Multicellular living organisms and unmodified parts thereof and – Method of introducing a polynucleotide molecule into or...
Reexamination Certificate
1999-12-17
2003-11-04
Bui, Phuong T. (Department: 1638)
Multicellular living organisms and unmodified parts thereof and
Method of introducing a polynucleotide molecule into or...
C435S069200, C435S183000, C435S410000, C435S419000, C435S252300, C435S320100, C530S350000, C530S370000, C536S023100, C536S023200, C536S023600, C536S024100, C536S024330, C800S295000, C800S281000
Reexamination Certificate
active
06642435
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 folate biosynthetic enzymes in plants and seeds.
BACKGROUND OF THE INVENTION
Tetrahydrofolic acid and its derivatives N
5
,N
10
-methylenetetrahydrofolate, N
5
,N
10
-methenyltetrahydrofolate, N
10
-formyltetrahydrofolate and N
5
-methyl-tetrahydrofolate are biologically active forms of folic acid. The tetrahydrofolates are coenzymes that function in a variety of enzyme catalyzed reactions as specialized cosubstrates for one-carbon metabolism. For example, tetrahydrofolate plays an important role in nucleic acid biosynthesis by serving as the immediate source of one-carbon units in purine and pyrimidine biosynthesis. The cellular tetrahydrofolate coenzyme pool must be maintained at specific levels to assure one-carbon metabolism operates efficiently. Thus, one of the most important reactions of the cell is the reduction of dihydrofolate to tetrahydrofolate by dihydrofolate reductase. The importance of this reaction in mammalian cells can be shown by the fact that methorexate, a very effective chemotherapy drug, is a potent inhibitor of dihydrofolate reductase (Zubay, G. (1983)
Biochemistry
, Addison-Wesley Publishing Co. Reading, Mass.). Other enzymes involved in the folic acid biosynthetic pathway to maintain the tetrahydrofolate coenzyme pool are tetrahydrofolypolyglutamate synthase, dihydropteroate synthase and dihydroneopterin aldolase.
There is a great deal of interest in identifying the genes that encode proteins required for tetrahydrofolate biosynthesis in plants. These genes may be used in plant cells to alter the tetrahydrofolate coenzyme pool concentration and modulate one-carbon metabolism. Accordingly, the availability of nucleic acid sequences encoding all or a portion of the tetrahydrofolypolyglutamate synthase, dihydropteroate synthase and dihydroneopterin aldolase enzymes would facilitate studies to better understand one-carbon metabolism in plants, provide genetic tools to one-carbon metabolism. The tetrahydrofolate biosynthetic enzymes may also provide targets to facilitate design and/or identification of inhibitors of cell cycle that may be useful as herbicides.
SUMMARY OF THE INVENTION
The present invention relates to isolated polynucleotides comprising a nucleotide sequence encoding a polypeptide of at least 131 amino acids that has at least 80% identity based on the Clustal method of alignment when compared to a polypeptide selected from the group consisting of a corn dihydroneopterin aldolase polypeptide of SEQ ID NO:2, a soybean dihydroneopterin aldolase polypeptide of SEQ ID NO:4 and a wheat dihydroneopterin aldolase polypeptide of SEQ ID NO:6. The present invention also relates to an isolated polynucleotide comprising the complement of the nucleotide sequences described above.
The present invention relates to isolated polynucleotides comprising a nucleotide sequence encoding a polypeptide of at least 75 amino acids that has at least 80% identity based on the Clustal method of alignment when compared to a polypeptide selected from the group consisting of a corn dihydropteroate synthase/dihydropteroate pyrophosphorylase polypeptide of SEQ ID NO:8, a rice dihydropteroate synthase/dihydropteroate pyrophosphorylase polypeptide of SEQ ID NO:10 and a soybean dihydropteroate synthase/dihydropteroate pyrophosphorylase polypeptide of SEQ ID NO:12. The present invention also relates to an isolated polynucleotide comprising the complement of the nucleotide sequences described above.
