CA2176236C

Tyrosinase-activator protein fusion enzyme

Abstract

The present invention relates to a nucleic acidsequence encoding a fusion enzyme comprising a nucleicacid sequence encoding for a tyrosinase and a nucleicacid sequence encoding for a tyrosinase activatorprotein. Activator proteins used in the presentinvention are ORF438 and URF402. Prokaryotictyrosinases that require a separate activator protein,particularly tyrosinases derived from Streptomyces arepreferred. It is also preferred that the activatorprotein sequence be positioned (5') relative to thetyrosinase sequence. The present invention alsorelates to a fusion enzyme which comprises an aminoacid sequence for tyrosinase and an amino acid sequencefor a tyrosinase activator protein, which may alsocontain a linker positioned between the amino acidsequences of the activator protein and the tyrosinase.The present invention also relates to melanin producedby a fusion enzyme of the present invention.

CA2176236C, drawing sheet 1 — Sheet 1 of 9

Term

Term ended

Expired 8 November 2014, 11.9 years ago.

41 claims: 6 independent, 35 dependent

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CA 02176236 2000-07-31 WO 95/13386 PCT7US94/12857 - 40 THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:1. A nucleic acid sequence encoding a fusion enzyme comprising a nucleic acid sequence encoding for a tyrosinase and a nucleic acid sequence encoding for a tyrosinase activator protein positioned 5’ relative to the tyrosinase sequence.
13
A vector for transforming an organism, said vector comprising :a nucleic acid sequence encoding for a fusion enzyme, said fusion enzyme comprising an amino acid sequence encoding for a tyrosinase and an amino acid sequence encoding for a tyrosinase activator protein that is positioned on the N-terminus end of the tyrosinase sequence;and a promoter sequence that regulates the transcription of fusion enzyme.
24
A fusion enzyme comprising an amino acid sequence for tyrosinase and an amino acid sequence for a tyrosinase activator protein that is positioned on the N-terminus end of the tyrosinase sequence.
33
An in vitro method of producing a melanin comprising the steps of contacting 25 a fusion enzyme comprising an amino acid sequence for tyrosinase and an amino acid sequence for a tyrosinase activator protein that is positioned on the N-terminus end of the tyrosinase sequence and a reaction substrate selected from the group consisting of L-tyrosine, 30 X/L-tyrosine, L-tyrosine/X and X/L-tyrosine/X where X is a single amino acid, a dipeptide or an oligopeptide bound to L-tyrosine, under suitable reaction conditions, to form melanin. CA 02176236 2001-02-08 WO 95/13386 PCT/US94/12857 - 44
34
A method of producing melanins, comprising the step of growing an organism which has been transformed with a vector comprising a nucleic acid sequence encoding for a fusion enzyme, said fusion enzyme comprising an amino acid sequence encoding for a tyrosinase and an amino acid sequence encoding for a tyrosinase activator protein that is positioned on the N-terminus end of the tyrosinase sequence ;and a promoter sequence that regulates the transcription of fusion enzyme.
38
A nucleic acid sequence encoding a fusion enzyme comprising a nucleic acid sequence encoding a tyrosinase with cresolase activity and catecholase activity;and a nucleic acid encoding a tyrosinase activator protein that is positioned 5' relative to the nucleic acid sequence encoding the tyrosinase with cresolase activity and catecholase activity.

Independent claims6

514 paragraphs in 142 sections, as filed

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TITLE OF THE INVENTION

TYROSINASE-ACTIVATOR PROTEIN FUSION ENZYME 5

BACKGROUND OF INVENTION

Melanogenesis, production of the biological polymer melanin, is a widespread phenomena in nature occurring in most phyla from fungi to mammals. The black, brown, buff and Tyndall-blue pigments found in feathers, hairs, eyes, insect cuticle, fruit and seeds are usually melanins. Melanins have been assigned a photoprotective role in the skin, their role in the eye and inner ear is unknown. Melanins

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-2are also found in the mammalian brain where they are referred to as neuromelanins. The biological function of neuromelanins is unknown.

The stepwise biosynthesis of melanins is 5 depicted in Figure 1. Tyrosinase (E.C. 1.14.18.1) catalyzes two types of reactions involved in the biosynthesis of melanin: the orthohydroxylation of monophenols to catechols, which is referred to as cresolase activity, and the dehydrogenation of catechols co o-quinones, designated as catecholase activity. Molecular oxygen is used for the hydroxylation reaction. For this reason, tyrosinase acting on a monophenol is referred to as a mixed function oxidase. Hayaishi, in Biological

Oxidation (Singer, ed.) p.581, Interscience

Publishers, New York (19€8) . As used herein tyrosinase refers to all enzymes possessing the above described enzymatic activity. Tyrosinases are also sometimes referred to as polyphenol oxidases.

As can be seen from the biosynthetic pathway of melanin depicted in Figure 1, tyrosinase is essential to the production of melanin. Therefore, the ability of an organism to express significant quantities of tyrosinase activity is essential to the organism'6 in vivo production of melanin.

Tyrosinase is not naturally found in all organisms. Rather, tyrosinase has been found to occur in a relatively limited number of prokaryotes, is absent in a variety of higher plants and is generally confined to specific cells of the skin in higher animals but may also occur in interior tissue, such as the substantia nigra, eye and inner ear.

Given the limited number of organisms that produce melanin, one objective of the present invention is to provide a means for introducing tyrosinase activity

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PCT/US94/12857 into organisms that are otherwise unable to produce melanin.

Even in those organisms where tyrosinase activity occurs naturally, such activity is generally present at low levels. As a result, melanin is generally produced in small quantities by those organisms that possess tyrosinase activity. It is therefore a further objective of the present invention to provide a means for enhancing the level of tyrosinase activity in organisms in order to enhance the in vivo production of melanin.

Several prior art references teach how to genetically engineer an organism to possess tyrosinase activity. In PCT application WO 92/00373, published January 9, 1992, Applicants teach a method for producing melanin from transformed microorganisms wherein a sequence encoding for tyrosinase is introduced into the organism. Microorganisms that have be genetically engineered to enhance their abilities to produce tyrosinases include, but are not limited to species of Streptomyces, Escherichia, Bacillus, Streptococcus, Salmonella, Staphylococcus , and Vibrio. For example, cloned tyrosinase genes from Steptomyces sp. have been shown to produce melanin pigments in culture. J. Gen. Microbiol. 129:2703-2714 (1983); Gene

37.:101-110 (1985). The cloned genes have also been expressed in Streptomyces and E. coli. della-Cioppa, Bio/Technology 8.:634-638 (1990) . In each case, both tyrosinase and ORF438 were required for melanin production.

U.S. Patent No. 4,898,814 issued to Kwon discloses a cDNA clone of human tyrosinase and claims

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-4a method of making human tyrosinase by expressing the cDNA in £. coli.

Many forms of tyrosinase from bacteria, such as Streptomvces, require an activator protein. For example, the mel locus of S, antibioticus has been shown to contain two open reading frames (ORF’s) that encode a putative ORF438 protein (Mt»14,754) and tyrosinase (Mr»30,612). ORF438 and tyrosinase are thought to be transcribed from the same promoter in S, antibioticus. Beman, et al., Gene 37:101 ¢1985). Both genes are required for melanin production.

Beman, et al-, Gene 37 tioi (1985) . Based on genetic evidence, ORF438 protein has been shown to function as a trans-activator of tyrosinase. Lee, et al., dene £5.:71 (1988) . It has been suggested that the ORF438 protein is involved in tyrosinase secretion, or it may function as a tnetallothionein-like protein that delivers copper to tyrosinase, Beman, et al., dene 37:101 (1985); Lee, et al., Gene 55:71 (1988). The me! locus of S._glaucescens has a nearly identical ORF sequence upstream of tyrosinase that probably serves a similar function. Huber, et al. BiflihsmifitiX 3J.Î6038 (1985); Huber, et al.. Nucleic Acids Res. 15:8106 (1987). The existence of an ORF438 protein, however, has never been confirmed in vivo.

The melanin opérons of S.. antibioticus and S. olaucescens have been isolated and sequenced, and both share sequence homology and similar gene arrangement. The polypeptide sequence encoded by ORF438 (146 amino acids) in S. antibioticus is structurally and functionally equivalent to URF402 (134 amino acids) from S, glaucesecens Huber, et al. Nucleic Acids Res. 15.:8106 (1987) . Disruption of the URF402 coding sequence abolishes the melanin

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- 5 phenotype similar to that already known for ORF438. Recent evidence suggest that the ORF438 protein (and by analogy the URF402 protein equivalent) functions as a molecular chaperone for tyrosinase. Chen, et al., J. Biol Chem. 268 :18710 (1993) .

Tyrosinase has also been isolated and employed to produce melanin in vitro. For example, in U.S. Patent No. 5,340,734, issued August 23, 1994, Applicants teach the production of melanin in vitro using a tyrosinase which is excreted from the microorganism during fermentation. In vitro production of melanin is dependant on the production of significant quantities of tyrosinase activity. Tyrosinase activity may be lost during isolation of the secreted tyrosinase due to a disruption of the tyrosinase - activator protein complex. It is therefore an objective of the present invention to stabilize the tyrosinase - activator protein complex in order to reduce tyrosinase activity loss during isolation and purification.

Fusion proteins have been synthesized to overcome instability and proteolytic degradation of a polypeptide of interest. Many eucaryotic proteins have been produced in E. coli as fusion proteins with E. coli polypeptides such as Beta-galactosidase. Beta-galactosidase can be used to protect the foreign passenger protein from degradation in E. coli. Somatostatin, the first eurcaryotic protein to be produced in E. coli was produced by fusing a synthetic gene to the entire Beta-galactosidase coding sequence (Itakura, et al., Science 198:1056 (1977)) and somatostatin was subsequently isolated by chemical cleavage of the fusion protein. When two independently functioning polypeptides are fused into

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PCT/ÜS94/J2857 a single polyprotein by way of a synthetic gene fusion, the resulting gene construct can be engineered for expression behind a single promoter, ribosome binding site, and initiation codon. A gene fusion constructed in such a way can be placed in a single location within a chromosome or episomal element for subsequent expression of the fusion, protein. Such fusion genes are inherently more stable because both gene sequences are coordinately expressed at high levels from a single promoter. Undesirable effects such as differential down regulation or rearrangement and deletion of one of the genes can thus be avoided.

Unfortunately, however, most fusion protein constructs are not functional. Proteins fold into conformationally active states as dictated by their primary amino acid sequence. Additional polypeptide sequences at the C- or N- terminus can interfere with proper folding, thereby preventing the formation of a biologically active protein. Enzymes typically fold into well defined three-dimensional conformations to allow the formation of a catalytic pocket that excludes potential substrate molecules of abnormal size or conformation, but allows access of substrate molecules with the correct three-dimensional structure. Many enzymes will not function as fusion polypeptides because the sequence extensions interfere with, proper folding, or interfere by sterically blocking the substrate’s access to the catalytic site.

