MATERIALS AND METHODS FOR INCREASING CORN SEED WEIGHT

Abstract: The subject invention pertains to novel variants of the maize gene, Shrunken2(Sh2) and a method of using that gene. The variant gene, Sh2-m1Rev6, encodes a subunit of the ADP-glucose pyrophosphorylase (AGP) enzyme that has additional amino acids inserted in or near the allosteric binding site of the protein. Corn seed expressing the Sh2-m1Rev6 gene has a 15 % weight increase over wild type seed. The increase in seed weight is not associated simply with an increase in percentage starch content of the seed.

Description: DESCRIPTION MATERIALS AND METHODS FOR INCREASING CORN SEED WEIGHT This invention was made with government support under National Science Foundation grant number 93052818. The government has certain rights in this invention. Cross-Reference to a Related Application This application is a continuation-in-part of co-pending application Serial No. 08/299,675 , filed September 1, 1994. Background of the Invention ADP-glucose pyrophosphorylase (AGP) catalyzes the conversion of ATP and α -glucose- 1- phosphate to ADP-glucose and pyrophosphate. ADP-glucose is used as a glycosyl donor in starch biosynthesis by plants and in glycogen biosynthesis by bacteria. The importance of ADP-glucose pyrophosphorylase as a key en2yme in the regulation of starch biosynthesis was noted in the study of starch deficient mutants of maize (Zea mays) endosperm (Tsai and Nelson, 1966; Dickinson and Preiss, 1969). AGP enzymes have been isolated from both bacteria and plants. Bacterial AGP consists of a homotetraraer, while plant AGP from photosynthetic and non-photosynthetic tissues is a heterotetramer composed of two different subunits. The plant enzyme is encoded by two different genes, with one subunit being larger than the other. This feature has been noted in a number of plants. The AGP subunits in spinach leaf have molecular weights of 54 kDa and 51 kDa, as estimated by SDS-PAGE. Both subunits are immunoreactive with antibody raised against purified AGP from spinach leaves (Copeland and Preiss, 1981; Morell et al., 1987). Immunological analysis using antiserum prepared against the small and large subunits of spinach leaf showed that potato tuber AGP is also encoded by two genes (Okita et al., 1990). The cDNA clones of the two subunits of potato tuber (50 and 51 kDa) have also been isolated and sequenced (Muller-Rober et al., 1990; Nakata etal, 1991). As Hannah and Nelson (Hannah and Nelson, 1975 and 1976) postulated, both Shrunken-2 (Sh2) (Bhave et al, 1990) and Brittle-2 (Bt2) (Bae et al., 1990) are structural genes of maize endosperm ADP-glucose pyrophosphorylase. Sh2 and Bt2 encode the large subunit and small subunit of the αuyme, respectively. From cDNA sequencing, Sh2 and Bt2 proteins have predicted molecular weight of 57,179 Da (Shaw and Hannah, 1992) and 52,224 Da, respectively. The endosperm is the site of most starch deposition during kernel development in maize. Sh2 and bt2 maize endosperm mutants have greatly reduced starch levels corresponding to deficient levels of AGP activity. Mutations of either gene have been shown to reduce AGP activity by about 95% (Tsai and Nelson, 1966; Dickinson and Preiss, 1969). Furthermore, it has been observed that enzymatic activities increase with the dosage of functional wild type Sh2 and Bt2 alleles, whereas mutant enzymes have altered kinetic properties. AGP is the rate limiting step in starch biosynthesis in plants. Stark et al. placed a mutant form of £ coli AGP in potato tuber and obtained a 35% increase in starch content (Stark, 1992). The cloning and characterization of the genes encoding the AGP enzyme subunits have been reported for various plants. These include Sh2 cDNA (Bhave et al., 1990), Sh2 genomic DNA (Shaw and Hannah, 1992), and Bt2 cDNA (Bae et al., 1990) from maize; small subunit cDNA (Anderson et al., 1989) and genomic DNA (Anderson et al., 1991) from rice; and small and large subunit cDNAs from spinach leaf (Morell etai, 1987) and potato tuber (Muller-Rober et al., 1990; Nakata et al, 1991). In addition, cDNA clones have been isolated from wheat endosperm and leaf tissue (Olive et al, 1989) and Arabidopsis thaliana leaf (Lin et al, 1988). AGP functions as an allosteric enzyme in all tissues and organisms investigated to date. The allosteric properties of AGP were first shown to be important in E. coli. A glycogen-overproducing E. coli mutant was isolated and the mutation mapped to the structural gene for AGP, designated as glyC. The mutant E. coli, known as g/yC-16, was shown to be more sensitive to the activator, fructose 1,6 bisphosphate, and less sensitive to the inhibitor, cAMP (Preiss, 1984). Although plant AGP's are also allosteric, they respond to different effector molecules than bacterial AGP's. In plants, 3-phosphoglyceric acid (3-PGA) functions as an activator while phosphate (PO 4 ) serves as an inhibitor (Dickinson and Preiss, 1969). In view of the fact that endosperm starch content comprises approximately 70% of the dry weight of the seed, alterations in starch biosynthesis correlate with seed weight. Unfortunately, the undesirable effect associated with such alterations has been an increase in the relative starch content of the seed. Therefore, the development of a method for increasing seed weight in plants without increasing the relative starch content of the seed is an object of the subject invention. Brief Summary of the Invention The subject invention