Chemistry: molecular biology and microbiology – Process of mutation – cell fusion – or genetic modification – Introduction of a polynucleotide molecule into or...
Reexamination Certificate
1999-03-15
2001-03-20
Fox, David T. (Department: 1638)
Chemistry: molecular biology and microbiology
Process of mutation, cell fusion, or genetic modification
Introduction of a polynucleotide molecule into or...
C435S419000, C435S069100, C435S485000, C435S486000, C435S487000, C435S488000, C435S252300, C536S023100, C536S023200, C536S023600
Reexamination Certificate
active
06204063
ABSTRACT:
FIELD OF THE INVENTION
This invention is in the field of plant molecular biology. More specifically, this invention pertains to nucleic acid fragments encoding proteins involved in glycolysis and respiration in plants and seeds.
BACKGROUND OF THE INVENTION
Glycolysis is the main pathway for carbohydrate catabolism. It is a process in which monosaccharides are broken down to pyruvic acid, two molecules of which are formed per monosaccharide residue. In plants D-glucose and D-fructose are the main monosaccharides catabolized by glycolysis although other monosaccharides that can be converted to glucose or fructose can be handled by this catabolic pathway. In cells where photosynthesis is not taking place glycolysis is a key metabolic component of the respiratory process which generates energy in the form of ATP. Typically the cells of germinating seedlings and non-photosynthetic cells of mature plants utilize this metabolic pathway. The glycolytic pathway is controlled in part by the potent allosteric regulator fructose-2,6-bisphosphate (F2,6P). This regulatory molecule activates the enzymatic activity of phosphofructosekinase (PFK) which stimulates the flow of carbon through the glycolytic pathway to pyruvate. PFK plays a central role in the control of glycolysis because it catalyzes one of the pathway's rate-determining reactions. F2,6P also inhibits the activity of fructose bisphosphatase (FBPase) which stimulates the flow of carbon through gluconeogenesis, to form glucose. The concentration of F2,6P in the cell depends on the action of 6-phosphofructo 2-kinase/fructose 2,6-bisphosphatase (PFK-2/FBPase). The formation and degradation of F2,6P is catalyzed by PFK-2 and FBPase-2, two enzyme activities that occur on different domains of the same protein molecule (Algaier, J. et al. (1988)
Biochem Biophys Res Commun
153(1):328-333). Thus, 6-phosphofructo 2-kinase/fructose 2,6-bisphosphatase is a key regulatory enzyme that controls carbon flux through glycolysis vs. gluconeogenesis. Because PFK-2/FBPase regulates the abundance of a key allosteric regulator, manipulating the activity of this enzyme either by controlling expression or by directed mutagenesis could be used to control carbon flux through the glycolytic of gluconeogenic pathways. This could be very important in bioprocessing in plants.
Respiration (aerobic metabolism) takes place in the mitochondria in most eukaryotes. The ubiquinol-cytochrome C reductase (bc1) complex is an important component of the mitochondrial electron transport system. The BCS1 gene encodes a product that has been shown to be necessary for the expression of the Rieske iron-sulfur protein a component of the bc1 complex (Nobrega, F. G. et al. (1992)
EMBO
11:3821-3829). By controlling the expression of BCS1 it may be possible to modulate the level of the Rieske iron-sulfur protein in plant cells which it turn would regulate the amount of functional ubiquinol-cytochrome C reductase complexes in mitochondria.
Few of the genes encoding the 6-phosphofructo 2-kinase/fructose 2,6-bisphosphatase and BCS1 proteins in corn, Momordica, rice and wheat, have been isolated and sequenced. For example, no corn, Momordica, rice or wheat genes have been reported for 6-phosphofructo 2-kinase/fructose 2,6-bisphosphatase and no plant genes have been reported for BCS1. Accordingly, the availability of nucleic acid sequences encoding all or a portion of these proteins would facilitate studies to better understand carbon flux and respiration, provide genetic tools for the manipulation of these metabolic pathways, and provide a means to control glycolysis and respiration in plant cells.
SUMMARY OF THE INVENTION
The instant invention relates to isolated nucleic acid fragments encoding proteins involved in glycolysis and respiration. Specifically, this invention concerns an isolated nucleic acid fragment encoding a BCS1 or 6-phosphofructo 2-kinase/fructose 2,6-bisphosphatase protein. In addition, this invention relates to a nucleic acid fragment that is complementary to the nucleic acid fragment encoding a BCS1 or 6-phosphofructo 2-kinase/fructose 2,6-bisphosphatase protein.
An additional embodiment of the instant invention pertains to a polypeptide encoding all or a substantial portion of a protein involved in glycolysis or respiration selected from the group consisting of BCS1 and 6-phosphofructo 2-kinase/fructose 2,6-bisphosphatase.
In another embodiment, the instant invention relates to a chimeric gene encoding a BCS1 or 6-phosphofructo 2-kinase/fructose 2,6-bisphosphatase protein, or to a chimeric gene that comprises a nucleic acid fragment that is complementary to a nucleic acid fragment encoding a BCS1 or 6-phosphofructo 2-kinase/fructose 2,6-bisphosphatase protein, operably linked to suitable regulatory sequences, wherein expression of the chimeric gene results in production of levels of the encoded protein in a transformed host cell that is altered (i.e., increased or decreased) from the level produced in an untransformed host cell.
In a further embodiment, the instant invention concerns a transformed host cell comprising in its genome a chimeric gene encoding a BCS1 or 6-phosphofructo 2-kinase/fructose 2,6-bisphosphatase protein, operably linked to suitable regulatory sequences. Expression of the chimeric gene results in production of altered levels of the encoded protein in the transformed host cell. The transformed host cell can be of eukaryotic or prokaryotic origin, and include cells derived from higher plants and microorganisms. The invention also includes transformed plants that arise from transformed host cells of higher plants, and seeds derived from such transformed plants.
An additional embodiment of the instant invention concerns a method of altering the level of expression of a BCS1 or 6-phosphofructo 2-kinase/fructose 2,6-bisphosphatase protein in a transformed host cell comprising: a) transforming a host cell with a chimeric gene comprising a nucleic acid fragment encoding a BCS1 or 6-phosphofructo 2-kinase/fructose 2,6-bisphosphatase protein; and b) growing the transformed host cell under conditions that are suitable for expression of the chimeric gene wherein expression of the chimeric gene results in production of altered levels of BCS1 or 6-phosphofructo 2-kinase/fructose 2,6-bisphosphatase protein in the transformed host cell.
An addition embodiment of the instant invention concerns a method for obtaining a nucleic acid fragment encoding all or a substantial portion of an amino acid sequence encoding a BCS1 or 6-phosphofructo 2-kinase/fructose 2,6-bisphosphatase protein.
REFERENCES:
patent: 5387756 (1995-02-01), Burrell et al.
NCBI General Identifier No. 3170230.
NCBI General Identifier No. 3309583.
NCBI General Identifier No. 2506091.
Algarier et al., Biochemical and Biophysical Research Comm., (1988), 153 No. 1, 328-333.
Nobrega et al., EMBO J., (1992), 11 No. 11, 3821-3829.
Folsch et al., EMBO J., (1996), 15, 479-487.
Allen Stephen M.
Lee Jian-Ming
Lightner Jonathan E.
Odell Joan T.
E. I. Du Pont de Nemours and Company
Fox David T.
Ibrahim Medina A.
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