The present invention relates to Applicants' recognition that it might be possible to construct a biologically active fusion enzyme coupling tyrosinase with an activator protein. It has been shown that histidine residues #102 and # 117 of the ORF438

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activator protein are critical for copper binding and delivery to the active site of tyrosinase. Chen, et al., J.. Biol, Chem.. 2€B:16710 (1S93) . In order to form a biologically active fusion enzyme between tyrosinase and an activator protein, both functional domains of tyrosinase and the activator protein must be correctly folded. Further, the fusion enzyme must be able to assume a structural conformation enabling the activator protein to intermolecularly deliver copper to the active site of the tyrosinase to form a biologically active tyrosinase. Based on the existing prior art, it is unclear whether such a fusion enzyme would be biologically active.

SUMMARY OP INVENTION

The present invention, relates to a nucleic acid sequence encoding a fusion enzyme comprising a nucleic acid sequence encoding for a tyrosinase and a nucleic acid sequence encoding for a tyrosinase activator protein. Activator proteins used, in the present invention include but are not limited to

ORF438 and URF402. Tyrosinases used in the present invention include both prokaryotic and eucaryotic tyrosinases. Prokaryotic tyrosinases that require a separate activator protein, particularly tyrosinases derived from Streptomvces are preferred. It is also preferred that the activator protein sequence be positioned 5' relative to the tyrosinase sequence.

The present invention also relates to a vector useful for introducing a nucleic acid sequence encoding a fusion enzyme into an organism. The vector comprises a nucleic acid sequence encoding for a fusion enzyme of the present invention and a promoter sequence that regulates the transcription of fusion enzyme. The present invention also relates to

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95/13386 - 8a - PCT/US94/12857 organisms, such as bacteria, yeast fungi, plants and animals which have been transformed by the vector of the present invention.

The present invention also relates a fusion enzyme which comprises an amino acid sequence for tyrosinase and an amino acid sequence for a tyrosinase activator protein. The fusion enzyme may also contain a linker positioned between the amino acid sequences of the activator protein and the tyrosinase.

The present invention also relates to melanin produced by a fusion enzyme of the present invention. The melanin may be produced in vitro by contacting a fusion enzyme with an enzyme substrate under suitable reaction conditions to form melanin. Melanin may also be produced in vivo by an organism transformed with a vector of the present invention.

The present invention also relates to a nucleic acid sequence encoding a fusion enzyme comprising a nucleic acid sequence encoding for a tyrosinase and a nucleic acid sequence encoding for a tyrosinase activator protein positioned 5’ relative to the tyrosinase sequence .

The present invention also relates to a vector for transforming an organism, the vector comprising: a nucleic acid sequence encoding for a fusion enzyme, the fusion enzyme comprising an amino acid sequence encoding for a tyrosinase and an amino acid sequence encoding for a tyrosinase activator protein that is positioned on the N-terminus end of the tyrosinase

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WO 95/13386 - 8b - PCT/US94/12857 sequence; and a promoter sequence that regulates the transcription of fusion enzyme.

The present invention also relates to a fusion enzyme comprising an amino acid sequence for tyrosinase and an amino acid sequence for a tyrosinase activator protein that is positioned on the N-terminus end of the tyrosinase sequence.

The present invention also relates to a 10 melanin wherein the melanin is produced in vitro by a fusion enzyme comprising an amino acid sequence for tyrosinase and an amino acid sequence for a tyrosinase activator protein that is positioned on the N-terminus end of the tyrosinase sequence.

The present invention also relates to an in vitro method of producing a melanin comprising the steps of contacting a fusion enzyme comprising an amino acid sequence for tyrosinase and an amino acid sequence for a tyrosinase activator protein and a reaction substrate selected from the group consisting of L-tyrosine, X/L-tyrosine, L-tyrosine/X and X/L-tyrosine/X where X is a single amino acid, a dipeptide or an oligopeptide bound to

L-tyrosine, under suitable reaction conditions, to form melanin.

The present invention also relates to a method of producing melanins, comprising the step of growing an organism which has been transformed with a vector comprising a nucleic acid sequence encoding for a fusion enzyme, the fusion enzyme comprising an amino acid sequence encoding for a tyrosinase and an amino acid

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95/13386 - 8 c - PCT/US94/12857 sequence encoding for a tyrosinase activator protein that is positioned on the N-terminus end of the tyrosinase sequence; and a promoter sequence that regulates the transcription of fusion enzyme.

The present invention also relates to a nucleic acid sequence encoding a fusion enzyme comprising a nucleic acid sequence encoding for a protein that has cresolase activity and catecholase activity; and a nucleic acid encoding for a gene product that functions as a tyrosinase activator protein.

The present invention also relates to an amino acid sequence for a fusion enzyme comprising: an amino acid sequence for a protein that has cresolase activity and catecholase activity; and an amino acid sequence for a protein that has a tyrosinase activator function .

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 depicts the stepwise biosynthesis of melanins.

Figure 2 provides the plasmid map of pBGC623.

Figure 3 provides the plasmid map of pBGCG35.

Figure 4 provides the plasmid map of pBGC636.

Figure 5 provides the plasmid map of pBGC646.

Figure 6 provides the plasmid map of pBGC648.

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Figure 7 provides a comparison of the melanin production capabilities of E. coli when transformed by plasmids pBGC623, pBGC635, pBGC636, pBGC646 and pBGC648.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a nucleic acid sequence encoding for a fusion enzyme of tyrosinase and an activator protein for tyrosinase. The present

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-9invention also relates to a vector useful for transforming a host organism that contains the nucleic acid sequence of the present invention.

The present invention also relates to the 5 expression of a fusion enzyme of a tyrosinase and an activator protein by the transformed organism. Host organisms that may be transformed to express the fusion enzyme of the present invention include but are not limited to bacteria, yeast fungi, plants and animals. Expression of the fusion enzyme by the host organism enables and/or enhances tyrosinase activity in the host thereby enabling and/or enhancing melanin production by the host.

The present invention also relates to a fusion enzyme comprising an amino acid sequence for tyrosinase and an amino acid sequence for an activator protein of tyrosinase.

Finally, the present invention relates to the in vivo and in vitro production of melanin using the fusion enzyme of the present invention.

The fusion enzyme of the present invention comprises an amino acid sequence for a tyrosinase and an amino acid sequence for an activator protein. The nucleic acid sequences for both proteins are under the same promoter control and are thus expressed as a single peptide.

Tyrosinases used in the present invention include both prokaryotic and eucaryotic tyrosinases. In some organisms, tyrosinases are referred to as polyphenol oxidases. Tyrosinases from prokaryotes are preferred, particularly those tyrosinases that require a separate activator protein such as those that have been obtained from Streptomvces.

Activator proteins used in the present invention include but are not limited to ORF438 and URF402 .

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-10The activator protein employed in the present invention need not naturally occur in the organism from which the tyrosinase is derived. Rather, activator proteins from a variety of sources should function with a given tyrosinase in view of the similarity of the copper binding sites of different tyrosinases. See Chen, et al., J. Biol. Chem, 2£S. 18710 (1993) .

The fusion enzymes of the present invention are preferably constructed such that the amino acid sequence for the activator protein is positioned on the N- terminus of the tyrosinase amino acid sequence. As can be seen from Example 5, fusion enzymes where the activator protein is positioned on the N- terminus of tyrosinase exhibit equivalent melanin production capabilities as where tyrosinase and the activator protein are expressed separately.

Eucaryotic tyrosinases tend to comprise higher molecular weight (50-70 kD) single polypeptide chains without defined activator proteins. Applicants speculate that the activator equivalent of the ORF438 protein is built into the polypeptide backbone of eucaryotic tyrosinases. Based on the amino acid homology at the active sites between eucaryotic and Streptomyces tyrosinases, it appears that excess polypeptide sequences occur in the C- terminal region of the larger eucaryotic enzymes. Based on this observation, it might be predicted that ORF438 would function best if fused to the C- terminus rather than the N- terminus of the 30 kD Streptomyces tyrosinase since the fusion protein would then mimic the eucaryotic tyrosinases with respect tp placement of the catalytic site. However, since many newly synthesized polypeptides undergo three dimensional folding into their preferred active conformation

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-11beginning with the free C- terminus, it might also be expected that C- terminal additions are disruptive to proper folding and, hence, normal catalytic activity. Hence, prior to preparing the fusion enzymes of the present invention, it was unclear whether a fusion enzyme at either the N- or C- terminus would be functional.

The amino acid sequence encoding the activator protein need not bedirectly attached to the tyrosinase sequence. In plasmid. pBGC648, a His residue has been inserted between the activator protein and the tyrosinase sequence. A single amino acid and a repeating Pro-Thr amino acid sequence, which behaves as a natural hinge, are preferred as linking sequences between tyrosinase and the activator protein.

Determination of the wide variety of amino acid sequences that may be interposed between the tyrosinase and activator protein sequences can be routinely determined by one of ordinary skill in the art in view of the teachings of the present invention.

Other polypeptide linkers that are known from the literature to provide a high degree of flexibility between two polypeptide sub-domains might work equally as well as linking sequences between the tyrosinase sequence and the activator protein sequence. One example of this type of hinge polypeptide is a proline - threonine repeating unit such as those known form cellulose binding proteins. Ong, et al., Bio/Technoloov 7 €04(1989).

Construction of synthetic oligonucleotide linkers that encode hinge polypeptides of known flexibility is within the level of ordinary skill. In addition, synthetic oligonucleotide linkers could be used to

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-12construct random polypeptide linker sequences to join the tyrosinase - activator protein domains together. Linkers with a high degree of flexibility and sufficient length might be expected to work best for intermolecular cis-activation of tyrosinase.

Inflexible linker polypeptides, or extremely short linker polypeptides, might be expected to permit only intramolecular trans-activation of neighboring fusion enzymes ,

In order to provide a clear and consistent understanding of the specification and the claims, including the scope given to such terms, the following definitions are provided:

Activator protein: a gene product that alters, activates or enhances the activity of tyrosinase. The activator protein may function as a trans-activator, as a metallothionein-like protein that delivers an ion to a tyrosinase apoenzyme or it may function in assisting the secretion of tyrosinase. ORF438 gene, URF402 and ORF{s)

3' to the tyrosinase coding sequence code for activator proteins that enhance melanogenesis in all of the ways described.

Melanin: Melanins are polymers produced by polymerization of reactive intermediates.

