Multicellular living organisms and unmodified parts thereof and – Method of introducing a polynucleotide molecule into or...
Reexamination Certificate
1999-05-12
2002-05-14
Bui, Phuong T. (Department: 1638)
Multicellular living organisms and unmodified parts thereof and
Method of introducing a polynucleotide molecule into or...
C800S286000, C800S295000, C800S298000, C800S320100, C800S320000, C800S320200, C800S320300, C800S306000, C800S312000, C800S314000, C800S322000, C435S069100, C435S419000, C435S468000, C435S320100, C536S023200, C536S023600, C536S024100, C536S024500
Reexamination Certificate
active
06388169
ABSTRACT:
TECHNICAL FIELD
The present invention relates generally to plant molecular biology. More specifically, it relates to nucleic acids and methods for modulating their expression in plants.
BACKGROUND OF THE INVENTION
Transgenic plant product development by conventional transformation and breeding efforts is a slow and unpredictable process. Gene targeting systems can overcome problems with expression variability, the unpredictable impact of random gene insertion on agronomic performance, and the large number of experiments that need to be conducted. Such systems can also provide approaches to manipulating endogeneous genes. Of course, a targeting system requires the ability to focus the recombination process to favor recovery of desired targeting events.
The bacterial recA gene and the cognate product RecA protein have been a subject of intense research for the past two decades. The recA gene and RecA protein play a crucial role in genetic recombination (Clark, A. J. and Margulies, A. D. (1965) Proc. Natl. Acad. Sci. USA 53, 451-459; Clark A. J. (1973) Annu. Rev. Genet. 7, 67-86; Cox, M. M. and Lehman, I. R. (1987) Annu. Rev. Biochem. 56, 229-262). Homologues of RecA have been reported in many prokaryotes (Kowalczykowaski S.C. and Eggleston, A. K. (1994) Annu. Rev. Biochem. 63, 991-1043; Kowalczykowaski, et al. (1994) Microbiol. Rev. (1994) 58, 401-465) as well as eukaryotes including humans (reviewed in Kowalczykowaski, et al. (1994) Annu. Rev. Biochem. 63, 991-1043; Kowalczykowaski, et al. (1994) Microbiol. Rev. 58: 401-465; Shinohara, et al. (1993) Nature Genet. 4: 239-243; Yoshimura, et al. (1993) Nucleic Acid Res. 21: 1665). In plants, a RecA homologue from
Arabidopsis thaliana
has been reported (Cerutti, et a.,
Proc Natl Acad Sci USA
89:8068-8072). No maize RecA homologues have been previously identified.
The eukaryotic homologues of RecA are typically grouped with the yeast Rad51 protein. The
E.coli
RecA and yeast Rad51 proteins and their respective genes have been investigated extensively and serve as prokaryotic and eukaryotic models for further studies (Kowalczykowaski, et al. (1994) Annu. Rev. Biochem. 63, 991-1043; Kowalczykowaski, et al. (1994) Microbiol. Rev. 58: 401-465; Shinohara, et al. (1993) Nature Genet. 4: 239-243; Yoshimura, et al. (1993) Nucleic Acid Res. 21: 1665).
It is well known that RecA binds single stranded DNA and promotes pairing and strand exchange between homologous DNA molecules. Reports of the use of bacterial RecA in association with DNA sequences to manipulate homologous target DNA, including improvement of the efficiency of gene targeting in non-plant systems, have been published (see, e.g., PCT published Patent Application Nos. WO 87/01730 and WO 93/22443).
The bacterial RecA as well as the yeast and human homologues also exhibit DNA dependent ATPase activity. However, the precise mechanism(s) by which RecA recognizes homology within a duplex DNA molecule remains unknown. The role of the Rad51 group of proteins in DNA recombination of higher eukaryotes is also ill defined.
To date, work with recombinase enzymes in plants has been very limited. Accordingly, there is an ongoing need for the identification and characterization of the functional activities of recombinase enzymes which may offer improved and expanded methods for use in plant systems, particularly agriculturally important crop species such as maize.
