JPWO2001025425A1 - Nuclear-localized RecQ5-type DNA helicase - Google Patents

Abstract

(57) [Abstract] Novel human RecQ5-type DNA helicases β and γ, as well as the genes encoding them, have been isolated. It has been revealed that three helicases, including the known RecQ-type DNA helicase (α), are generated as a result of alternative splicing. In particular, RecQ5-type DNA helicase β has the novel characteristic of being localized in the nucleus. The isoform β provided by this invention may be closely associated with chromosomal instability disorders.

Description

[Detailed Description of the Invention] TECHNICAL FIELD [0001] The present invention relates to nuclear-localized isoforms of RecQ5 helicase, genes encoding them, and their uses. BACKGROUND ART [0002] DNA helicases (EC 3.6.1.-) are important enzymes involved in various biological reactions involving DNA in living organisms or microbial cells, and there are many different types. The existence of a wide variety of DNA helicases, particularly in multicellular organisms, is easily predicted given the wide range of DNA-related reactions, including cell replication and proliferation, individual development and growth, and even life support. DNA helicases, also known as DNA unwinding enzymes, are known to unwind double-stranded DNA into single-stranded DNA. The energy required for this action is thought to be obtained through ATP hydrolysis. In fact, the existence of at least five types of biochemical reactions catalyzed by DNA helicases at the cellular level has been suggested: Among the many DNA helicases involved in DNA replication, repair, transcription, DNA segregation, and recombination, the recently discovered RecQ-type DNA helicase has attracted attention due to its association with human diseases and aging phenomena. The RecQ-type DNA helicase is a name given to helicases that have a helicase domain that shows approximately 40% or more homology at the amino acid level with the helicase domain of the Escherichia coli recQ gene. The RecQ-type helicase was first described by Nakayama et al. (Mol. Gen. Genet., 195, pp. 474-474).
-480, 1984) in Escherichia coli. Subsequently, genes encoding proteins highly homologous to this helicase were discovered in yeast and human cancer cells, and named sgs1 (Gangloff et al., Mol. C 1999).
Biol. 14, pp. 8391-8398 (1994)) and Rec
Q1 (Seki et al., Nuc. Acida Res. 22, pp45
66-4573 (1994)). Furthermore, BLM helicase was identified as the causative gene for Bloom syndrome, a disease characterized by the frequent onset of various cancers at a young age (Cell, 83, pp. 655-666, 1995). Furthermore, it was revealed that the direct cause of Werner syndrome, a hereditary disease that induces premature aging and abnormal cancers, is a mutation in WRN helicase, resulting in loss of nuclear transport activity (Science, 272, pp. 258-262, 1996). The only known helicases belonging to this family are the E. coli RecQ helicase, which was first discovered in single-cell organisms such as E. coli and yeast, and sgs1, a homologue of the E. coli RecQ helicase discovered in yeast. On the other hand, in multicellular humans, three types of helicases have been known to date: two types of helicases, BLM helicase and WRN helicase, which are associated with the aforementioned diseases, and RecQ1 helicase, which has not yet been found to be associated with any disease. In addition, the present inventors have identified two types of RecQ-type DNases, RecQ4 and RecQ5.
A helicase was reported (Genomics 54, 443-452, 1998
The RecQ4 and RecQ5 discovered by the present inventors are unique compared to other RecQ-type DNA helicases in that, as a result of homology searches, no known amino acid sequences resembling nuclear localization signals were found near the C-terminus.
It has been revealed that mutations in the ecQ4 gene cause Rothmund-Thomson syndrome (S. Kitao et al., 1998, Genomics,
54, pp443-452;S. Kitao et al. , 1999, Nat.
Genet. 22:82-84; WO99/05284; Japanese Patent Application No. 11-112
No. 18). Rothmund-Thomson syndrome is a chromosomal instability disorder accompanied by a high incidence of cancer, with symptoms similar to those of Bloom syndrome and Werner syndrome. Against this background, elucidating the function of the RecQ family and the mechanism of chromosome stabilization is an important key to understanding the symptoms of these RecQ gene-related diseases, including the high incidence of cancer. Disclosure of the Invention The present invention aims to isolate a novel RecQ-type DNA helicase. More specifically, the present invention aims to provide a nuclear-localized isoform of human RecQ5-type DNA helicase, a polynucleotide encoding the same, and uses thereof. The present inventors have analyzed the structure and expression of the previously reported RecQ4- and RecQ5-type DNA helicases. They then synthesized recombinant RecQ5.
We focused on the fact that observations of intracellular localization using helicases indicated that RecQ5 helicase is localized in the cytoplasm. Many previously reported RecQ-type helicases, such as RecQ1, BLM, and WRN, are localized to the nucleus and have been suggested to function on chromosomal DNA. Even RecQ4-type DNA helicases, for which no nuclear localization signal could be found in homology searches, were observed to be localized to the nucleus in similar experiments. Based on these findings, we predicted the existence of nuclear-localized isoforms of RecQ5-type DNA helicases. To support this prediction, we screened a human cDNA library using RecQ5 cDNA as a probe and successfully isolated two RecQ5 isoforms.
These isoforms of RecQ5-type DNA helicases, including previously reported RecQ5-type DNA helicases, have been shown to be expression products resulting from alternative splicing. The present inventors then discovered that one of these isoforms is a novel RecQ5-type DNA helicase with nuclear localization, leading to the completion of the present invention. Specifically, the present invention relates to the following nuclear-localized RecQ5-type DNA helicases, their genes, and uses thereof: [1] A polynucleotide set forth in any of the following (a) to (d): (a) a polynucleotide comprising a protein-coding region of the nucleotide sequence set forth in SEQ ID NO: 2; (b) a polynucleotide encoding the amino acid sequence set forth in SEQ ID NO: 2; (c) a polynucleotide encoding a nuclear-localized protein with helicase activity, comprising the amino acid sequence set forth in SEQ ID NO: 2 modified by deletion, addition, insertion, and/or substitution of one or several amino acids with other amino acids. (d) A polynucleotide encoding a protein encoded by a DNA that hybridizes under stringent conditions with a DNA consisting of the base sequence set forth in SEQ ID NO: 1, the polynucleotide encoding a protein localized in a nucleus and having helicase activity. [2] A protein encoded by the polynucleotide set forth in [1]. [3] A vector into which the polynucleotide set forth in [1] has been inserted. [4] A transformant harboring the polynucleotide set forth in [1] or the vector set forth in [3]. [5] A method of culturing the transformant set forth in [5], comprising the steps of culturing the transformant set forth in [5] and recovering an expression product.
[6] A method for producing the protein according to [2]. [7] A polynucleotide comprising a nucleotide sequence corresponding to 1390 to 3703 in the nucleotide sequence set forth in SEQ ID NO: 1, or a nucleotide sequence complementary to the complementary strand thereof,
A polynucleotide having a chain length of at least 15 nucleotides. [7] The primer for polynucleotide synthesis according to [1], which comprises the polynucleotide according to [6]. [8] The probe for polynucleotide detection according to [1], which comprises the polynucleotide according to [6].

[9] An antisense DNA against the polynucleotide according to [1] or a part thereof. [10] A partial peptide consisting of the amino acid sequence corresponding to positions 411 to 991 in the amino acid sequence of SEQ ID NO: 2. [11] An antibody that recognizes the partial peptide consisting of the amino acid sequence corresponding to positions 411 to 991 in the amino acid sequence of SEQ ID NO: 2. [12] An immunoassay method comprising the steps of contacting the antibody according to [11] with a protein consisting of the amino acid sequence of SEQ ID NO: 2 or a partial peptide thereof, and detecting an immunological reaction between the two. [13] A reagent for detecting the protein consisting of the amino acid sequence of SEQ ID NO: 2 or a partial peptide thereof, comprising the antibody according to [11]. [14] A transgenic non-human vertebrate in which the expression level of the polynucleotide according to [1] is altered. [15] The transgenic non-human vertebrate according to [14], which is a knockout animal in which the function of the polynucleotide according to [1] is deleted. The RecQ5 DNA helicase isoform provided by the present invention is a protein consisting of the amino acid sequence of SEQ ID NO: 2. The present inventors have identified the first reported RecQ5-type DNA helicase as α, the Re
The isoform of RecQ5-type DNA helicase was named β. Hereafter, the β-form of RecQ5-type DNA helicase will be referred to as RecQ5β, and the α-form as RecQ
RecQ5β, which was first discovered by the present inventors, has been confirmed to have nuclear transport activity and to be localized in the nucleus through experiments on intracellular localization using recombinant cells. On the other hand, since the known RecQ5α is localized in the cytoplasm, RecQ5β is
It can be said that cQ5β is a protein with novel characteristics.
Like known RecQ-type DNA helicases, Q5β has the following amino acid sequence:
It contains seven helicase motifs that are well conserved among E. coli, yeast, and humans. Furthermore, the gene for RecQ5α is located in the same genome as human 17th gene.
