AU750363B2 - Fatty acid desaturases and mutant sequences thereof - Google Patents

Description

WO 98/56239 PCT/US98/12332 1 FATTY ACID DESATURASES AND MUTANT SEQUENCES THEREOF Technical Field This invention relates to fatty acid desaturases and nucleic acids encoding desaturase proteins. More particularly, the invention relates to nucleic acids encoding delta-12 and delta-15 fatty acid desaturase proteins that affect fatty acid composition in plants, polypeptides produced from such nucleic acids and plants expressing such nucleic acids.

Background of the Invention Many breeding studies have been conducted to improve the fatty acid profile of Brassica varieties.

Pleines and Freidt, Fat Sci. Technol., 90(5), 167-171 (1988) describe plant lines with reduced C 18 levels combined with high oleic content Rakow and McGregor, J. Amer. Oil Chem. Soc., 50, 400-403 (Oct.

1973) discuss problems associated with selecting mutants for linoleic and linolenic acids. In. Can. J. Plant Sci., 68, 509-511 (Apr. 1988) Stellar summer rape producing seed oil with 3% linolenic acid and 28% linoleic acid is disclosed. Roy and Tarr, Z.

Pflanzenzuchtg, 95(3), 201-209 (1985) teaches transfer of genes through an interspecific cross from Brassica juncea into Brassica napus resulting in a reconstituted line combining high linoleic with low linolenic acid content.

Roy and Tarr, Plant Breeding, 98, 89-96 (1987) discuss prospects for development of B. napus L. having improved linolenic and linolenic acid content. European Patent application 323,753 published July 12, 1989 discloses seeds and oils having greater than 79% oleic acid combined with less than 3.5% linolenic acid. Canvin, SWO 98/56239 PCT/US98/1 2332 2 Can. J. Botany, 43, 63-69 (1965) discusses the effect of temperature on the fatty acid composition of oils from several seed crops including rapeseed.

Mutations typically are induced with extremely high doses of radiation and/or chemical mutagens (Gaul, H. Radiation Botany (1964) 4:155-232). High dose levels which exceed LD50, and typically reach LD90, led to maximum achievable mutation rates. In mutation breeding of Brassica varieties high levels of chemical mutagens alone or combined with radiation have induced a limited number of fatty acid mutations (Rakow, G.Z.

Pflanzenzuchtg (1973) 69:62-82). The low a-linolenic acid mutation derived from the Rakow mutation breeding program did not have direct commercial application because of low seed yield. The first commercial cultivar using the low a-linolenic acid mutation derived in 1973 was released in 1988 as the variety Stellar (Scarth, R.

et al., Can. J. Plant Sci. (1988) 68:509-511). Stellar was 20% lower yielding than commercial cultivars at the time of its release.

Alterations in fatty acid composition of vegetable oils is desirable for meeting specific food and industrial uses. For example, Brassica varieties with increased monounsaturate levels (oleic acid) in the seed oil, and products derived from such oil, would improve lipid nutrition. Canola lines which are low in polyunsaturated fatty acids and high in oleic acid tend to have higher oxidative stability, which is a useful trait for the retail food industry.

Delta-12 fatty acid desaturase (also known as oleic desaturase) is involved in the enzymatic conversion of oleic acid to linoleic acid. Delta-15 fatty acid desaturase (also known as linoleic acid desaturase) is involved in the enzymatic conversion of linoleic acid to a-linolenic acid. A microsomal delta-12 desaturase has WO 98/56239 PCT/US98/12332 3 been cloned and characterized using T-DNA tagging.

Okuley, et al., Plant Cell 6:147-158 (1994). The nucleotide sequences of higher plant genes encoding microsomal delta-12 fatty acid desaturase are described in Lightner et al., W094/11516. Sequences of higher plant genes encoding microsomal and plastid fatty acid desaturases are disclosed in Yadav, et al., Plant Physiol., 103:467-476 (1993), WO 93/11245 and Arondel, V. et al., Science, 258:1353-1355 (1992).

However, there are no teachings that disclose mutations in delta-12 or delta-15 fatty acid desaturase coding sequences from plants. There is a need in the art for more efficient methods to develop plant lines that contain delta-12 or delta-15 fatty acid desaturase gene sequence mutations effective for altering the fatty acid composition of seeds.

Summary of the Invention The invention comprises Brassicaceae or Helianthus seeds, plants and plant lines having at least one mutation that controls the levels of unsaturated fatty acids in plants. One embodiment of the invention is an isolated nucleic acid fragment comprising a nucleotide sequence encoding a mutation from a mutant delta-12 fatty acid desaturase conferring altered fatty composition in seeds when the fragment is present in a plant. A preferred sequence comprises a mutant sequence as shown in Fig. 2. Another embodiment of the invention is an isolated nucleic acid fragment comprising a nucleotide sequence encoding a mutation from a mutant delta-15 fatty acid desaturase. A plant in this embodiment may be soybean, oilseed Brassica species, sunflower, castor bean or corn. The mutant sequence may be derived from, for example, a Brassica napus, Brassica rapa, Brassica juncea or Helianthus delta-12 or delta-15 desaturase gene.

SWO 98/56239 PCT/US98/1 2332 4 Another embodiment of the invention involves a method of producing a Brassicaceae or Helianthus plant line comprising the steps of: inducing mutagenesis in cells of a starting variety of a Brassicaceae or Helianthus species; obtaining progeny plants from the mutagenized cells; identifying progeny plants that contain a mutation in a delta-12 or delta-15 fatty acid desaturase gene; and producing a plant line by selfing or crossing. The resulting plant line may be subjected to mutagenesis in order to obtain a line having both a delta-12 desaturase mutation and a desaturase mutation.

Yet another embodiment of the invention involves a method of producing plant lines containing altered fatty acid composition comprising: crossing a first plant with a second plant having a mutant delta-12 or fatty acid desaturase; obtaining seeds from the cross of step growing fertile plants from such seeds; obtaining progeny seed from the plants of step and identifying those seeds among the progeny that have altered fatty acid composition. Suitable plants are soybean, rapeseed, sunflower, safflower, castor bean and corn. Preferred plants are rapeseed and sunflower.

The invention is also embodied in vegetable oil obtained from plants disclosed herein, which vegetable oil has an altered fatty acid composition.

Brief Description of the Sequence Listing SEQ ID NO:1 shows a hypothetical DNA sequence of a Brassica Fad2 gene. SEQ ID NO:2 is the deduced amino acid sequence of SEQ ID NO:1.

SEQ ID NO:3 shows a hypothetical DNA sequence of a Brassica Fad2 gene having a mutation at nucleotide 316.

SEQ ID NO:4 is the deduced amino acid sequence of SEQ ID NO:3.

WO 98/56239 PCT/US98/12332 5 SEQ ID NO:5 shows a hypothetical DNA sequence of a Brassica Fad2 gene. SEQ ID NO:6 is the deduced amino acid sequence of SEQ ID SEQ ID NO:7 shows a hypothetical DNA sequence of a Brassica Fad2 gene having a mutation at nucleotide 515.

SEQ ID NO:8 is the deduced amino acid sequence of SEQ ID NO:7.

SEQ ID NO:9 shows the DNA sequence for the coding region of a wild type Brassica Fad2-D gene. SEQ ID is the deduced amino acid sequence for SEQ ID NO:9.

SEQ ID NO:11 shows the DNA sequence for the coding region of the IMC 129 mutant Brassica Fad2-D gene. SEQ ID NO:12 is the deduced amino acid sequence for SEQ ID NO:11.

SEQ ID NO:13 shows the DNA sequence for the coding region of a wild type Brassica Fad2-F gene. SEQ ID NO:14 is the deduced amino acid sequence for SEQ ID NO:13.

SEQ ID NO:15 shows the DNA sequence for the coding region of the Q508 mutant Brassica Fad2-F gene. SEQ ID NO:16 is the deduced amino acid sequence for SEQ ID SEQ ID NO:17 shows the DNA sequence for the coding region of the Q4275 mutant Brassica Fad2-F gene. SEQ ID NO:18 is the deduced amino acid sequence for SEQ ID NO:17.

SEQ ID NOS:19-27 show oligonucleotide sequences.

SEQ ID NO:28 shows the genomic DNA sequence for the Fad2-U gene from Brassica.

SEQ ID NOS:30-31 show genomic sequences located upstream from the start codon of Brassica Fad2-D genes.

Brief Description of the Figures Figure 1 is a histogram showing the frequency distribution of seed oil oleic acid (C 18 content in a segregating population of a Q508 X Westar cross. The bar labeled WSGA 1A represents the C 18 content of the Westar WO 98/56239 PCT/US98/12332 6 parent. The bar labeled Q508 represents the content of the Q508 parent.

Figure 2 shows the nucleotide sequences for a Brassica Fad2-D wild type gene (Fad2-D wt), IMC129 mutant gene (Fad2-D GA316 IMC129), Fad2-F wild type gene (Fad2-F wt), Q508 mutant gene (Fad2-F TA515 Q508) and Q4275 mutant gene (Fad2-F GA908 Q4275).

Figure 3 shows the deduced amino acid sequences for the polynucleotides of Figure 2.

Description of the Preferred Embodiments All percent fatty acids herein are percent by weight of the oil of which the fatty acid is a component.

As used herein, a "line" is a group of plants that display little or no genetic variation between individuals for at least one trait. Such lines may be created by several generations of self-pollination and selection, or vegetative propagation from a single parent using tissue or cell culture techniques. As used herein, the term "variety" refers to a line which is used for commercial production.

The term "mutagenesis" refers to the use of a mutagenic agent to induce random genetic mutations within a population of individuals. The treated population, or a subsequent generation of that population, is then screened for usable trait(s) that result from the mutations. A "population" is any group of individuals that share a common gene pool. As used herein "Mo" is untreated seed. As used herein, is the seed (and resulting plants) exposed to a mutagenic agent, while "M 2 is the progeny (seeds and plants) of self-pollinated M, plants, "M3" is the progeny of self-pollinated M 2 plants, and "M4" is the progeny of self-pollinated M 3 plants.

"Ms" is the progeny of self-pollinated M, plants. "M 6 etc. are each the progeny of self-pollinated plants WO 98/56239 PCT/US98/12332 7 of the previous generation. The term "selfed" as used herein means self-pollinated.

"Stability" or "stable" as used herein means that with respect to a given fatty acid component, the component is maintained from generation to generation for at least two generations and preferably at least three generations at substantially the same level, e.g., preferably The method of invention is capable of creating lines with improved fatty acid compositions stable up to from generation to generation. The above stability may be affected by temperature, location, stress and time of planting. Thus, comparison of fatty acid profiles should be made from seeds produced under similar growing conditions. Stability may be measured based on knowledge of prior generation.

Intensive breeding has produced Brassica plants whose seed oil contains less than 2% erucic acid. The same varieties have also been bred so that the defatted meal contains less than 30 gmol glucosinolates/gram.

"Canola" as used herein refers to plant variety seed or oil which contains less than 2% erucic acid (C 22 1 and meal with less than 30 Amol glucosinolates/gram.

Applicants have discovered plants with mutations in a delta-12 fatty acid desaturase gene. Such plants have useful alterations in the fatty acid compositions of the seed oil. Such mutations confer, for example, an elevated oleic acid content, a decreased, stabilized linoleic acid content, or both elevated oleic acid and decreased, stabilized linoleic acid content.

Applicants have further discovered plants with mutations in a delta-15 fatty acid desaturase gene. Such plants have useful alterations in the fatty acid composition of the seed oil, a decreased, stabilized level of a-linolenic acid.

WO 98/56239 PCT/US98/12332 8 Applicants have further discovered isolated nucleic acid fragments (polynucleotides) comprising sequences that carry mutations within the coding sequence of delta-12 or delta-15 fatty acid desaturases. The mutations confer desirable alterations in fatty acid levels in the seed oil of plants carrying such mutations.

Delta-12 fatty acid desaturase is also known as omega-6 fatty acid desaturase and is sometimes referred to herein as Fad2 or 12-DES. Delta-15 fatty acid desaturase is also known on omega-3 fatty acid desaturase and is sometimes referred to herein as Fad3 or A nucleic acid fragment of the invention may be in the form of RNA or in the form of DNA, including cDNA, synthetic DNA or genomic DNA. The DNA may be doublestranded or single-stranded, and if single-stranded, can be either the coding strand or non-coding strand. An RNA analog may be, for example, mRNA or a combination of ribo- and deoxyribonucleotides. Illustrative examples of a nucleic acid fragment of the invention are the mutant sequences shown in Fig. 3.

A nucleic acid fragment of the invention contains a mutation in a microsomal delta-12 fatty acid desaturase coding sequence or a mutation in a microsomal fatty acid desaturase coding sequence. Such a mutation renders the resulting desaturase gene product nonfunctional in plants, relative to the function of the gene product encoded by the wild-type sequence. The nonfunctionality of the delta-12 desaturase gene product can be inferred from the decreased level of reaction product (linoleic acid) and increased level of substrate (oleic acid) in plant tissues expressing the mutant sequence, compared to the corresponding levels in plant tissues expressing the wild-type sequence. The non-functionality of the delta-15 desaturase gene product can be inferred from the decreased level of reaction product (a-linolenic )WO 98/56239 PCT/US98/12332 9 acid) and the increased level of substrate (linoleic acid) in plant tissues expressing the mutant sequence, compared to the corresponding levels in plant tissues expressing the wild-type sequence.

A nucleic acid fragment of the invention may comprise a portion of the coding sequence, at least about 10 nucleotides, provided that the fragment contains at least one mutation in the coding sequence. The length of a desired fragment depends upon the purpose for which the fragment will be used, PCR primer, sitedirected mutagenesis and the like. In one embodiment, a nucleic acid fragment of the invention comprises the full length coding sequence of a mutant delta-12 or mutant fatty acid desaturase, the mutant sequences of Fig. 3. In other embodiments, a nucleic acid fragment is about 20 to about 50 nucleotides (or base pairs, bp), or about 50 to about 500 nucleotides, or about 500 to about 1200 nucleotides in length.

In another embodiment, the invention relates to an isolated nucleic acid fragment of at least 50 nucleotides in length that has at least 70% sequence identity to the nucleotide sequences of SEQ ID NO:30 or SEQ ID NO:31. In some embodiments, such nucleic acid fragments have at least 80% or 90% sequence identity to SEQ ID NO:30 or SEQ ID NO:31. Sequence identity for these and other nucleic acids disclosed herein can be determined, for example, using Blast 2.0.4 (Feb. 24, 1998) to search the nr database (non-redundant GenBank, EMBL, DDBT and PDB).

BLAST 2.0.4 is provided by the National Center for Biotechnology (http://www.ncbi.nlm.nih.gov). Altschul, S.F. et al., Nucleic Acids Res., 25:3389-3402 (1997).

Alternatively, MEGALIGN® (DNASTAR, Madison, WI) sequence alignment software can be used to determine sequence identity by the Clustal algorithm. In this method, sequences are grouped into clusters by examining the SWO 98/56239 PCT/US98/12332 10 distance between all pairs. Clusters are aligned pairwise, then as groups. The Jotun Hein algorithm is also available in MEGALIGN®. The nucleotide sequences of SEQ ID NO:30 and NO:31 are about 85% identical using the Clustal algorithm with default parameters.

The nucleotide sequences of SEQ ID NO:30 and SEQ ID NO:31 are located upstream of the ATG start codon for the fad2-D gene and can be isolated from Bridger and Westar canola plants, respectively. These upstream elements contain intron-like features.

The invention also relates to an isolated nucleic acid fragment that includes a sequence of at least 200 nucleotides. The fragment has at least 70% identity to nucleotides 1 to about 1012 of SEQ ID NO:28. In some embodiments, the fragment has 80% or at least sequence identity to nucleotides 1 to about 1012 of SEQ ID NO:28. This portion of SEQ ID NO:28 is located upstream of the ATG start codon and has intron-like features.

A mutation in a nucleic acid fragment of the invention may be in any portion of the coding sequence that renders the resulting gene product non-functional.

Suitable types of mutations include, without limitation, insertions of nucleotides, deletions of nucleotides, or transitions and transversions in the wild-type coding sequence. Such mutations result in insertions of one or more amino acids, deletions of one or more amino acids, and non-conservative amino acid substitutions in the corresponding gene product. In some embodiments, the sequence of a nucleic acid fragment may comprise more than one mutation or more than one type of mutation.

Insertion or deletion of amino acids in a coding sequence may, for example, disrupt the conformation of essential alpha-helical or beta-pleated sheet regions of the resulting gene product. Amino acid insertions or WO 98/56239 PCT/US98/12332 11 deletions may also disrupt binding or catalytic sites important for gene product activity. It is known in the art that the insertion or deletion of a larger number of contiguous amino acids is more likely to render the gene product non-functional, compared to a smaller number of inserted or deleted amino acids.

Non-conservative amino acid substitutions may replace an amino acid of one class with an amino acid of a different class. Non-conservative substitutions may make a substantial change in the charge or hydrophobicity of the gene product. Non-conservative amino acid substitutions may also make a substantial change in the bulk of the residue side chain, substituting an alanyl residue for a isoleucyl residue.

Examples of non-conservative substitutions include the substitution of a basic amino acid for a non-polar amino acid, or a polar amino acid for an acidic amino acid. Because there are only 20 amino acids encoded in a gene, substitutions that result in a non-functional gene product may be determined by routine experimentation, incorporating amino acids of a different class in the region of the gene product targeted for mutation.

Preferred mutations are in a region of the nucleic acid encoding an amino acid sequence motif that is conserved among delta-12 fatty acid desaturases or deltafatty acid desaturases, such as a His-Xaa-Xaa-Xaa-His motif (Tables An example of a suitable region has a conserved HECGH motif that is found, for example, in nucleotides corresponding to amino acids 105 to 109 of the Arabidopsis and Brassica delta-12 desaturase sequences, in nucleotides corresponding to amino acids 101 to 105 of the soybean delta-12 desaturase sequence and in nucleotides corresponding to amino acids 111 to 115 of the maize delta-12 desaturase sequence. See e.g., WO 94/115116; Okuley et al., Plant Cell 6:147-158 (1994).

WO 98/56239 PCT/US98/12332 12 The one letter amino acid designations used herein are described in Alberts, B. et al., Molecular Biology of the Cell, 3rd edition, Garland Publishing, New York, 1994.

Amino acids flanking this motif are also highly conserved among delta-12 and delta-15 desaturases and are also suitable candidates for mutations in fragments of the invention.

An illustrative embodiment of a mutation in a nucleic acid fragment of the invention is a Glu to Lys substitution in the HECGH motif of a Brassica microsomal delta-12 desaturase sequence, either the D form or the F form. This mutation results in the sequence HECGH being changed to HKCGH as seen by comparing SEQ ID NO:10 (wildtype D form) to SEQ ID NO:12 (mutant D form). A similar mutation in other Fad-2 sequences is contemplated to result in a non-functional gene product. (Compare SEQ ID NO:2 to SEQ ID NO:4).

A similar motif may be found at amino acids 101 to 105 of the Arabidopsis microsomal delta-15 fatty acid desaturase, as well as in the corresponding rape and soybean desaturases (Table See, WO 93/11245; Arondel, V. et al., Science, 258:1153-1155 (1992); Yadav, N. et al., Plant Physiol., 103:467-476 (1993). Plastid fatty acids have a similar motif (Table Among the types of mutations in an HECGH motif that render the resulting gene product non-functional are non-conservative substitutions. An illustrative example of a non-conservative substitution is substitution of a glycine residue for either the first or second histidine.

Such a substitution replaces a charged residue (histidine) with a non-polar residue (glycine). Another type of mutation that renders the resulting gene product non-functional is an insertion mutation, insertion of a glycine between the cysteine and glutamic acid residues in the HECGH motif.

WO 98/56239 PCT/US98/12332 13 Other regions having suitable conserved amino acid motifs include the HRRHH motif shown in Table 2, the HRTHH motif shown in Table 6 and the HVAHH motif shown in Table 3. See, WO 94/115116; Hitz, W. et al., Plant Physiol., 105:635-641 (1994); Okuley, et al., supra; and Yadav, N. et al., supra. An illustrative example of a mutation in the region shown in Table 3 is a mutation at nucleotides corresponding to the codon for glycine (amino acid 303 of B. napus). A non-conservative Gly to Glu substitution results in the amino acid sequence DRDYGILNKV being changed to sequence DRDYEILNKV (compare wild-type F form SEQ ID NO:14 to mutant Q4275 SEQ ID NO:18, Fig. 3).

Another region suitable for a mutation in a delta- 12 desaturase sequence contains the motif KYLNNP at nucleotides corresponding to amino acids 171 to 175 of the Brassica desaturase sequence. An illustrative example of a mutation is this region is a Leu to His substitution, resulting in the amino acid sequence (Table 4) KYHNN (compare wild-type Fad2-F SEQ ID NO:14 to mutant SEQ ID NO:16). A similar mutation in other Fad-2 amino acid sequences is contemplated to result in a nonfunctional gene product. (Compare SEQ ID NO:6 to SEQ ID NO:8).

WO 98/56239 WO 9856239PCT/US98/1 2332 14 TABLE 1 Alignment of Amino Acid Sequences from Microsomal Delta-12 Fatty Acid Desaturases Species Arabidopsis thaliana Glycine max Zea mays Ricinus cofluniflsa Brass ica napus D Brassica napus F Position 100-129 96- 125 106-13S 1- 29 100-128 100-128 Amino Acid Sequence IWVIAHECGH HAFSDYQWLD DTVGLIFHSF VWVIAHECGH HAFSKYQWVD DVVGLTLHST VWVIAHECGH HAFSDYSLLD DVVGLVLH-SS WVMAHDCGH HAFSDYQLLD DVVGLILHSC VWVIAHECGH HAFSDYQWLD DTVGLIFHS VWVIAHECGH HAFSDYQWLD DTVGLIFHS from plasmid pRF2-1C TABLE 2 Alignment of Amino Acid Sequences from Microsomal Delta-12 Fatty Acid Desaturases Species Arabidopsis thaliana Glycine max Zea mays Ricinus communiSa Brassica napus D Brassica napus F Position 130- 158 126- 154 136- 164 30- 58 130-158 130-158 Amino Acid Sequence LLVPYFSWKY SHRRHHSNTG SLERDEVFV LLVPYFSWKI SHRRHHSNTG SLDRDEVFV LMVPYFSWKY SHRRHHSNTG SLERDEVFV LLVPYFSWKH SHRRHHSNTG SLERDEVFV LLVPYFSWKY SHRRHHSMTG SLERDEVFV LLVPYFSWKY SHRRHHSNTG SLERDEVFV from plasmid pRF2-1C TABLE 3 Alignment of Amino Acid Sequences from Microsomal Delta-12 Fatty Acid Desaturases Species Position Amino Acid Sequence Arabidopsis thaliana 298-333

NAMEAT

Glycine max 294-329

HANEAT

Zea mays 305-340

HAMEAT

Ricinus communiSa 198-224 Brassica napus D 299-334

HAMEAT

Brassica napus F 299-334

HAMEAT

DRDYGILNKV FHINITDTHVA HHLFSTMPHY DRDYGILNKV FHHITDTHVA HHLFSTMPHY DRDYGILNRV FHINITDTHVA HHLFSTMPHY DRDYGILNKV FHNITDTQVA HHLF TMP DRDYGILNKV FHNITDTHVA HHLFSTMPHY DRDYGILNKV FHNITDTHVA HHLFSTMPHY a from plasmid pRF2-1C WO 98/56239 WO 9856239PCT/US98/1 2332 15 TABLE 4 Alignment of Conserved Amino Acids from Microsomal Delta-12 Fatty Acid Desaturases Species Arabidopsis thaliana Glycine max Zea mays Ricinus cornmuniSa Brassica napus D Brassica napus F Position 165-180 161-176 172-187 65- 80 165- 180 165- 180 Amino Acid Sequence IKWYGKYLNN PLGRIM VAWFSLYLNN PLGRAV PWYTPYVYNN PVGRVV IRWYSKYLNN PPGRIM IKWYGKYLNN PLGRTV IKWYGKYLNN PLGRTV from plasmid pRF2-IC TABLE Alignment of Conserved Amino Acids from Plastid and Microsomal Delta-is Fatty Acid Desaturases Species Position Amino Acid Sequence Arabi dopsi s thai anaa Bra salca napus' Glycine max' Arabidopsi s thailana Brassica napus Glycine max a Plastid sequences 156-177 114-135 164-185 94-115 87-10 9 93-114 WALFVLGHD CGHGSFSNDP KLN WALFVLGHD CGHGSFSNDP RLN WALFVLGHD CGHGSFSNNS KLN WAIFVLGHD CGHGSFSDIP LLN WALFVLGHD CGHGSFSNDP RLN WALFVLGHD CGHGSFSDSP PLN TABLE 6 Alignment of Conserved Amino Acids from Plastid and Microsomal Det-1S atty cid Desaturases Species Position Amino Acid Sequence A. thaiianaa B. napusa Glycine maxa A. thaliana Bra ssica napus Glycine max 188-216 146-174 196-224 126-154 117-145 125-153

ILVPYHGWRI

ILVPYHGWRI

ILVPYHGWRI

IL VPYHGWR I I LVPYHGWRI

ILVPYHGWRI

SHRTHHQNHG

SHRTHHQN{G

SHRTHHQHHG

SHRTHHQNHG

SHRTHHQNHG

SHRTHHQNIIG

HVENDESWH

HVENDESWH

HAENDESWH

HVENDESWV

HVENDESWV

HIEKDESWV

a Plastid sequences The conservation of amino acid motifs and their relative positions indicates that regions of a delta-12 or delta-15 fatty acid desaturase that can be mutated in one species to generate a non-functional desaturase can WO 98/56239 PCT/US98/12332 16 be mutated in the corresponding region from other species to generate a non-functional delta-12 desaturase or desaturase gene product in that species.