The present invention relates to isolated polynucleotides comprising a nucleotide sequence encoding a polypeptide of at least 553 amino acids that has at least 80% identity based on the Clustal method of alignment when compared to a corn tetrahydrofolypolyglutamate synthase/folylpolyglutamate synthase polypeptide of SEQ ID NO:14. The present invention also relates to an isolated polynucleotide comprising the complement of the nucleotide sequences described above.
The present invention relates to isolated polynucleotides comprising a nucleotide sequence encoding a polypeptide of at least 133 amino acids that has at least 80% identity based on the Clustal method of alignment when compared to a polypeptide selected from the group consisting of a corn tetrahydrofolypolyglutamate synthase/folylpolyglutamate synthase polypeptide of SEQ ID NO:16 and a soybean tetrahydrofolypolyglutamate synthase/folylpolyglutamate synthase polypeptide of SEQ ID NO:18. The present invention also relates to an isolated polynucleotide comprising the complement of the nucleotide sequences described above.
It is preferred that the isolated polynucleotides of the claimed invention consists of a nucleic acid sequence selected from the group consisting of SEQ ID NOs:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29 and 31 that codes for the polypeptide selected from the group consisting of SEQ ID NOs:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30 and 32. The present invention also relates to an isolated polynucleotide comprising a nucleotide sequences of at least one of 60 (preferably at least one of 40, most preferably at least one of 30) contiguous nucleotides derived from a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31 and the complement of such nucleotide sequences.
The present invention relates to a chimeric gene comprising an isolated polynucleotide of the present invention operably linked to suitable regulatory sequences.
The present invention relates to an isolated host cell comprising a chimeric gene of the present invention or an isolated polynucleotide of the present invention. The host cell may be eukaryotic, such as a yeast or a plant cell, or prokaryotic, such as a bacterial cell. The present invention also relates to a virus, preferably a baculovirus, comprising an isolated polynucleotide of the present invention or a chimeric gene of the present invention.
The present invention relates to a process for producing an isolated host cell comprising a chimeric gene of the present invention or an isolated polynucleotide of the present invention, the process comprising either transforming or transfecting an isolated compatible host cell with a chimeric gene or isolated polynucleotide of the present invention.
The present invention relates to a dihydroneopterin aldolase polypeptide of at least 131 amino acids comprising at least 80% homology based on the Clustal method of alignment compared to a polypeptide selected from the group consisting of SEQ ID NOs:2, 4 and 6.
The present invention relates to a dihydropteroate synthase/dihydropteroate pyrophosphorylase polypeptide of at least 75 amino acids comprising at least 80% homology based on the Clustal method of alignment compared to a polypeptide selected from the group consisting of SEQ ID NOs:8, 10 and 12.
The present invention relates to a tetrahydrofolypolyglutamate synthase/folylpolyglutamate synthase polypeptide of at least 553 amino acids comprising at least 80% homology based on the Clustal method of alignment compared to a polypeptide of SEQ ID NO:14.
The present invention relates to a tetrahydrofolypolyglutamate synthase/folylpolyglutamate synthase polypeptide of at least 133 amino acids comprising at least 80% homology based on the Clustal method of alignment compared to a polypeptide selected from the group consisting of SEQ ID NOs: 16 and 18.
The present invention relates to a method of selecting an isolated polynucleotide that affects the level of expression of a dihydroneopterin aldolase, dihydropteroate synthase/dihydropteroate pyrophosphorylase or tetrahydrofolypolyglutamate synthase/folylpolyglutamate synthase polypeptide in a host cell, preferably a plant cell, the method comprising the steps of: (a) constructing an isolated polynucleotide of the present invention or an isolated chimeric gene of the present invention; (b) introducing the isolated polynucleotide or the isolated chimeric ge
Harvell Leslie T.
Rafalski J. Antoni
Weng Zude
Bui Phuong T.
E. I. du Pont de Nemours and Company
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