concerns a novel variant of the Shrunken-2 {Shi) gene from maize. The Sh2 gene encodes ADP-glucose pyrophosphorylase (AGP), an important enzyme involved in starch synthesis in the major part of the corn seed, the endosperm. In a preferred embodiment, the novel gene of the subject invention encodes a variant AGP protein which has two additional amino acids inserted into the sequence. The variant g-sne described herein has been termed the Sh2-mlRev6 gene. Surprisingly, the presence of the Sh2-mlRev6 gene in a corn plant results in a substantial increase in com seed weight when compared to wild type seed weight, but does so in the absence of an increase in the relative starch content of the kernel. The subject invention further concerns a method of using the variant sh2 gene in maize to increase seed weight. The subject invention also concerns plants having the variant sh2 gene and expressing the mutant protein in the seed endosperm. As described herein, the shl variant, Sh2-mlRev6, can be produced using in vivo, site- specific mutagenesis. A transposable element was used to create a series of mutations in the sequence of the gene that encodes the enzyme. As a result, the Sh2-mlRev6 gene encodes an additional amino acid pair within or close to the allosteric binding site of the protein. Brief Description of the Sequences SEQ ID NO. 1 is the genomic nucleotide sequence of the Sh2-mlRev6 gene. SEQ ID NO. 2 is the nucleotide sequence of the Sh2-mlRev6 cDNA. SEQ ID NO.3 is the amino acid sequence of the protein encoded by nucleotides 87 through I640 of SEQ ID NO. 2. SEQ ID NO.4 is a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO. 5. SEQ ID NO. 5 is the amino acid sequence of an ADP-glucose pyrophosphorylase (AGP) enzyme subunit containing a single serine insertion. Detailed Disclosure of the Invention The subject invention provides novel variants of the Shrunken-2 (Sh2) gene and a method for increasing seed weight in a plant through the expression of the variant sh2 gene. The Sh2 gene encodes a subunit of the enzyme ADP-glucose pyrophosphorylase (AGP) in maize endosperm. One variant gene, denoted herein as Sh2-mlRev6, contains an insertion mutation that encodes an additional tyrosine:serine or serine:tyrosine amino acid pair that is not present in the wild type protein. The sequences of the wild type DNA and protein are disclosed in Shaw and Hannah, 1992. The in vivo, site-specific mutation which resulted in the tyrosine:serine or serine.tyrosine insertion, was generated in Sh2 using the transposable element, dissociation (Ds), which can insert into, and be excised from, the Sh2 gene under appropriate conditions. Ds excision can alter gene expression through the addition of nucleotides to a gene at the site of excision of the element. In a preferred embodiment, insertion mutations in the Sh2 gene were obtained by screening for germinal revertants after excision of the Ds transposon from the gene. The revertants were generated by self-pollination of a stock containing the DsSh2 mutant allele, the Activator (Ac) element of this transposable element system, and appropriate outside markers. The Ds element can transpose when the Ac element is present. Wild type seed were selected, planted, self-pollinated and crossed onto a tester stock. Results from this test cross were used to remove wild type alleles due to pollen contamination. Seeds homozygous for each revertant allele were obtained from the self- progeny. Forty-four germinal revertants of the Ds-induced shl mutant were collected. Cloning and sequencing of the Ds insertion site showed that the nucleotide insertion resides in the area of the gene that encodes the binding site for the AGP activator, 3-PGA (Morrell, 1988). Of the 44 germinal revertants obtained, 28 were sequenced. The sequenced revertants defined 5 isoalleles of shl: 13 restored the wild type sequence, 11 resulted in the insertion of the amino acid tyrosine, two contained an additional serine (inserted between amino acid residues 494 and 495, respectively, of the native protein sequence), one revertant contained a two amino acid insertion, tyrosine:tyrosine, and the last one, designated as Shl-mlRev6, contained the two amino acid insertion, tyrosine:serine or serine:tyrosine. The Sh2-mlRev6 variant encodes an AGP enzyme subunit that has either the serine.tyrosine amino acid pair inserted between the glycine and tyrosine at amino acid residues 494 and 495, respectively, of the native protein, or the serine.tyrosine amino acid pair inserted between the two tyrosine residues located at position 495 and 496 of the native protein sequence. Due to the sequence of the amino acids in the area of the insertions, the Sh2- mlRevό variant amino acid sequences encoded by each of these insertions are identical to each other. Surprisingly, the expression of the Sh2-mlRev6 gene in maize resulted in a significant increase in seed weight over that obtained from maize expressing the wild-type Sh2 allele. Moreover, seeds from plants having the Sh2-mlRev6 gene contained approximately the same percentage starch content relative to any of the other revertants generated. In a preferred embodiment, the Sh2-mlRev6 gene is contained in homozygous form within the genome of a plant seed. The subject invention further concerns a plant that has the Sh2-mlRev6 gene incorporated into its genome. Other alleles disclosed herein can also be incorporated into a plant genome. In a preferred embodiment, the plant is a