Production of gamma linolenic acid by a .delta.6-desaturase

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1¹ PRODUCTION OF GAMMA LINOLENIC ACID BY A Δ6-DESATURASE

Linoleic acid (18:2) (LA) is transformed into gamma linolenic acid (18:3) (GLA) by the enzyme

Δβ-desaturase. When this enzyme, or the nucleic acid encoding it, is transferred into LA-producing cells, GLA is produced. The present invention provides nucleic acids comprising the Δδ-desaturase gene. More specifically, the nucleic acids comprise the promoters, coding regions and termination regions of the Δό-desaturase genes. The present invention is further directed to recombinant constructions comprising a Δδ-desaturase coding region in functional combination with heterologous regulatory sequences.

The nucleic acids and recombinant constructions of the instant invention are useful in the production of GLA in transgenic organisms .

Unsaturated fatty acids such as linoleic (C_igA⁹·-²) and ûf-linolenic (Ο₁₈Δ^9,12,15) acids are essential dietary constituents that cannot be synthesized by vertebrates since vertebrate cells can introduce double bonds at the Δ⁹ position of fatty acids but cannot introduce additional double bonds between the Δ⁹ double bond and the methyl-terminus of the fatty , acid chain. Because they are precursors of other products, linoleic and α-linolenic acids are essential fatty acids, and are usually obtained from plant sources. Linoleic acid can be converted by mammals into γ-linolenic acid (GLA, C-_SA⁶'^9,12) which can in turn be converted to arachidonic acid (20:4), a critically

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-21 important fatty acid since it is an essential precursor of most prostaglandins.

The dietary provision of linoleic acid, by virtue of its resulting conversion to GLA and arachidonic acid, satisfies the dietary need for GLA and arachidonic acid. However, a relationship has been demonstrated between consumption of saturated fats and health risks such as hypercholesterolemia, atherosclerosis and other clinical disorders which correlate with susceptibility to coronary disease, while the consumption of unsaturated fats has been associated with decreased blood cholesterol concentration and reduced risk of atherosclerosis.

The therapeutic benefits of dietary GLA may result from GLA being a precursor to arachidonic acid and thus subsequently contributing to prostaglandin synthesis. Accordingly, consumption of the more unsaturated GLA, rather than linoleic acid, has potential health benefits. However, GLA is not present in virtually any commercially grown crop plant.

Linoleic acid is converted into GLA by the enzyme A6-desaturase. Δβ-desaturase, an enzyme of more than 350 amino acids, has a membrane-bound domain and an active site for desaturation of fatty acids. When this enzyme is transferred into cells which endogenously produce linoleic acid but not GLA, GLA is produced. The present invention, by providing the gene encoding Δβ-desaturase, allows the production of transgenic organisms which contain functional Δ6desaturase and which produce GLA. In addition to

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-31 allowing production of large amounts of GLA, the present invention provides new dietary sources of GLA.

The present invention is directed to isolated Δβ-desaturase genes. Specifically, the isolated genes comprises the Δβ-desaturase promoters, coding regions, and termination regions.

The present invention is further directed to expression vectors comprising the Δβ-desaturase promoter, coding region and termination region.

Yet another aspect of this invention is directed to expression vectors comprising a Δβdesaturase coding region in functional combination with heterologous regulatory regions, i.e. elements not derived from the Δβ-desaturase gene.

Cells and organisms comprising the vectors of the present invention, and progeny of such organisms, are also provided by the present invention.

A further aspect of the present invention provides isolated bacterial Δβ-desaturase. An isolated plant Δβ-desaturase is also provided.

Yet another aspect of this invention provides a method for producing plants with increased gamma linolenic acid content.

A method for producing chilling tolerant plants is also provided by the present invention.

Fig. 1 depicts the hydropathy profiles of the deduced amino acid sequences of Synechocvstis Δβdesaturase (Panel A) and Al2-desaturase (Panel B) . Putative membrane spanning regions are indicated by solid bars. Hydrophobic index was calculated for a

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-41 window size of 19 amino acid residues [Kyte, et al. (1982) J. Molec. Biol. 157].

Fig. 2 provides gas liquid chromatographyprofiles of wild type (Panel A) and transgenic (Panel

B) Anabaena.

Fig. 3 is a diagram of maps of cosmid cSy75, cSyl3 and Csy7 with overlapping regions and subclones. The origins of subclones of Csy75, Csy75-3.5 and Csy7 are indicated by the dashed diagonal lines.

Restriction sites that have been inactivated are in parentheses .

Fig. 4 provides gas liquid chromatography profiles of wild type (Panel A) and transgenic (Panel B) tobacco.

Fig. 5A depicts the DNA sequence of a Δ-6 desaturase cDNA isolated from borage.

Fig. 5B depicts the protein sequence of the open reading frame in the isolated borage Δ-6 desaturase cDNA. Three amino acid motifs characteristic of desaturases are indicated and are, in order, lipid box, metal box 1, and metal box 2.

Fig. 6 is a dendrogram showing similarity of the borage Δ6-desaturase to other membrane-bound desaturases. The amino acid sequence of the borage

Δ6-desaturase was compared to other known desaturases using Gene Works (IntelliGenetics). Numerical values correlate to relative phylogenetic distances between subgroups compared.

Fig. 7 is a restriction map of 221.Δ6.NOS and 121.Δ6.NOS. In 221.Δ6.NOS, the remaining portion

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-51 of the plasmid is pBI221 and in 121.Δ6.NOS, the remaining portion of the plasmid is pBI121.

Fig. 8 provides gas liquid chromatography profiles of mock transfected (Panel A) and 221.Δ6.NOS transfected (Panel B) carrot cells. The positions of 18:2, 18:3 of, and 18:3 γ (GLA) are indicated.

Fig. 9 provides gas liquid chromatography profiles of an untransformed tobacco leaf (Panel A) and a tobacco leaf transformed with 121.Δ6.NOS. The positions of 18:2, 18:3 ot, 18:3y(GLA), and 18:4 are indicated.

Fig. 10 provides gas liquid chromotography profiles for untransformed tobacco seeds (Panel A) and seeds of tobacco transformed with 121.Δ6.NOS. The positions of 18:2, 18:3a and 18:3y(GLA) are indicated.

The present invention provides isolated nucleic acids encoding Δβ-desaturase. To identify a nucleic acid encoding Δ6-desaturase, DNA is isolated from an organism which produces GLA. Said organism can be, for example, an animal cell, certain fungi (e.g. Mortierella), certain bacteria (e.g. Synechocystis) or certain plants (borage, Oenothera, currants). The isolation of genomic DNA can be accomplished by a variety of methods well-known to one of ordinary skill in the art, as exemplified by Sambrook et al. (1989) in Molecular Cloning: A

Laboratory Manual, Cold Spring Harbor, NY. The isolated DNA is fragmented by physical methods or enzymatic digestion and cloned into an appropriate βθ vector, e.g. a bacteriophage or cosmid vector, by any of a variety of well-known methods which can be found

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-61 in references such as Sambrook et al. (1989) .

Expression vectors containing the DNA of the present invention are specifically contemplated herein. DNA encoding Δδ-desaturase can be identified by gain of function analysis. The vector containing fragmented DNA is transferred, for example by infection, transconjugation, transfection, into a host organism that produces linoleic acid but not GLA. As used herein, transformation refers generally to the incorporation of foreign DNA into a host cell.

Methods for introducing recombinant DNA into a host organism are known to one of ordinary skill in the art and can be found, for example, in Sambrook et al. (1989). Production of GLA by these organisms (i.e., gain of function) is assayed, for example by gas chromatography or other methods known to the ordinarily skilled artisan. Organisms which are induced to produce GLA, i.e. have gained function by the introduction of the vector, are identified as expressing DNA encoding Δ6-desaturase, and said DNA is recovered from the organisms. The recovered DNA can again be fragmented, cloned with expression vectors, and functionally assessed by the above procedures to define with more particularity the DNA encoding δ625 desaturase.

As an example of the present invention, random DNA is isolated from the cyanobacteria Synechocvstis Pasteur Culture Collection (PCC) 6803, American Type Culture Collection (ATCC) 27184, cloned

3Q into a cosmid vector, and introduced by transconjugation into the GLA-deficient cyanobacterium

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-Ί1 Anabaena strain PCC 7120, ATCC 27893. Production of GLA from Anabaena linoleic acid is monitored by gas chromatography and the corresponding DNA fragment is isolated.

The isolated DNA is sequenced by methods well-known to one of ordinary skill in the art as found, for example, in Sambrook et al. (1989).

In accordance with the present invention,

DNA molecules comprising Δβ-desaturase genes have been isolated. More particularly, a 3.588 kilobase (kb)

DNA comprising a Δβ-desaturase gene has been isolated from the cyanobacteria Synechocystis. The nucleotide sequence of the 3.588 kb DNA was determined and is shown in SEQ ID NO:1. Open reading frames defining potential coding regions are present from nucleotide 317 to 1507 and from nucleotide 2002 to 3081. To define the nucleotides responsible for encoding δ6desaturase, the 3.588 kb fragment that confers Δβdesaturase activity is cleaved into two subfragments, each of which contains only one open reading frame.’ Fragment ORF1 contains nucleotides 1 through 1704, while fragment ORF2 contains nucleotides 1705 through 3588. Each fragment is subcloned in both forward and reverse orientations into a conjugal expression vector (AM542, Wolk et al. [1984] Proc. Natl. Acad. Sci. USA

81, 1561) that contains a cyanobacterial carboxylase promoter. The resulting constructs (i.e. ORF1(F),

ORF1(R), ORF2(F) and ORF2(R) ] are conjugated to wildtype Anabaena PCC 7120 by standard methods (see, for βθ example, Wolk et al. (1984) Proc. Natl. Acad. Sci. USA

81, 1561) . Conjugated cells of Anabaena are

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-81 identified as Neo” green colonies on a brown background of dying non-conjugated cells after two weeks of growth on selective media (standard mineral media BG11N + containing 30^g/ml of neomycin according to Rippka et al., (1979) J. Gen Microbiol, ill. l).

The green colonies are selected and grown in selective liquid media (BG11N + with 15/ig/ml neomycin) . Lipids are extracted by standard methods (e.g. Dahmer et al., (1989) Journal of American Oil Chemical Society 66,

543) from the resulting transconjugants containing the forward and reverse oriented ORF1 and ORF2 constructs. For comparison, lipids are also extracted from wildtype cultures of Anabaena and Synechocystis. The fatty acid methyl esters are analyzed by gas liquid chromatography (GLC), for example with a Tracor-560 gas liquid chromatograph equipped with a hydrogen flame ionization detector and a capillary column. The results of GLC analysis are shown in Table 1.

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-91 Table 1: Occurrence of C18 fatty acids in wild-type and transgenic cyanobacteria

<td> SOURCE</td><td> 18:0</td><td> 18:1</td><td> 18:2</td><td> γ18:3</td><td> α18:3</td><td> 18:4</td>

<td> Anabaena (wild type)</td><td> +</td><td> +</td><td> +</td><td> -</td><td> +</td><td> -</td>

<td> Anabaena + ORF1(F)</td><td> +</td><td> +</td><td> +</td><td> -</td><td> +</td><td> -</td>

<td> Anabaena + ORF1(R)</td><td> +</td><td> +</td><td> +</td><td> -</td><td> +</td><td> -</td>

<td> Anabaena + ORF2(F)</td><td> +</td><td> +</td><td> +</td><td> +</td><td> +</td><td> +</td>

<td> Anabaena + ORF2(R)</td><td> +</td><td> +</td><td> +</td><td> -</td><td> +</td><td> -</td>

<td> Synechocystis (wild type)</td><td> +</td><td> +</td><td> +</td><td> +</td><td> -</td><td> -</td>

As assessed by GLC analysis, GLA deficient Anabaena gain the function of GLA production when the construct containing ORF2 in forward orientation is introduced by transconjugation. Transconjugants containing constructs with ORF2 in reverse orientation to the carboxylase promoter, or ORF1 in either orientation, show no GLA production. This analysis demonstrates that the single open reading frame (ORF2) within the 1884 bp fragment encodes Δβ-desaturase.

The 1884 bp fragment is shown as SEQ ID NO:3. This is substantiated by the overall similarity of the hydropathy profiles between Δβ-desaturase and δ12desaturase [Wada et al. (1990) Nature 347] as shown in

Fig. 1 as (A) and (B), respectively.

Also in accordance with the present invention, a cDNA comprising a Δβ-desaturase gene from borage (Borago officinalis) has been isolated. The nucleotide sequence of the 1.685 kilobase (kb) cDNA

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-ΙΟΙ was determined and is shown in Fig. 5A (SEQ ID NO: 4) . The ATG start codon and stop codon are underlined.

The amino acid sequence corresponding to the open reading frame in the borage delta 6-desaturase is shown in Fig. 5B (SEQ ID NO: 5).

Isolated nucleic acids encoding Δβdesaturase can be identified from other GLA-producing organisms by the gain of function analysis described above, or by nucleic acid hybridization techniques using the isolated nucleic acid which encodes Synechocystis or borage Δβ-desaturase as a hybridization probe. Both genomic and cDNA cloning methods are known to the skilled artisan and are contemplated by the present invention. The hybridization probe can comprise the entire DNA sequence disclosed as SEQ. ID NO:1 or SEQ. ID NO:4, or a restriction fragment or other DNA fragment thereof, including an oligonucleotide probe. Methods for cloning homologous genes by cross-hybridization are known to the ordinarily skilled artisan and can be found, for example, in Sambrook (1989) and Beltz et al. (1983) Methods in Enzymolocry 100, 266.

In another method of identifying a delta 6desaturase gene from an organism producing GLA, a cDNA library is made from poly-A* RNA isolated from polysomal RNA. In order to eliminate hyper-abundant expressed genes from the cDNA population, cDNAs or fragments thereof corresponding to hyper-abundant cDNAs genes are used as hybridization probes to the

3Q cDNA library. Non hybridizing plaques are excised and the resulting bacterial colonies are used to inoculate

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-111 liquid cultures and sequenced. For example, as a means of eliminating other seed storage protein cDNAs from a cDNA library made from borage polysomal RNA, cDNAs corresponding to abundantly expressed seed storage proteins are first hybridized to the cDNA library. The subtracted DNA library is then used to generate expressed sequence tags (ETSs) and such tags are used to scan a data base such as GenBank to identify potential desaturates.

Transgenic organisms which gain the function of GLA production by introduction of DNA encoding δdesaturase also gain the function of octadecatetraeonic acid (18: 4^aS'⁹'¹²'¹⁵) production. Octadecatetraeonic acid is present normally in fish oils and in some plant species of the Boraginaceae family (Craig et al·. [1964] J. Amer. Oil Chem. Soc,

41, 209-211; Gross et al. [1976] Can. J. Plant Sci.