The polymerization mechanisms include but are not limited to autoxidacion, enzyme catalyzed oxidation and free radical initiated polymerization. The reactive intermediates are produced chemically or enzymatically from precursors. Suitable enzymes include, but are not limited to

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-13peroxidase and catalases, polyphenol oxidases, tyrosinases, tyrosine hydroxylases or laccases. The precursors which are converted to the reactive intermediates are hydroxylated aromatic compounds. Suitable hydroxylated aromatic compounds include, but are not limited to

1) phenols, polyphenols, aminophenols and thiophenols of· aromatic or polycyclic aromatic hydrocarbons, including but not limited to phenol, tyrosine, pyrogallol, 3aminotyrosine, thiophenol and a-naphthol;

2) phenols, polyphenols, aminophenols, and thiophenols of aromatic heterocyclic or heteropolycyclic hydrocarbons such as but not limited to 2-hydroxypyrrole, 4-hydroxy1,2-pyrazole, 4-hydroxypyridine, 8hydroxyquinoline, and 4,5dihydroxybenzothiazole. Suitable hydroxylated aromatic compounds also include X/L-tyrosine, L-tyrosine/X and X/Ltyrosine/X where X is a single amino acid, a dipeptide or an oligopeptide bound to Ltyrosine.

The nucleic acid sequences encoding for the fusion enzymes of the present invention may be inserted into a wide variety of vector constructs known in the art for transforming a host organism. Suitable techniques include those described in Maniatis, et al., Molecular Cloning. 1st Ed., Cold Spring Harbor Laboratory, New York (1982); Molecular Cloning. 2nd Ed., Cold Spring Harbor Laboratory, New York (1989); Methods in Enzymolocry. Vols. 68 (1979), 100 (1983), 101 (1983), 118 (1986) and Vols. 152-154

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- 14 (1987) DNA Cloning. Glover, Ed., IRL Press, Oxford (1985); and Plant Molecular Biology: Manual, Gelvin, et al., Eds., Kluwer Academic Publishers, Podrecht (1988). Medium compositions have been described in Miller, Experiments in Molecular Genetics, Cold Spring Harbor Laboratory, New York (1972), as well as the references previously identified. Hopewood, et al., Genetic Manipulation of Streptomyces: A Laboratory Manual, The John Innés Foundation, Norwich, England (1985).

With regard to the expression of the tyrosinaseactivator protein fusion enzyme of the present invention in a transformed microorganism, it is preferred that the transformed organism be grown under the conditions described in PCT application WO 92/00373, published January 9, 1992.

With regard to the expression of the tyrosinaseactivator protein fusion enzyme of the present invention in plants, it is preferred that the nucleic acid cassette encoding the fusion enzyme be inserted into one of the viral constructs described in U.S. Patent No. 5,316,931, issued May 31, 1994, or PCT application WO 94/16089, published July 21, 1994.

Vectors encoding the tyrosinase-activator protein fusion enzyme of the present invention may be produced by standard techniques. Appropriate vectors which can be utilized as starting materials are known in the art.

The DNA sequence coding for the fusion enzyme is inserted into an appropriate vector in such a manner that the enzyme is correctly expressed. In other words, the DNA sequence is positioned in the proper

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-15orientation and reading frame so that the correct amino acid sequence is produced upon expression of the DNA sequence in the host. In accordance with conventional techniques, a chimeric DNA sequence is generally constructed which contains a promoter operable in the specific host and the DNA sequence coding for the desired enzyme. The chimeric DNA sequence may further contain 3' non-coding sequences operable in the host. The chimeric DNA sequence can be prepared in situ within a suitable vector by inserting the DNA sequence coding for the enzyme into a restriction site of a known host transformation vector. Alternatively, the chimeric gene could be first constructed and then inserted into a vector to produce a transformation vector. The vector can be further modified by utilizing an enhancer sequence and/or a strong promoter, which leads to an increased production of the fusion enzyme.

The typical vector is a plasmid having one or more marker genes for antibiotic resistance, an origin of replication, and a variety of useful restriction sites for cloning or subcloning restriction fragments. A large number of vectors have been described which are useful for transforming many microorganisms including but not limited to Streptomyces and £. See, for example, Cloning

Vectors. Pouwels, et al. ed. Elsevier Science Publishers Amsterdam (1985).

A large number of naturally occurring Streptomyces plasmids have been described, many of which are conjugally proficient. Two such isolates, SLP1.2 and pIJlOl, have formed the basis of a series of useful plasmid vectors. Thompson, et al., Gene 211:51 (1982). The plasmids of the SLP1 family, of which SLP1.2 is the largest detected member, were

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-16discovered as autonomous replicons in 5. lividans 66 after interspecific matings with S, coelicolor A3(2). The SLPI replicon is integrated in the S, coelicolor genome but can be excised together with various lengths of neighboring DNA to become autonomous in S.

lividans. The SLPi plasmids exist stably at a copy number of 4-5 per chromosome in S. lividans and have a narrow host range.

The 8.9 kb plasmid pIJlOl was discovered in 5 .

lividans ISP5434 (Kieser, et al., Mol. Gen. Genet.

185:223 (1932)) but can be conjugally transferred to a wide variety of Streptomyces species. Derivatives (e.g. pIJ102) have been isolated from the plasmid which have similar properties but are smaller.

Kieser, et al. (1982), supra. Plasmid pIJlOl has a copy number of 100-300 per chromosome equivalent in most hosts and a minimum replicon of less than 2.1 kb. Derivatives carrying drug-resistance determinants have been constructed to act as vectors, and a chimeric plasmid which can be used as a shuttle vector between £. coli and Streptomvces is available.

The temperate phage $>C31 has a wide host range within the Streotomycetes and lysogenizes S, coelicolor A-3(2) via a site-specific integration event. Lomovshaya, et al., Bacteriol Rev. 44. 206 (1980} . Up to 42.4 kb of DNA can be packaged within a viable phage particle, but only 32 kb (at the most) of'the DNA contains the genetic information essential for plaque formation. Derivatives of 0C31 containing deletions can be used as vectors, and recombinant phages can either be grown lytically or used to lysogenize suitable streptomyces strains.

pBR322-derived plasmids are very common for use in £. coli transformation. They possess a pair of antibiotic resistance genes which confer antibiotic

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-17resistance when Escherichia coli are successfully transformed. Typically, the insertion of a DNA segment is made so that one of the antibiotic resistance genes is inactivated. Selection then is accomplished by selecting for £. coli exhibiting antibiotic resistance conferred by the second gene. Bolivar, et al.. Gene 2:95 (1977); and Sutcliff, J.. Proc. -Natl, Acad^-Sci. , USA 75:3737 (1978).

Another example, of transforming vectors is the 10 bacteriophage. The M13 series are modified filamentous £. coli bacteriophage containing single stranded circular DNA. The M13 series carry the lacZ gene for β-galactosidase and will metabolize the galactose analog Xgal to produce a blue color.

Placing a cloned insert into the polylinker sequence located in the amino terminus of the lacZ gene inactivates the gene. Microorganisms carrying an M13 with an inactivated lacZ (representing a cloned insert) are distinguishable from those carrying an

Mil with an active lacZ gene by their lack of blue color. Messing, et al., Proc. Natl. Acad, Sci.. USA 24:3642 (1977); and Messing, Methods χη.-ΕπζντΰοΙοαν 101:20 (1983) .

Other transforming vectors are the pUC series of plasmids. They contain the ampicillin resistance gene and origin of replication from pBR322, and a portion of the lacZ gene of £. coll. The lac region contains a polylinker sequence of restriction endonuclease recognition sites identical to those in the M13 series. The pUC series have the advantage that they can be amplified by chloramphenicol. When a DNA fragment is cloned into the lac region the lac gene is inactivated. When £. coli containing a pUC plasmid with an inactivated lacZ gene is grown in the presence of isopropylthiogalactoside (IPTG) and

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- 18 5-bromo-4-chloro-3-indolyl β-D-galactopyranoside (Xgal) its colonies are white. If it carries a pUC plasmid with an active lacZ gene its colonies are blue. Vieira, et al., Gene 19:259 (1982). Bacteria are transformed by means conventional in the art.

The genus Streptomyces is one of three aerobic genera of bacteria of the order Actinomycetales. Streptomyces are Gram-positive, mycelial, sporeforming bacteria. Several naturally occurring Streptomyces plasmids have been described. Streptomyces lividans TK64 has no tyrosinase gene and produces no melanin. Applicants teach the transformation of Streptomyces lividans TK64 with plasmid pIJ702 which encodes for the tyrosinase gene in PCT application WO 92/00373, published January 9, 1992. Transformation is carried out by means standard in the art. Similarly, transformation of Streptomyces can be performed using a plasmid encoding the fusion enzyme of the present invention. Transformation vectors of the present invention may also be used to transform a variety of microorganisms after insertion into vectors which are useful for transforming the corresponding host microorganism.

Bluescript (obtained from Stratagene, LaJolla, CA) is a pUC derivative having a β-galactosidase color indicator and a lac promoter. In PCT application WO 92/00373, published January 9, 1992, Applicants teach modification of the Bluescript plasmid by inserting a tyrosinase gene. This modified plasmid was used to successfully transform E. coli which formed pigmented colonies. Similarly, the Bluescript may be modified by inserting a nucleic acid sequence encoding for the fusion enzyme of the present invention.

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-19Melanin has been purified from bacterial cells with 0.5N NaOH at room temperature and at 100®C. Pigmented fractions were found to be: (l) soluble in acid and base; (2) soluble in ethyl alcohol and base,· and (3) soluble base only. Pavlenko, et al.,

MicroblflloçnL-USSR 539 (1981)

Soluble melanin can be extracted from the medium and purified. This is done by first removing cells and particulate matter using, for example, filtration or centrifugation. A variety of filtration methods are known in the art including filtration through glass wool. If centrifugation is used, 5,000 X gravity is usually sufficient. The melanin is then precipitated at between pH 2-4, preferably about 3.

Precipitated melanin is removed by either filtration or centrifugation. The melanin is washed, by successive resolubilization at high pH, i.e. about pH 7.0 to about pH 9.0, preferably about pH 8.0, and precipitation at low pH followed by filtration or centrifugation. The melanin may also be concentrated using molecular weight filtration, such as reverse osmosis. Salt precipitation can be as effective in precipitating the melanin as low pH.

The invention is further illustrated by the following non-limiting examples.

EXAMPLES

1. Preparation-Of Plasmids OBGC623, PBGC635 and

Plasmid pBGC€23 is a plasmid containing the nucleic acid sequence for ORF43B. Figure 2 provides the plasmid map of pBGC623. Plasmid pBGC635 is a plasmid containing a nucleic acid sequence for tyrosinase. Figure 3 provides the plasmid map of pBGC635. Plasmid pBGC636 is a plasmid encoding for

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- 20 both ORF438 and tyrosinase under separate promoters. Figure 4 provides the plasmid map of pBGC636. Preparation of plasmids pBGC623, pBGC635 and pBGC636 are taught in PCT application WO 92/00373, published January 9, 1992.

. Preparation of pBGC646

In order to prepare pBGC646, the Bel I site at the stop codon of the tyrosinase gene (in pBGC188Nde) was blunted with mung bean nuclease and ligated with a synthetic Eco RI linker (dGGAATTCC; SEQ. NO. 1). The tyrosinase gene was removed from plasmid pBGC188Nde as a 822bp Nde I/Eco RI fragment and gel purified in low melt agarose. The purified fragment was ligated into an ORF438 containing plasmid (pBGC623) that was modified as follows. Plasmid pBGC623 was cleaved with Neo I, blunted with mung bean nuclease, and ligated with a synthetic Eco RI I linker (dGGAATTCC). The plasmid was gel purified in low melt agarose and ligated with the Nde I/Eco RI fragment containing the modified tyrosinase gene. Figure 5 provides the plasmid map of pBGC646 [SEQ. NO. 2]. The translated amino acid sequence for the fusion enzyme encoded for by plasmid pBGC646 is provided as SEQ. NO. 3.