monocotyledonous species. More preferably, the plant may be Zea mays. Plants having the Sh2-mlRev6 gene can be grown from seeds that have the gene in their genome. In addition, techniques for transforming plants with a gene are known in the art. Because of the degeneracy of the genetic code, a variety of different polynucleotide sequences can encode the variant AGP polypeptide disclosed herein. In addition, it is well within the skill of a person trained in the art to create alternative polynucleotide sequences encoding the same, or essentially the same, polypeptide of the subject invention. These variant or alternative polynucleotide sequences are within the scope of the subject invention. As used herein, references to "essentially the same" sequence refers to sequences which encode amino acid substitutions, deletions, additions, or insertions which do not materially alter the functional activity of the polypeptide encoded by Sh2-mlRev6 or the other alleles. The subject invention also contemplates those polynucleotide molecules having sequences which are sufficiently homologous with the wild type Sh2 DNA sequence so as to permit hybridization with that sequence under standard high- stringency conditions. Such hybridization conditions are conventional in the art (see, e.g. , Maniatis et a , 1989). The polynucleotide molecules of the subject invention can be used to transform plants to express the Sh2-mlRev6 allele, or other alleles of the subject invention, in those plants. In addition, the polynucleotides of the subject invention can be used to express the recombinant variant AGP enzyme. They can also be used as a probe to detect related enzymes. The polynucleotides can also be used as DNA sizing standards. The polypeptides encoded by the polynucleotides of the subject invention can be used to catalyze the conversion of ATP and α -glucose- 1 -phosphate to ADP-glucose and pyrophosphate, or to raise an immunogenic response to the AGP enzymes and variants thereof. They can also be used as molecular weight standards, or as an inert protein in an assay. The following are examples which illustrate procedures and processes, including the best mode, for practicing the invention. These examples should not be construed as limiting, and are not intended to be a delineation of all possible modifications to the technique. All percentages are by weight and all solvent mixture proportions are by volume unless otherwise noted. Example 1 - Expression oiSh2-mlRev6 Gene in Maize Endosperm. Homozygous plants of each revertant obtained after excision of the Ds transposon were crossed onto the FI hybrid com, "Florida Stay Sweet." This sweet com contains a null allele for the Shl gene, termed sh2-R. Resulting endosperms contained one dose of the functional allele from a revertant and two female-derived null alleles, denoted by the following genotype Sh2-mlRevX/sh2- R/sh2-R, where X represents one of the various isoalleles of the revertants. Crosses were made during two growing seasons. Resulting seed weight data for each revertant and wild type seed are shown in Table 1. The first column shows the amino acid insertion in the AGP enzyme obtained after the in vivo, site- specific mutagenesis. Table 1. Sequence # of revertants Average Seed weight Standard deviation alteration wild type 13 0.250 grams 0.015 tyrosine 11 0.238 grams 0.025 serine 2 0.261 grams 0.014 tyr. tyr 1 0.223 grams nd* tyr, ser 1 0.289 grams 0.022 (Rev ^^^ *nd = not determined The data shown in Table 1 represents the average kernel seed weight for each revertant over the course of two growing seasons. The expression of the Sh2-mlRev6 gene to produce the Rev6 mutant AGP subunit gave rise to an almost 16% increase in seed weight in comparison to the wild type revertant. The revertants having the single serine insertion also showed an increase in average seed weight over wild type seed weight. In addition, starch content was determined on the kernels analyzed above using various methodologies. The analysis showed that Shl-mlRev6 containing kernels were no higher in percentage starch relative to kernels expressing the other alleles shown in the table above. Therefore, it appears that the increase in seed weight is not solely a function of starch content. Corn seeds that contain at least one functional Sh2-mlRev6 allele (the tyrosine, serine insertion) have been deposited with the American Type Culture Collection (ATCC), 12301 Parklawn Drive, Rockville, Maryland 20852 USA, on May 20, 1996 and assigned ATCC accesion number ATCC 97624. Seeds having at least one functional Sh2-mIRev20 allele (serine insertion) have also been deposited with ATCC on May 20, 1996 and assigned ATCC accession number ATCC 97625. The seeds have been deposited under conditions that assure that access to the biological material will be available during the pendency of this patent application to one determined by the Commissioner of Patents and Trademarks to be entitled thereto under 37 CFR 1.14 and 35 U.S.C. 122. The deposit will be available as required by foreign patent laws in countries wherein counterparts of the subject application, or its progeny, are filed. However, it should be understood that the availability of a deposit does not constitute a license to practice the subject invention in derogation of patent rights granted by governmental action. Further, the subject seed deposit will be stored and made available to the public in accord with the provisions of the Budapest Treaty