2">Ala Vai</td><td colspan="3">Gly Asp Vai</td>
<td></td><td></td><td> 115</td><td></td><td></td><td></td><td></td><td> 120</td><td></td><td></td><td></td><td></td><td> 125</td><td></td><td></td><td></td>
<td>Go</td><td>Lys</td><td>Thr</td><td colspan="2">Trp Arg</td><td>Phe</td><td>To be</td><td>Asn</td><td>Met</td><td>Arg</td><td>Gin</td><td>Trp</td><td>Asn</td><td>Val</td><td>Asn</td><td>Trp</td>
<td></td><td> 130</td><td></td><td></td><td></td><td></td><td> 135</td><td></td><td></td><td></td><td></td><td> 140</td><td></td><td></td><td></td><td></td>
<td>Asp</td><td>Ile</td><td>Arg</td><td>Xaa</td><td>Go</td><td>Allah</td><td>Ile</td><td>Glu</td><td>Phe</td><td>Asp</td><td>Glu</td><td>His</td><td>Ile</td><td>Asn</td><td>Val</td><td>Allah</td>
<td> 145</td><td></td><td></td><td></td><td></td><td> 150</td><td></td><td></td><td></td><td></td><td> 155</td><td></td><td></td><td></td><td></td><td> 160</td>
<td>Phe</td><td>To be</td><td>Cys</td><td>Go</td><td>To be</td><td>Allah</td><td>To be</td><td>Cys</td><td>Arg</td><td>Ile</td><td>Go</td><td>His</td><td>Glu</td><td>Tyr</td><td>Ile</td><td>Gly</td>
<td></td><td></td><td></td><td></td><td> 165</td><td></td><td></td><td></td><td></td><td> 170</td><td></td><td></td><td></td><td></td><td> 175</td><td></td>
<td colspan="2">Gly tyr</td><td>Ile</td><td>Phe</td><td>Read</td><td>To be</td><td>Thr</td><td>Arg</td><td>Glu</td><td>Xaa</td><td>Allah</td><td colspan="2">Arg Gly</td><td>Glu</td><td>Glu</td><td>Read</td>
<td></td><td></td><td></td><td> 180</td><td></td><td></td><td></td><td></td><td> 185</td><td></td><td></td><td></td><td></td><td> 190</td><td></td><td></td>
<td colspan="3">Asp Glu Asp</td><td>Read</td><td>Phe</td><td>Read</td><td>Gin</td><td>Read</td><td>Thr</td><td colspan="2">Gly Gly</td><td>His</td><td>Glu</td><td>Allah</td><td>Phe</td><td>Xaa</td>
<td></td><td></td><td> 195</td><td></td><td></td><td></td><td></td><td> 200</td><td></td><td></td><td></td><td></td><td> 205</td><td></td><td></td><td></td>
(2) INFORMATION FOR SEQ ID NO: 81:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 43 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: B1:
<td>Met</td><td>Ile</td><td>Phe</td><td>Read</td><td>Read</td><td>Phe</td><td>Read</td><td>Thr</td><td>Pro</td><td>Read</td><td>Trp</td><td>Read</td><td>Gin</td><td>Lys</td><td>Gly</td><td>To be</td>
<td> 1</td><td></td><td></td><td></td><td> 5</td><td></td><td></td><td></td><td></td><td> 10</td><td></td><td></td><td></td><td></td><td> 15</td><td></td>
<td>Allah</td><td>Gly</td><td>Lys</td><td>Met</td><td>To be</td><td>Gly</td><td>Glu</td><td>Phe</td><td>Read</td><td>Tyr</td><td>Allah</td><td>To be</td><td>Read</td><td>Phe</td><td>Gin</td><td>Trp</td>
<td></td><td></td><td></td><td> 20</td><td></td><td></td><td></td><td></td><td> 25</td><td></td><td></td><td></td><td></td><td> 30</td><td></td><td></td>
<td>Asn</td><td>Tyr</td><td>Phe</td><td>Trp</td><td>Arg</td><td>Asn</td><td>Lys</td><td>Lys</td><td>Val</td><td>Cys</td><td>Xaa</td><td></td><td></td><td></td><td></td><td></td>
40
267 (2) INFORMATION FOR SEQ ID NO: 82:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 146 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 82:
<td rowspan="2">Met 1</td><td rowspan="2">Pro</td><td colspan="2" rowspan="2">Be gly</td><td colspan="11">Phe Gin Thr Cys Read Leu Phe Thr Leu Ser Pro Phe</td>
<td colspan="2"> 5</td><td colspan="7"> 10</td><td colspan="2"> 15</td>
<td>To be</td><td>Read</td><td>To be</td><td>Lys</td><td colspan="2">Ile Vai</td><td>Gly</td><td>Go</td><td>Pro</td><td>To be</td><td>Gin</td><td>Gin</td><td>Read</td><td>Pro gly</td><td>Gin</td>
<td></td><td></td><td></td><td> 20</td><td></td><td></td><td></td><td></td><td> 25</td><td></td><td></td><td></td><td></td><td> 30</td><td></td>
<td>Read</td><td>To be</td><td>Glu</td><td>Gin</td><td colspan="2">Gly Gly</td><td>Read</td><td>Cys</td><td>Gly</td><td>His</td><td>Glu</td><td>Gly</td><td>Glu</td><td>To the wing</td><td>Arg</td>
<td></td><td></td><td> 35</td><td></td><td></td><td></td><td></td><td> 40</td><td></td><td></td><td></td><td></td><td> 45</td><td></td><td></td>
<td>Thr</td><td>Go</td><td>Pro</td><td>Glu</td><td>Thr</td><td>Gin</td><td>Read</td><td>Pro</td><td>Read</td><td>Pro</td><td>Phe</td><td>Asn</td><td>To be</td><td>Gly wing</td><td>Pro</td>
<td></td><td> 50</td><td></td><td></td><td></td><td></td><td> 55</td><td></td><td></td><td></td><td></td><td> 60</td><td></td><td></td><td></td>
<td>Pro</td><td>His</td><td>Read</td><td>Lys</td><td>Cys</td><td>Thr</td><td colspan="5">Gly Wing Gly Lys Arg</td><td>Go</td><td>Trp</td><td>To be pro</td><td>Pro</td>
<td> 65</td><td></td><td></td><td></td><td></td><td> 70</td><td></td><td></td><td></td><td></td><td> 75</td><td></td><td></td><td></td><td> 80</td>
<td>Arg</td><td>Arg</td><td>Allah</td><td>Allah</td><td>Gin</td><td>Glu</td><td>Go</td><td>To be</td><td>Read</td><td>Gin</td><td>Read</td><td>Go</td><td>To be</td><td>Cys Xaa</td><td>Pro</td>
<td></td><td></td><td></td><td></td><td> 85</td><td></td><td></td><td></td><td></td><td> 90</td><td></td><td></td><td></td><td> 95</td><td></td>
<td>Cys</td><td>Arg</td><td>Gin</td><td>Xaa</td><td>Thr</td><td>To be</td><td>Arg</td><td>Allah</td><td>Phe</td><td>To be</td><td>Read</td><td>Allah</td><td>Thr</td><td>Asp Arg</td><td>Thr</td>
