DNA molecules encoding human NHL a DNA helicase

Details DNA molecules encoding human NHL a DNA helicase DNA molecules encoding human NHL a DNA helicase

2002-06-05

2004-07-13

Hutson, Richard (Department: 1652)

Chemistry: molecular biology and microbiology

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

C435S193000, C435S006120, C435S320100, C435S252300, C435S325000, C435S410000, C536S023100, C536S023200, C536S023500

Reexamination Certificate

active

06762042

ABSTRACT:

STATEMENT REGARDING FEDERALLY-SPONSORED R&D
Not Applicable
REFERENCE TO MICROFICHE APPENDIX
Not Applicable
FIELD OF THE INVENTION
The present invention relates in part to isolated nucleic acid molecules (polynucleotides) which encode NHL, a putative DNA helicase. The present invention also relates to recombinant vectors and recombinant hosts which contain a DNA fragment encoding NHL, substantially purified forms of associated NHL, associated mutant proteins, and methods associated with identifying compounds which modulate NHL, which will be useful in the treatment of various neoplastic disorders, given that this gene is located at 20q13.3 and immediately adjacent to M68/DcR3, which is involved in tumor growth. Also included within the present invention is a human genomic fragment representing this portion of the human genome, along with three additional genes (M68/DcR3, SCLIP, and ARP).
BACKGROUND OF THE INVENTION
Naumovski et al. (1985,
Mol. Cell Biol.
5:17-26; Reynolds et al. (1985
Nucleic Acid Res
13:2357-2372) and Weber et al. (1990
EMBO J.
9:1437-1447) disclose members of the RAD3/FRCC2 gene family of DNA helicases.
It is known that several chemotherapeutic agents inhibit helicases, including actinomycin C1, daunorubicin and nogalamycin (Tuteja, et al., 1997,
Biochem. Biophys. Res. Comm.
236(3):636-640), and a prostate cancer drug, C1-958 (Lun, et al., 1998,
Cancer Chemother. Pharmacol.
42(6):447-453). In addition, some topoisomerases have been shown to have anti-cancer activity.
Despite the identification of the aforementioned helicase-encoding genes and chemotherapeutic agents, it would be advantageous to identify additional genes which reside within chromosomal regions associated with a disease state such as cancer as well as a gene which encodes a type of protein which may be associated with that disease. The present invention addresses and meets this need by disclosing a DNA molecule encoding a DNA helicase with a chromosomal location suggestive of association with cancer.
SUMMARY OF THE INVENTION
The present invention relates to an isolated or purified nucleic acid molecule (polynucleotide) which encodes a novel mammalian DNA helicase.
The present invention also relates to an isolated nucleic acid molecule (polynucleotide) which encodes mRNA which expresses a novel human DNA helicase, NHL.
A preferred aspect of the present invention relates to an isolated or purified DNA molecule which encodes human NHL, the nucleotide sequence as set forth in
FIGS. 1A-B
and SEQ ID NO: 1.
The present invention also relates to biologically active fragments or mutants of SEQ ID NO: 1 which encode a mRNA molecule expressing a novel DNA helicase, NHL. Any such biologically active fragment and/or mutant will encode either a protein or protein fragment which at least substantially mimics the biological properties of the human NHL protein disclosed herein in FIG.
2
and as set forth as SEQ ID NO: 2. Any such polynucleotide includes but is not necessarily limited to nucleotide substitutions, deletions, additions, amino-terminal truncations and carboxy-terminal truncations such that these mutations encode mRNA which express a functional NHL protein in a host cell, so as to be useful for screening for agonists and/or antagonists of NHL activity.
The present invention also relates to recombinant vectors and recombinant hosts, both prokaryotic and eukaryotic, which contain the substantially purified nucleic acid molecules disclosed throughout this specification.
The present invention also relates to a substantially purified form of a human NHL protein which comprises the amino acid sequence disclosed in FIG.
2
and set forth as SEQ ID NO: 2.
A preferred aspect of this portion of the present invention is a NHL protein which consists of the amino acid sequence disclosed in FIG.
2
and set forth as SEQ ID NO: 2.
Another preferred aspect of the present invention relates to a substantially purified NHL protein, preferably a human NHL protein, obtained from a recombinant host cell containing a DNA expression vector comprises a nucleotide sequence as set forth in SEQ ID NO: 1 and expresses the respective NHL protein. It is especially preferred is that the recombinant host cell be a eukaryotic host cell, such as a mammalian cell line.
The present invention also relates to biologically active fragments and/or mutants of a NHL protein comprising the amino acid sequence as set forth in SEQ ID NO: 2, including but not necessarily limited to amino acid substitutions, deletions, additions, amino terminal truncations and carboxy-terminal truncations such that these mutations provide for proteins or protein fragments of diagnostic, therapeutic or prophylactic use and would be useful for screening for selective modulators, including but not limited to agonists and/or antagonists for human NHL pharmacology.
A preferred aspect of the present invention is disclosed in FIG.
2
and is set forth as SEQ ID NO: 2, a respective amino acid sequence which encodes human NHL. Characterization of one or more of these DNA helicase-like proteins allows for screening methods to identify novel NHL modulators that may be useful in the treatment of human neoplastic disorders. The modulators selected through such screening and selection protocols may be used alone or in conjunction with other cancer therapies. As noted above, heterologous expression of a NHL protein will allow the pharmacological analysis of compounds which modulate NHL activity and hence may be useful in various cancer therapies. To this end, heterologous cell lines expressing a NHL protein can be used to establish functional or binding assays to identify novel NHL modulators.
The present invention also relates to polyclonal and monoclonal antibodies raised in response to either the NHL or a biologically active fragment of NHL.
The present invention relates to transgenic mice comprising altered genotypes and phenotypes in relation to NHL and its in vivo activity.
The present invention also relates to NHL fusion constructs, including but not limited to fusion constructs which express a portion of the NHL protein linked to various markers, including but in no way limited to GFP (Green fluorescent protein), the MYC epitope, and GST. Any such fusion constructs may be expressed in the cell line of interest and used to screen for NHL modulators.
Therefore, the present invention relates to methods of expressing mammalian NHL, and preferably human NHL, biological equivalents disclosed herein, assays employing these gene products, recombinant host cells which comprise DNA constructs which express these proteins, and compounds identified through these assays which act as agonists or antagonists of NHL activity.
The present invention also relates to the isolated genomic sequence which comprises SEQ ID NO: 1, a 115 kb genomic fragment set forth herein as SEQ ID NO: 3. As especially preferred aspect of this portion of the invention is the region of the genomic fragment of SEQ ID NO: 3 which comprises the regulatory and coding regions of human NHL, as well as intervening sequences (introns). This 115 kb fragment contains at least the coding region of four genes, NHL, M68/DcR3, SCLIP and ARP. As discussed herein, it has been shown that this region of chromosome 20 is associated with tumor growth. Therefore, an aspect of this invention also comprises the use of one or more regions of this 115 kb genomic sequence to identify compounds which up or downregulate expression of one or more of the genes localized within this 115 kb region, wherein this up or down regulation results in an interference of tumor growth. For example, a transcription element of one of these four genes may be responsible for M68/DcR3 (and/or NHL) overexpression in tumors, and if M68 or NHL overexpression in tumors has a caustic role, blockage of M68/DcR3 or NHL overexpression in tumors by interfering with this transcription site will be useful.
It is an object of the present invention to provide an isolated nucleic acid molecule (e.g., SEQ ID NO: 1) which encodes novel form of human NHL, or f

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