Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues – Blood proteins or globulins – e.g. – proteoglycans – platelet...
Reexamination Certificate
2000-10-27
2002-10-29
Chan, Christina (Department: 1644)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
Blood proteins or globulins, e.g., proteoglycans, platelet...
C530S388100, C530S388150, C530S388900, C530S389100
Reexamination Certificate
active
06472513
ABSTRACT:
TECHNICAL FIELD
The present invention relates to a gene encoding a protein having a helicase activity, a protein encoded by the gene, a method for producing the protein, and use of the gene and the protein.
BACKGROUND ART
DNA helicases are important enzymes that act on various biological reactions involving DNA in living bodies or microbial cells, and there are a number of types of DNA helicases.
The occurrence of a wide variety of DNA helicases is clearly demonsrated by the fact that the reactions involving DNA include a variety of reactions, such as replication and proliferation of cells, development and growth of an individual, and sustainment of life. As the recognized biochemical reactions occurring at the cellular level, there are assumed at least five reactions including replication, repairing, transcription, segregation and recombination of DNAs. DNA helicases are generally known to act to unwind a duplex DNA into single-stranded forms, and the energy required to such an action is considered to be provided by the hydrolysis of ATPs.
Among many types of DNA helicases, RecQ-type DNA helicases have recently been found, which individually have a helicase domain with at least about 40% homology to the helicase domain of
Escherichia coli
(
E. coli
) recQ gene at the amino acid level. The RecQ-type helicases have been focused on due to their involvement in various diseases and aging of humans. For example, Bloom's syndrome is a disease frequently inducible various cancers in younger age, and Werner's syndrome is a genetic disease inducible premature aging and abnormal cancer. It has recently been found that these syndromes are caused by mutation of different genes respectively encoding different human RecQ-type DNA helicases (
Cell,
83, pp.655-666, 1995; and Science, 272, pp.258-262, 1996).
A RecQ-type helicase was originally found in
E. coli
by Nakayama et al. (
Mol. Gen. Genet.,
195, pp.474-480, 1984). Two kinds of genes encoding proteins having high homology to the helicase have been found in an yeast cell and a human cancer cell, which were designated sgsl (Gangloff et at.,
Mol. Cell. Biol.
14, pp.8391-8398, 1994) and RecQ1 (Seki et al.,
Nuc. Acids Res.,
22, pp.4566-4573, 1994), respectively.
Known helicases belonging to this family are, in uni-cellular organisms such as
E. coli
and yeast, only the above-mentioned
E. coli
RecQ helicase and sgs1; and in multi-cellular organisms (i.e., human), Bloom DNA helicase (Ellis et al,
Cell,
83, pp.655-666, 1995) and Werner DNA helicase (Yu et al.,
Science,
272, pp.258-262, 1996) both responsible for the above-mentioned diseases, and RecQ1 helicase whose involvement in diseases is as yet unknown.
DISCLOSURE OF THE INVENTION
The object of the present invention is to provide a human RecQ4 DNA helicase gene, a protein encoded by the gene, a method for producing the protein, and use of the protein and the gene.
The present inventors assumed if there would be many RecQ family of DNA helicases other than the above three kinds in human. The inventors also assumed that, when genes of such helicases underwent mutation, the genes might induce various diseases as observed in Bloom's syndrome and Werner's syndrome and even be causative genes for refractory diseases of which etiologies have been undetermined yet. The inventors have made intensive and extensive studies for solving the above-mentioned problems. As a result, the inventors have succeeded in the cloning of a novel human RecQ4 DNA helicase gene by so-called RACE method. This success led to the achievement of the invention.
That is, the present invention provides a gene encoding:
(a) a protein comprising an amino acid sequence of SEQ ID NO: 2; or
(b) a protein having deletion, substitution or addition of at least one amino acid residue in the amino acid sequence of SEQ ID NO: 2, which has a helicase activity.
The present invention further provides a gene comprising:
(c) DNA comprising a nucleotide sequence of SEQ ID NO: 1; or
(d) DNA hybridizing to the DNA comprising the nucleotide sequence of SEQ ID NO: 1 under stringent conditions, which encodes a protein having a helicase activity.
The present invention further provides an oligonuleotide probe hybridizing to at least a portion of the gene.
The present invention further provides a recombinant vector containing the gene.
The present invention further provides a transformant containing the recombinant vector.
The present invention further provides a method for producing a protein having a helicase activity, comprising culturing the transformant and then collecting the protein from the resultant culture.
The present invention further provides a recombinant protein of:
(a) a protein comprising an amino acid sequence of SEQ ID NO: 2; or
(b) a protein having deletion, substitution or addition of at least one amino acid residue in the amino acid sequence of SEQ ID NO: 2, which has a helicase activity.
The present invention further provides a mouse- or rat-derived protein having a helicase activity, comprising an amino acid sequence having at least 70% homology to the amino acid sequence of the protein; and a mouse- or rat-derived gene encoding the protein.
The present invention further provides a method for producing a protein having a helicase activity, comprising culturing the transformant and then collecting the protein from the resultant culture.
The present invention further provides a monoclonal or polyclonal antibody specifically reacting with the protein.
The present invention further provides a hybridoma producing the monoclonal antibody, which is prepared by cell fusion of an antibody-producing cell immunized with the protein with a myeloma cell.
The present invention further provides a reagent for detecting a gene encoding helicase, comprising the oligonucleotide probe.
The present invention further provides a kit for diagnosing a disease caused by the genetic abnormality of a gene encoding a protein with a helicase activity, the kit comprising the protein and the monoclonal antibody and/or the polyclonal antibody.
The present invention further provides a transgenic animal having the gene introduced therein in the modified form, the modification being made such that the expression level of the gene is increased or decreased; and a knockout mouse in which the function of the gene has been treated to be lost.
Hereinafter, the present invention is described in detail.
The gene according to the present invention encodes a novel human RecQ4 DNA helicase, and encodes a protein which comprises an amino acid sequence of SEQ ID NO: 2 or a protein which comprises an amino acid sequence having deletion, substitution or addition of at least one amino acid residue in the amino acid sequence of SEQ ID NO: 2 and has a helicase activity.
The protein according to the present invention contains seven helicase motifs of the known RecQ-type DNA helicase, which are well-conserved between
E. coli
, yeast and human at the amino acid level, as shown in FIG.
2
. As is clearly demonstrated by the results of the radiation hybrid mapping shown in
FIG. 3
, it is confirmed that the human RecQ-type DNA helicase gene of the present invention is located on the long arm of human chromosome 8, 8q24.3, as shown in FIG.
4
. Genes derived from other species which correspond to the human-derived gene of the present invention can also be cloned by known techniques.
From the results of the multi-tissue northern blot analysis for determining the expression level of the gene of the present invention in different organs (see FIG.
5
), it is found that the expression of the gene is observed in all of the tissues examined, and a remarkably intensive expression is particularly observed in thymus and testis.
These results strongly suggest that the gene of the present invention is one of the genes responsible for the maintenance of the fundamental homeostasis of living bodies. Accordingly, the gene is useful for studying on the relation with development and aging of individuals. The elucidation of the expression control of the gene is
Furuichi Yasuhiro
Kitao Saori
Shimamoto Akira
Agene Research Institute Co., Ltd.
Chan Christina
Gray Cary Ware & Freidenrich LLP
Huynh Phuong N.
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