<td></td><td></td><td></td><td> 100</td><td></td><td></td><td></td><td></td><td> 105</td><td></td><td></td><td></td><td></td><td> 110</td><td></td>
<td>Allah</td><td>To be</td><td>Allah</td><td>Arg</td><td>Go</td><td>Cys</td><td colspan="2">Cys arg</td><td>Phe</td><td>Pro</td><td>Phe</td><td>Lys</td><td>His</td><td>Thr his</td><td>To be</td>
<td></td><td></td><td> 115</td><td></td><td></td><td></td><td></td><td> 120</td><td></td><td></td><td></td><td></td><td> 125</td><td></td><td></td>
<td>Pro</td><td>His</td><td>Pro</td><td colspan="2">Arg Arg</td><td>Pro</td><td>Glu</td><td>Go</td><td>Gin</td><td>Gly</td><td>Allah</td><td>Trp</td><td>Allah</td><td>Go Go</td><td>Pro</td>
130 135 140
Read Xaa
145
268 (2) INFORMATION FOR SEQ ID NO: 83:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 83:
Met Pro Trp Arg Arg Wing Gly Leu Met Met Leu Pro Ile Ile Thr Gly 15 10 15
Cys Cys Pro Cys Ser Ala Ser Ile Xaa
25 (2) INFORMATION FOR SEQ ID NO: 84:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 31 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear
269 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 84:
Met Lys Thr Leu Phe Leu Gly Will Thr Leu Gly Leu Wing Leu Pro Cys 15 10 15
Pro Be Pro Trp Xaa Arg Arg Ile Be Gin Gly Pro Gly Thr Xaa
25 30 (2) INFORMATION FOR SEQ ID NO: 85:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 374 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 85:
270
<td rowspan="2">Met 1</td><td colspan="4" rowspan="2">Be go to the Wing 5th</td><td rowspan="2">Phe</td><td rowspan="2">lie</td><td rowspan="2">Asp</td><td colspan="8">Ile Ser Glu Glu Asp Gin Wing Wing</td>
<td colspan="6"> 10</td><td colspan="2"> 15</td>
<td>Glu</td><td>Read</td><td>Arg</td><td>Allah 20</td><td>Tyr</td><td>Read</td><td>Lys</td><td>To be</td><td>Lys 25</td><td>Gly</td><td>Allah</td><td>Glu</td><td>ile</td><td>To be 30</td><td>Glu</td><td>Glu</td>
<td>Asn</td><td>To be</td><td>Glu 35</td><td colspan="2">Gly Gly</td><td>Read</td><td>His</td><td>Go 40</td><td>Asp</td><td>Read</td><td>Allah</td><td>Gin</td><td>Ile 45</td><td>Ile</td><td>Glu</td><td>Allah</td>
<td>Cys</td><td>Asp 50</td><td>Go</td><td>Cys</td><td>Read</td><td>Lys</td><td>Glu 55</td><td>Asp</td><td>Asp</td><td>Lys</td><td>Asp</td><td>go 60</td><td>Glu</td><td>To be</td><td>Go</td><td>Met</td>
<td>Asn 65</td><td>To be</td><td>Go</td><td>Go</td><td>To be</td><td>Read 70</td><td>Read</td><td>Read</td><td>Ile</td><td>Read</td><td>Glu 75</td><td>Pro</td><td>Asp</td><td>Lys</td><td>Gin</td><td>Glu 30</td>
<td>Allah</td><td>Read</td><td>Ile</td><td>Glu</td><td>To be 85</td><td>Read</td><td>cys</td><td>Glu</td><td>Lys</td><td>Read 90</td><td>Go</td><td>Lys</td><td>Phe</td><td>Arg</td><td>Glu 95</td><td>Gly</td>
<td>Glu</td><td>Arg</td><td>Pro</td><td>To be 100</td><td>Read</td><td>Arg</td><td>Read</td><td>Gin</td><td>Read 105</td><td>Read</td><td>To be</td><td>Asn</td><td>Read</td><td>Phe 110</td><td>His</td><td>Gly</td>
<td>Met</td><td>Asp</td><td>Lys 115</td><td>Asn</td><td>Thr</td><td>Pro</td><td>Go</td><td>Arg 120</td><td>Tyr</td><td>Thr</td><td>Go</td><td>Tyr</td><td>Cys 125</td><td>To be</td><td>Read</td><td>Ile</td>
<td>Lys</td><td>Go 130</td><td>Allah</td><td>Allah</td><td>To be</td><td>Cys</td><td colspan="2">Gly Wing 135</td><td>Ile</td><td>Gin</td><td>Tyr</td><td>Ile 140</td><td>Pro</td><td>Thr</td><td>Glu</td><td>Read</td>
<td>Asp 145</td><td>Gin</td><td>Go</td><td>Arg</td><td>Lys</td><td>Trp 150</td><td>Ile</td><td>To be</td><td>Asp</td><td>Trp</td><td>Asn 155</td><td>Read</td><td>Thr</td><td>Thr</td><td>Glu</td><td>Lys 160</td>
<td>Lys</td><td>His</td><td>Thr</td><td>Read</td><td>Read 165</td><td>Arg</td><td>Read</td><td>Read</td><td>Tyr</td><td>Glu 170</td><td>Allah</td><td>Read</td><td>Go</td><td>Asp</td><td>Cys 175</td><td>Lys</td>
<td>Lys</td><td>To be</td><td>Asp</td><td>Allah 180</td><td>Allah</td><td>To be</td><td>Lys</td><td>Go</td><td>Met 185</td><td>Go</td><td>Glu</td><td>Read</td><td>Read</td><td>Gly 190</td><td>To be</td><td>Tyr</td>
<td>Thr</td><td>Glu</td><td>Asp 195</td><td>Asn</td><td>Allah</td><td>To be</td><td>Gin</td><td>Allah 200</td><td>Arg</td><td>Go</td><td>Asp</td><td>Allah</td><td>His 205</td><td>Arg</td><td>Cys</td><td>Ile</td>
<td>Go</td><td>Arg</td><td>Allah</td><td>Read</td><td>Lys</td><td>Asp</td><td>Pro</td><td>Asn</td><td>Allah</td><td>Phe</td><td>Read</td><td colspan="2">Phe asp</td><td>His</td><td>Read</td><td>Read</td>
<td rowspan="2">Thr 225</td><td rowspan="2">210 Read</td><td rowspan="2">Lys</td><td colspan="2" rowspan="2">Pro Vai</td><td colspan="3" rowspan="2">215 Lys Phe Leu 230</td><td colspan="8">220 Glu Gly Glu Leu Ile His Asp Leu</td>
<td colspan="4"> 235</td><td colspan="4"> 240</td>
<td>Read</td><td>Thr</td><td>Ile</td><td>Phe</td><td>Go</td><td>To be</td><td>Allah</td><td>Lys</td><td>Read</td><td>Allah</td><td>To be</td><td>Tyr</td><td>Go</td><td>Lys</td><td>Phe</td><td>Tyr</td>
<td></td><td></td><td></td><td></td><td> 245</td><td></td><td></td><td></td><td></td><td> 250</td><td></td><td></td><td></td><td></td><td> 255</td><td></td>
<td>Gin</td><td>Asn</td><td>Asn</td><td>Lys</td><td>Asp</td><td>Phe</td><td>Ile</td><td>Asp</td><td>To be</td><td>Read</td><td>Gly</td><td>Read</td><td>Read</td><td>His</td><td>Glu</td><td>Gin</td>