Transformants were screened in HB101 and two independent transformants were identified as having the correct orientation. These plasmids are pBS646.9 and pBS646.19, and both are identical. Both plasmids were transformed into E. coli strain K38 that harbors plasmid PGP1-2 and plated on agar containing tyrosine and copper. Both transformants gave a melanin phenotype as described previously (della, Cioppa, et al.

Bio/Technology 8.:634-638 (1990)), and were shown by

SDS-PAGE to give a ~4 5kD band by SDS-PAGE. The

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-21hybrid tyrosinase/ORF438 fusion enzyme is predicted to encode a 45,806 MW protein with four additional amino acids linking the two functional domains (NH2tyrosinase-trp-asn-ser-ala-ORF438-COOH).

3, Preparation of OBSC648

To construct a second hybrid fusion gene between ORP438 and tyrosinase, the two synthetic oligonucleotides shown below ’-dCCAGGGCGCCCGGCTÇCTCqCCTTCCCCTCCAACCA-3 ' [SEQ. NO. 4] '-CTCGAGGTCCCGCGGGCCGAGGAGGGGAAGGGGAGGTTGGTAT-S ’ [SEQ. NO. 5J were annealed, kinased, and used to clone into the Sac 1 site of the ORF438 gene. This replacement oligonucleotide sequence results in the destruction of the TGA stop codon of ORF438 and creates an inframe Ndel site in its place for insertion of tyrosinase. A triple ligation reaction was set up that included the annealed oligonucleotide shown above, a 2,934 bp Hind III/Sac I fragment form pBGC623, and a 1,107 bp Ndel/Hind III fragment from PBGC635. The new plasmid (pBGC648) creates an ORF43B/tyrosinase in-frame gene fusion that encodes a single polypeptide chain of 46,230 Daltons. The introduction of the new Ndel site introduces a single histidine residue between the two coding sequences upon translation. Figure 6 provides the plasmid map of'pBGC648. The nucleic acid sequence for pBGC648 is provided as SEQ. NO. 6. The translated amino acid sequence for the fusion enzyme encoded for by plasmid pBGC648 is provided as SEQ. HO. 7.

The dna at the junction of the ORF438 and tyrosinase genes in plasmid pBGC64B were sequenced. Approximately 180 bp 3' of the Sac I site in the fusion were found to be correct, and the translated amino acid sequence is as predicted (Sss SEQ. No. 7) .

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-22Plasmid pBGC648 was transformed into E. coli strain K38 that harbors plasmid pGPl-2 and plated on agar containing tyrosine and copper. The transformants gave a black melanin phenotype as described previously (della-Cioppa, et al.. Bio/Technology £:634-638 (1990)), and were shown by SDS-PAGB to give a -46kD band by SDS-PAGE. Transformants harboring PBGC648 give rise to.the black melanin phenotype as rapidly as that seen when 0RF438 and tyrosinase are expressed as single polypeptide chains from single genes. Phenotypically, the ORF438/tyrosinase gene fusion retains full catalytic activity similar to the wild type -30kD tyrosinase holoenzyme.

4. Transformation-gf-E, coli with pBGC623^_ pBG-C635_, pBGC63S, P3GC646 and PBGC648

Plasmids pBGC623, pBGC635, pBGC636, pBGC646 and

PBGC648 were introduced into B. coli by pretreating exponentially growing cultures of the E. coli with CaCl2 as described in Maniatis, et al., Molecular so Cloning (1982).

. Comparison of Melanin-Production Bv E. coli transformed with PBGC623, PBGC635, pBGC636,

PBGC646- and-pBGC£-4B

Plasmids pBGC623, pBGC635, pBGC636, pBGC€46 and pBGC648 were each introduced into E. coll. The transformed strains of B. coli were then grown on agar plates containing tyrosine and copper as described previously in della-Cioppa, et al., Bio/Technolocrv £ 634 (1990) in order to evaluate each strains ability to produce melanin. Figure 7 provides a comparison of the melanin production capabilities of B, coli when transformed by these five different plasmids.

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The amount of melanin formed was determined by the size and color intensities of black melanin halos that formed around the transformed E. coli colonies. The rate of color development on agar plates, and the intensity of the black halo formation, is directly proportional to the level of tyrosinase enzymatic activity in each of the different plasnid bearing S. coli colonies. Melanin formation was quantitated as (-) none, (+) very weak, (++) weak, (+++) moderate, (++++) strong and (+++++} very strong.

E, coli transformed with either pBGC623 or pBGC635 do not produce a positive black melanin phenotype. E... coli transformed with pBGC63€ produced a positive black melanin phenotype.

E. coli transformed with pBGC646 gave rise to a positive black melanin phenotype. However, E, coli transformed with pBGC646 produced melanin at a slower rate than E. coli transformed with pBGC63€ in which ORF438 and tyrosinase are expressed as single polypeptide chains from single genes.

E. coli transformed with pBGC648 produced a positive black melanin phenotype at a comparable rate as pBGC536, indicating that the fusion enzyme produced by pBGC€48 functions equally well ae when ORF438 and tyrosinase are expressed independently.

6. Expressipn Of. Tvrosinase/QRF438 Fusion Enzyme

Containing Chloroplast Targeting Sequence In

Plants

For expression of a tyrosinase - activator protein fusion enzyme in higher plants, it may be advantageous to target the fusion enzyme to the chloroplast. Chloroplasts are known to contain the enzymatic pathway for production of L-tyrosine (the

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- 24 primary substrate for tyrosinase), and the oxidative environment inside the chloroplast may be well suited for achieving optimal enzymatic activity.

In order to target a tyrosinase - activator protein fusion enzyme to chloroplasts, the nucleotide sequence encoding the chloroplast transit peptide (CPT) from ribulose bisphosphate carboxylase small subunit (RuBPCase SSU) from Nicotiana tabacum was cloned (as an Neo Ι/Sph I fragment) and fused by way of its naturally occurring Sph I site (at the cys-met cleavage site of the CTP) to the naturally occurring Sph I site at the N-terminus of QRF438. The CTP/ORF438 nucleotide fusion was then exchanged as an Neo I/Sac I fragment in the plasmid BlueScript™ that contained the

ORF438/tyrosinase sequence as described in plasmid pBS648. The resulting nucleotide sequence and translated amino acid sequence of the CTP/ORF438/tyrosinase fusion are shown in SEQ. No. 8 and SEQ. No. 9 respectively. The CTP/ORF438 tyrosinase fusion gene encodes 478 amino acids residues of which the 57 at the N-terminus direct the ORF438/tyrosinase fusion into the chloroplast. Upon import into the chloroplast compartment, the 57 amino acid CTP is proteolytically removed thus resulting in the identical ORF438/tyrosinase fusion enzyme of ^45 kD as previously produced in E. Coli. The CTP/ORF438/tyrosinase fusion enzyme has a deduced molecular weight of 51,461 Daltons .

The CTP/ORF438/tyrosinase nucleotide sequence may then be inserted into a viral construct such as those described in U.S. Patent No. 5,316,931, issued May 31, 1994, or PCT application WO 94/16089, published July 21, 1994 and used to systemically infect higher plants.

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-25While the invention has been disclosed by reference to the details of preferred embodiments, the disclosure is intended in an illustrative rather than in a limiting sense, as it is contemplated that modifications will readily occur to those skilled in the art, within the spirit of the invention and the scope of the appended claims.

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-26SBQUEHCB LISTING (1} GENERAL INFORMATION:

(i) APPLICANT: Della-Cioppa, Guy

Kumagai, Monto (Ü) TITLE OP INVENTION: TYROSINASE-ACTIVATOR

PROTEIN FUSION ENZYME (iii) NUMBER OF SEQUENCES: 9 (iv) CORRESPONDENCE ADDRESS:

(A} ADDRESSEE: Pennie t Edmonds (B) STREET: 2730 Sand Kill Road (C) CITY: Menlo Park (D) STATE: California (E) COUNTRY: U.S.A.

(F) ZIP: 94025 (v) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS (D) SOFTWARE : Patent in Release #1.0,

Version #1.25 (vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER: US 08/152,483 (B) FILING DATE: November 12, 1993 (C, CLASSIFICATION:

(vii) PRIOR APPLICATION DATA:

(A) APPLICATION NUMBER: 7/857,602 (B) FILING DATE: March 30, 1992 (vii) PRIOR APPLICATION DATA:

(A) APPLICATION NUMBER: 923,692 (B) FILING DATE: July 31, 1992 (vii) PRIOR APPLICATION DATA:

(A) APPLICATION NUMBER: 600,244 (B) FILING DATE: October 22, 1990 (vii) PRIOR APPLICATION DATA:

(A) APPLICATION NUMBER: 641,617 (B) FILING DATE: January 16, 1991 (vii) PRIOR APPLICATION DATA:

(A) APPLICATION NUMBER: 737,899 (B) FILING DATE: July 26, 1991 (viii) ATTORNEY/AGENT INFORMATION:

(A) NAME: Halluin, Albert P.