Mutations in any of the regions of Tables 1-6 are specifically included within the scope of the invention and are substantially identical to those mutations exemplified herein, provided that such mutation (or mutations) renders the resulting desaturase gene product non-functional, as discussed hereinabove.

A nucleic acid fragment containing a mutant sequence can be generated by techniques known to the skilled artisan. Such techniques include, without limitation, site-directed mutagenesis of wild-type sequences and direct synthesis using automated DNA synthesizers.

A nucleic acid fragment containing a mutant sequence can also be generated by mutagenesis of plant seeds or regenerable plant tissue by, ethyl methane sulfonate, X-rays or other mutagens. With mutagenesis, mutant plants having the desired fatty acid phenotype in seeds are identified by known techniques and a nucleic acid fragment containing the desired mutation is isolated from genomic DNA or RNA of the mutant line. The site of the specific mutation is then determined by sequencing the coding region of the delta-12 desaturase or desaturase gene. Alternatively, labeled nucleic acid probes that are specific for desired mutational events can be used to rapidly screen a mutagenized population.

The disclosed method may be applied to all oilseed Brassica species, and to both Spring and Winter maturing types within each species. Physical mutagens, including but not limited to X-rays, UV rays, and other physical treatments which cause chromosome damage, and other chemical mutagens, including but not limited to ethidium bromide, nitrosoguanidine, diepoxybutane etc. may also be .1 WO 98/56239 PCT/US98/12332 17 used to induce mutations. The mutagenesis treatment may also be applied to other stages of plant development, including but not limited to cell cultures, embryos, microspores and shoot apices.

"Stable mutations" as used herein are defined as

M

s or more advanced lines which maintain a selected altered fatty acid profile for a minimum of three generations, including a minimum of two generations under field conditions, and exceeding established statistical thresholds for a minimum of two generations, as determined by gas chromatographic analysis of a minimum of 10 randomly selected seeds bulked together.

Alternatively, stability may be measured in the same way by comparing to subsequent generations. In subsequent generations, stability is defined as having similar fatty acid profiles in the seed as that of the prior or subsequent generation when grown under substantially similar conditions.

Mutation breeding has traditionally produced plants carrying, in addition to the trait of interest, multiple, deleterious traits, reduced plant vigor and reduced fertility. Such traits may indirectly affect fatty acid composition, producing an unstable mutation; and/or reduce yield, thereby reducing the commercial utility of the invention. To eliminate the occurrence of deleterious mutations and reduce the load of mutations carried by the plant, a low mutagen dose is used in the seed treatments to create an LD30 population. This allows for the rapid selection of single gene mutations for fatty acid traits in agronomic backgrounds which produce acceptable yields.

The seeds of several different plant lines have been deposited with the American Type Culture Collection and have the following accession numbers.

WO 98/56239 PCT/US98/12332 18 Line Accession No. Deposit Date A129.5 40811 May 25, 1990 A133.1 40812 May 25, 1990 M3032.1 75021 June 7, 1991 M3062.8 75025 June 7, 1991 M3028.10 75026 June 7, 1991 IMC130 75446 April 16, 1993 Q4275 97569 May 10, 1996 In some plant species or varieties more than one form of endogenous microsomal delta-12 desaturase may be found. In amphidiploids, each form may be derived from one of the parent genomes making up the species under consideration. Plants with mutations in both forms have a fatty acid profile that differs from plants with a mutation in only one form. An example of such a plant is Brassica napus line Q508, a doubly-mutagenized line containing a mutant D-form of delta-12 desaturase (SEQ ID NO:11) and a mutant F-form of delta-12 desaturase (SEQ ID Another example is line Q4275, which contains a mutant D-form of delta-12 desaturase (SEQ ID NO:11) and a mutant F-form of delta-12 desaturase (SEQ ID NO:17). See Figs. 2-3.

Preferred host or recipient organisms for introduction of a nucleic acid fragment of the invention are the oil-producing species, such as soybean (Glycine max), rapeseed Brassica napus, B. rapa and B.

juncea), sunflower (Helianthus annus), castor bean (Ricinus communis), corn (Zea mays), and safflower (Carthamus tinctorius).

A nucleic acid fragment of the invention may further comprise additional nucleic acids. For example, a nucleic acid encoding a secretory or leader amino acid sequence can be linked to a mutant desaturase nucleic acid fragment such that the secretory or leader sequence is fused in-frame to the amino terminal end of a mutant delta-12 or delta-15 desaturase polypeptide. Other .WO 98/56239 PCT/US98/12332 19 nucleic acid fragments are known in the art that encode amino acid sequences useful for fusing in-frame to the mutant desaturase polypeptides disclosed herein. See, U.S. 5,629,193 incorporated herein by reference. A nucleic acid fragment may also have one or more regulatory elements operably linked thereto.

The present invention also comprises nucleic acid fragments that selectively hybridize to mutant desaturase sequences. Such a nucleic acid fragment typically is at least 15 nucleotides in length. Hybridization typically involves Southern analysis (Southern blotting), a method by which the presence of DNA sequences in a target nucleic acid mixture are identified by hybridization to a labeled oligonucleotide or DNA fragment probe. Southern analysis typically involves electrophoretic separation of DNA digests on agarose gels, denaturation of the DNA after electrophoretic separation, and transfer of the DNA to nitrocellulose, nylon, or another suitable membrane support for analysis with a radiolabeled, biotinylated, or enzyme-labeled probe as described in sections 9.37- 9.52 of Sambrook et al., (1989) Molecular Cloning, second edition, Cold Spring Harbor Laboratory, Plainview; NY.

A nucleic acid fragment can hybridize under moderate stringency conditions or, preferably, under high stringency conditions to a mutant desaturase sequence.

High stringency conditions are used to identify nucleic acids that have a high degree of homology to the probe.

High stringency conditions can include the use of low ionic strength and high temperature for washing, for example, 0.015 M NaCl/0.0015 M sodium citrate (0.1X SSC); 0.1% sodium lauryl sulfate (SDS) at 50-65oC.

Alternatively, a denaturing agent such as formamide can be employed during hybridization, 50% formamide with 0.1% bovine serum albumin/0.1% Ficoll/0.1% polyvinylpyrrolidone/50 mM sodium phosphate buffer at pH WO 98/56239 PCT/US98/12332 20 with 750 mM NaCl, 75 mM sodium citrate at 420C.

Another example is the use of 50% formamide, 5 x SSC (0.75 M NaC1, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 0.1% sodium pyrophosphate, 5 x Denhardt's solution, sonicated salmon sperm DNA tg/ml), 0.1% SDS, and 10% dextran sulfate at 420C, with washes at 42°C in 0.2 x SSC and 0.1% SDS.

Moderate stringency conditions refers to hybridization conditions used to identify nucleic acids that have a lower degree of identity to the probe than do nucleic acids identified under high stringency conditions. Moderate stringency conditions can include the use of higher ionic strength and/or lower temperatures for washing of the hybridization membrane, compared to the ionic strength and temperatures used for high stringency hybridization. For example, a wash solution comprising 0.060 M NaCl/0.0060 M sodium citrate (4X SSC) and 0.1% sodium lauryl sulfate (SDS) can be used at 50 0 C, with a last wash in lX SSC, at 65 0

C.

Alternatively, a hybridization wash in 1X SSC at 37°C can be used.

Hybridization can also be done by Northern analysis (Northern blotting), a method used to identify RNAs that hybridize to a known probe such as an oligonucleotide, DNA fragment, cDNA or fragment thereof, or RNA fragment. The probe is labeled with a radioisotope such as 32 P, by biotinylation or with an enzyme. The RNA to be analyzed can be usually electrophoretically separated on an agarose or polyacrylamide gel, transferred to nitrocellulose, nylon, or other suitable membrane, and hybridized with the probe, using standard techniques well known in the art such as those described in sections 7.39-7.52 of Sambrook et al., supra.

WO 98/56239 PCT/US98/12332 21 A polypeptide of the invention comprises an isolated polypeptide having a mutant amino acid sequence, as well as derivatives and analogs thereof. See, e.g., the mutant amino acid sequences of Fig. 3. By "isolated" is meant a polypeptide that is expressed and produced in an environment other than the environment in which the polypeptide is naturally expressed and produced. For example, a plant polypeptide is isolated when expressed and produced in bacteria or fungi. A polypeptide of the invention also comprises variants of the mutant desaturase polypeptides disclosed herein, as discussed above.

In one embodiment of the claimed invention, a plant contains both a delta-12 desaturase mutation and a delta-15 desaturase mutation. Such plants can have a fatty acid composition comprising very high oleic acid and very low alpha-linolenic acid levels. Mutations in delta-12 desaturase and delta-15 desaturase may be combined in a plant by making a genetic cross between delta-12 desaturase and delta-15 desaturase single mutant lines. A plant having a mutation in delta-12 fatty acid desaturase is crossed or mated with a second plant having a mutation in delta-15 fatty acid desaturase. Seeds produced from the cross are planted and the resulting plants are selfed in order to obtain progeny seeds.

These progeny seeds are then screened in order to identify those seeds carrying both mutant genes.

Alternatively, a line possessing either a delta-12 desaturase or a delta-15 desaturase mutation can be subjected to mutagenesis to generate a plant or plant line having mutations in both delta-12 desaturase and desaturase. For example, the IMC 129 line has a mutation in the coding region (Glu 106 to Lys10 6 of the D form of the microsomal delta-12 desaturase structural gene. Cells seeds) of this line can be WO 98/56239 PCT/US98/12332 22 mutagenized to induce a mutation in a delta-15 desaturase gene, resulting in a plant or plant line carrying a mutation in a delta-12 fatty acid desaturase gene and a mutation in a delta-15 fatty acid desaturase gene.

Progeny includes descendants of a particular plant or plant line, seeds developed on an instant plant are descendants. Progeny of an instant plant include seeds formed on F 2

F

3 and subsequent generation plants, or seeds formed on BC,, BC 2

BC

3 and subsequent generation plants.

Plants according to the invention preferably contain an altered fatty acid composition. For example, oil obtained from seeds of such plants may have from about 69 to about 90% oleic acid, based on the total fatty acid composition of the seed. Such oil preferably has from about 74 to about 90% oleic acid, more preferably from about 80 to about 90% oleic acid. In some embodiments, oil obtained from seeds produced by plants of the invention may have from about 2.0% to about 5.0% saturated fatty acids, based on total fatty acid composition of the seeds. In some embodiments, oil obtained from seeds of the invention may have from about to about 14.0% linoleic acid, or from about 0.5% to about 10.0% a-linolenic acid.

Oil composition typically is analyzed by crushing and extracting fatty acids from bulk seed samples seeds). Fatty acid triglycerides in the seed are hydrolyzed and converted to fatty acid methyl esters.

Those seeds having an altered fatty acid composition may be identified by techniques known to the skilled artisan, gas-liquid chromatography (GLC) analysis of a bulked seed sample or of a single half-seed. Half-seed analysis is well known in the art to be useful because the viability of the embryo is maintained and thus those seeds having a desired fatty acid profile may be planted WO 98/56239 PCT/US98/12332 23 to from the next generation. However, half-seed analysis is also known to be an inaccurate representation of genotype of the seed being analyzed. Bulk seed analysis typically yields a more accurate representation of the fatty acid profile of a given genotype. Fatty acid composition can also be determined on larger samples, oil obtained by pilot plant or commercial scale refining, bleaching and deodorizing of endogenous oil in the seeds.

The nucleic acid fragments of the invention can be used as markers in plant genetic mapping and plant breeding programs. Such markers may include restriction fragment length polymorphism (RFLP), random amplification polymorphism detection (RAPD), polymerase chain reaction (PCR) or self-sustained sequence replication (3SR) markers, for example. Marker-assisted breeding techniques may be used to identify and follow a desired fatty acid composition during the breeding process.

Marker-assisted breeding techniques may be used in addition to, or as an alternative to, other sorts of identification techniques. An example of marker-assisted breeding is the use of PCR primers that specifically amplify a sequence containing a desired mutation in delta-12 desaturase or delta-15 desaturase.

Methods according to the invention are useful in that the resulting plants and plant lines have desirable seed fatty acid compositions as well as superior agronomic properties compared to known lines having altered seed fatty acid composition. Superior agronomic characteristics include, for example, increased seed germination percentage, increased seedling vigor, increased resistance to seedling fungal diseases (damping off, root rot and the like), increased yield, and improved standability.

WO 98/56239 PCT/S98/1 2332 24 While the invention is susceptible to various modifications and alternative forms, certain specific embodiments thereof are described in the general methods and examples set forth below. For example the invention may be applied to all Brassica species, including B.

rapa, B. juncea, and B. hirta, to produce substantially similar results. It should be understood, however, that these examples are not intended to limit the invention to the particular forms disclosed but, instead the invention is to cover all modifications, equivalents and alternatives falling within the scope of the invention.

This includes the use of somaclonal variation; physical or chemical mutagenesis of plant parts; anther, microspore or ovary culture followed by chromosome doubling; or self- or cross-pollination to transmit the fatty acid trait, alone or in combination with other traits, to develop new Brassica lines.

EXAMPLE 1 Mutagenesis Seeds of Westar, a Canadian (Brassica napus) spring canola variety, were subjected to chemical mutagenesis. Westar is a registered Canadian spring variety with canola quality. The fatty acid composition of field-grown Westar, 3.9% C 16 0 1.9% C 18 0 67.5% C 18 1 17.6% C 18 :2 7.4% C 8 3 C20:1 C 22 1 has remained stable under commercial production, with deviation, since 1982.

Prior to mutagenesis, 30,000 seeds of B. napus cv.

Westar seeds were preimbibed in 300-seed lots for two hours on wet filter paper to soften the seed coat. The preimbibed seeds were placed in 80 mM ethylmethanesulfonate (EMS) for four hours. Following mutagenesis, the seeds were rinsed three times in distilled water. The seeds were sown in 48-well flats WO 98/56239 PCT/US98/12332 25 containing Pro-Mix. Sixty-eight percent of the mutagenized seed germinated. The plants were maintained at 25oC/150C, 14/10 hr day/night conditions in the greenhouse. At flowering, each plant was individually self-pollinated.

M

2 seed from individual plants were individually catalogued and stored, approximately 15,000 M 2 lines was planted in a summer nursery in Carman, Manitoba. The seed from each selfed plant were planted in 3-meter rows with 6-inch row spacing. Westar was planted as the check variety. Selected lines in the field were selfed by bagging the main raceme of each plant. At maturity, the selfed plants were individually harvested and seeds were catalogued and stored to ensure that the source of the seed was known.

Self-pollinated M 3 seed and Westar controls were analyzed in 10-seed bulk samples for fatty acid composition via gas chromatography. Statistical thresholds for each fatty acid component were established using a Z-distribution with a stringency level of 1 in 10,000. Mean and standard deviation values were determined from the non-mutagenized Westar control population in the field. The upper and lower statistical thresholds for each fatty acid were determined from the mean value of the population the standard deviation, multiplied by the Z-distribution. Based on a population size of 10,000, the confidence interval is 99.99%.

The selected M 3 seeds were planted in the greenhouse along with Westar controls. The seed was sown in 4-inch pots containing Pro-Mix soil and the plants were maintained at 250C/150C, 14/10 hr day/night cycle in the greenhouse. At flowering, the terminal raceme was self-pollinated by bagging. At maturity, selfed M 4 seed was individually harvested from each plant, labelled, and stored to ensure that the source of the seed was known.

WO 98/56239 PCT/US98/12332 26 The M 4 seed was analyzed in 10-seed bulk samples.

Statistical thresholds for each fatty acid component were established from 259 control samples using a Zdistribution of 1 in 800. Selected M 4 lines were planted in a field trial in Carman, Manitoba in 3-meter rows with 6-inch spacing. Ten M 4 plants in each row were bagged for self-pollination. At maturity, the selfed plants were individually harvested and the open pollinated plants in the row were bulk harvested. The M, seed from single plant selections was analyzed in 10-seed bulk samples and the bulk row harvest in 50-seed bulk samples.

Selected M. lines were planted in the greenhouse along with Westar controls. The seed was grown as previously described. At flowering the terminal raceme was self-pollinated by bagging. At maturity, selfed M 6 seed was individually harvested from each plant and analyzed in 10-seed bulk samples for fatty acid composition.

Selected M 6 lines were entered into field trials in Eastern Idaho. The four trial locations were selected for the wide variability in growing conditions. The locations included Burley, Tetonia, Lamont and Shelley (Table The lines were planted in four 3-meter rows with an 8-inch spacing, each plot was replicated four times. The planting design was determined using a Randomized Complete Block Designed. The commercial cultivar Westar was used as a check cultivar. At maturity the plots were harvested to determine yield.

Yield of the entries in the trial was determined by taking the statistical average of the four replications.

The Least Significant Difference Test was used to rank the entries in the randomized complete block design.

WO 98/56239 PCT/US98/12332 27 TABLE 7 Trial Locations for Selected Fatty Acid Mutants LOCATION SITE CHARACTERIZATIONS BURLEY Irrigated. Long season. High temperatures during flowering.

TETONIA Dryland. Short season. Cool temperatures.

LAMONT Dryland. Short season. Cool temperatures.

SHELLEY Irrigated. Medium season. High temperatures during flowering.

To determine the fatty acid profile of entries, plants in each plot were bagged for self-pollination.

The M 7 seed from single plants was analyzed for fatty acids in ten-seed bulk samples.

To determine the genetic relationships of the selected fatty acid mutants crosses were made. Flowers of M 6 or later generation mutations were used in crossing.

F1 seed was harvested and analyzed for fatty acid composition to determine the mode of gene action. The F 1 progeny were planted in the greenhouse. The resulting plants were self-pollinated, the F 2 seed harvested and analyzed for fatty acid composition for allelism studies.

The F 2 seed and parent line seed was planted in the greenhouse, individual plants were self-pollinated. The

F

3 seed of individual plants was tested for fatty acid composition using 10-seed bulk samples as described previously.

In the analysis of some genetic relationships dihaploid populations were made from the microspores of the F, hybrids. Self-pollinated seed from dihaploid plants were analyzed for fatty acid analysis using methods described previously.

For chemical analysis, 10-seed bulk samples were hand ground with a glass rod in a 15-mL polypropylene tube and extracted in 1.2 mL 0.25 N KOH in 1:1 ether/methanol. The sample was vortexed for 30 sec. and WO 98/56239 PCT/US98/12332 28 heated for 60 sec. in a 60 0 C water bath. Four mL of saturated NaC1 and 2.4 mL of iso-octane were added, and the mixture was vortexed again. After phase separation, 600 yL of the upper organic phase were pipetted into individual vials and stored under nitrogen at -5 0 C. One AL samples were injected into a Supelco SP-2330 fused silica capillary column (0.25 mm ID, 30 M length, 0.20 ym df).

The gas chromatograph was set at 180 0 C for minutes, then programmed for a 2oC/minute increase to 212 0 C, and held at this temperature for 1.5 minutes.

Total run time was 23 minutes. Chromatography settings were: Column head pressure 15 psi, Column flow (He) 0.7 mL/min., Auxiliary and Column flow 33 mL/min., Hydrogen flow 33 mL/min., Air flow 400 mL/min., Injector temperature 250 0 C, Detector temperature 300 0 C, Split vent 1/15.

Table 8 describes the upper and lower statistical thresholds for each fatty acid of interest.

WO 98/56239 PCT/US98/12332 29 TABLE 8 Statistical Thresholds for Specific Fatty Acids Derived from Control Westar Plantings Percent Fatty Acids Genotype C 16 0

C

18 i 0

C

1 :1 C 18 2

C

18 :3 Sats*

M

3 Generation(l in 10,000 rejection rate) Lower 3.3 1.4 13.2 5.3 Upper 4.3 2.5 71.0 21.6 9.9 8.3

M

4 Generation(l in 800 rejection rate) Lower 3.6 0.8 12.2 3.2 5.3 Upper 6.3 3.1 76.0 32.4 9.9 11.2 Ms Generation (1 in 755 rejection rate) Lower 2.7 0.9 9.6 2.6 Upper 5.7 2.7 80.3 26.7 9.6 10.0 *Sats=Total Saturate Content EXAMPLE 2 High Oleic Acid Canola Lines In the studies of Example 1, at the M 3 generation, 31 lines exceeded the upper statistical threshold for oleic acid Line W7608.3 had 71.2% oleic acid.

At the M 4 generation, its selfed progeny (W7608.3.5, since designated A129.5) continued to exceed the upper statistical threshold for C,, 8 with 78.8% oleic acid. M s seed of five self-pollinated plants of line A129.5 (ATCC 40811) averaged 75.0% oleic acid. A single plant selection, A129.5.3 had 75.6% oleic acid. The fatty acid composition of this high oleic acid mutant, which was stable under both field and greenhouse conditions to the

M

7 generation, is summarized in Table 9. This line also stably maintained its mutant fatty acid composition to the M generation in field trials in multiple locations.

WO 98/56239 PCT/US98/12332 30 Over all locations the self-pollinated plants (A129) averaged 78.3% oleic acid. The fatty acid composition of the A129 for each Idaho trial location are summarized in Table 10. In multiple location replicated yield trials, A129 was not significantly different in yield from the parent cultivar Westar.

The canola oil of A129, after commercial processing, was found to have superior oxidative stability compared to Westar when measured by the Accelerated Oxygen Method (AOM), American Oil Chemists' Society Official Method Cd 12-57 for fat stability; Active Oxygen Method (revised 1989). The AOM of Westar was 18 AOM hours and for A129 was 30 AOM hours.

TABLE 9 Fatty Acid Composition of a High Oleic Acid Canola Line Produced by Seed Mutaqenesis Percent Fatty Acids Genotype C 16 0 C 18 0

C

18 :1 C 1 :2 C 18 :3 Sats Westar 3.9 1.9 67.5 17.6 7.4 W7608.3 3.9 2.4 71.2 12.7 6.1 7.6

(M

3 W7608.3.5 3.9 2.0 78.8 7.7 3.9 7.3 A129.5.3 3.8 2.3 75.6 9.5 4.9 7.6 (Ms) Sats=Total Saturate Content WO 98/56239 PCT/US98/12332 31 TABLE Fatty Acid Composition of a Mutant High Oleic Acid Line at Different Field Locations in Idaho Percent Fatty Acids Location C 16 0

C

18 :0 C 18 :I C18:2 C18 3 Sats Burley 3.3 2.1 77.5 8.1 6.0 Tetonia 3.5 3.4 77.8 6.5 4.7 Lamont 3.4 1.9 77.8 7.4 6.5 6.3 Shelley 3.3 2.6 80.0 5.7 4.5 7.7 Sats=Total Saturate Content The genetic relationship of the high oleic acid mutation A129 to other oleic desaturases was demonstrated in crosses made to commercial canola cultivars and a low linolenic acid mutation. A129 was crossed to the commercial cultivar Global (C16: 0

C

18 0 C18 62.9%,C18:2 20.0%, C18:3 Approximately 200 F 2 individuals were analyzed for fatty acid composition.

