Multicellular living organisms and unmodified parts thereof and – Plant – seedling – plant seed – or plant part – per se
Reexamination Certificate
1999-11-09
2001-11-13
McGarry, Sean (Department: 1635)
Multicellular living organisms and unmodified parts thereof and
Plant, seedling, plant seed, or plant part, per se
C435S006120, C435S091100, C435S091300, C435S325000, C435S375000, C435S419000, C536S023100, C536S023400, C536S023600
Reexamination Certificate
active
06316698
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 alpha-glucosidase II subunits in plants and seeds.
BACKGROUND OF THE INVENTION
Catabolism is a key determinant of the actual steady-state level of a particular metabolite. For example, levels of oligosaccharides and complex carbohydrates such as starch in plants are determined by both enzymes involved in their synthesis as well as enzymes involved in their degradation. Among the enzynes involved in carbohydrate catabolism is alpha-glucosidase. An exo-carbohydrase, the enzyme hydrolyzes the terminal, non-reducing 1,4-linked D-glucose residues in malto-oligosaccharides and starch releasing D-glucose. Different isoforms of alpha-glucosidase that vary in substrate specificities and pH optima have been characterized in plants. In spinach, isoforms I and II use starch and a range of malto-oligosaccharides as substrates whereas isoforms III and IV only use malto-oligosaccharides (Sugimoto et al. (1997)
Plant Mol Biol
33:765-768). In mammalian systems, glucosidase I and glucosidase II are known to be sequentially involved in the trimming of glucose residues from N-linked glycans on newly synthesized glycoproteins in the endoplasmic reticulum (ER) (Kalz-Fuller et al. (1995)
Eur J Biochem
231:344-351; Trombetta et al. (1996)
J Biol Chem
271:27509-27516). Alpha-glucosidase II is composed of two subunits, alpha and beta, with the alpha subunit harboring the catalytic activity, while the smaller subunit which contains a putative ER retention signal (HDEL) at the C terminus, may be involved in localizing the enzyme to the ER (Trombetta et al. (1996)
J Biol Chem
271:27509-27516).
Plant glucosidase genes have been isolated from various species, including spinach (Sugimoto et al. (1997)
Plant Mol Biol
33:765-768), potato (Taylor et al. (1998)
Plant J
13:419-425), and barley (Tibbot and Skadsen (1996)
Plant Mol Biol
30:229-241; U.S. Pat. No. 5,763,252). Expectedly, high gene expression is observed during germination stages, when starch reserves are broken down to supply the energy needed for growth (Tibbot and Skadsen (1996)
Plant Mol Biol
30:229-241; Taylor et al. (1998)
Plant J
13:419-425). There is a great deal of interest in identifying the genes that encode alpha-glucosidases in plants. These genes may be used in plants to control carbohydrate levels in plants and seeds or to manipulate glycoprotein processing. Accordingly, the availability of nucleic acid sequences encoding all or a portion of these alpha-glucosidase II enzymes would facilitate studies to better understand plant metabolism, and provide genetic and biochemical tools for the manipulation of the starch/sucrose content in plants and seeds, and mass-production of the enzyme for post-harvest starch modification.
SUMMARY OF THE INVENTION
The present invention relates to isolated polynucleotides comprising a nucleotide sequence encoding a first polypeptide of at least 50 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 alpha-glucosidase II alpha subunit polypeptide of SEQ ID NO:2, a rice alpha-glucosidase II alpha subunit polypeptide of SEQ ID NO:4, a soybean alpha-glucosidase II alpha subunit polypeptide of SEQ ID NO:6, a soybean alpha-glucosidase II alpha subunit polypeptide of SEQ ID NO:8, and a wheat alpha-glucosidase II alpha subunit polypeptide of SEQ ID NO:10. The present invention relates to isolated polynucleotides comprising a nucleotide sequence encoding a first polypeptide of at least 50 amino acids that has at least 70% identity based on the Clustal method of alignment when compared to a polypeptide selected from the group consisting of a corn alpha-glucosidase II beta subunit polypeptide of SEQ ID NO:12, a wheat alpha-glucosidase II beta subunit polypeptide of SEQ ID NO:14, and a wheat alpha-glucosidase II beta subunit polypeptide of SEQ ID NO:16. 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 consist of a nucleic acid sequence selected from the group consisting of SEQ ID NOs:1, 3, 5, 7, 9, 11, 13, and 15 that codes for the polypeptide selected from the group consisting of SEQ ID NOs:2, 4, 6, 8, 10, 12, 14, and 16. The present invention also relates to an isolated polynucleotide comprising a nucleotide sequences of at least one of 40 (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, and 15 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 an alpha-glucosidase II alpha subunit polypeptide of at least 50 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, 6, 8, and 10. The present invention also relates to an alpha-glucosidase II beta subunit polypeptide of at least 50 amino acids comprising at least 70% homology based on the Clustal method of alignment compared to a polypeptide selected from the group consisting of SEQ ID NOs:12, 14, and 16.
The present invention relates to a method of selecting an isolated polynucleotide that affects the level of expression of an alpha-glucosidase II alpha or beta subunit polypeptide in a host cell, preferably a plant cell, the method comprising the steps of:
constructing an isolated polynucleotide of the present invention or an isolated chimeric gene of the present invention;
introducing the isolated polynucleotide or the isolated chimeric gene into a host cell;
measuring the level of an alpha-glucosidase II alpha or beta subunit polypeptide in the host cell containing the isolated polynucleotide; and
comparing the level of an alpha-glucosidase II alpha or beta subunit polypeptide in the host cell containing the isolated polynucleotide with the level of an alpha-glucosidase II alpha or beta subunit polypeptide in a host cell that does not contain the isolated polynucleotide.
The present invention relates to a method of obtaining a nucleic acid fragment encoding a substantial portion of an alpha-glucosidase II alpha or beta subunit polypeptide gene, preferably a plant alpha-glucosidase II alpha or beta subunit polypeptide gene, comprising the steps of: synthesizing an oligonucleotide primer comprising a nucleotide sequence of at least one of 40 (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, and 15 and the complement of such nucleotide sequences; and amplifying a nucleic acid fragment (preferably a cDNA inserted in a cloning vector) using the oligonucleotide primer. The amplified nucleic acid fragment preferably will encode a portion of an alpha-glucosidase II alpha or beta subunit amino acid sequence.
The present invention
Allen Stephen M.
Kinney Anthony J.
E. I. du Pont de Nemours & Company
McGarry Sean
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