for the Deposit of Microorganisms, i. e. , it will be stored with all the care necessary to keep it viable and uncontaminated for a period of at least five years after the most recent request for the furnishing of a sample of the deposit, and in any case, for a period of at least thirty (30) years after the date of deposit or for the enforceable life of any patent which may issue disclosing the seed. The depositor acknowledges the duty to replace the deposit should the depository be unable to furnish a sample when requested, due to the condition of the deposit. All restrictions on the availability to the public of the subject seed deposit will be irrevocably removed upon the granting of a patent disclosing it. As would be apparent to a person of ordinary skill in the art, seeds and plants that are homozygous for the Sh2-mlRev6 or the Sh2-mlRev20 allele can be readily prepared from heterozygous seeds using techniques that are standard in the art. In addition, the Sh2-mlRev6 and Sh2-mlRev20 genes can be readily obtained from the deposited seeds. The skilled artisan, using standard techniques known in the art, can also prepare polynucleotide molecules that encode additional amino acid residues, such as serine, at the location of the insertions in the subject revertants. Such polynucleotide molecules are included within the scope of the subject invention. It should be understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the scope and purview of this application and the scope of the appended claims. References Anderson, J.M, J. Hnilo, R Larson, t.W. Okita, M. Morell, J. Preiss (1989) "The encoded primary sequence of a rice seed ADP-glucose pyrophosphorylase subunit and its homology to the bacterial enzyme," J Biol Chem. 264:12238-12242. Anderson, J.M., R. Larson, D. Landencia, W.T. Kim, D. Morrow, T.W. Okita, J. Preiss (1991) "Molecular characterization of the gene encoding a rice endosperm-specific ADP-glucose pyrophosphorylase subunit and its developmental pattern of transcription," Gene 97: 199- 205. Bae, J.M., M. Giroux, L.C. Hannah (1990) "Cloning and characterization of the Brittle-2 gene of maize," Maydica 35:317-322. Bhave, M.R., S. Lawrence, C. Barton, L.C. Hannah (1990) "Identification and molecular characterization oϊShrunken-2 cDNA clones of maize," Plant Cell 2:581-588. Copeland, L., J. Preiss (1981) "Purification of spinach leaf ADP-glucose pyrophosphorylase," Plant Physiol 68:996-1001. Dickinson, D.B., J. Preiss (1969) "Presence of ADP-glucose pyrophosphorylase in Shrunken-2 and Brittle-2 mutants of maize endosperm," Plant Physiol 44:1058-1062. Hannah, L.C, O.E. Nelson (1975) "Characterization of adenosine diphosphate glucose pyrophosphorylase from developing maize seeds," Plant Physiol. 55:297-302. Hannah, L.C, O.E. Nelson (1976) "Characterization of adenosine diphosphate glucose pyrophosphorylase from Shrunken-2 and Brittle-2 mutants of maize," Biochem. Genet. 14:547-560. Lin, T., T. Caspar, C. Somerville, J. Preiss (1988) "A starch deficient mutant of Arabidopsis thaliana with low ADP-glucose pyrophosphorylase activity lacks one of the two subunits of the enzyme," PlantPhysiol 88: 1175-1181. Maniatis, T., E.F. Fritsch, J. Sambrook (1989) Molecular Cloning: A Laboratory Manual, 2d Edition, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York. Morell, M., M. Bloon, V. Knowles, J. Preiss (1988) "Subunit structure of spinach leaf ADP-glucose pyrophosphorylase," J. Bio. Chem. 263:633. Muller-Rober, B.T., J. Kossmann, L.C. Hannah, L. Willmitzer, U. Sounewald (1990) "One of the two different ADP-glucose pyrophosphorylase genes from potato responds strongly to elevated levels of sucrose," Mol. Gen. Genet. 224:136-146. Nakata, P.A., T.W. Greene, J.M. Anderson, B.J. Smith-White, T.W. Okita, J. Preiss (1991) "Comparison of primary sequences of two potato tuber ADP-glucose pyrophosphorylase subunits," Plant Mol. Biol. 17:1089-1093. Okita, T.W., P.A. Nakata, J.M. Anderson, J. Sowokinos, M. Morell, J. Preiss (1990) "The subunit structure of potato tuber ADP-glucose pyrophosphorylase," Plant Physiol. 93 :785-790. Olive, M.R., R.J. Ellis, W.W. Schuch (1989) "Isolation and nucleotide sequences of cDNA clones encoding ADP-glucose pyrophosphorylase polypeptides from wheat leaf and endoosperm," Plant Physiol. Mol. Biol. 12:525-538. Preiss, J. (1984) "Bacterial glycogen synthesis and it regulation," Ann. Rev. Microbwl 419-458. Shaw, J.R., L.C. Hannah (1992) "Genomic nucleotide sequence of a wild type Shrunken-2 allele of Zea mays " Plant Physiol. 98:1214-1216. Starke, et al. (1992) "Regulation of the amount of starch in plant tissues by ADP-glucose pyrophosphorylase," Science 258:287. Tsai, C, O.E. Nelson (1966) "Starch-deficient maize mutant lacking adenosine diphosphate glucose pyrophosphorylase activity," Science 151 :341-343. NOT FURNISHED UPON FILING (A) LENGTH: 7745 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS : single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID Nθ:l: TAAGAGGGGT GCACCTAGCA TAGATTTTTT GGGCTCCCTG GCCTCTCCTT TCTTCCGCCT 60 GAAAACAACC TACATGGATA CATCTGCAAC CAGAGGGAGT ATCTGATGCT TTTTCCTGGG 120 CAGGGAGAGC TATGAGACGT ATGTCCTCAA AGCCACTTTG CATTGTGTGA AACCAATATC 180 GATCTTTGTT ACTTCATCAT GCATGAACAT TTGTGGAAAC TACTAGCTTA CAAGCATTAG 240 TGACAGCTCA GAAAAAAGTT ATCTCTGAAA GGTTTCATGT GTACCGTGGG AAATGAGAAA 300 TGTTGCCAAC TCAAACACCT TCAATATGTT GTTTGCAGGC AAACTCTTCT GGAAGAAAGG 360 TGTCTAAAAC TATGAACGGG TTACAGAAAG GTATAAACCA CGGCTGTGCA TTTTGGAAGT 420 ATCATCTATA