Multicellular living organisms and unmodified parts thereof and – Method of introducing a polynucleotide molecule into or... – The polynucleotide alters carbohydrate production in the plant
Reexamination Certificate
1998-11-19
2001-02-06
Fox, David T. (Department: 1638)
Multicellular living organisms and unmodified parts thereof and
Method of introducing a polynucleotide molecule into or...
The polynucleotide alters carbohydrate production in the plant
C800S320100, C435S101000, C435S194000, C435S419000, C435S468000, C536S023600
Reexamination Certificate
active
06184438
ABSTRACT:
BACKGROUND OF THE INVENTION
ADP-glucose pyrophosphorylase (AGP) catalyzes the conversion of ATP and &agr;-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 enzyme 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 homotetramer, 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 et al., 1991).
As Hannah and Nelson (Hannah and Nelson, 1975 and 1976) postulated, maize endosperm ADP-glucose pyrophosphorylase is encoded by two unlinked genes, Shrunken-2 (Sh2) (Bhave et al., 1990) and Brittle-2 (Bt2) (Bae et al., 1990). Sh2 and Bt2 encode the large subunit and small subunit of the enzyme, 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
E. 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 et al., 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).
While introns in nuclear genes are ubiquitous in nature, the signals required to precisely define and recognize exon-intron borders are not fully understood. Studies from all eukaryotes point to splicing as being essentially a two-step cleavage-ligation reaction. The first step involves the cleavage at the 5′ splice site that leads to the formation of an intron lariat with the adenosine residue of the branch point sequence located upstream to the 3′ splice site. This is followed by the ligation of the exon and release of the intron lariat. (Moore and Sharp, 1993; Brown, 1996; Simpson and Filipowicz, 1996) This complex set of events is carried out by pre-mRNA association with a conglomeration of small nuclear RNA (snRNAs) and nuclear proteins that forms a dynamic large ribonucleosome protein complex termed a spliceosome (reviewed by Moore et al., 1993; Sharp, 1994). This fundamental process, common to all eurkaryotic gene expression, can have a diverse impact on the regulation of gene expression. For example, imprecise or inaccurate pre-mRNA splicing often imparts a mutant phenotype (reviewed by Weil and Wessler, 1990) whereas alternative splicing is sometimes important in the regulation of gene expression (Gorlach et al., 1995; Nishihama et al., 1997; Golovkin and Reddy, 1996). Interestingly, certain introns dramatically enhance gene expression in transient and stably transformed callus tissue (Callis et al., 1987; Clancy et al., 1994; Vasil et al., 1989). Finally, intron splicing is required for some plant viruses to be pathogenic (Kiss-Laszlo et al., 1995).
Unlike yeast and vertebrates, the lack of a plant in vitro system capable of efficiently splicing introns has hindered our understanding of the mechanism of splicing in plants. Despite the universal nature of the splicing pathway, primary transcripts of animal origin are not efficiently or accurately spliced in plants cells. Conversely, very few plant pre-mRNA are faithfully spliced in animal cells (Barta et al., 1986; van Santen and Spritz, 1987; Pautot et al., 1989). This species barrier between the heterologous splicing of pre-mRNA is also observed between monocots and dicots. Some monocot introns are not spliced in dicots (Keith and Chua, 1986; Goodall and Filipowicz, 1991). In contrast, introns of dicot origin are efficiently spliced in monocots, suggesting that monocot splicing machinery is more flexible or complex. There are also fundamental structural/sequence differences that differentiate plant introns from those of vertebrate and yeast introns (Goodall and Filipowicz, 1991; reviewed by Simpson and Filipowicz, 1996). Vertebrate introns possess a polypyramidine track and a somewhat conserved 3′ sequence needed for lariat formation that is not found in plant introns (Simpson et al., 1996). A feature distinguishing plant introns from those of other organisms is their AU richness. This has been implicated to be essential for intron processing and for definition of the intron/exon junction (Lou et al., 1993; McCullough et al., 1993; Carle-Urioste et al., 1994; Luehrsen and Walbot, 1994). Interestingly, the requirement of AU rich region are more stringent in dicots than in monocots (Goodall and Filipowicz, 1989; 1991), and some monocot introns are, in fact, GC-rich.
Sweet corn is a major vegetable crop grown worldwide. Within the last twenty years the identification of a mutant allele of the shrunken-2 gene, termed sh2-R, was an important development for this crop. The advantage of the sh2-R gene is that it confers enhanced sweetness compared to older sweet corn varieties that do not contain the mutant form of the gene. Unfortunately, however, the use of this mutant form of the gene in corn also results in reduced germination and seedling vigor of the corn. Thus, there remains a need in the art for enhancing germination rate and/or seedling vigor in maize without negatively impacting the taste or sweetness of the corn. Improved growth characteristics would confer a major advantage in the commercial production of this corn.
BRIEF SUMMARY OF THE INVENTION
The subject invention concerns mutant alleles of the gene that encodes the large subunit of ADP-glucose pyrophosphorylase (AGP) in plants. These mutant polynucleotides, when expressed in a plant such as maize, provide for enhanced growth characteristics such as germination and seedling vigor, with little or no reduction in food or taste quality of vegetables or fruit from these plants. The mutant polynucleotides of the subject invention comprise a substitution of the wild-type terminal base in an intron from a G to another base, such as A, C or T. In one embodiment, the Shrunken-2 gene of maize contains a G to A mutation of the terminal nucleotide of an intron, thereby changing the AG nucleotide sequence that is found at the termi
Fox David T.
Saliwanchik Lloyd & Saliwanchik
University of Florida
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