IL103407A - Isolated nucleic acid encoding a cyanobacterial delta6-desaturase and uses thereof

20 claims: 6 independent, 14 dependent

1
CLAIMS:1. An isolated nucleic acid encoding a cyanobacterial Δό-desaturase comprising the sequence of SEQ ID No:3,
2
The isolated nucleic acid of Claim 1 wherein said nucleic acid is contained in a vector.
6
A transgenic organism containing an isolated nucleic acid of any one of claims 1-5.
10
A method of producing a plant with increased gamma linolenic acid (GLA) content which comprises:(a) transforming a plant cell with the isolated nucleic acid of any one of Claims 3-5 and (b) regenerating a plant with increased GLA content from said plant cell.
12
A method of inducing production of gamma linolenic acid (GLA) in an organism deficient or lacking in GLA with comprises transforming said organism with the isolated nucleic acid of any one of claims 3-5.
14
The method of Claim 13 wherein said isolated nucleic acid encoding Δό-desaturase comprises nucleotides 317 to 1507 of SEQ. ID. No:l.
15
A method of inducing production of octadecatetraeonic acid in an organism deficient or lacking in gamma linolenic acid which comprises transforming said organism with the isolated nucleic acid of any of Claims 35־.
16
The method of Claim 15 wherein said organism is a bacterium or a plant.
17
A method of use of the isolated nucleic acid of any one of claims 3-5 to produce a plant with improved chilling resistance which comprises:(a) transforming a plant cell with the isolated nucleic acid of any one of claims 35־;and (b) regenerating said plant with improved chilling resistance from said transformed plant cell.

Independent claims9

353 paragraphs in 186 sections, as filed

ISOLATED NUCLEIC ACID ENCODING A CYANOBACTERIAL A6-DESATURASE AND USES THEREOF

Linoleic acid (18:2) (LA) is transformed into gamma linolenic acid (18:3) (GLA) by the enzyme Δ65 desaturase. When this enzyme, or the nucleic acid encoding it, is transferred into LA-producing calls, GLA is produced. The present invention provides a nucleic acid comprising the &6&#1470;desaturase gene. More specifically, the nucleic acid comprises the promoter, 3-0 coding region and termination regions of the Δ6desaturase gene. The present invention is further directed to recombinant constructions comprising a Δ6desaturase coding region in functional combination with heterologous regulatory sequences. The nucleic acids 15 and recombinant constructions of the instant invention are useful in the production of GLA in transgenic organisms.

Unsaturated fatty acids such as linoleic (Cie4B1&#1523;a) and a-linolenic (CxeA&#1524;'ia*18)acids are 20 essential dietary constituents that cannot be synthesized by vertebrates since vertebrate cells can introduce double bonds at the AB position of fatty acids but cannot Introduce additional double bonds between the ΔΒ double bond and the methyl-terminus of the fatty acid 25 chain. Because they are precursors of other products, linoleic and a-linolenic acids are essential fatty acids, and are usually obtained from plant sources. Linoleic acid can be converted by mammals into γlinolenic acid (GLA, CxbA6'*12&#1523;) which can in turn be 30 converted to arachidonic acid (20:4), a critically 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 chemical 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 Δ6-desaturase. A6-desaturase, an enzyme of about 359 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 Δ6-desaturase, allows the production of transgenic organisms which contain functional AB-desaturase and which produce GLA. in addition to allowing production of large amounts of GLA, the present invention provides new dietary sources of GLA.

The present invention is directed to an isolated Δβ-desaturase gene. Specifically, the isolated gene e

. 3 .

The present invention is directed to an isolated nucleic acid encoding a cyanobacterial A6-desaturase comprising the sequence of SEQ ID No:3. The isolated gene comprises the Δ&#972;-desaturase promoter, coding region, and termination region.

The present invention is further directed to an isolated nucleic acid encoding a cyanobacterial Δ&#972;-desaturase comprising the sequence of SEQ ID No:3, wherein said nucleic acid is contained in a vector. The said isolated nucleic acid contained in a vector may be operably linked to a promoter and/or a termination signal capable of effecting expression of the gene product of said isolated nucleic acid.

The said vectors may comprise a cyanobacterial Δ&#972;-desaturase coding region in functional combination with heterologous regulatory regions, i.e. elements not derived from the Δ&#972;-desaturase gene.

Transgenic plant cells and organisms comprising an isolated nucleic acid of the present invention, and progeny of such organisms, are also provided by the present invention.

The present invention further provides bacterial Δ&#972;-desaturase, isolated from the transgenic organism of the invention.

The present invention further provides a method of producing plants with increased gamma linolenic acid (GLA) content which comprises transforming a plant cell with an isolated nucleic acid of the present invention and regenerating a plant with increased GLA content from said plant cell.

A method of using of the isolated nucleic acid of the invention to produce a plant with improved chilling resistance is also provided by the present invention.