56, 659-664). In the transgenic organisms of the present invention, octadecatetraenoic acid results from further desaturation of α-linolenic acid by δ6desaturase or desaturation of GLA by δ15-desaturase.

The 359 amino acids encoded by ORF2, i.e. the open reading frame encoding Synechocystis δ6desaturase, are shown as SEQ. ID NO:2. The open reading frame encoding the borage Δ6-desaturase is shown in SEQ ID NO: 5. The present invention further contemplates other nucleotide sequences which encode the amino acids of SEQ ID NO:2 and SEQ ID NO: 5. It is within the ken of the ordinarily skilled artisan to βθ identify such sequences which result, for example, from the degeneracy of the genetic code. Furthermore,

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-12one of ordinary skill in the art can determine, by the gain of function analysis described hereinabove, smaller subfragments of the fragments containing the open reading frames which encode A6-desaturases.

The present invention contemplates any such polypeptide fragment of δ6-desaturase and the nucleic acids therefor which retain activity for converting LA to GLA.

In another aspect of the present invention, a vector containing a nucleic acid of the present invention or a smaller fragment containing the promoter, coding sequence and termination region of a Δ6-desaturase gene is transferred into an organism, for example, cyanobacteria, in which the Δ6-desaturase promoter and termination regions are functional. Accordingly, organisms producing recombinant Δ6desaturase are provided by this invention. Yet another aspect of this invention provides isolated Δ6desaturase, which can be purified from the recombinant organisms by standard methods of protein purification. (For example, see Ausubel et al. [1987] Current

Protocols in Molecular Biology, Green Publishing Associates, New York).

Vectors containing DNA encoding δ6desaturase are also provided by the present invention. It will be apparent to one of ordinary skill in the art that appropriate vectors can be constructed to direct the expression of the Δ6-desaturase coding sequence in a variety of organisms. Replicable expression vectors are particularly preferred. Replicable expression vectors as described herein are

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-131 DNA or RNA molecules engineered for controlled expression of a desired gene, i.e. the Δβ-desaturase gene. Preferably the vectors are plasmids, bacteriophages, cosmids or viruses. Shuttle vectors,

e.g. as described by Wolk et al. (1984) Proc. Natl.

Acad. Sci. USA, 1561-1565 and Bustos et al. (1991) J.

Bacteriol. 174, 7525-7533, are also contemplated in accordance with the present invention. Sambrook et al. (1989), Goeddel, ed. (1990) Methods in Enzymolocrv

185 Academic Press, and Perbal (1988) A Practical

Guide to Molecular Cloning, John Wiley and Sons, Inc., provide detailed reviews of vectors into which a nucleic acid encoding the present δ6-desaturase can be inserted and expressed. Such vectors also contain nucleic acid sequences which can effect expression of nucleic acids encoding Δβ-desaturase. Sequence elements capable of effecting expression of a gene product include promoters, enhancer elements, upstream activating sequences, transcription termination

2q signals and polyadenylation sites. Both constitutive and tissue specific promoters are contemplated. For transformation of plant cells, the cauliflower mosaic virus (CaMV) 35S promoter and promoters which are regulated during plant seed maturation are of particular interest. All such promoter and transcriptional regulatory elements, singly or in combination, are contemplated for use in the present replicable expression vectors and are known to one of ordinary skill in the art. The CaMV 355 promoter is

2q. described, for example, by Restrepo et al. (1990)

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-141 Plant Cell 2, 987. Genetically engineered and mutated regulatory sequences are also contemplated.

The ordinarily skilled artisan can determine vectors and regulatory elements suitable for expression in a particular host cell. For example, a vector comprising the promoter from the gene encoding the carboxylase of Anabaena operably linked to the coding region of Δβ-desaturase and further operably linked to a termination signal from Synechocystis is appropriate for expression of Δβ-desaturase in cyanobacteria. Operably linked in this context means that the promoter and terminator sequences effectively function to regulate transcription. As a further example, a vector appropriate for expression

3.5 of Δβ-desaturase in transgenic plants can comprise a seed-specific promoter sequence derived from helianthinin, napin, or glycinin operably linked to the Δβ-desaturase coding region and further operably linked to a seed termination signal or the nopaline synthase termination signal. As a still further example, a vector for use in expression of Δ 6desaturase in plants can comprise a constitutive promoter or a tissue specific promoter operably linked to the Δ 6-desaturase coding region and further operably linked to a constitutive or tissue specific terminator or the nopaline synthase termination signal.

In particular, the helianthinin regulatory elements disclosed in applicant's copending U.S.

3Q Application Serial No. 682,354, filed April 8, 1991 and incorporated herein by reference, are contemplated

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-1510 as promoter elements to direct the expression of the Δδ-desaturase of the present invention.

Modifications of the nucleotide sequences or regulatory elements disclosed herein which maintain the functions contemplated herein are within the scope of this invention. Such modifications include insertions, substitutions and deletions, and specifically substitutions which reflect the degeneracy of the genetic code.

Standard techniques for the construction of such hybrid vectors are well-known to those of ordinary skill in the art and can be found in references such as Sambrook et al. (1989), or any of the myriad of laboratory manuals on recombinant DNA technology that are widely available. A variety of strategies are available for ligating fragments of DNA, the choice of which depends on the nature of the termini of the DNA fragments. It is further contemplated in accordance with the present invention to include in the hybrid vectors other nucleotide sequence elements which facilitate cloning, expression or processing, for example sequences encoding signal peptides, a sequence encoding KDEL, which is required for retention of proteins in the endoplasmic reticulum or sequences encoding transit peptides which direct Δδ-desaturase to the chloroplast. Such sequences are known to one of ordinary skill in the art. An optimized transit peptide is described, for example, by Van den Broeck et al. (1985) Nature 313, 358.

Prokaryotic and eukaryotic signal sequences are

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-161 disclosed, for example, by Michaelis et al. (1982)

Ann. Rev. Microbiol. 36, 425.

A further aspect of the instant invention provides organisms other than cyanobacteria or plants which contain the DNA encoding the ù6-desaturase of the present invention. The transgenic organisms contemplated in accordance with the present invention include bacteria, cyanobacteria, fungi, and plants and animals. The isolated DNA of the present invention

IQ can be introduced into the host by methods known in the art, for example infection, transfection, transformation or transconjugation. Techniques for transferring the DNA of the present invention into such organisms are widely known and provided in references such as Sambrook et al. (1989).

A variety of plant transformation methods are known. The A6-desaturase gene can be introduced into plants by a leaf disk transformation-regeneration procedure as described by Horsch et al. (1985) Science ²²⁷ &lt; 1229. Other methods of transformation, such as protoplast culture (Horsch et al. (1984) Science 223,

496; DeBlock et al. (1984) EMBO J. 2, 2143; Barton et al. (1983) Cell 32, 1033) can also be used and are within the scope of this invention. In a preferred embodiment plants are transformed with Agrobacteriumderived vectors. However, other methods are available to insert the A6-desaturase genes of the present invention into plant cells. Such alternative methods include biolistic approaches (Klein et al. (1987) gO Nature 327, 70), electroporation, chemically-induced DNA uptake, and use of viruses or pollen as vectors.

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-171 When necessary for the transformation method, the Δβ-desaturase genes of the present invention can be inserted into a plant transformation vector, e.g. the binary vector described by Bevan (1984) Nucleic Acids Res. 12. 8111. Plant transformation vectors can be derived by modifying the natural gene transfer system of Agrobacterium tumefaciens. The natural system comprises large Ti (tumor-inducing)-plasmids containing a large segment, known as T-DNA, which is transferred to transformed plants. Another segment of the Ti plasmid, the vir region, is responsible for T-DNA transfer. The T-DNA region is bordered by terminal repeats. In the modified binary vectors the tumor-inducing genes have been deleted and the functions of the vir region are utilized to transfer foreign DNA bordered by the T-DNA border sequences. The T-region also contains a selectable marker for antibiotic resistance, and a multiple cloning site for inserting sequences for

2Q transfer. Such engineered strains are known as disarmed A. tumefaciens strains, and allow the efficient transformation of sequences bordered by the T-region into the nuclear genomes of plants.

Surface-sterilized leaf disks are inoculated with the disarmed foreign DNA-containing A. tumefaciens, cultured for two days, and then transferred to antibiotic-containing medium. Transformed shoots are selected after rooting in medium containing the appropriate antibiotic, βθ transferred to soil and regenerated.

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-181 Another aspect of the present invention provides transgenic plants or progeny of these plants containing the isolated DNA of the invention. Both monocotyledenous and dicotyledenous plants are contemplated. Plant cells are transformed with the isolated DNA encoding Δβ-desaturase by any of the plant transformation methods described above. The transformed plant cell, usually in a callus culture or leaf disk, is regenerated into a complete transgenic plant by methods well-known to one of ordinary skill in the art (e.g. Horsch et al. (1985) Science 227.

1129). In a preferred embodiment, the transgenic plant is sunflower, oil seed rape, maize, tobacco, peanut or soybean. Since progeny of transformed plants inherit the DNA encoding Δβ-desaturase, seeds or cuttings from transformed plants are used to maintain the transgenic plant line.

The present invention further provides a method for providing transgenic plants with an increased content of GLA. This method includes introducing DNA encoding Δβ-desaturase into plant cells which lack or have low levels of GLA but contain LA, and regenerating plants with increased GLA content from the transgenic cells. In particular, commercially grown crop plants are contemplated as the transgenic organism, including, but not limited to, sunflower, soybean, oil seed rape, maize, peanut and tobacco.

The present invention further provides a method for providing transgenic organisms which contain GLA. This method comprises introducing DNA

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-191 encoding δ 6-desaturase into an organism which lacks or has low levels of GLA, but contains LA. In another embodiment, the method comprises introducing one or more expression vectors which comprise DNA encoding δ12-desaturase and δ6-desaturase into organisms which are deficient in both GLA and LA. Accordingly, organisms deficient in both LA and GLA are induced to produce LA by the expression of δ12-desaturase, and GLA is then generated due to the expression of δ610 desaturase. Expression vectors comprising DNA encoding Al2-desaturase, or δ12-desaturase and δ6desaturase, can be constructed by methods of recombinant technology known to one of ordinary skill in the art (Sambrook et al., 1989) and the published sequence of δ12-desaturase (Wada et al [1990] Nature (London) 347, 200-203. In addition, it has been discovered in accordance with the present invention that nucleotides 2002-3081 of SEQ. ID NO:1 encode cyanobacterial δ12-desaturase. Accordingly, this sequence can be used to construct the subject expression vectors. In particular, commercially grown crop plants are contemplated as the transgenic organism, including, but not limited to, sunflower, soybean, oil seed rape, maize, peanut and tobacco.

The present invention is further directed to a method of inducing chilling tolerance in plants. Chilling sensitivity may be due to phase transition of lipids in cell membranes. Phase transition temperature depends upon the degree of unsaturation of fatty acids in membrane lipids, and thus increasing the degree of unsaturation, for example by introducing

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-201 δ6-desaturase to convert LA to GLA, can induce or improve chilling resistance. Accordingly, the present method comprises introducing DNA encoding δ6desaturase into a plant cell, and regenerating a plant with improved chilling resistance from said transformed plant cell. In a preferred embodiment, the plant is a sunflower, soybean, oil seed rape, maize, peanut or tobacco plant.

The following examples further illustrate 10 the present invention.

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-21! EXAMPLE 1

Strains and Culture Conditions

Synechocystis (PCC 6803, ATCC 27184),

Anabaena (PCC 7120, ATCC 27893) and Synechococcus (PCC

7942, ATCC 33912) were grown photoautotrophically at 30°C in BG11N+ medium (Rippka et al. [1979] J. Gen,

Microbiol. 111, 1-61) under illumination of incandescent lamps jq ( 60μΕ. m'². S'¹ ) . Cosmids and plasmids were selected and propagated in Escherichia coli strain DH5a on LB medium supplemented with antibiotics at standard concentrations as described by Maniatis et al. (1982)

Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring, New York.

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-221 EXAMPLE 2

Construction of Synechocystis Cosmid Genomic LibraryTotal genomic DNA from Synechocystis (PCC 5 6803) was partially digested with Sau3A and fractionated on a sucrose gradient (Ausubel et al. [1987] Current Protocols in Molecular Biology, Greene Publishing Associates and Wiley Interscience, New York), Fractions containing 30 to 40 kb DNA fragments were selected and ligated into the dephosphorylated

BamHI site of the cosmid vector, pDUCA7 (Buikema et al. [1991] J. Bacteriol. 173, 1879-1885). The ligated DNA was packaged in vitro as described by Ausubel et al. (1987), and packaged phage were propagated in L coli DH5a containing the Aval and Eco4711 methylase helper plasmid, pRL528 as described by Buikema et al. (1991) . A total of 1152 colonies were isolated randomly and maintained individually in twelve 96-well microtiter plates.

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-231 EXAMPLE 3

Gain-of-Function Expression of GLA in Anabaena

Anabaena (PCC 7120), a filamentous 5 cyanobacterium, is deficient in GLA but contains significant amounts of linoleic acid, the precursor for GLA (Figure 2; Table 2). The Synechocystis cosmid library described in Example 2 was conjugated into Anabaena (PCC 7120) to identify transconjugants that produce GLA. Anabaena cells were grown to mid-log phase in BG11N+ liquid medium and resuspended in the same medium to a final concentration of approximately 2xl0^? cells per ml. A mid-log phase culture of E. coli RP4 (Burkardt et al. [1979] J. Gen. Microbiol.

114, 341-348) grown in LB containing ampicillin was washed and resuspended in fresh LB medium. Anabaena and RP4 were then mixed and spread evenly on BG11N+ plates containing 5¾ LB. The cosmid genomic library was replica plated onto LB plates containing 50 //g/ml kanamycin and 17.5 μg/ml chloramphenicol and was subsequently patched onto BG11N+ plates containing Anabaena and RP4. After 24 hours of incubation at 30°C, 3 0 ^g/ml of neomycin was underlaid; and incubation at 30°C was continued until transconjugants appeared.

Individual transconjugants were isolated after conjugation and grown in 2 ml BG11N+ liquid medium with 15 /ig/ml neomycin. Fatty acid methyl esters were prepared from wild type cultures and

3Q, cultures containing pools of ten transconjugants as follows. Wild type and transgenic cyanobacterial

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-241 cultures were harvested by centrifugation and washed twice with distilled water. Fatty acid methyl esters were extracted from these cultures as described by Dahmer et al. (1989) J. Amer. Oil. Chem. Soc. 66, 5435 548 and were analyzed by Gas Liquid Chromatography (GLC) using a Tracor-560 equipped with a hydrogen flame ionization detector and capillary column (30 m x 0.25 mm bonded FSOT Superox II, Alltech Associates Inc., IL). Retention times and co-chromatography of standards (obtained from Sigma Chemical Co.) were used for identification of fatty acids. The average fatty acid composition was determined as the ratio of peak area of each C18 fatty acid normalized to an internal standard.