The results are summarized in Table 11. The segregation fit 1:2:1 ratio suggesting a single co-dominant gene controlled the inheritance of the high oleic acid phenotype.

TABLE 11 Genetic Studies of A129 X Global Frequency C18:0 Genotype Content(%) Observed Expected od-od- 77.3 43 47 od-od+ 71.7 106 94 od+od+ 66.1 49 47 A cross between A129 and IMC 01, a low linolenic acid variety (C,,16 C o:0 C,8:1 66.4%, C18:2 18.1%, CI8:3 was made to determine the inheritance of the oleic acid desaturase and linoleic acid WO 98/56239 PCT/US98/12332 32 desaturase. In the F, hybrids both the oleic acid and linoleic acid desaturase genes approached the mid-parent values indicating a co-dominant gene actions. Fatty acid analysis of the F 2 individuals confirmed a 1:2:1:2:4:2:1:2:1 segregation of two independent, codominant genes (Table 12). A line was selected from the cross of A129 and IMC01 and designated as IMC130 (ATCC deposit no. 75446) as described in U.S. Patent Application No. 08/425,108, incorporated herein by reference.

TABLE 12 Genetic Studies of A129 X IMC 01 Frequency Genotype Ratio Observed Expected od-od-ld-ld- 1 11 12 od-od-ld-ld+ 2 30 24 od-od-ld+ld+ 1 10 12 od-od+ld-ld- 2 25 24 od-od+ld-ld+ 4 54 47 od-od+ld+ld+ 2 18 24 od+od+ld-ld- 1 7 12 od+od+ld-ld+ 2 25 24 od+od+ld+ld+ 1 8 12 An additional high oleic acid line, designated A128.3, was also produced by the disclosed method. A seed bulk analysis of this line showed the following fatty acid composition: C 16 0

C

18 0 Cg8:1 77.3%, C 18 :2 C 18 :3 FDA Sats Total Sats This line also stably maintained its mutant fatty acid composition to the M 7 generation. In multiple locations replicated yield trials, A128 was not significantly different in yield from the parent cultivar Westar.

A129 was crossed to A128.3 for allelism studies.

Fatty acid composition of the F 2 seed showed the two lines to be allelic. The mutational events in A129 and A128.3 although different in origin were in the same gene.

WO 98/56239 PCT/US98/12332 33 An additional high oleic acid line, designated M3028.-10 (ATCC 75026), was also produced by the disclosed method in Example 1. A 10-seed bulk analysis of this line showed the following fatty acid composition:

C

16 :0 C 18 :0 C 18 :1 77.3%, C 18 :2 C 18 :3 FDA Saturates Total Saturates In a single location replicated yield trial M3028.10 was not significantly different in yield from the parent cultivar Westar.

EXAMPLE 3 Low Linoleic Acid Canola In the studies of Example 1, at the M 3 generation, lines exceeded the lower statistical threshold for linoleic acid Line W12638.8 had 9.4% linoleic acid. At the M 4 and M s generations, its selfed progenies [W12638.8, since designated A133.1 (ATCC 40812)] continued to exceed the statistical threshold for low C 18 :2 with linoleic acid levels of 10.2% and 8.4%, respectively. The fatty acid composition of this low linoleic acid mutant, which was stable to the M 7 generation under both field and greenhouse conditions, is summarized in Table 13. In multiple location replicated yield trials, A133 was not significantly different in yield from the parent cultivar Westar. An additional low linoleic acid line, designated M3062.8 (ATCC 75025), was also produced by the disclosed method. A 10-seed bulk analysis of this line showed the following fatty acid composition: C 16 C o 18 C18,: 77.1%, C18:2 C18:3 FDA Sats-6.1%. This line has also stably maintained its mutant fatty acid composition in the field and greenhouse.

"WO 98/56239 PCT/US98/12332 34 TABLE 13 Fatty Acid Composition of a Low Linoleic Acid Canola Line Produced by Seed Mutagenesis Percent Fatty Acids Genotype C 16 0 Cie: 0

C

18 C8: 2

C

18 3 Satsb Westar 3.9 1.9 67.5 17.6 7.4 W12638.8 3.9 2.3 75.0 9.4 6.1

(M

3 W12638.8.1 4.1 1.7 74.6 10.2 5.9 7.1

(M

4 A133.1.8 3.8 2.0 77.7 8.4 5.0 (Ms) aLetter and numbers up to second decimal point indicate the plant line. Number after second decimal point indicates an individual plant.

bSats=Total Saturate Content EXAMPLE 4 Low Linolenic and Linoleic Acid Canola In the studies of Example 1, at the M 3 generation, 57 lines exceeded the lower statistical threshold for linolenic acid Line W14749.8 had 5.3% linolenic acid and 15.0% linoleic acid. At the M 4 and M generations, its selfed progenies [W14749.8, since designated M3032 (ATCC 75021)] continued to exceed the statistical threshold for low with linolenic acid levels of 2.7% and respectively, and for a low sum of linolenic and linoleic acids with totals of 11.8% and 12.5% respectively. The fatty acid composition of this low linolenic acid plus linoleic acid mutant, which was stable to the M s generation under both field and greenhouse conditions, is summarized in Table 14. In a single location replicated yield trial M3032 was not significantly different in yield from the parent cultivar (Westar).

WO 98/56239 PCT/US98/12332 35 TABLE 14 Fatty Acid Composition of a Low Linolenic Acid Canola Line Produced by Seed Mutagenesis Percent Fatty Acids Genotype C 16 :0 C 18 :0 C 18 1

C

18 :2 C 18 3 Sats Westar 3.9 1.9 67.5 17.6 7.4 W14749.8 4.0 2.5 69.4 15.0 5.3

(M

3 M3032.8 3.9 2.4 77.9 9.1 2.7 6.4

(M

4 M3032.1 3.5 2.8 80.0 10.2 2.3 (Ms) Sats=Total Saturate Content EXAMPLE Canola Lines Q508 and 04275 Seeds of the B. napus line IMC-129 were mutagenized with methyl N-nitrosoguanidine (MNNG). The MNNG treatment consisted of three parts: pre-soak, mutagen application, and wash. A 0.05M Sorenson's phosphate buffer was used to maintain pre-soak and mutagen treatment pH at 6.1. Two hundred seeds were treated at one time on filter paper (Whatman #3M) in a petri dish (100mm x 15mm). The seeds were pre-soaked in mis of 0.05M Sorenson's buffer, pH 6.1, under continued agitation for two hours. At the end of the pre-soak period, the buffer was removed from the plate.

A 10mM concentration of MNNG in 0.05M Sorenson's buffer, pH 6.1, was prepared prior to use. Fifteen ml of MNNG was added to the seeds in each plate. The seeds were incubated at 22 0 C+3 0 C in the dark under constant agitation for four hours. At the end of the incubation period, the mutagen solution was removed.

The seeds were washed with three changes of distilled water at 10 minute intervals. The fourth wash WO 98/56239 PCT/US98/1 2332 36 was for thirty minutes. This treatment regime produced an LD60 population.

Treated seeds were planted in standard greenhouse potting soil and placed into an environmentally controlled greenhouse. The plants were grown under sixteen hours of light. At flowering, the racemes were bagged to produce selfed seed. At maturity, the M2 seed was harvested. Each M2 line was given an identifying number. The entire MNNG-treated seed population was designated as the Q series.

Harvested M2 seeds was planted in the greenhouse.

The growth conditions were maintained as previously described. The racemes were bagged at flowering for selfing. At maturity, the selfed M3 seed was harvested and analyzed for fatty acid composition. For each M3 seed line, approximately 10-15 seeds were analyzed in bulk as described in Example 1.

High oleic-low linoleic M3 lines were selected from the M3 population using a cutoff of >82% oleic acid and linoleic. From the first 1600 M3 lines screened for fatty acid composition, Q508 was identified.

The Q508 M3 generation was advanced to the M4 generation in the greenhouse. Table 15 shows the fatty acid composition of Q508 and IMC 129. The M4 selfed seed maintained the selected high oleic-low linoleic acid phenotype (Table 16).

TABLE Fatty Acid Composition of A129 and High Oleic Acid M3 Mutant 0508 Line 16:0 18:0 18:1 18:2 18:3 A129* 4.0 2.4 77.7 7.8 4.2 Q508 3.9 2.1 84.9 2.4 2.9 *Fatty acid composition of A129 is the average of 50 self-pollinated plants grown with the M3 population WO 98/56239 PCTIUS98/12332 37

M

4 generation Q508 plants had poor agronomic qualities in the field compared to Westar. Typical plants were slow growing relative to Westar, lacked early vegetative vigor, were short in stature, tended to be chlorotic and had short pods. The yield of Q508 was very low compared to Westar.

The M 4 generation Q508 plants in the greenhouse tended to be reduced in vigor compared to Westar.

However, Q508 yields in the greenhouse were greater than Q508 yields in the field.

TABLE 16 Fatty Acid Composition of Seed Oil from Greenhouse-Grown 0508, IMC 129 and Westar.

FDA

Line 16:0 18:0 18:1 18:2 18:3 Sats .IMC 4.0 2.4 77.7 7.8 4.2 6.4 129a Westarb 3.9 1.9 67.5 17.6 7.4 >5.8 Q508c 3.9 2.1 84.9 2.4 2.9 aAverage of 50 self-pollinated plants bData from Example 1 CAverage of 50 self-pollinated plants Nine other M4 high-oleic low-linoleic lines were also identified: Q3603, Q3733, Q4249, Q6284, Q6601, Q6761, Q7415, Q4275, and Q6676. Some of these lines had good agronomic characteristics and an elevated oleic acid level in seeds of about 80% to about 84%.

Q4275 was crossed to the variety Cyclone. After selfing for seven generations, mature seed was harvested from 93GS34-179, a progeny line of the Q4275 Cyclone cross. Referring to Table 17, fatty acid composition of a bulk seed sample shows that 93GS34 retained the seed WO 98/56239 PCT/US98/12332 38 fatty acid composition of Q4275. 93GS34-179 also maintained agronomically desirable characteristics.

After more than seven generations of selfing of Q4275, plants of Q4275, IMC 129 and 93GS34 were field grown during the summer season. The selections were tested in 4 replicated plots (5 feet X 20 feet) in a randomized block design. Plants were open pollinated.

No selfed seed was produced. Each plot was harvested at maturity, and a sample of the bulk harvested seed from each line was analyzed for fatty acid composition as described above. The fatty acid compositions of the selected lines are shown in Table 17.

TABLE 17 Fatty Acid Composition of Field Grown IMC 129, Q4275 and 93GS34 Seeds Line Fatty Acid Composition

C

16 0

C

18 0

C

1

B:

1 C18: 2

C

18 3 FDA Sats IMC 129 3.3 2.4 76.7 8.7 5.2 5.7 Q4275 3.7 3.1 82.1 4.0 3.5 6.8 93GS34-179 2.6 2.7 85.0 2.8 3.3 5.3 The results shown in Table 17 show that Q4275 maintained the selected high oleic low linoleic acid phenotype under field conditions. The agronomic characteristics of Q4275 plants were superior to those of Q508.

M

4 generation Q508 plants were crossed to a dihaploid selection of Westar, with Westar serving as the female parent. The resulting F1 seed was termed the 92EF population. About 126 Fl individuals that appeared to have better agronomic characteristics than the Q508 parent were selected for selfing. A portion of the F 2 seed from such individuals was replanted in the field.

I, WO 98/56239 PCT/US98/1 2332 39 Each F2 plant was selfed and a portion of the resulting F3 seed was analyzed for fatty acid composition. The content of oleic acid in F 3 seed ranged from 59 to 79%.

No high oleic individuals were recovered with good agronomic type.

A portion of the F 2 seed of the 92EF population was planted in the greenhouse to analyze the genetics of the Q508 line. F 3 seed was analyzed from 380 F2 individuals. The C 8 levels of F 3 seed from the greenhouse experiment is depicted in Figure 1. The data were tested against the hypothesis that Q508 contains two mutant genes that are semi-dominant and additive: the original IMC 129 mutation as well as one additional mutation. The hypothesis also assumes that homozygous Q508 has greater than 85% oleic acid and homozygous Westar WO 98/56239 PCT/US98/12332 40 has 62-67% oleic acid. The possible genotypes at each gene in a cross of Q508 by Westar may be designated as: AA Westar Fad2a BB Westar Fad2 b aa Q508 Fad2abb Q508 Fad2 b Assuming independent segregation, a 1:4:6:4:1 ratio of phenotypes is expected. The phenotypes of heterozygous plants are assumed to be indistinguishable and, thus, the data were tested for fit to a 1:14:1 ratio of homozygous Westar: heterozygous plants: homozygous Q508.

Phenotypic of Ratio Westar Alleles Genotype 1 4 AABB(Westar) 4 3 AABb,AaBB,AABb,AaBB 6 2 AaBb,AAbb,AaBb,AaBb,aaBB,AaBb 4 1 Aabb,aaBb,Aabb,aaBb 1 0 aabb (Q508) Using Chi-square analysis, the oleic acid data fit a 1:14:1 ratio. It was concluded that Q508 differs from Westar by two major genes that are semi-dominant and additive and that segregate independently. By comparison, the genotype of IMC 129 is aaBB.

The fatty acid composition of representative F3 individuals having greater than 85% oleic acid in seed oil is shown in Table 18. The levels of saturated fatty acids are seen to be decreased in such plants, compared to Westar.

WO 98/56239 PCT/US98/12332 41 TABLE 18 92EF F 3 Individuals with >85% C. in Seed Oil F3 Plant Fatty Acid Composition Identifier Id iier C16:0 C18:0 C18:1 C18:2 C18:3 FDASA +38068 3.401 1.582 85.452 2.134 3.615 4.983 +38156 3.388 1.379 85.434 2.143 3.701 4.767 +38171 3.588 1.511 85.289 2.367 3.425 5.099 +38181 3.75 1.16 85.312 2.968 3.819 4.977 +38182 3.529 0.985 85.905 2.614 3.926 4.56 +38191 3.364 1.039 85.737 2.869 4.039 4.459 +38196 3.557 1.182 85.054 2.962 4.252 4.739 +38202 3.554 1.105 86.091 2.651 3.721 4.713 +38220 3.093 1.16 86.421 1.931 3.514 4.314 +38236 3.308 1.349 85.425 2.37 3.605 4.718 +38408 3.617 1.607 85.34 2.33 3.562 5.224 +38427 3.494 1.454 85.924 2.206 3.289 4.948 +38533 3.64 1.319 85.962 2.715 3.516 4.959 EXAMPLE 6 Leaf and Root Fatty Acid Profiles of Canola Lines IMC-129, 0508, and Westar Plants of Q508, IMC 129 and Westar were grown in the greenhouse. Mature leaves, primary expanding leaves, petioles and roots were harvested at the 6-8 leaf stage, frozen in liquid nitrogen and stored at -70 0 C. Lipid extracts were analyzed by GLC as described in Example 1.

The fatty acid profile data are shown in Table 19.

The data in Table 19 indicate that total leaf lipids in Q508 are higher in content than the C 18 :2 plus C 18 :3 content. The reverse is true for Westar and IMC 129. The difference in total leaf lipids between Q508 and IMC 129 is consistent with the hypothesis that a second Fad2 gene is mutated in Q508.

WO 98/56239 PCT/US98/12332 42 The C 16 :3 content in the total lipid fraction was about the same for all three lines, suggesting that the plastid FadC gene product was not affected by the Q508 mutations. To confirm that the FadC gene was not mutated, chloroplast lipids were separated and analyzed.

No changes in chloroplast C16i, C 16 :2 or C 16 3 fatty acids were detected in the three lines. The similarity in plastid leaf lipids among Q508, Westar and IMC 129 is consistent with the hypothesis that the second mutation in Q508 affects a microsomal Fad2 gene and not a plastid FadC gene.

TABLE 19 MATURE EXPANDING LEAF LEAF PETIOLE ROOT West. 129 3Q508 West. 129 3Q508 West. 129 3Q508 West. 129 3Q508 16:0 12.1 11.9 10.1 16.4 16.1 11.3 21.7 23.5 11.9 21.1 21.9 12.0 16:1 0.8 0.6 1.1 0.7 0.6 1.1 1.0 1.3 1.4 16:2 2.3 2.2 2.0 2.8 3.1 2.8 1.8 2.2 1.8 16:3 14.7 15.0 14.0 6.3 5.4 6.9 5.7 4.6 5.7 18:0 2.2 1.6 1.2 2.5 2.8 1.5 3.7 4.0 1.6 3.6 2.9 18:1 2.8 4.9 16.7 3.8 8.3 38.0 4.9 12.9 46.9 3.5 6.1 68.8 18:2 12.6 11.5 6.8 13.3 13.8 4.9 20.7 18.3 5.2 28.0 30.4 4.4 18:3 50.6 50.3 46.0 54.2 50.0 33.5 40.4 33.2 25.3 43.8 38.7 12.3 EXAMPLE 7 Sequences of Mutant and Wild-Type Delta-12 Fatty Acid Desaturases from B. napus Primers specific for the FAD2 structural gene were used to clone the entire open reading frame (ORF) of the D and F delta-12 desaturase genes by reverse transcriptase polymerase chain reaction (RT-PCR). RNA from seeds of IMC 129, Q508 and Westar plants was isolated by standard methods and was used as template.

The RT-amplified fragments were used for nucleotide sequence determination. The DNA sequence of each gene WO 98/56239 PCT/US98/1 2332 43 from each line was determined from both strands by standard dideoxy sequencing methods.

Sequence analysis revealed a G to A transversion at nucleotide 316 (from the translation initiation codon) of the D gene in both IMC 129 and Q508, compared to the sequence of Westar. The transversion changes the codon at this position from GAG to AAG and results in a nonconservative substitution of glutamic acid, an acidic residue, for lysine a basic residue. The presence of the same mutation in both lines was expected since the Q508 line was derived from IMC 129. The same base change was also detected in Q508 and IMC 129 when RNA from leaf tissue was used as template.

The G to A mutation at nucleotide 316 was confirmed by sequencing several independent clones containing fragments amplified directly from genomic DNA of IMC 129 and Westar. These results eliminated the possibility of a rare mutation introduced during reverse transcription and PCR in the RT-PCR protocol. It was concluded that the IMC 129 mutant is due to a single base transversion at nucleotide 316 in the coding region of the D gene of rapeseed microsomal delta 12-desaturase.

A single base transition from T to A at nucleotide 515 of the F gene was detected in Q508 compared to the Westar sequence. The mutation changes the codon at this position from CTC to CAC, resulting in the nonconservative substitution of a non-polar residue, leucine, for a polar residue, histidine, in the resulting gene product. No mutations were found in the F gene sequence of IMC 129 compared to the F gene sequence of Westar.

These data support the conclusion that a mutation in a delta-12 desaturase gene sequence results in alterations in the fatty acid profile of plants containing such a mutated gene. Moreover, the data show WO 98/56239 PCT/US98/12332 44 that when a plant line or species contains two delta-12 desaturase loci, the fatty acid profile of an individual having two mutated loci differs from the fatty acid profile of an individual having one mutated locus.

The mutation in the D gene of IMC 129 and Q508 mapped to a region having a conserved amino acid motif (His-Xaa-Xaa-Xaa-His) found in cloned delta-12 and deltamembrane bound-desaturases (Table TABLE Alignment of Amino Acid Sequences of Cloned Canola Membrane Bound-Desaturases Desaturase Gene Sequencea Position Canola-fad2-D(mutant) AHKCGH 109-114 Canola-Fad2-D AHECGH 109-114 Canola-Fad2-F AHECGH 109-114 Canola-FadC GHDCAH 170-175 Canola-fad3 (mutant) GHKCGH 94-99 Canola-Fad3 GHDCGH 94-99 Canola-FadD GHDCGH 125-130 (FadD Plastid delta 15, Fad3 Microsomal (FadC Plastid delta-12, Fad2 Microsomal delta-12) a One letter amino acid code; conservative substitutions are underlined; non-conservative substitutions are in bold.

EXAMPLE 8 Transcription and Translation of Microsomal Delta-12 Fatty Acid Desaturases Transcription in vivo was analyzed by RT-PCR analysis of stage II and stage III developing seeds and leaf tissue. The primers used to specifically amplify delta-12 desaturase F gene RNA from the indicated tissues WO 98/56239 PCT/US98/12332 45 were sense primer 5'-GGATATGATGATGGTGAAAGA-3' and antisense primer 5'-TCTTTCACCATCATCATATCC-3'. The primers used to specifically amplify delta-12 desaturase D gene RNA from the indicated tissues were sense primer 5'-GTTATGAAGCAAAGAAGAAAC-3' and antisense primer GTTTCTTCTTTGCTTCATAAC-3'. The results indicated that mRNA of both the D and F gene was expressed in seed and leaf tissues of IMC 129, Q508 and wild type Westar plants.

In vitro transcription and translation analysis showed that a peptide of about 46 kD was made. This is the expected size of both the D gene product and the F gene product, based on sum of the deduced amino acid sequence of each gene and the cotranslational addition of a microsomal membrane peptide.

These results rule out the possibility that nonsense or frameshift mutations, resulting in a truncated polypeptide gene product, are present in either the mutant D gene or the mutant F gene. The data, in conjunction with the data of Example 7, support the conclusion that the mutations in Q508 and IMC 129 are in delta-12 fatty acid desaturase structural genes encoding desaturase enzymes, rather than in regulatory genes.

EXAMPLE 9 Development of Gene-Specific PCR Markers Based on the single base change in the mutant D gene of IMC 129 described in above, two 5' PCR primers were designed. The nucleotide sequence of the primers differed only in the base (G for Westar and A for IMC 129) at the 3' end. The primers allow one to distinguish between mutant fad2-D and wild-type Fad2-D alleles in a DNA-based PCR assay. Since there is only a single base difference in the 5' PCR primers, the PCR assay is very sensitive to the PCR conditions such as annealing WO 98/56239 PCT/US98/12332 46 temperature, cycle number, amount, and purity of DNA templates used. Assay conditions have been established that distinguish between the mutant gene and the wild type gene using genomic DNA from IMC 129 and wild type plants as templates. Conditions may be further optimized by varying PCR parameters, particularly with variable crude DNA samples. A PCR assay distinguishing the single base mutation in IMC 129 from the wild type gene along with fatty acid composition analysis provides a means to simplify segregation and selection analysis of genetic crosses involving plants having a delta-12 fatty acid desaturase mutation.

EXAMPLE Transformation with Mutant and Wild Type Fad3 Genes B. napus cultivar Westar was transformed with mutant and wild type Fad3 genes to demonstrate that the mutant Fad3 gene for canola cytoplasmic linoleic desaturase delta-15 desaturase is nonfunctional.

Transformation and regeneration were performed using disarmed Agrobacterium tumefaciens essentially following the procedure described in WO 94/11516.

Two disarmed Agrobacterium strains were engineered, each containing a Ti plasmid having the appropriate gene linked to a seed-specific promoter and a corresponding termination sequence. The first plasmid, pIMC110, was prepared by inserting into a disarmed Ti vector the full length wild type Fad3 gene in sense orientation (nucleotides 208 to 1336 of SEQ ID 6 in WO 93/11245), flanked by a napin promoter sequence positioned 5' to the Fad3 gene and a napin termination sequence positioned 3' to the Fad3 gene. The rapeseed napin promoter is described in EP 0255378.

The second plasmid, pIMC205, was prepared by inserting a mutated Fad3 gene in sense orientation into a disarmed Ti vector. The mutant sequence contained WO 98/56239 PCT/US98/12332 47 mutations at nucleotides 411 and 413 of the microsomal Fad3 gene described in W093/11245, thus changing the sequence for codon 96 from GAC to AAG. The amino acid at codon 96 of the gene product was thereby changed from aspartic acid to lysine. See Table 20. A bean (Phaseolus vulgaris) phaseolin (7S seed storage protein) promoter fragment of 495 base pairs, starting with TGGTCTTTTGGT-3', was placed 5' to the mutant Fad3 gene and a phaseolin termination sequence was placed 3' to the mutant Fad3 gene. The phaseolin sequence is described in Doyle et al., (1986) J. Biol. Chem. 261:9228-9238) and Slightom et al., (1983) Proc. Natl. Acad. Sci. USA 80:1897-1901.