Fig. 1 depicts the hydropathy profiles of the deduced amino acid sequence of Svnechocystis Δ&#972;-desaturase (Panel A) and A12-desaturase (Panel B). Putative membrane spanning regions are indicated by solid bars. Hydrophobic index was calculated for a widow size of 19 amino acid residues [Kyle, et al. (1982) J. Molec. Biol. 1571.

Fig. 2 provides gas liquid chromatography profiles 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.

The present invention provides an isolated nucleic acid encoding Δ6-desaturase. To identify a nucleic acid encoding A6-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. synechocystls) 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 vector, e.g. a bacteriophage or cosmid vector, by any of a variety &#9633;f well-known methods which can be found in references such as Sambrook et al. (1989). Expression vectors containing the DNA of the present invention are specifically contemplated herein. DNA encoding a6-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 Δ6desaturase, 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 a6-desaturase.

As an example of the present invention, random DNA is isolated from the cyanobacteria Svnechocvstis Pasteur Culture Collection (PCC) 6803, American Type Culture Collection (ATCC) 27184, cloned into a cosmid vector, and introduced by transconjugation into the GLAdeficient cyanobacterium 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 wellknown to one of ordinary skill in the art as found, for example, in Sarobrook et .al. (1989).

In accordance with the present Invention, a DNA comprising a A6-desaturase gene has been isolated. More particularly, a 3.588 kilobase (kb) DNA comprising a Δ6desaturase gene has been isolated from the cyanobacteria Synechocystls. The nucleotide sequence of the 3.588 kb DNA was determined and is shown in SEQ ID NO .I. Open 3 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 A6&#1470;desaturase, the 3.588 kb fragment that confers aS-desaturase activity is cleaved into two subfragments, each of which contains only one open reading frame. Fragment ORF1 contains nucleotides 1&#1523; 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 (&#902;Μ542, 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)J are conjugated to wild-type Anabaena FCC 7120 by standard methods (see, for example, Wolk et al. (1984) Proc. Natl. Acad, Sci, USA Bl, 1561). Conjugated cells of Anabaena are identified as Neon green colonies on a brown background of dying non-conjugated cells after two weeks of growth on selective media (standard mineral media BQ11N + containing 30ug/ml of neomycin according to Rippka et al., (1979) J. Gen Microbiol, Ill, 1). The green colonies are selected and grown in selective liquid media (BG11N + with 15ug/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 0RF1 and ORF2 constructs.

For comparison, lipids are also extracted from wild-type cultures of Anabaena and Synechocystla. The fatty acid methyl esters are analysed 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,

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

<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> w</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 0RF2 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 (0RF2) within the 1884 bp fragment encodes Αβ-desaturase. The 1884 bp fragment is shown as SEQ ID NO;3. This is substantiated by the overall similarity of the hydropathy profiles between A6-desaturase and 412- &#1470;81 desaturase [Wada et al. (1990) Nature 347] as shown in Fig. 1 as (A) and (B), respectively.

Isolated nucleic acids encoding a6**desaturase 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 Anabaena a6-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 NOsl, or a restriction fragment or other DNA fragment thereof, including an oligonucleotide probe. Methods for cloning homologous genes by cross15 hybridization are known to the ordinarily skilled artisan and can be found, for example, in Sambrook (1989) and Beltz et al. (1983) Methods in Enzymology 100. 266.

Transgenic organisms which gain the function of

GLA production by introduction of DNA encoding adesaturase also gain the function of octadecatetraeonic acid (18.*4Aa1&#1524;’&#1524;a&#1524;iS) production. Octadecatetraeonic acid is present normally in fish oils and in some plant species of the Boraglnaceae family (Craig et al. [1964]

J, Amer, Oil Chem, Soc, 41, 209-211; Gross et al. [1976] Can. J. Plant Sei. 56 > 659-664). In the transgenic organisms of the present invention, octadecatetraenoic acid results from further desaturation of a-linolenic acid by A6־desaturase or desaturation of GLA by a15־ desaturase.

The 359 amino acids encoded by ORF2, i.e. the open reading frame encoding A6-desaturase, are shown as &#1470;9SEQ. ID NO:2. The present invention further contemplates other nucleotide sequences which encode the amino acids of SEQ ID NO:2. It is within the ken of the ordinarily skilled artisan to identify such sequences which result, for example, from the degeneracy of the genetic code. Furthermore, one of ordinary skill in the art can determine, by the gain of function analysis described hereinabove, smaller subfragments of the 1884 bp fragment containing 0RF2 which encode a6-desaturase.