Representative GLC profiles are shown in

Fig. 2. C18 fatty acid methyl esters are shown.

Peaks were identified by comparing the elution times with known standards of fatty acid methyl esters and were confirmed by gas chromatography-mass spectrometry. Panel A depicts GLC analysis of fatty acids of wild type Anabaena. The arrow indicates the migration time of GLA. Panel B is a GLC profile of fatty acids of transconjugants of Anabaena with pAM542+l.8F. Two GLA producing pools (of 25 pools representing 250 transconjugants) were identified that produced GLA. Individual transconjugants of each GLA positive pool were analyzed for GLA production; two independent transconjugants, AS13 and AS75, one from each pool, were identified which expressed significant

2Q levels of GLA and which contained cosmids, cSyl3 and cSy75, respectively (Figure 3). The cosmids overlap

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-251 in a region approximately 7.5 kb in length. A 3.5 kb Nhel fragment of cSy75 was recloned in the vector pDUCA7 and transferred to Anabaena resulting in gainof-function expression of GLA (Table 2).

Two Nhel/Hind III subfragments (1.8 and 1.7 kb) of the 3.5 kb Nhe I fragment of cSy75-3.5 were subcloned into pBLUESCRIPT (Stratagene) (Figure 3) for sequencing. Standard molecular biology techniques were performed as described by Maniatis et al. (1982) and Ausubel et al. (1987). Dideoxy sequencing (Sanger et al. [1977] Proc. Natl. Acad. Sci. USA 74, 54635467) of pBS1.8 was performed with SEQUENASE (United States Biochemical) on both strands by using specific oligonucleotide primers synthesized by the Advanced

DNA Technologies Laboratory (Biology Department, Texas A &amp; M University) . ' DNA sequence analysis was done with the GCG (Madison, Wl) software as described by Devereux et al. (1984) Nucleic Acids Res. 12. 387-395.

Both NheI/HindiII subfragments were transferred into a conjugal expression vector, AM542, in both forward and reverse orientations with respect to a cyanobacterial carboxylase promoter and were introduced into Anabaena by conjugation.

Transeonjugants containing the 1.8 kb fragment in the forward orientation (AM542-1.8F) produced significant quantities of GLA and octadecatetraenoic acid (Figure 2; Table 2). Transconjugants containing other constructs, either reverse oriented 1.8 kb fragment or forward and reverse oriented 1.7 kb fragment, did not

3Q produce detectable levels of GLA (Table 2).

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-261 Figure 2 compares the C18 fatty acid profile of an extract from wild type Anabaena (Figure 2A) with that of transgenic Anabaena containing the 1.8 kb fragment of cSy75-3.5 in the forward orientation (Figure 2B). GLC analysis of fatty acid methyl esters from AM542-1.8F revealed a peak with a retention time identical to that of authentic GLA standard. Analysis of this peak by gas chromatography-mass spectrometry (GC-MS) confirmed that it had the same mass fragmentation pattern as a GLA reference sample. Transgenic Anabaena with altered levels of polyunsaturated fatty acids were similar to wild type in growth rate and morphology.

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-271 Table 2 Composition of C18 Fatty Acids in Wild Type and Transgenic Cyanobacteria

<td rowspan="2"> Strain</td><td colspan="6"> Fatty Acid (%)</td>

<td> 18:0</td><td> 18:1</td><td> 18:2</td><td> 18.3(a)</td><td> 18.3{γ)</td><td> 18.4</td>

<td> Wild Type</td><td></td><td></td><td></td><td></td><td></td><td></td>

<td> Synechocystis</td><td> 13.6</td><td> 4.5</td><td> 54.5</td><td> -</td><td> 27.3</td><td> -</td>

<td> (sp.PCC6803)</td><td></td><td></td><td></td><td></td><td></td><td></td>

<td> Anabaena</td><td> 2.9</td><td> 24.8</td><td> 37.1</td><td> 35.2</td><td></td><td></td>

<td> (sp.PCC7120)</td><td></td><td></td><td></td><td></td><td></td><td></td>

<td> Synechococcus</td><td> 20.6</td><td> 79.4</td><td> -</td><td> -</td><td></td><td></td>

<td> (sp.PCC7942)</td><td></td><td></td><td></td><td></td><td></td><td></td>

<td> Anabaena Transconjugants</td><td></td><td></td><td></td><td></td><td></td><td></td>

<td> cSy75</td><td> 3.8</td><td> 24.4</td><td> 22.3</td><td> 9.1</td><td> 27.9</td><td> 12.5</td>

<td> cSy75-3.5</td><td> 4.3</td><td> 27.6</td><td> 18.1</td><td> 3.2</td><td> 40.4</td><td> 6.4</td>

<td> pAM542 - 1.8F</td><td> 4.2</td><td> 13.9</td><td> 12.1</td><td> 19.1</td><td> 25.4</td><td> 25.4</td>

<td> pAM542 - 1.8R</td><td> 7.7</td><td> 23.1</td><td> 38.4</td><td> 30.8</td><td> -</td><td> -</td>

<td> pAM542 - 1.7F</td><td> 2.8</td><td> 27,8</td><td> 36.1</td><td> 33.3</td><td> -</td><td> -</td>

<td> pAM542 - 1.7R</td><td> 2.8</td><td> 25.4</td><td> 42.3</td><td> 29.6</td><td> -</td><td> -</td>

Synechococcus Transformants

<td> pAM854</td><td> 27.8</td><td> 72.2</td><td> -</td><td> -</td><td> -</td>

<td> pAM854 -Δ¹²</td><td> 4.0</td><td> 43.2</td><td> 46.0</td><td> -</td><td> -</td>

<td> pAM854 -Δ⁶</td><td> 18.2</td><td> 81.8</td><td> -</td><td> -</td><td> -</td>

<td> pAM854 -Δ⁶&amp;Δ¹²</td><td> 42.7</td><td> 25.3</td><td> 19.5</td><td> 16.5</td><td> -</td>

<td> 18:0, stearic acid;</td><td> 18:1, oleic</td><td> acid;</td><td> 18:2, linoleic</td><td> acid;</td><td></td>

18:3(a), linolenic acid; 18:3(γ), γ-linolenic acid; 18:4, octadecatetraenoic acid

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-281 EXAMPLE 4

Transformation of Synechococcus with a6 and a!2 Desaturase Genes

A third cosmid, cSy7, which contains a δ12desaturase gene, was isolated by screening the Synechocystis genomic library with a oligonucleotide synthesized from the published Synechocystis δ12desaturase gene sequence (Wada et al. [1990] Nature (London) 347, 200-203). A 1.7 kb Aval fragment from this cosmid containing the δ12-desaturase gene was identified and used as a probe to demonstrate that cSyl3 not only contains a Δβ-desaturase gene but also a δ12-desaturase gene (Figure 3). Genomic Southern blot analysis further showed that both the Δβ-and δ12desaturase genes are unique in the Synechocystis genome so that both functional genes involved in Cl8 fatty acid desaturation are linked closely in the Synechocystis genome.

The unicellular cyanobacterium Synechococcus (PCC 7942) is deficient in both linoleic acid and GLA(3). The δ12 and Δβ-desaturase genes were cloned individually and together into pAM854 (Bustos et al. [1991] J. Bacteriol. 174, 7525-7533), a shuttle vector that contains sequences necessary for the integration of foreign DNA into the genome of Synechococcus (Golden et al. [1987] Methods in Enzymol. 153, 215231). Synechococcus was transformed with these gene constructs and colonies were selected. Fatty acid methyl esters were extracted from transgenic Synechococcus and analyzed by GLC.

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-291 Table 2 shows that the principal fatty acids of wild type Synechococcus are stearic acid (18:0) and oleic acid (18:1). Synechococcus transformed with ρΑΜ854-Δΐ2 expressed linoleic acid (18:2) in addition to the principal fatty acids. Transformants with ρΑΜ854-Δ6 and δ12 produced both linoleate and GLA (Table 1) . These results indicated that Synechococcus containing both δ12- and Δδ-desaturase genes has gained the capability of introducing a second double bond at the δ12 position and a third double bond at the δ6 position of C18 fatty acids. However, no changes in fatty acid composition was observed in the transformant containing ρΑΜ854-Δβ, indicating that in the absence of substrate synthesized by the δ12 desaturase, the Δδ-desaturase is inactive. This experiment further confirms that the 1.8 kb Nhel/HindXII fragment (Figure 3) contains both coding and promoter regions of the Synechocystis Δδdesaturase gene. Transgenic Synechococcus with altered levels of polyunsaturated fatty acids were similar to wild type in growth rate and morphology.

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-301 EXAMPLE 5

Nucleotide Sequence of δ6-Desaturase

The nucleotide sequence of the 1.8 kb 5 fragment of cSy75-3.5 including the functional δ6desaturase gene was determined. An open reading frame encoding a polypeptide of 359 amino acids was identified (Figure 4). A Kyte-Doolittle hydropathy analysis (Kyte et al. [1982] J. Mol. Biol. 157, 10510 132) identified two regions of hydrophobic amino acids that could represent transmembrane domains (Figure 1A); furthermore, the hydropathic profile of the δ6desaturase is similar to that of the δ12-desaturase gene (Figure IB; Wada et al.) and A9-desaturases (Thiede et al. [1986] J. Biol. Chem. 261, 1323013235). However, the sequence similarity between the Synechocystis δ6- and δ12-desaturases is less than 40% at the nucleotide level and approximately 18% at the amino acid level.

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-311 EXAMPLE 6

Transfer of Cyanobacterial A⁶-Desaturase into Tobacco

The cyanobacterial δ⁶-desaturase gene was 5 mobilized into a plant expression vector and transferred to tobacco using Agrobacterium mediated gene transfer techniques. To ensure that the transferred desaturase is appropriately expressed in leaves and developing seeds and that the desaturase gene product is targeted to the endoplasmic reticulum or the chloroplast, various expression cassettes with Synechocystis Δ-desaturase open reading frame (ORF) were constructed. Components of these cassettes include: (i) a 35S promoter or seed specific promoter derived from the sunflower helianthinin gene to drive A^s-desaturase gene expression in all plant tissues or only in developing seeds respectively, (ii) a putative signal peptide either from carrot extensin gene or sunflower helianthinin gene to target newly synthesized A^e-desaturase into the ER, (iii) an ER lumen retention signal sequence (KDEL) at the COOHterminal of the A⁶-desaturase ORF, and (iv) an optimized transit peptide to target δ⁶ desaturase into the chloroplast. The 35S promoter is a derivative of pRTL2 described by Restrepo et al. (1990). The optimized transit peptide sequence is described by Van de Broeck et al. (1985). The carrot extensin signal peptide is described by Chen et al (1985) EMBO J. 9, 2145.

βθ Transgenic tobacco plants were produced containing a chimeric cyanobacterial desaturase gene,

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-321 comprised of the Synechocystis δ⁶ desaturase gene fused to an endoplasmic reticulum retention sequence (KDEL) and extensin signal peptide driven by the CaMV 35S promoter. PCR amplifications of transgenic tobacco genomic DNA indicate that the δ⁵ desaturase gene was incorporated into the tobacco genome. Fatty acid methyl esters of leaves of these transgenic tobacco plants were extracted and analyzed by Gas Liquid Chromatography (GLC). These transgenic tobacco accumulated significant amounts of GLA (Figure 4).

Figure 4 shows fatty acid methyl esters as determined by GLC. Peaks were identified by comparing the elution times with known standards of fatty acid methyl ester. Accordingly, cyanobacterial genes

3_5 involved in fatty acid metabolism can be used to generate transgenic plants with altered fatty acid compositions.

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-331 EXAMPLE 7

Construction of Borage cDNA library

Membrane bound polysomes were isolated from 5 borage seeds 12 days post pollination (12 DPP) using the protocol established for peas by Larkins and Davies (1975 Plant Phys. 55:749-756). RNA was extracted from the polysomes as described by Mechler (1987 Methods in Enzymology 152:241-248 . Academic

Press).

Poly-A+ RNA was isolated from the membrane bound polysomal RNA by use of Oligotex-dT beads (Qiagen) . Corresponding cDNA was made using Stratagene's ZAP cDNA synthesis kit. The cDNA library was constructed in the lambda ZAP IX vector (Stratagene) using the lambda ZAP II vector kit. The primary library was packaged in Gigapack II Gold packaging extract (Stratagene). The library was used to generate expressed sequence tags (ESTs), and sequences corresponding to the tags were used to scan the GenBank database.

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-341 EXAMPLE 8

Hybridization Protocol

Hybridization probes for screening the 5 borage cDNA library were generated by using random primed DNA synthesis as described by Ausubel et al (1994 Current Protocols in Molecular Biology, Wiley Interscience, N.Y.) and corresponded to previously identified abundantly expressed seed storage protein

3_0 cDNAs. Unincorporated nucleotides were removed by use of a G-50 spin column (Boehringer Manheim) . Probe was denatured for hybridization by boiling in a water bath for 5 minutes, then quickly cooled on ice. Filters for hybridization were prehybridized at 60°C for 2-4 hours in prehybridization solution (6XSSC [Maniatis et al 1984 Molecular Gloning A Laboratory Manual, Cold Spring Harbor Laboratory], IX Denharts Solution, 0.05% sodium pyrophosphate, 100 /zg/ml denatured salmon sperm DNA) . Denatured probe was added to the hybridization solution (6X SSC, IX Denharts solution, 0.05% sodium pyrophosphate, 100 ^g/ml denatured salmon sperm DNA) and incubated at 60°C with agitation overnight.

Filters were washed in 4x, 2x, and lx SET washes for 15 minutes each at 60°C. A 20X SET stock solution is

3M NaCl, 0.4 M Tris base, 20 mM Na₂EDTA-2H₂0. The 4X SET wash was 4X SET, 12.5 mM PO₄, pH 6.8 and 0.2% SDS. The 2X SET wash was 2X SET, 12.5 mM PO₄, pH 6.8 and 0.2% SDS. The IX SET wash was IX SET, 12.5 mM P0₄, pH 6.8 and 0.2% SDS. Filters were allowed to air dry and

2q were then exposed to X-ray film for 24 hours with intensifying screens at -80°C.

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-351 EXAMPLE 9

Random sequencing of cDNAs from a borage seed (12 DPP) membrane-bound polysomal library

The borage cDNA library was plated at low density (500 pfu on 150 mm petri dishes). Highly prevalent seed storage protein cDNAs were subtracted by screening with the previously identified corresponding cDNAs. Non-hybridizing plaques were excised using Stratagene's excision protocol and reagents. Resulting bacterial colonies were used to inoculate liquid cultures and were either sequenced manually or by an ABI automated sequencer. Each cDNA was sequenced once and a sequence tag generated from 200-300 base pairs. All sequencing was performed by cycle sequencing (Epicentre) . Over 300 ESTs were generated. Each sequence tag was compared to GenBank database by BLASTX computer program and a number of lipid metabolism genes, including the Δβ-desaturase were identified.