GATGTCTGTT GAGGGGAAAG CCGTACGCCA ACGTTATTTA CTCAGAAACA 480 GCTTCAACAC ACAGTTGTCT GCTTTATGAT GGCATCTCCA CCCAGGCACC CACCATCACC 540 TATTCACCTA TCTCTCGTGC CTGTTTATTT TCTTGCCCTT TCTGATCATA AAAAATCATT 600 AAGAGTTTGC AAACATGCAT AGGCATATCA ATATGCTCAT TTATTAATTT GCTAGCAGAT 660 CATCTTCCTA CTCTTTACTT TATTTATTGT TTGAAAAATA TGTCCTGCAC CTAGGGAGCT 720 CGTATACAGT ACCAATGCAT CTTCATTAAA TGTGAATTTC AGAAAGGAAG TAGGAACCTA 780 TGAGAGTATT TTTCAAAATT AATTAGCGGC TTCTATTATG TTTATAGCAA AGGCCAAGGG 840 CAAAATCGGA ACACTAATGA TGGTTGGTTG CATGAGTCTG TCGATTACTT GCAAGAAATG 900 TGAACCTTTG TTTCTGTGCG TGGGCATAAA ACAAACAGCT TCTAGCCTCT TTTACGGTAC 960 TTGCACTTGC AAGAAATGTG AACTCCTTTT CATTTCTGTA TGTGGACATA ATGCCAAAGC 1020 ATCCAGGCTT TTTCATGGTT GTTGATGTCT TTACACAGTT CATCTCCACC AGTATGCCCT 1080 CCTCATACTC TATATAAACA CATCAACAGC ATCGCAATTA GCCACAAGAT CACTTCGGGA 1140 GGCAAGTGTG ATTTCGACCT TGCAGCCACC TTTTTTTGTT CTGTTGTAAG TATACTTTCC 1200 CTTACCATCT TTATCTGTTA GTTTAATTTG TAATTGGGAA GTATTAGTGG AAAGAGGATG 1260 AGATGCTATC ATCTATGTAC TCTGCAAATG CATCTGACGT TATATGGGCT GCTTCATATA 1320 ATTTGAATTG CTCCATTCTT GCCGACAATA TATTGCAAGG TATATGCCTA GTTCCATCAA 1380 AAGTTCTGTT TTTTCATTCT AAAAGCATTT TAGTGGCACG CAATTTTGTC CATGAGGGAA 1440 AGGAAATCTG TTTTGGTTAC TTTGCTTGAG GTGCATTCTT CATATGTCCA GTTTTATGGA 1500 AGTAATAAAC TTCAGTTTGG TCATAAGATG TCATATTAAA GGGCAAACAT ATATTCAATG 1560 TTCAATTCAT CGTAAATGTT CCCTTTTTGT AAAAGATTGC ATACTCATTT ATTTGAGTTG 1620 CAGGTGTATC TAGTAGTTGG AGGAGATATG CAGTTTGCAC TTGCATTGGA CACGAACTCA 1680 GGTCCTCACC AGATAAGATC TTGTGAGGGT GATGGGATTG ACAGGTTGGA AAAATTAAGT 1740 ATTGGGGGCA GAAAGCAGGA GAAAGCTTTG AGAAATAGGT GCTTTGGTGG TAGAGTTGCT 1800 GCAACTACAC AATGTATTCT TACCTCAGAT GCTTGTCCTG AAACTCTTGT AAGTATCCAC 1860 CTCAATTATT ACTCTTACAT GTTGGTTTAC TTTACGTTTG TCTTTTCAAG GGAAATTTAC 1920 TGTATTTTTT GTGTTTTGTG GGAGTTCTAT ACTTCTGTTG GACTGGTTAT TGTAAAGATT 1980 TGTTCAAATA GGGTCATCTA ATAATTGTTT GAAATCTGGG AACTGTGGTT TCACTGCGTT 2040 CAGGAAAAAG TGAATTATTG GTTACTGCAT GAATAACTTA TGGAAATAGA CCTTAGAGTT 2100 GCTGCATGAT TATCACAAAT CATTGCTACG ATATCTTATA ATAGTTCTTT CGACCTCGCA 2160 TTACATATAT AACTGCAACT CCTAGTTGCG TTCAAAAAAA AAAATGCAAC TCTTAGAACG 2220 CTCACCAGTG TAATCTTTCC TGAATTGTTA TTTAATGGCA TGTATGCACT ACTTGTATAC 2280 TTATCTAGGA TTAAGTAATC TAACTCTAGG CCCCATATTT GCAGCATTCT CAAACACAGT 2340 CCTCTAGGAA AAATTATGCT GATGCAAACC GTGTATCTGC TATCATTTTG GGCGGAGGCA 2400 CTGGATCTCA GCTCTTTCCT CTGACAAGCA CAAGAGCTAC GCCTGCTGTA AGGGATAACA 2460 CTGAACATCC AACGTTGATT ACTCTATTAT AGTATTATAC AGACTGTACT TTTCGAATTT 2520 ATCTTAGTTT TCTACAATAT TTAGTGGATT CTTCTCATTT TCAAGATACA CAATTGATCC 2580 ATAATCGAAG TGGTATGTAA GACAGTGAGT TAAAAGATTA TATTTTTTGG GAGACTTCCA 2640 GTCAAATTTT CTTAGAAGTT TTTTTGGTCC AGATGTTCAT AAAGTCGCCG CTTTCATACT 2700 TTTTTTAATT TTTTAATTGG TGCACTATTA GGTACCTGTT GGAGGATGTT ACAGGCTTAT 2760 TGATATCCCT ATGAGTAACT GCTTCAACAG TGGTATAAAT AAGATATTTG TGATGAGTCA 2820 GTTCAATTCT ACTTCGCTTA ACCGCCATAT TCATCGTACA TACCTTGAAG GCGGGATCAA 2880 CTTTGCTGAT GGATCTGTAC AGGTGATTTA CCTCATCTTG TTGATGTGTA ATACTGTAAT 2940 TAGGAGTAGA TTTGTGTGGA GAGAATAATA AACAGATGCC GAGATTCTTT TCTAAAAGTC 3000 TAGATCCAAA GGCATTGTGG TTCAAAACAC TATGGACTTC TACCATTTAT GTCATTACTT 3060 TGCCTTAATG TTCCATTGAA TGGGGCAAAT TATTGATTCT ACAAGTGTTT AATTAAAAAC 3120 TAATTGTTCA TCCTGCAGGT ATTAGCGGCT ACACAAATGC CTGAAGAGCC AGCTGGATGG 3180 TTCCAGGGTA CAGCAGACTC TATCAGAAAA TTTATCTGGG TACTCGAGGT AGTTGATATT 3240 TTCTCGTTTA TGAATGTCCA TTCACTCATT CCTGTAGCAT TGTTTCTTTG TAATTTTGAG 3300 TTCTCCTGTA TTTCTTTAGG ATTATTACAG TCACAAATCC ATTGACAACA TTGTAATCTT 3360 GAGTGGCGAT CAGCTTTATC GGATGAATTA CATGGAACTT GTGCAGGTAT GGTGTTCTCT 3420 TGTTCCTCAT GTTTCACGTA ATGTCCTGAT TTTGGATTAA CCAACTACTT TTGGCATGCA 3480 TTATTTCCAG AAACATGTCG AGGACGATGC TGATATCACT ATATCATGTG CTCCTGTTGA 3540 TGAGAGGTAA TCAGTTGTTT ATATCATCCT AATATGAATA TGTCATCTTG TTATCCAACA 3600 CAGGATGCAT ATGGTCTAAT CTGCTTTCCT TTTTTTTCCC TTCGGAAGCC GAGCTTCTAA 3660 AAATGGGCTA GTGAAGATTG ATCATACTGG ACGTGTACTT CAATTCTTTG AAAAACCAAA 3720 GGGTGCTGAT TTGAATTCTA TGGTTAGAAA TTCCTTGTGT AATCCAATTC TTTTGTTTTC 3780 CTTTCTTTCT TGAGATGAAC CCCTCTTTTA GTTATTTCCA TGGATAACCT GTACTTGACT 3840 TATTCAGAAA TGATTTTCTA TTTTGCTGTA GAATCTGACA CTAAAGCTAA TAGCACTGAT 3900 GTTGCAGAGA GTTGAGACCA ACTTCCTGAG CTATGCTATA GATGATGCAC AGAAATATCC 3960 ATACCTTGCA TCAATGGGCA TTTATGTCTT CAAGAAAGAT GCACTTTTAG ACCTTCTCAA 4020 GTAATCACTT TCCTGTGACT TATTTCTATC CAACTCCTAG TTTACCTTCT AACAGTGTCA 4080 ATTCTTAGGT CAAAATATAC TCAATTACAT GACTTTGGAT CTGAAATCCT CCCAAGAGCT 4140 GTACTAGATC ATAGTGTGCA GGTAAGTCTG ATCTGTCTGG AGTATGTGTT CTGTAAACTG 4200 TAAATTCTTC ATGTCAAAAA GTTGTTTTTG TTTCCAGTTT CCACTACCAA TGCACGATTT 4260 ATGTATTTTC GCTTCCATGC ATCATACATA CTAACAATAC ATTTTACGTA TTGTGTTAGG 4320 CATGCATTTT TACGGGCTAT TGGGAGGATG TTGGAACAAT CAAATCATTC TTTGATGCAA 4380 ACTTGGCCCT CACTGAGCAG GTACTCTGTC ATGTATTCTG TACTGCATAT ATATTACCTG 4440 GAATTCAATG CATAGAATGT GTTAGACCAT CTTAGTTCCA TCCTGTTTTC TTCAATTAGC 4500 TTATCATTTA ATAGTTGTTG GCTAGAATTT AAACACAAAT TTACCTAATA TGTTTCTCTC 4560 TTCAGCCTTC CAAGTTTGAT TTTTACGATC CAAAAACACC TTTCTTCACT GCACCCCGAT 4620 GCTTGCCTCC GACGCAATTG GACAAGTGCA AGGTATATGT CTTACTGAGC ACAATTGTTA 4680 CCTGAGCAAG ATTTTGTGTA CTTGACTTGT TCTCCTCCAC AGATGAAATA TGCATTTATC 47 0 TCAGATGGTT GCTTACTGAG AGAATGCAAC ATCGAGCATT CTGTGATTGG AGTCTGCTCA 800 CGTGTCAGCT CTGGATGTGA ACTCAAGGTA CATACTCTGC CAATGTATCT ACTCTTGAGT 4860 ATACCATTTC AACACCAAGC ATCACCAAAT CACACAGAAC AATAGCAACA AAGCCTTTTA 4920 GTTCCAAGCA ATTTAGGGTA GCCTAGAGTT GAAATCTAAC AAAACAAAAG TCAAAGCTCT 4980 ATCACGTGGA TAGTTGTTTT CCATGCACTC TTATTTAAGC TAATTTTTTG GGTATACTAC 5040 ATCCATTTAA TTATTGTTTT ATTGCTTCTT CCCTTTGCCT TTCCCCCATT