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

In another aspect of the present invention, a vector containing the 1884 bp fragment or a smaller fragment containing the promoter, coding sequence and termination region of the 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 A6&#1470;desaturase are provided by this invention. Yet another aspect of this invention provides Isolated A6-desaturase, 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 Δβ-desaturase 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 DNA or RNA molecules engineered for controlled expression of a desired gene, i.e. the δ6-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, sal, USA, 1561-1565 and Bustos et al. (1991) J. Bacteriol, 174, 7525-7533, are also contemplated in accordance with the present invention. Sambrook et a^l, (1989), Goeddel, ed. (1990) Methods in Enzymology 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 A6-desaturase can be inserted and expressed. Such vectors also contain nucleic acid sequences which can effect expression of nucleic acids encoding a6-desaturase. Sequence elements capable of----effecting expression of a gene product Include promoters, enhancer elements, upstream activating sequences, transcription termination 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 described, for example, by Restrepo et al. (1990) &#1470;11Plant 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 Δδ-desaturase and further operably linked to a termination signal from Synechocystis is appropriate for expression of Δ6-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 of A6-desaturase in transgenic plants can comprise a seed-specific promoter sequence derived from helianthinin, napin, or glycinin operably linked to the Δβ-desaturase ceding region and further operably linked to a seed termination signal or the nopalina synthase termination signal.

In particular, the helianthinin regulatory elements disclosed in wo 92/17580, incorporated herein by reference, are contemplated as promoter elements to direct the expression of the Δ6desaturase 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 &6&#1470;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 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 which contain the DNA encoding the a6-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 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).

λ 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, 1229. Other methods of transformation, such as protoplast culture (Horsch et al. (1984) Science 223&#1524; 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 gene of the present invention into plant cells. Such alternative methods include biolistic approaches (Klein et al. (1987) Nature 327, 70), electroporation, chemically-induced DNA uptake, and use of viruses or pollen as vectors.

When necessary for the transformation method, the a6-desaturase gene 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 vlr region are utilized to transfer foreign DNA bordered by the T-DNA border sequences. The T&#1470;region also contains a selectable marker for antibiotic resistance, and a multiple cloning site for inserting sequences for transfer. Such engineered strains are known as disarnied A. tumefaclens strains, and allow the efficient transformation of sequences bordered by the Tregion into the nuclear genomes of plants.

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

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 A6&#1470;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 46&#1470; desaturase, seeds or cuttings-from ±ransformed_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 46-desaturasa 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 encoding a6desaturase 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 412-desaturase and 46-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 412-desaturase, and GLA is then generated due to the expression of A6-desaturase. Expression vectors comprising DNA encoding 412-desaturase, or a12desaturase and 46-desaturase, 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 A12-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 412&#1470;desaturase. Accordingly, this sequence can be used to construct the subject expression vectors. In particular, eommerci.lly grown crop plants are contemplate« 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 traneition of lipids in cell membranes. Phase transition temperature depends upon the degree of unsaturation of fatty acids 10 in membrane lipids, and thus increasing the degree of unsaturation, for example by introducing Δβ-desaturaee to convert LA to GLA, can induce or Improve chilling resistance. Accordingly, the present method comprises introducing DNA encoding 46-desaturase into a plant 15 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 the 20 present invention.

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&#1523; at 30eC in BG11N+ medium (Rippka et al. [1979] J._ Gen, Microbiol, ill. 161) under illumination of incandescent lamps (60μΕ.3-&#1493;&#1494;&#1494;.s3&#1470;). Cosmids and plasmids were selected and propagated in Escherichia coll strain DH5a on LB medium supplemented with antibiotics at standard concentrations as described by Maniatis et ^1. (1982) Molecular Cloning: a Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring, New York.

EXAMPLE 2

Construction of Synechocystls Cosmld Genomic Library

Total genomic DNA from Synechocystlj (pcc 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, S*°teriol. Γ73, 1879-1885). The ligated DNA was packaged in vitro as described by Ausubel et al. (1987), and packaged phage were propagated in E. coll DH5a Containing the Aval and EC04711 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.

EXAMPLE 3 Gain-of-Funotion Expression of GLA in Anabaena Anabaena (PCC 7120), a filamentous 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 (?cc 7120) to identify transcon jugants 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 2x10 cells per ml. a mid-log phase culture of E. coll R?4 (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 5% LB, The cosmid genomic library was replica plated onto LB plates containing 50 ug/ml kanamycin and 17.5 ug/ml chloramphenicol and was subsequently patched onto BG11N+ plates containing Anabaena and RP4. After 24 hours of incubation at 30’C, 30 ug/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 ug/ml neomycin. Fatty acid methyl esters were prepared from wild type cultures and cultures containing pools of ten transconjugants as follows. Wild type and transgenic cyanobacteria! 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. Chern., Soc. 66, 543-548 and were analyzed by &#9633;as 1 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 11, Alltech

Associates Inc., IL). Retention times and cochromatography 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 Cl8 fatty 10 acid normalized to an internal standard.

Representative GLC profiles are shown in Fig. 2. Cl9 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 15 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+1.8F. Two GLA 20 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 25 identified which expressed significant levels of GLA and which contained cosmids, 0SV13 and cSy7R, rocpoetivoly (Figure 3). The cosmids overlap in a region approximately 7.5 kb in length. A 3.5 kb Nhel fragment of cSy75 was recloned in the vector pDUCA7 and 30 transferred to Anabaena resulting in gain-of-functlon expression of GLA (Table 2).