(B) REGISTRATION NUMBER: 25,227 (C) REFERENCE/DOCKET NUMBER: BIOG-20240/8129-040 (ix) TELECOMMUNICATION INFORMATION:

(A) TELEPEONE: (415) 854-3660 (B) TELEFAX; (415) 854-3694 (Cl TELEX: €6141 PEMNIE

PCT/US94/12857

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<td> (2)</td><td> INFORMATION FOR SEQ ID NO: 1:</td><td></td>

<td> (!)</td><td> SEQUENCE CHARACTERISTICS : (A) LENGTH: 8 (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear</td><td></td>

<td> (ii)</td><td> MOLECULE TYPE: (A) DESCRIPTION: DNA</td><td></td>

<td> (iii)</td><td> HYPOTHETICAL : NO</td><td></td>

<td> (iv)</td><td> ANTI-SENSE: NO</td><td></td>

<td> (vi)</td><td> ORIGINAL SOURCE: (A) ORGANISM:</td><td></td>

<td> (vii)</td><td> IMMEDIATE SOURCE: (B) CLONE:</td><td></td>

<td> (ix)</td><td> FEATURE:</td><td></td>

<td> (xi)</td><td> SEQUENCE DESCRIPTION: SEQ</td><td> ID NO: 1</td>

GGAATTCC g (2) INFORMATION FOR SEQ ID NO: 2:

(i) SEQUENCE CHARACTERISTICS :

(A) LENGTH: 42»4 (B) TYPE: nucleic acid (C) STRANDEDNESS : Single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:

(A) DESCRIPTION: DNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE:

(A) ORGANISM:

(vii) IMMEDIATE SOURCE:

(B) CLONE:

(ix) FEATURE:

(xi) SEQUENCE DESCRIPTION: SBQ ID NO: 2:

AAATCAATCT AAAGTATATA TGAGTAAACT TGGTCTGACA GTTACCAATG CTTAATCAGT6C

GAGGCACCTA TCTCAGCGAT CTGTCTATTT CGTTCATCCA TAGTTGCCTG ACTCCCCGTC120

GTGTAGATAA CTACGATACG GGAGGGCTTA CCATCTGGCC CAGTGCTGCA ATGATACCGC180

GAGACCCACG CTGACCGGCT CCAGATTTAT CAGCAATAAA CCAGCCAGCC GGAAGGGCCG240

AGCGCAGAAG TGGTCCTGCA ACTTTATCCG CCTCCATCCA GTCTATTAAT TGTTGCCGGG300

AAGCTAGAGT AAGTAGTTCG CCAGTTAATA GTTTGCGCAA CGTTGTTGCC ATTGCTACAG360

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<td> GCATCGTGGT</td><td> GTCACGCTCG</td><td> GCGTTTGGTA</td><td> TGGCTTCATT</td><td> CAGCTCCGGT</td><td> TCCCAACGAT420</td>

<td> CAAGGCGAGT</td><td> TACATGATCC</td><td> CCCATGTTGT</td><td> GCAAAAAAGC</td><td> GGTTAGCTCC</td><td> TTCGGTCCTC480</td>

<td> CGATCGTTGT</td><td> CAGAAGTAAG</td><td> TTGGCCGCAG</td><td> TGTTATCACT</td><td> CATGGTTATO</td><td> GCAGCACTCCS40</td>

<td> ATAATTCTCT</td><td> TACTGTCATG</td><td> CCATCCGTAA</td><td> GATGCTTTTC</td><td> TGTGACTGGT</td><td> GAGTACTCAASOO</td>

<td> CCAAGTATTT</td><td> OGAAGATGCG</td><td> CGACCGAGTT</td><td> GCTCTTGCCC</td><td> GGCGTCAACA</td><td> CGGGATAATA660</td>

<td> CCGCGCCACA</td><td> TAGCAGAACT</td><td> TTAAAASTGC</td><td> TCATCKTTGG</td><td> AAAACGTTCT</td><td> TCGGGGCGAA720</td>

<td> AACTCTCAAG</td><td> GATCTTACCG</td><td> CTGTTGAGAT</td><td> CCAGTTCGAT</td><td> GTAACCCACT</td><td> CGTGCACCCA78C</td>

<td> ACTGATCTTC</td><td> AGCATCTTTT</td><td> ACTTTCACCA</td><td> GCGTTTCTGG</td><td> GTGAGCAAAA</td><td> ACAGGAAGGC840</td>

<td> AAAATGCCGC</td><td> AAAAAAGGGA</td><td> ATAAGGGCGA</td><td> CACGGAAATG</td><td> TTGAATACTC</td><td> ATACTCTTCC900</td>

<td> TTTTTCAATA</td><td> TTATTGAAGC</td><td> ATTTATCAGG</td><td> GTTATTGTCT</td><td> CATGAGCGGA</td><td> TACATATTTG980</td>

<td> AATGTATTTA</td><td> GAAAAATAAA</td><td> CAAATAGGGG</td><td> TTCCGCGCAC</td><td> ATTTCCCCGA</td><td> AAAGTGCCAC1020</td>

<td> CTGACGTCTA</td><td> AGAAACCATT</td><td> ATTATCATGK</td><td> CATTAACCTA</td><td> TAAAAATAGG</td><td> CGTATCACGA10 8 0</td>

<td> GGCCCTTTCG</td><td> TCTTCAAGAA</td><td> ttaaaaggat</td><td> ctaggtgaag</td><td> ateetttttg</td><td> ataatctcatlL4 0</td>

<td> gaeeaaaatc</td><td> ccttaacgtg</td><td> agttttcgtt</td><td> ceactgagcg</td><td> tcagaccccg</td><td> tagaaaagatl2 0 0</td>

<td> caaaggatct</td><td> tettgagatc</td><td> ctttttctet</td><td> gcgcgtaatc</td><td> tgctgcttgc</td><td> aaacaaaaaal2€ 0</td>

<td> accaccgcta</td><td> ccagcggtgg</td><td> tttgtttgcc</td><td> ggatcaagag</td><td> ctaccaaetc</td><td> tttttccgaal320</td>

<td> ggtaactggc</td><td> ttcagcagag</td><td> cgcagatacc</td><td> aaatactgtc</td><td> cttctagtgt</td><td> agccgtagttlîBQ</td>

<td> aggccaccac</td><td> ttcaagaact</td><td> ctgtagcacc</td><td> gcctacatac</td><td> ctcgctctgc</td><td> taatcctgttl44C</td>

<td> accagtgget</td><td> gctgccagtg</td><td> gcgataagtc</td><td> gtgtcttaee</td><td> gggttggact</td><td> caagaegatalSOO</td>

<td> gttaccggat</td><td> aaggcgcagc</td><td> ggtcgggetg</td><td> «•«cggggggt</td><td> tcgtgcacac</td><td> agcccagcttisso</td>

<td> ggagcgaacg</td><td> acctacaccg</td><td> aactgagata</td><td> cctacagcgt</td><td> gagctatgag</td><td> aaagcgccacl€20</td>

<td> gcttcccgaa</td><td> gggagaaagg</td><td> cggacaggta</td><td> tccggtaagc</td><td> ggcagggtcg</td><td> g&acaggagaieeo</td>

<td> gcgcaegagg</td><td> gagcttccag</td><td> ggggaaacgc</td><td> ctggtatctt</td><td> tatagtcctg</td><td> tcgggtttcgi740</td>

<td> ccacctctga</td><td> cttgagcgtc</td><td> gatttttgtg</td><td> atgctcgtca</td><td> 533993=99*</td><td> gcctatggaalflOO</td>

<td> aaacgccagc</td><td> aacgcggect</td><td> ttttacggtt</td><td> cctggccttt</td><td> tgctggcctt</td><td> ttgctcaeatieeo</td>

<td> gttctttcct</td><td> gcgttatccc</td><td> ctgattctgt</td><td> ggataaccgt</td><td> attaccgcct</td><td> ttgagtgagcl920</td>

<td> tgataecgct</td><td> cgccgcagcc</td><td> gaacgaccga</td><td> gcgcagcgag</td><td> tcagtgagcg</td><td> aggaageggal98O</td>

<td> agagcgcctg</td><td> atgcggtatt</td><td> ttcteettac</td><td> gcatctgtgc</td><td> ggtatttcac</td><td> accgcaCAGA2040</td>

<td> TCTGtggtgc</td><td> actctcagta</td><td> caatctgctc</td><td> tgatgccgca</td><td> tagttaagcc</td><td> agtatacact.2100</td>

<td> ccgctatcge</td><td> tacgtgactg</td><td> ggtcatggct</td><td> gcgccccgac</td><td> acccgccaac</td><td> acccgctgac216O</td>

<td> gcgccctgac</td><td> gggcttgtct</td><td> gctcccggca</td><td> tcegcttaca</td><td> gacaagctgt</td><td> gaccgtctcc2220</td>

<td> gggagetgca</td><td> tgtgtcagag</td><td> gttttcaccg</td><td> tcatcaccga</td><td> aacgcgcgag</td><td> gCCcagctgC2280</td>

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<td> GATTCGAACC</td><td> tetcgattcg</td><td> aacttetgat</td><td> agaettçgaa</td><td> attaatacga</td><td> ctcaetatag2340</td>

<td> ggagaccaca</td><td> aeggtetecc</td><td> tetagaaata</td><td> attttgttta</td><td> actttaagaa</td><td> ggagatat*c24ûo</td>

<td> atatgACCGT</td><td> CCGCAAGAAC</td><td> CAGGCGTCCC</td><td> TGACCGCCGA</td><td> GGAGAKGCGC</td><td> CGCTTCGTCG2460</td>

<td> CCGCCCTGCT</td><td> CGAACTCAAG</td><td> CGCACCGGCC</td><td> GCTACGACGC</td><td> CTTCGTCACC</td><td> ACGCACAACG2520</td>

<td> CGTTCATCCT</td><td> GGGCGACACC</td><td> GACAACGGCG</td><td> AGCGCACCGG</td><td> CCACCGTTCG</td><td> CCGTCCTTCC25S0</td>

<td> TGCCCTGGCA</td><td> CCGCAGATTT</td><td> CTGCTGGAGT</td><td> TCGAGCGGGC</td><td> GCTCCAGTCG</td><td> GTGGACGCGT2640</td>

<td> CGGTGGCGCT</td><td> GCCGTACTGG</td><td> GACTGGTCCG</td><td> CCGACCGGTC</td><td> CACCCGGTCC</td><td> TCGCTGTGGG2700</td>

<td> CGCCGGACTT</td><td> CCTCGGCGGC</td><td> ACCGGGCGCA</td><td> GCCGGGACGG</td><td> CCAGGTGATG</td><td> GACGGGCCGT2760</td>

<td> TCGCCGCGTC</td><td> GGCCGGCAAC</td><td> TGGCCGATCA</td><td> ATGTGCGGGT</td><td> GGACGGCCGT</td><td> ACGTTCCTGC2820</td>

<td> GGCGGGCGCT</td><td> CGGCGCGGGC</td><td> GTGAGCGAAC</td><td> TGCCCACGCG</td><td> TGCCGAGGTC</td><td> GACTCGGTGC28BO</td>

<td> TGGCGATGGC</td><td> GACGTACGAC</td><td> ATGGCGCCCT</td><td> GGAACAGCGG</td><td> CTCCGACGGC</td><td> TTCCGCAACC2940</td>

<td> ATCTCGAAGG</td><td> GTGGCGCGGG</td><td> GTCAATCTGC</td><td> ACAACCGGGT</td><td> GCATGTCTGG</td><td> GTCGGCGGCC3000</td>

<td> AGATGGCGAC</td><td> CGGGGTCTCC</td><td> CCCAACGACC</td><td> CGGTGTTCTG</td><td> GCTGCACCAC</td><td> GCCTACATCG3060</td>

<td> ACAAGCTGTG</td><td> GGCCGAGTGG</td><td> CAGCGGCGGC</td><td> ACCCCTCGTC</td><td> CCCGTATCTG</td><td> CCGGGCGGCG3120</td>

<td> GCACGCCGAA</td><td> CGTCGTCGAC</td><td> CTCAACGAGA</td><td> CGATGAAGCC</td><td> GTGGAACGAC</td><td> ACCACCCCGG31S0</td>

<td> CGGCCCTGCT</td><td> GGACCACACC</td><td> CGGCACTACA</td><td> CCÏTCGACGT</td><td> Ctggaattcc</td><td> GCGGAACTCA3240</td>