The appropriate plasmids were engineered and transferred separately to Agrobacterium strain LBA4404.

Each engineered strain was used to infect 5 mm segments of hypocotyl explants from Westar seeds by cocultivation.

Infected hypocotyls were transferred to callus medium and, subsequently, to regeneration medium. Once discernable stems formed from the callus, shoots were excised and transferred to elongation medium. The elongated shoots were cut, dipped in Rootone", rooted on an agar medium and transplanted to potting soil to obtain fertile Tl plants. T2 seeds were obtained by selfing the resulting T1 plants.

Fatty acid analysis of T2 seeds was carried out as described above. The results are summarized in Table 21.

Of the 40 transformants obtained using the pIMC110 plasmid, 17 plants demonstrated wild type fatty acid profiles and 16 demonstrated overexpression. A proportion of the transformants are expected to display an overexpression phenotype when a functioning gene is transformed in sense orientation into plants.

Of the 307 transformed plants having the pIMC205 gene, none exhibited a fatty acid composition indicative WO 98/56239 PCT/US98/12332 48 of overexpression. This result indicates that the mutant fad3 gene product is non-functional, since some of the transformants would have exhibited an overexpression phenotype if the gene product were functional.

WO 98/56239 PCT/US98/12332 49 TABLE 21 Overexpression and Co-suppression Events in Westar Populations Transformed with pIMC205 or pIMCll0.

Construct Number of a-Linolenic Overexpression Co-Suppression Wild Type Transformants Acid Events Events Events Range(%) linolenic) linolenic) pIMC110 40 2.4 20.6 16 7 17 pIMC205 307 4.6 10.4 0 0 307 Fatty acid compositions of representative transformed plants are presented in Table 22. Lines 652- 09 and 663-40 are representative of plants containing pIMC110 and exhibiting an overexpression and a cosuppression phenotype, respectively. Line 205-284 is representative of plants containing pIMC205 and having the mutant fad3 gene.

TABLE 22 Fatty Acid Composition of T2 Seed From Westar Transformed With pIMC205 or pIMC110.

Line Fatty Acid Composition C16:0 C18 C1 818:1 C1B:2 C18:3 652-09 pIMC110 4.7 3.3 65.6 8.1 14.8 overexpression 663-40 4.9 2.1 62.5 23.2 3.6 pIMC11O co-suppression 205-284 3.7 1.8 68.8 15.9 6.7 pIMC205 EXAMPLE 11 Sequences of Wild Type and Mutant Fad2-D and Fad2-F High molecular weight genomic DNA was isolated from leaves of Q4275 plants (Example 5) and from Westar and Bridger canola plants. This DNA was used as template for amplification of Fad2-D and Fad2-F genes by polymerase chain reaction (PCR). PCR amplifications were carried out in a total volume of 100 il and contained 0.3 Ag genomic DNA, 200 pM deoxyribonucleoside triphosphates, 3 mM MgSO 4 1-2 Units DNA polymerase and iX Buffer WO 98/56239 PCT/US98/12332 50 (supplied by the DNA polymerase manufacturer). Cycle conditions were: 1 cycle for 1 min at 950C, followed by cycles of 1 min at 94°C, 2 min at 550C and 3 min at 730C.

The Fad2-D gene was amplified once using Elongase® (Gibco-BRL). PCR primers were: CAUCAUCAUCAUCTTCTTCGTAGGGTTCATCG (SEQ ID NO:23) and CUACUACUACUATCATAGAAGAGAAAGGTTCAG (SEQ ID NO:24) for the and 3' ends of the gene, respectively.

The Fad2-F gene was independently amplified 4 times, twice with Elongase® and twice with Taq polymerase (Boehringer Mannheim). The PCR primers used were: 5'CAUCAUCAUCAUCATGGGTGCACGTGGAAGAA3' (SEQ ID NO:25) and 5'CUACUACUACUATCTTTCACCATCATCATATCC3' (SEQ ID NO:26) for the 5' and 3' ends of the gene, respectively.

Amplified DNA products were resolved on an agarose gel, purified by JetSorb® and then annealed into pAMP1 (Gibco-BRL) via the (CAU) 4 and (CUA), sequences at the ends of the primers, and transformed into E. coli The Fad2-D and Fad2-F inserts were sequenced on both strands with an ABI PRISM 310 automated sequencer (Perkin-Elmer) following the manufacturer's directions, using synthetic primers, AmpliTaq® DNA polymerase and dye terminator.

The Fad2-D gene was found to have intron-like sequences upstream of the ATG start codon (SEQ ID and SEQ ID NO:31). As expected, the coding sequence of the gene derived from IMC 129 contained a G to A mutation at nucleotide 316 (Fig. 2).

A single base transversion from G to A at nucleotide 908 was detected in the F gene sequence of the Q4275 amplified products, compared to the wild type F gene sequence (Fig. This mutation changes the codon at amino acid 303 from GGA to GAA, resulting in the nonconservative substitution of a glutamic acid residue for WO 98/56239 PCT/US98/12332 51 a glycine residue (Table 3 and Fig. Expression of the mutant Q4275 Fad2-F delta-12 desaturase gene in plants alters the fatty acid composition, as described hereinabove.

EXAMPLE 12 Sequence of Wild Type Fad2-U High molecular weight genomic DNA was isolated from the leaves of Bridger and Westar Brassica plants by standard methods. The Fad2-U gene was amplified in a 100 il total reaction containing 1 AM of each primer, 0.3 pg genomic DNA, 200 pM dNTP, 3 mM MgSO 4 Ix Buffer (supplied by the manufacturer of the DNA polymerase), and 1-2 units of Elongase DNA polymerase (BRL). The amplification conditions included one cycle for 1 min at 95 0 C, cycles of denaturation at 94 0 C for 1 min, annealing at 0 C for 2 min, and elongation at 72 0 C for 3 min.

Subsequently, the reaction was incubated at 72 0 C for an additional 10 min. Fad2U gene was amplified twice from Westar and twice from Bridger genomic DNAs using the following primers: end primer 5'(CAU) CTTCTTCGTAGGGTTCATCG3' (SEQ ID NO:23) 3' end primer 5' (CUA) CATAACTTATTGTTGTACCAG3' (SEQ ID NO:27) Amplified DNA products were purified and sequenced as described in Example 11. The Fad2-U sequence contains an intron-like sequence upstream of the ATG start codon (SEQ ID NO:28).

To the extent not already indicated, it will be understood by those of ordinary skill in the art that any one of the various specific embodiments herein described and illustrated may be further modified to incorporate features shown in other of the specific embodiments.

WO 98/56239 PCT/US98/12332 52 The foregoing detailed description has been provided for a better understanding of the invention only and no unnecessary limitation should be understood therefrom as some modifications will be apparent to those skilled in the art without deviating from the spirit and scope of the appended claims.

WO 98/56239 PCTIUS98/12332 53 SEQUENCE LISTING GENERAL INFORMATION APPLICANT: CARGILL, INCORPORATED (ii) TITLE OF THE INVENTION: FATTY ACID DESATURASES AND MUTANT SEQUENCES THEREOF (iii) NUMBER OF SEQUENCES: 31 (iv) CORRESPONDENCE ADDRESS: ADDRESSEE: Fish Richardson P.A.

STREET: 60 South Sixth Street, Suite 3300 CITY: Minneapolis STATE: MN COUNTRY: USA ZIP: 55402 COMPUTER READABLE FORM: MEDIUM TYPE: Diskette COMPUTER: IBM Compatible OPERATING SYSTEM: DOS SOFTWARE: FastSEQ for Windows Version (vi) CURRENT APPLICATION DATA: APPLICATION NUMBER: FILING DATE: 11-JUN-97

CLASSIFICATION:

(viii) ATTORNEY/AGENT INFORMATION: NAME: Lundquist, Ronald C REGISTRATION NUMBER: 37,875 REFERENCE/DOCKET NUMBER: 07148/067WO1 (ix) TELECOMMUNICATION INFORMATION: TELEPHONE: 612-335-5070 TELEFAX: 612-288-9696

TELEX:

INFORMATION FOR SEQ ID NO:1: SEQUENCE CHARACTERISTICS: LENGTH: 1155 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (iii) HYPOTHETICAL: YES (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: WO 98/56239 PCT/US98/12332 t.

54 ORGANISM: Brassica napus (ix) FEATURE: OTHER INFORMATION: Wild type Fad2.

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:

ATG

Met 1 GGT GCA GGT GGA AGA ATG CAA GTG Gly Ala Gly Gly Arg Met Gin Val 5 CCT CCC TCC AAG Pro Pro Ser Lys AAG TCT Lys Ser GAA ACC GAC Glu Thr Asp GTC GGA GAA Val Gly Glu

ACC

Thr ATC AAG CGC GTA Ile Lys Arg Val

CCC

Pro 25 TGC GAG ACA CCC Cys Glu Thr Pro CCC TTC ACT Pro Phe Thr AAA CCC TCG Lys Arg Ser CTC AAC AAA GCA Leu Lys Lys Ala

ATC

Ile CCA CCG CAC TGT Pro Pro His Cys ATC CCT Ile Pro CGC TCT TTC TCC Arg Ser Phe Ser CTC ATC TOG GAC Leu Ile Trp Asp ATC ATA GCC TCC Ile Ile Ala Ser

TGC

Cys TTC TAC TAC NTC Phe Tyr Tyr Xaa

GCC

Ala 70 ACC ACT TAC TTC Thr Thr Tyr Phe

CCT

Pro 75 CTC CTC CCT CAC Leu Leu Pro His

CCT

Pro CTC TCC TAC TTC Leu Ser Tyr Phe TGC CCT CTC TAC Trp Pro Leu Tyr CCC TGC CAA GGG Ala Cys Gin Gly TGC GTC Cys Val CTA ACC GGC Leu Thr Cly AGC CAC TAC Ser Asp Tyr 115

GTC

Val 100 TGG GTC ATA GCC Trp Val Ile Ala

CAC

His 105 GAA TGC GGC CAC Glu Cys Cly His CAC CCC TTC His Ala Phe 110 TTC CAC TCC Phe His Ser 336 384 CAG TGG CTT GAC Gin Trp Leu Asp

CAC

Asp 120 ACC CTC GGT CTC Thr Val Gly Leu

ATC

Ile 125 TTC CTC Phe Leu 130 CTC GTC CCT TAC Leu Val Pro Tyr

TTC

Phe 135 TCC TGG AAG TAC Ser Trp Lys Tyr

AGT

Ser 140 CAT CCC AGC CAC His Arg Ser His

CAT

His 145 TCC AAC ACT GGC TCC CTC GAG AGA GAC Ser Asn Thr Gly Ser Leu Glu Arg Asp

GAA

Glu 155 GTC TTT CTC CCC Vai Phe Val Pro

AAG

Lys 160 AAG AAG TCA GAC Lys Lys Ser Asp ATC AAC TGG TAC GGC AAG TAC CTC AAC AAC CCT TTC Ile Lys Trp Tyr Cly Lys Tyr Leu Asn Asn Pro Leu 165 170 175 OGA CCC ACC Gly Arg Thr TAC TTA GCC Tyr Leu Ala 195

GTG

Va1 180 ATG TTA ACG OTT Met Leu Thr Val

CAG

Gin 185 TTC ACT CTC GGC Phe Thr Leu Gly TGO CCC TTC Trp Pro Leu 190 GGC TTC CGT Gly Phe Arg TTC AAC GTC TCG Phe Asn Val Ser

GGA

Cly 200 AGA CCT TAC CAC Arg Pro Tyr Asp

GGC

Gly 205 TGC CAT Cys His 210 TTC CAC CCC AAC Phe His Pro Asn

GCT

Ala 215 CCC ATC TAC AAC Pro Ile Tyr Asn

CAC

Asp 220 CGC GAG CGT CTC Arg Glu Arg Leu WO 98/56239 PCT/US98/12332 55 CAG ATA TAC ATC TCC GAC GCT Gin Ile Tyr Ile Ser Asp Ala 225 230

TTC

Phe

GGA

Gly

TTG

Leu

GAT

Asp

TTG

Leu 305

CCG

Pro

ATA

Ile

GTT

Val

GAC

Asp

CGT

Arg

GTC

Val

CAG

Gin

TGG

Trp 290

AAC

Asn

TTC

Phe

AAG

Lys

AAG

Lys

AGG

Arg 370

TAC

Tyr

CCG

Pro

CAC

His 275

TTC

Phe

AAG

Lys

TCC

Ser

CCG

Pro

GCG

Ala 355

CAA

Gin

GCC

Ala

CTT

Leu 260

ACG

Thr

AGG

Arg

GTC

Val

ACG

Thr

ATA

Ile 340

ATG

Met

GGT

Gly

GCC

Ala 245

CTG

Leu

CAT

His

GGA

Gly

TTC

Phe

ATG

Met 325

CTG

Leu

TGG

Trp

GAG

Glu

GGC

Gly

ATT

Ile

CCT

Pro

GCT

Ala

CAC

His 310

CCG

Pro

GGA

Gly

AGG

Arg

AAG

Lys

CAG

Gin

GTC

Val

TCC

Ser

TTG

Leu 295

AAT

Asn

CAT

His

GAG

Glu

GAG

Glu

AAA

Lys 375 GGC ATC CTC GCC Gly Ile Leu Ala 235 GGA GTG GCC TCG Gly Val Ala Ser 250 AAT GGT TTC CTC Asn Gly Phe Leu 265 CTG CCT CAC TAC Leu Pro His Tyr 280 GCT ACC GTT GAC Ala Thr Val Asp ATT ACC GAC ACG Ile Thr Asp Thr 315 TAT CAC GCG ATG Tyr His Ala Met 330 TAT TAT CAG TTC Tyr Tyr Gin Phe 345 GCG AAG GAG TGT Ala Lys Glu Cys 360 Val

ATG

Met

GTG

Val

GAT

Asp

AGA

Arg 300

CAC

His

GAA

Glu

GAT

Asp

ATC

Ile GTC TGC TAC Cys

GTC

Val

TTG

Leu

TCG

Ser 285

GAC

Asp

GTG

Val

GCT

Ala

GGG

Gly

TAT

Tyr 365 Tyr

TGC

Cys

ATC

Ile 270

TCC

Ser

TAC

Tyr

GCC

Ala

ACC

Thr

ACG

Thr 350

GTG

Val GGT CTC Gly Leu 240 TTC TAC Phe Tyr 255 ACT TAC Thr Tyr GAG TGG Glu Trp GGA ATC Gly Ile CAT CAT His His 320 AAG GCG Lys Ala 335 CCG GTG Pro Val GAA CCG Glu Pro 720 768 816 864 912 960 1008 1056 1104 GGT GTG TTC TGG TAC AAC AAT AAG TTA T 1153 Gly Val Phe Trp Tyr 380 Asn Asn Lys Leu 1155 INFORMATION FOR SEQ ID NO:2: SEQUENCE CHARACTERISTICS: LENGTH: 384 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: Met Gly Ala Gly Gly Arg Met Gin Val Ser Pro Pro Ser Lys Lys Ser 1 5 10 Glu Thr Asp Thr Ile Lys Arg Val Pro Cys Glu Thr Pro Pro Phe Thr 25 Val Gly Glu Leu Lys Lys Ala Ile Pro Pro His Cys Phe Lys Arg Ser 40 WO 98/56239 WO 9856239PCT/US98/1 2332 56 Ile Pro Arg Ser Phe Ser Cys Leu Leu Ser Phe His 145 Lys Gly Tyr Cys Gin 225 Phe Gly Leu Asp Leu 305 Pro Ile Val Asp Phe Ser Thr Asp Leu 130 Ser Lys Arg Leu His 210 Ile Arg Val Gin Trp, 290 Asn Phe Lys Lys Arg 3170 Tyr Tyr Gly Tyr 115 Leu Asn Ser Thr Ala 195 Phe Tyr Tyr Pro His 275 Phe Lys Ser Pro Ala 355 Gin Tyr Phe Vai 100 Gin Vai Thr Asp Val 180 Phe His Ile Ala Leu 260 Thr Arg Vai Thr Ile 340 Met Gly Xaa Ala Trp, Trp Pro Gly Ile 165 Met Asn Pro Ser Ala 245 Leu His Giy Phe Met 325 Leu Trp Glu Ala 70 Trp Val Leu Tyr Ser 150 Lys Leu Val Asn Asp 230 Gly Ile Pro Ala His 310 Pro Gly Arg Lys Tyr Leu Ile 55 Thr Thr Tyr Pro Leu Tyr Ile Ala His 105 Asp Asp Thr 120 Phe Ser Trp, 135 Leu Giu Arg Trp Tyr Gly Thr Vai Gin 185 Ser Gly Arg 200 Ala Pro Ile 215 Ala Gly Ile Gin Gly Val Vai Asn Gly 265 Ser Leu Pro 280 Leu Ala Thr 295 Asn Ile Thr His Tyr His Glu Tyr Tyr 345 Giu Ala Lys 360 Lys Gly Val 375 Trp Phe Trp 90 Giu Vai Lys Asp Lys 170 Phe Pro Tyr Leu Ala 250 Phe His Val Asp Ala 330 Gin Giu Phe Asp Pro 75 Ala Cys Gly Tyr Giu 155 Tyr Thr Tyr Asn Ala 235 Ser Leu Tyr Asp Thr 315 Met Phe Cys Trp Ile Leu Cys Gly Leu Ser 140 Val Leu Leu Asp Asp 220 Val Met Val Asp Arg 300 His Glu Asp Ile Tyr 380 Ile Leu Gin His Ile 125 His Phe Asn Gly Gly 205 Arg Cys Val Leu Ser 285 Asp Val Ala Gly Tyr 365 Asn Ile Pro Gly His 110 Phe Arg Val Asn Trp 190 Gly Giu Tyr Cys Ile 270 Ser Tyr Ala Thr Thr 350 Val Asn Ala His Cys Al a His Ser Pro Pro 175 Pro Phe Arg Gly Phe 255 Thr Giu Gly His Lys 335 Pro Giu Lys Ser Pro Val1 Phe Ser His Lys 160 Leu Leu Arg Leu Leu 240 Tyr Tyr Trp Ile His 320 Ala Val Pro Leu WO 98/56239 PCT/US98/12332 57 INFORMATION FOR SEQ ID NO:3: SEQUENCE CHARACTERISTICS: LENGTH: 1155 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (iii) HYPOTHETICAL: YES (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: Brassica napus (ix) FEATURE: OTHER INFORMATION: G to A transversion mutation at nucleotide 316.

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: ATG GGT GCA GGT Met Gly Ala Gly 1 AGA ATG CAA GTG Arg Met Gin Val

TCT

Ser 10 CCT CCC TCC AAG Pro Pro Ser Lys AAG TCT Lys Ser GAA ACC GAC Glu Thr Asp GTC GGA GAA Val Gly Glu

ACC

Thr ATC AAG CGC GTA Ile Lys Arg Val TGC GAG ACA CCG Cys Glu Thr Pro CCC TTC ACT Pro Phe Thr AAA CGC TCG Lys Arg Ser CTC AAG AAA GCA Leu Lys Lys Ala

ATC

Ile 40 CCA CCG CAC TGT Pro Pro His Cys

TTC

Phe ATC CCT Ile Pro CGC TCT TTC TCC Arg Ser Phe Ser CTC ATC TGG GAC Leu Ile Trp Asp ATC ATA GCC TCC Ile Ile Ala Ser

TGC

Cys TTC TAC TAC NTC Phe Tyr Tyr Xaa

GCC

Ala 70 ACC ACT TAC TTC Thr Thr Tyr Phe CTC CTC CCT CAC Leu Leu Pro His

CCT

Pro 192 240 288 CTC TCC TAC TTC Leu Ser Tyr Phe

GCC

Ala TGG CCT CTC TAC Trp Pro Leu Tyr GCC TGC CAA GGG Ala Cys Gin Gly TGC GTC Cys Val CTA ACC GGC Leu Thr Gly AGC GAC TAC Ser Asp Tyr 115

GTC

Val 100 TGG GTC ATA GCC Trp Val Ile Ala

CAC

His 105 AAG TGC GGC CAC Lys Cys Gly His CAC GCC TTC His Ala Phe 110 TTC CAC TCC Phe His Ser CAG TGG CTT GAC Gin Trp Leu Asp ACC GTC GGT CTC Thr Val Gly Leu TTC CTC Phe Leu 130 CTC GTC CCT TAC Leu Val Pro Tyr

TTC

Phe 135 TCC TGG AAG TAC AGT CAT CGC AGC CAC Ser Trp Lys Tyr Ser His Arg Ser His 140

CAT

His 145 TCC AAC ACT GGC Ser Asn Thr Gly TCC CTC Ser Leu 150 GAG AGA GAC Glu Arg Asp GTG TTT GTC CCC Val Phe Val Pro WO 98/56239 PCT/US98/12332 58 AAG AAG TCA GAC Lys Lys Ser Asp

ATC

Ile 165 AAG TGG TAC GGC Lys Trp Tyr Gly AAG TAC CTC AAC AAC CCT TTG Lys Tyr Leu Asn Asn Pro Leu 170 175 GGA CGC ACC Gly Arg Thr TAC TTA GCC Tyr Leu Ala 195

GTG

Val 180 ATG TTA ACG GTT Met Leu Thr Val

CAG

Gin 185 TTC ACT CTC GGC Phe Thr Leu Gly TGG CCG TTG Trp Pro Leu 190 GGC TTC CGT Gly Phe Arg 576 624 TTC AAC GTC TCG Phe Asn Val Ser

GGA

Gly 200 AGA CCT TAC GAC Arg Pro Tyr Asp

GGC

Gly 205 TGC CAT Cys His 210 TTC CAC CCC AAC Phe His Pro Asn

GCT

Ala 215 CCC ATC TAC AAC Pro Ile Tyr Asn CGC GAG CGT CTC Arg Glu Arg Leu

CAG

Gin 225 ATA TAC ATC TCC Ile Tyr Ile Ser

GAC

Asp 230 GCT GGC ATC CTC Ala Gly Ile Leu

GCC

Ala 235 GTC TGC TAC GGT Val Cys Tyr Gly

CTC

Leu 240 TTC CGT TAC GCC Phe Arg Tyr Ala GCC GGC CAG GGA GTG GCC TCG ATG GTC TGC TTC TAC Ala Gly Gin Gly Val Ala Ser Met Val Cys Phe Tyr 245 250 255 GGA GTC CCG Gly Val Pro TTG CAG CAC Leu Gin His 275

CTT

Leu 260 CTG ATT GTC AAT Leu Ile Val Asn

GGT

Gly 265 TTC CTC GTG TTG Phe Leu Val Leu ATC ACT TAC Ile Thr Tyr 270 TCC GAG TGG Ser Glu Trp ACG CAT CCT TCC Thr His Pro Ser

CTG

Leu 280 CCT CAC TAC GAT Pro His Tyr Asp

TCG

Ser 285 GAT TGG Asp Trp 290 TTC AGG GGA GCT Phe Arg Gly Ala

TTG

Leu 295 GCT ACC GTT GAC Ala Thr Val Asp

AGA

Arg 300 GAC TAC GGA ATC Asp Tyr Gly Ile

TTG

Leu 305 AAC AAG GTC TTC Asn Lys Val Phe AAT ATT ACC GAC Asn Ile Thr Asp CAC GTG GCC CAT His Val Ala His

CAT

His 320 CCG TTC TCC ACG Pro Phe Ser Thr

ATG

Met 325 CCG CAT TAT CAC Pro His Tyr His

GCG

Ala 330 ATG GAA GCT ACC Met Glu Ala Thr AAG GCG Lys Ala 335 912 960 1008 1056 1104 ATA AAG CCG Ile Lys Pro GTT AAG GCG Val Lys Ala 355

ATA

Ile 340 CTG GGA GAG TAT Leu Gly Glu Tyr

TAT

Tyr 345 CAG TTC GAT GGG Gin Phe Asp Gly ACG CCG GTG Thr Pro Val 350 GTG GAA CCG Val Glu Pro ATG TGG AGG GAG Met Trp Arg Glu