Database searches with a cDNA clone designated mbp-65 using BLASTX with the GenBank database resulted in a significant match to the Synechocystis Δβ-desaturase. It was determined however, that this clone was not a full length cDNA.

A full length cDNA was isolated using mbp-65 to screen the borage membrane-bound polysomal library. The sequence of the isolated cDNA was determined (Fig. 5A, SEQ ID NO:4) and the protein sequence of the open reading frame (Fig. 5B, SEQ ID NO:5) was compared to other known desaturases using Geneworks

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-361 (IntelligGenetics) protein alignment program (Fig. 2). This alignment indicated that the cDNA was the borage A6-desaturase gene.

Although similar to other known plant desaturases, the borage delta 6-desaturase is distinct as indicated in the dendrogram shown in Fig. 6. Furthermore, comparison of the amino acid sequences characteristic of desaturases, particularly those proposed to be involved in metal binding (metal box 1 and metal box 2), illustrates the differences between the borage delta 6-desaturase and other plant desaturases (Table 3).

The borage delta 6-desaturase is distinguished from the cyanobacterial form not only in over all sequence (Fig. 6) but also in the lipid box, metal box 1 and metal box 2 amino acid motifs (Table 3). As Table 3 indicates, all three motifs are novel in sequence. Only the borage delta 6-desaturase metal box 2 shown some relationship to the Synechocystis go delta-6 desaturase metal box 2.

In addition, the borage delta 6-desaturase is also distinct from another borage desaturase gene, the delta-12 desaturase. Pl-81 is a full length cDNA that was identified by EST analysis and shows high similarity to the Arabidopsis delta-12 desaturase (Fad 2). A comparison of the lipid box, metal box l and metal box 2 amino acid motifs (Table 3) in borage delta 6 and delta-12 desaturases indicates that little homology exists in these regions. The placement of βθ the two sequences in the dendrogram in Fig. 6 indicates how distantly related these two genes are.

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<td> X i</td><td> X X &gt;1</td><td> X X Eh</td><td> X ' X Eh</td><td> X X EH</td><td> X X Eh</td><td> X X EH</td><td> X X X</td><td> X X X</td><td> X X X</td><td> X X X</td><td> X X OI o</td><td> X X SB</td><td> X X X</td><td> t-» co ω</td>

<td></td><td> z</td><td> K</td><td> X</td><td> X</td><td> X</td><td> X</td><td> X</td><td> X</td><td> a</td><td> Q</td><td> X</td><td> Û</td><td> z</td><td></td>

<td> X</td><td> X</td><td> X</td><td> X</td><td> X</td><td> X</td><td> X</td><td> X</td><td> X</td><td> X</td><td> X</td><td></td><td> X</td><td> X</td><td> &gt;1</td>

<td></td><td colspan="9"></td><td></td><td> Ο</td><td colspan="2"></td><td> •o</td>

<td></td><td> ·***</td><td></td><td></td><td> x“&gt;»</td><td></td><td></td><td></td><td> Λ</td><td> O</td><td> o</td><td></td><td> »4</td><td></td><td></td>

<td> LD</td><td> Γ</td><td> 00</td><td> 00</td><td> 00</td><td> 00</td><td> 00</td><td> es</td><td> σι</td><td> i“L</td><td></td><td></td><td> rL</td><td> co</td><td> •H</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></td>

<td> o</td><td> o</td><td> o</td><td> o</td><td> e&gt;</td><td> o</td><td> o</td><td> o</td><td> ο</td><td> o</td><td> o</td><td> Ο ζ</td><td> d</td><td> d</td><td> •rl 4J</td>

<td> z</td><td> z</td><td> z</td><td> z</td><td> z</td><td> z</td><td> z</td><td> z</td><td> z</td><td> z</td><td> z</td><td></td><td> z</td><td> z</td><td> c</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> y</td>

<td> d</td><td> d</td><td> d</td><td> a</td><td> Q</td><td> d</td><td> a</td><td> a</td><td> Q</td><td> o</td><td> Q</td><td> Η</td><td> Q</td><td> a</td><td> Ό Ή</td>

<td> PL</td><td> H</td><td> H</td><td> H</td><td> H</td><td> H</td><td> H</td><td> M</td><td> H</td><td> H</td><td> H</td><td></td><td> H</td><td> H</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></td><td> to</td>

<td> ci</td><td> ci</td><td> ci</td><td> ex</td><td> ci</td><td> Cf</td><td> ex</td><td> o</td><td> O</td><td> ci</td><td> ex</td><td> Ο» ω</td><td> σ</td><td> ex</td><td> /0 3</td>

<td> Ex3</td><td></td><td> ω</td><td> ω</td><td> ω</td><td> ω</td><td> ω</td><td> ω</td><td> Cx3</td><td> U</td><td> ω</td><td> CO</td><td> ω</td><td> Cx3</td><td></td>

<td> CO</td><td> co</td><td> to</td><td> co</td><td> co</td><td> co</td><td> to</td><td> co</td><td> CO</td><td> CO</td><td> CO</td><td> —*</td><td> co</td><td> CO</td><td> X</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> U</td>

<td> X</td><td> X</td><td> X</td><td> X</td><td> X</td><td> X</td><td> X</td><td> X</td><td> X</td><td> 3</td><td> 3</td><td> §</td><td> X</td><td> X</td><td> •r| X</td>

<td> 3</td><td> z</td><td> 0</td><td> 0</td><td> o</td><td> EE)</td><td> o</td><td> 0</td><td> 0</td><td> 3</td><td> 3</td><td> ι&gt;</td><td> 0</td><td> 0</td><td> 2</td>

<td> «5</td><td> Λ,</td><td> Γ)</td><td> ()</td><td> E)</td><td> &lt;&gt;</td><td> E)</td><td> υ</td><td> 0</td><td> υ</td><td> 0</td><td> D</td><td> 0</td><td> 0</td><td></td>

<td> Q</td><td> Q</td><td> Q</td><td> Q</td><td> o</td><td> a</td><td> a</td><td> ω</td><td> ω</td><td> u</td><td> u</td><td> X</td><td> ο</td><td> U</td><td></td>

<td> X</td><td> X</td><td> X</td><td> X</td><td> X</td><td> X</td><td> X</td><td> 3</td><td> X</td><td> X</td><td> X</td><td> 0</td><td> X</td><td> X</td><td> z</td>

<td> 0</td><td> 0</td><td> 0</td><td> to</td><td> E9</td><td> EO</td><td> EE)</td><td> 2</td><td> 3</td><td> 0</td><td> 0</td><td> W</td><td> 0</td><td> 0</td><td> Q</td>

<td> pl</td><td></td><td></td><td> Pl</td><td> P)</td><td> iP</td><td> h!</td><td> Μ</td><td> H</td><td> W</td><td> K</td><td> &gt;</td><td></td><td> p-l</td><td> U</td>

<td> £</td><td></td><td> &gt;</td><td> ;&gt;</td><td> &gt;</td><td></td><td> &gt;</td><td> &gt;</td><td></td><td> &gt;</td><td> ï&gt;</td><td></td><td></td><td> &gt;</td><td></td>

<td></td><td></td><td></td><td></td><td> *n</td><td></td><td></td><td></td><td></td><td></td><td></td><td> KD</td><td></td><td></td><td> P</td>

<td></td><td></td><td></td><td></td><td> &lt;</td><td></td><td></td><td></td><td></td><td></td><td></td><td> I</td><td></td><td></td><td> en</td>

<td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td> 2</td><td></td><td> S</td><td></td><td></td><td> c</td>

<td></td><td></td><td> &lt;</td><td></td><td> 4J</td><td></td><td></td><td></td><td></td><td> &lt;</td><td></td><td></td><td></td><td></td><td> y</td>

<td></td><td></td><td></td><td></td><td> to</td><td></td><td></td><td> ♦</td><td></td><td></td><td></td><td> Η</td><td></td><td></td><td> H</td>

<td></td><td></td><td> 4-)</td><td></td><td> 10</td><td></td><td></td><td> —»</td><td></td><td> 4-&gt;</td><td> &lt;3</td><td> «0</td><td></td><td></td><td></td>

<td></td><td> «</td><td> CO</td><td></td><td> rL</td><td></td><td> &lt;</td><td> T-L</td><td></td><td> to</td><td></td><td> Ή</td><td></td><td></td><td> Ή</td>

<td></td><td> &lt;3</td><td> (0</td><td></td><td> Ü</td><td></td><td></td><td> 00</td><td></td><td> (0</td><td> Έ3</td><td> Ό</td><td> &lt;3</td><td></td><td> H</td>

<td></td><td></td><td></td><td></td><td> o</td><td></td><td></td><td> 1</td><td></td><td> rL</td><td> •H</td><td> Ή</td><td></td><td></td><td> 3</td>

<td></td><td> (0</td><td> Û,</td><td></td><td> H</td><td> &lt;</td><td> ΓΌ</td><td> rH</td><td> &lt;3</td><td> Û4</td><td> P</td><td> Ρ</td><td> 10</td><td></td><td> ML</td>

<td></td><td></td><td> 0</td><td></td><td> 0</td><td></td><td> Ό</td><td> CL</td><td></td><td> ü</td><td> (0</td><td> (0</td><td> -Η</td><td></td><td></td>

<td></td><td> 4-)</td><td> W</td><td></td><td></td><td></td><td> &lt;0</td><td></td><td></td><td> M</td><td> td</td><td> (0</td><td> 4J</td><td></td><td> (0</td>

<td></td><td> to</td><td> O</td><td></td><td> JZ</td><td> co</td><td> ML</td><td></td><td> CN</td><td> o</td><td> rL</td><td> rL</td><td> to</td><td> &lt;3</td><td></td>

<td></td><td> &gt;.</td><td> rL</td><td></td><td> υ</td><td> .TJ</td><td></td><td></td><td> Ό</td><td> r-1</td><td> Û4</td><td> ο,</td><td> &gt;1</td><td></td><td> to</td>

<td> &lt;3</td><td> 0</td><td> X</td><td></td><td></td><td> f0</td><td> (0</td><td> &lt;</td><td> &lt;0</td><td> X!</td><td></td><td></td><td> Ü</td><td> &lt;0</td><td> •H</td>

<td></td><td> o</td><td> u</td><td></td><td> Φ</td><td> M-l</td><td> u</td><td></td><td> ML</td><td> O</td><td> 0)</td><td> χ:</td><td> Ο</td><td> G</td><td></td>

<td> 0)</td><td> X</td><td></td><td> &lt;3</td><td> a</td><td></td><td> •H</td><td> t)</td><td></td><td></td><td> c</td><td> ο</td><td> X</td><td> y</td><td> rL</td>

<td> t7»</td><td> υ</td><td> *</td><td></td><td> •H</td><td> «</td><td> to</td><td> cjt</td><td> •</td><td> •</td><td> Ή</td><td> (0</td><td> Ü</td><td> &lt;0</td><td> 00</td>

<td> (0</td><td> QJ</td><td> XI</td><td> 0</td><td> u</td><td> Λ</td><td> to</td><td> 10</td><td> Λ</td><td> Λ</td><td> υ</td><td> C</td><td> y</td><td> X</td><td> 1</td>

<td> M</td><td> a</td><td> (0</td><td> u</td><td></td><td> &lt;0</td><td> (0</td><td> M</td><td> &lt;0</td><td> (0</td><td> &gt;1</td><td> Ή</td><td> G</td><td> &lt;0</td><td> H</td>

<td> 0</td><td></td><td> IL</td><td></td><td> rH</td><td> M</td><td> M</td><td> o</td><td> M</td><td> M</td><td> rH</td><td> (X</td><td></td><td> c</td><td> CM</td>

<td> CÛ</td><td> co</td><td></td><td></td><td> 0</td><td></td><td> X</td><td> X</td><td> &lt;</td><td></td><td> 0</td><td> CO</td><td> CO</td><td> 3</td><td> 4c</td>

Arbidopsis Δ12 desaturase (fad2)

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-38EXAMPLE 10

Construction of 222.1A⁶NOS for transient and expression

The vector pBI221 (Jefferson et al. 1987 EMBO J. £:3901-3907) was prepared for ligation by’ digestion with BamHI and EcoICR I (Promega) which excises the GUS coding region leaving the 35S promoter and NOS terminator intact. The borage Δ 6-desaturase cDNA was excised from the Bluescript plasmid (Stratagene) by digestion with BamHI and Xhol. The Xhol end was made blunt by use of the Klenow fragment. This fragment was then cloned into the BamHI/EcoICR I sites of pBI221, yielding 221.A⁶NOS (Fig. 7). In 221. Δ⁶.NOS, the remaining portion (backbone) of the restriction map depicted in Fig. 7 is pBI221.

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-392 EXAMPLE 11

Construction of 121.Δ⁶.NOS for stable transformation

The vector pBI121 (Jefferson et al. 1987 5 EMBO J. 6:3901-3907) was prepared for ligation by digestion with BamHI and EcoICR I (Promega) which excises the GUS coding region leaving the 35S promoter and NOS terminator intact. The borage Δ 6-desaturase cDNA was excised from the Bluescript plasmid

IQ (Stratagene) by digestion with BamHI and Xhol. The

Xhol end was made blunt by use of the Klenow fragment. This fragment was then cloned into the BamHI/EcoICR I sites of pBI121, yielding 121.1A⁶NOS (Fig. 7). In 121.A⁶.NOS, the remaining portion (backbone) of the restriction map depicted in Fig. 7 is pBI121.

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-402 EXAMPLE 12

Transient Expression

All work involving protoplasts was performed 5 in a sterile hood. One ml of packed carrot suspension cells were digested in 30 mis plasmolyzing solution (25 g/1 KC1, 3.5 g/1 CaCl₂-H₂O, lOmM MES, pH 5.6 and 0.2 M mannitol) with 1% cellulase, 0.1% pectolyase, and 0.1% dreisalase overnight, in the dark, at room temperature. Released protoplasts were filtered through a 150 μπι mesh and pelleted by centrifugation (lOOx g, 5 min.) then washed twice in plasmolyzing solution. Protoplasts were counted using a double chambered hemocytometer. DNA was transfected into the protoplasts by PEG treatment as described by Nunberg and Thomas (1993 Methods in Plant Molecular Biology and Biotechnology, B.R. Glick and J.E. Thompson, eds. pp. 241-248) using 10^s protoplasts and 50-70 ug of plasmid DNA (221.Δ6.NOS). Protoplasts were cultured

2q in 5 mis of MS media supplemented with 0.2M mannitol and 3 μτα 2,4-D for 48 hours in the dark with shaking.