ACTATCGCGT 5100 CTTAAGATCA TACTACGCAC TAGTGTCTTT AGAGGTCTCT GGTGGACATG TTCAAACCAT 5160 CTCAATCGGT GTTGGACAAG TTTTTCTTGA ATTTGTGCTA CACCTAACCT ATCACGTATG 5220 TCATCGTTTC AAACTCGATC CTTCCTGTAT CATCATAAAT CCAATGCAAC ATACGCATTT 5280 ATGCAACATT TATCTGTTGA ACATGTCATC TTTTTGTAGG TTAACATTAT GCACCATACA 5340 ATGTAGCATG TCTAATCATC ATCCTATAAA ATTTACATTT TAGCTTATGT GGTATCCTCT 5400 TGCCACTTAG AACACCATAT GCTTGATGCC ATTTCATCCA CCCTGCTTTG ATTCTATGGC 5460 TAACATCTTC ATTAATATCC TCGCCTCTCT GTATCATTGG TCCTAAATAT GGAAATACAT 5520 TCTTTCTGGG CACTACTTGA CCTTCCAAAC TAACGTCTCC TTTGCTCCTT TCTTGTGTGT 5580 AGTAGTACCG AAGTCACATC TCATATATTC GGTTTTAGTT CTACTAAGTC CCGGGTTCGA 5640 TCCCCCTCAG GGGTGAATTT CGGGCTTGGT AAAAAAAATC CCCTCGCTGT GTCCCGCCCG 5700 CTCTCGGGGA TCGATATCCT GCGCGCCACC CTCCGGCTGG GCATTGCAGA GTGAGCAGTT 5760 GATCGGCTCG TTAGTGATGG GGAGCGGGGT TCAAGGGTTT TCTCGGCCGG GACCATGTTT 5820 CGGTCTCTTA ATATAATGCC GGGAGGGCAG TCTTTCCCTC CCCGGTCGAG TTTTAGTTCT 5880 ACCGAGTCTA AAACCTTTGG ACTCTAGAGT CCCCTGTCAC AACTCACAAC TCTAGTTTTC 5940 TATTTACTTC TACCTAGCGT TTATTAATGA TCACTATATC GTCTGTAAAA AGCATACACC 6000 AATGTAATCC CCTTGTATGT CCCTTGTAAT ATTATCCATC ACAAGAAAAA AAGGTAAGGC 6060 TCAAAGTTGA CTTTTGATAT AGTCCTATTC TAATCGAGAA GTCATCTGTA TCTTCGTCTC 6120 TTGTTCGAAC ACTAGTCACA AAATTTTTTG TACATGTTCT TAATGAGTCC AACGTAATAT 6180 TCCTTGATAT TTTGTCATAA GCCCTCATCA AGTCAATGAA AATCACGTGT AGGTCCTTCA 6240 TTTGTTCCTT ATACTGCTCC ATCACTTGTC TC ..TTAAGAA AATCTCTCTC ATAGTTAACC 6300 TTTTGGCATG AAACAAAATC ACACAGAAGT TGTTTCCTTT TTTTAAGATC CCACACAAAA 6360 GAGGTTTGAT CTAAGGAATC TGGATCCCTG ACAGGTTTAT CAAAATCCTT TGTGTTTTTC 6420 TTAAAACTGA ATATTCCTCC AGCTTCTAGT ATTGATGTAA TATTCAATCT GTTTAGCAAG 6480 TGAACACCTT GGTTCTTGTT GTTACTGTAC CCCCCCCCCC CCCCCCCCCC CGAGGCCCAG 6540 ATTACCACGA CATGAATACA AGAATATTGA ACCCAGATCT AGAGTTTGTT TGTACTGTTG 6600 AAAATCGGTG ACAATTCATT TTGTTATTGC GCTTTCTGAT AACGACAGGA CTCCGTGATG 6660 ATGGGAGCGG ACACCTATGA AACTGAAGAA GAAGCTTCAA AGCTACTGTT AGCTGGGAAG 6720 GTCCCAGTTG GAATAGGAAG GAACACAAAG ATAAGGTGAG TATGGATGTG GAACCACCGG 6780 TTAGTTCCC AAAATATCAC TCACTGATAC CTGATGGTAT CCTCTGATTA TTTTCAGGAA 6840 CTGTATCATT GACATGAATG CTAGGATTGG GAAGAACGTG GTGATCACAA ACAGTAAGGT 6900 GAGCGAGCGC ACCTACATGG GTGCAGAATC TTGTGTGCTC ATCTATCCTA ATTCGGTAAT 6960 TCCTATCCAG CGCTAGTCTT GTGACCATGG GGCATGGGTT CGACTCTGTG ACAGGGCATC 7020 CAAGAGGCTG ATCACCCGGA AGAAGGGTAC TCGTACTACA TAAGGTCTGG AATCGTGGTG 7080 ATCTTGAAGA ATGCAACCAT CAACGATGGG TCTGTCATAT AGATCGGCTG CGTGTGCGTC 7140 TACAAAACAA GAACCTACAA TGGTATTGCA TCGATGGATC GTGTAACCTT GGTATGGTAA 7200 GAGCCGCTTG ACAGAAAGTC GAGCGTTCGG GCAAGATGCG TAGTCTGGCA TGCTGTTCCT 7260 TGACCATTTG TGCTGCTAGT ATGTACTGTT ATAAGCTGCC CTAGAAGTTG CAGCAAACCT 7320 TTTTATGAAC CTTTGTATTT CCATTACCTG CTTTGGATCA ACTATATCTG TCATCCTATA 7380 TATTACTAAA TTTTTACGTG TTTTTCTAAT TCGGTGCTGC TTTTGGGATC TGGCTTCGAT 7440 GACCGCTCGA CCCTGGGCCA TTGGTTCAGC TCTGTTCCTT AGAGCAACTC CAAGGAGTCC 7500 TAAATTTTGT ATTAGATACG AAGGACTTCA GCCGTGTATG TCGTCCTCAC CAAACGCTCT 7560 TTTTGCATAG TGCAGGGGTT GTAGACTTGT AGCCCTTGTT TAAAGAGGAA TTTGAATATC 7620 AAATTATAAG TATTAAATAT ATATTTAATT AGGTTAACAA ATTTGGCTCG TTTTTAGTCT 7680 TTATTTATGT AATTAGTTTT AAAAATAGAC CTATATTTCA ATACGAAATA TCATTAACAT 77 0 CGATA 7745 (2) INFORMATION FOR SEQ ID NO: 2: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1919 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS : single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: CDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2: ACAAGATCAC TTCGGGAGGC AAGTGCGATT TTGATCTTGC AGCCACCTTT TTTTGTTCTG 60 TTGTGTATCT AGTAGTTGGA GGAGATATGC AGTTTGCACT TGCATTGGAC ACGAACTCAG 120 GTCCTCACCA GATAAGATCT TGTGAGGGTG ATGGGATTGA CAGGTTGGAA AAATTAAGTA 180 TTGGGGGCAG AAAGCAGGAG AAAGCTTTGA GAAATAGGTG CTTTGGTGGT AGAGTTGCTG 240 CAACTACACA ATGTATTCTT ACCTCAGATG CTTGTCCTGA AACTCTTCAT TCTCAAACAC 300 AGTCCTCTAG GAAAAATTAT GCTGATGCAA ACCGTGTATC TGCGATCATT TTGGGCGGAG 360 GCACTGGATC TCAGCTCTTT CCTCTGACAA GCACAAGAGC TACGCCTGCT GTACCTGTTG 420 GAGGATGTTA CAGGCTTATT GATATCCCTA TGAGTAACTG CTTCAACAGT GGTATAAATA 480 AGATATTTGT GATGAGTCAG TTCAATTCTA CTTCGCTTAA CCGCCATATT CATCGTACAT 540 ACCTTGAAGG CGGGATCAAC TTTGCTGATG GATCTGTACA GGTATTAGCG GCTACACAAA 600 TGCCTGAAGA GCCAGCTGGA TGGTTCCAGG GTACAGCAGA CTCTATCAGA AAATTTATCT 660 GGGTACTCGA GGATTATTAC AGTCACAAAT CCATTGACAA CATTGTAATC TTGAGTGGCG 720 ATCAGCTTTA TCGGATGAAT TACATGGAAC TTGTGCAGAA ACATGTCGAG GACGATGCTG 780 ATATCACTAT ATCATGTGCT CCTGTTGATG AGAGCCGAGC TTCTAAAAAT GGGCTAGTGA 840 AGATTGATCA TACTGGACGT GTACTTCAAT TCTTTGAAAA ACCAAAGGGT GCTGATTTGA 900 ATTCTATGAG AGTTGAGACC AACTTCCTGA GCTATGCTAT AGATGATGCA CAGAAATATC 960 CATACCTTGC ATCAATGGGC ATTTATGTCT TCAAGAAAGA TGCACTTTTA GACCTTCTCA 1020 AGTCAAAATA TACTCAATTA CATGACTTTG GATCTGAAAT CCTCCCAAGA GCTGTACTAG 1080 ATCATAGTGT GCAGGCATGC ATTTTTACGG GCTATTGGGA GGATGTTGGA ACAATCAAAT 1140 CATTCTTTGA TGCAAACTTG GCCCTCACTG AGCAGCCTTC CAAGTTTGAT TTTTACGATC 1200 CAAAAACACC TTTCTTCACT GCACCCCGAT GCTTGCCTCC GACGCAATTG GACAAGTGCA 1260 AGATGAAATA TGCATTTATC TCAGATGGTT GCTTACTGAG AGAATGCAAC ATCGAGCATT 1320 CTGTGATTGG AGTCTGCTCA CGTGTCAGCT CTGGATGTGA ACTCAAGGAC TCCGTGATGA 1380 TGGGAGCGGA CATCTATGAA ACTGAAGAAG AAGCTTCAAA GCTACTGTTA GCTGGGAAGG 1440 TCCCGATTGG AATAGGAAGG AACACAAAGA TAAGGAACTG TATCATTGAC ATGAATGCTA 1500 GGATTGGGAA GAACGTGGTG ATCACAAACA GTAAGGGCAT CCAAGAGGCT GATCACCCGG 1560 AAGAAGGGTA CTCGTACTAC ATAAGGTCTG GAATCGTGGT GATCCTGAAG AATGCAACCA 1620 TCAACGATGG GTCTGTCATA TAGATCGGCT GCGTTTGCGT CTACAAAACA AGAACCTACA 1680 ATGGTATTGC ATCGATGGAT CGTGTAACCT TGGTATGGTA AGAGCCGCTT GACAGGAAGT 1740 CGAGCTTCGG GCGAAGATGC TAGTCTGGCA