Drosophila recombination-associated protein and methods for use

Details Drosophila recombination-associated protein and methods for use Drosophila recombination-associated protein and methods for use

2002-07-19

2004-12-14

Hutson, Richard (Department: 1652)

Chemistry: molecular biology and microbiology

Enzyme , proenzyme; compositions thereof; process for...

Transferase other than ribonuclease

C435S183000, C435S233000, C435S440000, C435S471000, C435S455000, C435S477000, C435S091100, C435S015000, C536S023100, C536S023200, C536S023500, C536S025300, C536S024500, C530S350000, C530S358000

Reexamination Certificate

active

06830910

ABSTRACT:

FIELD OF THE INVENTION
This invention encompasses the Drosophila Recombination Associated Protein (DRAP), nucleic acid sequences encoding DRAP, and methods of using DRAP.
BACKGROUND
One common method for introducing exogenous genes in eukaryotic cells and organisms is by direct transfection. Transfection is relatively efficient but genomic integration tends to be random and regulation of gene expression can be difficult. Various strategies have been developed to enhance the control of transgene expression, limit expression to a specific tissue or link expression to a specific time during development in transgenic animals. The direct transgenic approach suffers primarily from an inability to directly modify a specific genetic locus.
Another approach for introducing exogenous genes into eukaryotic cells utilizes homologous recombination directed at a specific locus in specialized embryonic stem cells. A number of genes and gene products that participate in general and site-specific recombination in viruses, prokaryotes and eukaryotes have been identified. (Camerini-Otero, R. D., et al., Ann. Rev. Genetics, 1995, 29:509-522.) These gene products and/or proteins encoded by these genes have proven useful in the molecular genetic manipulation of DNA in vitro and in vivo with applications in cloning, gene mapping and manipulation of genomes in living organisms. (Grindley, N. D., et al., 1995, Cell 83: 1063-1066; and Wang, J. C., et al., 1990, Cell 62: 403-406.) The use of such proteins in these applications relies upon their activity in homologous recombination.
Homologous recombination is routinely used to create “knock out” mutations in the production of mutant animals. (Nomura, T., 1997, Lab Anim. Sci. 47: 113-117; and Torres, M., 1998, Cur. Top. Dev. Biol. 36: 99-114.) This approach results in gene-directed, i.e. sequence directed, insertions that “knock out” gene function to produce such mutations. Although this approach is generally an inefficient process, selection methods and screening permit facilitated identification of cells bearing specific genomic modifications. The enzymatic machinery that completes this low efficiency process in specialized embryonic stem cells is unknown. It would thus be beneficial to identify one or a number of genes or gene products involved so that homologous recombination could be performed with a higher efficiency. Such site specific homologous recombination might prove useful for therapeutic purposes. The use of these specific genes and/or gene products could improve the efficiency of the knock-out process and extend the range of cell types where homologous recombination could be accomplished.
It would thus be highly desirable to develop a method for efficient, homology directed genome modification that would permit modification of genes ranging from knockouts to subtle modifications. Such methods would rely upon the use of an efficient homology dependent DNA pairing protein capable of directing a mutagenic oligonucleotide to its cognate gene within a complex genome. The aim of this approach would be to promote DNA strand exchange and force a gene conversion event that can be identified at the molecular level and is heritable. There are several, general, homology-dependent strand transferases that might be suitable for such a purpose that have been used for related purposes in vitro (e.g., the RARE technique). (Ferrin, L. J. et al., Nature Genetics, 1994, 6:379-383; and Ferrin, L. J., Genet. Eng., 1995, 17:21-30.
General recombinases currently used in the promotion of DNA strand transfers and gene conversions include UV Sensitive X (“UVSX”) from T4 phage, Recombination Protein A (“RecA”) from
E. coli
