JPH11178578A - Human rnf5 gene - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain the subject new gene comprising a gene comprising a specific amino acid sequence and containing a base sequence encoding a protein having binding properties to an RNF5 and useful for elucidating the morbid state of diseases such as genetic disease or cancer, gene diagnosis, the development, etc., of a therapeutic agent. SOLUTION: This new human RNF5 gene comprises a base sequence capable of encoding a protein comprising an amino acid sequence represented by the formula or a protein comprising an amino acid sequence in which one or several amino acids are deleted, replaced or added in the amino acid sequence and having a binding activity to an RNF5 and is useful for the production, etc., of a human RNF5 protein useful for elucidation or research on the canceration, DNA repair, rearrangement, etc., of immunoglobulin and T cell receptor genes, elucidation of the morbid state of genetic disease, cancer, etc., associated therewith and diagnosis, therapy, etc., thereof. The gene is obtained by carrying out the sequence analysis of a cDNA clone optionally selected from a human fetal brain cDNA library and search for the database and providing the gene from a clone having a high homology with a ring-finger protein gene of a nematode.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a gene useful as a guide for the prevention, diagnosis and treatment of human diseases, and more specifically, to a novel gene having similarity to rat, mouse, yeast, nematode, known human genes and the like. The present invention relates to a gene that can be used for gene diagnosis and development of a new therapeutic drug using the gene by analyzing the cDNA of the gene, mapping it to a chromosome, and analyzing the function of the cDNA.

[0002]

2. Description of the Related Art Genetic information of an organism is accumulated as a sequence (DNA) of four types of bases A, C, G and T existing in the nucleus of a cell. Reserved for ontogeny. In the case of humans, the number of bases is said to be about 3 billion (3 × 10 ⁹ ), of which 5 to 1
It is estimated that there are 10,000 genes. Such genetic information is involved in the maintenance of life phenomena through the creation of regulatory proteins, structural proteins, enzymes, etc. along with the flow of mRNA transcribed from genes (DNA) and then translated into proteins.

[0003] Abnormalities in the flow from the above genes to protein translation cause abnormalities in life support systems such as cell proliferation and differentiation, and are thought to cause various diseases. From the results of gene analysis to date, various receptors such as insulin receptor and LDL receptor, and genes related to cell growth and differentiation, such as proteases, ATPase, and metabolic enzymes such as superoxide dismutase, have been identified for drug development. It was thought to be a useful material.

[0004] However, the analysis of human genes and the relationship between the functions of the analyzed genes and various diseases has only just begun, and there are many unclear points. There is a need in the art for functional analysis of the gene and research on the relationship with the disease, and further, gene diagnosis using the analyzed gene and research on application of the gene to medical use.

[0005]

As described above, if a new human gene can be provided, the expression level in each cell, its structure and function can be analyzed, and its expression can be analyzed by analyzing its expression. It is considered that elucidation of pathological conditions, diagnosis, treatment, and the like of diseases, for example, genetic diseases, cancers, and the like, are possible, and the present invention aims to provide such new human genes.

[0006]

Means for Solving the Problems The present inventors have intensively studied as follows in view of the above objects. That is, the present inventors first synthesized cDNA from mRNA extracted from various tissues of human fetal brain, adult blood vessels, and placenta, incorporated this into a vector to construct a library, and transformed the library. Escherichia coli colonies were formed on an agar medium, and the colonies were randomly picked up and transferred to a 96-well microplate to prepare and register Escherichia coli clones containing various human genes. Next, after culturing each of these clones, the DNA is extracted and purified, and the resulting cDN
Using A as a template, an elongation reaction for terminating the four bases specifically was performed by the deoxyterminator method, and about 400 base sequences were determined from the 5 'end of the human gene of each registered clone by an automatic DNA sequencer. Based on the nucleotide sequence information of the obtained human genes, novel family genes having similarity to various animal and plant species such as known bacteria, yeast, nematodes, mice, and humans were searched. The above-mentioned cDNA analysis method is described in detail in the literature of Fujiwara et al. (Riki Fujiwara, Cell Engineering, 14 , 645-65).
4 (1995)).

As a result, the cDNA clone arbitrarily selected from the human fetal brain cDNA library contains CE0, a C. elegans ring-finger protein.
Cosmid C16C10.7, a known gene believed to encode 1498 [Wilson, R., et al.
l., Nature, 368 , 32-38 (1994)], and have completed the present invention.

That is, according to the present invention, a protein having the amino acid sequence represented by SEQ ID NO: 1 or an amino acid sequence in which one or more amino acids are deleted, substituted or added in said amino acid sequence and has an activity of binding to RNF5 A human RNF5 gene comprising a nucleotide sequence encoding a protein having the formula: SEQ ID NO: 2 or a human comprising a nucleotide sequence hybridizable therewith under stringent conditions The RNF5 gene;
The human RNF having the nucleotide sequence of SEQ ID NO: 3
Five genes are provided.