GCG

Ala 360 AAG GAG TGT ATC Lys Glu Cys Ile GAC AGG Asp Arg 370 CAA GGT GAG AAG Gin Gly Glu Lys GGT GTG TTC TGG Gly Val Phe Trp

TAC

Tyr 380 AAC AAT AAG TTA T 1153 Asn Asn Lys Leu 1155 INFORMATION FOR SEQ ID NO:4: SEQUENCE CHARACTERISTICS: LENGTH: 384 amino acids TYPE: amino acid WO 98/56239 WO 9856239PCT/US98/1 2332 59 TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: Met Giy Ala Gly Giu Val Ile Cys Leu Leu Ser Phe His 145 Lys Gly Tyr Cys Gin 225 Phe Gly Leu Thr Gly Pro Phe Ser Thr Asp Leu 130 Ser Lys Arg Leu His 210 Ile Arg Val Gin Asp Giu Arg Tyr Tyr Gly Tyr 115 Leu Asn Ser Thr Aia 195 Phe Tyr Tyr Pro His 275 Thr Leu Ser Tyr Phe Vai 100 Gin Val Thr Asp Val 180 Phe His Ile Ala Leu 260 Thr Gly Ile Lys Phe Xaa Ala Trp, Trp Pro Gly Ile 165 Met Asn Pro Ser Ala 245 Leu His Arg Met Gin Val Ser Pro Pro Ser Lys Lys Lys Ser Ala 70 Trp Val Leu Tyr Ser 150 Lys Leu Val Asn Asp 230 Gly Ile Pro Arg Ala Tyr Thr Pro Ile Asp Phe 135 Leu Trp Thr Ser Ala 215 Ala Gin Val Ser Val Ile 40 Leu Thr Leu Ala Asp 120 Ser Glu Tyr Val Giy 200 Pro Gly Gly Asn Leu 280 Pro 25 Pro Ile Tyr Tyr His 105 Thr Trp Arg Gly Gin 185 Arg Ile Ile Val Gly 265 Pro Cys Pro Trp Phe Trp, 90 Lys Val Lys Asp Lys 170 Phe Pro Tyr Leu Ala 250 Phe His Giu His Asp Pro 75 Al a Cys Gly Tyr Glu 155 Tyr Thr Tyr Asn Ala 235 Ser Leu Tyr Pro Phe Ile Leu Gin His Ile 125 His Phe Asn Gly Gly 205 Arg Cys Val Leu Ser 285 Pro Lys Ile Pro Gly His 110 Phe Arg Val Asn Trp 190 Gly Giu Tyr Cys I le 270 Ser Lys Ser Phe Thr Arg Ser Ala Ser His Pro Cys Val Ala Phe His Ser Ser His Pro Lys 160 Pro Leu 175 Pro Leu Phe Arg Arg Leu Giy Leu 240 Phe Tyr 255 Thr Tyr Giu Trp, Asp Trp 290 Phe Arg Gly Ala Leu Ala Thr Val Asp Asp Tyr Gly Ile WO 98/56239 PCT/US98/12332 60 Leu Asn Lys Val Phe His Asn Ile Thr Asp Thr His Val Ala His His 305 310 315 320 Pro Phe Ser Thr Met Pro His Tyr His Ala Met Glu Ala Thr Lys Ala 325 330 335 Ile Lys Pro Ile Leu Gly Glu Tyr Tyr Gin Phe Asp Gly Thr Pro Val 340 345 350 Val Lys Ala Met Trp Arg Glu Ala Lys Glu Cys Ile Tyr Val Glu Pro 355 360 365 Asp Arg Gin Gly Glu Lys Lys Gly Val Phe Trp Tyr Asn Asn Lys Leu 370 375 380 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 1155 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (iii) HYPOTHETICAL: YES (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: Brassica napus (ix) FEATURE: OTHER INFORMATION: Wild type Fad2.

(xi) SEQUENCE DESCRIPTION: SEQ ID ATG GGT GCA GGT GGA AGA ATG CAA GTG TCT CCT CCC TCC AAA AAG TCT 48 Met Gly Ala Gly Gly Arg Met Gin Val Ser Pro Pro Ser Lys Lys Ser 1 5 10 GAA ACC GAC AAC ATC AAG CGC GTA CCC TGC GAG ACA CCG CCC TTC ACT 96 Glu Thr Asp Asn Ile Lys Arg Val Pro Cys Glu Thr Pro Pro Phe Thr 25 WO 98/56239 PCT/US98/12332 61 GTC GGA GAA CTC AAG AAA GCA ATC CCA CCG CAC TGT TTC AAA CGC TCG 144 Val Gly Glu Leu Lys Lys Ala Ile Pro Pro His Cys Phe Lys Arg Ser 40 ATC CCT CGC TCT TTC TCC TAC CTC ATC TGG GAC ATC ATC ATA GCC TCC 192 Ile Pro Arg Ser Phe Ser Tyr Leu Ile Trp Asp Ile Ile Ile Ala Ser 55 TGC TTC TAC TAC GTC GCC ACC ACT TAC TTC CCT CTC CTC CCT CAC CCT 240 Cys Phe Tyr Tyr Val Ala Thr Thr Tyr Phe Pro Leu Leu Pro His Pro 70 75 CTC TCC TAC TTC GCC TGG CCT CTC TAC TGG GCC TGC CAG GGC TGC GTC 288 Leu Ser Tyr Phe Ala Trp Pro Leu Tyr Trp Ala Cys Gin Gly Cys Val 90 CTA ACC GGC GTC TGG GTC ATA GCC CAC GAG TGC GGC CAC CAC GCC TTC 336 Leu Thr Gly Val Trp Val Ile Ala His Glu Cys Gly His His Ala Phe 100 105 110 AGC GAC TAC CAG TGG CTG GAC GAC ACC GTC GGC CTC ATC TTC CAC TCC 384 Ser Asp Tyr Gin Trp Leu Asp Asp Thr Val Gly Leu Ile Phe His Ser 115 120 125 TTC CTC CTC GTC CCT TAC TTC TCC TGG AAG TAC AGT CAT CGA CGC CAC 432 Phe Leu Leu Val Pro Tyr Phe Ser Trp Lys Tyr Ser His Arg Arg His 130 135 140 CAT TCC AAC ACT GGC TCC CTC GAG AGA GAC GAA GTG TTT GTC CCC AAG 480 His Ser Asn Thr Gly Ser Leu Glu Arg Asp Glu Val Phe Val Pro Lys 145 150 155 160 AAG AAG TCA GAC ATC AAG TGG TAC GGC AAG TAC CTC AAC AAC CCT TTG 528 Lys Lys Ser Asp Ile Lys Trp Tyr Gly Lys Tyr Leu Asn Asn Pro Leu 165 170 175 GGA CGC ACC GTG ATG TTA ACG GTT CAG TTC ACT CTC GGC TGG CCT TTG 576 Gly Arg Thr Val Met Leu Thr Val Gin Phe Thr Leu Gly Trp Pro Leu 180 185 190 TAC TTA GCC TTC AAC GTC TCG GGG AGA CCT TAC GAC GGC GGC TTC GCT 624 Tyr Leu Ala Phe Asn Val Ser Gly Arg Pro Tyr Asp Gly Gly Phe Ala 195 200 205 TGC CAT TTC CAC CCC AAC GCT CCC ATC TAC AAC GAC CGC GAG CGT CTC 672 Cys His Phe His Pro Asn Ala Pro Ile Tyr Asn Asp Arg Glu Arg Leu 210 215 220 CAG ATA TAC ATC TCC GAC GCT GGC ATC CTC GCC GTC TGC TAC GGT CTC 720 Gin Ile Tyr Ile Ser Asp Ala Gly Ile Leu Ala Val Cys Tyr Gly Leu 225 230 235 240 TAC CGC TAC GCT GCT GTC CAA GGA GTT GCC TCG ATG GTC TGC TTC TAC 768 Tyr Arg Tyr Ala Ala Val Gin Gly Val Ala Ser Met Val Cys Phe Tyr 245 250 255 GGA GTT CCG CTT CTG ATT GTC AAT GGG TTC TTA GTT TTG ATC ACT TAC 816 Gly Val Pro Leu Leu Ile Val Asn Gly Phe Leu Val Leu Ile Thr Tyr 260 265 270 TTG CAG CAC ACG CAT CCT TCC CTG CCT CAC TAT GAC TCG TCT GAG TGG 864 Leu Gin His Thr His Pro Ser Leu Pro His Tyr Asp Ser Ser Glu Trp 275 280 285 .WO 98/56239 PCT/US98/12332

GAT

Asp

TTG

Leu 305

CTG

Leu

ATA

Ile

GTT

Val

GAC

Asp

GA

TGG

Trp 290

AAC

Asn

TTC

Phe

AAG

Lys

AAG

Lys

AGG

Arg 370

AGG

Arg

GTC

Val

ACC

Thr

ATA

Ile 340

ATG

Met

GGT

Gly

GCT

Ala

CAC

His 310

CCG

Pro

GGA

Gly

AGG

Arg

AAG

Lys

TTG

Leu 295

AAT

Asn

CAT

His

GAG

Glu

GAG

Glu

AAA

Lys 375 62 GCC ACC GTT Ala Thr Val ATC ACG GAC Ile Thr Asp TAT CAT GCG Tyr His Ala 330 TAT TAT CAG Tyr Tyr Gin 345 GCG AAG GAG Ala Lys Glu 360 GGT GTG TTC Gly Val Phe

GAC

Asp

ACG

Thr 315

ATG

Met

TTG

Leu

TGT

Cys

TGG

Trp

AGA

Arg 300

CAC

His

GAA

Glu

CAT

His

ATC

Ile

TAC

Tyr 380

GAC

Asp

GTG

Val

GCT

Ala

GGG

Gly

TAT

Tyr 365

AAC

Asn

TAC

Tyr

GCG

Ala

ACG

Thr

ACG

Thr 350

GTG

Val

AAT

Asn

GGA

Gly

CAT

His

AAG

Lys 335

CCG

Pro

GAA

Glu

AAG

Lys ATC 912 Ile CAC 960 His 320 GCG 1008 Ala GTG 1056 Val CCG 1104 Pro TTA T 1153 Leu 1155 INFORMATION FOR SEQ ID NO:6: SEQUENCE CHARACTERISTICS: LENGTH: 384 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein Met 1 Glu Val Ile Cys Leu Leu Ser Phe (xi) SEQUENCE Gly Ala Gly Gly 5 Thr Asp Asn Ile Gly Glu Leu Lys Pro Arg Ser Phe Phe Tyr Tyr Val Ser Tyr Phe Ala Thr Gly Val Trp 100 Asp Tyr Gin Trp 115 Leu Leu Val Pro 130 DESCRIPTION: SEQ ID Arg Met Gin Val Ser 10 Lys Arg Val Pro Cys 25 Lys Ala Ile Pro Pro 40 Ser Tyr Leu Ile Trp 55 Ala Thr Thr Tyr Phe 70 Trp Pro Leu Tyr Trp 90 Val Ile Ala His Glu 105 Leu Asp Asp Thr Val 120 Tyr Phe Ser Trp Lys 135 NO:6: Pro Pro Glu Thr His Cys Asp Ile Pro Leu 75 Ala Cys Cys Gly Gly Leu Tyr Ser 140 Ser Pro Phe Ile Leu Gin His Ile 125 His Lys Pro Lys Ile Pro Gly His 110 Phe Arg Lys Phe Arg Ala His Cys Ala His Arg Ser Thr Ser Ser Pro Val Phe Ser His WO 98/56239 PCT/US98/12332 63 His Ser Asn Thr Gly 145 Ser Leu Glu Arg Asp Lys Gly Tyr Cys Gin 225 Tyr Gly Leu Asp Leu 305 Leu Ile Val Lys Arg Leu His 210 Ile Arg Val Gin Trp 290 Asn Phe Lys Lys Ser Thr Ala 195 Phe Tyr Tyr Pro His 275 Leu Lys Ser Pro Ala 355 Asp Val 180 Phe His Ile Ala Leu 260 Thr Arg Val Thr Ile 340 Met Ile 165 Met Asn Pro Ser Ala 245 Leu His Gly Phe Met 325 Leu Trp Lys Leu Val Asn Asp 230 Val Ile Pro Ala His 310 Pro Gly Arg Trp Thr Ser Ala 215 Ala Gin Val Ser Leu 295 Asn His Glu Glu Gly Gin 185 Arg Ile Ile Val Gly 265 Pro Thr Thr His Tyr 345 Lys Glu 155 Tyr Thr Tyr Asn Ala 235 Ser Leu Tyr Asp Thr 315 Met Leu Cys Val Leu Leu Asp Asp 220 Val Met Val Asp Arg 300 His Glu His Ile Tyr 380 Phe Asn Gly Gly 205 Arg Cys Val Leu Ser 285 Asp Val Ala Gly Tyr 365 Val Asn Trp 190 Gly Glu Tyr Cys Ile 270 Ser Tyr Ala Thr Thr 350 Val Lys 160 Leu Leu Ala Leu Leu 240 Tyr Tyr Trp Ile His 320 Ala Val Pro Asp Arg 370 Gin Gly Glu Lys Lys Gly Val Phe Trp 375 Asn Asn Lys Leu INFORMATION FOR SEQ ID NO:7: SEQUENCE CHARACTERISTICS: LENGTH: 1155 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (iii) HYPOTHETICAL: YES (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: Brassica napus (ix) FEATURE: WO. 98/56239 PCT/US98/12332 64 OTHER INFORMATION: T to A transversion mutation at nucleotide 515.

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:

ATG

Met 1 GGT GCA GGT Gly Ala Gly GGA AGA ATG CAA GTG TCT CCT CCC TCC AAA AAG TCT Gly Arg Met Gin Val Ser Pro Pro Ser Lys Lys Ser 5 10 GAA ACC GAC Glu Thr Asp GTC GGA GAA Val Gly Glu ATC AAG CGC GTA Ile Lys Arg Val

CCC

Pro TGC GAG ACA CCG CCC TTC ACT Cys Glu Thr Pro Pro Phe Thr CTC AAG AAA GCA Leu Lys Lys Ala CCA CCG CAC TGT Pro Pro His Cys

TTC

Phe AAA CGC TCG Lys Arg Ser ATC CCT Ile Pro CGC TCT TTC TCC Arg Ser Phe Ser CTC ATC TGG GAC Leu Ile Trp Asp

ATC

Ile ATC ATA GCC TCC Ile Ile Ala Ser

TGC

Cys TTC TAC TAC GTC Phe Tyr Tyr Val ACC ACT TAC TTC Thr Thr Tyr Phe CTC CTC CCT CAC Leu Leu Pro His

CCT

Pro CTC TCC TAC TTC Leu Ser Tyr Phe

GCC

Ala TGG CCT CTC TAC Trp Pro Leu Tyr GCC TGC CAG GGC Ala Cys Gin Gly TGC GTC Cys Val 144 192 240 288 336 384 432 480 CTA ACC GGC Leu Thr Gly AGC GAC TAC Ser Asp Tyr 115 GTC TGG GTC ATA GCC CAC GAG TGC GGC CAC CAC GCC TTC Val Trp Val Ile Ala His Glu Cys Gly His His Ala Phe 100 105 110 CAG TGG CTG GAC Gin Trp Leu Asp

GAC

Asp 120 ACC GTC GGC CTC Thr Val Gly Leu

ATC

Ile 125 TTC CAC TCC Phe His Ser TTC CTC Phe Leu 130 CTC GTC CCT TAC Leu Val Pro Tyr

TTC

Phe 135 TCC TGG AAG TAC Ser Trp Lys Tyr

AGT

Ser 140 CAT CGA CGC CAC His Arg Arg His

CAT

His 145 TCC AAC ACT GGC Ser Asn Thr Gly CTC GAG AGA GAC Leu Glu Arg Asp

GAA

Glu 155 GTG TTT GTC CCC Val Phe Val Pro

AAG

Lys 160 AAG AAG TCA GAC ATC AAG TGG TAC GGC AAG Lys Lys Ser Asp Ile Lys Trp Tyr Gly Lys 165 170 TAC CAC AAC AAC Tyr His Asn Asn CCT TTG Pro Leu 175 GGA CGC ACC Gly Arg Thr TAC TTA GCC Tyr Leu Ala 195

GTG

Val 180 ATG TTA ACG GTT Met Leu Thr Val

CAG

Gin 185 TTC ACT CTC GGC Phe Thr Leu Gly TGG CCT TTG Trp Pro Leu 190 GGC TTC GCT Gly Phe Ala TTC AAC GTC TCG Phe Asn Val Ser AGA CCT TAC GAC Arg Pro Tyr Asp

GGC

Gly 205 TGC CAT Cys His 210 TTC CAC CCC AAC Phe His Pro Asn

GCT

Ala 215 CCC ATC TAC AAC Pro Ile Tyr Asn

GAC

Asp 220 CGC GAG CGT CTC Arg Glu Arg Leu WO 98/56239 PCT/US98/12332 65 CAG ATA TAC ATC TCC GAC GCT GGC ATC CTC Gin 225

TAC

Tyr

GGA

Gly

TTG

Leu

GAT

Asp

TTG

Leu 305

CTG

Leu

ATA

Ile

GTT

Val

GAC

Asp Ile

CGC

Arg

GTT

Val

CAG

Gin

TGG

Trp 290

AAC

Asn

TTC

Phe

AAG

Lys

AAG

Lys

AGG

Arg 370 Tyr

TAC

Tyr

CCG

Pro

CAC

His 275

TTG

Leu

AAG

Lys

TCG

Ser

CCG

Pro

GCG

Ala 355

CAA

Gin Ile

GCT

Ala

CTT

Leu 260

ACG

Thr

AGG

Arg

GTC

Val

ACC

Thr

ATA

Ile 340

ATG

Met

GGT

Gly Ser

GCT

Ala 245

CTG

Leu

CAT

His

GGA

Gly

TTC

Phe

ATG

Met 325

CTG

Leu

TGG

Trp

GAG

Glu Asp 230

GTC

Val

ATT

Ile

CCT

Pro

GCT

Ala

CAC

His 310

CCG

Pro

GGA

Gly

AGG

Arg

AAG

Lys Ala Gly Ile Leu

CAA

Gin

GTC

Val

TCC

Ser

TTG

Leu 295

AAT

Asn

CAT

His

GAG

Glu

GAG

Glu

AAA

Lys 375

GGA

Gly

AAT

Asn

CTG

Leu 280

GCC

Ala

ATC

Ile

TAT

Tyr

TAT

Tyr

GCG

Ala 360

GGT

Gly

GTT

Val

GGG

Gly 265

CCT

Pro

ACC

Thr

ACG

Thr

CAT

His

TAT

Tyr 345

AAG

Lys

GTG

Val

GCC

Ala 250

TTC

Phe

CAC

His

GTT

Val

GAC

Asp

GCG

Ala 330

CAG

Gin

GAG

Glu

TTC

Phe

GCC

Ala 235

TCG

Ser

TTA

Leu

TAT

Tyr

GAC

Asp

ACG

Thr 315

ATG

Met

TTG

Leu

TGT

Cys

TGG

Trp

GTC

Val

ATG

Met

GTT

Val

GAC

Asp

AGA

Arg 300

CAC

His

GAA

Glu

CAT

His

ATC

Ile

TGC

Cys

GTC

Val

TTG

Leu

TCG

Ser 285

GAC

Asp

GTG

Val

GCT

Ala

GGG

Gly

TAT

Tyr 365

TAC

Tyr

TGC

Cys

ATC

Ile 270

TCT

Ser

TAC

Tyr

GCG

Ala

ACG

Thr

ACG

Thr 350

GTG

Val

GGT

Gly

TTC

Phe 255

ACT

Thr

GAG

Glu

GGA

Gly

CAT

His

AAG

Lys 335

CCG

Pro

GAA

Glu

CTC

Leu 240

TAC

Tyr

TAC

Tyr

TGG

Trp

ATC

Ile

CAC

His 320

GCG

Ala

GTG

Val

CCG

Pro 720 768 816 864 912 960 1008 1056 1104 TAC AAC AAT AAG TTA T 1153 Tyr 380 Asn Asn Lys Leu 1155 INFORMATION FOR SEQ ID NO:8: SEQUENCE CHARACTERISTICS: LENGTH: 384 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: Met Gly Ala Gly Gly Arg Met Gin Val Ser Pro Pro Ser Lys Lys Ser 1 5 10 Glu Thr Asp Asn Ile Lys Arg Val Pro Cys Glu Thr Pro Pro Phe Thr 25 Val Gly Glu Leu Lys Lys Ala Ile Pro Pro His Cys Phe Lys Arg Ser 40 WO 98/56239 WO 9856239PCTIUS98/1 2332 66 Ile Pro Arg Ser Phe Ser Cys Leu Leu Ser Phe His 145 Lys Gly Tyr Cys Gin 225 Tyr Gly Leu Asp Leu 305 Leu Ile Val Phe Ser Thr Asp Leu 130 Ser Lys Arg Leu His 210 Ile Arg Vai Gin Trp 290 Asn Phe Lys Lys Tyr Tyr Giy Tyr Leu Asn Ser Thr Al a 195 Phe Tyr Tyr Pro His 275 Leu Lys Ser Pro Ala Tyr Phe Val 100 Gin Val Thr Asp Val 180 Phe His Ile Ala Leu 260 Thr Arg Val Thr Ile 340 Met Val Ala Trp Trp Pro Gly Ile 165 Met Asn Pro Ser Ala 245 Leu His Gly Phe Met 325 Leu Trp Ala 70 Trp Val Leu Tyr Ser 150 Lys Leu Val Asn Asp 230 Val Ile Pro Al a His 310 Pro Gly Arg Tyr 55 Thr Pro Ile Asp Phe 135 Leu Trp Thr Ser Ala 215 Ala Gin Val Ser Leu 295 Asn His Giu Glu Leu Ile Trp Thr Tyr Leu Tyr Ala His 105 Asp Thr 120 Ser Trp Giu Arg Tyr Gly Vai Gin 185 Gly Arg 200 Pro Ile Gly Ile Gly Val Asn Gly 265 Leu Pro 280 Ala Thr Ile Thr Tyr His Tyr Tyr 345 Ala Lys 360 Phe Trp 90 Glu Val Lys Asp Lys 170 Phe Pro Tyr Leu Ala 250 Phe His Val1 Asp Ala 330 Gin Giu Asp Pro 75 Ala Cys Gly Tyr Glu 155 Tyr Thr Tyr Asn Ala 235 Ser Leu Tyr Asp Thr 315 Met Leu Cys Ile Ile Leu Leu Cys Gin Gly His Leu Ile 125 Ser His 140 Val Phe His Asn Leu Gly Asp Gly 205 Asp Arg 220 Val Cys Met Val Val Leu Asp Ser 285 Arg Asp 300 His Val Giu Ala His Gly Ile Tyr 365 Ile Pro Gly His 110 Phe Arg Val1 Asn Trp 190 Gly Glu Tyr Cys Ile 270 Ser Tyr Ala Thr Thr 350 Val Ala His Cys Ala His Arg Pro Pro 175 Pro Phe Arg Gly Phe 255 Thr Glu Gly His Lys 335 Pro Glu Ser Pro Val Phe Ser His Lys 160 Leu Leu Ala Leu Leu 240 Tyr Tyr Trp Ile His 320 Ala Val Pro 355 Asp Arg Gin Gly Glu Lys 370 Lys Giy Vai Phe Trp Tyr 375 380 Asn Asn Lys Leu WO 98/56239 PCTIUS98/12332 67 INFORMATION FOR SEQ ID NO:9: SEQUENCE CHARACTERISTICS: LENGTH: 1155 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: Genomic DNA (ix) FEATURE: NAME/KEY: Coding Sequence LOCATION: 1...1152 OTHER INFORMATION: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: ATG GGT GCA GGT GGA AGA ATG CAA GTG TCT CCT CCC TCC AAA Met Gly Ala Gly Gly Arg Met Gin Val Ser Pro Pro Ser Lys AAG TCT Lys Ser GAA ACC GAC Glu Thr Asp GTC GGA GAA Val Gly Glu