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-412 EXAMPLE 13

Stable transformation of tobacco

121. Δ⁶.NOS plasmid construction was used to 5 transform tobacco (Nicotiana tabacum cv. xanthi) via

Agrobacterium according to standard procedures (Horsh et al., 1985 Science 227: 1229-1231; Bogue et al., 1990 Mol. Gen. Genet. 221:49-57). except that initial transformants were selected on 100 ug/ml kanamycin.

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-42j EXAMPLE 14

Preparation and analysis of fatty acid methyl esters (FAMEs)

Tissue from transfected protoplasts and 5 transformed tobacco plants was frozen in liquid nitrogen and lyophilized overnight. FAMEs were prepared as described by Dahmer et al (1989 J. Amer. Oil Chem. Soc. 66:543-548). In some cases, the solvent was evaporated again, and the FAMEs were resuspended in ethyl acetate and extracted once with deionized water to remove any water soluble contaminants. The FAMEs were analyzed by gas chromatography (GC) on a J&amp;W Scientific DB-wax column (30 m length, 0.25 mm ID, 0.25 urn film).

An example of a transient assay is shown in

Fig. 8 which represents three independent transfections pooled together. The addition of the borage Δ6-desaturase cDNA corresponds with the appearance of gamma linolenic acid (GLA) which is one of the possible products of Δ6-desaturase.

Figures 9 and 10 depict GC profiles of the

FAMES derived from leaf and seed tissue, respectively, of control and transformed tobacco plants. Figure 9A provides the profile of leaf tissue of wild-type tobacco (xanthi); Figure 9B provides the profile of leaf tissue from a tobacco plant transformed with the borage Δ-6 desaturase under the transcriptional control of the 35S CaMV promoter (pBI 121A⁶NOS) .

Peaks correspond to 18:2, 18:3γ (GLA), 18:3a and 18:4 (octadecanonic acid).· Figure 10A shows the GC profile of seeds of a wild-type tobacco; Figure 10B shows the

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-433. profile of seed tissue of a tobacco plant transformed with pBI 121A⁶NOS. Peaks correspond to 18:2,

18:3y(GLA) and 18:3a.

The relative distribution of the C₁₈ fatty acids in control and transgenic tobacco seeds is shown in Table 4.

TABLE 4

<td> Fatty Acid</td><td> Xanthi</td><td> pBH21A^fiN0S</td>

<td> 18:0</td><td> 4.0%</td><td> 2.5%</td>

<td> 18 :1</td><td> 13%</td><td> 13%</td>

<td> 18:2</td><td> 82%</td><td> 82%</td>

<td> 18:3y(GLA)</td><td> -</td><td> 2.7%</td>

<td> 18:3a</td><td> 0.82%</td><td> 1,4%</td>

The foregoing results demonstrate that GLA is incorporated into the triacylglycerides of transgenic tobacco leaves and seeds containing the

2q borage Δ6-desaturase.

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-44SEQUENCE LISTING (1) GENERAL INFORMATION:

(i) APPLICANT: Rhone-Poulenc Agrochimie (ii) TITLE OF INVENTION: PRODUCTION OF GAMMA LINOLENIC ACID BY A DELTA 6-DESATURASE (iii) NUMBER OF SEQUENCES: 25 (iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: Scully, Scott, Murphy &amp; Presser (B) STREET: 400 Garden City Plaza (C) CITY: Garden City (D) STATE: New York (E) COUNTRY: United States (F) ZIP: 11530 (v) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS (D) SOFTWARE: Patentln Release #1.0, Version #1.25 (vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER:

(B) FILING DATE: 30-DEC-1994 (C) CLASSIFICATION:

(viii) ATTORNEY/AGENT INFORMATION:

(A) NAME: Presser, Leopold (B) REGISTRATION NUMBER: 19,827 (C) REFERENCE/DOCKET NUMBER: 8383ZYXW (ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: (516) 742-4343 (B) TELEFAX: (516) 742-4366 (C) TELEX: 230 901 SANS UR (2) INFORMATION FOR SEQ ID NO:1:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 3588 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: both (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic)

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-45(ix) FEATURE:

(A) NAME/KEY: CDS (B) LOCATION: 2002..3081 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:

<td> GCTAGCCACC</td><td> AGTGACGATG</td><td> CCTTGAATTT</td><td> GGCCATTCTG</td><td> ACCCAGGCCC</td><td> GTATTCTGAA</td><td> 60</td>

<td> TCCCCGCATT</td><td> CGCATTGTTA</td><td> ATCGTTTGTT</td><td> CAACCATGCC</td><td> CTGGGTAAAC</td><td> GTTTAGACAC</td><td> 120</td>

<td> CACCTTGCCA</td><td> GACCACGTTA</td><td> GTTTGAGTGT</td><td> TTCCGCCCTG</td><td> GCGGCCCCGA</td><td> TTTTTTCCTT</td><td> 180</td>

<td> TGCGGCTTTG</td><td> GGCAATCAGG</td><td> CGATCGGGCA</td><td> ATTGCGTTTG</td><td> TTTGACCAGA</td><td> CTTGGCCCAT</td><td> 240</td>

<td> TCAGGAAATT</td><td> GTCATTCACC</td><td> AAGACCATCC</td><td> CTGGCTCAAT</td><td> TTACCCCTGG</td><td> CGGATTTATG</td><td> 300</td>

<td> GGATGATCCG</td><td> AGCCGAATGT</td><td> TGATCTATTA</td><td> CCTACCGGCC</td><td> CACAGTGAAA</td><td> CGGATTTAGT</td><td> 360</td>

<td> AGGCGCAGTG</td><td> GTGAATAATT</td><td> TAACGTTGCA</td><td> ATCTGGGGAC</td><td> CATTTAATAG</td><td> TGGGACAAAA</td><td> 420</td>

<td> ACCCCAACCC</td><td> AAGACCAAAC</td><td> GGCGATCGCC</td><td> TTGGCGCAAA</td><td> TTTTCCAAAC</td><td> TGATTACCAA</td><td> 480</td>

<td> CCTGCGGGAG</td><td> TATCAGCGGT</td><td> ATGTCCAACA</td><td> GGTGATATGG</td><td> GTGGTGTTGT</td><td> TTTTATTGTT</td><td> 540</td>

<td> GATGATTTTT</td><td> CTGGCCACCT</td><td> TCATCTACGT</td><td> TTCCATTGAT</td><td> CAACATATTG</td><td> CCCCAGTGGA</td><td> 600</td>

<td> CGCGTTGTAT</td><td> TTTTCCGTGG</td><td> GCATGATTAC</td><td> CGGGGCCGGT</td><td> GGCAAGGAAG</td><td> AGGTGGCCGA</td><td> 660</td>

<td> AAAGTCCCCC</td><td> GATATCATCA</td><td> AAGTATTCAC</td><td> AGTGGTGATG</td><td> ATGATCGCCG</td><td> GGGCGGGGGT</td><td> 720</td>

<td> GATTGGTATT</td><td> TGTTATGCCC</td><td> TACTGAATGA</td><td> TTTCATCCTT</td><td> GGCAGTCGCT</td><td> TTAGTCAGTT</td><td> 780</td>

<td> TTTGGATGCG</td><td> GCCAAGTTAC</td><td> CCGATCGCCA</td><td> TCACATCATC</td><td> ATTTGTGGGC</td><td> TGGGGGGAGT</td><td> 840</td>

<td> GAGCATGGCC</td><td> ATTATTGAAG</td><td> AGTTAATTCA</td><td> CCAGGGCCAT</td><td> GAAATTGTGG</td><td> TAATCGAAAA</td><td> 900</td>

<td> GGATACAGAT</td><td> AATCGTTTCT</td><td> TGCATACGGC</td><td> CCGCTCCCTG</td><td> GGGGTGCCCG</td><td> TAATTGTGGA</td><td> 960</td>

<td> GGATGCCCGC</td><td> CTAGAAAGAA</td><td> CGTTGGCCTG</td><td> CGCCAATATC</td><td> AACCGAGCCG</td><td> AAGCCATTGT</td><td> 1020</td>

<td> GGTGGCCACC</td><td> AGCGACGACA</td><td> CCGTTAACTT</td><td> GGAAATTGGC</td><td> CTAACTGCCA</td><td> AGGCGATCGC</td><td> 1080</td>

<td> CCCTAGCCTG</td><td> CCAGTGGTGT</td><td> TGCGTTGCCA</td><td> GGATGCCCAG</td><td> TTTAGCCTGT</td><td> CCCTGCAGGA</td><td> • 1140</td>

<td> AGTATTTGAA</td><td> TTTGAAACGG</td><td> TGCTTTGTCC</td><td> GGCGGAATTG</td><td> GCCACCTATT</td><td> CCTTTGCGGC</td><td> 1200</td>

<td> GGCGGCCCTG</td><td> GGGGGCAAAA</td><td> TTTTGGGCAA</td><td> CGGCATGACC</td><td> GATGATTTGC</td><td> TGTGGGTAGC</td><td> 1260</td>

<td> CCTAGCCACC</td><td> TTAATCACTC</td><td> CTAACCATCC</td><td> CTTTGCCGAC</td><td> CAATTGGTTA</td><td> AAATTGCAGC</td><td> 1320</td>

<td> CCAAAAGTCT</td><td> GATTTCGTTC</td><td> CCCTCTATCT</td><td> AGAACGGGGT</td><td> GGCAAAACCA</td><td> TCCATAGCTG</td><td> 1380</td>

<td> GGAATTATTG</td><td> GGTACCCATC</td><td> TCGACTCTGG</td><td> AGACGTGTTG</td><td> TATTTAACCA</td><td> TGCCCGCCAC</td><td> 1440</td>

<td> TGCCCTAGAG</td><td> CAACTTTGGC</td><td> GATCGCCCCG</td><td> TGCCACTGCT</td><td> GATCCTCTGG</td><td> ACTCTTTTTT</td><td> 1500</td>

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PCT/IB95/01167

-46GGTTTAGCAT GGGGGGATGG AACTCTTGAC TCGGCCCAAT GGTGATCAAG AAAGAACGCT 1560

TTGTCTATGT TTAGTATTTT TAAGTTAACC AACAGCAGAG GATAACTTCC AAAAGAAATT 1620

AAGCTCAAAA AGTAGCAAAA TAAGTTTAAT TCATAACTGA GTTTTACTGC TAAACAGCGG 1680

TGCAAAAAAG TCAGATAAAA TAAAAGCTTC ACTTCGGTTT TATATTGTGA CCATGGTTCC 1740

CAGGCATCTG CTCTAGGGAG TTTTTCCGCT GCCTTTAGAG AGTATTTTCT CCAAGTCGGC 1800

TAACTCCCCC ATTTTTAGGC AAAATCATAT ACAGACTATC CCAATATTGC CAGAGCTTTG 1860

ATGACTCACT GTAGAAGGCA GACTAAAATT CTAGCAATGG ACTCCCAGTT GGAATAAATT 1920

TTTAGTCTCC CCCGGCGCTG GAGTTTTTTT GTAGTTAATG GCGGTATAAT GTGAAAGTTT 1980

<td colspan="2" rowspan="2"> TTTATCTATT TAAATTTATA A ATG Met</td><td colspan="5"> CTA ACA GCG GAA AGA ATT AAA</td><td rowspan="3"> TTT ACC Phe Thr 10</td>

<td rowspan="2"> Leu</td><td rowspan="2"> Thr</td><td rowspan="2"> Ala Glu 5</td><td rowspan="2"> Arg</td><td rowspan="2"> He Lys</td>

<td></td><td> 1</td>

<td> CAG</td><td> AAA CGG GGG TTT CGT CGG</td><td> GTA</td><td> CTA</td><td> AAC CAA</td><td> CGG</td><td> GTG GAT</td><td> GCC TAC</td>

<td> Gin</td><td> Lys Arg Gly Phe Arg Arg 15</td><td> Val</td><td> Leu</td><td> Asn Gin 20</td><td> Arg</td><td> Val Asp</td><td> Ala Tyr 25</td>

<td> TTT</td><td> GCC GAG CAT GGC CTG ACC</td><td> CAA</td><td> AGG</td><td> GAT AAT</td><td> CCC</td><td> TCC ATG</td><td> TAT CTG</td>

<td> Phe</td><td> Ala Glu His Gly Leu Thr 30</td><td> Gin</td><td> Arg 35</td><td> Asp Asn</td><td> Pro</td><td> Ser Met 40</td><td> Tyr Leu</td>

<td> AAA</td><td> ACC CTG ATT ATT GTG CTC</td><td> TGG</td><td> TTG</td><td> TTT TCC</td><td> GCT</td><td> TGG GCC</td><td> TTT GTG</td>

<td> Lys</td><td> Thr Leu Ile Ile Val Leu 45</td><td> Trp 50</td><td> Leu</td><td> Phe Ser</td><td> Ala</td><td> Trp Ala 55</td><td> Phe Val</td>

<td> CTT</td><td> TTT GCT CCA GTT ATT TTT</td><td> CCG</td><td> GTG</td><td> CGC CTA</td><td> CTG</td><td> GGT TGT</td><td> ATG GTT</td>

<td> Leu</td><td> Phe Ala Pro Val He Phe 60 65</td><td> Pro</td><td> Val</td><td> Arg Leu</td><td> Leu 70</td><td> Gly Cys</td><td> Met Val</td>

<td> TTG</td><td> GCG ATC GCC TTG GCG GCC</td><td> TTT</td><td> TCC</td><td> TTC AAT</td><td> GTC</td><td> GGC CAC</td><td> GAT GCC</td>

<td> Leu 75</td><td> Ala He Ala Leu Ala Ala 80</td><td> Phe</td><td> Ser</td><td> Phe Asn 85</td><td> Val</td><td> Gly His</td><td> Asp Ala 90</td>

<td> AAC</td><td> CAC AAT GCC TAT TCC TCC</td><td> AAT</td><td> CCC</td><td> CAC ATC</td><td> AAC</td><td> CGG GTT</td><td> CTG GGC</td>

<td> Asn</td><td> His Asn Ala Tyr Ser Ser 95</td><td> Asn</td><td> Pro</td><td> His He 100</td><td> Asn</td><td> Arg Val</td><td> Leu Gly 105</td>

<td> ATG</td><td> ACC TAC GAT TTT GTC GGG</td><td> TTA</td><td> TCT</td><td> AGT TTT</td><td> CTT</td><td> TGG CGC</td><td> TAT CGC</td>

<td> Met</td><td> Thr Tyr Asp Phe Val Gly 110</td><td> Leu</td><td> Ser 115</td><td> Ser Phe</td><td> Leu</td><td> Trp Arg 120</td><td> Tyr Arg</td>

<td> CAC</td><td> AAC TAT TTG CAC CAC ACC</td><td> TAC</td><td> ACC</td><td> AAT ATT</td><td> CTT</td><td> GGC CAT</td><td> GAC GTG</td>