Two Nhel/Hind II! 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. t1977&#1504; Proo._Natl. Acad, Sci, USA 74] 5463-5467) of pBSl.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 λ & M University), dna sequence analysis was done with the GCG (Madison, WI) software as described by Devereux et <1. (1984) Nucleic Acids Res, 12, 387-395.

Both Nhel/Hindlll subfragments were transferred into a conjugal expression vector, AM542, in both forward and reverse orientations with respect to a cyanobacterlal carboxylase promoter and were introduced into Anabaena by conjugation. Transconjugants 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 produce detectable levels of GLA (Table 2).

Figure 2 compares the C18 fatty acid profile of an extract from wild type Anabaena (Figure 2&#902;) 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.

Table 2 Cnmposlllun of CIUFnlly Ac ids In Wild Type nnil Trnn!t||cnlc

Cj&#1523;n11&#1524;bnc(<!rln

<td rowspan="2"> 10</td><td rowspan="2"> <S tin In</td><td colspan="6"> Fnlly ndd (%)</td>

<td> 15</td><td> Wild type • HynfxhnryMix (r.p.l'CCfiHO.1)</td><td> Ι.1.Γ1</td><td> 4.3</td><td> 54.5</td><td> •</td><td> 27.3</td><td></td>

<td></td><td> Aiinbnena (sp. l’CC7l?0)</td><td> 2.9</td><td> 24.R</td><td> 37.1</td><td> 35.2</td><td> -</td><td> •</td>

<td></td><td> Synedwcoceiu (sp. I’CC7942)</td><td> 20.fi</td><td> 79.4</td><td> •</td><td> -</td><td> «</td><td> •</td>

<td> 20</td><td> ΛιιηΙιπρππ Ίϊ!υι.1ζ0ι1]ι1|1ηη|,$ cSy75</td><td> 3.11</td><td> 24.4</td><td> 22.1</td><td> 9.1</td><td> 7.7.9</td><td> 12.5</td>

<td></td><td> Jj'ifcc/incwccii.T Ίϊ»ιι5Γ0γιιι»1ιΙλ ρΛΜΙΙ,Ή</td><td> 27.8</td><td> 72.7.</td><td></td><td> .</td><td> •</td><td> •</td>

18:0, slcnric &#1524;eld; 18:1, nlclc nchl; 111:2, lln&#1524;lclc &#1524;cld; 18:3(1;.), <1-lhu»lcnlc ndd; 18:.&#906;(γ). γ-llmdcnlc ndd;IH:&#1523;l, (icfndeailclrncnolc ncld

EXAMPLE 4

Transformation of Synechococcus with a6 and a!2 Desaturase Genes & third cosmid, cSy7, which contains a a12desaturase gene, was isolated by screening the Synechocystis genomic library with a oligonucleotide synthesized from the published Synechocystis a12desaturase gene sequence (Wada et al. [1990] Nature !London), 347, 200-203). A 1.7 kb Aval fragment from this cosmid containing the A12-desaturase gene was identified and used as a probe to demonstrate that cSy!3 not only contains a Αδ-desaturase gene but also a a 12desaturase gene (Figure 3). Genomic Southern blot analysis further showed that both the A6-and a12desaturase 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. The a 12 and Αδ-desaturase genes were cloned individually and together into pAM854 (Bustos et al. [1991] J. Bagteriol. 174, 7525-7533), a shuttle vector that contains sequences necessary for the integration of foreign DNA into the genome of Synechococcus (Golden et 11. £1987] Methods in Enzymol, 153. 215-231). Synechococcus was transformed with these gene constructs and colonies were selected. Fatty acid methyl esters were extracted from transgenic Synechococcus and analyzed by glc.

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 PAM854-a12 expressed linoleic acid (18:2) in addition to the principal fatty acids. Transformants with pAM854-46 and 412 produced both linoleate and GLA (Table 1). These results indicated that Synechococcus containing both a12- and a6-desaturase genes has gained the capability of introducing a second double bond at the a12 position and a third double bond at the a6 position of C18 fatty acids. However, no changes in fatty acid composition was observed in the transformant containing PAM854-a6, indicating that in the absence of substrate synthesized by the a12 desaturase, the A6-desaturase is inactive. This experiment further confirms that the 1.8 kb Nhel/HindHI fragment (Figure 3) contains both coding and promoter regions of the Synechocvstis a6-desaturase gene. Transgenic Synechococcus with altered levels of polyunsaturated fatty acids were similar to wild type in growth rate and morphology.

EXAMPLE 5

Nucleotide Sequence of A6-Deeaturaee

The nucleotide sequence of the 1.8 kb fragment of cSy75-3.5 including the functional 46-desaturase 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. 105-132) identified two regions of hydrophobic amino acids that could represent transmembrane domains (Figure 1A)&#1503; furthermore, the hydropathic profile of the 46-desaturase is similar to that of the 412-desaturase gene (Figure IB; Wada et al.) and a9-desaturases (Thlede et al. [1986] J. Biol, Chem, 261, 13230-13235). However, the sequence similarity between the Synechocystis a6- and a12-desaturases is less than 40% at the nucleotide level and approximately 18% at the amino acid level.