AAC

Asn ATC AAG CGC GTA Ile Lys Arg Val

CCC

Pro 25 TGC GAG ACA CCG Cys Glu Thr Pro CCC TTC ACT Pro Phe Thr AAA CGC TCG Lys Arg Ser CTC AAG AAA GCA Leu Lys Lys Ala

ATC

Ile 40 CCA CCG CAC TGT Pro Pro His Cys

TTC

Phe 96 144 192 240 ATC CCT Ile Pro CGC TCT TTC TCC Arg Ser Phe Ser

TAC

Tyr 55 CTC ATC TGG GAC Leu Ile Trp Asp

ATC

Ile ATC ATA GCC TCC Ile Ile Ala Ser

TGC

Cys TTC TAC TAC GTC Phe Tyr Tyr Val

GCC

Ala 70 ACC ACT TAC TTC Thr Thr Tyr Phe

CCT

Pro 75 CTC CTC CCT CAC Leu Leu Pro His

CCT

Pro CTC TCC TAC TTC Leu Ser Tyr Phe GCC TGG CCT CTC TAC TGG GCC TGC CAG GGC TGC GTC Ala Trp Pro Leu Tyr Trp Ala Cys Gin Gly Cys Val 90 CTA ACC GGC Leu Thr Gly AGC GAC TAC Ser Asp Tyr 115

GTC

Val 100 TGG GTC ATA GCC Trp Val Ile Ala

CAC

His 105 GAG TGC GGC CAC Glu Cys Gly His CAC GCC TTC His Ala Phe 110 TTC CAC TCC Phe His Ser CAG TGG CTG GAC Gin Trp Leu Asp

GAC

Asp 120 ACC GTC GGC CTC Thr Val Gly Leu

ATC

Ile 125 TTC CTC Phe Leu 130 CTC GTC CYT TAC Leu Val Xaa Tyr

TTC

Phe 135 TCC TGG AAG TAC Ser Trp Lys Tyr

AGT

Ser 140 CAT CGA CGC CAC His Arg Arg His

CAT

His 145 TCC AAC ACT GGC Ser Asn Thr Gly CTC GAG AGA GAC Leu Glu Arg Asp GTG TTT GTC CCC Val Phe Val Pro

AAG

Lys 160 AAG AAG TCA GAC Lys Lys Ser Asp ATC AAG TGG TAC GGC AAG TAC CTC AAC AAC CCT TTG Ile Lys Trp Tyr Gly Lys Tyr Leu Asn Asn Pro Leu 165 170 175 WO 98/56239 PCT/US98/12332 68 GGA CGC ACC Gly Arg Thr TAC TTR GCC Tyr Leu Ala 195 ATG TTA ACG GTT Met Leu Thr Val

CAG

Gin 185 TTC ACT CTC GGC Phe Thr Leu Gly TGG CCT TTG Trp Pro Leu 190 GGC TTC GCT Gly Phe Ala TTC AAC GTC TCG Phe Asn Val Ser

GGG

Gly 200 AGA CCT TAC GAC Arg Pro Tyr Asp TGC CAT Cys His 210 TTC CAC CCC AAC Phe His Pro Asn

GCT

Ala 215 CCC ATC TAC AAC Pro Ile Tyr Asn

GAC

Asp 220 CGT GAG CGT CTC Arg Glu Arg Leu

CAG

Gin 225 ATA TAC ATC TCC Ile Tyr Ile Ser GCT GGC ATC CTC Ala Gly Ile Leu GTC TGC TAC GGT Val Cys Tyr Gly

CTC

Leu 240 624 672 720 768 816 864 TAC CGC TAC GCT Tyr Arg Tyr Ala

GCT

Ala 245 RTC CAA GGA GTT Xaa Gin Gly Val

GCC

Ala 250 TCG ATG GTC TGC Ser Met Val Cys TTC TAC Phe Tyr 255 GGA GTT CCT Gly Val Pro TTG CAG CAC Leu Gin His 275 CTG RTT GTC AAC Leu Xaa Val Asn

GGG

Gly 265 TTC TTA GTT TTG Phe Leu Val Leu ATC ACT TAC Ile Thr Tyr 270 TCT GAG TGG Ser Glu Trp ACG CAT CCT TCC Thr His Pro Ser CCT CAC TAT GAC Pro His Tyr Asp GAT TGG Asp Trp 290 TTG AGG GGA GCT Leu Arg Gly Ala

TTG

Leu 295 GCC ACC GTT GAC Ala Thr Val Asp

AGA

Arg 300 GAC TAC GGA ATC Asp Tyr Gly Ile

TTG

Leu 305 AAC AAG GTC TTC Asn Lys Val Phe AAT ATC ACG GAC Asn Ile Thr Asp

ACG

Thr 315 CAC GTG GCG CAT His Val Ala His

CAC

His 320 CTG TTC TCG ACC Leu Phe Ser Thr

ATG

Met 325 CCG CAT TAT CAT Pro His Tyr His ATG GAA GCT ACG Met Glu Ala Thr AAG GCG Lys Ala 335 912 960 1008 1056 1104 ATA AAG CCG Ile Lys Pro GTT AAG GCG Val Lys Ala 355

ATA

Ile 340 CTG GGA GAG TAT Leu Gly Glu Tyr

TAY

Tyr 345 CAG TTC GAT GGG Gin Phe Asp Gly ACG CCG GTG Thr Pro Val 350 GTG GAA CCG Val Glu Pro ATG TGG AGG GAG Met Trp Arg Glu

GCG

Ala 360 AAG GAG TGT ATC Lys Glu Cys Ile

TAT

Tyr 365 GAC AGG Asp Arg 370 CAA GGT GAG AAG Gin Gly Glu Lys

AAA

Lys 375 GGT GTG TTC TGG Gly Val Phe Trp AAC AAT AAG TTA T 1153 Asn Asn Lys Leu 1155 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 384 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein WO 98/56239 WO 9856239PCTIUS98/1 2332 69 FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ Met Gly Ala Gly Giu Val Ile Cys Leu Leu Ser Phe His 145 Lys Gly Tyr Cys Gin 225 Tyr Gly Leu Asp Thr Giy Pro Phe Ser Thr Asp Leu 130 Ser Lys Arg Leu His 210 Ile Arg Val Gin Trp 290 Asp Giu Arg Tyr Tyr Giy Tyr 115 Leu Asn Ser Thr Ala 195 Phe Tyr Tyr Pro His 275 Leu Asn Leu Ser Tyr Phe Val 100 Gin Val Thr Asp Val 180 Phe His Ile Ala Leu 260 Thr Arg Giy Ile Lys Phe Val Ala Trp Trp Xaa Gly Ile 165 Met Asn Pro Ser Ala 245 Leu His Gly Lys Lys Ser Ala 70 Trp Val Leu Tyr Ser 150 Lys Leu Val Asn Asp 230 Xaa Xaa Pro Al a Arg Ala Tyr 55 Thr Pro Ile Asp Phe 135 Leu Trp Thr Ser Ala 215 Ala Gin Val Ser Leu 295 Arg Met Gin Val S Val Ile 40 Leu Thr Leu Al a Asp 120 Ser Glu Tyr Val Gly 200 Pro Gly Gly Asn Leu 280 Ala Pro 25 Pro Ile Tyr Tyr His 105 Thr Trp Arg Gly Gin 185 Arg Ile Ile Val Gly 265 Pro Thr

C

P

T

P

T

G

V

L

P

L

ID NO:l0: er Pro Pro ys Glu Thr ro His Cys 'rp, Asp Ile he Pro Leu 75 rp Ala Cys 90 iu Cys Gly 'al Gly Leu .ys Tyr Ser 140 hsp Glu Val 155 ys Tyr Leu .70 ~he Thr Leu ~ro Tyr Asp yr Asn Asp 220 jeu Ala Val 235 kl1a Ser Met so0 ?he Leu Val iis Tyr Asp 7al Asp Arg 300 Ser Pro Phe Ile Leu Gin His Ile 125 His Phe Asn Gly Gly 205 Arg Cys Val Leu Ser 285 Asp Lys Pro Lys Ile Pro Gly His 110 Phe Arg Val Asn Trp 190 Gly Giu Tyr Cys Ile 270 Ser Tyr Lys Phe Arg Ala His Cys Ala His Arg Pro Pro 175 Pro Phe Arg Gly Phe 255 Thr Glu Gly Ser Thr Ser Ser Pro Val Phe Ser His Lys 160 Leu Leu Ala Leu Leu 240 Tyr Tyr Trp Ile Leu Asn Lys Val Phe His Asn Ile Thr Asp Thr His Val Ala His His 305 310 315 320 WO 98/56239 PCT/US98/12332 Leu Phe Ser Thr Pro His Tyr His Ala 330 Met Glu Ala Thr Lys Ala 335 Ile Lys Pro Val Lys Ala 355 Ile 340 Leu Gly Glu Tyr Gin Phe Asp Gly Thr Pro Val 350 Val Glu Pro Met Trp Arg Glu Ala 360 Lys Glu Cys Ile Asp Arg 370 Gin Gly Glu Lys Lys Gly Val Phe Trp Tyr Asn Asn Lys Leu 375 380 INFORMATION FOR SEQ ID NO:11: SEQUENCE CHARACTERISTICS: LENGTH: 1155 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: Genomic DNA (ix) FEATURE: NAME/KEY: Coding Sequence LOCATION: 1...1152 OTHER INFORMATION: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:

ATG

Met 1 GGT GCA GGT Gly Ala Gly

GGA

Gly 5 AGA ATG CAA GTG Arg Met Gin Val

TCT

Ser 10 CCT CCC TCC AAA Pro Pro Ser Lys AAG TCT Lys Ser GAA ACC GAC Glu Thr Asp GTC GGA GAA Val Gly Glu ATC AAG CGC GTA CCC TGC GAG ACA CCG Ile Lys Arg Val Pro Cys Glu Thr Pro CCC TTC ACT Pro Phe Thr AAA CGC TCG Lys Arg Ser CTC AAG AAA GCA Leu Lys Lys Ala CCA CCG CAC TGT Pro Pro His Cys ATC CCT Ile Pro CGC TCT TTC TCC Arg Ser Phe Ser

TAC

Tyr CTC ATC TGG GAC Leu Ile Trp Asp ATC ATA GCC TCC Ile Ile Ala Ser

TGC

Cys TTC TAC TAC GTC Phe Tyr Tyr Val

GCC

Ala 70 ACC ACT TAC TTC Thr Thr Tyr Phe CTC CTC CCT CAC Leu Leu Pro His

CCT

Pro CTC TCC TAC TTC Leu Ser Tyr Phe

GCC

Ala TGG CCT CTC TAC Trp Pro Leu Tyr

TGG

Trp 90 GCC TGC CAG GGC Ala Cys Gin Gly TGC GTC Cys Val CTA ACC GGC Leu Thr Gly AGC GAC TAC Ser Asp Tyr 115 TGG GTC ATA GCC Trp Val Ile Ala AAG TGC GGC CAC Lys Cys Gly His CAC GCC TTC His Ala Phe 110 TTC CAC TCC Phe His Ser 336 384 CAG TGG CTG GAC Gin Trp Leu Asp

GAC

Asp 120 ACC GTC GGC CTC Thr Val Gly Leu WO 98/56239 PCT/US98/12332 71 TTC CTC Phe Leu 130 CAT TCC His Ser 145 CTC GTC CYT TAC TTC TCC TGG AAG Leu Val Xaa Tyr Phe Ser Trp Lys 135 TAC AGT Tyr Ser 140 GAA GTG Glu Val 155 CAT CGA CGC CAC His Arg Arg His AAC ACT GGC Asn Thr Gly TCC CTC Ser Leu 150 GAG AGA GAC Glu Arg Asp TTT GTC CCC Phe Val Pro

AAG

Lys 160 AAG AAG TCA GAC Lys Lys Ser Asp AAG TGG TAC GGC Lys Trp Tyr Gly

AAG

Lys 170 TAC CTC AAC AAC Tyr Leu Asn Asn CCT TTG Pro Leu 175 GGA CGC ACC Gly Arg Thr TAC TTR GCC Tyr Leu Ala 195

GTG

Val 180 ATG TTA ACG GTT Met Leu Thr Val TTC ACT CTC GGC Phe Thr Leu Gly TGG CCT TTG Trp Pro Leu 190 GGC TTC GCT Gly Phe Ala TTC AAC GTC TCG Phe Asn Val Ser

GGG

Gly 200 AGA CCT TAC GAC Arg Pro Tyr Asp

GGC

Gly 205 432 480 528 576 624 672 720 768 816 864 TGC CAT Cys His 210 TTC CAC CCC AAC Phe His Pro Asn

GCT

Ala 215 CCC ATC TAC AAC Pro Ile Tyr Asn

GAC

Asp 220 CGT GAG CGT CTC Arg Glu Arg Leu

CAG

Gin 225 ATA TAC ATC TCC Ile Tyr Ile Ser

GAC

Asp 230 GCT GGC ATC CTC Ala Gly Ile Leu

GCC

Ala 235 GTC TGC TAC GGT Val Cys Tyr Gly

CTC

Leu 240 TAC CGC TAC GCT Tyr Arg Tyr Ala

GCT

Ala 245 RTC CAA GGA GTT Xaa Gin Gly Val

GCC

Ala 250 TCG ATG GTC TGC Ser Met Val Cys TTC TAC Phe Tyr 255 GGA GTT CCT Gly Val Pro TTG CAG CAC Leu Gin His 275

CTT

Leu 260 CTG RTT GTC AAC Leu Xaa Val Asn

GGG

Gly 265 TTC TTA GTT TTG Phe Leu Val Leu ATC ACT TAC Ile Thr Tyr 270 TCT GAG TGG Ser Glu Trp ACG CAT CCT TCC Thr His Pro Ser

CTG

Leu 280 CCT CAC TAT GAC Pro His Tyr Asp

TCG

Ser 285 GAT TGG Asp Trp 290 TTG AGG GGA GCT Leu Arg Gly Ala GCC ACC GTT GAC Ala Thr Val Asp

AGA

Arg 300 GAC TAC GGA ATC Asp Tyr Gly Ile

TTG

Leu 305 AAC AAG GTC TTC Asn Lys Val Phe

CAC

His 310 AAT ATC ACG GAC ACG CAC GTG GCG CAT Asn Ile Thr Asp Thr His Val Ala His 315

CAC

His 320 CTG TTC TCG ACC Leu Phe Ser Thr

ATG

Met 325 CCG CAT TAT CAT Pro His Tyr His ATG GAA GCT ACG Met Glu Ala Thr AAG GCG Lys Ala 335 ATA AAG CCG Ile Lys Pro GTT AAG GCG Val Lys Ala 355

ATA

Ile 340 CTG GGA GAG TAT Leu Gly Glu Tyr

TAY

Tyr 345 CAG TTC GAT GGG Gin Phe Asp Gly ACG CCG GTG Thr Pro Val 350 GTG GAA CCG Val Glu Pro 1008 1056 1104 ATG TGG AGG GAG Met Trp Arg Glu

GCG

Ala 360 AAG GAG TGT ATC Lys Glu Cys Ile

TAT

Tyr 365 WO 98/56239 PCT/US98/12332 72 GAC AGG CAA GGT GAG AAG AAA GGT GTG TTC TGG TAC AAC AAT AAG TTA T 1153 Asp Arg Gin Gly Glu Lys Lys Gly Val Phe Trp Tyr Asn Asn Lys Leu 370 375 380 GA 1155 INFORMATION FOR SEQ ID NO:12: SEQUENCE CHARACTERISTICS: LENGTH: 384 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12: Met Gly Ala Gly Gly Arg Met Gin Val Ser Pro Pro Ser Lys Lys Ser 1 5 10 Glu Thr Asp Asn Ile Lys Arg Val Pro Cys Glu Thr Pro Pro Phe Thr 25 Val Gly Glu Leu Lys Lys Ala Ile Pro Pro His Cys Phe Lys Arg Ser 40 Ile Pro Arg Ser Phe Ser Tyr Leu Ile Trp Asp Ile Ile Ile Ala Ser 55 Cys Phe Tyr Tyr Val Ala Thr Thr Tyr Phe Pro Leu Leu Pro His Pro 70 75 Leu Ser Tyr Phe Ala Trp Pro Leu Tyr Trp Ala Cys Gin Gly Cys Val 90 Leu Thr Gly Val Trp Val Ile Ala His Lys Cys Gly His His Ala Phe 100 105 110 Ser Asp Tyr Gin Trp Leu Asp Asp Thr Val Gly Leu Ile Phe His Ser 115 120 125 Phe Leu Leu Val Xaa Tyr Phe Ser Trp Lys Tyr Ser His Arg Arg His 130 135 140 His Ser Asn Thr Gly Ser Leu Glu Arg Asp Glu Val Phe Val Pro Lys 145 150 155 160 Lys Lys Ser Asp Ile Lys Trp Tyr Gly Lys Tyr Leu Asn Asn Pro Leu 165 170 175 Gly Arg Thr Val Met Leu Thr Val Gin Phe Thr Leu Gly Trp Pro Leu 180 185 190 Tyr Leu Ala Phe Asn Val Ser Gly Arg Pro Tyr Asp Gly Gly Phe Ala 195 200 205 Cys His Phe His Pro Asn Ala Pro Ile Tyr Asn Asp Arg Glu Arg Leu 210 215 220 Gin Ile Tyr Ile Ser Asp Ala Gly Ile Leu Ala Val Cys Tyr Gly Leu 225 230 235 240 WO 98/56239 PCTIUS98/1 2332 73 Tyr Arg Tyr Ala Ala Xaa Gin Gly Val Ala Ser Met Val Cys Phe Tyr 245 250 255 Gly Val Pro Leu Leu Xaa Val Asn Gly Phe Leu Val Leu Ile Thr Tyr 260 265 270 Leu Gin His Thr His Pro Ser Leu Pro His Tyr Asp Ser Ser Glu Trp 275 280 285 Asp Trp Leu Arg Gly Ala Leu Ala Thr Val Asp Arg Asp Tyr Gly Ile 290 295 300 Leu Asn Lys Val Phe His Asn Ile Thr Asp Thr His Val Ala His His 305 310 315 320 Leu Phe Ser Thr Met Pro His Tyr His Ala Met Glu Ala Thr Lys Ala 325 330 335 Ile Lys Pro Ile Leu Gly Glu Tyr Tyr Gin Phe Asp Gly Thr Pro Val 340 345 350 Val Lys Ala Met Trp Arg Glu Ala Lys Glu Cys Ile Tyr Val Glu Pro 355 360 365 Asp Arg Gin Gly Glu Lys Lys Gly Val Phe Trp Tyr Asn Asn Lys Leu 370 375 380 INFORMATION FOR SEQ ID NO:13: SEQUENCE CHARACTERISTICS: LENGTH: 1155 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: Genomic DNA (ix) FEATURE: NAME/KEY: Coding Sequence LOCATION: 1...1152 OTHER INFORMATION: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13: ATG GGT GCA GGT GGA AGA ATG CAA GTG TCT CCT CCC TCC AAG AAG TCT 48 Met Gly Ala Gly Gly Arg Met Gin Val Ser Pro Pro Ser Lys Lys Ser 1 5 10 GAA ACC GAC ACC ATC AAG CGC GTA CCC TGC GAG ACA CCG CCC TTC ACT 96 Glu Thr Asp Thr Ile Lys Arg Val Pro Cys Glu Thr Pro Pro Phe Thr 25 GTC GGA GAA CTC AAG AAA GCA ATC CCA CCG CAC TGT TTC AAA CGC TCG 144 Val Gly Glu Leu Lys Lys Ala Ile Pro Pro His Cys Phe Lys Arg Ser 40 ATC CCT CGC TCT TTC TCC TAC CTC ATC TGG GAC ATC ATC ATA GCC TCC 192 Ile Pro Arg Ser Phe Ser Tyr Leu Ile Trp Asp Ile Ile Ile Ala Ser 55 WO 98/56239 PCTIUS98/1 2332 74

TGC

Cys TTC TAC TAC GTC Phe Tyr Tyr Val

GCC

Ala 70 ACC ACT TAC TTC Thr Thr Tyr Phe

CCT

Pro CTC CTC CCT CAC Leu Leu Pro His CTC TCC TAC TTC Leu Ser Tyr Phe GCC TGG CCT CTC TAC TGG GCC TGC CAA GGG TGC GTC Ala Trp Pro Leu Tyr Trp Ala Cys Gin Gly Cys Val 90 CTA ACC GGC Leu Thr Gly AGC GAC TAG Ser Asp Tyr 115

GTC

Val 100 TGG GTC ATA GCC Trp Val Ile Ala GAG TGC GGC CAC Glu Cys Gly His CAC GCC TTC His Ala Phe 110 TTC CAC TCC Phe His Ser GAG TGG CTT GAG Gin Trp Leu Asp

GAG

Asp 120 ACC GTC GGT CTC Thr Val Gly Leu

ATC

Ile 125 TTC CTC Phe Leu 130 CTC GTC CCT TAG Leu Val Pro Tyr TCC TGG AAG TAG Ser Trp Lys Tyr CAT CGA CGC CAC His Arg Arg His

CAT

His 145 TCC AAC ACT GGC Ser Asn Thr Gly

TCC

Ser 150 CTC GAG AGA GAG Leu Giu Arg Asp GTG TTT GTG CCC Vai Phe Val Pro AAG AAG TCA GAG Lys Lys Ser Asp

ATC

Ile 165 AAG TGG TAG GGG Lys Trp Tyr Gly

AAG

Lys 170 TAC CTC AAC AAC Tyr Leu Asn Asn CCT TTG Pro Leu 175 GGA CGC ACC Gly Arg Thr TAG TTA GCC Tyr Leu Ala 195

GTG

Val 180 ATG TTA ACG OTT Met Leu Thr Val

GAG

Gin 185 TTC ACT CTC GGC Phe Thr Leu Gly TGG CCG TTO Trp Pro Leu 190 GGC TTC OCT Gly Phe Ala 432 480 528 576 624 672 720 768 TTG AAC GTC TCG Phe Asn Val Ser

GGA

Oly 200 AGA GCT TAG GAG Arg Pro Tyr Asp TGC CAT Cys His 210 TTC CAC CCC AAC Phe His Pro Asn

GCT

Ala 215 CCC ATC TAG AAG Pro Ile Tyr Asn

GAG

Asp 220 CGC GAG COT GTC Arg Glu Arg Leu

GAG

Gin 225 ATA TAG ATG TCC Ile Tyr Ile Ser

GAG

Asp 230 OCT GOC ATC CTC Ala Gly Ile Leu

GCC

Ala 235 GTC TGC TAG GGT Vai Cys Tyr Gly

CTC

Leu 240 TTC CGT TAC GCC Phe Arg Tyr Ala 0CC Ala 245 GCG GAG OGA GTG Ala Gin Oly Val 0CC Ala 250 TCG ATG GTC TGC Ser Met Val Cys TTC TAG Phe Tyr 255 GGA GTC CCO Gly Val Pro TTG GAG CAC Leu Gin His 275

CTT

Leu 260 CTG ATT OTC AAT Leu Ile Val Asn

GGT

Oly 265 TTG CTC GTG TTG Phe Leu Val Leu ATC ACT TAG Ile Thr Tyr 270 TCC GAG TGG Ser Glu Trp ACG CAT CCT TCG Thr His Pro Ser

CTG

Leu 280 CCT CAC TAG GAT Pro His Tyr Asp

TCG

Ser 285 GAT TGG Asp Trp 290 TTG AGG GGA OCT Leu Arg Gly Ala

TTG

Leu 295 OCT ACC GTT GAG Ala Thr Val Asp GAG TAG GGA ATC Asp Tyr Oly Ile

TTG

Leu 305 AAC AAG GTC TTC Asn Lys Val Phe

CAC

His 310 AAT ATT ACC GAG Asn Ile Thr Asp CAC GTG GCG CAT His Val Ala His WO 98/56239 PCT/US98/12332