<td> His</td><td> Asn Tyr Leu His His Thr 125</td><td> Tyr 130</td><td> Thr</td><td> Asn He</td><td> Leu</td><td> Gly His 135</td><td> Asp Val</td>

<td> GAA</td><td> ATC CAT GGA GAT GGC GCA</td><td> GTA</td><td> CGT</td><td> ATG AGT</td><td> CCT</td><td> GAA CAA</td><td> GAA CAT</td>

<td> Glu</td><td> He His Gly Asp Gly Ala 140 145</td><td> Val</td><td> Arg</td><td> Met Ser</td><td> Pro 150</td><td> Glu Gin</td><td> Glu His</td>

2031

2079

2127

2175

2223

2271

2319

2367

2415

2463

CA 02207906 1997-06-16

WO 96/21022 PCT/ÏB95/O1167

<td colspan="2" rowspan="2"> GTT GGT Val Gly 155</td><td colspan="14"> ATT TAT CGT TTC CAG CAA TTT TAT ATT TGG GGT TTA TAT CTT</td><td rowspan="2"> 2511</td>

<td colspan="2"> He Tyr</td><td> Arg</td><td> Phe 160</td><td> Gin</td><td> Gin</td><td colspan="2"> Phe Tyr</td><td> He 165</td><td colspan="3"> Trp Gly Leu</td><td> Tyr</td><td> Leu 170</td>

<td> TTC</td><td> ATT</td><td> CCC</td><td> TTT</td><td> TAT</td><td> TGG</td><td> TTT</td><td> CTC</td><td> TAC</td><td> GAT</td><td> GTC</td><td> TAC</td><td> CTA</td><td> GTG</td><td> CTT</td><td> AAT</td><td> 2559</td>

<td> Phe</td><td> He</td><td> Pro</td><td> Phe</td><td> Tyr</td><td> Trp</td><td> Phe</td><td> Leu</td><td colspan="2"> Tyr Asp</td><td> Val</td><td> Tyr</td><td> Leu</td><td> Val</td><td> Leu</td><td> Asn</td><td></td>

<td></td><td></td><td></td><td></td><td> 175</td><td></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> AAA</td><td> GGC</td><td> AAA</td><td> TAT</td><td> CAC</td><td> GAC</td><td> CAT</td><td> AAA</td><td> ATT</td><td> CCT</td><td> CCT</td><td> TTC</td><td> CAG</td><td> CCC</td><td> CTA</td><td> GAA</td><td> 2607</td>

<td> Lys</td><td> Gly</td><td> Lys</td><td> Tyr</td><td> His</td><td> Asp</td><td> His</td><td> Lys</td><td> He</td><td> Pro</td><td> Pro</td><td> Phe</td><td> Gin</td><td> Pro</td><td> Leu</td><td> Glu</td><td></td>

<td></td><td></td><td></td><td> 190</td><td></td><td></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> TTA</td><td> GCT</td><td> AGT</td><td> TTG</td><td> CTA</td><td> GGG</td><td> ATT</td><td> AAG</td><td> CTA</td><td> TTA</td><td> TGG</td><td> CTC</td><td> GGC</td><td> TAC</td><td> GTT</td><td> TTC</td><td> 2655</td>

<td> Leu</td><td> Ala</td><td> Ser</td><td> Leu</td><td> Leu</td><td> Gly</td><td> He</td><td> Lys</td><td> Leu</td><td> Leu</td><td> Trp</td><td> Leu</td><td> Gly</td><td> Tyr</td><td> Val</td><td> Phe</td><td></td>

<td></td><td></td><td> 205</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> GGC</td><td> TTA</td><td> CCT</td><td> CTG</td><td> GCT</td><td> CTG</td><td> GGC</td><td> TTT</td><td> TCC</td><td> ATT</td><td> CCT</td><td> GAA</td><td> GTA</td><td> TTA</td><td> ATT</td><td> GGT</td><td> 2703</td>

<td> Gly</td><td> Leu</td><td> Pro</td><td> Leu</td><td> Ala</td><td> Leu</td><td> Gly</td><td> Phe</td><td> Ser</td><td> He</td><td> Pro</td><td> Glu</td><td> Val</td><td> Leu</td><td> He</td><td rowspan="2"> Gly</td><td></td>

<td></td><td> 220</td><td></td><td></td><td></td><td></td><td> 225</td><td></td><td></td><td></td><td></td><td> 230</td><td></td><td></td><td></td><td></td>

<td> GCT</td><td> TCG</td><td> GTA</td><td> ACC</td><td> TAT</td><td> ATG</td><td> ACC</td><td> TAT</td><td> GGC</td><td> ATC</td><td> GTG</td><td> GTT</td><td> TGC</td><td> ACC</td><td> ATC</td><td> TTT</td><td> 2751</td>

<td> Ala</td><td> Ser</td><td> Val</td><td> Thr</td><td> Tyr</td><td> Met</td><td> Thr</td><td> Tyr</td><td> Gly</td><td> He</td><td> Val</td><td> Val</td><td> Cys</td><td> Thr</td><td> lie</td><td> Phe</td><td></td>

<td> 235</td><td></td><td></td><td></td><td></td><td> 240</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> ATG</td><td> CTG</td><td> GCC</td><td> CAT</td><td> GTG</td><td> TTG</td><td> GAA</td><td> TCA</td><td> ACT</td><td> GAA</td><td> TTT</td><td> CTC</td><td> ACC</td><td> CCC</td><td> GAT</td><td> GGT</td><td> 2799</td>

<td> Met</td><td> Leu</td><td> Ala</td><td> His</td><td> Val</td><td> Leu</td><td> Glu</td><td> Ser</td><td> Thr</td><td> Glu</td><td> Phe</td><td> Leu</td><td> Thr</td><td> Pro</td><td> Asp</td><td rowspan="2"> Gly</td><td></td>

<td></td><td></td><td></td><td></td><td> 255</td><td></td><td></td><td></td><td></td><td> 260</td><td></td><td></td><td></td><td></td><td> 265</td><td></td>

<td> GAA</td><td> TCC</td><td> GGT</td><td> GCC</td><td> ATT</td><td> GAT</td><td> GAC</td><td> GAG</td><td> TGG</td><td> GCT</td><td> ATT</td><td> TGC</td><td> CAA</td><td> ATT</td><td> CGT</td><td> ACC</td><td> 2847</td>

<td> Glu</td><td> Ser</td><td> Gly</td><td> Ala</td><td> He</td><td> Asp</td><td> Asp</td><td> Glu</td><td> Trp</td><td> Ala</td><td> He</td><td> Cys</td><td> Gin</td><td> He</td><td> Arg</td><td> Thr</td><td></td>

<td></td><td></td><td></td><td> 270</td><td></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> ACG</td><td> GCC</td><td> AAT</td><td> TTT</td><td> GCC</td><td> ACC</td><td> AAT</td><td> AAT</td><td> CCC</td><td> TTT</td><td> TGG</td><td> AAC</td><td> TGG</td><td> TTT</td><td> TGT</td><td> GGC</td><td> 2895</td>

<td> Thr</td><td> Ala</td><td> Asn</td><td> Phe</td><td> Ala</td><td> Thr</td><td> Asn</td><td> Asn</td><td> Pro</td><td> Phe</td><td> Trp</td><td> Asn</td><td> Trp</td><td> Phe</td><td rowspan="2"> Cys</td><td rowspan="2"> Gly</td><td></td>

<td></td><td></td><td> 285</td><td></td><td></td><td></td><td></td><td> 290</td><td></td><td></td><td></td><td></td><td> 295</td><td></td><td></td>

<td> GGT</td><td> TTA</td><td> AAT</td><td> CAC</td><td> CAA</td><td> GTT</td><td> ACC</td><td> CAC</td><td> CAT</td><td> CTT</td><td> TTC</td><td> CCC</td><td> AAT</td><td> ATT</td><td> TGT</td><td> CAT</td><td> 2943</td>

<td> Gly</td><td> Leu</td><td> Asn</td><td> His</td><td> Gin</td><td> Val</td><td> Thr</td><td> His</td><td> His</td><td> Leu</td><td> Phe</td><td> Pro</td><td> Asn</td><td> He</td><td rowspan="2"> Cys</td><td> His</td><td></td>

<td></td><td> 300</td><td></td><td></td><td></td><td></td><td> 305</td><td></td><td></td><td></td><td></td><td> 310</td><td></td><td></td><td></td><td></td>

<td> ATT</td><td> CAC</td><td> TAT</td><td> CCC</td><td> CAA</td><td> TTG</td><td> GAA</td><td> AAT</td><td> ATT</td><td> ATT</td><td> AAG</td><td> GAT</td><td> GTT</td><td> TGC</td><td> CAA</td><td> GAG</td><td> 2991</td>

<td> He</td><td> His</td><td> Tyr</td><td> Pro</td><td> Gin</td><td> Leu</td><td> Glu</td><td> Asn</td><td> He</td><td> He</td><td> Lys</td><td> Asp</td><td> Val</td><td> Cys</td><td> Gin</td><td> Glu</td><td></td>

<td> 315</td><td></td><td></td><td></td><td></td><td> 320</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> TTT</td><td> GGT</td><td> GTG</td><td> GAA</td><td> TAT</td><td> AAA</td><td> GTT</td><td> TAT</td><td> CCC</td><td> ACC</td><td> TTC</td><td> AAA</td><td> GCG</td><td> GCG</td><td> ATC</td><td> GCC</td><td> 3039</td>

<td> Phe</td><td> Gly</td><td> Val</td><td> Glu</td><td> Tyr</td><td> Lys</td><td> Val</td><td> Tyr</td><td> Pro</td><td> Thr</td><td> Phe</td><td> Lys</td><td> Ala</td><td> Ala</td><td> He</td><td> Ala</td><td></td>

<td></td><td></td><td></td><td></td><td> 335</td><td></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> TCT</td><td> AAC</td><td> TAT</td><td> CGC</td><td> TGG</td><td> CTA</td><td> GAG</td><td> GCC</td><td> ATG</td><td> GGC</td><td> AAA</td><td> GCA</td><td> TCG</td><td colspan="3"> TGACATTGCC</td><td> 3088</td>

<td> Ser</td><td> Asn</td><td> Tyr</td><td> Arg</td><td> Trp</td><td> Leu</td><td> Glu</td><td> Ala</td><td> Met</td><td> Gly</td><td> Lys</td><td> Ala</td><td> Ser</td><td></td><td></td><td></td><td></td>

350 355 360

TTGGGATTGA AGCAAAATGG CAAAATCCCT CGTAAATCTA TGATCGAAGC CTTTCTGTTG 3148

CCCGCCGACC AAATCCCCGA TGCTGACCAA AGGTTGATGT TGGCATTGCT CCAAACCCAC 3208

CA 02207906 1997-06-16

PCT/IB95/01167

WO 96/21022

<td> TTTGAGGGGG</td><td> TTCATTGGCC</td><td> GCAGTTTCAA</td><td> GCTGACCTAG</td><td> GAGGCAAAGA</td><td> TTGGGTGATT</td><td> 3268</td>

<td> TTGCTCAAAT</td><td> CCGCTGGGAT</td><td> ATTGAAAGGC</td><td> TTCACCACCT</td><td> TTGGTTTCTA</td><td> CCCTGCTCAA</td><td> 3328</td>

<td> TGGGAAGGAC</td><td> AAACCGTCAG</td><td> AATTGTTTAT</td><td> TCTGGTGACA</td><td> CCATCACCGA</td><td> CCCATCCATG</td><td> 3388</td>

<td> TGGTCTAACC</td><td> CAGCCCTGGC</td><td> CAAGGCTTGG</td><td> ACCAAGGCCA</td><td> TGCAAATTCT</td><td> CCACGAGGCT</td><td> 3448</td>

<td> AGGCCAGAAA</td><td> AATTATATTG</td><td> GCTCCTGATT</td><td> TCTTCCGGCT</td><td> ATCGCACCTA</td><td> CCGATTTTTG</td><td> 3508</td>

<td> AGCATTTTTG</td><td> CCAAGGAATT</td><td> CTATCCCCAC</td><td> TATCTCCATC</td><td> CCACTCCCCC</td><td> GCCTGTACAA</td><td> 3568</td>

<td> AATTTTATCC</td><td> ATCAGCTAGC</td><td></td><td></td><td></td><td></td><td> 3588</td>

(2) INFORMATION FOR SEQ ID NO:2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 359 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein

<td></td><td colspan="4"> (xi) SEQUENCE</td><td colspan="3"> DESCRIPTION:</td><td colspan="2"> SEQ ID</td><td> NO :2</td><td></td><td></td><td></td><td></td><td></td>

<td> Met 1</td><td> Leu</td><td> Thr</td><td> Ala</td><td> Glu 5</td><td> Arg</td><td> He</td><td> Lys</td><td> Phe</td><td> Thr 10</td><td> Gin</td><td> Lys</td><td> Arg</td><td> Gly</td><td> Phe 15</td><td> Arg</td>

<td> Arg</td><td> Val</td><td> Leu</td><td> Asn 20</td><td> Gin</td><td> Arg</td><td> Val</td><td> Asp</td><td> Ala 25</td><td> Tyr</td><td> Phe</td><td> Ala</td><td> Glu</td><td> His 30</td><td> Gly</td><td> Leu</td>

<td> Thr</td><td> Gin</td><td> Arg 35</td><td> Asp</td><td> Asn</td><td> Pro</td><td> Ser</td><td> Met 40</td><td> Tyr</td><td> Leu</td><td> Lys</td><td> Thr</td><td> Leu 45</td><td> He</td><td> He</td><td> Val</td>

<td> Leu</td><td> Trp 50</td><td> Leu</td><td> Phe</td><td> Ser</td><td> Ala</td><td> Trp 55</td><td> Ala</td><td> Phe</td><td> Val</td><td> Leu</td><td> Phe 60</td><td> Ala</td><td> Pro</td><td> Val</td><td> He</td>

<td> Phe 65</td><td> Pro</td><td> Val</td><td> Arg</td><td> Leu</td><td> Leu 70</td><td> Gly</td><td> Cys</td><td> Met</td><td> Val</td><td> Leu 75</td><td> Ala</td><td> He</td><td> Ala</td><td> Leu</td><td> Ala 80</td>

<td> Ala</td><td> Phe</td><td> Ser</td><td> Phe</td><td> Asn 85</td><td> Val</td><td> Gly</td><td> His</td><td> Asp</td><td> Ala 90</td><td> Asn</td><td> His</td><td> Asn</td><td> Ala</td><td> Tyr 95</td><td> Ser</td>