It was confirmed to be located on the long arm of chromosome 17q25. RecQ5β of the present invention is a protein consisting of 991 amino acid residues, and its structure is shown in Figure 3. In Figure 3, the structure of RecQ5β of the present invention is compared with the structure of the known RecQ5α (410 amino acids) and RecQ5γ (435 amino acids), which was also confirmed along with RecQ5β of the present invention. The region indicated by the lines is the portion common to the three isoforms. Within this common region, the region indicated by the black box corresponds to the helicase domain. The region indicated by the diagonal lines is the R
This region is present only in ecQ5β and shares homology with the C-terminal region of E. coli RecQ. The region located further C-terminally is specifically found in RecQ5β, and as described below, a portion of this region is predicted to be essential for the nuclear import activity of RecQ5β. Genomic analysis confirmed that these three isoforms are alternative splicing forms. Figure 2 shows the exon usage by each isoform. The three isoforms share exons 1 to 6. Regarding the large exon 7, which includes the 3'UTR, RecQ5α uses the entire exon, while RecQ5β uses only a portion of the 5' end, 7a, and RecQ5γ uses the same exon 7a and 7b, which includes part of the 3'UTR. Furthermore, RecQ5β uses exons 8 to 19, which are not used in other isoforms. RecQ5α(1-6)-7/3715nt. RecQ5β(1-6)-7a-(8-19)/3703 nt. RecQ5γ(1-6)-7a-7b/1749 nt. The genomic structure encoding the RecQ5 isoforms is shown in Figure 1. Figure 1 shows that the human RecQ5 gene is composed of 19 exons and 18 introns. The lengths of the introns indicated by dashed lines in the figure have not been determined. The protein of the present invention, having the amino acid sequence set forth in SEQ ID NO: 2, can be extracted from human tissues that express this protein. Specifically, the protein can be extracted by known methods from tissues such as human testis where high expression is observed, and the RecQ5β of the present invention can be purified by techniques such as immunoaffinity chromatography using antibodies against RecQ5β. The present invention also relates to a nuclear-localized protein that has helicase activity and is composed of the amino acid sequence set forth in SEQ ID NO: 2, modified by deletion, addition, insertion, and/or substitution of one or more amino acids with other amino acids. A nuclear-localized protein refers to a protein that has nuclear translocation activity. Therefore, even if a portion of the protein is distributed in the cytoplasm, if the distribution in the nucleus is relatively strong, the protein can be said to be nuclear-localized. When observing the localization of WRN helicase in actual cultured cells, some cells are not found to be localized in the nucleus. This is a phenomenon caused by WRN helicase being localized in the cytoplasm in M-phase cells. In fact, the proportion of M-phase cells in cultured cells (5% to less than 10%) is about 10%.
This corresponds to the proportion of cells in which nuclear localization of the helicase was not observed. Because WRN helicase is not involved in nuclear division (chromosome segregation), it is thought to be in the cytoplasm during the M phase. Like WRN helicase, RecQ5β of the present invention is also localized in the cytoplasm of 90% of cultured cells.
Nuclear localization is observed in 90% or more of the cells observed. Therefore, if nuclear localization is observed in 90% or more of the cells observed, the protein can be said to be nuclear-localized. In the present invention, a nuclear-localized protein that contains a mutation in the amino acid sequence set forth in SEQ ID NO: 2 and has helicase activity is referred to as a RecQ5β homolog. A RecQ5β homolog is a protein that is functionally equivalent to human RecQ5β. A RecQ5β homolog consists of an amino acid sequence that does not show significant homology to other known RecQ-type DNA helicases. In the present invention, significant homology means at least 50%, preferably 60%, to the amino acid sequence set forth in SEQ ID NO: 2.
The homology of an amino acid sequence is determined by BLAST (Altschul, S.F., et al., J. Amino Acid Sequences, vol. 1, pp. 111-115, 2002).
al. 1990 J Mol Biol. 215:403-410;Altsc
hul, S. F. , etc. al. 1997 Nucleic Acids Re
s. 25:3389-3402). Since the amino acid sequence homology of human RecQ-type helicases is generally 40% or less, the RecQ5 homolog of the present invention is clearly distinguishable from known RecQ-type DNA helicases. The RecQ5β protein of the present invention was not only localized in the nucleus but also observed to interact with topoisomerases 3α and 3β.
Topoisomerases (SGS1) have been identified as a group of enzymes that relieve twists and tangles in DNA molecules by cutting the DNA, or maintain the proper state. They are classified into type I and type II topoisomerases based on their function, with type I topoisomerases 1 and 3, and type II topoisomerases known as topoisomerase 2. Among them, topoisomerase 3 has been shown to potentially contribute to chromosome stabilization in yeast through its interaction with sgs1. (Gangloff, S. et al. 1994 Mol Ce
As mentioned above, yeast sgs1 is a homolog of human RecQ, and the fact that the interaction between RecQ-type helicase and topoisomerase 3 has also been confirmed in humans is indicative of the R
This supports the finding that the ecQ5β protein is closely related to chromosome stabilization in human cells. Therefore, proteins functionally equivalent to the RecQ5β protein of the present invention include:
In addition to being a nuclear-localized protein with helicase activity, it is desirable that the protein interacts with topoisomerase 3. The interaction between a RecQ5β homolog and topoisomerase 3 can be confirmed by detecting the binding of the two. There are no limitations on the number or sites of amino acid mutations in a RecQ5β homolog, as long as the function is maintained. The number of mutations is typically within 10% of all amino acids, preferably within 5% of all amino acids, and more preferably within 1% of all amino acids.
For example, at least one amino acid sequence of SEQ ID NO: 2 is within 1%.
1, preferably about 1 to 10, more preferably 1 to 5 amino acids may be deleted. Alternatively, at least 1, preferably about 1 to 10, more preferably 1 to 5 amino acids may be added to the amino acid sequence represented by SEQ ID NO: 2. Also, at least 1, preferably about 1 to 10, more preferably 1 to 5 amino acids in the amino acid sequence represented by SEQ ID NO: 2 may be substituted with other amino acids. These mutations may include not only one type of mutation, but also several mutations at the same time. Therefore, proteins resulting from this change in amino acid sequence also include those in which the first methionine (Met) in the amino acid sequence represented by SEQ ID NO: 2 is deleted. In addition, Re
Polymorphisms are often observed in the base sequences of genes encoding eukaryotic proteins such as cQ-type DNA helicases. Polymorphisms are small base substitutions found in the base sequence of a gene, and the effect of base substitutions on protein activity is usually small. Proteins with small mutations in the base sequence or amino acids due to polymorphisms are also included in the proteins of the present invention, as long as they have nuclear-localized DNA helicase activity. Proteins consisting of an amino acid sequence modified by deletion, addition, insertion, and/or substitution with other amino acids with respect to the amino acid sequence set forth in SEQ ID NO: 2,
That is, RecQ5β homologs can be obtained, for example, by the following method. RecQ5β homologs are isolated using hybridization techniques, gene amplification techniques, and the like, which are well known to those skilled in the art. That is, hybridization techniques (Current Protocols in Molecular Biol.
ogy edit. Ausubel et al. (1987)Publish
Using the DNA sequence (SEQ ID NO: 1) encoding human RecQ5β or a portion thereof, DNA highly homologous to this sequence can be isolated using the DNA sequence (SEQ ID NO: 1) encoding human RecQ5β.
A. It is within the ordinary skill of those skilled in the art to obtain a protein functionally equivalent to human RecQ5β from A. Thus, proteins encoded by DNA that hybridizes with DNA encoding human RecQ5β and functionally equivalent to these proteins are also included in the proteins of the present invention. Organisms from which RecQ5β homologs of the present invention can be isolated by hybridization techniques include, in addition to humans, rats, mice, rabbits, chickens, pigs, and cows. Furthermore, the isolation of RecQ5β homologs is not limited to vertebrates, and can also be carried out from yeast and the nematode C. elegans.
The RecQ5β homologs according to the present invention can also be found in organisms such as S. ans. The stringency of hybridization for isolating DNA encoding a RecQ5β homolog is generally set to 1x SSC, 0.1% SDS,
37°C. Stringent conditions include "0.5x SSC, 0.
For example, conditions such as "0.1% SDS, 42°C" can be used. Even more stringent conditions can be "0.2xSSC, 0.1% SDS, 65°C." The stricter the stringency, the more likely it is that DNA having a high homology to the probe sequence will be isolated.
The above combination of conditions of concentration and temperature is merely an example, and a person skilled in the art would be able to achieve the same stringency as above by appropriately combining other factors that determine the stringency of hybridization (e.g., probe concentration, probe length, hybridization reaction time, etc.). In general, DNA isolated using such hybridization techniques
The protein encoded by Re is composed of the amino acid sequence of SEQ ID NO: 2.
The DNA of the present invention has a high degree of homology in amino acid sequence to RecQ5β. High homology refers to sequence identity of at least 50% or more, preferably 60% or more, and more preferably 70% or more. Amino acid sequence homology can be determined using the BLAST search algorithm. The base sequences constituting the DNA of the present invention have a high degree of homology to the base sequence of RecQ5β consisting of SEQ ID NO: 1. In the present invention, high homology in base sequence refers to at least 70% or more,
Preferably, it means a sequence identity of 80% or more, more preferably 90% or more. In addition, it also means a sequence identity of 80% or more, more preferably 90% or more.
in Molecular Biology edit. Ausubel et
al. (1987) Publish. John Wiley & Sons
Section 6.1-6.4) to identify D encoding a RecQ5 homologue.
NA (or a fragment thereof) can also be isolated.
If PCR is performed using primers designed based on a portion of the DNA encoding the RecQ5β of the present invention, DNA highly homologous to SEQ ID NO: 1 will be amplified, at least in the region where the primers hybridize. The protein encoded by the resulting amplification product can also be obtained based on the product. Like proteins obtained by hybridization techniques, proteins obtained in this manner are likely to be composed of an amino acid sequence highly homologous to the RecQ5β of the present invention. The DNA helicase activity of proteins modified with the amino acid sequence set forth in SEQ ID NO: 2 can be confirmed by known methods (N. Suzuki et al.,
al. , 1997, Nucleic Acids Res. 25:2973-2
978;M. D. Gray et al. , 1997, Nature Gene
17:100-103). Nuclear localization also appears to be a key factor in the development of Re2+ in eukaryotic cells.
This can be confirmed by expressing a gene encoding a RecQ5β homolog and observing its intracellular localization. In this case, fusing a marker protein such as EGFP to the gene encoding the RecQ5β homolog makes it easier to observe the localization state (T. Matsumoto et al., 2004).
al. , 1997 Nature Genet. 16:335-336;N. S
uzuki et al. , 1997, Nucleic Acids Res.
25:2973-2978). Alternatively, as shown in the Examples, an HA tag can be fused to RecQ5β, which can be detected immunochemically using an anti-HA antibody. Based on the disclosure of the present invention, partial peptides of RecQ5β (or its homologs) can be obtained. Partial peptides based on the present invention include antigen peptides for antibody preparation. In order for the amino acid sequence constituting the partial peptide to be specific to RecQ5β, the amino acid sequence from positions 441 to 99 in the amino acid sequence set forth in SEQ ID NO: 2 should be selected.