or RecA-derived peptides, or Radiation Induced Mutant 51 (“RAD51”) from yeast or RAD 51 homologues from Drosophila, mouse and human. These proteins are each part of a large superfamily of recombination-related proteins. These recombinases typically require accessory proteins such as Singles. Strand Binding Protein (“SSB”), Replication Protein A (“RPA”) and Radiation Induced Mutant 52 (“RAD 52”) in order to achieve maximal efficiency. There are also a host of site directed recombinases (of the integrase and resolvase super-families) that might be modified for such a purpose.
Drosophila embryos provide a rich source of enzymes that are involved in homologous recombination. (Eisen, A., et al, 1988, PNAS 85:7481-85.) It was shown that purified protein fractions possessed an efficient ATP-independent, homology-dependent strand transferase activity similar to RecA. The active fractions appeared to work catalytically as opposed to stoichiometrically. (Eisen, A, et al., 1988.) The Drosophila embryonic cells are rapidly dividing and thus provide a large quantity of enzymes involved in mitotic recombination and DNA processing. A similar potent homology-dependent strand transferase activity was demonstrated to be present in nuclear extracts from Drosophila. (Eisen, A. et al., 1988.) The apparent catalytic nature of this Drosophila protein activity distinguishes it from most general recombinases, typified by proteins in the RecA/Rad 51 superfamily of gene products which operate in a stoichiometric fashion (Camerini-Otero, R. D., et al., 1995; and Yancey-Wrona, J. E., et al., 1995, Current Biol. 5: 1149-1158; Baumann, P., et al., 1996, Cell 87: 757-766; Benson, F. E., et al., 1998, Nature 391: 401-404; Shinohara, A., et al., 1998, Nature 391: 404-407; New, J. H., et al., 1998, Nature 391: 407-410; Plasterk, R. H. A., 1993, Cell 74:781-786; and O.N. Voloshin, O. N., et al., 1996, Science 272: 868-872.) The unique Drosophila homology-dependent stand transferase activity offers certain theoretical advantages for performing high efficiency gene targeting. It would thus be advantageous to utilize this Drosophila activity in the promotion of homologous recombination and homology directed gene conversion.
SUMMARY OF THE INVENTION
The present invention provides an isolated cDNA clone containing the coding region for Drosophila Recombination Associated Protein (DRAP) [SEQ ID NO: 1], a DNA encoding DRAP corresponding to nt 134-610 of the isolated cDNA clone [SEQ ID NO: 2] and the longest Open Reading Frame (ORF) contained within the isolated cDNA clone sequence which corresponds to nt 104-610 of the isolated cDNA clone [SEQ ID NO: 3] (see FIG.
1
). The invention also provides an isolated DRAP polypeptide as depicted in [SEQ ID NO: 4], as well as antigenic peptides, examples of which are defined by [SEQ ID NO: 5] [SEQ ID NO: 6] and [SEQ ID NO: 7] (see FIGS.
2
and
4
B). The invention further provides DNA vectors and transformed cells suitable for recombinant expression of DRAP.
The Drosophila Recombination Associated Protein, its homologues from other organisms or active peptides derived therefrom, as well as DNA encoding such protein are useful for homology-dependent pairing of three DNA stands. The combination of strand-transfer and topoisomerase activities associated with DRAP permits directed pairing and cleavage at defined site(s) within DNA. This in turn makes possible the isolation and/or removal of a defined segment of DNA. DRAP is also useful in cloning, genomic cloning and gene mapping.
DRAP is also useful in promoting gene disruptions or “knockout” mutations, carrying out targeted mutagenesis of specific genes and in generating transgenic animals.
Thus, in one aspect, the present invention provides methods for DNA cloning, gene isolation and gene mapping.
In another aspect, the invention provides a method for targeted mutagenesis.
In yet a further aspect, the invention provides a method for experimental and therapeutic application of DRAP driven knockouts or other modifications of genes responsible for genetic diseases and the use of DRAP driven genetic manipulation of genes in gene therapy.
These and other aspects of the present invention will be apparent to those of ordinary skill in the art in light of the present description, claims and drawings.


REFERENCES:
patent: 5763240 (1998-

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