<td> CCCGTCGTCG</td><td> CGCGCTCGGC</td><td> GCCGCAGCCG</td><td> TCGTCGCCGC</td><td> CGGTGTCCCG</td><td> CTGGTCGCCC33OO</td>

<td> TTCCCGCCGC</td><td> CCGCGCGGAC</td><td> GATCGGGGGC</td><td> ACCACACCCC</td><td> CGAGGTCCCC</td><td> GGGAACCCGG336O</td>

<td> CCGCGTCCGG</td><td> CGCCCCCGCC</td><td> GCCTTCGACG</td><td> AGATCTACAA</td><td> GGGCCGCCGG</td><td> ATACAGGGCC3420</td>

<td> GGACGGTCAC</td><td> CGACGGCGGG</td><td> GGCCACCACG</td><td> GCGGCGGTCA</td><td> CGGCGGTGAC</td><td> GGTCACGGCG3480</td>

<td> GCGGCCATCA</td><td> CGGCGGCGGT</td><td> TACGCCGTGT</td><td> TCGTGGACGG</td><td> CGTCGAACTG</td><td> CATGTGATGC3540</td>

<td> GCAACGCCGA</td><td> COGCTCGTGG</td><td> ATCAGCGTCG</td><td> TCAGCCACTA</td><td> CGAGCCGGTG</td><td> GACACCCCGC3600</td>

<td> GCGCCGCGGC</td><td> CCGCGCTGCG</td><td> GTCGACGAGC</td><td> TCCAGGGCGC</td><td> CCGGCTCCTC</td><td> CCCTTCCCCT3660</td>

<td> CCAACtgaCC</td><td> TTCTCCCCCG</td><td> CACTTTTGGA</td><td> GCACCCGCAC</td><td> atgACCGTCC</td><td> GCAAGAACCA3720</td>

<td> GGCGTCCCTG</td><td> ACCGCCGAGG</td><td> AGAAGCGCCG</td><td> CTTCGTCGCC</td><td> GCCCTGCTCG</td><td> AACTCAAGCG3780</td>

<td> CACCGGCCGC</td><td> TACGACGCCT</td><td> TCGTCACCAC</td><td> GCACAACGCG</td><td> TTCATCCTGG</td><td> GCGACACCGA3840</td>

<td> CAACGGCGAG</td><td> CGCACCGGCC</td><td> ACCGTTCGCC</td><td> GTCCTTCCTG</td><td> CCCTGGCACC</td><td> GCAGATTTCT39OO</td>

<td> GCTGGAGTTC</td><td> GAGCGGGCGC</td><td> TCCAGTCGGT</td><td> GGACGCGTCG</td><td> GTGGCGCTGC</td><td> CGTACTGGGA3960</td>

<td> CTGGTCCGCC</td><td> GACCGGTCCK</td><td> CCCGGTCCTC</td><td> GCTGTGGGCG</td><td> CCGGACTTCC</td><td> TCGGCGGCAC4020</td>

<td> CGGGCGCAGC</td><td> CGGGACGGCC</td><td> AGGTGATGGA</td><td> CGGGCCGTTC</td><td> GCCGCGTCGG</td><td> CCGGCAACTG4Q80</td>

<td> GCCGATCAAT</td><td> GTGCGGGTGG</td><td> ACGGCCGTAC</td><td> GTTCCTGCGG</td><td> CGGGCGCTCG</td><td> GCGCGGGCGT414D</td>

<td> GAGCGAACTG</td><td> CCCACGCGTG</td><td> CCGAGgtcga</td><td> cCTCAACGAG</td><td> ACGATGAAGC</td><td> CGTGGAACGA4200</td>

WO 95/13386

2Î76236 ,

PCT/US94/12857

-30CACCACCCCG GCGGCCCTGC TGGACCACAC CCGGCACTAC ACCTTCGACG TCtgaTCcaa4260 gcttATCGAT GATAAGCTGT CAAACATGAG AATT 4294 (2} INFORMATION FOR SEQ ID NO: 3:

(X) SEQUENCE CHARACTERISTICS :

(A) LENGTH: 422 (B) TYPE: Amino acid (C) STRANDEDNESS:

(D) TOPOLOGY·, linear (ii) MOLECULE TYPE:

(Al DESCRIPTION: protein (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE:

(A) ORGANISM:

(vii) IMMEDIATE SOURCE:

(B) CLONE:

(ix) FEATURE:

<td> (xi)</td><td> SEQUENCE DESCRIPTION:</td><td> SEQ ID *</td><td> IO: 3:</td>

WO 95/13386

PCT/ÜS94/12857

225 σι y 240 Asn. 255 Thr 270 Leu 285 Leu 300 Val 315 Glu 330 Gly 345 Gly 360 Glu 375 Val 390 Ala 40S Ser 420 (2) INFORMATION FOR SEQ ID NO: 4:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 36 (B) TYPE: nucleic acid (C) STRANDEDNESS : single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:

(A) DESCRIPTION: DNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE:

(A) ORGANISM:

(vü) IMMEDIATE SOURCE:

(B) CLONE:

(ix) FEATURE:

(Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:

CCASGGCGCC CGGCTCCTCC CCTTCCCCTC CAACCA 36 (2) INFORMATION FOR SEQ ID NO: 5:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 42 (B) TÏPE: nucleic acid

WO 95/13386

2Î/6236

PCT/US94/12857

-32(C) STRANDEDNESS : single <D> TOPOLOGY: linear (±±> MOLECULE TYPE:

(A) DESCRIPTION: DNA (iii) HYPOTHETICAL: KO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE:

(A) ORGANISM;

(vii) IMMEDIATE SOURCE:

(B) CLONE:

(ix) FEATURE:

(Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:

TCGAGGTCCC GCGGGCCGAG GAGGGGAAGG GGAGGTTGGT AT 42

<td> (2)</td><td> INFORMATION FOR SEQ ID NO: 6:</td>

<td> (i)</td><td> SEQUENCE CHARACTERISTICS: (A) LENGTH: 4009 (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: circular</td>

<td> (ii)</td><td> MOLECULE TYPE: (A) DESCRIPTION: DNA</td>

<td> (iii)</td><td> HYPOTHETICAL: NO</td>

<td> (iv)</td><td> ANTI-SENSE: NO</td>

<td> (vi)</td><td> ORIGINAL SOURCE: (A) ORGANISM:</td>

<td> (vii)</td><td> IMMEDIATE SOURCE: (B) CLONE:</td>

<td> (ix)</td><td> FEATURE:</td>

<td> (xi)</td><td> SEQUENCE DESCRIPTION'. SEQ ID NO</td>

AAATCAATCT AAAGTATATA TGAGTAAACT TGGTCTGACA GTTACCAATG CTTAATCAGTSO GACGCÂCCTA TCTCAGCGAT CTGTCTATTT CGTTCATCCA TAGTTGCCTG ACTCCCCGTC120

GTGTAGATAA CTACGATACG GGASGGCTTA CCATCTGGCC CAGTGCTGCA ATGATACCGC180

GAGACCCACG CTGACCGGCT CCAGATTTAT CAGCAATAAA CCAGCCAGCC GGAAGGGCCG240

AGCGCAGAAG TGGTCCTGCA ACTTTATCCG CCTCCATCCA GTCTATTAAT TGTTGCCGGG3Q0

AAGCTAGAGT AAGTAGTTCG CCAGTTAATA GTTTGCGCAA CGTTGTTGCC ATTGCTACAG360

GCATCGTGGT GTCACGCTCG GCGTTTGGTA TGGCTTCATT CAGCTCCGGT TCCCAACGAT420 CAAOGCGAGT TACATGATCC CCCATGTTGT GCAAAAAAGC GGTTAGCTCC TTCGGTCCTC4B0

WO 95/13386

PCT/ÜS94/12857

-33TTGGCCGCAG TGTTATCACT

CCATCCGTAA GATGCTTTTC

CGACCGAGTT GCTCTTGCCC

TTAAAAGTGC TCATCATTOO

CTGTTGAGAT CCAGTTCGAT

ACTTTCACCK GCGTTTCTGG

ATAAGGGCGA CACGGAAATG

ATTTATCAGG GTTATTGTCT

CAAATAGGGG TTCCGCGCAC

ATTATCATGA CATTAACCTA ttaaaaggat ctaggtgaag agttttegtt eeaetgageg ctttttttct gcgcgtaatc tttgtttgcc ggatcaagag cgcagatacc aaatactgte ctgtagcacc gcctacatac gcgataagte gtgtcttacc ggtcgggctg aaeggggggt aactgagata cctacagcgt cggaeaggta teeggtaagc ggggaaacgc ctggtatctt gatttttgtg atgctcgtca ttttacggtt ectggccttt ctgattctgt ggataaccgt gaacgaccga gcgcagcgag ttctccttac gcatctgtgc eaatctgcte cgatgecgca ggtcatggct gcgececgac gctcccggca tccgcttaca gttttcaceg tcatcaccga aacttctgat agacttcgaa TCTAGAaata attttgttta

CGATCGTTQT CAGAAGTAAG

ATAATTCTCT TACTGTCATG

CCAAGTATTT OGAAGATGCG

CCGCGCCACA TAGCAGAACT

AACTCTCAAG GATCTTACCG

ACTGATCTTC AGCATCTTTT

AAAATGCCGC AAAAAAGGGA 'XTTXTCAATA TTA1TGAAGC

AATGTATTTA GAAAAATAAA

CTGACGTCTA AGAAACCATT

GGCCCTTTCG TCTTCAAGAA gaccaaaatc cctLaacgtg eaaaggatct tcttgagatc accaccgcta ccagcggtgg .ggtaactggc ttcagcagag aggccaecac ttcaagaact accagtggct gctgccagtg gttaccggat aaggcgcago ggagegaacg aeetaeaeeg gcttcccgaa gggagaaagg gegcacgagg gagcttccag ccacctctga ettgagegte aaacgccagc aacgcggcct gttctttcct gcgttatece tgataccgct cgccgcagcc agagcgcctg aegcggtatt TCTGtggtgc actctcagta ccgctatcgc tacgtgactg gcgccctgac gggcttgtct gggagctgca tgtgtcagag GATTCGAAet tctcgattcg ggagaccaca acggtttcec

CATGGTTATG GCAGCACTGC540

TGTGACTGGT GAGTACTCAA6 0 0

GGCGTCAKCA CGGGATAATA660

AAAACGTTCT TCGGGGCGAA720

GTAACCCACT CGTGCACCCA7B0

GTGAGCAAAA ACAGGAAGGC840

TTGAATACTC ATACTCTTCC900

CATGAGCGGA TACATATTTG960

ATTTCCCCGA AAAGTGCCAC1020

TAAAAATAQG CGTATCACGA1080 atcctttttg ataatctcatll40 tcagaccceg tagaaaagatl200 tgctgcttgc aaacaaaaaaX2€0 ctaccaactc tttttccgaal320 cttccagtgt agccgtagttl360 ctcgctctgc taatcctgttl440 gggttggact caagacgataisoo tcgtgcacac *gcccagctt!560 gagctatgag aaagcgccacl620 ggcagggtcg gaacaggagaX6S0 tatagtcctg tcgggtttcgl740

