Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Reexamination Certificate
1999-10-29
2003-11-25
Ketter, James (Department: 1636)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C435S419000, C435S468000, C536S023200, C536S023600, C800S284000, C800S295000
Reexamination Certificate
active
06653099
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 hemicellulose production in plants and seeds.
BACKGROUND OF THE INVENTION
UDP-glucuronic acid a precursor of hemicellulose. Hemicellulose is a major component of primary plant cell walls. Many of the glycosyl residues found in hemicellulose are derived from the sugar precursor UDP-glucuronic acid, which can be converted into UDP-arabinose, UDP-apiose, UDP-galacturonic acid, and UDP-xylose. The synthesis of non-cellulosic cell wall polysaccharides, a major component of plant cell walls, is directed through the enzyme, UDP-glucose dehydrogenase. This enzyme has been shown to be the rate limiting step in the biosynthesis of non-cellulosic polysaccharides, suggesting it is a key regulatory step in cell wall synthesis. It has been shown that over expression of UDP-glucose dehydrogenase in transgenic plants caused a 50% increase in leaf elongation rate (LER) compared to wildtype (Cramer, G. R., et al., (1998) The American Society of Plant Physiologists Annual Meeting, Session #39, Abstract #332). The enzyme is highly expressed in young roots, but lower expression levels have been observed in expanding tissues of the epicotyl and in young leaves. The expression pattern of the enzyme in different developmental stages strengthens the argument that UDP-glucose dehydrogenase is a key regulator for the availability of hemicellulose precursors. Thus, it appears that biosynthesis of cell wall components is a rate-limiting step for cell expansion and plant growth and that UDP-glucose dehydrogenase may be powerful new tool to manipulate the growth of plants.
SUMMARY OF THE INVENTION
The present invention relates to isolated polynucleotides comprising a nucleotide sequence encoding a first polypeptide of at least 70 amino acids, preferably 480 amino acids, that has at least 96% identity based on the Clustal method of alignment when compared to a polypeptide selected from the group consisting of a rice UDP-glucose dehydrogenase polypeptide of SEQ ID NO:6, and a wheat UDP-glucose dehydrogenase polypeptide of SEQ ID NO:8. 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 first polypeptide of at least 70 amino acids, preferably 480 amino acids, that has at least 94%, preferably 96%, identity based on the Clustal method of alignment when compared to a polypeptide selected from the group consisting of UDP-glucose dehydrogenase polypeptides of SEQ ID NOs:2, 4, 6 and 8. 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 and 7 that codes for the polypeptide selected from the group consisting of SEQ ID NOs:2, 4, 6 and 8. 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 and 7 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 UDP-glucose dehydrogenase polypeptide of at least 480 amino acids comprising at least 94%, preferably 96% homology based on the Clustal 25 method of alignment compared to a polypeptide selected from the group consisting of SEQ ID NOs:2, 4, 6 and 8 .
The present invention relates to a method of selecting an isolated polynucleotide that affects the level of expression of a UDP-glucose dehydrogenase 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 a UDP-glucose dehydrogenase polypeptide in the host cell containing the isolated polynucleotide; and
comparing the level of a UDP-glucose dehydrogenase polypeptide in the host cell containing the isolated polynucleotide with the level of a UDP-glucose dehydrogenase 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 a UDP-glucose dehydrogenase polypeptide gene, preferably a plant UDP-glucose dehydrogenase 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 and 7 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 a UDP-glucose dehydrogenase amino acid sequence.
The present invention also relates to a method of obtaining a nucleic acid fragment encoding all or a substantial portion of the amino acid sequence encoding a UDP-glucose dehydrogenase polypeptide comprising the steps of: probing a cDNA or genomic library with an isolated polynucleotide of the present invention; identifying a DNA clone that hybridizes with an isolated polynucleotide of the present invention; isolating the identified DNA clone; and sequencing the cDNA or genomic fragment that comprises the isolated DNA clone.
A further embodiment of the instant invention is a method for evaluating at least one compound for its ability to inhibit the activity of a UDP-glucose dehydrogenase, the method comprising the steps of: (a) transforming a host cell with a chimeric gene comprising a nucleic acid fragment encoding a UDP-glucose dehydrogenase, operably linked to suitable regulatory sequences; (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 UDP-glucose dehydrogenase in the transformed host cell; (c) optionally purifying the UDP-glucose dehydrogenase expressed by the transformed host cell; (d) treating the UDP-glucose dehydrogenase with a compound to be tested; and (e) comparing the activity of the UDP-glucose dehydrogenase that has been treated with a test compound to the activity of an untreated UDP-glucose dehydrogenase, thereby selecting compounds with potential for inhibitory activity.
The present invention also relates to a composition comprising an isolated polynucleotide comprising a nucleotide sequence encoding a first polypeptide of at least 70 amino acids that has at least 94% identity based on the Clustal method of alignme
Famodu Omolayo O.
Orozco, Jr. Emil M.
E. I. du Pont de Nemours and Company
Ketter James
Sullivan Daniel M
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