The amino acid sequence should be selected from an amino acid sequence corresponding to position 1 (C-terminus), or an amino acid sequence containing at least a portion of this amino acid sequence. Furthermore, it is desirable that the number of amino acids constituting the partial peptide be a contiguous amino acid sequence consisting of at least 7 amino acids, preferably 8 or more amino acids, and more preferably 9 or more amino acids. As described below, this region contains the amino acid sequence necessary for the expression of nuclear transport activity. For example, the inventors' analysis suggested that a region of 246 amino acids located on the C-terminus (positions 746 to 991 counting from the N-terminus) is necessary for the expression of nuclear transport activity. A partial peptide consisting of an amino acid sequence selected from the amino acid sequence constituting this region is particularly desirable as an immunogen for obtaining antibodies to RecQ5β. The partial peptide of the present invention can be produced, for example, by genetic engineering techniques, known peptide synthesis methods, or by cleaving the protein of the present invention with an appropriate peptidase. Additionally, the present invention relates to a polynucleotide encoding the protein of the present invention. In the present invention, a polynucleotide refers to a molecule composed of a large number of polymerized nucleotides. While the number of polymerized nucleotides is not particularly limited, a polynucleotide with a relatively low degree of polymerization may also be referred to as an oligonucleotide. The polynucleotides or oligonucleotides of the present invention may be naturally occurring or chemically synthesized.
The polynucleotide of the present invention may be synthesized by an enzymatic reaction such as CR. The polynucleotide of the present invention includes not only natural polynucleotides such as DNA and RNA, but also derivatives thereof. Examples of polynucleotide derivatives include structures that are nuclease-resistant due to the introduction of phosphorothioate bonds, and polynucleotides into which labeling molecules have been introduced. The polynucleotide of the present invention is not particularly limited in its form as long as it can encode the protein of the present invention, and includes cDNA, genomic DNA, chemically synthesized DNA, and the like. Furthermore, polynucleotides having any nucleotide sequence based on the degeneracy of the genetic code are included, so long as they can encode the protein of the present invention. As described above, the polynucleotide of the present invention can be synthesized by hybridization techniques using the full-length or a portion of the DNA nucleotide sequence (SEQ ID NO: 1) encoding human RecQ5β as a probe, or by PCR using primers synthesized based on these nucleotide sequences.
For example, human genomic DNA encoding RecQ5β can be obtained from the long arm of chromosome 17, 17q25.
NA can be obtained, for example, from a human sperm cDNA library. Furthermore, as already mentioned, it is possible to obtain homologs of RecQ5β in other species. Mutations can also be artificially introduced into DNA consisting of the base sequence shown in SEQ ID NO: 1. Techniques for artificially introducing mutations are known (for example, site-directed mutagenesis (Current Protocols in Molecules)).
lar Biology edit. Ausubcl et al. (1987
) Publish. Jhon Wily & Sons Section 8.
1-8.5) Furthermore, kits for introducing point mutations therein are commercially available (for example, Takara Shuzo's TAKARA LA PCR in vitro Mutag).
The DNA encoding RecQ5β (or a homolog thereof) according to the present invention is
RecQ5β (or its homologue) can be expressed by incorporating it into an appropriate vector.
The vector can be used as an expression vector. Furthermore, by transforming a host capable of expression with this vector, a transformant according to the present invention can be obtained. The vector DNA into which the DNA fragment is inserted is not particularly limited as long as it can be replicated in the host cell, and examples thereof include plasmid DNA, phage DNA, etc. Examples of the plasmid DNA include the plasmid pUC118 (manufactured by Takara Shuzo Co., Ltd.)
, pBluescript SK ⁺ (Stratagene), pGEM-
T (Promega), and examples of phage DNA include M1
When a plasmid DNA is used as the vector DNA, for example, EcoRI
When inserting the DNA fragment, the plasmid DNA is digested with the restriction enzyme EcoRI (NEB). The DNA fragment and the digested vector DNA are then mixed and treated with, for example, T4 DNA ligase (Takara Shuzo) to obtain a recombinant vector. The host is not particularly limited as long as it can express the target gene.
Both eukaryotic and prokaryotic cells can be used.
Examples of prokaryotic cells include animal cells such as yeast, COS cells, and CHO cells, and insect cells. On the other hand, examples of prokaryotic cells include bacteria such as Escherichia coli and Bacillus subtilis. When bacteria such as Escherichia coli are used as hosts, it is preferable that the recombinant vector of the present invention is capable of autonomous replication in the host and is configured to include a promoter, the gene of the present invention, and a transcription termination sequence. Examples of Escherichia coli include XL1-Blue (Stratagene), JM
109 (manufactured by Takara Shuzo Co., Ltd.), and the expression vector is, for example, pBTrp
2, etc. In addition, any promoter may be used as long as it can be expressed in a host such as E. coli. For example, promoters derived from E. coli or phages, such as trp promoter, lac promoter, PL promoter, and PR promoter, are used. In the present invention, transformation can be carried out, for example, by the Hanahan method [Technique
s for Transformation of E. coli In DN
A Cloning, Vol. 1, Glover, D. M. (ed.), pp.
109-136, IRL Press (1985)]. When yeast is used as a host, the expression vector can be, for example, Yep13, Y
Examples of promoters include gal 1 promoter and gal 10 promoter. Methods for introducing recombinant vectors into yeast include electroporation [Methods. Enzyme
mol., 194, pp. 182-187 (1990)], spheroplast method [
Proc. Natl. Acad. Sci. USA, 84, pp1929-193
3 (1978)], lithium acetate method [J. Bacteriol., 153, 16
3-168 (1983)]. When animal cells are used as hosts, the expression vector may be, for example, pcDNAI.
, pcDNAI/Amp (Invitrogen), etc. are used. Methods for introducing recombinant vectors into animal cells include, for example, electroporation, lipofection, calcium phosphate precipitation, etc. When insect cells are used as hosts, the expression vector is, for example, pVL139.
2, pVL1393 (both from Invitrogen), pMBac (Str
pBacPAK8/9 (Clontech), etc. are used. Methods for introducing recombinant vectors into insect cells include, for example, the calcium phosphate method, lipofection, and electroporation. Screening of the above-mentioned transformants can be carried out by colony hybridization using a DNA fragment containing a part of the target gene as a probe, or by synthesizing a 5' primer (FP: forward primer) based on the base sequence of the target gene and then using a 3' primer (RP: rev primer) based on the base sequence of the complementary strand DNA.
Colonies (transformants) containing the gene of interest can be selected and collected by PCR using these primers. Furthermore, by culturing the transformant according to the present invention, the RecQ5 (
The culture method is not particularly limited, but by employing a liquid culture method, operations such as induction of expression, separation of cells, and isolation of the expression product can be easily performed. The method for culturing the transformant of the present invention in a medium is carried out according to a conventional method used for culturing a host. Media for culturing a transformant using a microorganism such as Escherichia coli or yeast as a host include:
Either a natural medium or a synthetic medium can be used. Specifically, there is no particular limitation as long as the medium contains carbon sources, nitrogen sources, inorganic salts, etc. that can be assimilated by the microorganism and allows the transformant to be cultured efficiently. The culture is usually carried out under aerobic conditions such as shaking culture or aeration and agitation culture for 35 to 40 minutes.
The incubation is carried out at about 37°C, preferably at about 37°C, for 14 to 20 hours.
The pH is adjusted to 7.4. During the culture, antibiotics such as ampicillin and tetracycline, vitamins, etc. can be added to the medium as needed. When culturing a microorganism transformed with an expression vector using an inducible promoter, an inducer can be added to the medium as needed. For example, when culturing a microorganism transformed with an expression vector using a Lac promoter, isopropyl-β-D-thiogalactopyranoside (IPTG) or the like can be added to the medium.
When culturing a microorganism transformed with an expression vector using the rp promoter, indoleacrylic acid (IAA) or the like may be added to the culture medium. When culturing a transformant using an animal cell as the host, commonly used RPMI-1640 medium, DMEM medium, or a medium supplemented with fetal bovine serum or the like to these media is used. Culture is usually carried out in the presence of 5% _CO2 at 37°C.
During the culture, antibiotics such as kanamycin and penicillin may be added to the medium as needed.
e's Insect Medium [Grace, T. C. C. Naturc
, 195:788, (1962)] to which additives such as 10% fetal bovine serum are appropriately added. The pH of the medium is adjusted to 6.6 to 7.0, and the medium is usually kept at 25°C.