<td> Ser</td><td> Asn</td><td> Pro</td><td> His 100</td><td> He</td><td> Asn</td><td> Arg</td><td> Val</td><td> Leu 105</td><td> Gly</td><td> Met</td><td> Thr</td><td> Tyr</td><td> Asp 110</td><td> Phe</td><td> Val</td>

<td> Gly</td><td> Leu</td><td> Ser 115</td><td> Ser</td><td> Phe</td><td> Leu</td><td> Trp</td><td> Arg 120</td><td> Tyr</td><td> Arg</td><td> His</td><td> Asn</td><td> Tyr 125</td><td> Leu</td><td> His</td><td> His</td>

<td> Thr</td><td> Tyr 130</td><td> Thr</td><td> Asn</td><td> He</td><td> Leu</td><td> Gly 135</td><td> His</td><td> Asp</td><td> Val</td><td> Glu</td><td> He 140</td><td> His</td><td> Gly</td><td> Asp</td><td> Gly</td>

<td> Ala 145</td><td> Val</td><td> Arg</td><td> Met</td><td> Ser</td><td> Pro 150</td><td> Glu</td><td> Gin</td><td> Glu</td><td> His</td><td> Val 155</td><td> Gly</td><td> He</td><td> Tyr</td><td> Arg</td><td> Phe 160</td>

CA 02207906 1997-06-16

WO 96/21022 PCT/IB95/01167

<td> Gin</td><td> Gin</td><td> Phe</td><td> Tyr</td><td> He</td><td> Trp</td><td> Gly</td><td> Leu</td><td> Tyr</td><td> Leu</td><td> Phe</td><td> He</td><td> Pro</td><td> Phe</td><td> Tyr</td><td> Trp</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> Phe</td><td> Leu</td><td> Tyr</td><td> Asp</td><td> Val</td><td> Tyr</td><td> Leu</td><td> Val</td><td> Leu</td><td> Asn</td><td> Lys</td><td> Gly</td><td> Lys</td><td> Tyr</td><td> His</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> His</td><td> Lys</td><td> He</td><td> Pro</td><td> Pro</td><td> Phe</td><td> Gin</td><td> Pro</td><td> Leu</td><td> Glu</td><td> Leu</td><td> Ala</td><td> Ser</td><td> Leu</td><td> Leu</td><td rowspan="2"> Gly</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> He</td><td> Lys</td><td> Leu</td><td> Leu</td><td> Trp</td><td> Leu</td><td> Gly</td><td> Tyr</td><td> Val</td><td> Phe</td><td> Gly</td><td> Leu</td><td> Pro</td><td> Leu</td><td> Ala</td><td> Leu</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> Gly</td><td> Phe</td><td> Ser</td><td> He</td><td> Pro</td><td> Glu</td><td> Val</td><td> Leu</td><td> He</td><td> Gly</td><td> Ala</td><td> Ser</td><td> Val</td><td> Thr</td><td rowspan="2"> Tyr</td><td> Met</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> 240</td>

<td> Thr</td><td> Tyr</td><td> Gly</td><td> He</td><td> Val</td><td> Val</td><td> Cys</td><td> Thr</td><td> He</td><td> Phe</td><td> Met</td><td> Leu</td><td> Ala</td><td> His</td><td> Val</td><td> Leu</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> Glu</td><td> Ser</td><td> Thr</td><td> Glu</td><td> Phe</td><td> Leu</td><td> Thr</td><td> Pro</td><td> Asp</td><td> Gly</td><td> Glu</td><td> Ser</td><td> Gly</td><td> Ala</td><td> He</td><td rowspan="2"> Asp</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> Asp</td><td> Glu</td><td> Trp</td><td> Ala</td><td> He</td><td> Cys</td><td> Gin</td><td> He</td><td> Arg</td><td> Thr</td><td> Thr</td><td> Ala</td><td> Asn</td><td> Phe</td><td> Ala</td><td> Thr</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> Asn</td><td> Pro</td><td> Phe</td><td> Trp</td><td> Asn</td><td> Trp</td><td> Phe</td><td> Cys</td><td> Gly</td><td> Gly</td><td> Leu</td><td> Asn</td><td> His</td><td> Gin</td><td> Val</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> Thr</td><td> His</td><td> His</td><td> Leu</td><td> Phe</td><td> Pro</td><td> Asn</td><td> He</td><td> Cys</td><td> His</td><td> He</td><td> His</td><td> Tyr</td><td> Pro</td><td> Gin</td><td> Leu</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> Glu</td><td> Asn</td><td> He</td><td> He</td><td> Lys</td><td> Asp</td><td> Val</td><td> Cys</td><td> Gin</td><td> Glu</td><td> Phe</td><td> Gly</td><td> Val</td><td> Glu</td><td> Tyr</td><td rowspan="2"> Lys</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> Val</td><td> Tyr</td><td> Pro</td><td> Thr</td><td> Phe</td><td> Lys</td><td> Ala</td><td> Ala</td><td> He</td><td> Ala</td><td> Ser</td><td> Asn</td><td> Tyr</td><td> Arg</td><td> Trp</td><td> Leu</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> Glu</td><td> Ala</td><td> Met</td><td> Gly</td><td> Lys</td><td> Ala</td><td> Ser</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

355 (2) INFORMATION FOR SEQ ID NO:3:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1884 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: both (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:

AGCTTCACTT CGGTTTTATA TTGTGACCAT GGTTCCCAGG CATCTGCTCT AGGGAGTTTT 60

TCCGCTGCCT TTAGAGAGTA TTTTCTCCAA GTCGGCTAAC TCCCCCATTT TTAGGCAAAA 120

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<td> TCATATACAG</td><td> ACTATCCCAA</td><td> TATTGCCAGA</td><td> GCTTTGATGA</td><td> CTCACTGTAG</td><td> AAGGCAGACT</td><td> 180</td>

<td> AAAATTCTAG</td><td> CAATGGACTC</td><td> CCAGTTGGAA</td><td> TAAATTTTTA</td><td> GTCTCCCCCG</td><td> GCGCTGGAGT</td><td> 240</td>

<td> TTTTTTGTAG</td><td> TTAATGGCGG</td><td> TATAATGTGA</td><td> AAGTTTTTTA</td><td> TCTATTTAAA</td><td> TTTATAAATG</td><td> 300</td>

<td> CTAACAGCGG</td><td> AAAGAATTAA</td><td> ATTTACCCAG</td><td> AAACGGGGGT</td><td> TTCGTCGGGT</td><td> ACTAAACCAA</td><td> 360</td>

<td> CGGGTGGATG</td><td> CCTACTTTGC</td><td> CGAGCATGGC</td><td> CTGACCCAAA</td><td> GGGATAATCC</td><td> CTCCATGTAT</td><td> 420</td>

<td> CTGAAAACCC</td><td> TGATTATTGT</td><td> GCTCTGGTTG</td><td> TTTTCCGCTT</td><td> GGGCCTTTGT</td><td> GCTTTTTGCT</td><td> 480</td>

<td> CCAGTTATTT</td><td> TTCCGGTGCG</td><td> CCTACTGGGT</td><td> TGTATGGTTT</td><td> TGGCGATCGC</td><td> CTTGGCGGCC</td><td> 540</td>

<td> TTTTCCTTCA</td><td> ATGTCGGCCA</td><td> CGATGCCAAC</td><td> CACAATGCCT</td><td> ATTCCTCCAA</td><td> TCCCCACATC</td><td> 600</td>

<td> AACCGGGTTC</td><td> TGGGCATGAC</td><td> CTACGATTTT</td><td> GTCGGGTTAT</td><td> CTAGTTTTCT</td><td> TTGGCGCTAT</td><td> 660</td>

<td> CGCCACAACT</td><td> ATTTGCACCA</td><td> CACCTACACC</td><td> AATATTCTTG</td><td> GCCATGACGT</td><td> GGAAATCCAT</td><td> 720</td>

<td> GGAGATGGCG</td><td> CAGTACGTAT</td><td> GAGTCCTGAA</td><td> CAAGAACATG</td><td> TTGGTATTTA</td><td> TCGTTTCCAG</td><td> 780</td>

<td> CAATTTTATA</td><td> TTTGGGGTTT</td><td> ATATCTTTTC</td><td> ATTCCCTTTT</td><td> ATTGGTTTCT</td><td> CTACGATGTC</td><td> 840</td>

<td> TACCTAGTGC</td><td> TTAATAAAGG</td><td> CAAATATCAC</td><td> GACCATAAAA</td><td> TTCCTCCTTT</td><td> CCAGCCCCTA</td><td> 900</td>

<td> GAATTAGCTA</td><td> GTTTGCTAGG</td><td> GATTAAGCTA</td><td> TTATGGCTCG</td><td> GCTACGTTTT</td><td> CGGCTTACCT</td><td> 960</td>

<td> CTGGCTCTGG</td><td> GCTTTTCCAT</td><td> TCCTGAAGTA</td><td> TTAATTGGTG</td><td> CTTCGGTAAC</td><td> CTATATGACC</td><td> 1020</td>

<td> TATGGCATCG</td><td> TGGTTTGCAC</td><td> CATCTTTATG</td><td> CTGGCCCATG</td><td> TGTTGGAATC</td><td> AACTGAATTT</td><td> 1080</td>

<td> CTCACCCCCG</td><td> ATGGTGAATC</td><td> CGGTGCCATT</td><td> GATGACGAGT</td><td> GGGCTATTTG</td><td> CCAAATTCGT</td><td> 1140</td>

<td> ACCACGGCCA</td><td> ATTTTGCCAC</td><td> CAATAATCCC</td><td> TTTTGGAACT</td><td> GGTTTTGTGG</td><td> CGGTTTAAAT</td><td> 1200</td>

<td> CACCAAGTTA</td><td> CCCACCATCT</td><td> TTTCCCCAAT</td><td> ATTTGTCATA</td><td> TTCACTATCC</td><td> CCAATTGGAA</td><td> 1260</td>

<td> AATATTATTA</td><td> AGGATGTTTG</td><td> CCAAGAGTTT</td><td> GGTGTGGAAT</td><td> ATAAAGTTTA</td><td> TCCCACCTTC</td><td> 1320</td>

<td> AAAGCGGCGA</td><td> TCGCCTCTAA</td><td> CTATCGCTGG</td><td> CTAGAGGCCA</td><td> TGGGCAAAGC</td><td> ATCGTGACAT</td><td> 1380</td>

<td> TGCCTTGGGA</td><td> TTGAAGCAAA</td><td> ATGGCAAAAT</td><td> CCCTCGTAAA</td><td> TCTATGATCG</td><td> AAGCCTTTCT</td><td> 1440</td>

<td> GTTGCCCGCC</td><td> GACCAAATCC</td><td> CCGATGCTGA</td><td> CCAAAGGTTG</td><td> ATGTTGGCAT</td><td> TGCTCCAAAC</td><td> 1500</td>

<td> CCACTTTGAG</td><td> GGGGTTCATT</td><td> GGCCGCAGTT</td><td> TCAAGCTGAC</td><td> CTAGGAGGCA</td><td> AAGATTGGGT</td><td> 1560</td>

<td> GATTTTGCTC</td><td> AAATCCGCTG</td><td> GGATATTGAA</td><td> AGGCTTCACC</td><td> ACCTTTGGTT</td><td> TCTACCCTGC</td><td> 1620</td>

<td> TCAATGGGAA</td><td> GGACAAACCG</td><td> TCAGAATTGT</td><td> TTATTCTGGT</td><td> GACACCATCA</td><td> CCGACCCATC</td><td> 1680</td>

<td> CATGTGGTCT</td><td> AACCCAGCCC</td><td> TGGCCAAGGC</td><td> TTGGACCAAG</td><td> GCCATGCAAA</td><td> TTCTCCACGA</td><td> 1740</td>

<td> GGCTAGGCCA</td><td> GAAAAATTAT</td><td> ATTGGCTCCT</td><td> GATTTCTTCC</td><td> GGCTATCGCA</td><td> CCTACCGATT</td><td> 1800</td>

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<td> TTTGAGCATT TTTGCCAAGG AATTCTATCC CCACTATCTC CATCCCACTC CCCCGCCTGT</td><td> 18G0</td>

<td> ACAAAATTTT ATCCATCAGC TAGC</td><td> 1884</td>

<td> (2) INFORMATION FOR SEQ ID NO:4:</td><td></td>

<td> (i) SEQUENCE CHARACTERISTICS:</td><td></td>

<td> (A) LENGTH: 1685 base pairs</td><td></td>

<td> (B) TYPE: nucleic acid</td><td></td>

<td> (C) STRANDEDNESS: both</td><td></td>

<td> (D) TOPOLOGY: linear</td><td></td>

<td> (ii) MOLECULE TYPE: DNA (genomic)</td><td></td>

<td> (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:</td><td></td>

<td> AATATCTGCC TACCCTCCCA AAGAGAGTAG TCATTTTTCA TCAATGGCTG CTCAAATCAA</td><td> 60</td>

<td> GAAATACATT ACCTCAGATG AACTCAAGAA CCACGATAAA CCCGGAGATC TATGGATCTC</td><td> 120</td>

<td> GATTCAAGGG AAAGCCTATG ATGTTTCGGA TTGGGTGAAA GACCATCCAG GTGGCAGCTT</td><td> 180</td>

<td> TCCCTTGAAG AGTCTTGCTG GTCAAGAGGT AACTGATGCA TTTGTTGCAT TCCATCCTGC</td><td> 240</td>

<td> CTCTACATGG AAGAATCTTG ATAAGTTTTT CACTGGGTAT TATCTTAAAG ATTACTCTGT</td><td> 300</td>

<td> TTCTGAGGTT TCTAAAGATT ATAGGAAGCT TGTGTTTGAG TTTTCTAAAA TGGGTTTGTA</td><td> 360</td>

<td> TGACAAAAAA GGTCATATTA TGTTTGCAAC TTTGTGCTTT ATAGCAATGC TGTTTGCTAT</td><td> 420</td>

<td> GAGTGTTTAT GGGGTTTTGT TTTGTGAGGG TGTTTTGGTA CATTTGTTTT CTGGGTGTTT</td><td> 480</td>

<td> GATGGGGTTT CTTTGGATTC AGAGTGGTTG GATTGGACAT GATGCTGGGC ATTATATGGT</td><td> 540</td>

<td> AGTGTCTGAT TCAAGGCTTA ATAAGTTTAT GGGTATTTTT GCTGCAAATT GTCTTTCAGG</td><td> 600</td>

<td> AATAAGTATT GGTTGGTGGA AATGGAACCA TAATGCACAT CACATTGCCT GTAATAGCCT</td><td> 660</td>

<td> TGAATATGAC CCTGATTTAC AATATATACC ATTCCTTGTT GTGTCTTCCA AGTTTTTTGG</td><td> 720</td>

<td> TTCACTCACC TCTCATTTCT ATGAGAAAAG GTTGACTTTT GACTCTTTAT CAAGATTCTT</td><td> 780</td>

<td> TGTAAGTTAT CAACATTGGA CATTTTACCC TATTATGTGT GCTGCTAGGC TCAATATGTA</td><td> 840</td>

<td> TGTACAATCT CTCATAATGT TGTTGACCAA GAGAAATGTG TCCTATCGAG CTCAGGAACT</td><td> 900</td>

<td> CTTGGGATGC CTAGTGTTCT CGATTTGGTA CCCGTTGCTT GTTTCTTGTT TGCCTAATTG</td><td> 960</td>

<td> GGGTGAAAGA ATTATGTTTG TTATTGCAAG TTTATCAGTG ACTGGAATGC AACAAGTTCA</td><td> 1020</td>

<td> GTTCTCCTTG AACCACTTCT CTTCAAGTGT TTATGTTGGA AAGCCTAAAG GGAATAATTG</td><td> 1080</td>

<td> GTTTGAGAAA CAAACGGATG GGACACTTGA CATTTCTTGT CCTCCTTGGA TGGATTGGTT</td><td> 1140</td>

<td> TCATGGTGGA TTGCAATTCC AAATTGAGCA TCATTTGTTT CCCAAGATGC CTAGATGCAA</td><td> 1200</td>

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<td> CCTTAGGAAA</td><td> ATCTCGCCCT</td><td> ACGTGATCGA</td><td> GTTATGCAAG</td><td> AAACATAATT</td><td> TGCCTTACAA</td><td> 1260</td>