<td></td><td></td><td></td><td> 260</td><td></td><td></td><td></td><td></td><td> 265</td><td></td><td></td><td></td><td></td><td> 270</td><td></td><td></td>
<td>Asn</td><td>Met</td><td>Allah</td><td>Lys</td><td>Met</td><td>Arg</td><td>Read</td><td>Read</td><td>Thr</td><td>Phe</td><td>Met</td><td>Gly</td><td>Met</td><td>Allah</td><td>Go</td><td>Glu</td>
<td></td><td></td><td> 275</td><td></td><td></td><td></td><td></td><td> 280</td><td></td><td></td><td></td><td></td><td> 285</td><td></td><td></td><td></td>
271
<td>Asn</td><td colspan="2">Lys glu</td><td>Ile</td><td>To be</td><td>Phe</td><td>Asp</td><td>Thr</td><td>Met</td><td>Gin</td><td>Gin</td><td>Glu</td><td>Read</td><td>Gin</td><td>Ile</td><td>Gly</td>
<td></td><td> 290</td><td></td><td></td><td></td><td></td><td> 295</td><td></td><td></td><td></td><td></td><td> 300</td><td></td><td></td><td></td><td></td>
<td>Allah</td><td colspan="2">Asp Asp</td><td>Go</td><td>Glu</td><td>Allah</td><td>Phe</td><td>Go</td><td>Ile</td><td>Asp</td><td>Allah</td><td>Go</td><td>Arg</td><td>Thr</td><td>Lys</td><td>Met</td>
<td> 305</td><td></td><td></td><td></td><td></td><td> 310</td><td></td><td></td><td></td><td></td><td> 315</td><td></td><td></td><td></td><td></td><td> 320</td>
<td>Go</td><td>Tyr</td><td>Cys</td><td>Lys</td><td>Ile</td><td>Asp</td><td>Gin</td><td>Thr</td><td>Gin</td><td>Arg</td><td>Lys</td><td>Go</td><td>Go</td><td>Go</td><td>To be</td><td>His</td>
<td></td><td></td><td></td><td></td><td> 325</td><td></td><td></td><td></td><td></td><td> 330</td><td></td><td></td><td></td><td></td><td> 335</td><td></td>
<td>To be</td><td>Thr</td><td>His</td><td>Arg</td><td>Thr</td><td>Phe</td><td>Gly</td><td>Lys</td><td>Gin</td><td>Gin</td><td>Trp</td><td>Gin</td><td>Gin</td><td>Read</td><td colspan="2">Tyr asp</td>
<td></td><td></td><td></td><td> 340</td><td></td><td></td><td></td><td></td><td> 345</td><td></td><td></td><td></td><td></td><td> 350</td><td></td><td></td>
<td>Thr</td><td>Read</td><td>Asn</td><td>Allah</td><td>Trp</td><td>Lys</td><td>Gin</td><td>Asn</td><td>Read</td><td>Asn</td><td>Lys</td><td>Go</td><td>Lys</td><td>Asn</td><td>To be</td><td>Read</td>
<td></td><td></td><td> 355</td><td></td><td></td><td></td><td></td><td> 360</td><td></td><td></td><td></td><td></td><td> 365</td><td></td><td></td><td></td>
Read Be Read Read Be Asp Thr
370 (2) INFORMATION FOR SEQ ID NO: 86:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 13 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 86:
Met Ser Goes To Ala Phe Ile Asp Ile Ser Glu Glu Asp 15 10 (2) SEQ ID: 87:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear
272 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 87:
Gin Wing Glu Wing Leu Arg Wing Tyr Leu Lys Ser Lys Gly Wing Glu 15 10 15 (2) SEQ ID: 88:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 17 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (x) SEQUENCE DESCRIPTION: SEQ ID NO: 88:
ile Be Glu Glu Asn Be Glu Gly Gly Read His Go Asp Read Wing Gin 15 10 15
Ile (2) INFORMATION FOR SEQ ID NO: 89:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 89:
Ile Glu Wing Cys Asp Going Cys Leu Lys Glu Asp Going Lys Asp Going Glu 15 10 15
Will be
273 (2) INFORMATION FOR SEQ ID NO: 90:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 16 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 90:
Go Ala Arg Pro Be Ser Leu Phe Arg Ser Ala Trp Ser Cys Glu Trp 15 10 15 (2) SEQ ID: 91:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 12 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 91:
Leu Arg Leu Gin Leu Leu Ser Asn Leu Phe His Gly 15 10 (2) INFORMATION FOR SEQ ID NO: 92:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 17 amino acids (B) TYPE: amino acid
274 (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 92:
Lys Asp Val Valu Ser Val Val Asn Val Val Ser Leu Leu Leu Ile 15 10 15
Leu (2) INFORMATION FOR SEQ ID NO: 93:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 26 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 93:
Asp Wing Wing Ser Lys Val Met Val Glu Leu Leu Gly Ser Tyr Thr Glu 15 10 15
Asp Asn Wing Be Gin Wing Arg Val Asp Wing
25 (2) INFORMATION FOR SEQ ID NO: 94:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear
275 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 94:
Vai Glu Ala Phe Vai Ile Asp Ala Vai Arg 15 10 (2) INFORMATION FOR SEQ ID NO: 95:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 35 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 95:
<td>Met</td><td>To be</td><td>Glu</td><td>Ile Tyr Read Arg Cys</td><td>Gin</td><td>Asp</td><td>Glu Gin</td><td>Gin</td><td>Tyr</td><td>Allah</td><td>Arg</td>
<td> 1</td><td></td><td></td><td> 5</td><td></td><td> 10</td><td></td><td></td><td></td><td> 15</td><td></td>
<td>Trp</td><td>Met</td><td>Allah</td><td>Gly Cys Arg Read Wing</td><td>To be</td><td>Lys</td><td>Gly arg</td><td>Thr</td><td>Met</td><td>Allah</td><td>Asp</td>
<td></td><td></td><td></td><td> 20</td><td> 25</td><td></td><td></td><td></td><td> 30</td><td></td><td></td>
<td>To be</td><td>To be</td><td>Tyr</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>
<td></td><td></td><td> 35</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>