The culture is performed for 1 to 7 days at RT, with aeration and stirring as necessary. After the culture is completed, the protein of the present invention can be collected from the culture (cell lysate, culture broth, or their supernatant) using conventional protein purification methods. If the protein of the present invention is produced intracellularly or intracellularly after the culture, the cells are disrupted by lysis using an enzyme such as lysozyme, ultrasonic disruption, or trituration, and the protein of the present invention is excreted outside the cells. Next, insoluble matter is removed by filtration or centrifugation to obtain a crude protein solution. On the other hand, if the protein of the present invention is produced extracellularly or extracellularly, the culture broth is used as is, or the cells or cells are removed by centrifugation or the like to obtain a supernatant. The protein of the present invention can then be isolated and purified from the culture by common biochemical methods used for protein isolation and purification, such as ammonium sulfate precipitation, ion exchange chromatography, hydrophobic chromatography, gel filtration chromatography, affinity chromatography, electrophoresis, etc., either alone or in combination. Alternatively, if the RecQ5β protein of the present invention is expressed as a fusion protein with a binding ligand, it can be combined with the receptor for this binding ligand and purified by affinity chromatography. For example, a method is known in which a fusion protein with a histidine tag consisting of a series of histidine residues is purified using a nickel column. Histidine-tag-containing vectors for this purpose are also commercially available. Fusing the binding ligand to RecQ5β via a linker that can be cleaved with a protease or the like is convenient because the expression product adsorbed by affinity chromatography can be easily recovered. DNA encoding the RecQ5β of the present invention can be used in gene therapy. The RecQ5β of the present invention has nuclear transport activity and can be described as an enzyme capable of expressing in vivo the chromosome stabilizing effect, which can be said to be the original function of DNA helicase. Therefore, inoculation of a vector incorporating the RecQ5β of the present invention in an expressible manner can be expected to be effective in treating chromosomal instability disorders caused by RecQ5 deficiency or mutation. Vectors that can be used to introduce the DNA encoding the RecQ5β of the present invention into patients include viral vectors derived from retroviruses or adenoviruses, and non-viral vectors using liposomes, etc. The present invention also provides antibodies against the proteins of the present invention. The antibodies of the present invention are directed against RecQ5β homologs including RecQ5β, or the aforementioned RecQ5β
Using a partial peptide of the above as an immunogen, monoclonal or polyclonal antibodies can be obtained according to known methods. Monoclonal antibodies according to the present invention can be obtained, for example, as follows. (1) Antigen Preparation: A RecQ5β homolog containing RecQ5β, or a partial peptide thereof, is dissolved in a buffer solution and then mixed with an adjuvant to form an immunogen. Examples of adjuvants include commercially available Freund's complete adjuvant and Freund's incomplete adjuvant, and any of these may be mixed. (2) Immunization and Collection of Antibody-Producing Cells: The immunogen is administered to mammals, such as rats and mice. An immunizing dose of 10 to 500 μg of antigen per animal is used per administration. The immunization site is typically intravenous, subcutaneous, or intraperitoneal. The immunization interval is not particularly limited; immunizations are carried out 2 to 5 times, preferably 3 to 4 times, at intervals of several days to several weeks, preferably 1 to 3 weeks. Two to seven days, preferably four to five days, after the final immunization, antibody-producing cells are collected after confirming a sufficient increase in antibody titer. Antibody-producing cells include B cells derived from spleen cells, lymph node cells, peripheral blood cells, etc., with B cells derived from spleen cells or local lymph node cells being preferred. (3) Cell Fusion: Commonly available established cell lines from animals such as mice are used as myeloma cells to be fused with antibody-producing cells. The cell lines used should have drug selectivity and
Myeloma cells are preferably those that cannot survive in a selective medium (HAT medium: containing hypoxanthine, aminopterin, and thymine) in the unfused state, but can survive only when fused with antibody-producing cells. Specific examples of myeloma cells include mouse myeloma cell lines such as P3U-1 (manufactured by Dainippon Pharmaceutical Co., Ltd.) and P3x63Ag8.653. Next, the myeloma cells and antibody-producing cells are fused. Cell fusion is carried out by mixing equal volumes of 10 ⁸ cells/ml of antibody-producing cells and 2 x 10 ⁵ cells/ml of myeloma cells in an animal cell culture medium such as serum-free DMEM or RPMI-1640 medium, and carrying out the fusion reaction in the presence of a fusion promoter. Polyethylene glycol with an average molecular weight of 1,500 daltons or the like can be used to promote cell fusion. Electrical stimulation (e.g., electroporation) can also be used.
Antibody-producing cells and myeloma cells can also be fused using a commercially available cell fusion device that utilizes a hybridoma fusion system. (4) Hybridoma Selection and Cloning The desired hybridoma is selected from the cells after the cell fusion treatment. To do this, the cell suspension is appropriately diluted with, for example, RPMI-1640 medium containing fetal bovine serum, and then seeded on a microtiter plate at 5 to 10 cells per well. A selective medium is added to each well, and the selective medium is then replaced as appropriate for subsequent cultivation. As a result,
After the start of culturing in the selective medium, cells that grow from about 14 days can be obtained as hybridomas. The culture supernatant of the grown hybridomas is screened for the presence of the target antibody. Hybridoma screening can be performed according to conventional methods and is not particularly limited. For example, a portion of the culture supernatant contained in the well in which the hybridomas have grown is collected and the antibody is screened by ELISA, RIA, or the like. The fused cells that have been confirmed to produce the target antibody are cloned by limiting dilution or the like, and finally, hybridomas, which are monoclonal antibody-producing cells, are established. (5) Collection of Monoclonal Antibodies Conventional cell culture methods or ascites formation methods can be used to collect monoclonal antibodies from the established hybridomas. In the cell culture method, the hybridomas are cultured in RPMI-containing 10% fetal bovine serum.
The hybridoma cells are cultured in an animal cell culture medium such as 1640 medium, MEM medium, or serum-free medium under normal culture conditions (e.g., 37°C, 5% _CO2 concentration) for 10 to 14 days, and the antibody can be obtained from the culture supernatant. In the case of ascites formation, approximately 5 x ¹⁰⁶ hybridoma cells are administered into the peritoneal cavity of an animal of the same species as the mammal from which the myeloma cells were derived,
The hybridomas are grown in large quantities. Then, after 1 to 2 weeks, ascites fluid is collected. In the above antibody collection method, if antibody purification is required, it can be performed by appropriately selecting known methods such as ammonium sulfate precipitation, ion exchange chromatography, affinity chromatography, gel chromatography, or a combination of these. The antibody of the present invention can also be obtained as a polyclonal antibody, for example, based on the following method: (1) Antigen Preparation: An immunogen similar to that for monoclonal antibodies is prepared. (2) Immunization: Rabbits, guinea pigs, goats, sheep, etc. are usually used as animals. For example, in the case of rabbits, 100 μg to 500 μg of polypeptide is usually injected subcutaneously into the footpad of the rabbit together with Freund's complete adjuvant. Two weeks later, the same amount of antigen mixed with Freund's incomplete adjuvant is injected intramuscularly. Two weeks later, the intramuscular injection is repeated, and one week after the final immunization, partial blood is collected from the ear and E.
Measure the antibody titer using the IA method, etc. If the antibody titer reaches the target value, take a whole blood sample.
If the antibody titer is low, intramuscular injections are repeated, and immunization is repeated until the antibody titer reaches the desired value. Purification of antibodies from serum by ammonium sulfate fractionation can be achieved using the method described in the monoclonal antibody section. When the entire RecQ5β molecule is used as the immunogen, an antibody specific to RecQ5β can be obtained by further absorption using a region common to other RecQ5-type DNA helicases, including the helicase domain. Mutations in known human RecQ-type DNA helicase genes often cause chromosomal instability, leading to a high incidence of cancer and premature aging. These genes are naturally expressed in a variety of human tissues. Therefore,
The human RecQ gene can be said to be one of the genes encoding DNA helicases involved in maintaining basic homeostasis in the body. Meanwhile, the RecQ5β of the present invention is a RecQ5-type DNA helicase with nuclear localization activity, where DNA helicases should express their original functions. Therefore, elucidating the regulation of human RecQ5β expression is useful for elucidating the mechanisms governing homeostasis in the body, as well as for creating new pharmaceuticals for maintaining basic homeostasis in the body and for research into its relationship with aging. Furthermore, various reagents based on the present invention for detecting RecQ5β protein or the gene encoding it are useful for detecting and diagnosing diseases caused by abnormalities in genes encoding proteins with helicase activity. The present invention relates to a polynucleotide comprising a nucleotide sequence corresponding to 1390 to 3703 in the nucleotide sequence set forth in SEQ ID NO: 1, or a DNA comprising a nucleotide sequence of at least 15 nucleotides complementary to the complementary strand thereof. In the present invention, "complementary" refers not only to a polynucleotide that is completely complementary to a target nucleotide sequence, but also to a polynucleotide that is completely complementary to the target nucleotide sequence, i.e., to a polynucleotide that is completely complementary to the target nucleotide sequence but also to a polynucleotide that is completely complementary to the target nucleotide sequence.
The base sequence of the target DNA is at least 80%, preferably 90% or more, more preferably 95% or more identical to the completely complementary base sequence. The base sequence of the target DNA is at least 80%, preferably 90% or more, more preferably 95% or more identical to the completely complementary base sequence. The base sequence of the target DNA is at least 80%, preferably 90% or more, more preferably 95% or more identical to the completely complementary base sequence.
The DNA of the present invention is useful as a probe for detecting a target base sequence or as a primer for synthesis. Hybridization can be performed using the DNA of the present invention as an oligonucleotide probe to detect RNA or DNA by Southern or Northern blotting. Examples of oligonucleotide probes include DNA probes and RNA probes. When designing an oligonucleotide, any region corresponding to positions 1390 to 3703 of the cDNA encoding RecQ5β is selected. The open reading frame region is particularly preferred.
To design the base sequence of a probe specific to ecQ5β, not only a base sequence selected from the base sequence corresponding to 1390 to 3703 in SEQ ID NO: 1 can be selected, but also a region containing a portion contiguous to 1390 to 3703 can be selected.
Even if the probe recognizes the region including nucleotides 390 to 3703, a RecQ5β-specific nucleotide sequence can be selected. The length of the oligonucleotide used as the probe is not particularly limited, as long as it is at least 15 bases long and can specifically hybridize with the target nucleotide sequence under stringent conditions. Gene amplification methods such as PCR can also be used to detect the gene.
The primers required for CR can be designed based on the base sequence set forth in SEQ ID NO: 1. The segment to be amplified by PCR is designed to include the base sequence corresponding to 1390 to 3703 in the base sequence set forth in SEQ ID NO: 1, similar to the target base sequence of the probe. DNA encoding a region corresponding to approximately 580 amino acid residues on the C-terminus that is not found in RecQ5α but is found specifically in RecQ5β of the present invention is important as an amplified segment for PCR. Specifically, the portion encoding this region is used as the reverse primer, and the forward primer is used as the R
The combination of selecting from regions common to RecQα and RecQ5 of the present invention is
The length of the primers used in PCR is not particularly limited as long as they can specifically hybridize with the target base sequence under stringent conditions. Generally, oligonucleotides having a chain length of 15 bp to 100 bp, preferably 15 bp to 35 bp, are used as primers. Hybridization assays and PCR are performed on D-type DNA fragments such as human tissues and blood cells.
This method can be applied to any sample that may contain DNA or RNA. Methods for extracting DNA or RNA from these samples are known.
When amplifying RNA such as RT-PCR, cDNA is synthesized in advance using reverse transcriptase and PCR is performed using this as a template. Alternatively, hybridization assays or PCR (RT-PCR) can be performed in fixed tissues (in situ).
It is also possible to carry out hybridization assays (including PCR). When detecting the presence of a target nucleotide sequence by hybridization assay or PCR, many methods are known in which probes or primers are labeled and signals are obtained by various detection methods. All of these known assay formats can be applied to the present invention. Furthermore, the antibodies against RecQ5β or RecQ5β homologs of the present invention are useful for analyzing expression levels at the protein level and detecting mutations. For example,
The expression level of RecQ5β in tissues can be determined by Western blotting. Alternatively, the intracellular localization of RecQ5β can be elucidated by immunochemical staining. Because nuclear localization activity is an important characteristic of the RecQ5β of the present invention or its homologs, reagents that can reveal intracellular localization are of great importance. Furthermore, the protein of the present invention contained in body fluids can be detected by ELISA.
