Multicellular living organisms and unmodified parts thereof and – Plant – seedling – plant seed – or plant part – per se
Reexamination Certificate
1999-12-14
2001-08-21
Carlson, Karen Cochrane (Department: 1653)
Multicellular living organisms and unmodified parts thereof and
Plant, seedling, plant seed, or plant part, per se
C800S260000, C435S069100, C435S320100, C435S252200, C435S325000, C530S350000
Reexamination Certificate
active
06278042
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 heavy metal transporters in plants and seeds.
BACKGROUND OF THE INVENTION
Arsenite extrusion pumps have been identified in bacteria, mice and humans. A hydrophilic transporter belonging to the ATPase superfamily, having no transmembrane domain and having two potential adenylate-binding sites has been studied in arsenite-resistant bacteria (Chen, C. M. et al. (1986)
J. Biol. Chem
. 261:15030-15038). Lower molecular weight homologs have been found in mice and humans where the different domains may be encoded by two different genes (Kurdi-Haidar, B. et al. (1996)
Genomics
36:486-491).
Plants require certain essential elements for completing their life cycle. Energy from sunlight and availability of the essential elements allows plants to synthesize the compounds necessary for their normal growth. One of these essential elements is zinc which is absorbed from the soil, is required for the activity of many enzymes and is required for chloroplast biosynthesis in some plants. Zinc deficiency results in small plants with distorted leaves; in some species such as corn, sorghum and beans the leaves show necrotic spots and chlorosis. Excess zinc is also detrimental for plants. To prevent zinc toxic buildup in the cytosol, plants may sequester it in the vacuole. The zinc transporter ZnT-2 protects cells from zinc toxicity by facilitating vacuolar zinc transport into an endosomal/lysosomal compartment (Palmiter, R. D. et al. (1996)
EMBO J
. 15:1784-1791).
SUMMARY OF THE INVENTION
The present invention relates to isolated polynucleotides comprising a nucleotide sequence encoding a polypeptide of at least 60 amino acids that has at least 55% identity based on the Clustal method of alignment when compared to a polypeptide selected from the group consisting of a corn arsenite transporter polypeptide of SEQ ID NO:2, a soybean arsenite transporter polypeptide of SEQ ID NO:4, and a wheat arsenite transporter polypeptide of SEQ ID NO:6, and isolated polynucleotides comprising a nucleotide sequence encoding a polypeptide of at least 157 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 zinc transporter polypeptide of SEQ ID NO:8, a rice zinc transporter polypeptide of SEQ ID NO:10, a soybean zinc transporter polypeptide of SEQ ID NO:12, and a wheat zinc transporter polypeptide of SEQ ID NO:14. 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, and 13 that codes for the polypeptide selected from the group consisting of SEQ ID NOs:2, 4, 6, 8, 10, 12, and 14. 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, and 13, 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 arsenite transporter polypeptide of at least 60 amino acids comprising at least 55% 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, and a zinc transporter polypeptide of at least 157 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, 12, and 14.
The present invention relates to a method of selecting an isolated polynucleotide that affects the level of expression of an arsenite or zinc transporter 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 gene into a host cell; (c) measuring the level an arsenite or zinc transporter polypeptide in the host cell containing the isolated polynucleotide; and (d) comparing the level of an arsenite or zinc transporter polypeptide in the host cell containing the isolated polynucleotide with the level of an arsenite or zinc transporter polypeptide in the 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 arsenite or zinc transporter polypeptide gene, preferably a plant arsenite or zinc transporter polypeptide gene, comprising the steps of: synthesizing an oligonucleotide primer comprising a nucleotide sequence 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, and 13, 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 arsenite or zinc transporter 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 an arsenite or zinc transporter 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.
The present invention relates to a composition, such as a hybridization mixture, comprising an isolated polynucleotide of the present invention.
The present invention relates to an isolated polynucleotide of the present invention comprising at least one of 30 contiguous nucleotides derived from a nucleic acid sequence selected from the group consisting of SEQ ID NOs:1, 3, 5, 7, 9, 11, and 13.
The present invention relates to an expression cassette comprising an isolated polynucleotide of the present invention operably linked to a promoter.
The present invention relates to a method for positive selection of a transformed cell comprising: (a) transforming a host cell with the chimeric gene of the present invention or an expression cassette of the present invention; and (b) growing the transformed host cell, preferably plant cell, such as a monocot or a dicot, under conditions which allow expression of the arsenite or zinc transporters polynucleotide in an amount sufficient to complement a sensitivity to high levels of heavy metals to provide a
Allen Stephen M.
Rafalski J. Antoni
Sakai Hajime
Carlson Karen Cochrane
E.I. Du Pont de Nemours and Company
Li Kening
LandOfFree
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