<td> 2)</td><td>INF</td><td colspan="2">ORCHEMENT FOR SEQ</td><td>ID</td><td>No.</td><td> 96 :</td><td></td><td></td><td></td><td></td>
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 45 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear
276 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 96:
<img file="PT1015477E_D0005.tif" />
<td colspan="5">Read Go Ala Pro Arg</td><td colspan="5" rowspan="2">Phe Gin Arg Lys Phe 10</td><td colspan="2" rowspan="2">Lys Wing</td><td colspan="2" rowspan="2">Lys gin</td>
<td colspan="2"> 1</td><td colspan="3"> 5</td>
<td>Pro</td><td>Arg</td><td>Ile</td><td>Read</td><td>Glu</td><td>Allah</td><td>His</td><td>Gin</td><td>Asn</td><td>Go</td><td>Allah</td><td>Gin</td><td>Read</td><td>To be</td>
<td></td><td></td><td></td><td> 20</td><td></td><td></td><td></td><td></td><td> 25</td><td></td><td></td><td></td><td></td><td> 30</td>
<td>Glu</td><td>Allah</td><td>Gin</td><td>Read</td><td>Arg</td><td>Phe</td><td>Ile</td><td>Gin</td><td>Allah</td><td>Trp</td><td>Gin</td><td>To be</td><td>Read</td><td></td>
<td></td><td></td><td> 35</td><td></td><td></td><td></td><td></td><td> 40</td><td></td><td></td><td></td><td></td><td> 45</td><td></td>
<img file="PT1015477E_D0006.tif" />
(2) INFORMATION FOR SEQ ID NO: 97:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 23 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 97:
Go Gly Asp Go Go Lys Thr Trp Arg Phe Be Asn Met Arg Gin Trp 15 10 15
Asn Go Asn Trp Asp Ile Arg (2) INFORMATION FOR SEQ ID NO: 98:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 26 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear
277 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 98:
Glu Glu Ile Asp Cys Thr Glu Glu Glu Met Met Go Phe Wing Wing Leu 15 10 15
Gin Tyr His Ile Asn Lys Read Ser Ser Gin
25 (2) INFORMATION FOR SEQ ID NO: 99:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 26 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 99:
Glu Glu Ile Asp Cys Thr Glu Glu Glu Met Met Go Phe Wing Wing Leu 15 10 15
Gin Tyr His Ile Asn Lys Read Ser Ser Gin
25 (2) INFORMATION FOR SEQ ID NO: 100:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 26 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear
278 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 100:
<td colspan="5">Lys Glu Read Ser Phe Wing Arg Ile Lys</td><td colspan="2">Val Glu Wing Cys Val Glu Ser</td>
<td rowspan="2">1 Thr</td><td colspan="4">5th Gly Arg His Ile Tyr Phe Thr</td><td rowspan="2">10 Val</td><td rowspan="2"> 15</td>
<td> 20</td><td colspan="3"> 25</td>
<td> 2)</td><td colspan="2">INFORMATION</td><td>FOR SEQ</td><td>ID</td><td>No .: 101:</td><td></td>
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 17 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 101:
Gly Trp Asn Gin Wing Ile Thr Read Gly Leu Val Lys Phe Lys Asn Gin 15 10 15
Gin (2) INFORMATION FOR SEQ ID NO: 102:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 16 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear
279 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 102:
Leu Val Leu Gly Leu Ser Xaa Leu Asn Asn Ser Tyr Asn Phe Ser Phe 15 10 15 (2) SEQ ID: 103:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 17 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 103:
His Val Val Ile Gly Ser Gin Wing Glu Glu Gly Gin Tyr Ser Leu Asn 15 10 15
Phe (2) INFORMATION FOR SEQ ID NO: 104:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 19 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 104:
His Asn Cys Asn Asn Ser Val Pro Gly Lys Glu His Pro Phe Asp Ile 15 10 15
Thr val met
280 (2) INFORMATION FOR SEQ ID NO: 105:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 17 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 105:
Phe Ile Lys Tyr Goes To Be Asp Lys Glu Lys Lys Goes To Phe Gly Ile 15 10 15
Vai (2) INFORMATION FOR SEQ ID NO: 106:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 13 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 106:
Ile Pro Met Gin Goes Wing Asn Goes Wing Tyr Ile Ile 15 10 (2) SEQ ID: 107:
(i) SEQUENCE CHARACTERISTICS:
281 (A) LENGTH: 13 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 107:
Ile Pro Met Gin Goes Leu Wing Asn Goes Wing Tyr Ile Ile 1 5 10 (2) SEQ ID: 108:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 108:
Asp Gly Lys Go Ala Go Asn Leu Ala Lys Leu Lys Leu Phe Arg 15 10 15 (2) SEQ ID: 109:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 13 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear
282 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 109:
Ile Arg Glu Lys Asn Pro Asp Gly Phe Leu Ser Ala Wing 15 10 (2) SEQ ID: 110:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 110:
Met Met Phe Gly Gly Gly Tyr Glu Thr Ile
5th (2) INFORMATION FOR SEQ ID NO: 111:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 111:
Tyr Arg Asp Be Glu Be Be Glu Read Le Be Val Asp Be Glu Val Glu
10 15
Phe Gin Read Tyr Ser Gin Ile His
283 (2) INFORMATION FOR SEQ ID NO: 112:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 136 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 112:
<td rowspan="2">Tyr 1</td><td rowspan="2">Allah</td><td colspan="2" rowspan="2">Gin Asp</td><td colspan="12">Leu Asp Asp Go Ile Arg Glu Glu Glu His Glu Glu</td>