Hereinafter, the abbreviations of amino acids, peptides, base sequences, nucleic acids and the like in this specification are referred to as IUPA.
C, IUB rules, "Guidelines for the preparation of specifications containing base sequences or amino acid sequences" (Japan Patent Office), and commonly used symbols in the art.

[0010]

BEST MODE FOR CARRYING OUT THE INVENTION As a specific example of the gene of the present invention, “GEN-086B1
The sequence deduced from the DNA sequence of the clone named "2" can be mentioned, and its nucleotide sequence is as shown in the sequence listing.

The gene possessed by this clone has an open reading frame of 540 nucleotides (nucleic acid) encoding 180 amino acid residues shown in the sequence listing as SEQ ID NO: 1 (the sequence is shown in SEQ ID NO: 2). The nucleic acid sequence of the full-length cDNA clone is 1074 nucleotides as shown in SEQ ID NO: 3. In the sequence shown in SEQ ID NO: 3, the initiation codon is 67
It is located at position -69, and the polyadenylation signal (AATAA) is located at 1056-1061.

Hereinafter, the human RNF5 gene of the present invention will be described in detail. As described above, the gene of the present invention has similarity to the gene encoding the ring-finger protein of C. elegans, and a human gene based on the gene information thereof. Can be used for research on the relationship between the function of the analyzed gene and various diseases, and can be used for gene diagnosis of diseases related to the gene and research on application of the gene to pharmaceutical use. . That is, the function of the protein (gene product) encoded by the gene of the present invention can be inferred from that of a known homologous gene, and according to the provision of the gene of the present invention, the candidate gene is incorporated into an expression vector, and the recombinant is used. It can also be prepared and examined for functions such as enzyme activity and binding activity.

[0013] Thus, the ring finger protein is
Cysteine and histidine residues form a site for binding zinc ions. However, this is another zinc
Unlike finger proteins, they do not have DNA-binding ability but are thought to be involved in binding between proteins.

CE0149 having homology to human RNF5
Although the function of Escherichia coli 8 is not yet known, a protein having a ring-finger has been identified as a cancerous [Blake, TJ, et al., EMBO
J., 12 , 2017-2026 (1993); Miki, Y., Et al., Scienc
e, 266 , 66-71 (1994); Borden, KLB, et al., EMBO
J., 14 , 1532-1541 (1995); Kanno, M., et al., EMBO
J., 14 , 5212-5222 (1994)], rearrangement of immunoglobulin and T cell receptor genes (Leu, TM and Schatz, DG,
Mol. Cell. Biol., 15 , 5657-5670 (1995)], and it is expected that human RNF5 protein also plays an important function in vivo.

Further, the fact that it is highly conserved between C. elegans and humans, and that its expression is observed in all tissues as far as it is examined, also suggests the importance of human RNF5 protein.

Furthermore, the chromosomal locus of the gene of the present invention is 6p
At chromosome 6p21.3, a chromosomal deletion (LOH) has been identified in colorectal cancer [Honchel, R., et al., Cancer Res., 56 , 145-149.
(1996)], it is conceivable that a compound that suppresses the activity of human RNF5 protein in vivo may be used for treating tumors and the like.

The gene of the present invention is represented by a single-stranded DNA sequence as shown in, for example, SEQ ID NO: 2. The gene of the present invention includes a DNA complementary to the single-stranded DNA sequence.
Components that include both arrays and components are also included. The sequence of the gene of the present invention shown in SEQ ID NO: 2 is an example of one combination of codons of each amino acid residue encoded thereby. The gene of the present invention is not limited to this, Selected by combining arbitrary codons
It is of course possible to have an NA base sequence. The selection of the codon can be performed according to a conventional method, for example, considering the codon usage of the host to be used [Ncl.
s., 9 , 43-74 (1981)].

Further, the gene of the present invention may have a partial amino acid to amino acid sequence substitution in the amino acid sequence shown above,
A DNA sequence which is modified by deletion, addition, etc. and has the same function and encodes the same compound is also included. The production, modification (mutation), etc. of such modified products may occur naturally, and the natural gene (the gene of the present invention) may be prepared by post-translational modification or by genetic engineering techniques, for example, by site-specific mutagenesis. (Methods in Enzymol
ogy, 154 , p350, 367-382 (1987); same 100 , p468 (198
3); Nucleic Acids Research, 12 , p9441 (1984);
p105 (1986)] or chemical synthesis means such as the phosphate triester method and the phosphate amidite method (J.
Am. Chem. Soc., 89 , p4801 (1967); id. 91 , p3350 (19
69); Science, 150 , p178 (1968); Tetrahedron Lett.,
22 , p1859 (1981); ibid., 24 , p245 (1983)], and can be obtained by a combination thereof.