<td> TTATGCATCT</td><td> TTCTCCAAGG</td><td> CCAATGAAAT</td><td> GACACTCAGA</td><td> ACATTGAGGA</td><td> ACACAGCATT</td><td> 1320</td>

<td> GCAGGCTAGG</td><td> GATATAACCA</td><td> AGCCGCTCCC</td><td> GAAGAATTTG</td><td> GTATGGGAAG</td><td> CTCTTCACAC</td><td> 1380</td>

<td> TCATGGTTAA</td><td> AATTACCCTT</td><td> AGTTCATGTA</td><td> ATAATTTGAG</td><td> ATTATGTATC</td><td> TCCTATGTTT</td><td> 1440</td>

<td> GTGTCTTGTC</td><td> TTGGTTCTAC</td><td> TTGTTGGAGT</td><td> CATTGCAACT</td><td> TGTCTTTTAT</td><td> GGTTTATTAG</td><td> 1500</td>

<td> ATGTTTTTTA</td><td> ATATATTTTA</td><td> GAGGTTTTGC</td><td> TTTCATCTCC</td><td> ATTATTGATG</td><td> AATAAGGAGT</td><td> 1560</td>

<td> TGCATATTGT</td><td> CAATTGTTGT</td><td> GCTCAATATC</td><td> TGATATTTTG</td><td> GAATGTACTT</td><td> TGTACCACTG</td><td> 1620</td>

<td> tgttttcagt</td><td> TGAAGCTCAT</td><td> GTGTACTTCT</td><td> ATAGACTTTG</td><td> TTTAAATGGT</td><td> TATGTCATGT</td><td> 1680</td>

<td> ΤΑΤΤΤ</td><td></td><td></td><td></td><td></td><td></td><td> 1685</td>

(2) INFORMATION FOR SEQ ID NO:5:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 448 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:

<td> Met 1</td><td> Ala</td><td> Ala</td><td> Gin</td><td> lie 5</td><td> Lys</td><td> Lys</td><td> Tyr</td><td> He</td><td> Thr 10</td><td> Ser</td><td> Asp</td><td> Glu</td><td> Leu</td><td> Lys 15</td><td> Asn</td>

<td> His</td><td> Asp</td><td> Lys</td><td> Pro 20</td><td> Gly</td><td> Asp</td><td> Leu</td><td> Trp</td><td> He 25</td><td> Ser</td><td> He</td><td> Gin</td><td> Gly</td><td> Lys 30</td><td> Ala</td><td> Tyr</td>

<td> Asp</td><td> Val</td><td> Ser 35</td><td> Asp</td><td> Trp</td><td> Val</td><td> Lys</td><td> Asp 40</td><td> His</td><td> Pro</td><td> Gly</td><td> Gly</td><td> Ser 45</td><td> Phe</td><td> Pro</td><td> Leu</td>

<td> Lys</td><td> Ser 50</td><td> Leu</td><td> Ala</td><td> Gly</td><td> Gin</td><td> Glu 55</td><td> Val</td><td> Thr</td><td> Asp</td><td> Ala</td><td> Phe 60</td><td> Val</td><td> Ala</td><td> Phe</td><td> His</td>

<td> Pro 65</td><td> Ala</td><td> Ser</td><td> Thr</td><td> Trp</td><td> Lys 70</td><td> Asn</td><td> Leu</td><td> Asp</td><td> Lys</td><td> Phe 75</td><td> Phe</td><td> Thr</td><td> Gly</td><td> Tyr</td><td> Tyr 80</td>

<td> Leu</td><td> Lys</td><td> Asp</td><td> Tyr</td><td> Ser 85</td><td> Val</td><td> Ser</td><td> Glu</td><td> Val</td><td> Ser 90</td><td> Lys</td><td> Asp</td><td> Tyr</td><td> Arg</td><td> Lys 95</td><td> Leu</td>

<td> Val</td><td> Phe</td><td> Glu</td><td> Phe 100</td><td> Ser</td><td> Lys</td><td> Met</td><td> Gly</td><td> Leu 105</td><td> Tyr</td><td> Asp</td><td> Lys</td><td> Lys</td><td> Gly 110</td><td> His</td><td> He</td>

<td> Met</td><td> Phe</td><td> Ala 115</td><td> Thr</td><td> Leu</td><td> Cys</td><td> Phe</td><td> He 120</td><td> Ala</td><td> Met</td><td> Leu</td><td> Phe</td><td> Ala 125</td><td> Met</td><td> Ser</td><td> Val</td>

<td> Tyr</td><td> Gly 130</td><td> Val</td><td> Leu</td><td> Phe</td><td> Cys</td><td> Glu 135</td><td> Gly</td><td> Val</td><td> Leu</td><td> Val</td><td> His 140</td><td> Leu</td><td> Phe</td><td> Ser</td><td> Gly</td>

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<td> Cys 145</td><td> Leu</td><td> Met</td><td> Gly</td><td> Phe</td><td> Leu 150</td><td> Trp</td><td> He</td><td> Gin</td><td> Ser</td><td> Gly 155</td><td> Trp</td><td> He</td><td> Gly</td><td> His</td><td> Asp 160</td>

<td> Ala</td><td> Gly</td><td> His</td><td> Tyr</td><td> Met 165</td><td> Val</td><td> Val</td><td> Ser</td><td> Asp</td><td> Ser 170</td><td> Arg</td><td> Leu</td><td> Asn</td><td> Lys</td><td> Phe 175</td><td> Met</td>

<td> Gly</td><td> He</td><td> Phe</td><td> Ala 180</td><td> Ala</td><td> Asn</td><td> Cys</td><td> Leu</td><td> Ser 185</td><td> Gly</td><td> He</td><td> Ser</td><td> He</td><td> Gly 190</td><td> Trp</td><td> Trp</td>

<td> Lys</td><td> Trp</td><td> Asn 195</td><td> His</td><td> Asn</td><td> Ala</td><td> His</td><td> His 200</td><td> He</td><td> Ala</td><td> Cys</td><td> Asn</td><td> Ser 205</td><td> Leu</td><td> Glu</td><td> Tyr</td>

<td> Asp</td><td> Pro 210</td><td> Asp</td><td> Leu</td><td> Gin</td><td> Tyr</td><td> He 215</td><td> Pro</td><td> Phe</td><td> Leu</td><td> Val</td><td> Val 220</td><td> Ser</td><td> Ser</td><td> Lys</td><td> Phe</td>

<td> Phe 225</td><td> Gly</td><td> Ser</td><td> Leu</td><td> Thr</td><td> Ser 230</td><td> His</td><td> Phe</td><td> Tyr</td><td> Glu</td><td> Lys 235</td><td> Arg</td><td> Leu</td><td> Thr</td><td> Phe</td><td> Asp 240</td>

<td> Ser</td><td> Leu</td><td> Ser</td><td> Arg</td><td> Phe 245</td><td> Phe</td><td> Val</td><td> Ser</td><td> Tyr</td><td> Gin 250</td><td> His</td><td> Trp</td><td> Thr</td><td> Phe</td><td> Tyr 255</td><td> Pro</td>

<td> He</td><td> Met</td><td> Cys</td><td> Ala 260</td><td> Ala</td><td> Arg</td><td> Leu</td><td> Asn</td><td> Met 265</td><td> Tyr</td><td> Val</td><td> Gin</td><td> Ser</td><td> Leu 270</td><td> He</td><td> Met</td>

<td> Leu</td><td> Leu</td><td> Thr 275</td><td> Lys</td><td> Arg</td><td> Asn</td><td> Val</td><td> Ser 280</td><td> Tyr</td><td> Arg</td><td> Ala</td><td> Gin</td><td> Glu 285</td><td> Leu</td><td> Leu</td><td> Gly</td>

<td> Cys</td><td> Leu 290</td><td> Val</td><td> Phe</td><td> Ser</td><td> He</td><td> Trp 295</td><td> Tyr</td><td> Pro</td><td> Leu</td><td> Leu</td><td> Val 300</td><td> Ser</td><td> Cys</td><td> Leu</td><td> Pro</td>

<td> Asn 305</td><td> Trp</td><td> Gly</td><td> Glu</td><td> Arg</td><td> He 310</td><td> Met</td><td> Phe</td><td> Val</td><td> He</td><td> Ala 315</td><td> Ser</td><td> Leu</td><td> Ser</td><td> Val</td><td> Thr 320</td>

<td> Gly</td><td> Met</td><td> Gin</td><td> Gin</td><td> Val 325</td><td> Gin</td><td> Phe</td><td> Ser</td><td> Leu</td><td> Asn 330</td><td> His</td><td> Phe</td><td> Ser</td><td> Ser</td><td> Ser 335</td><td> Val</td>

<td> Tyr</td><td> Val</td><td> Gly</td><td> Lys 340</td><td> Pro</td><td> Lys</td><td> Gly</td><td> Asn</td><td> Asn 345</td><td> Trp</td><td> Phe</td><td> Glu</td><td> Lys</td><td> Gin 350</td><td> Thr</td><td> Asp</td>

<td> Gly</td><td> Thr</td><td> Leu 355</td><td> Asp</td><td> He</td><td> Ser</td><td> Cys</td><td> Pro 360</td><td> Pro</td><td> Trp</td><td> Met</td><td> Asp</td><td> Trp 365</td><td> Phe</td><td> His</td><td> Gly</td>

<td> Gly</td><td> Ser 370</td><td> Gin</td><td> Phe</td><td> Gin</td><td> He</td><td> Glu 375</td><td> His</td><td> His</td><td> Leu</td><td> Phe</td><td> Pro 380</td><td> Lys</td><td> Met</td><td> Pro</td><td> Arg</td>

<td> Cys 385</td><td> Asn</td><td> Leu</td><td> Arg</td><td> Lys</td><td> He 390</td><td> Ser</td><td> Pro</td><td> Tyr</td><td> Val</td><td> He 395</td><td> Glu</td><td> Leu</td><td> Cys</td><td> Lys</td><td> Lys 400</td>

<td> His</td><td> Asn</td><td> Leu</td><td> Pro</td><td> Tyr 405</td><td> Asn</td><td> Tyr</td><td> Ala</td><td> Ser</td><td> Phe 410</td><td> Ser</td><td> Lys</td><td> Ala</td><td> Asn</td><td> Glu 415</td><td> Met</td>

<td> Thr</td><td> Leu</td><td> Arg</td><td> Thr 420</td><td> Leu</td><td> Arg</td><td> Asn</td><td> Thr</td><td> Ala 425</td><td> Leu</td><td> Gin</td><td> Ala</td><td> Arg</td><td> Asp 430</td><td> He</td><td> Thr</td>

<td> Lys</td><td> Pro</td><td> Leu 435</td><td> Pro</td><td> Lys</td><td> Asn</td><td> Leu</td><td> Val 440</td><td> Trp</td><td> Glu</td><td> Ala</td><td> Leu</td><td> His 445</td><td> Thr</td><td> His</td><td> Gly</td>

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-54(2) INFORMATION FOR SEQ ID NO:6:

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(A) LENGTH: 8 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:

Trp lie Gly His Asp Ala Gly His 1 5 (2) INFORMATION FOR SEQ ID NO:7:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 8 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:

Asn Val Gly His Asp Ala Asn His 1 5 (2) INFORMATION FOR SEQ ID NO:8:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 8 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:

Val Leu Gly His Asp Cys Gly His 1 5 (2) INFORMATION FOR SEQ ID NO:9:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 8 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:

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-55Val He Ala His Glu Cys Gly His 1 5 (2) INFORMATION FOR SEQ ID NO:10:

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(A) LENGTH: 8 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:

Val He Gly His Asp Cys Ala His l 5 (2) INFORMATION FOR SEQ ID NO:11:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 8 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:

Val Val Gly His Asp Cys Gly His 1 5 (2) INFORMATION FOR SEQ ID NO:12:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 5 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:

His Asn Ala His His 1 5 (2) INFORMATION FOR SEQ ID NO:13:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 6 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear

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-56(ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:

His Asn Tyr Leu His His 1 5 (2) INFORMATION FOR SEQ ID NO:14:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 5 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:

His Arg Thr His His 1 5 (2) INFORMATION FOR SEQ ID NO:15:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 5 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:

His Arg Arg His His 1 5

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-57(2) INFORMATION FOR SEQ ID NO:16:

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(A) LENGTH: 5 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:

His Asp Arg His His 1 5 (2) INFORMATION FOR SEQ ID NO-.17:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 5 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17;

His Asp Gin His His 1 5 (2) INFORMATION FOR SEQ ID NO:18:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 5 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (Xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:

His Asp His His His 1 5 (2) INFORMATION FOR SEQ ID NO:19:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 5 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic)

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-58(xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:

His Asn His His His 1 5 (2) INFORMATION FOR SEQ ID NO:20:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 6 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:

Phe Gin Ile Glu His His 1 5 (2) INFORMATION FOR SEQ ID NO:21:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 6 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:

His Gin Val Thr His His 1 5 (2) INFORMATION FOR SEQ ID NO:22:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 5 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:

His Val lie His His 1 5 (2) INFORMATION FOR SEQ ID NO:23:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 5 amino acids (B) TYPE: amino acid

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-59(D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:

His Val Ala His His 1 5 (2) INFORMATION FOR SEQ ID NO:24:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 5 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:

His He Pro His His 1 5 (2) INFORMATION FOR SEQ ID NO:25:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 5 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:

His Val Pro His His 1 5