<td colspan="2"> 5</td><td colspan="7"> 10</td><td colspan="3"> 15</td>
<td>Lys</td><td>Asn</td><td>To be</td><td>Gly</td><td>Asn</td><td>To be</td><td>Glu</td><td>To be</td><td>To be</td><td>To be</td><td>To be</td><td>Lys</td><td>Pro</td><td>Asn</td><td>Gin</td><td>Lys</td>
<td></td><td></td><td></td><td> 20</td><td></td><td></td><td></td><td></td><td> 25</td><td></td><td></td><td></td><td></td><td> 30</td><td></td><td></td>
<td>Lys</td><td>Read</td><td>Ile</td><td>Go</td><td>Read</td><td>To be</td><td>Asp</td><td>To be</td><td>Glu</td><td>Go</td><td>Ile</td><td>Gin</td><td>Read</td><td>To be</td><td>Asp</td><td>Gly</td>
<td></td><td></td><td> 35</td><td></td><td></td><td></td><td></td><td> 40</td><td></td><td></td><td></td><td></td><td> 45</td><td></td><td></td><td></td>
<td>To be</td><td>Glu</td><td>Go</td><td>Ile</td><td>Thr</td><td>Read</td><td>To be</td><td>Asp</td><td>Glu</td><td>Asp</td><td>To be</td><td>Ile</td><td>Tyr</td><td>Arg</td><td>Cys</td><td>Lys</td>
<td></td><td> 50</td><td></td><td></td><td></td><td></td><td> 55</td><td></td><td></td><td></td><td></td><td> 60</td><td></td><td></td><td></td><td></td>
<td colspan="2">Gly lys</td><td>Asn</td><td>Go</td><td>Arg</td><td>Go</td><td>Gin</td><td colspan="2">Gin Wing</td><td>Glu</td><td colspan="2">Asn wing</td><td>His</td><td>Gly</td><td>Read</td><td>To be</td>
<td> 65</td><td></td><td></td><td></td><td></td><td> 70</td><td></td><td></td><td></td><td></td><td> 75</td><td></td><td></td><td></td><td></td><td> 80</td>
<td>To be</td><td>To be</td><td>Read</td><td>Gin</td><td>To be</td><td>Asn</td><td>Glu</td><td>Read</td><td>Go</td><td>Asp</td><td>Lys</td><td>Lys</td><td>Cys</td><td>Lys</td><td>To be</td><td>Asp</td>
<td></td><td></td><td></td><td></td><td> 85</td><td></td><td></td><td></td><td></td><td> 90</td><td></td><td></td><td></td><td></td><td> 95</td><td></td>
<td>Ile</td><td>Glu</td><td>Lys</td><td>Pro</td><td>Lys</td><td>To be</td><td>Glu</td><td>Glu</td><td>Arg</td><td>To be</td><td>Gly</td><td>Go</td><td>He</td><td>Arg</td><td>Glu</td><td>Go</td>
<td></td><td></td><td></td><td> 100</td><td></td><td></td><td></td><td></td><td> 105</td><td></td><td></td><td></td><td></td><td> 110</td><td></td><td></td>
<td>Met</td><td>Ile</td><td>Ile</td><td>Glu</td><td>Go</td><td>To be</td><td>To be</td><td>To be</td><td>Glu</td><td>Glu</td><td>Glu</td><td>Glu</td><td>To be</td><td>Thr</td><td>Ile</td><td>To be</td>
<td></td><td></td><td> 115</td><td></td><td></td><td></td><td></td><td> 120</td><td></td><td></td><td></td><td></td><td> 125</td><td></td><td></td><td></td>
<td>Glu</td><td colspan="2">Gly asp</td><td>Asn</td><td>Go</td><td>Glu</td><td>To be</td><td>Trp</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>
130 135 (2) INFORMATION FOR SEQ ID NO: 113:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 37 amino acids (B) TYPE: amino acid
284 (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 113:
Met Leu Leu Gly Cys Glu Go Asp Asp Lys Asp Asp Asp Ile Leu Leu 15 10 15
Asn Leu Goes Gly Cys Glu Asn Be Goes Thr Glu Gly Glu Asp Gly Ile 20 25 30
Asn Trp Ser Ile Ser (2) INFORMATION FOR SEQ ID NO: 114:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 114:
Asp Lys Asp Ile Glu Wing Gin Ile Wing Asn Asn Arg Thr Pro Gly Arg 15 10 15
Trp Thr (2) INFORMATION FOR SEQ ID NO: 115:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 31 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear
285 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 115:
Gin Arg Tyr Tyr Ser Asa Asn Lys Asn
5
Lys Arg Gly His Reads Being Lys Asn Cys
Ile Ile Cys Arg Asn Cys Asp
15
Pro Leu Pro Arg Lys Vai (2) INFORMATION FOR SEQ ID NO: 116:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 179 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 116:
<td>Arg</td><td>Arg</td><td>Cys</td><td>Phe</td><td>Read</td><td>Cys</td><td>To be</td><td>Arg</td><td>Arg</td><td>Gly</td><td>His</td><td>Read</td><td>Read</td><td>Tyr</td><td>To be</td><td>Cys</td>
<td> 1</td><td></td><td></td><td></td><td> 5</td><td></td><td></td><td></td><td></td><td> 10</td><td></td><td></td><td></td><td></td><td> 15</td><td></td>
<td>Pro</td><td>Allah</td><td>Pro</td><td>Read</td><td>Cys</td><td>Glu</td><td>Tyr</td><td>Cys</td><td>Pro</td><td>Go</td><td>Pro</td><td>Lys</td><td>Met</td><td>Read</td><td>Asp</td><td>His</td>
<td></td><td></td><td></td><td> 20</td><td></td><td></td><td></td><td></td><td> 25</td><td></td><td></td><td></td><td></td><td> 30</td><td></td><td></td>
<td>To be</td><td>Cys</td><td>Read</td><td>Phe</td><td>Arg</td><td>His</td><td>To be</td><td>Trp</td><td>Asp</td><td>Lys</td><td>Gin</td><td>Cys</td><td>Asp</td><td>Arg</td><td>Cys</td><td>His</td>
<td></td><td></td><td> 35</td><td></td><td></td><td></td><td></td><td> 40</td><td></td><td></td><td></td><td></td><td> 45</td><td></td><td></td><td></td>
<td>Met</td><td>Read</td><td>Gly</td><td>His</td><td>Tyr</td><td>Thr</td><td>Asp</td><td>Allah</td><td>Cys</td><td>Thr</td><td>Glu</td><td>Ile</td><td>Trp</td><td>Arg</td><td>Gin</td><td>Tyr</td>
<td></td><td> 50</td><td></td><td></td><td></td><td></td><td> 55</td><td></td><td></td><td></td><td></td><td> 60</td><td></td><td></td><td></td><td></td>