The amount of RecQ5β can be quantified by known immunoassay techniques such as SA and RIA. The present invention also relates to transgenic animals into which a gene modified to increase or decrease the expression level of RecQ5β or its homolog gene has been introduced. The transgenic animals of the present invention can be analyzed for their phenotypes to determine whether they are RcQ5β or its homolog gene.
It provides information necessary for functional analysis of ecQ5β. Furthermore, knockout animals lacking the function of RecQ5β or its homolog genes are useful as model animals for chromosomal instability disorders caused by RecQ5β mutations. By introducing mutations such as deletions, substitutions, additions, and insertions into some of the key sites on the genome that regulate gene expression, it is possible to modify the expression level of the RecQ5 (or its homolog) gene so that it is artificially increased or decreased compared to the original level. Sites that affect gene expression include enhancers, promoters, introns, etc. As vectors carrying the mutated gene, two types of vectors are considered: vectors that overexpress the RecQ5-type gene of each animal species, and, conversely, antisense vectors that suppress its expression. Examples of these vectors include pcDNA, pRC/RSV (Invitrogen), and others.
In either case, a drug resistance gene (e.g., neomycin) for positive selection is linked to select the gene. Based on the present invention, antisense DNA can be designed against DNA of RecQ5β or a RecQ5β homolog, or a part thereof.
Antisense DNA consisting of a 5-bp antisense sequence and targeting a predicted hairpin loop region on mRNA can be expected to have an antisense effect. Methods for introducing genes into cells are known. For example, vector DNA can be directly introduced into cells.
Microinjection, in which A is injected, or electroporation can be used. Fertilized eggs or ES cells are used as cells for introducing vector DNA. Among them, ES cells have the advantage that they can be cultured outside the body and can generate complete individuals from these cells, so methods using various ES cells are more efficient and preferable. An example of an ES cell is TT2 cells (Aizawa Shinichi, Gene Targeting, 1995, Yodosha). The above vector DNA containing the mouse RecQ5 gene is introduced into ES cells by electroporation, and positive selection is performed with neomycin to produce the desired mutant ES cells.
Cells are obtained. The ES cells are injected into blastocysts or 8-cell embryos using a capillary tube or other device. The blastocysts or 8-cell embryos are then directly transplanted into the oviducts of the surrogate mother, or cultured for one day and developed into blastocysts, which are then transplanted into the uterine tracts of the surrogate mother. Chimeric animals are selected from the offspring born to the surrogate mother. Animals with a high chimeric contribution rate are likely to be germline chimeric animals, but by mating chimeric animals with normal animals, it is possible to confirm that they are germline chimeric animals. Heterozygotes can be obtained by mating germline chimeric animals with normal animals, and homozygotes can be obtained by mating heterozygotes with each other. Based on the present invention, knockout animals in which the function of the RecQ5β (or its homolog) gene has been lost can also be created. The creation of knockout mice is described below using mice as an example. First, genomic DNA containing the mouse RecQ5 gene is obtained, and a vector is constructed in which a neo resistance gene is inserted into one of its exons. Genomic DNA can be obtained by PCR from genomic DNA prepared from mouse ES cells or from a genomic library. This procedure disrupts the function of this exon. At the same time, the vector contains a thymidine kinase (tk) gene or diphtheria (DT) gene for negative selection.
The toxin gene is then ligated into the vector DNA by electroporation.
The cells are then transfected with neomycin for positive selection and the nucleic acid analog FIAU (fluoroiodoadenosyl) for negative selection.
The cells are cultured in the presence of uracil, or diphtheria toxin. This procedure eliminates diphtheria toxin-sensitive cells that have undergone non-homologous recombination, and G418-sensitive cells that have not undergone recombination at all, leaving only cells that have undergone homologous recombination to survive.
In cells where this homologous recombination has occurred, the gene containing the disrupted exon is knocked out, and the resulting cells are then injected into mouse blastocysts or eight-cell embryos.
Thereafter, knockout mice can be produced using the same techniques as for producing transgenic animals. BEST MODE FOR CARRYING OUT THE INVENTION The present invention will be specifically explained below using examples, but the present invention is not limited to these examples. [Example 1] Isolation of RecQ5 isoform cDNA (preparation of library filter) 1 x ¹⁰⁶ clones from a human testis-derived cDNA library (Takara Shuzo) constructed based on the pAP3-neo vector were spread on an LB agar plate (containing 50 μg/ml
Nitrocellulose filter (HA) attached to ampicillin
The E. coli colonies on the filter were transferred to a nylon filter (Biodyne A, Palo Alto Networks, Calif.) and cultured at 37°C for approximately 10 hours.
The original nitrocellulose filter and the replica nylon filter were then cultured on an LB agar plate at 37°C for 3 hours. The original nitrocellulose filter was stored at 4°C, and the replica nylon filter was cultured on an LB agar plate (containing 50 µg/ml ampicillin, 170
The nylon filter was cultured on 0.5 μg/ml chloramphenicol at 37° C. for 12 hours. The nylon filter was treated with a denaturing solution (0.5 N NaOH, 1.5 M NaCl) for 5 minutes, and then neutralized with a neutralizing solution (0.5 M Tris-HCl (pH 7.4), 1.5 M
After neutralization with NaCl, the filters were immersed in 2xSSC, air-dried, and then heat-treated at 80°C for 2 hours.
The mixture was shaken at 50°C for 20 minutes in 1 mM EDTA and air-dried. (First screening) Human RecQ5 cDNA fragment (1.3 kb, S. Kitao et al.,
, 1998, Genomics, 54, pp. 443-452)
The probe was labeled with [α- ³² P]-dCTP using Primer DNA Labeling Kit Ver. 2 (Takara Shuzo).
The library filter that had been prehybridized for 3 hours was
The probes were hybridized in a hybridization solution at 65°C for 20 hours.
30 min, 0.5 x SSC/0.1% SDS solution at 65°C, 30 min, 0.1
1x SSC/0.1% SDS solution at 65°C for 30 minutes, and then 0.1x S
The colonies on the original nitrocellulose filter corresponding to the positive signals on the developed X-ray film were suspended in LB medium and plated on an LB agar plate (
Nitrocellulose coated on a substrate containing 50 μg/ml ampicillin
The filter was cultured at 37°C for approximately 10 hours. The E. coli colonies on the filter were transferred to a nylon filter, and the original and replica filters were cultured on an LB agar plate at 37°C for 3 hours. The original nitrocellulose filter was stored at 4°C, and the replica nylon filter was cultured on an LB agar plate (
Contains 50μg/ml ampicillin, 170μg/ml chloram
The nylon filter was treated with a denaturing solution (0.5 N NaOH, 1.5 M NaCl) for 5 minutes, and then neutralized with a neutralizing solution (0.
After neutralizing with 5 M Tris-Cl (pH 7.4), 1.5 M NaCl,
These filters were immersed in a pre-washing solution (3 x SSC, 0.5% SDS, 1 mM EDTA) for 5 min, then air-dried, and then heat-treated at 80°C for 2 hours.
The mixture was shaken at 0°C for 20 minutes and then air-dried.
Hybridization was carried out using the Q5 cDNA fragment labeled with [α- ³² P]-dCTP as a probe. (Determination of base sequence) An independent E. coli colony was selected from the obtained positive signal, cultured in 3 ml of LB medium (containing 100 μg/ml ampicillin), and then subjected to alkaline-SDS
Plasmid DNA was prepared by the method to obtain 100 μl of DNA solution.
Using the DNA as a template, the 5' end of the resulting clone was amplified using primers for the T7 and T3 sequences located upstream and downstream of the cloning site of the pAP3-neo vector.
The nucleotide sequences of the nucleotides at the 5' and 3' ends were determined. The nucleotide sequence determination was performed using PCR cycle sequencing. Specifically, PCR was performed in a reaction system containing unlabeled primers, four types of fluorescently labeled nucleotide 5'-triphosphates, and Taq polymerase. The composition of the reaction mixture was as follows: Dye Terminator Cycle Sequencing Ready Reaction (Applied Biosystems) 8.0 μl Template DNA 3.0 μl Primer (3.2 pmol/μl) 1.0 μl DMSO 1.0 μl dH ₂ O 7.0 μl Total volume 20 μl PCR was performed at 96°C for 30 seconds (denaturation), 55°C for 15 seconds (annealing), and 60°C for 10 seconds (annealing).
This reaction was repeated 25 times, with one cycle consisting of a 4-minute (extension) reaction at 0°C. This reaction randomly synthesized DNA fragments containing fluorescent dyes, which could then be analyzed using a sequencer to ultimately determine the continuous base sequence.
The PCR reaction products were analyzed using an automated DNA analyzer manufactured by Applied Biosystems.
The screening was performed using a sequencer (model ABI 373). Five independent clones were isolated through the above screening procedures. These five clones obtained from the 5' and 3' base sequences contained nearly full-length RecQ5α, β, and γ genes, including the entire open reading frame (ORF).
One clone each of the cDNAs and one clone each of the partial sequence cDNAs of RecQ5β and γ were obtained. The nucleotide sequences of the primers used to determine the nucleotide sequences of the full-length RecQ5α, β, and γ cDNAs are as follows. The sequence names are followed by the sequence numbers in parentheses. Example 2 Determination of the Genomic Structure of the Human RecQ5 Gene (Isolation of P1 Phage Clones of Human Genomic DNA Containing the RecQ5 Gene) P1 DNA clones #15033 and #21570 were isolated by PCR-based screening at Genomc Systems Inc.
P1#15033 was screened using primers 611 and 612, and P1#21570 was screened using primers 613 and 614. The nucleotide sequences of each primer are as follows. The sequence names are followed by the sequence numbers in parentheses. (Preparation of P1 DNA) 120 ml of E. coli harboring the P1 clone containing the human RecQ5 gene was
The cells were cultured in LB medium (containing 100 μg/ml ampicillin) for 12 hours, and 0.5 mM IPTG was added and cultured for another 5 hours. Plasmid DNA was then prepared by the alkaline-SDS method to obtain 800 μl of DNA solution.