999999^99« gcctatggaal800 tgceggcctt ttgctcacatl86O attaccgcct ttgagtgagcl920 tcagtgagcg aggaagcgg<19S0 ggtatttcac accgcaCAGA2040 tagttaagcc agtatac*ct2100 accegccaac acccgctgac2160 gacaagctgt gacegtetec2220 aacgcgcgag gCCcagctgC2280 attaatacga ctcactatag2340 actttaagaa ggagatatac2400

PCT/US94/12857

WO 95/13386

<td> atATGGCTAG</td><td> AATTGCCatg</td><td> GCGGAACTCA</td><td> CCCGTCGTCG</td><td> CGCGCTCGGC</td><td> GCCGCAGCCG2460</td>

<td> TCGTCGCCGC</td><td> CGGTGTCCCG</td><td> CTGGTCGCCC</td><td> TTCCCGCCGC</td><td> CCGCGCGGAC</td><td> GATCGGGGGC2S20</td>

<td> ACCACACCCC</td><td> CGAGGTCCCC</td><td> GGGAACCCGG</td><td> CCGCGTCCGG</td><td> CGCCCCCGCC</td><td> GCCTTCGACG2580</td>

<td> AGATCTACAA</td><td> GGGCCGCCGG</td><td> ATACAGGGCC</td><td> GGACGGTCAC</td><td> CGACGGCGGG</td><td> GGCCACCACG264C</td>

<td> GCGGCGGTCA</td><td> CGGCGGTGAC</td><td> GGTCACGGCG</td><td> GCGGCCATCA</td><td> CGGCGGCGGT</td><td> TACGCCGTGT2700</td>

<td> TCGTGGACGG</td><td> CGTCGAACTG</td><td> CATGTGATGC</td><td> GCAACGCCGA</td><td> CGGCTCGTGG</td><td> ATCAGCGTCG2760</td>

<td> TCAGCCACTA</td><td> CGAGCCGGTG</td><td> GACACCCCGC</td><td> GCGCCGCGGC</td><td> CCGCGCTGCG</td><td> GTCGACGAGC2620</td>

<td> TCCAGGGCGC</td><td> CCGGCTCCTC</td><td> CCCTTCCCCT</td><td> CCAACeatAT</td><td> GACCGTCCGC</td><td> AAGAACCAGG2BS0</td>

<td> CGTCCCTGAC</td><td> CGCCGAGGAG</td><td> AAGCGCCGCT</td><td> TCGTCGCCGC</td><td> CCTGCTCGAA</td><td> CTCAAGCGCA2940</td>

<td> CCGGCCGCTA</td><td> CGACGCCTTC</td><td> GTCACCACGC</td><td> ACAACGCGTT</td><td> CATCCTGGGC</td><td> GACACCGACA3000</td>

<td> ACGGCGAGCG</td><td> CACCGGCCAC</td><td> CGTTCGCCGT</td><td> CCTTCCTGCC</td><td> CTGOCACCGC</td><td> AGATTTCTGC3 060</td>

<td> TGGAGTTCGA</td><td> GCGGGCGCTC</td><td> CAGTCGGTGG</td><td> ACGCGTCGGT</td><td> GGCGCTGCCG</td><td> TACTGGGACT3120</td>

<td> GGTCCGCCGA</td><td> CCGGTCCACC</td><td> CGGTCCTCGC</td><td> TGTGGGCGCC</td><td> GGACTTCCTC</td><td> GGCGGCACCG3180</td>

<td> GGCGCAGCCG</td><td> GGACGGCCAG</td><td> GTGKTGGACG</td><td> GGCCGTTCGC</td><td> CGCGTCGGCC</td><td> GGCAACTGGC3240</td>

<td> CGATCAATGT</td><td> GCGGGTGGAC</td><td> OGCCGTACGT</td><td> TCCTGCGGCG</td><td> GGCGCTCGGC</td><td> GCGGGCGTGA3300</td>

<td> GCGAACTGCC</td><td> 'CACGCGTGCC</td><td> GAGGTCGACT</td><td> CGGTGCTGGC</td><td> GATGGCGACG</td><td> TACGACATGG3360</td>

<td> CGCCCTGGAA</td><td> CAGCGGCTCC</td><td> GACGGCTTCC</td><td> GCAACCATCT</td><td> CGAAGGGTGG</td><td> CGCGGGGTCA3420</td>

<td> ATCTGCACAA</td><td> CCGOGTGCAT</td><td> GTCTGÔGTCG</td><td> GCGGCCAGAT</td><td> GGCGACCGGG</td><td> GTCTCCCCCA3480</td>

<td> ACGACCCGGT</td><td> GTTCTGGCTG</td><td> CACCACGCCT</td><td> ACATCGACAA</td><td> GCTGTGGGCC</td><td> GAGTGGCAGC3S4C</td>

<td> GGCGGCACCC</td><td> CTCGTCCCCG</td><td> TATCTGCCGG</td><td> GCGGCGGCAC</td><td> GCCGAACGTC</td><td> GTCGACCTCA3600</td>

<td> ACGAGACGAT</td><td> GAAGCCGTGG</td><td> AACGACACCA</td><td> CCCCGGCGGC</td><td> CCTGCTGGAC</td><td> CACACCCGGC3660</td>

<td> ACTACACCTT</td><td> CGACGTCtga</td><td> tcatcactga</td><td> cgaatcgagg</td><td> tcgaggaacc</td><td> gagcgtccga3720</td>

<td> ggaacagagg</td><td> cgcttatcgg</td><td> ttggccgcga</td><td> gattcctgtc</td><td> gatcctetcg</td><td> tgcagcgega3780</td>

<td> ttccgaggga</td><td> aacggaaacg</td><td> ttgagagact</td><td> cggtctggct</td><td> catcatgggg</td><td> atggaaaccg3840</td>

<td> aggcggaaga</td><td> cgcctcctcg</td><td> aacaggtcgg</td><td> aaggcccacc</td><td> cttttcgctg</td><td> ccgaacagca3 900</td>

<td> aggccagecg</td><td> atccggattg</td><td> tccccgagtt</td><td> ccttcacgga</td><td> aatgtcgcca</td><td> tecgccttga3960</td>

<td> gcgtcatcag</td><td> ATCaagcttA</td><td> TCGATGATAA</td><td> GCTGTCAAAC</td><td> ATGAGAATT</td><td> 4009</td>

(2) INFORMATION FOR SEQ ID NO: 7:

(ij SEQUENCE CHARACTERISTICS:

(A) LENGTH: 426 (B) TYPE: amino acid (C) STRANDEDNESS:

(D) TOPOLOGY: circular

WO 95/13386

PCIYUS94/128S7

-35- -

<td> (ii)</td><td> MOLECULE TYPE: (A) DESCRIPTION: protein</td>

<td> (iii)</td><td> HYPOTHETICAL: KO</td>

<td> (iv)</td><td> ANTI-SENSE: NO</td>

<td> (vi)</td><td> ORIGINAL SOURCE: (A) ORGANISM:</td>

<td> (vii)</td><td> IMMEDIATE SOURCE: (B) CLONE:</td>

<td> (ix)</td><td> FEATURE :</td>

<td> (xi)</td><td> SEQUENCE DESCRIPTION: SEQ</td>

WO 95/13386

PCTYUS94/12857

425 (2) INFORMATION FOR SBQ ID NO: 8;

(i) SEQUENCE CHARACTERISTICS;

(A) LENGTH: 1442 (B) TYPE: nucleic acid (Ci STRANDEDNESS; single CD) TOPOLOGY: linear (ii) MOLECULE TYPE:

(A) DESCRIPTION: DNA (iii) HYPOTHETICAL ; NO (iv) ANTI-SSNSE: NO (vi) ORIGINAL SOURCE:

(A) ORGANISM:

(vii) IMMEDIATE SOURCE:

(B) CLONE :

(ix) FEATURE:

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8:

<td> ctcgagccAT</td><td> GGCTTCCTCA</td><td> GTTCTTTCCT</td><td> CTGCAGCAGT</td><td> TGCCACCCGC</td><td> AGCAATGTTG60</td>

<td> CTCAAGCTAA</td><td> CATGGTTGCA</td><td> CCTTTCACTG</td><td> GCCTTAAGTC</td><td> AGCTGCCTCA</td><td> TTCCCTGTTT120</td>

<td> CAAGGAAGCA</td><td> AAACCTTGAC</td><td> ATCACTTCCA</td><td> TTGCCAGCAA</td><td> CGGCGGAAGA</td><td> GTGCAATGCA180</td>

<td> TGCCGGAACT</td><td> CACCCGTCGT</td><td> CGCGCGCTCG</td><td> GCGCCGCAGC</td><td> CGTCGTCGCC</td><td> GCCGGTGTCC240</td>

<td> CGCTGGTCGC</td><td> CCTTCCCGCC</td><td> GCCCGCGCGG</td><td> ACGATCGGGG</td><td> GCACCACACC</td><td> CCCGAGGTCC300</td>

<td> CCGGGAACCC</td><td> GGCCGCGTCC</td><td> GGCGCCCCCG</td><td> CCGCCTTCGA</td><td> CGAGATCTAC</td><td> AAGGGCCGCC360</td>

<td> GGATACAGGG</td><td> CCGGACGGTC</td><td> ACCGACGGCG</td><td> GGGGCCACCA</td><td> CGGCGGCGGT</td><td> CACGGCGGTG420</td>

<td> ACGGTCACGG</td><td> CGGCGGCCAT</td><td> CACGGCGGCG</td><td> GTTACGCCST</td><td> GTTCGTGGAC</td><td> GGCGTCGAAC40O</td>

<td> TGCATGTGAT</td><td> GCGCAACGCC</td><td> GACGGCTCGT</td><td> GGATCAGCGT</td><td> CGTCAGCCAC</td><td> TACGASCCGG540</td>

<td> TGGACACCCC</td><td> GCGCGCCGCG</td><td> GCCCGCGCTG</td><td> CGGTCGACGA</td><td> GCTCCAGGGC</td><td> GCCCGGCTCC600</td>

<td> TCCCCTTCCC</td><td> CTCCAACCAT</td><td> ATGACCGTCC</td><td> GCAAGAACCA</td><td> GGCGTCCCTG</td><td> ACCGCCGAGG660</td>

<td> AGAAGCGCCG</td><td> CTTCGTCGCC</td><td> GCCCTGCTCG</td><td> AACTCAAGCG</td><td> CACCGGCCGC</td><td> TACGACGCCT720</td>

WO 95/13386

PCTAJS94/12857

<td> TCGTCACCAC</td><td> GCACAACGCG</td><td> TTCATCCTGG</td><td> GCGACACCGA</td><td> CAACGGCGAG</td><td> CGCACCGGCC70O</td>