<td>His</td><td>Read</td><td>Thr</td><td>Thr</td><td>Lys</td><td>Pro</td><td>Gly</td><td>Pro</td><td>Pro</td><td>Lys</td><td>Lys</td><td>Pro</td><td>Lys</td><td>Thr</td><td>Pro</td><td>To be</td>
<td> 65</td><td></td><td></td><td></td><td></td><td> 70</td><td></td><td></td><td></td><td></td><td> 75</td><td></td><td></td><td></td><td></td><td> 80</td>
<td>Arg</td><td>Pro</td><td>To be</td><td>Allah</td><td>Read</td><td>Allah</td><td>Tyr</td><td>Cys</td><td>Tyr</td><td>His</td><td>Cys</td><td>Allah</td><td>Gin</td><td>Lys</td><td>Gly</td><td>His</td>
<td></td><td></td><td></td><td></td><td> 85</td><td></td><td></td><td></td><td></td><td> 90</td><td></td><td></td><td></td><td></td><td> 95</td><td></td>
286
<td colspan="2">Tyr gly</td><td>His</td><td>Glu</td><td>Cys</td><td>Pro</td><td>Glu</td><td>Arg</td><td>Glu</td><td>Val</td><td>Tyr</td><td>Asp</td><td>Pro</td><td>To be</td><td>Pro</td><td>Val</td>
<td></td><td></td><td></td><td> 100</td><td></td><td></td><td></td><td></td><td> 105</td><td></td><td></td><td></td><td></td><td> 110</td><td></td><td></td>
<td>To be</td><td>Pro</td><td>Phe</td><td>Ile</td><td>cys</td><td>Tyr</td><td>Tyr</td><td>Xaa</td><td>Asp</td><td>Lys</td><td>Tyr</td><td>Glu</td><td>Ile</td><td>Gin</td><td>Glu</td><td>Arg</td>
<td></td><td></td><td> 115</td><td></td><td></td><td></td><td></td><td> 120</td><td></td><td></td><td></td><td></td><td> 125</td><td></td><td></td><td></td>
<td>Glu</td><td>Lys</td><td>Arg</td><td>Read</td><td>Lys</td><td>Gin</td><td>Lys</td><td>Ile</td><td>Lys</td><td>Val</td><td>Xaa</td><td>Lys</td><td>Lys</td><td>Asn</td><td>Gly</td><td>Val</td>
<td></td><td> 130</td><td></td><td></td><td></td><td></td><td> 135</td><td></td><td></td><td></td><td></td><td> 140</td><td></td><td></td><td></td><td></td>
<td>Ile</td><td>Pro</td><td>Glu</td><td>Pro</td><td>To be</td><td>Lys</td><td>Read</td><td>Pro</td><td>Tyr</td><td>Ile</td><td>Lys</td><td colspan="2">Wing wing</td><td>Asn</td><td>Glu</td><td>Asn</td>
<td> 145</td><td></td><td></td><td></td><td></td><td> 150</td><td></td><td></td><td></td><td></td><td> 155</td><td></td><td></td><td></td><td></td><td> 160</td>
<td>Pro</td><td>His</td><td>His</td><td>Asp</td><td>Ile</td><td>Arg</td><td>Lys</td><td colspan="2">Gly arg</td><td>Allah</td><td>To be</td><td colspan="2">Trp lys</td><td>To be</td><td>Asn</td><td>Arg</td>
<td></td><td></td><td></td><td></td><td> 165</td><td></td><td></td><td></td><td></td><td> 170</td><td></td><td></td><td></td><td></td><td> 175</td><td></td>
Trp Pro Gin (2) INFORMATION FOR SEQ ID NO: 117:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 17 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 117:
Leu Ser Ile Ile Phe Leu Wing Phe Will Be Ile Asp Arg Cys Leu Gin 15 10 15
Read (2) INFORMATION FOR SEQ ID NO: 118:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 67 amino acids (B) TYPE: amino acid
287 (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 118:
<td>Gly</td><td>To be</td><td>Cys</td><td>Phe</td><td>Allah</td><td>Thr</td><td>Trp</td><td>Allah</td><td>Phe</td><td>Ile</td><td>Gin</td><td>Lys</td><td>Asn</td><td>Thr asn</td><td>His</td>
<td> 1</td><td></td><td></td><td></td><td> 5</td><td></td><td></td><td></td><td></td><td> 10</td><td></td><td></td><td></td><td> 15</td><td></td>
<td>Arg</td><td>Cys</td><td>Go</td><td>To be</td><td>Ile</td><td>Tyr</td><td>Read</td><td>Ile</td><td>Asn</td><td>Read</td><td>Read</td><td>Thr</td><td>Allah</td><td>Asp Phe</td><td>Read</td>
<td></td><td></td><td></td><td> 20</td><td></td><td></td><td></td><td></td><td> 25</td><td></td><td></td><td></td><td></td><td> 30</td><td></td>
<td>Read</td><td>Thr</td><td>Read</td><td>Allah</td><td>Read</td><td>Pro</td><td>Go</td><td>Lys</td><td>Ile</td><td>Go</td><td>Go</td><td>Asp</td><td>Read</td><td>Gly Vai</td><td>Allah</td>
<td></td><td></td><td> 35</td><td></td><td></td><td></td><td></td><td> 40</td><td></td><td></td><td></td><td></td><td> 45</td><td></td><td></td>
<td>Pro</td><td>Trp</td><td>Lys</td><td>Read</td><td>Lys</td><td>Ile</td><td>Phe</td><td>His</td><td>Cys</td><td>Gin</td><td>Go</td><td>Thr</td><td>Allah</td><td>Cys leu</td><td>Ile</td>
<td></td><td> 50</td><td></td><td></td><td></td><td></td><td> 55</td><td></td><td></td><td></td><td></td><td> 60</td><td></td><td></td><td></td>
Tyr ile Asn (2) INFORMATION FOR SEQ ID NO: 119:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 60 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 119:
<td>Allah 1</td><td>Pro leu</td><td>Glu</td><td>Thr 5th</td><td>Met</td><td>Gin</td><td>Asn</td><td>Lys</td><td>Pro 10</td><td>Arg</td><td>Allah</td><td>Pro</td><td>Gin</td><td>Lys 15</td><td>Arg</td>
<td>Allah</td><td>Read pro</td><td>Phe 20</td><td>Pro</td><td>Glu</td><td>Read</td><td>Glu</td><td>Read 25</td><td>Arg</td><td>Asp</td><td>Tyr</td><td>Allah</td><td>To be 30</td><td>Go</td><td>Read</td>
<td>Thr</td><td>Arg tyr 35</td><td>To be</td><td>Read</td><td>Gly</td><td>Read</td><td>Arg 40</td><td>Asn</td><td>Lys</td><td>Glu</td><td>Pro</td><td>To be 45</td><td>Read</td><td>Gly</td><td>His</td>
<td>Arg</td><td>Trp gly</td><td>Thr</td><td>Gin</td><td>Lys</td><td>Read</td><td colspan="2">Gly arg</td><td>To be</td><td>Pro</td><td>Cys</td><td></td><td></td><td></td><td></td>
55 60
288 (2) INFORMATION FOR SEQ ID NO: 120:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 166 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 120:
<td>Asn</td><td>Arg</td><td>Glu</td><td colspan="2">Arg Gly Gly</td><td>Allah</td><td>Gly</td><td>Allah</td><td>Thr</td><td>Phe</td><td colspan="2">Glu Cys</td><td>Asn</td><td>Ile</td><td>Cys</td>
<td> 1</td><td></td><td></td><td> 5</td><td></td><td></td><td></td><td></td><td> 10</td><td></td><td></td><td></td><td></td><td> 15</td><td></td>
<td>Read</td><td>Glu</td><td>Thr</td><td>Arg Wing</td><td>Glu</td><td>Allah</td><td>Val</td><td>Val</td><td>To be</td><td>Val</td><td colspan="2">Cys gly</td><td>His</td><td>Read</td><td>Tyr</td>
<td></td><td></td><td></td><td> 20</td><td></td><td></td><td></td><td> 25</td><td></td><td></td><td></td><td></td><td> 30</td><td></td><td></td>
<td>Cys</td><td>Trp</td><td>Pro</td><td>Cys leu</td><td>His</td><td>Gin</td><td>Trp</td><td>Read</td><td>Glu</td><td>Thr</td><td>Arg</td><td>Pro</td><td>Glu</td><td>Arg</td><td>Gin</td>
<td></td><td></td><td> 35</td><td></td><td></td><td></td><td> 40</td><td></td><td></td><td></td><td></td><td> 45</td><td></td><td></td><td></td>
<td>Glu</td><td>Cys</td><td>Pro</td><td>Val cys</td><td>Lys</td><td>Allah</td><td>Gly</td><td>Ile</td><td>To be</td><td>Arg</td><td>Glu</td><td>Lys</td><td>Val</td><td>Val</td><td>Pro</td>
<td></td><td> 50</td><td></td><td></td><td></td><td> 55</td><td></td><td></td><td></td><td></td><td> 60</td><td></td><td></td><td></td><td></td>
<td>Read</td><td>Tyr</td><td colspan="2">Gly Arg Gly</td><td>To be</td><td>Gin</td><td>Lys</td><td>Pro</td><td>Gin</td><td>Asp</td><td>Pro</td><td>Arg</td><td>Read</td><td>Lys</td><td>Thr</td>
<td> 65</td><td></td><td></td><td></td><td> 70</td><td></td><td></td><td></td><td></td><td> 75</td><td></td><td></td><td></td><td></td><td> 80</td>
<td>Pro</td><td>Pro</td><td>Arg</td><td>Pro gin</td><td>Gly</td><td>Gin</td><td>Arg</td><td>Pro</td><td>Allah</td><td>Pro</td><td>Glu</td><td>To be</td><td>Arg</td><td colspan="2">Gly Gly</td>
<td></td><td></td><td></td><td> 85</td><td></td><td></td><td></td><td></td><td> 90</td><td></td><td></td><td></td><td></td><td> 95</td><td></td>
<td>Phe</td><td>Gin</td><td>Pro</td><td colspan="2">Phe Gly Asp</td><td>Thr</td><td colspan="2">Gly Gly</td><td>Phe</td><td>His</td><td>Phe</td><td>To be</td><td>Phe</td><td colspan="2">Gly val</td>
<td></td><td></td><td></td><td> 100</td><td></td><td></td><td></td><td> 105</td><td></td><td></td><td></td><td></td><td> 110</td><td></td><td></td>
<td>Gly</td><td>Allah</td><td>Phe</td><td>Pro phe</td><td>Gly</td><td>Phe</td><td>Phe</td><td>Thr</td><td>Thr</td><td>Val</td><td>Phe</td><td>Asn</td><td>Allah</td><td>His</td><td>Glu</td>
<td></td><td></td><td> 115</td><td></td><td></td><td></td><td> 120</td><td></td><td></td><td></td><td></td><td> 125</td><td></td><td></td><td></td>
<td>Pro</td><td>Phe</td><td colspan="2">Arg Arg Gly</td><td>Thr</td><td>Gly</td><td>val</td><td>Asp</td><td>Read</td><td>Gly</td><td>Gin</td><td>Gly</td><td>His</td><td>Pro</td><td>Allah</td>
<td></td><td> 130</td><td></td><td></td><td></td><td> 135</td><td></td><td></td><td></td><td></td><td> 140</td><td></td><td></td><td></td><td></td>
<td>To be</td><td>To be</td><td colspan="2">Trp Gin Asp</td><td>To be</td><td>Read</td><td>Phe</td><td>Read</td><td>Phe</td><td>Read</td><td>Allah</td><td>Ile</td><td>Phe</td><td>Phe</td><td>Phe</td>
<td> 145</td><td></td><td></td><td></td><td> 150</td><td></td><td></td><td></td><td></td><td> 155</td><td></td><td></td><td></td><td></td><td> 160</td>
Phe Trp Leu Leu Ser Ile
165
Lisbon, 21st December 2010
Contents465
6 sheets
Sheet 1 Sheet 2 Sheet 3 Sheet 4 Sheet 5 Sheet 6
1,441 members in 12 offices
Priority claims36
| Document | Office | Kind | Date |
|---|---|---|---|
| 4403997 | United States of America | P | |
| 4403997 | United States of America | P | |
| 4809397 | United States of America | P | |
| 4809397 | United States of America | P | |
| 4810197 | United States of America | P | |
| 4810197 | United States of America | P | |
| 4819097 | United States of America | P | |
| 4819097 | United States of America | P | |
| 4835697 | United States of America | P | |
| 4835697 | United States of America | P | |
| 5093597 | United States of America | P | |
| 5093597 | United States of America | P | |
| 5625097 | United States of America | P | |
| 5625097 | United States of America | P | |
| 5629397 | United States of America | P | |
| 5629397 | United States of America | P | |
| 5629697 | United States of America | P | |
| 5629697 | United States of America | P | |
| 44039P | – | – | – |
| 48093P | – | – | – |
| 48101P | – | – | – |
| 48190P | – | – | – |
| 48356P | – | – | – |
| 50935P | – | – | – |
| 56250P | – | – | – |
| 56293P | – | – | – |
| 56296P | – | – | – |
| US19970044039P | – | – | – |
| US19970048093P | – | – | – |
| US19970048101P | – | – | – |
| US19970048190P | – | – | – |
| US19970048356P | – | – | – |
| US19970050935P | – | – | – |
| US19970056250P | – | – | – |
| US19970056293P | – | – | – |
| US19970056296P | – | – | – |
Members1,441
| Document | Office | Kind | |
|---|---|---|---|
| US5700047A | United States of America | A | |
| CA2278118A1 | Canada | A1 | |
| CA2278154A1 | Canada | A1 | |
| CA2278248A1 | Canada | A1 | |
| CA2278349A1 | Canada | A1 | |