To determine the exon/intron boundary sequences of the gene, the above-mentioned two P1 D
The nucleotide sequence was determined using the DNA as a template with primers corresponding to the RecQ5 cDNA sequence. The cycle sequencing method was used for nucleotide sequencing as described above. The composition of the reaction mixture was as follows: Dye Terminator Cycle Sequencing Ready Reaction (Applied Biosystems) 8.0 μl Template DNA 8.0 μl Primer (3.2 pmol/μl) 1.0 μl DMSO 1.0 μl _dH2O 2.0 μl Total volume 20 μl The nucleotide sequences of the primers used for nucleotide sequencing are as follows: The sequence name is followed by the sequence number in parentheses. The obtained nucleotide sequence was compared with the nucleotide sequences of RecQ5α, β, and γ cDNA to determine the exon/intron boundary sequences. The results are shown in Table 1. The exon/intron structure of the human RecQ5 gene is shown in Figure 1. These results revealed that the human RccQ5 gene is composed of 19 exons and 18 introns, and that the RecQ5α, β, and γ isoforms are generated by selective use of these exons through splicing. RecQ5α
The different exons used for the β and γ isoforms are shown in Figure 2, and the structures of the proteins they encode are shown in Figure 3. Example 3 Expression Analysis of Human RecQ5 Gene β Isoform mRNA (Probe Preparation) A RecQ5β-specific cDNA fragment of approximately 1.7 kb was prepared by digesting the 6HA-RecQ5β plasmid (described below) with the restriction enzymes EcoRV and XhoI, and 30 ng of this cDNA fragment was labeled with [α- ³² P]dCTP (NEN).
Ver. 2 (Takara Shuzo) was used and labeling was carried out according to the attached protocol.
A Multiple Tissue Northern Blot membrane (Clontech) on which 2 μg of poly A ⁺ RNA from various human tissues treated with 5× SSPE solution was blotted was placed in 10 ml of hybridization solution (500 μg).
x SSPE, 10 x Denhardt's, 50% Formamide, 2%
The DNA was immersed in a 100 μg/ml salmon sperm DNA solution (SDS, 100 μg/ml salmon sperm DNA) and prehybridized at ⁴² ° C. for 2 hours.
Hybridization was carried out in 5 ml of fresh hybridization solution containing an ecQ5β-specific probe at 42°C for 20 hours.
15 minutes in 2 x SSC/0.1% SDS solution at room temperature, 30 minutes in 2 x SSC/0.1% SDS solution at 65°C, 15 minutes in 0.2 x SSC/0.1% SDS solution at 65°C.
The fragments were incubated at 4°C for 15 minutes, and then washed in 0.2x SSC/0.1% SDS solution at 65°C for 15 minutes, and then analyzed using a BAS1500 system (Fujifilm). The results showed that mRNA for the β isoform of the human RecQ5 gene was expressed in all 16 types of tissues examined, with significantly higher expression observed in the testis (Figure 4). [Example 4] Construction of an expression vector for human RecQ5 isoforms in mammalian cells (construction of an expression vector carrying an influenza hemagglutinin (HA) epitope) The NotI-
A synthetic oligo DNA (5'-GCGGCCGCCACCATGGCCTACCCATAC) encoding the HA epitope with a translation initiation ATG codon at the XhoI site
A synthetic oligo DNA encoding the HA epitope (5'-GCTAGTCGGATTACGCTAGCCTCCTCGAG-3'/SEQ ID NO: 57) was further inserted into the NheI site present on the 3' side of the HA epitope.
TCTACCCATACGATGTTCCGGATTACGCTAGC-3'/
By repeatedly incorporating the amino acid sequence (MAYPYDVPDYASLYPYDVPDYASLYPY) of the HA epitope, six repeats of the amino acid sequence (MAYPYDVPDYASLYPYDVPDYASLYPY) of the HA epitope are obtained.
DVPDYASLYPYDVPDYASLYPYDVPDYASLYPYDVP
Expression plasmid pcDNA-6HA encoding DYASL (SEQ ID NO: 67)
(Modification of the 5' Proximal Region of the Translation Initiation ATG Codon of the RecQ5 Isoform) A RecQ5 isoform containing the full-length ORF cloned into the pAP3-neo vector was constructed.
Q5α, β, and γ cDNAs were prepared by digestion with the restriction enzymes EcoRI and NotI and subcloned into the EcoRI-NotI site of the plasmid vector pBluescriptII KS+. The 5′-EcoRI-NheI-3′ site was located adjacent to the 5′ side of the translation initiation ATG codon common to the three isoforms.
A sense primer (503: 5'-ATA
GAATTCGCTAGCATGAGCAGCCACCATACCACCTT
CC-3'/SEQ ID NO: 59) and 27 common to the three isoforms
Antisense primer downstream of NcoI at position 3 (Q6V: 5′-GTCCAC
TTGGTCCTGAGTCAAAGC-3'/SEQ ID NO: 60) was used for PCR
The amplified DNA fragments were digested with restriction enzymes EcoRI and NcoI, and the resulting common cDNA fragment was inserted into the plasmid vector pBluescriptI.
The three isoforms EcoRI-Nco subcloned into IKS+
By the above manipulation, all ORs of these three isoforms were obtained.
F can be excised with the restriction enzymes NheI and XhoI. (Construction of an expression vector for a RecQ5 isoform having an HA epitope) The vector was subcloned into pBluescript II KS+, and the translation initiation AT
The cDNAs of the three isoforms with altered 5' ends of the G codon were cloned using NheI and X
The fragments were excised with hoI and subcloned into the NheI-XhoI sites of the above-mentioned pcDNA-6HA to obtain 6HA-RecQ5α, 6HA-RecQ5β, and 6HA-RecQ5β.
HA-RecQ5γ was constructed (Fig. 5a).
The ecQ5 isoform contains a six-repeated HA epitope at the N-terminus.
The in-frame fusion protein is expressed in mammalian cells and is detectable with an anti-HA antibody. The β isoform also contains an endogenous NheI sequence at position 2396, allowing for the 6H
A-RecQ5β was completely digested with the restriction enzyme NheI and ligated to construct 6HA-RecQ5βc, which removes approximately 2.4 kb between the NheI site located adjacent to the 5' side of the translation initiation ATG codon and the internal NheI site (Fig. 5a). This expression vector contains the C-terminal 246 residues of RecQ5β, with six repeated HA epitopes fused in frame to the N-terminus.
The amino acids (746-991) are expressed. [Example 5] Expression of HA epitope-human RecQ5 isoform fusion protein in mammalian cells (gene transfection) Human fetal kidney-derived 293EBNA cells (Invitrogen) were cultured in Doulbecco's modified Eagle's medium containing 10% fetal bovine serum.
The cells were cultured in DMEM at 37°C. When the cells reached confluence, they were washed with phosphate buffered saline (PBS) and treated with 0.25% trypsin to detach them from the culture dish, and then passaged at 4-fold dilutions. Plasmid D of the RecQ5 isoform expression vector and the control vector pcDNA-6HA (described above) were used.
5 μg of NA was mixed with 0.2 ml of OPTI-MEM culture medium (Gibco BRL), and 18 μl of Lipofectamine was mixed with 0.2 ml of OPTI-MEM culture medium.
The mixture was further mixed with the culture medium and allowed to stand at room temperature for 45 minutes.
The cells were washed with TI-MEM culture medium, left to stand for 45 minutes, and then 1.6 ml of OPTI-MEM culture medium was added to the DNA-Lipofectamine solution, which was then incubated at 37°C for 5 hours. An equal volume of DMEM culture medium containing 20% fetal bovine serum was added, and the cells were then incubated for another 3 hours.
The cells were cultured at 7°C for 19 hours, and then the medium was replaced with 5 ml of DMEM culture medium containing 10% fetal bovine serum, followed by further culture for 24 hours. (Preparation of cell extract) The transfected cells were recovered by trypsin treatment, washed with PBS, and then extracted with 300 μl of elution solution [50 mM Tris-Cl (pH 8.0), 150 mM NaCl, 10 ...
The cells were suspended in a 0.5 mM NaCl, 0.5% NP-40, and 1 mM PMSF solution. After standing on ice for 30 minutes, the cells were centrifuged at 15,000 rpm in a microcentrifuge for 30 minutes, and the supernatant was used as the cell extract. (Western blotting) 10 μl of the cell extract was mixed with SDS/sample buffer and heated at 98°C for 5 minutes, after which SDS-PAGE was performed using a 7.5% acrylamide gel.
The proteins separated in the gel were transferred onto a PVDF membrane (Immobilon, M
The primary antibody was transferred to a PBS containing 0.05% Tween 20 (
Rabbit anti-influenza hemagglutinin (HA) antibody (Santa Cruz Biotechnology, Illinois) diluted to 2.0 μg/ml in PBS-T.
The PVDF membrane was gently shaken in PBS (E1000) at room temperature for 1 hour and then washed four times with PBS-T. The PVDF membrane was gently shaken in anti-rabbit immunoglobulin antibody (Amersham) labeled with horseradish peroxidase (HRP) diluted 2000-fold with 5% skim milk/PBS as the secondary antibody at room temperature for 1 hour and then washed four times with PBS-T. After immersing the PVDF membrane in PBS,
The protein was detected using the CL system (Amersham). As a result, all proteins were found to have the expected size (RecQ5α: 410 aa/
46kD, RecQ5β: 991aa/110kD, RecQ5γ: 435aa
A band was detected at a position larger than the RecQ5βc: 246 aa/27 kD bands (Figure 5b). This difference is thought to be primarily due to the six-repeat HA epitope (68 aa/8 kD) fused to the N-terminus of each protein. [Example 6] Analysis of the intracellular localization of HA epitope/human RecQ5 isoform fusion proteins by immunofluorescence. Using the same method as described above, RecQ5 isoforms, their derivatives, and four expression plasmids were each transfected into 293EBNA cells and expressed. 48 hours after transfection, the 293EBNA cells were washed with PBS, detached by trypsinization, and suspended in 5 ml of DMEM culture medium containing 10% fetal bovine serum. ⁵ x 104 cells were collected, washed with PBS, and then attached to non-fluorescent glass slides (Matsunami, s8111) by cytospin. The sections were fixed with 10% formalin at room temperature for 10 minutes, washed with PBS-T, and then resuspended in 0.1% Trilon X-1.