TGCTGTTCCT TGACCATTTG TGCTGCTAGT 1800 ATGTACCTGT TATAAGCTGC CCTAGAAGTT GCAGCAAACC TTTTTATGAA CCTTTGTATT 1860 TCCATTACCC TGCTTTGGAT CAACTATATC TGTCAGTCCT ATATATTACT AAATTTTTA 1919 (2) INFORMATION FOR SEQ ID NO: 3: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 518 amino acids (B) TYPE: amino acid (C) STRANDEDNESS : single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3: Met Gin Phe Ala Leu Ala Leu Asp Thr Asn Ser Gly Pro His Gin lie 1 5 10 15 Arg Ser Cys Glu Gly Asp Gly lie Asp Arg Leu Glu Lys Leu Ser lie 20 25 30 Gly Gly Arg Lys Gin Glu Lys Ala Leu Arg Asn Arg Cys Phe Gly Gly 35 40 45 Arg Val Ala Ala Thr Thr Gin Cys lie Leu Thr Ser Asp Ala Cys Pro 50 55 60 Glu Thr Leu His Ser Gin Thr Gin Ser Ser Arg Lys Asn Tyr Ala Asp 65 70 75 80 Ala Asn Arg Val Ser Ala He He Leu Gly Gly Gly Thr Gly Ser Gin 85 90 95 Leu Phe Pro Leu Thr Ser Thr Arg Ala Thr Pro Ala val Pro Val Gly 100 105 110 Gly cys Tyr Arg Leu He Asp He Pro Met Ser Asn Cys Phe Asn Ser 115 120 125 Gly He Asn Lys He Phe Val Met Ser Gin Phe Asn Ser Thr Ser Leu 130 135 140 Asn Arg His He His Arg Thr Tyr Leu Glu Gly Gly He Asn Phe Ala 145 150 155 160 Asp Gly ser Val Gin Val Leu Ala Ala Thr Gin Met Pro Glu Glu Pro 165 170 175 Ala Gly Trp Phe Gin Gly Thr Ala Asp Ser He Arg Lys he He Trp 180 185 190 val Leu Glu Asp Tyr Tyr Ser His Lys ser He Asp Asn He Val He 195 200 205 Leu ser Gly Asp Gin Leu Tyr Arg Met Asn Tyr Met Glu Leu Val Gin 210 215 220 Lys His Val Glu Asp Asp Ala Asp lie Thr He ser cys Ala Pro Val 225 230 235 240 Asp Glu Ser Arg Ala ser Lys Asn Gly Leu Val Lys He Asp His Thr 245 250 255 Gly Arg val Leu Gin Phe Phe Glu Lys Pro Lys Gly Ala Asp Leu Asn 260 265 270 Ser Met Arg Val Glu Thr Asn Phe Leu Ser Tyr Ala He Asp Asp Ala 275 280 285 Gin Lys Tyr Pro Tyr Leu Ala Ser Met Gly He Tyr Val Phe Lys Lys 290 295 300 Asp Ala Leu Leu Asp Leu Leu Lys Ser Lys Tyr Thr Gin Leu His Asp 305 310 315 320 Phe Gly ser Glu He Leu Pro Arg Ala Val Leu Asp His Ser Val Gin 325 330 335 Ala cys He Phe Thr Gly Tyr Trp Glu Asp Val Gly Thr lie Lys ser 340 345 350 Phe Phe Asp Ala Asn Leu Ala Leu Thr Glu Gin Pro Ser Lys Phe Asp 355 360 365 Phe Tyr Asp Pro Lys Thr Pro phe Phe Thr Ala Pro Arg cys Leu pro 370 375 380 Pro Thr Gin Leu Asp Lys cys Lys Met Lys Tyr Ala Phe He Ser Asp 385 390 395 400 Gly Cys Leu Leu Arg Glu Cys Asn He Glu His Ser Val lie Gly Val 405 410 415 Cys Ser Arg Val Ser Ser Gly Cys Glu Leu Lys Asp Ser Val Met Met 420 425 430 Gly Ala Asp He Tyr Glu Thr Glu Glu Glu Ala Ser Lys Leu Leu Leu 435 440 445 Ala Gly Lys val Pro He Gly He Gly Arg Asn Thr Lys He Arg Asn 450 455 460 Cys He He Asp Met Asn Ala Arg He Gly Lys Asn Val Val He Thr 465 470 475 480 Asn Ser Lys Gly He Gin Glu Ala Asp His Pro Glu Glu Gly Tyr ser 485 490 495 Tyr Tyr He Arg Ser Gly He Val Val He Leu Lys Asn Ala Thr He 500 505 510 Asn Asp Gly Ser val He 515 (2) INFORMATION FOR SEQ ID NO: 4: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1551 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: CDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4: ATGCAGTTTG CACTTGCATT GGACACGAAC TCAGGTCCTC ACCAGATAAG ATCTTGTGAG 60 GGTGATGGGA TTGACAGGTT GGAAAAATTA AGTATTGGGG GCAGAAAGCA GGAGAAAGCT 120 TTGAGAAATA GGTGCTTTGG TGGTAGAGTT GCTGCAACTA CACAATGTAT TCTTACCTCA 180 GATGCTTGTC CTGAAACTCT TCATTCTCAA ACACAGTCCT CTAGGAAAAA TTATGCTGAT 240 GCAAACCGTG TATCTGCGAT CATTTTGGGC GGAGGCACTG GATCTCAGCT CTTTCCTCTG 300 ACAAGCACAA GAGCTACGCC TGCTGTACCT GTTGGAGGAT GTTACAGGCT TATTGATATC 360 CCTATGAGTA ACTGCTTCAA CAGTGGTATA AATAAGATAT TTGTGATGAG TCAGTTCAAT 420 TCTACTTCGC TTAACCGCCA TATTCATCGT ACATACCTTG AAGGCGGGAT CAACTTTGCT 480 GATGGATCTG TACAGGTATT AGCGGCTACA CAAATGCCTG AAGAGCCAGC TGGATGGTTC 540 CAGGGTACAG CAGACTCTAT CAGAAAATTT ATCTGGGTAC TCGAGGATTA TTACAGTCAC 600 AAATCCATTG ACAACATTGT AATCTTGAGT GGCGATCAGC TTTATCGGAT GAATTACATG 660 GAACTTGTGC AGAAACATGT CGAGGACGAT GCTGATATCA CTATATCATG TGCTCCTGTT 720 GATGAGAGCC GAGCTTCTAA AAATGGGCTA GTGAAGATTG ATCATACTGG ACGTGTACTT 780 CAATTCTTTG AAAAACCAAA GGGTGCTGAT TTGAATTCTA TGAGAGTTGA GACCAACTTC 840 CTGAGCTATG CTATAGATGA TGCACAGAAA TATCCATACC TTGCATCAAT GGGCATTTAT 900 GTCTTCAAGA AAGATGCACT TTTAGACCTT CTCAAGTCAA AATATACTCA ATTACATGAC 960 TTTGGATCTG AAATCCTCCC AAGAGCTGTA CTAGATCATA GTGTGCAGGC ATGCATTTTT 1020 ACGGGCTATT GGGAGGATGT TGGAACAATC AAATCATTCT TTGATGCAAA CTTGGCCCTC 10BO ACTGAGCAGC CTTCCAAGTT TGATTTTTAC GATCCAAAAA CACCTTTCTT CACTGCACCC 1140 CGATGCTTGC CTCCGACGCA ATTGGACAAG TGCAAGATGA AATATGCATT TATCTCAGAT 1200 GGTTGCTTAC TGAGAGAATG CAACATCGAG CATTCTGTGA TTGGAGTCTG CTCACGTGTC 1260 AGCTCTGGAT GTGAACTCAA GGACTCCGTG ATGATGGGAG CGGACATCTA TGAAACTGAA 1320 GAAGAAGCTT CAAAGCTACT GTTAGCTGGG AAGGTCCCGA TTGGAATAGG AAGGAACACA 1380 AAGATAAGGA ACTGTATCAT TGACATGAAT GCTAGGATTG GGAAGAACGT GGTGATCACA 1440 AACAGTAAGG GCATCCAAGA GGCTGATCAC CCGGAAGAAG GGTCCTACTA CATAAGGTCT 1500 GGAATCGTGG TGATCCTGAA GAATGCAACC ATCAACGATG GGTCTGTCAT A 1551 (2) INFORMATION FOR SEQ ID NO: 5: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 517 amino acids (B) TYPE: amino acid (C) STRANDEDNESS : single ( D) TOPOLOGY : 1inear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5: Met Gin Phe Ala Leu Ala Leu Asp Thr Asn Ser Gly Pro His Gin He 1 5 10 15 Arg Ser Cys Glu Gly Asp Gly He Asp Arg Leu Glu Lys Leu Ser He 20 25 30 Gly Gly Arg Lys Gin Glu Lys Ala Leu Arg Asn Arg Cys Phe Gly Gly 35 40 45 Arg Val Ala Ala Thr Thr Gin Cys He Leu Thr Ser Asp Ala Cys Pro 50 55 60 Glu Thr Leu His Ser Gin Thr Gin Ser ser Arg Lys Asn Tyr Ala Asp 65 70 75 80 Ala Asn Arg Val Ser Ala He He Leu Gly Gly Gly Thr Gly Ser Gin 85 90 