By utilizing the gene of the present invention, that is, for example, by incorporating the gene into a microorganism vector and culturing the transformed microorganism, the protein (RNF5 protein) encoded by each of the above genes can be easily prepared. And it can be stably expressed.

The protein obtained by using the gene of the present invention can be used to prepare a specific antibody. As the component used as the antigen, a protein produced in large amounts according to the above-mentioned genetic engineering technique can be used, and the obtained antibody may be either a polyclonal antibody or a monoclonal antibody. , Measurement, identification and the like.

The production of the gene of the present invention can be easily carried out by general genetic engineering techniques according to the sequence information on the gene of the present invention disclosed by the present invention [Molecu
larCloning 2nd Ed, Cold Spring Harbor Laboratory P
ress (1989);
I, III ", edited by The Biochemical Society of Japan (1986)].

For example, a desired clone is selected from a human cDNA library (prepared from an appropriate source cell in which each gene is expressed) by using an appropriate probe or antibody specific to the gene of the present invention. Natl. Acad. Sci. USA., 78 , 661.
3 (1981); Science, 222 , 778 (1983), etc.].

In the above method, examples of the source cells include various cells expressing the target gene, cultured cells derived from tissues and the like, and the separation of total RNA therefrom.
Isolation and purification of mRNA, conversion to cDNA (synthesis), cloning thereof, and the like can all be carried out according to conventional methods. In addition, cDNA libraries are also commercially available. In the present invention, these cDNA libraries, for example, various cDNAs available from ClontechLab. Inc.
An NA library or the like can also be used.

The screening of the gene of the present invention from the cDNA library can be carried out according to the above-mentioned usual method. The screening method includes, for example, c
By performing an immunoscreening on a protein produced by DNA using the protein-specific antibody, the corresponding cDN
Examples of the method include selecting a clone A, plaque hybridization using a probe that selectively binds to a target DNA sequence, colony hybridization, and combinations thereof. As the probe used here, a DNA sequence or the like chemically synthesized based on information on the DNA sequence of the gene of the present invention is generally used. Of course, the gene of the present invention or a fragment thereof which has already been obtained is also used. Can be used as a probe.

Further, based on partial amino acid sequence information of a natural extract extracted, isolated and purified from each cell or tissue, sense primers and antisense primers can also be used as screening probes.

When obtaining the gene of the present invention, P
DN by CR method [Science, 230 , 1350-1354 (1985)]
The A / RNA amplification method can be suitably used. Particularly, when it is difficult to obtain a full-length cDNA from the library, the lace method (RACE: Rapid amplification of cDNA ends;
Experimental Medicine, 12 (6), 35-38 (1994)), especially the 5'-RACE method [Frohman, MA, et al., Proc.
Atl. Acad. Sci., USA., 8 , 8998-9002 (1988)] is preferred. Primers to be used when employing such a PCR method can be appropriately set based on the sequence information of the gene of the present invention which has already been revealed by the present invention, and can be synthesized according to a conventional method.

The isolation and purification of the amplified DNA / RNA fragment can be carried out by a conventional method as described above, for example, by gel electrophoresis.

The gene of the present invention or various DNs obtained above
The determination of the base sequence of the A fragment and the like can be performed according to a conventional method,
For example, the dideoxy method [Proc. Natl. Acad. Sci. USA., 7
4 , 5463-5467 (1977)) or the Maxam-Gilbert method (Met
hods in Enzymology, 65 , 499 (1980)] and the like. Such determination of the nucleotide sequence can be easily performed even using a commercially available sequence kit or the like.

According to the use of the gene of the present invention, conventional gene recombination techniques [for example, Science, 224 , p1431 (1984);
Biochem. Biophys. Res. Comm., 130 , p692 (1985); Pr
oc. Natl. Acad. Sci., USA., 80 , p5990 (1983) and the references cited above] can obtain each recombinant protein. More specifically, the production of the protein is carried out by using a recombinant DN in which the gene of the present invention can be expressed in a host cell.
A is prepared by introducing A into a host cell for transformation, and culturing the transformant.