CTG

Leu

ATA

Ile

GTT

Val

GAC

Asp

GA

TTC

Phe

AAG

Lys

AAG

Lys

AGG

Arg 370

TCC

Ser

CCG

Pro

GCG

Ala 355

CAA

Gin

ATG

Met 325

CTG

Leu

TGG

Trp

GAG

Glu

CAT

His

GAG

Glu

GAG

Glu

AAA

Lys 375 75 TAT CAC GCG Tyr His Ala 330 TAT TAT CAG Tyr Tyr Gin 345 GCG AAG GAG Ala Lys Glu 360 GGT GTG TTC Gly Val Phe ATG GAA GCT ACC AAG GCG 1008 Met Glu Ala Thr Lys Ala 335 TTC GAT GGG ACG CCG GTG 1056 Phe Asp Gly Thr Pro Val 350 TGT ATC TAT GTG GAA CCG 1104 Cys Ile Tyr Val Glu Pro 365 TGG TAC AAC AAT AAG TTA T 1153 Trp Tyr Asn Asn Lys Leu 380 1155 INFORMATION FOR SEQ ID NO:14: SEQUENCE CHARACTERISTICS: LENGTH: 384 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14: Met Gly Ala Gly Glu Val Ile Cys Leu Leu Ser Phe His 145 Thr Gly Pro Phe Ser Thr Asp Leu 130 Ser Asp Glu Arg Tyr Tyr Gly Tyr 115 Leu Asn Thr Leu Ser Tyr Phe Val 100 Gin Val Thr Gly Ile Lys Phe Val Ala Trp Trp Pro Gly Lys Lys Ser Ala 70 Trp Val Leu Tyr Ser 150 Arg Met Gin Val Ser Pro Pro Ser Lys Arg Ala Tyr 55 Thr Pro Ile Asp Phe 135 Leu Val Ile 40 Leu Thr Leu Ala Asp 120 Ser Glu Pro 25 Pro Ile Tyr Tyr His 105 Thr Trp Arg Cys Pro Trp Phe Trp 90 Glu Val Lys Asp Lys 170 Glu His Asp Pro 75 Ala Cys Gly Tyr Glu 155 Thr Cys Ile Leu Cys Gly Leu Ser 140 Val Pro Phe Ile Leu Gin His Ile 125 His Phe Pro Lys Ile Pro Gly His 110 Phe Arg Val Lys Ser Phe Thr Arg Ser Ala Ser His Pro Cys Val Ala Phe His Ser Arg His Pro Lys 160 Lys Lys Ser Asp Ile Lys Trp Tyr Gly Tyr Leu Asn Asn Pro Leu WO 98/56239 PCT/US98/12332 Gly Tyr Cys Gin 225 Phe Gly Leu Asp Leu 305 Leu Ile Val Arg Leu His 210 Ile Arg Val Gin Trp 290 Asn Phe Lys Lys Thr Ala 195 Phe Tyr Tyr Pro His 275 Leu Lys Ser Pro Ala 355 Val 180 Phe His Ile Ala Leu 260 Thr Arg Val Thr Ile 340 Met Met Asn Pro Ser Ala 245 Leu His Gly Phe Met 325 Leu Trp Leu Val Asn Asp 230 Ala Ile Pro Ala His 310 Pro Gly Arg 76 Thr Val Gin Phe Thr 185 Ser Gly Arg Pro Tyr 200 Ala Pro Ile Tyr Asn 215 Ala Gly Ile Leu Ala 235 Gin Gly Val Ala Ser 250 Val Asn Gly Phe Leu 265 Ser Leu Pro His Tyr 280 Leu Ala Thr Val Asp 295 Asn Ile Thr Asp Thr 315 His Tyr His Ala Met 330 Glu Tyr Tyr Gin Phe 345 Glu Ala Lys Glu Cys 360 Lys Gly Val Phe Trp 375 SFOR SEO ID Leu Asp Asp 220 Val Met Val Asp Arg 300 His Glu Asp Ile Tyr 380 Gly Gly 205 Arg Cys Val Leu Ser 285 Asp Val Ala Gly Tyr 365 Asn Pro Phe Arg Gly Phe 255 Thr Glu Gly His Lys 335 Pro Glu Lys Leu Ala Leu Leu 240 Tyr Tyr Trp Ile His 320 Ala Val Pro Leu Asp Arg 370 Gin Gly Glu Lys

INFORMATIO

SEQUENCE CHARACTERISTICS: LENGTH: 1155 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: Genomic DNA (ix) FEATURE: NAME/KEY: Coding Sequence LOCATION: 1...1152 OTHER INFORMATION: (xi) SEQUENCE DESCRIPTION: SEQ ID ATG GGT GCA GGT GGA AGA ATG CAA GTG TCT CCT CCC TCC AAG AAG TCT Met Gly Ala Gly Gly Arg Met Gin Val Ser Pro Pro Ser Lys Lys Ser 1 5 10 WO 98/56239 WO 9856239PCTIUS98/1 2332 77 GAA ACC GAC ACC ATC AAG CGC GTA CCC TGC GAG ACA CCG CCC TTC ACT Giu

GTC

Val

ATC

Ile

TGC

Cys

CTC

Leu

CTA

Leu

AGC

Ser

TTC

Phe

CAT

His 145

AAG

Lys

GGA

Gly

TAC

Tyr

TGC

Cys

CAG

Gin 225

TTC

Phe Thr

GGA

Gly

CCT

Pro 50

TTC

Phe

TCC

Ser

ACC

Thr

GAC

Asp

CTC

Leu 130

TCC

Ser

AAG

Lys

CGC

Arg

TTA

Leu

CAT

His 210

ATA

Ile

CGT

Arg Asp

GAA

Glu 35

CGC

Arg

TAC

Tyr

TAC

Tyr

GGC

Gly

TAC

Tyr 115

CTC

Leu

AAC

Asn

TCA

Ser

ACC

Thr

GCC

Al a 195

TTC

Phe

TAC

Tyr

TAC

Tyr Thr

CTC

Leu

TCT

Ser

TAC

Tyr

TTC

Phe

GTC

Val 100

CAG

Gin

GTC

Val

ACT

Thr

GAC

Asp

GTG

Val 180

TTC

Phe

CAC

His

ATC

Ile

GCC

Ala Ile

AAG

Lys

TTC

Phe

GTC

Val

GCC

Ala

TGG

Trp

TGG

Trp

CCT

Pro

GGC

Gly

ATC

Ile 165

ATG

Met

AAC

Asn

CCC

Pro

TCC

Ser

GCC

Ala 245 Lys

AAA

Lys

TCC

Ser

GCC

Ala 70

TGG

Trp

GTC

Val

CTT

Leu

TAC

Tyr

TCC

Ser 150

AAG

Lys

TTA

Leu

GTC

Vai

AAC

Asn

GAC

Asp 230

GCG

Ala Arg

GCA

Ala

TAC

Tyr 55

ACC

Thr

CCT

Pro

ATA

Ile

GAC

Asp

TTC

Phe 135

CTC

Leu

TGG

Trp

ACG

Thr

TCG

Ser

GCT

Ala 215

GCT

Ala

GAG

Gin Val

ATC

Ile 40

CTC

Leu

ACT

Thr

CTC

Leu

GCC

Ala

GAC

Asp 120

TCC

Ser

GAG

Glu

TAG

Tyr

GTT

Val

GGA

Gly 200

CCC

Pro

GGC

Gly

GGA

Gly Pro 25

CCA

Pro

ATC

Ile

TAC

Tyr

TAC

Tyr

GAG

His 105

ACC

Thr

TGG.

Trp

AGA

Arg

GGC

Gly

CAG

Gln 185

AGA

Arg

ATC

Ile

ATC

Ile

GTG

Val Cys Glu Thr CCG CAC TGT Pro His Cys TGG GAC ATC Trp Asp Ile TTC CCT CTC Phe Pro Leu 75 TGG GCC TGC Trp Ala Cys 90 GAG TGC GGC Glu Cys Gly GTC GGT CTC Val Gly Leu AAG TAC AGT Lys Tyr Ser 140 GAC GAA GTG Asp Giu Val 155 AAG TAC CAC Lys Tyr His 170 TTC ACT CTC Phe Thr Leu CCT TAC GAC Pro Tyr Asp TAC AAC GAC Tyr Asn Asp 220 CTC GCC GTC Leu Ala Val 235 GCC TCG ATG Ala Ser Met 250 Pro Pro Phe Thr

TTC

Phe

ATC

Ile

CTC

Leu

CAA

Gln

CAC

His

ATC

Ile 125

CAT

His

TTT

Phe

AAC

Asn

GGC

Gly

GGC

Gly 205

CGC

Arg

TGC

Gys

GTC

Val

AAA

Lys

ATA

Ile

CCT

Pro

GGG

Gly

CAG

His 110

TTC

Phe

CGA

Arg

GTC

Val

AAC

Asn

TGG

Trp 190

GGC

Gly

GAG

Glu

TAC

Tyr

TGC

Cys

CGC

Arg

GCC

Ala

CAC

His

TGG

Cys

GCC

Ala

CAC

His

CGC

Arg

CCC

Pro

CCT

Pro 175

CCG

Pro

TTC

Phe

CGT

Arg

GGT

Gly

TTC

Phe 255

TCG

Ser

TCC

Ser

CT

Pro s0

GTC

Val

TTC

Phe

TCC

Ser

CAC

His

AAG

Lys 160

TTG

Leu

TTG

Leu

GCT

Ala

CTC

Leu

CTC

Leu 240

TAC

Tyr 144 192 240 288 336 384 432 480 528 576 624 672 720 768 816 GGA GTG Gly Val CCG CTT Pro Leu 260 CTG ATT Leu Ile GTC AAT GGT Val Asn Gly 265 TTC CTC GTG TTG ATC ACT TAC Phe Leu Val Leu Ile Thr Tyr 270 WO 98/56239 PCT/US98/12332 78 TTG CAG CAC ACG CAT CCT TCC CTG CCT CAC TAC GAT TCG TCC GAG TGG 864 Leu Gin His Thr His Pro Ser Leu Pro His Tyr Asp Ser Ser Glu Trp 275 280 285 GAT TGG TTG AGG GGA GCT TTG GCT ACC GTT GAC AGA GAC TAC GGA ATC 912 Asp Trp Leu Arg Gly Ala Leu Ala Thr Val Asp Arg Asp Tyr Gly Ile 290 295 300 TTG AAC AAG GTC TTC CAC AAT ATT ACC GAC ACG CAC GTG GCG CAT CAT 960 Leu Asn Lys Val Phe His Asn Ile Thr Asp Thr His Val Ala His His 305 310 315 320 CTG TTC TCC ACG ATG CCG CAT TAT CAC GCG ATG GAA GCT ACC AAG GCG 1008 Leu Phe Ser Thr Met Pro His Tyr His Ala Met Glu Ala Thr Lys Ala 325 330 335 ATA AAG CCG ATA CTG GGA GAG TAT TAT CAG TTC GAT GGG ACG CCG GTG 1056 Ile Lys Pro Ile Leu Gly Glu Tyr Tyr Gin Phe Asp Gly Thr Pro Val 340 345 350 GTT AAG GCG ATG TGG AGG GAG GCG AAG GAG TGT ATC TAT GTG GAA CCG 1104 Val Lys Ala Met Trp Arg Glu Ala Lys Glu Cys Ile Tyr Val Glu Pro 355 360 365 GAC AGG CAA GGT GAG AAG AAA GGT GTG TTC TGG TAC AAC AAT AAG TTA T 1153 Asp Arg Gin Gly Glu Lys Lys Gly Val Phe Trp Tyr Asn Asn Lys Leu 370 375 380 GA 1155 INFORMATION FOR SEQ ID NO:16: SEQUENCE CHARACTERISTICS: LENGTH: 384 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16: Met Gly Ala Gly Gly Arg Met Gin Val Ser Pro Pro Ser Lys Lys Ser 1 5 10 Glu Thr Asp Thr Ile Lys Arg Val Pro Cys Glu Thr Pro Pro Phe Thr 25 Val Gly Glu Leu Lys Lys Ala Ile Pro Pro His Cys Phe Lys Arg Ser 40 Ile Pro Arg Ser Phe Ser Tyr Leu Ile Trp Asp Ile Ile Ile Ala Ser 55 Cys Phe Tyr Tyr Val Ala Thr Thr Tyr Phe Pro Leu Leu Pro His Pro 70 75 Leu Ser Tyr Phe Ala Trp Pro Leu Tyr Trp Ala Cys Gin Gly Cys Val 90 Leu Thr Gly Val Trp Val Ile Ala His Glu Cys Gly His His Ala Phe 100 105 110 WO 98/56239 PCT/US98/12332 79 Ser Phe His 145 Lys Gly Tyr Cys Gin 225 Phe Gly Leu Asp Leu 305 Leu Ile Val Asp Leu 130 Ser Lys Arg Leu His 210 Ile Arg Val Gin Trp 290 Asn Phe Lys Lys Tyr 115 Leu Asn Ser Thr Ala 195 Phe Tyr Tyr Pro His 275 Leu Lys Ser Pro Ala 355 Gin Val Thr Asp Val 180 Phe His Ile Ala Leu 260 Thr Arg Val Thr Ile 340 Met Trp Pro Gly Ile 165 Met Asn Pro Ser Ala 245 Leu His Gly Phe Met 325 Leu Trp Leu Tyr Ser 150 Lys Leu Val Asn Asp 230 Ala Ile Pro Ala His 310 Pro Gly Arg Asp Phe 135 Leu Trp Thr Ser Ala 215 Ala Gin Val Ser Leu 295 Asn His Glu Glu Lys 375 Asp Thr Val Gly 120 Ser Trp Lys Tyr Glu Arg Asp Glu 155 Tyr Gly Lys Tyr 170 Val Gin Phe Thr 185 Gly Arg Pro Tyr 200 Pro Ile Tyr Asn Gly Ile Leu Ala 235 Gly Val Ala Ser 250 Asn Gly Phe Leu 265 Leu Pro His Tyr 280 Ala Thr Val Asp Ile Thr Asp Thr 315 Tyr His Ala Met 330 Tyr Tyr Gin Phe 345 Ala Lys Glu Cys 360 Gly Val Phe Trp R SEO ID NO:17: Leu Ile 125 Ser His 140 Val Phe His Asn Leu Gly Asp Gly 205 Asp Arg 220 Val Cys Met Val Val Leu Asp Ser 285 Arg Asp 300 His Val Glu Ala Asp Gly Ile Tyr 365 Tyr Asn 380 Phe His Arg Arg Val Pro Asn Pro 175 Trp Pro 190 Gly Phe Glu Arg Tyr Gly Cys Phe 255 Ile Thr 270 Ser Glu Tyr Gly Ala His Thr Lys 335 Thr Pro 350 Val Glu Asn Lys Ser His Lys 160 Leu Leu Ala Leu Leu 240 Tyr Tyr Trp Ile His 320 Ala Val Pro Leu Asp Arg 370 Gin Gly Glu Lys INFORMATION FO SEQUENCE CHARACTERISTICS: LENGTH: 1155 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: Genomic DNA (ix) FEATURE: NAME/KEY: Coding Sequence WO 98/56239 PCT/US98/12332 80 LOCATION: 1...1152 OTHER INFORMATION: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:

ATG

Met 1 GGT GCA GGT Gly Ala Gly

GGA

Gly 5 AGA ATG CAA GTG Arg Met Gin Val

TCT

Ser 10 CCT CCC TCC AAG Pro Pro Ser Lys AAG TCT Lys Ser GAA ACC GAC Glu Thr Asp GTC GGA GAA Val Gly Glu

ACC

Thr ATC AAG CGC GTA Ile Lys Arg Val

CCC

Pro 25 TGC GAG ACA CCG Cys Glu Thr Pro CCC TTC ACT Pro Phe Thr AAA CGC TCG Lys Arg Ser CTC AAG AAA GCA Leu Lys Lys Ala CCA CCG CAC TGT Pro Pro His Cys ATC CCT Ile Pro CGC TCT TTC TCC Arg Ser Phe Ser

TAC

Tyr 55 CTC ATC TGG GAC Leu Ile Trp Asp

ATC

Ile ATC ATA GCC TCC Ile Ile Ala Ser 96 144 192 240 288

TGC

Cys TTC TAC TAC GTC Phe Tyr Tyr Val

GCC

Ala 70 ACC ACT TAC TTC Thr Thr Tyr Phe

CCT

Pro 75 CTC CTC CCT CAC Leu Leu Pro His

CCT

Pro CTC TCC TAC TTC GCC TGG CCT CTC TAC Leu Ser Tyr Phe Ala Trp Pro Leu Tyr

TGG

Trp 90 GCC TGC CAA GGG Ala Cys Gin Gly TGC GTC Cys Val CTA ACC GGC Leu Thr Gly AGC GAC TAC Ser Asp Tyr 115

GTC

Val 100 TGG GTC ATA GCC Trp Val Ile Ala

CAC

His 105 GAG TGC GGC CAC Glu Cys Gly His CAC GCC TTC His Ala Phe 110 TTC CAC TCC Phe His Ser CAG TGG CTT GAC Gin Trp Leu Asp

GAC

Asp 120 ACC GTC GGT CTC Thr Val Gly Leu TTC CTC Phe Leu 130 CTC GTC CCT TAC Leu Val Pro Tyr TCC TGG AAG TAC Ser Trp Lys Tyr

AGT

Ser 140 CAT CGA CGC CAC His Arg Arg His

CAT

His 145

AAG

Lys TCC AAC ACT GGC Ser Asn Thr Gly AAG TCA GAC ATC Lys Ser Asp Ile 165

TCC

Ser 150 CTC GAG AGA GAC Leu Glu Arg Asp

GAA

Glu 155 GTG TTT GTC CCC Val Phe Val Pro

AAG

Lys 160 AAG TGG TAC GGC Lys Trp Tyr Gly TAC CTC AAC AAC Tyr Leu Asn Asn CCT TTG Pro Leu 175 GGA CGC ACC Gly Arg Thr TAC TTA GCC Tyr Leu Ala 195

GTG

Val 180 ATG TTA ACG GTT Met Leu Thr Val

CAG

Gin 185 TTC ACT CTC GGC Phe Thr Leu Gly TGG CCG TTG Trp Pro Leu 190 GGC TTC GCT Gly Phe Ala TTC AAC GTC TCG Phe Asn Val Ser

GGA

Gly 200 AGA CCT TAC GAC Arg Pro Tyr Asp 576 624 672 TGC CAT Cys His 210 TTC CAC CCC AAC Phe His Pro Asn

GCT

Ala 215 CCC ATC TAC AAC Pro Ile Tyr Asn

GAC

Asp 220 CGC GAG CGT CTC Arg Glu Arg Leu WO 98/56239 PCT/US98/12332 81

CAG

Gin 225 ATA TAC ATC TCC Ile Tyr Ile Ser GCT GGC ATC CTC Ala Gly Ile Leu

GCC

Ala 235 GTC TGC TAC GGT Val Cys Tyr Gly

CTC

Leu 240 TTC CGT TAC GCC Phe Arg Tyr Ala

GCC

Ala 245 GCG CAG GGA GTG Ala Gin Gly Val

GCC

Ala 250 TCG ATG GTC TGC Ser Met Val Cys TTC TAC Phe Tyr 255 720 768 816 864 GGA GTC CCG Gly Val Pro TTG CAG CAC Leu Gin His 275

CTT

Leu 260 CTG ATT GTC AAT Leu Ile Val Asn

GGT

Gly 265 TTC CTC GTG TTG Phe Leu Val Leu ATC ACT TAC Ile Thr Tyr 270 TCC GAG TGG Ser Glu Trp ACG CAT CCT TCC Thr His Pro Ser

CTG

Leu 280 CCT CAC TAC GAT Pro His Tyr Asp

TCG

Ser 285 GAT TGG Asp Trp 290 TTG AGG GGA GCT Leu Arg Gly Ala

TTG

Leu 295 GCT ACC GTT GAC Ala Thr Val Asp GAC TAC GAA ATC Asp Tyr Glu Ile

TTG

Leu 305 AAC AAG GTC TTC Asn Lys Val Phe

CAC

His 310 AAT ATT ACC GAC Asn Ile Thr Asp

ACG

Thr 315 CAC GTG GCG CAT His Val Ala His

CAT

His 320 CTG TTC TCC ACG Leu Phe Ser Thr CCG CAT TAT CAC Pro His Tyr His

GCG

Ala 330 ATG GAA GCT ACC Met Glu Ala Thr AAG GCG Lys Ala 335 912 960 1008 1056 1104 ATA AAG CCG Ile Lys Pro GTT AAG GCG Val Lys Ala 355

ATA

Ile 340 CTG GGA GAG TAT Leu Gly Glu Tyr CAG TTC GAT GGG Gin Phe Asp Gly ACG CCG GTG Thr Pro Val 350 GTG GAA CCG Val Glu Pro ATG TGG AGG GAG Met Trp Arg Glu

GCG

Ala 360 AAG GAG TGT ATC Lys Glu Cys Ile

TAT

Tyr 365 GAC AGG Asp Arg 370 CAA GGT GAG AAG Gin Gly Glu Lys

AAA

Lys 375 GGT GTG TTC TGG Gly Val Phe Trp

TAC

Tyr 380 AAC AAT AAG TTA T 1153 Asn Asn Lys Leu 1155 INFORMATION FOR SEQ ID NO:18: SEQUENCE CHARACTERISTICS: LENGTH: 384 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18: Met Gly Ala Gly Gly Arg Met Gin Val Ser 1 5 10 Glu Thr Asp Thr Ile Lys Arg Val Pro Cys 25 Pro Pro Ser Lys Lys Ser Glu Thr Pro Pro Phe Thr Lys Arg Ser Val Gly Glu Leu Lys Lys Ala Ile Pro Pro His Cys Phe 40 WO 98/56239 WO 9856239PCTIUS98/1 2332 Ile Cys Leu Leu Ser Phe His 145 Lys Gly Tyr Cys Gin 225 Phe Giy Leu Asp Leu 305 Leu Ile Val Pro Phe Ser Thr Asp Leu 130 Ser Lys Arg Leu His 210 Ile Arg Val Gin Trp 290 Asn Phe Lys Lys Arg Tyr Tyr Gly Tyr 115 Leu Asn Ser Thr Al a 195 Phe Tyr Tyr Pro His 275 Leu Lys Ser Pro Ala 355 Ser Tyr Phe Val 100 Gin Val Thr Asp Val 180 Phe His Ile Ala Leu 260 Thr Arg Val Thr Ile 340 Met Phe Val Ala Trp Trp Pro Gly Ile 165 Met Asn Pro Ser Ala 245 Leu His Gly Phe Met 325 Leu Trp Ser Tyr 55 Ala Thr 70 Trp Pro Val Ile Leu Asp Tyr Phe 135 Ser Leu 150 Lys Trp Leu Thr Vai Ser Asn Ala 215 Asp Ala 230 Ala Gin Ile Val Pro Ser Ala Leu 295 His Asn 310 Pro His Gly Giu Arg Glu Leu Thr Leu Ala Asp 120 Ser Glu Tyr Val Gly 200 Pro Gly Gly Asn Leu 280 Ala Ile Tyr Tyr Ala 360 82 Ile Tyr Tyr His 105 Thr Trp Arg Gly Gin 185 Arg Ile Ile Val Gly 265 Pro Thr Thr His Tyr 345 Lys Trp Phe Trp 90 Glu Val Lys Asp Lys 170 Phe Pro Tyr Leu Ala 250 Phe His Val Asp Ala 330 Gin Giu Asp Pro 75 Ala Cys Gly Tyr Giu 155 Tyr Thr Tyr Asn Ala 235 Ser Leu Tyr Asp Thr 315 Met Phe Cys Ile Leu Cys Gly Leu Ser 140 Val Leu Leu Asp Asp 220 Val Met Val Asp Arg 300 His Giu Asp Ile Ile Leu Gin His Ile 125 His Phe Asn Gly Gly 205 Arg Cys Val Leu Ser 285 Asp Val Al a Gly Tyr 365 Ile Pro Gly His 110 Phe Arg Val Asn Trp 190 Gly Glu Tyr Cys Ile 270 Ser Tyr Al a Thr Thr 350 Val Ala His Cys Ala His Arg Pro Pro 175 Pro Phe Arg Gly Phe 255 Thr Giu Glu His Lys 335 Pro Glu Ser Pro Val Phe Ser His Lys 160 Leu Leu Ala Leu Leu 240 Tyr Tyr Trp Ile His 320 Ala Val Pro Asp Arg 370 Gln Gly Glu Lys Lys Gly Val Phe Trp Tyr Asn Asn Lys Leu 375 380 WO 98/56239 PCT/US98/12332 83 INFORMATION FOR SEQ ID NO:19: SEQUENCE CHARACTERISTICS: LENGTH: 21 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: Other Nucleic Acid (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19: GGATATGATG ATGGTGAAAG A 21 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 21 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: Other Nucleic Acid (xi) SEQUENCE DESCRIPTION: SEQ ID TCTTTCACCA TCATCATATC C 21 INFORMATION FOR SEQ ID NO:21: SEQUENCE CHARACTERISTICS: LENGTH: 21 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: Other Nucleic Acid (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21: GTTATGAAGC AAAGAAGAAA C 21 INFORMATION FOR SEQ ID NO:22: SEQUENCE CHARACTERISTICS: LENGTH: 26 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: Other Nucleic Acid (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22: GTTTCTTCTT TGCTTTGCTT CATAAC 26 INFORMATION FOR SEQ ID NO:23: SEQUENCE CHARACTERISTICS: LENGTH: 32 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear WO. 98/56239 PCT/US98/12332 84 (ii) MOLECULE TYPE: Other Nucleic Acid (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23: CAUCAUCAUC AUCTTCTTCG TAGGGTTCAT CG 32 INFORMATION FOR SEQ ID NO:24: SEQUENCE CHARACTERISTICS: LENGTH: 33 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: Other Nucleic Acid (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24: CUACUACUAC UATCATAGAA GAGAAAGGTT CAG 33 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 32 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: Other (xi) SEQUENCE DESCRIPTION: SEQ ID CAUCAUCAUC AUCATGGGTG CACGTGGAAG AA 32 INFORMATION FOR SEQ ID NO:26: SEQUENCE CHARACTERISTICS: LENGTH: 33 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: Other (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26: CUACUACUAC UATCTTTCAC CATCATCATA TCC 33 INFORMATION.FOR SEQ ID NO:27: SEQUENCE CHARACTERISTICS: LENGTH: 33 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: Other (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27: CUACUACUAC UACATAACTT ATTGTTGTAC CAG 11 WO 98/56239 PCT/US98/12332 85 INFORMATION FOR SEQ ID NO:28: SEQUENCE CHARACTERISTICS: LENGTH: 2168 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: Genomic DNA (ix) FEATURE: NAME/KEY: Coding Sequence LOCATION: 1014...2165 OTHER INFORMATION: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:

CTTCTTCGTA

AGCTTTCAGG

CCTGGTCTGT

TAGRTCTGCC

AATCTTGTGT

CATATGAAAT

AATAGAAAAG

CTCAAAAATT

TTTCAAAATT

GAACCATAAR

TTTAACTYTA

TTTTGACCTT

TTCAATATAA

CTTGCTACTG

CTTTGTACAA

TTTATAGATT

TCCGTCCTCC

GGGTTCATCG

GTCCCCTTCT

GTTCACCTCG

AGTCTTTATT

CATGTTTCTT

GTTGCAACTT

GTCAAATCTC

AAAATGACCA

TGAAATCTTA

CCCTAAACCC

AACCATAAGT

GAGTGCTAGT

AAATCACTTA

TTATTTCTTA

ATAAAACGGA

CAAATGCATG

ATAACATTAT

TTATTAACGT

TCTTCTTCTT

TCCATCTCTC

GCATTCAACT

TCATCTCACC

TCTATCTATT

CAAAATAGCA

AAATATTATT

TCCCCAAAAC

TAAACTCTAA

TTGTGACTTT

TTGGGAACAA

TTGTTGAACC

ATAAATGGAA

TGATTTAAAG

AAGAGTTGCA

TATTATTTTT

AAAATCTCTC

CTTCTCATTT

TAGCAGTCTA

AAAGATCTGT

GTTAAATAAT

CAGAAATCTT

ACTTTCTAAG

TTATCTTTTG

CTCATTTCTC

ACCCTAAACC

TGATAAAATA

AAACTTGGTT

TTTGATAGAT

GAACGTTTCA

TAGGTAGGTA

TATACAACTT

TGGTTTTCTC

TCCCCCCACC

TCCTCTTATT

GCATTTGGCA

TCCTCTGTTT

GATTACTGTC

TTTATTCAAT

TTTATATCAC

AAAATTTTAA

AACTCTAAAC

CTAAACCCTA

TTAAGTGATA

TAGTGCTATT

TTGACCGATT

TTGACTTATA

CTTCAGGGTT

TGATTAAAGG

TACAGAAACA

CTACGTCAGC

TTTATGAATT

TTTAAATCGA

CCATTTGACA

TATGGTCTAG

AGGTTGGTGA

AAAAATAGCA

TTTTTTTATT

CCTAAACTCT

AACCCCACCC

TTTTTGTGAC

TTTGTTTTTT

CCTACTGGTT

AGCTCATCAA

TAGATGTTCT

ATAAAAAGTC

AAC ATG Met 1 GGC GCA GRT Gly Ala Xaa ACC AAA ACC Thr Lys Thr

GGA

Gly AGA ATG CAA ATC Arg Met Gin Ile

TCT

Ser 10 CCT CCC TCC AGC TCC CCC GAA Pro Pro Ser Ser Ser Pro Glu 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1016 1064 1112 1160 1208 1256 1304 CTC AAA CGC GTC Leu Lys Arg Val

CCC

Pro 25 TGC GAG ACA CCA Cys Giu Thr Pro

CCC

Pro TTC ACT CTC Phe Thr Leu GGA GAC Gly Asp CTC GAG AAA GCA Leu Glu Lys Ala

ATC

Ile CCA CCT CAC TGC Pro Pro His Cys AAA CGC TCC ATC Lys Arg Ser Ile

CCT

Pro CGC TCC TTC TCC Arg Ser Phe Ser

TAC

Tyr CTC CTC TTC GAC Leu Leu Phe Asp CTC GTC TCC TCC Leu Val Ser Ser CTC TAC CAC CTC Leu Tyr His Leu ACA GCC TAC TTC Thr Ala Tyr Phe

CCT

Pro CTC CTC CCC CAC Leu Leu Pro His CCT CTC Pro Leu CCT TAC CTC Pro Tyr Leu

GCC

Ala TGG CCC CTC TAC Trp Pro Leu Tyr

TGG

Trp 90 GCC TGC CAA GGC Ala Cys Gin Gly TGC GTC CTA Cys Val Leu WO 98/56239 PCT/US98/12332 86 ACG GGC CTC Thr Gly Leu 100 TGG GTC ATC GCC Trp Val Ile Ala

CAC

His 105 GAA TGC GGC CAC Glu Cys Gly His GCC TTC AGC Ala Phe Ser GAC CAC Asp His 115 CAG TGG CTG GAC Gin Trp Leu Asp GCC GTG GGC CTC Ala Val Gly Leu

GTC

Val 125 TTC CAC TCC TTC Phe His Ser Phe

CTC

Leu 130 CTC GTC CCT TAC Leu Val Pro Tyr

TTC

Phe 135 TCC TGG AAG TAC Ser Trp Lys Tyr

AGC

Ser 140 CAT CGA CGC CAC His Arg Arg His

CAT

His 145 TCC AAC ACC GGA Ser Asn Thr Gly

TCC

Ser 150 CTC GAG AGG GAT Leu Glu Arg Asp

GAA

Glu 155 GTG TTC GTC CCC Val Phe Val Pro AAG AAG Lys Lys 160 AAA TCC GAC Lys Ser Asp CGC ACG GTG Arg Thr Val 180

ATC

Ile 165 AAG TGG TAC GGA Lys Trp Tyr Gly

AAG

Lys 170 TAC CTC AAC AAC Tyr Leu Asn Asn CCG CTA GGA Pro Leu Gly 175 CCG TTG TAC Pro Leu Tyr ATG CTA ACC GTC Met Leu Thr Val TTC ACG CTC GGC Phe Thr Leu Gly

TGG

Trp 190 TTA GCC Leu Ala 195 TTC AAC GTC TCT Phe Asn Val Ser

GGA

Gly 200 AGA CCT TAC AGC Arg Pro Tyr Ser GGT TTC GCT TGC Gly Phe Ala Cys

CAT

His 210 TTC CAC CCG AAC Phe His Pro Asn CCC ATC TAC AAC Pro Ile Tyr Asn

GAC

Asp 220 CGC GAG CGT CTC Arg Glu Arg Leu

CAG

Gin 225 1352 1400 1448 1496 1544 1592 1640 1688 1736 1784 1832 1880 1928 1976 2024 2072 ATA TAC ATC TCT Ile Tyr Ile Ser

GAC

Asp 230 GCT GGC GTC CTC Ala Gly Val Leu GTA TGT TAC GGT Val Cys Tyr Gly CTC TAC Leu Tyr 240 CGC TAC GCT Arg Tyr Ala GTT CCG CTT Val Pro Leu 260

GGT

Gly 245 TCG CGA GGA GTG Ser Arg Gly Val

GCC

Ala 250 TCG ATG GTC TGT Ser Met Val Cys GTC TAC GGA Val Tyr Gly 255 ACT TAC TTG Thr Tyr Leu ATG ATT GTC AAC Met Ile Val Asn

TGT

Cys 265 TTC CTC GTC TTG Phe Leu Val Leu

ATC

Ile 270 CAG CAC Gin His 275 ACG CAC CCT TCG Thr His Pro Ser

CTG

Leu 280 CCT CAC TAT GAT Pro His Tyr Asp

TCT

Ser 285 TCG GAG TGG GAT Ser Glu Trp Asp

TGG

Trp 290 TTG AGA GGA GCT Leu Arg Gly Ala

TTG

Leu 295 GCT ACT GTG GAT Ala Thr Val Asp

AGA

Arg 300 GAC TAT GGA ATC Asp Tyr Gly Ile

TTG

Leu 305 AAC AAG GTG TTT Asn Lys Val Phe TTC TCG ACG ATG Phe Ser Thr Met 32! AAG CCG ATA CTT Lys Pro Ile Leu 340

CAT

His 310

CCG

Pro 5 AAC ATC ACG GAC Asn Ile Thr Asp CAC GTG GCG CAT His Val Ala His CAT CTG His Leu 320 CAT TAT AAC His Tyr Asn GCG ATG Ala Met 330 GAA GCG ACC Glu Ala Thr AAG GCG ATA Lys Ala Ile 335 CCG GTG GTT Pro Val Val GGA GAG TAT Gly Glu Tyr CAG TTT GAT Gin Phe Asp GGA ACG Gly Thr 350 WO 98/56239 PCT/US98/12332 87 AAG GCG ATG TGG AGG GAG GCG AAG GAG TGT ATC TAT GTT GAA CCG GAT Lys Ala Met Trp Arg Glu Ala Lys Glu Cys Ile Tyr Val Glu Pro Asp 355 360 365 AGG CAA GGT GAG AAG AAA GGT GTG TTC TGG TAC AAC AAT AAG TTA TGA Arg Gin Gly Glu Lys Lys Gly Val Phe Trp Tyr Asn Asn Lys Leu 370 375 380 INFORMATION FOR SEQ ID NO:29: SEQUENCE CHARACTERISTICS: LENGTH: 384 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29: Met Gly Ala Xaa Gly Arg Met Gln Ile Ser Pro Pro Ser Ser Ser Pro 2120 2168 Glu Leu Ile Ser Leu Leu Ser Phe His 145 Lys Gly Tyr Thr Gly Pro Leu Pro Thr Asp Leu 130 Ser Lys Arg Leu Lys Thr Asp Leu Arg Ser Tyr His Tyr Leu Gly Leu 100 His Gln 115 Leu Val Asn Thr Ser Asp Thr Val 180 Ala Phe 195 Leu Glu Phe Leu Ala Trp Trp Pro Gly Ile 165 Met Asn Lys Lys Ser Ser 70 Trp Val Leu Tyr Ser 150 Lys Leu Val Arg Ala Tyr 55 Thr Pro Ile Asp Phe 135 Leu Trp Thr Ser Val Ile 40 Leu Ala Leu Ala Asp 120 Ser Glu Tyr Val Gly 200 Pro 25 Pro Leu Tyr Tyr His 105 Ala Trp Arg Gly Gin 185 Arg Cys Glu Pro His Phe Asp Phe Pro 75 Trp Ala 90 Glu Cys Val Gly Lys Tyr Asp Glu 155 Lys Tyr 170 Phe Thr Pro Tyr Thr Cys Ile Leu Cys Gly Leu Ser 140 Val Leu Leu Ser Pro Phe Leu Leu Gin His Val 125 His Phe Asn Gly Asp 205 Pro Phe Lys Arg Val Ser Pro His Gly Cys His Ala 110 Phe His Arg Arg Val Pro Asn Pro 175 Trp Pro 190 Gly Phe Thr Ser Ser Pro Val Phe Ser His Lys 160 Leu Leu Ala Cys His 210 Phe His Pro Asn Ala Pro Ile Tyr Asn Asp Arg Glu Arg Leu 215 220 WO 98/56239 WO 9856239PCTIUS98/1 2332 88 Gin 225 Ile Tyr Ile Ser Asp 230 Ala Gly Val Leu Ser 235 Val Cys Tyr Gly Tyr Arg Tyr Ala Ser Arg Giy Vai Ala 250 Ser Met Vai Cys Val Tyr 255 Gly Vai Pro Leu Met Ile Val Asn Cys Phe Leu Val Leu Ile Thr Tyr 260 265 270 Leu Gin His 275 Thr His Pro Ser Leu 280 Pro His Tyr Asp Ser 285 Ser Giu Trp Asp Trp 290 Leu Arg Gly Ala Leu 295 Ala Thr Val Asp Arg 300 Asp Tyr Gly Ile Leu 305 Asn Lys Vai Phe His 310 Asn Ile Thr Asp His Val Ala His His 320 Leu Phe Ser Thr Pro His Tyr Asn Ala 330 Met Giu Ala Thr Lys Ala 335 Ile Lys Pro Val Lys Ala 355 Ile 340 Leu Gly Glu Tyr Gin Phe Asp Gly Thr Pro Val 350 Val Giu Pro Met Trp Arg Glu Ala 360 Lys Giu Cys Ile Asp Arg 370 Gin Giy Glu Lys Gly Vai Phe Trp Tyr 380 Asn Asn Lys Leu INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 1132 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: Genomic DNA (xi) SEQUENCE DESCRIPTION: SEQ ID

GCGTAACCCT

TTTCTTCTTC

ATCGCTTTTC

AGTATTTATT

TTGATGCTGT

TTGCATTTTC

TTGATGGGTT

ATCCTTCCTA

TAAGTACATA

GGAGATTTGA

TTCATTTGGC

GATGTCCTTC

CAGTAAAAAA

TAGAGTTGCT

AGACATTGTG

TTGATATCCA

GTATTCTCAA

CTCTTTTGTC

AATTTGTTGG

TATTAACGTT

CATTTCTTCT

TATTCTATCT

GCATTAAACT

CTTTACCATT

ATTTGTCCGA

GGTGGAGTTG

TTCAAAGTTA

TTTGTTGAAT

CCGATTCCTA

CTATGCTCAC

ATAGATTCAG

GATTGTATTT

CTCAACTGTT

ATTATGACTT

CAAGAAAGAG

CGCTATCGTT

CCACGTACTA

TTTTTTTAAC

AAATCTTCAT

CATTTTTACG

ATCATTTTTG

ATAGATCTGG

AATCTGATTA

ATACAAACTG

AAAAATCACC

TATATTTGTT

CTTTGGGTAA

TTGGCTCTTG

TTCATGCTTA

ATGCAATAGA

TTGTTTGTTT

TCATTTAGCT

GTCTTCTCTA

ATGTAAGCTG

TATTTCTTTC

TCCATTTTTT

TTTGAGTCTT

CCCCCCCTAC

TTGTTTTCAA.

CATTTCAGTC

TCTTGATTCT

TATTGTCTAT

TTTGACTTTC

ATAGCAGTCT

TACTTGTCTT

AAAATTTATG

ATTCTGTAGT

CAAACTTTTC

TTTGCATGAA

GTTTATGTTT

TTTTGTTTTT

ACGTAGTTTA

TAACGTATCA

TTTGGTTTGC

TGAAACTTTA

TGCTTTTGGT

GTCAGCCAGC

TCTTGGTCTG

GATTTAATTC

CTGTTTTCAT

ACCGTGGAGA

AATCTTTTTT

CACGTCCTGG

AGATCTGGAC

TCTCTGGGTA

TACCTAATAC

TTTGCAAATT

GAAAATAATA

AAAAGTCTAT

GTCAAGTTGC

GTAATAAAAG

AATCTCATTA

CACTATATGC

ATAACGTAAC

TTATGCAGAA

TCAAGGTCCC

TTCTTTTCTT

TAGATCTGTT

GTGTGAAATC

ATATGAAATG

AATGATTTAT

TCTTAGAAAT

CTGAGACATG

AAATTTGCTT

ATGAAAAAGT

AATTGGATTA

GGATTCATGA

ATGTTGACAA

TTATTCTTAG

ACGAAAGAAA

ATAACTAGTA

CGCTTCTCTG

ACTGAATATT

AC

120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1132 0 0 WO 98/56239 PCTIUS98/1 2332 89 INFORMATION FOR SEQ ID NO:31: SEQUENCE CHARACTERISTICS: LENGTH: 1135 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: Genomic DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:31: 0@ 00 0 *00 0

S

006000 0 0@

S

OSS

0 00 0 0 .0 50

TTATTAACGT

CCATTTCTTC

CTGTTCTATC

TGCATTAAAC

CTTTACCATT

ATTTGTCCGA

GGGTTGGTGG

TCCTATTCAA

GTACCTATTT

TTTGACCGAT

CTCACGTCAT

ATTCAGATGC

TACATTTTTT

CTGTTTCATT

GACTTGTCGT

AAGAGATGTG

TCGTTTATTT

ACTATCCATT

ATTAATTTGT

TAAATCTTCA

TCATTTTTAC

TATCATTTTT

TACAGATCTG

AATCTGCTTA

ATACAAACTG

AGTTGAAAAA

AGTTATATAT

GTTGAATCTT

TCCTATTGGC

GCTTACAAAC

AATAGATTTG

TGTTTGTTTA

TAGCTTT TTG

CTTAACGTA

AGCTGTAGCG

CTTTCTrTGG

TTT'ITTGTGG

TGGTTTAATT

TCCCCCCCTA

GTTGTTTTCA

GCATTTCAGT

GTCTCGATTC

TATTGTATAT

TTTGACTTCC

TCACCATAGC

TTGTTTACTT

TGGGTAAAAT

TCTGGATTCT

TTTTCTTTGC

CATGAAGAAA

TGTTTAAAAG

TTTTTCTCAA

GTTTAGTAAT

TATCAAATCT

TTTGCCACTA

TAGTCCATTT

AACTTTGAGT

CGTCAGCCAG

ATCTTGGTCT

CGATTTAATT

TCTGTTTTCA

ACCGTGGAGA

AATCGTTTTT

AGTCTCACGT

TTGTTTTAGA

TTATGTCTCT

GTATACATGA

AAATTAATTC

ATAATAGGAT

TCTATATGTT

GTTGCTTATT

AAAAGACGAA

CATTAATAAC

TATGCCGCTT

TTTTGAAGCT

CTTTGCTTTT

CTCAAGGTCC

GTTCTTTTCT

CTAGATCTGT

TGTGTGAAAT.

ATATGAAATG

AATTATATAT

CCTGGTTTTA

TCTGGACCTG

GGGTAAAATT

AP.AAGTTTCA

GATTAGATGC

TCATGATAGT

GACAATAGGG

CTTAGAGACA

AGAAATTGAT

TAGTAGTATT

CTCTC!CTCTT

TIAATAACGT

GGTTTATGCA

CTTTCTTCTT

TATCGCTTTT

TAATATTTAT

CTGATGCTGT

TTGCATTTTC

ATTTTTTGAT

GAAATATCCT

AGACATGTAA

TGCTGAGAGA

TTGGCCTATG

TCCTTCATAG

AAAAAAATTG

TTGCTATCAA

TTGTGATTAT

ATCCACAAGA

CTCAACGCTA

TATCCCACGT

AACACTGAAT

GAAAC

120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 -1020 1080 1135 Whr0h em cmpie,"opie","opie"o copiig r sd Whaere tetes incomrs, compsecn risered ou "cmrng" aorelusedth 0505 presence or addition of one or more other feature, integer, step, component or *550 00 0 5@ 0 group thereof.

Claims (18)

1. An isolated nucleic acid fragment, wherein said nucleic acid fragment is selected from the group consisting of: SEQ ID SEQIDNO:31; an RNA analog of SEQ ID an RNA analog of SEQ ID NO:31; a nucleic acid fragment having a nucleic acid sequence complementary to c) or and a nucleic acid fragment of d) or at least 50 nucleotides in length that has at least 70% sequence identity to the nucleotide sequence of SEQ ID or SEQ ID NO:31.

2. The isolated nucleic acid fragment of Claim 1, wherein said nucleic acid 15 fragment is SEQ ID

3. The isolated nucleic acid fragment of Claim 1, wherein said nucleic acid fragment is SEQ ID NO:31.

4. The isolated nucleic acid fragment of Claim 1, wherein said nucleic acid fragment is at least 200 nucleotides in length and has at least 90% sequence identity to 20 SEQ ID

5. The isolated nucleic acid fragment of Claim 1, wherein said nucleic acid fragment is at least 200 nucleotides in length and has at least 90% sequence identity to SEQ ID NO:31.

6. The isolated nucleic acid fragment of Claims 4 or 5, wherein said nucleic acid fragment is at least 1000 nucleotides in length.

7. An isolated nucleic acid fragment, said nucleic acid fragment comprising a sequence of at least 200 nucleotides that has at least 70% sequence identity to SEQ ID NO:28.

8. The isolated nucleic acid fragment of Claim 7, wherein said sequence is at least 1000 nucleotides in length.

9. The isolated nucleic acid fragment of Claim 8, wherein said sequence has at least 80% sequence identity to SEQ ID NO:28. The isolated nucleic acid fragment of Claim 9, wherein said sequence has at least 90% sequence identity to SEQ ID NO:28. 18/04/02,mcl 1028.claims,90 -91

11. The isolated nucleic acid fragment of Claim 10, wherein said sequence is nucleotides 1014 to 2168 of SEQ ID NO:28.

12. The isolated nucleic acid fragment of Claim 11, wherein said fragment is SEQ ID NO:28.

13. An isolated nucleic acid fragment encoding a polypeptide having the amino acid sequence of SEQ ID NO:18.

14. An isolated nucleic acid fragment, wherein said nucleic acid fragment is selected from the group consisting of: SEQ ID NO:17; an RNA analog of SEQ ID NO: 17; a nucleic acid fragment having a nucleic acid sequence complementary to a) or and a nucleic acid fragment of b) or that is at least 10 nucleotides 15 in length and that hybridizes under stringent conditions to genomic DNA encoding the mutation in the polypeptide of SEQ ID NO:18.

15. The nucleic acid fragment of Claim 14, wherein said fragment has the nucleotide sequence of SEQ ID NO: 17.

16. An isolated polypeptide having the amino acid sequence of SEQ ID NO: 18

17. Canola seed designated Q4275 and represented by ATCC accession number 97569, deposited on 10 May 1996 at the American Type Culture Collection.

18. Progeny of the seed of Claim 17, said progeny having the mutant F-form of delta-12 fatty acid desaturase present in seed represented by ATCC accession number 97569, deposited on 10 May 1996 at the American Type Culture Collection. 25 19. Progeny of the seed of Claim 17, said progeny having two mutant delta-12 fatty acid desaturases present in seed represented by ATCC accession number 97569, deposited on 10 May 1996 at the American Type Culture Collection. The progeny of Claims 18 or 19, wherein said progeny are Brassica napus plants. 18/04/02,mcl 1028.claims,91 92

21. The isolated nucleic acid fragment of any one of Claims 1, 7 or 14 substantially as hereinbefore described in any one of the Examples. DATED this 18 t1h day of April, 2002 CARGILL, INCORPORATED By their Patent Attorneys: CALLINAN LAWRIE I 8/04/02,mcl I 028.claim,92