95 Leu Phe Pro Leu Thr Ser Thr Arg Ala Thr Pro Ala Val Pro Val Gly 100 105 110 Gly Cys Tyr Arg Leu He Asp He Pro Met Ser Asn Cys Phe Asn Ser 115 120 125 Gly He Asn Lys He Phe Val Met ser Gin Phe Asn ser Thr Ser Leu 130 135 140 Asn Arg His He His Arg Thr Tyr Leu Glu Gly Gly He Asn Phe Ala 145 150 155 160 Asp Gly Ser Val Gin Val Leu Ala Ala Thr Gin Met Pro Glu Glu Pro 165 170 175 Ala Gly Trp Phe Gin Gly Thr Ala Asp Ser He Arg Lys Phe He Trp 180 185 190 Val Leu Glu Asp Tyr Tyr Ser His Lys ser He Asp Asn He Val He 195 200 205 Leu Ser Gly Asp Gin Leu Tyr Arg Met Asn Tyr Met Glu Leu Val Gin 210 215 220 Lys His Val Glu Asp Asp Ala Asp He Thr He ser cys Ala Pro Val 225 230 235 240 Asp Glu Ser Arg Ala Ser Lys Asn Gly Leu Val Lys He Asp His Thr 245 250 255 Gly Arg Val Leu Gin Phe Phe Glu Lys Pro Lys Gly Ala Asp Leu Asn 260 265 270 Ser Met Arg Val Glu Thr Asn Phe Leu Ser Tyr Ala He Asp Asp Ala 275 280 285 Gin Lys Tyr Pro Tyr Leu Ala Ser Met Gly He Tyr Val Phe Lys Lys 290 295 300 Asp Ala Leu Leu Asp Leu Leu Lys Ser Lys Tyr Thr Gin Leu His Asp 305 310 315 320 Phe Gly Ser Glu He Leu Pro Arg Ala Val Leu Asp His Ser Val Gin 325 330 335 Ala Cys He Phe Thr Gly Tyr Trp Glu Asp Val Gly Thr He Lys Ser 340 345 350 Phe Phe Asp Ala Asn Leu Ala Leu Thr Glu Gin Pro Ser Lys Phe Asp 355 360 365 Phe Tyr Asp Pro Lys Thr Pro Phe Phe Thr Ala Pro Arg Cys Leu Pro 370 375 380 Pro Thr Gin Leu Asp Lys Cys Lys Met Lys Tyr Ala Phe He Ser Asp 385 390 395 400 Gly Cys Leu Leu Arg Glu Cys Asn He Glu His Ser Val He Gly Val 405 410 415 cys Ser Arg Val Ser Ser Gly cys Glu Leu Lys Asp Ser Val Met Met 420 425 430 Gly Ala Asp He Tyr Glu Thr Glu Glu Glu Ala Ser Lys Leu Leu Leu 435 440 445 Ala Gly Lys Val Pro He Gly He Gly Arg Asn Thr Lys He Arg Asn 450 455 460 Cys He He Asp Met Asn Ala Arg He Gly Lys Asn val Val He Thr 465 470 475 480 Asn Ser Lys Gly He Gin Glu Ala Asp His Pro Glu Glu Gly Ser Tyr 485 490 495 Tyr He Arg Ser Gly He Val Val He Leu Lys Asn Ala Thr He Asn 500 505 510 Asp Gly Ser Val He 515

Claims: Claims 1. A polynucleotide molecule, comprising a variant of the wild type shrunken-2 (Sh2) gene, wherein said variant codes for the insertion of at least one additional amino acid within or close to the allosteric binding site of the ADP-glucose pyrophosphorylase (AGP) enzyme subunit, whereby a plant expressing said polynucleotide molecule has increased seed weight relative to the seed weight of a plant expressing the wild type Sh2 gene. 2. The polynucleotide molecule, according to claim 1, wherein said polynucleotide molecule encodes at least one serine residue inserted between amino acids 494 and 495 of the native AGP enzyme subunit. 3. The polynucleotide molecule, according to claim 1, wherein said polynucleotide molecule encodes the amino acid pair tyrosine.serine, wherein said amino acid pair is inserted between amino acids 494 and 495 of the native AGP enzyme subunit. 4. The polynucleotide molecule, according to claim 1, wherein said polynucleotide molecule encodes the amino acid pair serine:tyrosine, wherein said amino acid pair is inserted between amino acids 495 and 496 of the native AGP enzyme subunit. 5. The polynucleotide molecule, according to claim 1, wherein the AGP enzyme encoded by said polynucleotide molecule consists essentially of an amino acid sequence selected from the group consisting of SEQ ID NO. 5 and SEQ ID NO. 3. 6. The polynucleotide molecule, according to claim 5, wherein the nucleotide sequence encoding SEQ ID NO. 3 comprises nucleotides 87 through 1640 of the sequence shown in SEQ ID NO. 2 or a degenerate fragment thereof. 7. A method for increasing the seed weight of a plant, comprising incorporating the polynucleotide molecule of claim 1 into the genome of said plant and expressing the protein encoded by said polynucleotide molecule. 8. The method, according to claim 7, wherein said plant is Zea mays. 9. A plant seed comprising the polynucleotide molecule of claim 1 within the genome of said seed. 10. A plant expressing the polynucleotide molecule of claim 1. 11. The plant, according to claim 10, wherein said plant is Zea mays. 12. The plant, according to claim 10, wherein said plant is grown from the seed of claim 9. 13. A variant ADP-glucose pyrophosphorylase (AGP) protein, wherein said protein has at least one additional amino acid inserted within or close to the allosteric binding site of the wild-type AGP protein. 14. The variant AGP protein, according to claim 13, wherein said protein has at least one serine residue inserted between amino acids 494 and 495 of the wild type AGP protein sequence. 15. The variant AGP protein, according to claim 11, wherein said protein has the amino acid pair tyrosine: serine inserted between amino acids 494 and 495 of the wild-type AGP protein sequence. 16. The variant AGP protein, according to claim 11, wherein said protein has the amino acid pair serine.tyrosine inserted between amino acids 495 and 496 of the wild-type AGP protein sequence. 17. The variant AGP protein, according to claim 13, wherein said protein consists essentially of an amino acid sequence selected from the group consisting of SEQ ID NO. 5 and SEQ ID NO. 3. 18. The variant AGP protein, according to claim 13, wherein said protein is expressed in the endosperm of a plant during seed development.

Inventors: GIROUX MICHAEL, HANNAH L CURTIS, GIROUX, MICHAEL, HANNAH, L., CURTIS
Applicants: UNIV FLORIDA, UNIVERSITY OF FLORIDA
Application year: 1996
Main classification: C12N15
IPC codes: C12N9, C12N15