| WO9831799A2 | World Intellectual Property Organization (WIPO) | A2 | |
| WO9831800A2 | World Intellectual Property Organization (WIPO) | A2 | |
| WO9831801A1 | World Intellectual Property Organization (WIPO) | A1 | |
| WO9831806A2 | World Intellectual Property Organization (WIPO) | A2 | |
| WO9831818A2 | World Intellectual Property Organization (WIPO) | A2 | |
| AU5923398A | Australia | A | |
| AU5927398A | Australia | A | |
| AU6030398A | Australia | A | |
| AU6241698A | Australia | A | |
| CA2283299A1 | Canada | A1 | |
| CA2284131A1 | Canada | A1 | |
| WO9839446A2 | World Intellectual Property Organization (WIPO) | A2 | |
| WO9839448A2 | World Intellectual Property Organization (WIPO) | A2 | |
| CA2283678A1 | Canada | A1 | |
| WO9840483A2 | World Intellectual Property Organization (WIPO) | A2 | |
| AU6545298A | Australia | A | |
| AU6545398A | Australia | A | |
| AU6552198A | Australia | A | |
| CA2284550A1 | Canada | A1 | |
| WO9842738A1 | World Intellectual Property Organization (WIPO) | A1 | |
| WO9843240A2 | World Intellectual Property Organization (WIPO) | A2 | |
| AU6564698A | Australia | A | |
| CA2286303A1 | Canada | A1 | |
| WO9845712A2 | World Intellectual Property Organization (WIPO) | A2 | |
| AU6562798A | Australia | A | |
| WO9839448A9 | World Intellectual Property Organization (WIPO) | A9 | |
| AU6952998A | Australia | A | |
| WO9831799A3 | World Intellectual Property Organization (WIPO) | A3 | |
| WO9831806A3 | World Intellectual Property Organization (WIPO) | A3 | |
| WO9840483A3 | World Intellectual Property Organization (WIPO) | A3 | |
| WO9831800A3 | World Intellectual Property Organization (WIPO) | A3 | |
| WO9831818A3 | World Intellectual Property Organization (WIPO) | A3 | |
| WO9839448A3 | World Intellectual Property Organization (WIPO) | A3 | |
| CA2291221A1 | Canada | A1 | |
| WO9845712A3 | World Intellectual Property Organization (WIPO) | A3 | |
| WO9854206A1 | World Intellectual Property Organization (WIPO) | A1 | |
| CA2291260A1 | Canada | A1 | |
| WO9854963A2 | World Intellectual Property Organization (WIPO) | A2 | |
| CA2294526A1 | Canada | A1 | |
| WO9856804A1 | World Intellectual Property Organization (WIPO) | A1 | |
| AU7812098A | Australia | A | |
| WO9839446A3 | World Intellectual Property Organization (WIPO) | A3 | |
| WO9843240A3 | World Intellectual Property Organization (WIPO) | A3 | |
| AU7801998A | Australia | A | |
| AU8066798A | Australia | A | |
| CA2294705A1 | Canada | A1 | |
| WO9901020A2 | World Intellectual Property Organization (WIPO) | A2 | |
| CA2295474A1 | Canada | A1 | |
| WO9902546A1 | World Intellectual Property Organization (WIPO) | A1 | |
| AU8379598A | Australia | A | |
| CA2296762A1 | Canada | A1 | |
| CA2296815A1 | Canada | A1 | |
| WO9903982A1 | World Intellectual Property Organization (WIPO) | A1 | |
| WO9903990A1 | World Intellectual Property Organization (WIPO) | A1 | |
| AU8474398A | Australia | A | |
| AU8404598A | Australia | A | |
| AU8571198A | Australia | A | |
| CA2298852A1 | Canada | A1 | |
| WO9906423A1 | World Intellectual Property Organization (WIPO) | A1 | |
| CA2299605A1 | Canada | A1 | |
| WO9907891A1 | World Intellectual Property Organization (WIPO) | A1 | |
| AU8763498A | Australia | A | |
| CA2301796A1 | Canada | A1 | |
| WO9909155A1 | World Intellectual Property Organization (WIPO) | A1 | |
| AU8768498A | Australia | A | |
| CA2302387A1 | Canada | A1 | |
| WO9910363A1 | World Intellectual Property Organization (WIPO) | A1 | |
| AU8910998A | Australia | A | |
| CA2302808A1 | Canada | A1 | |
| WO9911293A1 | World Intellectual Property Organization (WIPO) | A1 | |
| AU8921598A | Australia | A | |
| AU9130498A | Australia | A | |
| CA2305685A1 | Canada | A1 | |
| WO9918208A1 | World Intellectual Property Organization (WIPO) | A1 | |
| CA2305690A1 | Canada | A1 | |
| WO9919339A1 | World Intellectual Property Organization (WIPO) | A1 | |
| AU9679898A | Australia | A | |
| AU9790698A | Australia | A | |
| CA2307320A1 | Canada | A1 | |
| WO9921575A1 | World Intellectual Property Organization (WIPO) | A1 | |
| WO9922243A1 | World Intellectual Property Organization (WIPO) | A1 | |
| AU1118499A | Australia | A | |
| AU1273499A | Australia | A | |
| CA2308768A1 | Canada | A1 | |
| WO9924836A1 | World Intellectual Property Organization (WIPO) | A1 | |
| AU1303799A | Australia | A | |
| CA2314379A1 | Canada | A1 | |
| CA2315295A1 | Canada | A1 | |
| WO9931116A1 | World Intellectual Property Organization (WIPO) | A1 | |
| WO9931117A1 | World Intellectual Property Organization (WIPO) | A1 | |
| AU1931399A | Australia | A | |
| AU2306499A | Australia | A | |
| CA2317702A1 | Canada | A1 | |
| WO9935158A1 | World Intellectual Property Organization (WIPO) | A1 | |
| AU2451199A | Australia | A |
Numbers
- Publication, DOCDB
- 1015477
- Publication, EPODOC
- PT1015477E
- Application
- 98926105
- Application, DOCDB
- 98926105
- Application, EPODOC
- PT19980926105T
Titles2
- English
- 32 HUMAN SECRETED PROTEINS
- Portuguese
- 32 PROTEÍNAS HUMANAS SEGREGADAS
Classification
- CPC, 4
- C07K14/47
- A61K38/00
- C07K14/525
- G01N2500/04
- IPC, 14
- C07K14 525
- C12N15 09
- A61K38 00
- C07K14 47
- C07K16 18
- C12N1 15
- C12N1 19
- C12N1 21
- C12N5 02
- C12N5 10
- C12P21 02
- C12P21 08
- C12Q1 02
- C12R1 91