SUMMARY OF THE INVENTION
Generally, it is the object of the present invention to provide nucleic acids and proteins relating to genetic recombination. It is an object of the present invention to provide antigenic fragments of the proteins of the present invention. It is an object of the present invention to provide transgenic plants comprising the nucleic acids of the present invention. Additionally, it is an object of the present invention to provide methods for modulating, in a transgenic plant, the expression of the nucleic acids of the present invention.
Therefore, in one aspect, the present invention relates to an isolated nucleic acid comprising a member selected from the group consisting of (a) a polynucleotide having at least 60% identity to a polynucleotide encoding a polypeptide selected from the group consisting of SEQ ID NOS: 2, 4, 6, and 8 wherein the polypeptide when presented as an immunogen elicits the production of an antibody which is specifically reactive to the polypeptide; (b) a polynucleotide which is complementary to the polynucleotide of (a); and (c) a polynucleotide comprising at least 25 contiguous nucleotides from a polynucleotide of (a) or (b). In some embodiments, the polynucleotide has a sequence selected from the group consisting of SEQ ID NOS: 1, 3, 5, and 7. The isolated nucleic acid can be DNA.
In another aspect, the present invention relates to recombinant expression cassettes, comprising a nucleic acid as described, supra, operably linked to a promoter. In some embodiments, the nucleic acid is operably linked in antisense orientation to the promoter.
In another aspect, the present invention is directed to a host cell transfected with the recombinant expression cassette as described, supra. In some embodiments, the host cell is a sorghum (
Sorghum bicolor
), maize (
Zea mays
), rice (
Oryza sativa
), or wheat (
Triticum aestivum
) cell.
In a further aspect, the present invention relates to an isolated protein comprising a polypeptide of at least 10 contiguous amino acids encoded by the isolated nucleic acid referred to, supra. In some embodiments, the polypeptide has a sequence selected from the group consisting of SEQ ID NOS: 2, 4, 6 and 8.
In another aspect, the present invention relates to an isolated nucleic acid comprising a polynucleotide of at least 25 nucleotides in length which selectively hybridizes under stringent conditions to a nucleic acid selected from the group consisting of SEQ ID NOS: 1, 3, 5, and 7 or a complement thereof. In some embodiments, the isolated nucleic acid is operably linked to a promoter.
In yet another aspect, the present invention relates to an isolated nucleic acid comprising a polynucleotide, the polynucleotide having at least 80% sequence identity to an identical length of a nucleic acid selected from the group consisting of SEQ ID NOS: 1, 3, 5, and 7 or a complement thereof.
In another aspect, the present invention relates to an isolated nucleic acid comprising a polynucleotide having a sequence of a nucleic acid amplified from a
Zea mays
nucleic acid library using the primers designed from the 5′ and 3′ end of the polynucleotide selected from the group consisting of SEQ ID NOS: 1, 3, 5, and 7 or a complement thereof. In some embodiments, the nucleic acid library is a cDNA library.
In another aspect, the present invention relates to a recombinant expression cassette comprising a nucleic acid amplified from a library as referred to supra, wherein the nucleic acid is operably linked to a promoter. In some embodiments, the present invention relates to a host cell transfected with this recombinant expression cassette In some embodiments, the present invention relates to a protein of the present invention which is produced from this host cell.
In an additional aspect, the present invention is directed to an isolated nucleic acid comprising a polynucleotide encoding a polypeptide wherein: (a) the polypeptide comprises at least 10 contiguous amino acid residues from a first polypeptide selected from the group consisting of SEQ ID NOS: 2, 4, 6, and 8 wherein said polypeptide, when presented as an immunogen, elicits the production of an antibody which specifically binds to said first polypeptide; (b) the polypeptide does not bind to antisera raised against the first polypeptide which has been fully immunosorbed with the first polypeptide; (c) the polypeptide has a molecular weight in non-glycosylated form within 10% of the first polypeptide.
In a further aspect, the present invention relates to a heterologous promoter operably linked to a non-isolated polynucleotide of the present invention, wherein the polypeptide is encoded by a nucleic acid amplif
Baszczynski Christopher
Bowen Benjamin
Mahajan Pramod B.
McElver John
Shi Jinrui
Bui Phuong T.
Ibrahim Medina A.
Pioneer Hi-Bred International , Inc.
Pioneer Hi-Bred International Inc.
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