00 in PBS for 5 minutes, and then in PBS containing 3% skim milk at room temperature for 1 minute.
Blocking was performed for 2 h with PBS containing 1.0% BSA as the primary antibody.
The cells were treated overnight at 4°C in rabbit anti-HA antibody (described above) diluted to 5 µg/ml.
After washing with PBS-T at room temperature for 10 minutes three times, 200 μg of secondary antibody was added.
After treatment with 10-fold diluted biotinylated anti-rabbit IgG (Biomeda) at room temperature for 1 hour, the cells were washed three times with PBS-T at room temperature for 10 minutes, and then further treated with 5 μg/
The cells were treated with 1 ml of streptavidin-FITC (Pharmingen) at room temperature for 1 hour. After washing with PBS-T at room temperature for 10 minutes three times, the cells were washed with PBS-T for 30 minutes.
μg/ml DAPI (4',6-diamidino-2-phenylin
Nuclear staining was performed with a 50% glycerol solution containing HA (RecQ5α). The intracellular localization of the HA fusion protein was examined using a fluorescence microscope (Nikon). The results are shown in Figure 6. In the figure, the left side of each panel shows the localization of each RecQ5 isoform, and the right side shows a DAPI-stained image of the nucleus. RecQ5α and RecQ5γ were localized in the cytoplasm, while RecQ5β was localized in the nucleus. The C-terminal 246 amino acids of RecQ5β (RecQβc) were also localized in the nucleus. From the above, it was confirmed that the three RecQ5α and RecQ5γ are localized in the cytoplasm, while the C-terminal 246 amino acids of RecQ5β (RecQβc) are localized in the nucleus.
Among the cQ5 isoforms, the small ones (RecQ5α and RecQ5γ) are localized in the cytoplasm, and the large one (RecQ5β) is localized in the nucleus.
It was thought that a nuclear localization signal was present within the C-terminal 246 amino acids of this gene. [Example 7] Analysis of the interaction between human type I topoisomerase and RecQ5β (construction of an expression vector for human type I topoisomerase in mammalian cells)
Flag epitope (MADYKD) with translation initiation ATG codon at the XhoI site
A synthetic oligo DNA (5′-GCG) encoding DDDKAS (SEQ ID NO: 68)
GCCGCCACCATGGCCGACTACAAGGACGACGACGAC
pcDNA-Flag was constructed by incorporating the following sequence: AAGGCTAGCCTCCTCTCGAG-3'/SEQ ID NO: 69) Topoisomerase 1 (GenBank ACCESSION J03250)
, topoisomerase 3α (GenBank ACCESSION U43431
) and topoisomerase 3β (GenBank ACCESSION AF01
cDNA containing the entire ORF of (7146) was subjected to RT-PCR using the following primers:
It was amplified and cloned by The template cDNA from HeLa cells was prepared as follows: Approximately 1 μg of Poly(A) ⁺ RNA derived from HeLa cells, dithiothreitol, dNTP (dATP
, dCTP, dGTP, and dTTP), a buffer for reverse transcriptase, and a 25 μL reaction solution containing reverse transcriptase SuperScript II (Gibco BRL).
The resulting mixture was incubated at 42°C for 30 minutes and then treated with RNase to prepare template cDNA. The PCR reaction mixture had the following composition: Template DNA 10 μl, Sense primer (20 pmol/μl) 1.0 μl, Antisense primer (20 pmol/μl) 1.0 μl, 10x cDNA PCR Reaction Buffer 5.0 μl.
(Clontech) 2.5mM dNTP 4.0μl Advantage cDNA Polymerase Mix 1.0μl
(Clontech) dH ₂ O 28 μl Total volume 50 μl PCR was performed by repeating one cycle of 95°C for 2 minutes (denaturation) and 72°C for 3 minutes (annealing and extension), followed by five cycles of 94°C for 30 seconds (denaturation) and 70°C for 3 minutes (annealing and extension), and then repeating five cycles of 94°C for 30 seconds (denaturation) and 70°C for 3 minutes (annealing and extension).
A cycle of denaturation at 100°C for 2 seconds and annealing and extension at 68°C for 3 minutes was performed 30 times, followed by a final 10-minute extension reaction.
The ends of the NA fragment were ligated with restriction enzymes XbaI and SalI, SpeI and XhoI, and Nhe
The pcDNA3-Flag vector was digested with NheI and XhoI.
The expression vector Flag-T was constructed by subcloning into the -XhoI site.
In the case of type I topoisomerases op1, Flag-Top3α, and Flag-Top3β, proteins in which the Flag epitope is fused in-frame to the N-terminus of these type I topoisomerases are expressed in mammalian cells and can be detected with an anti-Flag antibody. (Gene transfection and immunoprecipitation into 293EBNA cells) 10 μg of 6HA-RecQ5β plasmid DNA alone or Flag-T
op1, Flag-Top3α, or Flag-Top3β plasmid DNA
A mixture of 10 μg of Lipofectamine and 0.6 ml of OPTI-MEM culture medium and a mixture of 54 μl of Lipofectamine and 0.6 ml of OPTI-MEM culture medium were further mixed and left to stand at room temperature for 45 minutes.
293EBNA cells at 100% confluence were washed with 5 ml of OPTI-MEM culture medium, and the DNA-Lipofectamine solution was left to stand for 45 minutes. 4.8 ml of OPT-MEM was added to the washed cells.
I-MEM culture medium was added and incubated at 37°C for 5 hours.
% fetal bovine serum was added to the DMEM culture medium, and the mixture was further cultured at 37°C for 19 hours.
The medium was replaced with 10 ml of DMEM containing 10% fetal bovine serum, and the cells were further cultured for 24 hours. The transfected cells were recovered by trypsin treatment, washed with PBS, and then eluted with 1 ml of elution solution [50 mM Tris-Cl (pH 8.0), 150 mM
The cells were then suspended in a 1000 ml solution of 0.5% NaCl, 0.5% NP-40, and 1 mM PMSF.
After standing for 10 minutes, the mixture was centrifuged at 15,000 rpm in a microcentrifuge for 30 minutes, and the supernatant was used as a cell extract. 10 μl of this cell extract was mixed with SDS/sample buffer and heated at 98°C for 5 minutes. The remaining cell extract was mixed with agarose beads (M2 agarose, Eas) on which mouse anti-Flag monoclonal antibody (M2) was immobilized.
20 μl of tman Kodak (RecQ5β) was added and the mixture was gently shaken overnight at 4°C. The agarose beads were washed four times with 1 ml of elution solution. The agarose beads were suspended in 20 μl of elution solution containing 100 μg/ml Flag peptide and gently shaken at 4°C for 30 minutes. After centrifugation at 15,000 rpm for 10 minutes in a microcentrifuge, the immunoprecipitates eluted from the agarose beads by Flag peptide were mixed with SDS/sample buffer and heated at 98°C for 5 minutes. 10 μl of cell extract or immunoprecipitates were fractionated by SDS-PAGE using a 7.5% acrylamide gel, and Western blotting was performed as described above. RecQ5β was detected using 2.0 μg/ml rabbit anti-HA antibody as the primary antibody and a 2000-fold diluted HRP-labeled anti-rabbit immunoglobulin antibody as the secondary antibody. For the detection of type I topoisomerase, 5.0 μg/ml of mouse anti-Flag monoclonal antibody was used as the primary antibody, and 20 μg/ml of mouse anti-Flag monoclonal antibody was used as the secondary antibody.
The HRP-labeled anti-mouse immunoglobulin antibody (DAKO) diluted 1:100 was used.
Rec in 293EBNA cells transfected with plasmid DNA of each combination of Flag-Top1, Flag-Top3α, or Flag-Top3β
The expression of Q5β is shown in Fig. 7a, and the expression of Top1,
The expression of Top3α or Top3β is shown in Figure 7b. RecQ5β was expressed at the same level in 293EBNA cells transfected with each gene, and specific expression of type I topoisomerase was confirmed in each combination.
The predicted molecular weights of the op1, Top3α, and Top3β proteins were 91 kD, 112 kD, and 97 kD, respectively, which were almost identical to the band sizes detected by Western blotting using anti-Flag antibody. Extracts were prepared from 293EBNA cells transfected with each combination of expression vectors, and immunoprecipitation was performed using anti-Flag antibody agarose.
-RecQ5β and Flag-Top3α, or 6HA-RecQ5β and Flag
RecQ5β protein was detected in immunoprecipitates derived from extracts of cells co-expressing Flag-Top3β. This result suggests that RecQ5β protein interacts with topoisomerase 3α protein or topoisomerase 3β protein in the cell nucleus. [Example 8] Analysis of the nuclear localization of RecQ5β, topoisomerase 3α, and topoisomerase 3β (gene transfection and expression into 293EBNA cells) 5 μg of 6HA-RecQ5β plasmid DNA was co-expressed with Flag-Top3α.
Alternatively, 5 μg of Flag-Top3β plasmid DNA was added to 0.2 ml of OPTI
- 0.2 ml of MEM culture medium and 18 μl of Lipofectamine
The resulting mixture was mixed with OPTI-MEM medium and allowed to stand at room temperature for 45 minutes. 293EBNA cells at 60-70% confluence in a 60-mm culture dish were washed with 2.5 ml of OPTI-MEM medium, and a solution of the DNA-Lipofectamine solution, which had been allowed to stand for 45 minutes, plus 1.6 ml of OPTI-MEM medium was added and incubated at 37°C for 5 hours. An equal volume of DMEM medium containing 20% fetal bovine serum was added, and the cells were further cultured at 37°C for 19 hours. The medium was then replaced with 10 ml of DMEM medium containing 10% fetal bovine serum and further cultured for 24 hours. (Immunofluorescence) 48 hours after gene transfer, the 293EBNA cells were washed with PBS, detached by trypsinization, and suspended in 5 ml of DMEM medium containing 10% fetal bovine serum.
5 × 10 ⁴ cells were collected and washed with PBS, and then adsorbed onto a non-fluorescent slide glass by the cytospin method. They were fixed with 10% formalin at room temperature for 10 minutes, and then PBS was added.
