Multicellular living organisms and unmodified parts thereof and – Method of introducing a polynucleotide molecule into or...
Reexamination Certificate
2000-06-07
2004-03-16
McElwain, Elizabeth F. (Department: 1638)
Multicellular living organisms and unmodified parts thereof and
Method of introducing a polynucleotide molecule into or...
C800S298000, C800S295000, C800S320000, C800S317000, C435S419000, C435S468000, C435S320100, C435S430100, C536S023100, C536S023600, C536S024100
Reexamination Certificate
active
06706949
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
Homologous recombination is a very important mechanism by which all living organisms exchange genetic information, thereby generating genetic diversity. Moreover, this mechanism also allows organisms to maintain genetic stability by protecting their genome against physico-chemical or environmental insults. Homologous recombination is a very complex process involving several well-defined biochemical steps, which are catalyzed by the concerted action of many cellular proteins. For example, in
E.coli
, dozens of genes and their cognate protein products have been implicated in homologous recombination (Kowalczykowski, S., Dixon, D., Eggleston, A., Lauder, S., and Rehrauer, W.,
Microb. Rev
. 58: 401-465, 1994). The picture is even more complex in eukaryotic organisms.
Despite the complexity of the process, recent genetic, biochemical, and molecular studies have revealed many mechanistic similarities in homologous recombination of prokaryotic and eukaryotic organisms (Camerini-Otero, D. and Hsieh, P.,
Ann. Rev. Biochem
. 29: 509-552, 1995). For the sake of understanding, this pathway can be divided into four discrete biochemical steps: 1) initiation 2) homologous pairing and DNA stand exchange 3) branch migration and 4) resolution. In bacteria, the action of various proteins such as those encoded by the genes recBCD, recE, recQ and recJ, initiate the recombination process, step 1, by generating single stranded DNA. The bacterial RecA protein, the product of the recA gene, catalyzes the next step of homology search, pairing, and strand exchange, whereas in eukaryotes, members of the RAD51 gene family are implicated in this process. The final step, resolution of the Holliday junction, is attributed to the actions of bacterial RuvC.
The third step in the process is branch migration, also known as heteroduplex extension. A complex of two proteins, RuvA and RuvB catalyzes this reaction. Extensive characterization of these proteins and their role of in this reaction has been performed in
E.coli
(West S.C.,
Ann. Rev. Gen
. 31: 213-244, 1997). The product of the ruvA gene, the 22 kDa RuvA protein, exists as a stable tetramer in solution and specifically recognizes and binds to the Holliday junction with very high affinity. This binding is structure specific, independent of the DNA sequence, and Mg
2+
dependent. Binding of RuvA to the Holliday junction by itself is not sufficient to cause branch migration either in vitro or in vivo. Presence of the second component, RuvB, is required.
RuvB is the product of the bacterial gene ruvB. This protein has two NTP binding motifs, known as Walker A and Walker B boxes, as well as other structural motifs common to DNA helicases (West S.C.,
Ann. Rev. Gen
. 31: 213-244, 1997). In the absence of metal ions, RuvB exists as a monomer or dimer in solution. However, the functional form of this enzyme is thought to be a hexamer. Two hexameric RuvB units bind to DNA in a symmetrical manner to form a ring, similar to the hexameric ring formed by other DNA helicases. Binding of ATP or DNA to RuvB is Mg
2+
dependent, such that at low concentration of Mg
2+
, RuvB has very low affinity for DNA and does not hydrolyze ATP. Raising Mg
2+
concentration >20 mM increases RuvB's affinity for DNA as well as it's ATPase activity. Circular duplex DNA also stimulates the ATPase activity. RuvA also increases the affinity of RuvB for DNA. RuvB alone is sufficient to promote branch migration in vitro. However, a RuvAB complex functions much more robustly, and requires less Mg
2+
. Thus, RuvB participates in one of the rate limiting steps in homologous recombination in all living organisms.
Recently, eukaryotic homologues of bacterial RuvB have been cloned and characterized. For example, Kanemaki et al. isolated a 49 kDa IBP interacting protein (Tip49) from rat liver. The full-length cDNA of Tip49 revealed its strong homology to bacterial RuvB protein (Kanemaki, M., Makino, Y., Yoshida, T., Kishimoto, T., Koga, A., Yamamoto, K., Moncollin, V., Egly, J-M, Muramnatsu, M. and Tamura, T.,
Arch. Biochem. Biophys
., 235: 64-68, 1997). The same scientists also reported the cloning of human Tip49 (Makino, Y., Minori, T., Koike, C., Kanemaki, M., Kurokawa Y., Inoue, S., Kishimoto, T., and Tamura, T.,
Biochem. Biophys. Res. Commun
., 245: 819-823, 1998). Similarly, cDNA for a human protein called RuvBL1 has been isolated employing the small (14 kDa) subunit of human Replication Protein A, a hetero-trimeric protein involved in DNA replication, recombination and repair, as bait in the yeast two-hybrid assays. Two yeast homologues, scRuvBL1 and ScRuvBL2, have also been identified in the yeast genome database. Gene knockout experiments in yeast indicate that scRuvBL1 is essential for growth (Qiu, X., Lin, Y., Thome, K., Pian, P., Schlegel, B., Weremowicz, S., Parvin, J. and Dutta, A.,
J. Biol Chem
. 273: 27786-27793, 1998).
Due to the involvement of RuvB in homologous recombination, modulating RuvB expression levels provides the means to modulate the efficiency with which nucleic acids of interest are incorporated into the genomes of a target plant cell. Control of these processes has important implications in the creation of novel recombinantly engineered crops such as maize. The present invention provides this and other advantages.
SUMMARY OF THE INVENTION
Generally, it is the object of the present invention to provide nucleic acids and proteins relating to maize RuvB. It is an object of the present invention to provide: 1) antigenic fragments of the proteins of the present invention; 2) transgenic plants comprising the nucleic acids of the present invention; 3) 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 a specified sequence identity to a polynucleotide encoding a polypeptide of the present invention; (b) a polynucleotide which is complementary to the polynucleotide of (a); and, (c) a polymucleotide comprising a specified number of contiguous nucleotides from a polynucleotide of (a) or (b). The isolated nucleic acid can be DNA.
In another aspect, the present invention relates to recombinant expression cassettes, comprising a nucleic acid of the present invention operably linked to a promoter.
In another aspect, the present invention is directed to a host cell into which has been introduced the recombinant expression cassette.
In a further aspect, the present invention relates to an isolated protein comprising a polypeptide having a specified number of contiguous amino acids encoded by an isolated nucleic acid of the present invention.
In another aspect, the present invention relates to an isolated nucleic acid comprising a polynucleotide of specified length which selectively hybridizes under stringent conditions to a polynucleotide of the present invention, or a complement thereof. In some embodiments, the isolated nucleic acid is operably linked to a promoter.
In another aspect, the present invention relates to a recombinant expression cassette comprising a nucleic acid amplified from a library as referred to sura, 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 that is produced from this host cell.
In yet another aspect, the present invention relates to a transgenic plant comprising a recombinant expression cassette comprising a plant promoter operably linked to any of the isolated nucleic acids of the present invention. The present invention also provides transgenic seed from the
Ibrahim Medina A.
McElwain Elizabeth F.
Pioneer Hi-Bred International , Inc.
Pioneer Hi-Bred International Inc.
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