<td> ACCGTTCGCC</td><td> GTCCTTCCTG</td><td> CCCTOGCACC</td><td> GCAGATTTCT</td><td> GCTGGAGTTC</td><td> GAGCGGGCGC840</td>

<td> TCCAGTCGGT</td><td> GGACGCGTCG</td><td> GTGGCGCTGC</td><td> CGTACTGGGA</td><td> CTGGTCCGCC</td><td> GACCGGTCCA900</td>

<td> CCCGGTCCTC</td><td> GCTGTGGGCG</td><td> CCGGACTTCC</td><td> TCGGCGGCAC</td><td> CGGGCGCASC</td><td> COGGACGGCC96O</td>

<td> AGGTGATGGA</td><td> CGGGCCGTTC</td><td> GCCGCGTCGG</td><td> CCGGCAACTG</td><td> GCCGATCAAT</td><td> GTGCGGGTGG1020</td>

<td> ACGGCCGTAC</td><td> GTTCCTGCGG</td><td> CGGGCGCTCG</td><td> GCGCGGGCGT</td><td> GAGCGAACTG</td><td> CCCACGCGTG1080</td>

<td> CCCAGGTCGA</td><td> CTCGGTGCTG</td><td> GCGATGGCGA</td><td> CGTACGACAT</td><td> GGCGCCCTGG</td><td> AACAGCGGCT1140</td>

<td> CCGACGGCTT</td><td> CCGCAACCAT</td><td> CTCGAAGGGT</td><td> GGCOCGGGGT</td><td> CAATCTGCAC</td><td> AACCGGGTGC1200</td>

<td> ATGTCTGGGT</td><td> CGGCGGCCAG</td><td> ATGGCGACCG</td><td> GGGTCTCCCC</td><td> CAACGACÇCG</td><td> GTGTTCTGGC12€0</td>

<td> TGCACCACGC</td><td> CTACATCGAC</td><td> AAGCTGTGGG</td><td> CCOAGTGGCA</td><td> GCGGCGGCAC</td><td> CCCTCGTCCC1320</td>

<td> CGTATCTGCC</td><td> GGGCGGCGGC</td><td> ACGCCGAACG</td><td> TCGTCGACCT</td><td> CAACGAGACG</td><td> ATGAAGCCST1380</td>

<td> GGAACCACAC</td><td> CACCCCGGCG</td><td> GCCCTGCTGG</td><td> ACCACACCCG</td><td> GCACTACACC</td><td> TTCGACGTCT1440</td>

(2) INFORMATION FOR SEQ ID NO: 9:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 470 (B) TYPE: amino acid {C) STRANDEDNES S: S ingle (D) TOPOLOGY: linear (Ü) MOLECULE TYPE:

(A) DESCRIPTION: DNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE:

(A) ORGANISM:

(vii) IMMEDIATE SOURCE:

(B) CLONE:

(ix) FEATURE:

<td> (Xi)</td><td> SEQUENCE DESCRIPTION:</td><td> SEQ</td><td> ID l</td><td> ÎO: 9:</td>

WO 95713386

PCTYUS5M/I2857

WO 95/13386

2î 76236

PCT/US94/I2857

International Application No: PCT/

MICROORGANISMS

Qotioml Sheet in connection with the tweraotgamscr τ«!*π·ύ to cn pee· lines _ ol the description *

A. IDENTIFICATION OF DEPOSIT

Further deposits ere identified on an additional sheet '

Name of depositary msomooQ * American Type Culture CoBectton

Address of depositary institution {including postal code and country} *

12301 Parklawn Driva Rockville, MD 20S52 US

Date of deposit ‘ December 23, 1993 Accession Number · 75532

B. ADDITIONAL INDICATIONS · flave blot ii IM »ppiie»ble). Tliii infonwioft « minute on t tcemt bkW ihni

C. DESIGNATED STATES FOR WHICH INDICATIONS ARE MADE '

D. SEPARATE FURNISHING OF INDICATIONS · Have οω* ir «« tht indiettient lutod bolgw will M luwniTHd to mo Mtunational Biroou m«r ' (Specify IM ponoral notuft ol (ht indications o.p.. •Aceastion MumOOl Of Popout*)

E. □ This sheet was received with the lotemanonal application when filed (to be checked by the receiving Office} (Authorized Officer) □ The date of receipt (from the applicant} by As lntcmitionil Bureau * was .

(Authorized Officer)

Form ÉCT/W/134~lJanuary 19Ô1}

Contents142

207 members in 18 offices

This recordMember, by publication datePriority claim8 more offices are in the list below

Priority claims9

Priority claims
Document	Office	Kind	Date
152483	United States of America	–	1993-11-12
15248393	United States of America	A	1993-11-12
15248393	United States of America	A	1993-11-12
9412857	United States of America	W	1994-11-08
9412857	United States of America	W	1994-11-08
152483	–	–	–
PCTUS9412857	–	–	–
US19930152483	–	–	–
WO1994US12857	–	–	–

Members207

Family members, oldest first
Document	Office	Kind
WO8908145A1	World Intellectual Property Organization (WIPO)	A1	1989-09-08
AU4072589A	Australia	A	1989-09-22
WO9000611A1	World Intellectual Property Organization (WIPO)	A1	1990-01-25
AU3975089A	Australia	A	1990-02-05
CA2000114A1	Canada	A1	1990-04-03
EP0363792A1	European Patent Office (EPO)	A1	1990-04-18
WO9004029A1	World Intellectual Property Organization (WIPO)	A1	1990-04-19
AU4418989A	Australia	A	1990-05-01
ZA897501B	South Africa	B	1990-06-27
WO9013654A1	World Intellectual Property Organization (WIPO)	A1	1990-11-15
AU5664090A	Australia	A	1990-11-29
KR900702037A	Republic of Korea	A	1990-12-05
EP0406267A1	European Patent Office (EPO)	A1	1991-01-09
JPH03502886A	Japan	A	1991-07-04
CA2086417A1	Canada	A1	1991-12-30
CA2166818A1	Canada	A1	1991-12-30
WO9200373A1	World Intellectual Property Organization (WIPO)	A1	1992-01-09
AU8295491A	Australia	A	1992-01-23
CA2114636A1	Canada	A1	1993-02-18
WO9303161A1	World Intellectual Property Organization (WIPO)	A1	1993-02-18
AU3351193A	Australia	A	1993-03-02
KR930701589A	Republic of Korea	A	1993-06-12
EP0547065A1	European Patent Office (EPO)	A1	1993-06-23
AU638411B2	Australia	B2	1993-07-01
EP0547065A4	European Patent Office (EPO)	A4	1993-09-15
JPH06500011A	Japan	A	1994-01-06
EP0596979A1	European Patent Office (EPO)	A1	1994-05-18
US5316931A	United States of America	A	1994-05-31
KR940005597B1	Republic of Korea	B1	1994-06-21
EP0596979A4	European Patent Office (EPO)	A4	1994-07-13
CA2152934A1	Canada	A1	1994-07-21
WO9416089A1	World Intellectual Property Organization (WIPO)	A1	1994-07-21
AU5987194A	Australia	A	1994-08-15
IL108241D0	Israel	D0	1994-10-07
EP0363792B1	European Patent Office (EPO)	B1	1995-01-18
IL111608D0	Israel	D0	1995-01-24
AT117376T	Austria	T	1995-02-15
ATE117376T1	Austria	T1	1995-02-15
ZA939798B	South Africa	B	1995-02-28
DE68920685D1	Germany	D1	1995-03-02
JPH07503361A	Japan	A	1995-04-13
ES2069560T3	Spain	T3	1995-05-16
CA2176236A1	Canada	A1	1995-05-18
WO9513386A2	World Intellectual Property Organization (WIPO)	A2	1995-05-18
DE68920685T2	Germany	T2	1995-05-24
AU1173495A	Australia	A	1995-05-29
CA2000114C	Canada	C	1995-06-20
WO9513386A3	World Intellectual Property Organization (WIPO)	A3	1995-06-22
IL113955D0	Israel	D0	1995-08-31
EP0677113A1	European Patent Office (EPO)	A1	1995-10-18
CA2193094A1	Canada	A1	1995-12-21
CA2309028A1	Canada	A1	1995-12-21
WO9534668A2	World Intellectual Property Organization (WIPO)	A2	1995-12-21
AU2653495A	Australia	A	1996-01-05
IL115578D0	Israel	D0	1996-01-19
KR960700344A	Republic of Korea	A	1996-01-19
WO9534668A3	World Intellectual Property Organization (WIPO)	A3	1996-02-01
ZA954451B	South Africa	B	1996-02-05
ZA948973B	South Africa	B	1996-02-08
AU3010695A	Australia	A	1996-02-22
CA2202652A1	Canada	A1	1996-04-25
WO9612028A1	World Intellectual Property Organization (WIPO)	A1	1996-04-25
ZA958659B	South Africa	B	1996-04-29
AU3763795A	Australia	A	1996-05-06
JPH08505289A	Japan	A	1996-06-11
US5529909A	United States of America	A	1996-06-25
EP0731843A1	European Patent Office (EPO)	A1	1996-09-18
CA2223493A1	Canada	A1	1996-12-19
WO9640867A1	World Intellectual Property Organization (WIPO)	A1	1996-12-19
AU6157296A	Australia	A	1996-12-30
US5589367A	United States of America	A	1996-12-31
US5631151A	United States of America	A	1997-05-20
MX9606476A	Mexico	A	1997-05-31
JPH09505468A	Japan	A	1997-06-03
MX9702714A	Mexico	A	1997-06-28
EP0787195A1	European Patent Office (EPO)	A1	1997-08-06
EP0804600A1	European Patent Office (EPO)	A1	1997-11-05
AU683412B2	Australia	B2	1997-11-13
KR970707291A	Republic of Korea	A	1997-12-01
IS4629A	Iceland	A	1997-12-05
JPH10501968A	Japan	A	1998-02-24
EP0832191A1	European Patent Office (EPO)	A1	1998-04-01
CA1339841C	Canada	C	1998-04-28
IL122412D0	Israel	D0	1998-06-15
AU694102B2	Australia	B2	1998-07-16
AU695044B2	Australia	B2	1998-08-06
KR0149181B1	Republic of Korea	B1	1998-08-17
JPH10508468A	Japan	A	1998-08-25
US5811653A	United States of America	A	1998-09-22
US5814495A	United States of America	A	1998-09-29
US5837505A	United States of America	A	1998-11-17
CA1340378C	Canada	C	1999-02-02
US5866785A	United States of America	A	1999-02-02
KR19990022818A	Republic of Korea	A	1999-03-25
US5889190A	United States of America	A	1999-03-30
US5889191A	United States of America	A	1999-03-30
JPH11507217A	Japan	A	1999-06-29
CA2086417C	Canada	C	1999-07-06
US5922602A	United States of America	A	1999-07-13
AU710588B2	Australia	B2	1999-09-23

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Events

Legal events, newest first
	Event	Code
2003-11-10	LapsedLapsedMKLA	MKLA
1997-11-06	Examination requestEEER	EEER