After washing with PBS-T, the sections were treated with PBS containing 0.1% Triton X-100 for 5 minutes, and then blocked with PBS containing 3% skim milk at room temperature for 1 hour. The sections were treated overnight at 4°C with rabbit anti-HA antibody diluted to 2.5 μg/ml in PBS containing 1.0% BSA, and mouse anti-Flag antibody diluted to 10 μg/ml as primary antibodies. After washing three times with PBS-T at room temperature for 10 minutes,
After treatment with 2000-fold diluted biotin-labeled anti-rabbit IgG and 40-fold diluted FITC-labeled anti-mouse IgG (Zymed) as secondary antibodies at room temperature for 1 hour,
The cells were washed three times with PBS-T at room temperature for 10 minutes. Then, the cells were treated with 20-fold diluted avidin-labeled Texas Red (Oncor) at room temperature for 1 hour.
After washing three times for 10 minutes at room temperature with 1000 kJ/ml, the localization of both proteins was examined using a scanning laser microscope (Olympus, Fluoview). Similar to the nuclear-localized RecQ5β protein, the topoisomerase 3α protein was also localized in the nucleus, and the localization of both proteins within the nucleus was consistent (Fig. 8a, b). Furthermore, the topoisomerase 3β protein was also localized in the nucleus, and the localization of both proteins within the nucleus was consistent with that of the RecQ5β protein (Fig. 8c, d). These results, combined with the immunoprecipitation results, demonstrate that the RecQ5β protein interacts with the topoisomerase 3α protein or the topoisomerase 3β protein within the nucleus. INDUSTRIAL APPLICABILITY : The present invention provides a nuclear-localized RecQ5-type DNA helicase. D
Based on the knowledge that NA helicase supports chromosome stability, the nuclear localization feature is an important factor that allows for the analysis of the original function of DNA helicase. Even if we assume that, for example, 0.1% of the DNA that makes up the chromosomes in the nucleus is used for gene expression, the concentration reaches 20 μM. Moreover, if several hundred times as many copies of mRNA coexist, the nucleus is in a state where the risk of "entanglement" between single-stranded DNA or between single-stranded DNA and RNA is significantly increased. Under these circumstances, the double-stranded DNA helicase, also known as a DNA unwinding enzyme, is required.
It is highly likely that DNA helicase, which has the ability to unwind NA into single-stranded DNA, plays an important role.
It is a very interesting fact that the RecQ5β helicase (RecQ5α) lacks the C-terminal structure characteristic of the RecQ5β of the present invention. Furthermore, immunoprecipitation experiments have shown that the RecQ5β of the present invention interacts with topoisomerases 3α and β, among the three type I topoisomerases. In addition, immunofluorescence in cells has shown that the localization of topoisomerases 3α and β coincides with that of RecQ5β. These findings, combined with the high expression of RecQ5β in the testis, suggest that the RecQ5β of the present invention interacts with topoisomerases 3α and β in the cell nucleus and is closely involved in DNA metabolic reactions such as recombination and repair. Furthermore, the cytoplasm-localized isoform (Re
Therefore, RecQ5β of the present invention, which includes RecQ5α, is expected to be a useful protein for the diagnosis and treatment of chromosomal instability diseases.

[Sequence List]

[Brief explanation of the drawings]

Figure 1 is a diagram showing the genomic structure of the human RecQ5 gene. The nucleotide sequence numbers of the cDNAs corresponding to the exons common to the three RecQ5 isoforms are shown in bold, the nucleotide sequence numbers of the cDNAs specific to RecQ5α are shown in thin letters, and the nucleotide sequence numbers of the RecQ5α-specific cDNAs are shown in bold.
The base sequence numbers of the cDNA specific to RecQ5β are shown in italics, and the base sequence numbers of the cDNA specific to RecQ5γ are shown in parentheses. Figure 2 is a diagram showing the schematic diagram of the exon usage of RecQ5 isoforms. Figure 3 is a diagram showing the schematic diagram of the structure of RecQ5 isoforms. Figure 4 is a photograph showing the results of a Northern blot analysis of RecQ5β mRNA expression in human organs. RecQ5β-specific cDNA was used as a probe. a; heart, b; brain, c; placenta, d; lung, e; liver, f; skeletal muscle, g; kidney, h;
Pancreas, i; spleen, j; thymus, k; prostate, l; testis, m; ovary, n; small intestine, o;
Colon, p: Peripheral blood lymphocytes. Figure 5 shows a schematic diagram illustrating the structure of the HA epitope-human RecQ5 isoform fusion protein expression vector, as well as a photograph showing the results of an analysis of the expression state of RecQ5 isoform proteins in human 293EBNA cells. a: Expression vector structure: RecQ5α based on the pcDNA3 plasmid.
Expression vectors for RecQ5β, RecQ5γ, and the C-terminal 246 amino acids of RecQ5β were constructed. The influenza hemagglutinin epitope (HA) was added to the N-terminus of each construct. b: Photograph showing the results of expression analysis by Western blot. pcD was used as a mock control.
The NA3 plasmid and four types of RecQ5 expression vectors were each
RecQ5 was expressed in human 293EBNA cells, and cell extracts were fractionated by SDS-PAGE and detected using an anti-HA antibody. Figure 6 is a photograph showing the results of an analysis of the localization of RecQ5 isoform proteins in human 293EBNA cells. The left panels (green) in each of a to h show the localization of RecQ5 isoform proteins detected with an anti-HA antibody. The right panels (blue) show nuclei stained with DAPI. Figure 7 is a photograph showing the results of an analysis of the interaction of RecQ5β with topoisomerase 3α and β by Western blotting. RecQ5β expression plasmids carrying the HA epitope were expressed alone or in combination with Flag
Type I topoisomerase (Topoisomerase) with epitopes
RecQ5β was expressed in human 293EBNA cells together with expression plasmids for topoisomerases 3α and 3β. RecQ5β coprecipitates with topoisomerases 3α and β. a: Expression of RecQ5β in each expressing cell was detected with an anti-HA antibody. b: Expression of type I topoisomerase in each expressing cell was detected with an anti-Flag antibody. c: Extracts from each expressing cell were immunoprecipitated with an anti-Flag antibody, and the precipitated proteins were detected with an anti-HA antibody. Figure 8 is a photograph showing the results of immunochemical detection performed to confirm the coincidence of the localization of RecQ5β with topoisomerases 3α and β. An expression plasmid for RecQ5β carrying the HA epitope was expressed in human 293EBNA cells together with expression plasmids for topoisomerase 3α and β carrying the Flag epitope, and localization was detected by immunofluorescence using anti-HA (RecQ5β/red) and anti-Flag (topoisomerase 3α or β/green). Panels a and b show the localization of RecQ5β and topoisomerase 3α, while panels c and d show the localization of RecQ5β and topoisomerase 3β. The left (green) part of each panel in a–d shows the localization of topoisomerase 3α or β, and the center (red) of each panel shows the localization of RecQ5β. The right panel shows an overlay of the respective localizations. The yellow areas in the right panels indicate the coincidence of the localization of RecQ5β with topoisomerase 3α or β.

─────────────────────────────────────────────────────── Continued from the front page (51) Int.Cl. ⁷ Identification symbol FIG G01N 33/53 G01N 33/53 D (Note) This publication has been created based on the gazette published internationally by the International Bureau of Patents (WIPO). The effect of the international publication of the Japanese language patent application (Japanese language utility model registration application) related to this publication arises pursuant to Article 184-10, Paragraph 1 of the Patent Act (Article 48-13, Paragraph 2 of the Utility Model Act), and is unrelated to this publication.

Claims (15)

[Claims] [Claim 1] A polynucleotide set forth in any one of the following (a) to (d): (a) a polynucleotide comprising a protein-coding region of the nucleotide sequence set forth in SEQ ID NO: 2; (b) a polynucleotide encoding the amino acid sequence set forth in SEQ ID NO: 2; (c) a polynucleotide encoding a nuclear-localized protein having helicase activity, the polynucleotide consisting of an amino acid sequence in which one or more amino acids in the amino acid sequence set forth in SEQ ID NO: 2 have been modified by deletion, addition, insertion, and/or substitution with other amino acids; (d) a polynucleotide encoding a nuclear-localized protein having helicase activity, the polynucleotide being a protein encoded by DNA that hybridizes under stringent conditions with DNA consisting of the nucleotide sequence set forth in SEQ ID NO: 1. 2. A protein encoded by the polynucleotide of claim 1. 3. A vector into which the polynucleotide according to claim 1 has been inserted. 4. A transformant harboring the polynucleotide according to claim 1 or the vector according to claim 3. 5. A method for producing the protein according to claim 2, which comprises the steps of culturing the transformant according to claim 5 and recovering the expression product. [Claim 6] A polynucleotide having a chain length of at least 15 nucleotides, which comprises a polynucleotide containing a base sequence corresponding to 1390 to 3703 in the base sequence set forth in SEQ ID NO: 1, or a base sequence complementary to the complementary strand thereof. 7. The primer for polynucleotide synthesis according to claim 1, which comprises the polynucleotide according to claim 6. 8. The probe for detecting the polynucleotide according to claim 1, which comprises the polynucleotide according to claim 6. 9. An antisense DNA to the polynucleotide of claim 1 or a part thereof. 10. A partial peptide consisting of an amino acid sequence corresponding to positions 411 to 991 in the amino acid sequence set forth in SEQ ID NO: 2. 11. An antibody that recognizes a partial peptide consisting of the amino acid sequence corresponding to positions 411 to 991 in the amino acid sequence set forth in SEQ ID NO: 2. 12. An immunoassay method comprising the steps of contacting the antibody of claim 11 with a protein consisting of the amino acid sequence set forth in SEQ ID NO: 2 or a partial peptide thereof, and detecting an immunological reaction between the two. 13. A reagent for detecting a protein consisting of the amino acid sequence set forth in SEQ ID NO: 2 or a partial peptide thereof, comprising the antibody set forth in claim 11. 14. A transgenic non-human vertebrate in which the expression level of the polynucleotide according to claim 1 is altered. 15. The transgenic non-human vertebrate according to claim 14, which is a knockout animal lacking the function of the polynucleotide according to claim 1.