EXAMPLE 6

Transfer of Cyanobaoterial 4.-Desaturase into Tobacco The cyanobaoterial 4®-desaturase gene was 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 Synechocvstis 4-desaturase open reading frame (ORF) were constructed. Components of these cassettes include: (1) a 35S promoter or seed specific promoter derived from the sunflower helianthinin gene to drive 4®-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 4®-desaturase into the ER, (iii) an ER lumen retention signal sequence (KDEL) at the coOH-terminal of the 4®-desaturase ORF, and (iv) an optimized transit peptide to target 4® 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. '

Transgenic tobacco plants were produced containing a chimeric cyanobaoterial desaturase gene, comprised of the Synechocystls 4e 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 4. 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, cyanobacteria! genes involved in fatty acid metabolism can be used to generate transgenic plants with altered fatty acid compositions.

Passages of the description which are out of ambit of the appended claims do not constitute part of the claimed invention.

SEQUENCE LISTING (1) GENERAL INFORMATION!

(i) applicant: Thomas, Terry L.

Reddy, Avutu S. Nuccio, Michael Freyssinet, Georges L.

(ii) TITLE OF INVENTION: PRODUCTION OF GAMMA LINOLENIC ACID BY A DELTA 6-DESATURASE (iii) NUMBER OF SEQUENCES: 3 (iv) CORRESPONDENCE ADDRESS:

!«! S^SE?;«Scully&#1523; Sc°tt, Murphy & Presser (B) STREET: 400 Garden City Plaza (C) CITY: Garden City (D) STATE: New York (E) COUNTRY! United States (Γ) 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: Patentin Release #1.0, Version #1.25 (vl) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER: To be assigned (B) FILING DATE: 08-JAN-1992 (viii) ATTORNEY/AGENT INFORMATION:

(λ) NAME: McNulty, William E.

(B) REGISTRATION NUMBER: 22,606 (C) REFERENCE/DOCKET NUMBER: 83832 (ix) TELECOMMUNICATION INFORMATION:

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

SEQUENCE CHARACTERISTICS:

(A) LENGTH: 3588 base pairs (B) type: nucleic acid (C) STRANDEDNESS: - both (D) TOPOLOGY: linear (D) TOPOLOGY:

(ii) MOLECULE TYPE: DNA (genomic) (ix) FEATURE:

(A) NAME/KEY: CDS (B) LOCATION: 2002.3081 (Xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:

GCTAGCCACC AGTGACGATG CCTTGAATTT GGCCATTCTG ACCCAGGCCC GTATTCTGAA60

TCCCCGCATT CGCATTGTTA ATCGITTGTT caaccatgcc ctgggtaaac GTTTAGACAC120

CACCTTGCCA GACCACGTTA GTTTGAGTGT TTCCGCCCTG GCGGCCCCGA TWTTTCCTT!80

TGCGGCTTTG GGCAATCAGG CGATCGGGCA ATTGCGTTTG TTTGACCAGA CTTiSCCMAIT04,

TCAGGAAAIT GTCATTCACC AAGACCATCC CTGGCTCAAT TTACCCCTGG CGGATTTATG300

GGATGATCCG AGCCGAATGT TGATCTATTA CCTACCGGCC CACAGTGAAA CGGATTTAGT360

AGGCGCAGTG GTGAATAATT TAACGTTGCA ATCTGGGGAC CATTOATAG TGGGACAAAA420

ACCCCAACCC AAGACCAAAC GGCGATCGCC TTGGCGCAAA TTTTCCAAAC TGATTACCAA480

CCTGCGGGAG TATCAGCGGT ATGTCCAACA GGTGATATGG GTGGTGTTGT ITTTATOGTT540

GATGATTTW CTGGCCACCT TCATCTACGT TTCCAITGAT CAACATATTG CCCCAGTGGA600

CGCGTTGTAT TWTCCGTGG GCATGATTAC CGGGGCCGGT GGCAAGGAAG AGGTGGCCGA660

AAAGTCCCCC GATATCATCA AAGTATTCAC AGTGGTGATG ATGATCGCCG GGGCGGGGGT720

GATTGGTATT TGTTATGCCC TACTGAATGA raTCATCMT GGCAGTCGCT TTAGTCAGTT780

TTTGGATOCG GCCAAGTTAC CCOATCGCCA TCACATCAIC ATTTGTGGGC TGGG6GGA5T840

GAGCATGGCC ATTATTGAAG AGTTAATTCA CCAGGGCCAT GAAATTGTGG TAATCGAAAA900

GGATACAGAT AATCGTTTCT TGCATACGGC CCGCTCCCTG GGGGTGCCCG TAATTGTGGA960

GGATGCCCGC CTAGAAAGAA CGTTGGCCTG G^pGCCACC AGCGACGACA CCGTTAACTT CCCTAGCCTG CCAGTGGTGT TGCGTTGCCA agtatttgaa tttgaaacgg tgctttgtcc GGCGGCCCTG GflGGGCAAAA TTTTGGGCAA CCTAGCCACC TTAATCACTC CTAACCATCC CCAAAAGTCT GATTTCGTTC CCCTCTATCT GGAATTATTG GGTACCCATC TCGACTCTGG TGCCCTAGAG CAACTTTGGC GATCGCCCCG GGTTTAGCAT GGGGGGATGG AACTCTTGAC TTGTCTATGT TTAGTATTTT TAAGTTAACC aagctcaaaa agtagcaaaa TAAGTTTAAT TGCAAAAAAG TCAGATAAAA TAAAAGCTTC CAGGCATCTG ctctagggag tttttccgct taactccccc ATTTTTAGGC aaaatcatat ATGACTCACT GTAGAAGGCA GACTAAAATT TTTAGTCTCC CCCGGCGCTG GAGTTTTTTT TTTATCTATT TAAATTTATA A ATG CTA A Met Leu &#906; 1

CGCCAATATC AACCGAGCCG AAGCCATTGT GGAAATTGGC CTAACTGCCA AGGCGATCGC GGATGCCCAG TTTAGCCTGT CCCTGCAGGA GGCGGAATTG GCCACCTATT CCTTTGCGGC CGGCATGACC GATGATTTGC TGTGGGTAGC CTTTGCCGAC CAATTGGTTA AAATTGCAGC AGAACGGGGT GGCAAAACCA tccatagctg AGACGTGTTG TATTTAACCA TGCCCGCCAC tgccactgct gatcctctgg actctttttt TCGGCCCAAT GGTGATCAAG AAAGAACGCT AACAGCAGAG GATAACTTCC AAAAGAAATT TCATAACTGA GTTTTACTGC TAAACAGCGG ACTTCGGTTT TATATTGTGA CCATGGTTCC GCCTTTAGAG AGTATTTTCT CCAAGTCGGC ACAGACTATC CCAATATTGC CAGAGCTTTG CTAGCAATGG ACTCCCAGTT GGAATAAATT GTAGTTAATG GCGGTATAAT GTGAAAGTTT CA GCG GAA AGA ATT AAA TTT ACC hr Ala Glu Arg He Lys Phe Thr 5 10

CAG

Gin

AAA CGG &#943;-ys Arg

GGG

Gly

TTT CGT CGQ GTA CTA AAC

Phe Arg Arg Vai Leu Asn 15 20

CAA CGG GTG Gin Arg Vai

GAT GCC TAG Asp Ala Tyr 25

1020

1080

1140

1200

1260

1320

1380

1440

1500

1560

1620

1680

1740

1800

1860

20&#1470;15&#1470;

1980

2031

2079

2127

TTT GCC GAG CAT GGC CTG ACC Phe Ala Glu His Gly Leu Thr 30

CAA AGG GAT AAT CCC TCC ATG Gin Arg Asp Asn Pro Ser Met

TAT CTG

Tyr Leu

31AAA ACC L^Thr

CTG

Leu

ATT lie

GTG CTC Vai Leu

CTT , Leu

TTT

Phe

GCT Ala

CCA

Pro

GTT

Vai

ATT TTT He

Phe 65

TTG

Leu

GCG

Ala

ATC He

GCC

Ala

TTG GCG Leu

Ala 80

GCC Ala

AAC

Asn

CAC His

AAT

Asn

GCC Ala

TAT

Tyr

TCC

Ser

TCC

Ser

ATG

Met

ACC

Thr

TAC

Tyr

GAT

Asp 110

TTT

Phe

GTC Vai

GGG

Gly

TTA Leu

TCT Ser 115

AAT

Asn

CCC

Pro

GTG

Vai

CCG

Pro

TCC Ser

TTT

Phe

TTG

Leu

TGG

TIP

2175

2223

2271

2319

2367

GGT

GAA

G1U 230

CTT

Leu

TTC Phe

GTA

Vai

GGC Gly 135

CTC Leu

GGC Gly 215

CAG

Gin

CTA

Leu

GAA

Glu

CCT Pro 150

TAC

Tyr

TGG

Trp Gly

CAT His

GAC

Asp

GTG

Vai

2415

CAA

Gin

TTA Leu

GTG

Vai

CCC Pro 200

TAC

Tyr

TTA

Leu

GAA Glu

CAT His

2463

TAT

Tyr

CTT Leu 185

CTA

Leu

GTT Vai

ATT lie

CTT Leu 170

AAT Asn

GAA

Glu

TTC

Phe

GGT

Gly

2511

2559

2607

2655

2703

&#1470;32“

GCT TCG GTA ACC TAT ATG ACC TAT AM Ser Vai Thr Tyr Met Thr Tyr 2W 240

ATG CTG GCC CAT GTG TTG GAA TCA Met Leu. Ala His Vai Leu Glu Ser 255

GAA TCC GGT GCC ATT GAT GAC GAG Glu Ser Gly Ala lie Asp Asp Glu

ACG GCC AAT TTT GCC ACC AAT AAT Thr Ala Asn Phe Ala Thr Asn Asn 285290

GGT TTA AAT CAC CAA GTT ACC CAC Gly Leu Asn His Gin Vai Thr His 300305

ATT CAC TAT CCC CAA TTG GAA AAT He His Tyr Pro Gin Leu Glu Asn 315320

TTT GGT GTG GAA TAT AAA GTT TAT

Phe Gly Vai Glu Tyr Lys Vai Tyr 335

TCT AAC TAT CGC TGG CTA GAG GCC ser Asn Tyr Arg Trp Leu Glu Ala 350

GGC ATC GTG GTT TGC ACC ATC TTT Gly lie Vai Vai Cys Thr He Phe 245250

ACT GAA TTT CTC ACC CCC GAT GGT Thr Glu Phe Leu Thr Pro Asp Gly 260265

TGG GCT ATT TGC CAA ATT CGT ACC Trp Ala lie Cys Gin lie Arg Thr 275290

CCC TTT TGG AAC TGG TTT TGT GGC

Pro Phe Trp Asn Trp Phe Cys Gly 295

CAT CTT TTC CCC AAT ATT TGT CAT His Leu Phe Pro Asn He Cys His 310

ATT ATT AAG GAT GTT TGC CAA GAG lie lie Lys Asp Vai Cys Gin Glu 325330

CCC ACC TTC AAA GCG GCG ATCGCC

Pro Thr Phe Lys Ala Ala HeAla 34O345

ATG GGC AAA GCA TCG TGACATTGCC

Met Gly Lys AlaSer 355360

2751

2799

2847

2895

2943

2991

3039

3088

TTGGGATTGA AGCAAAATGG CAAAATCCCT CGTAAATCTA TGATCGAAGC CTTTCTGTTG 3148 CCCGCCGACC AAATCCCCGA TGCTGACCAA AGGTTGATGT TGGCATTGCT CCAAACCCAC 3208 TTTGAGGGGG TTCATTGGCC GCAGTTTCAA GCTGACCTAG GAGGCAAAGA TTGGGTGATT 3268 TTGCTCAAAT CCGCTGGGAT ATTGAAAGGC TTCACCACCT TTGGTTTCTA CCCTGCTCAA 3328 TGGGAAGGAC AAACCGTCAG AATTGTTTAT TCTGGTGACA CCATCACCGA CCCATCCATG 3388 TGGTCTAACC CAGCCCTGGC CAAGGCTTGG ACCAAGGCCA TGCAAATTCT CCACGAGGCT 3448 ^ggccagaaa AATTATATTG GCTCCTGATT TCTTCCGGCT ATCGCACCTA CCGATTTTTG 3508 XGCATTTTTG CCAAGGAATT CTATCCCCAC tatctccatc CCACTCCCCC gcctgtacaa 3568

33aattttatcc atcagctagc

3588 (2) INFORMATION FOR SEQ ID NO:2i (i) SEQUENCE CHARACTERISTICS!

(A) LENGTH; 359 amino acids (B) TYPE: amino acid (D) topology: linear di) MOLECULE TYPE; protein (Xi) SEQUENCE DESCRIPTION: SEQ IDNO;2:

Met Leu Thr Ala Glu Arg lie Lys Phe Thr Gin Lys Arg Gly PheArg 1 5 1015

Are Val Lau Aan sin Arg Vai Asp Ala Tyr Phe Ala Glu Hla GlyLau

2530

Thr Gin Arg Asp Asn Pro Ser Met Tyr Leu Lys Thr Leu lie He Vai 35 4045

Leu Trp Leu Phe Ser Ala Trp Ala Phe val Leu Phe Ala Pro Vai He 50 555q

Phe Pro Val Arg Leu Leu Gly Cys Met Val Leu Ala He Ala LeuAla 65 70 7580

Ala Phe Ser Phe Asn Val Gly His Asp Ala Asn His Asn Ala TyrSer

9095

Ser Asn Pro His He Aan Arg Val Leu Gly Met Thr Tyr Asp Phe Val 100 !05HO

Gly Leu Ser Ser Phe Leu Trp Arg Tyr Arg His Asn Tyr Leu His Hia 115 120125

Thr Tyr Thr Asn He Leu Gly His Asp Val Glu He His Gly Asp Gly

Ala Val Arg Met Ser Pro Glu Gin Glu His Val Gly lie Tyr ArgPhe 145 150 155160

Gin Gin Phe Tyr He Trp Gly Leu Tyr Leu Phe He Pro Phe TyrTro

165 170175

Phe Leu Tyr Asp Vai Tyr Leu Val Leu Asn Lys Gly Lys Tyr His Asp

Hit Lys lie Pro Pro Phe Gin Pro Leu Glu Leu Ala Ser Leu Leu Gly

(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:

35AGCTTCACTT

CGGTTTTATA

TTGTGACCAT

TCCGCTGCCT

TTAGAGAGTA

TTTTCTCCAA

GTCGGCTAAC

TCATATACAG

ACTATCCCAA

TATTGCCAGA

GCTTTGATGA

AAAATTCTAG

CAATGGACTC

CCAGTTGGAA

TAAATTTTTA

TTTTTTGTAG

TTAATGGCGG

TATAATGTGA

AAGTTTTTTA

CTAACAGCGG

AAAGAATTAA

ATTTACCCAG

AAACGGGGGT

CGGGTGGATG

CCTACTTTGC

CGAGCATGGC

CTGACCCAAA

CTGAAAACCC

TGATTATTGT

GCTCTGGTTG

TTTTCCGCTT

CCAGTTATTT

TTCCGGTGCG

CCTACTGGGT

TGTATGGTTT

TTTTCCTTCA

ATGTCGGCCA

CGATGCCAAC

CACAATGCCT

AACCGGGTTC

TGGGCATGAC

CTACGATTTT

GTCGGGTTAT

CGCCACAACT

ATTTGCACCA

CACCTACACC aatattcttg

GGAGATGGCG

CAGTACGTAT

GAGTCCTGAA CAAGAACATG

CAATTTTATA

TTTGGGGTTT

ATATCTTTTC

ATTCCCTTTT

TACCTAGTGC

TTAATAAAGG

CAAATATCAC

GACCATAAAA

GAATTAGCTA

GTTTGCTAGG

GATTAAGCTA

TTATGGCTCG

CTGGCTCTGG

GCTTTTCCAT

TCCTGAAGTA

TTAATTGGTG

<td> י CATCTGCTCT AGGGAGTTTT</td><td> 60</td>

<td> I TCCCCCATTT TTAGGCAAAA</td><td> 120</td>

<td> : CTCACTGTAG AAGGCAGACT</td><td> 180</td>

<td> GTCTCCCCCG GCGCTGGAGT</td><td> 240</td>

<td> TCTATTTAAA TTTATAAATG</td><td> 300</td>

<td> TTCGTCGGGT ACTAAACCAA</td><td> 360</td>

<td> GGGATAATCC CTCCATGTAT</td><td> 420</td>

<td> GGGCCTTTGT GCTTTTTGCT</td><td> 480</td>

<td> TGGCGATCGC CTTGGCGGCC</td><td> 540</td>

<td> ATTCCTCCAA TCCCCACATC</td><td> 600</td>

<td> CTAGTTTTCT TTGGCGCTAT</td><td> 660</td>

<td> GCCATGACGT GGAAATCCAT</td><td> 720</td>

<td> TTGGTATTTA TCGTTTCCAG</td><td> 780</td>

<td> ATTGGTTTCT CTACGATGTC</td><td> 840</td>

<td> TTCCTCCTTT CCAGCCCCTA</td><td> 900</td>

<td> GCTACGTTTT CGGCTTACCT</td><td> 960</td>

<td> CTTCGGTAAC CTATATGACC</td><td> 1020</td>

TGTTGGAATC

AACTGAATTT

1080

TATGGCATCG

TGGTTTGCAC

CATCTTTATG

CTGGCCCATG

CTCACCCCCG

ATGGTGAATC

CGGTGCCATT

GATGACGAGT

GGGCTATTTG

CCAAATTCGT

1140

ACCACGGCCA

ATTTTGCCAC

CAATAATCCC

TTTTGGAACT

GGTI^TGTGG

CGGTTTAAAT

1200

CACCAAGTTA

CCCACCATCT

TTTCCCCAAT

ATTTGTCATA

TTCACTATCC

CCAATTGGAA

1260

AATATTATTA

AGGATGTTTG

CCAAGAGTTT

GGTGTGGAAT

ATAAAGTTTA

TCCCACCTTC

1320

AAAGCGGCGA

TCGCCTCTAA

CTATCGCTGG

CTAGAGGCCA

TGGGCAAAGC

ATCGTGACAT

1380

TGCCTTGGGA

TTGAAGCAAA

ATGGCAAAAT

CCCTCGTAAA

TCTATGATCG

AAGCCTTTCT

1440

&#1470;36&#1470;

GTTGCCCGCC GACCAAATCC cWcTTTGAG GGGGTTCATT GATTTTGCTC AAATCCGCTG TCAATGGGAA GGACAAACCG CATGTGGTCT AACCCAGCCC GGCTAGGCCA GAAAAATTAT TTTGAGCATT TTTGCCAAGG ACAAAA1&#1524;r1&#1470;r ATCCATCAGC

CCGATGCTGA CCAAAGGTTG GGCCGCAGTT TCAAGCTGAC GGATAITGAA AGGCTTCACC TCAGAATTGT TTATTCTGGT TGGCCAAGGC TTGGACCAAG ATTGGCTCCT GATTTCTTCC AATTCTATCC CCACTATCTC TAGC

ATGTTGGCAT TGCTCCAAAC

CTAGGAGGCA AAGATTGGGT

ACCTTTGGIT TCTACCCTGC

GACACCATCA CCGACCCATC

GCCATGCAAA TTCTCCACGA

GGCTATCGCA CCTACCGATT

CATCCCACTC CCCCGCCTGT