Here, as the host cells, both eukaryotes and prokaryotes can be used. The eukaryotic cells include cells such as vertebrates and yeast, and the vertebrate cells include, for example, COS cells [Cel cells]
l, 23 , 175-182 (1981)) and Chinese hamster ovary cells and their dihydrofolate reductase-deficient strains (Pr
oc. Natl. Acad. Sci., USA., 77 , 4216-4220 (1980)]
Are often used, but are not limited to these. As a vertebrate expression vector, a vector having a promoter, an RNA splice site, a polyadenylation site, a transcription termination sequence, and the like, which is usually located upstream of the gene to be expressed, can be used. You may have. Examples of such expression vectors include, for example, p40 carrying the early promoter of SV40.
SV2 dhfr [Mol. Cell. Biol., 1 , 854 (1981)] and the like. As eukaryotic microorganisms, yeasts are generally used, and among them, yeasts belonging to the genus Saccharomyces can be advantageously used. Examples of expression vectors for eukaryotic microorganisms such as the yeast include pAM82 having a promoter for the acid phosphatase gene [Proc. Natl. Acad. Sci., US
A., 80 , 1-5 (1983)].

A prokaryotic gene fusion vector can be preferably used as an expression vector for the gene of the present invention.

Escherichia coli and Bacillus subtilis are generally used as prokaryotic hosts. When these are used as a host, for example, using a plasmid vector that is replicable in the host bacterium,
An expression plasmid having a promoter and an SD (Shine and Dalgarno) base sequence upstream of the gene and an initiation codon (eg, ATG) necessary for initiation of protein synthesis is used in this vector so that the gene of the present invention can be expressed. Is preferred. As Escherichia coli as the host, Escheric hia coli K12
Strains are often used, and the vector is generally pBR3
22 and its improved vector are often used, but not limited thereto, and various known strains and vectors can also be used. As a promoter, for example, tryptophan (tr
p) Promoters, lpp promoters, lac promoters, PL / PR promoters and the like can be used.

As a method for introducing the desired recombinant DNA thus obtained into a host cell and a method for transforming the same, various general methods can be employed. The obtained transformant can be cultured according to a conventional method, and the target protein encoded by the gene of the present invention is produced and expressed by the culture. As the medium used for the culture, various types commonly used depending on the host cell employed can be appropriately selected and used,
The culture can also be performed under conditions suitable for the growth of the host cell.

As described above, the desired recombinant protein is expressed, produced, accumulated or secreted inside, outside, or on the cell membrane of the transformant.

Each recombinant protein may be subjected to various separation operations utilizing its physical properties, chemical properties and the like [Biochemical Data Book II], pages 1175-1259, 1st edition, 1st edition, if desired.
Printing, June 23, 1980, published by Tokyo Chemical Dojin; Bioche
mistry, 25 (25), 8274-8277 (1986); Eur. J. Bioche
m., 163 , 313-321 (1987)]. Examples of the method include, for example, ordinary reconstitution treatment, treatment with a protein precipitant (salting out method), centrifugation,
Osmotic shock method, sonication, ultrafiltration, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, affinity chromatography, high-performance liquid chromatography (HPL
Examples of such methods include various types of liquid chromatography such as C), dialysis methods, combinations thereof, and the like. Particularly preferred examples of the above method include affinity chromatography using a column to which a desired protein is bound.

Further, based on the sequence information of the gene of the present invention revealed by the present invention, the expression of the gene of the present invention in various human tissues can be carried out, for example, by utilizing a part or all of the nucleotide sequence of the gene. Can be detected. This can be performed according to a conventional method, for example, RT-PCR (Reverse transcribed-Polymerase chain).
reaction) (Kawasaki, ES, et al., Amplification
of RNA.In PCR Protocol, A Guide to methods and a
pplications, Academic Press, Inc., SanDiego, 21-27
(1991)], and Northern blotting analysis [Molecular Cloning, Cold Spring Harbo
r Laboratory (1989)].

When the PCR method is employed, the primers used are not particularly limited as long as they are specific to the gene of the present invention capable of specifically amplifying only the gene of the present invention. Thus, the arrangement can be appropriately set. Usually this is 20-
It may have a partial sequence of about 30 nucleotides.

[0038]

According to the present invention, a novel human RNF5 gene is provided, and by using the gene, the expression of the gene in various tissues can be detected and its structure and function can be analyzed.
In addition, it is possible to produce a human RNF5 protein encoded by the gene by genetic engineering, and thereby, by analyzing the protein, elucidation of canceration, DNA repair, rearrangement of immunoglobulin and T cell receptor genes, and the like. , Research, and diseases involving them, such as genetic diseases, elucidation of pathological conditions such as cancer, and diagnosis and treatment.

[0039]

The present invention will now be described in further detail with reference to examples.

[0040]

Example 1 RNF5 gene (1) Cloning and DNA sequencing of RNF5 gene c arbitrarily selected from a human fetal brain cDNA library
By sequence analysis of DNA clones and database search, one clone (GEN-086B12) having high homology to a gene considered to encode a ring-finger protein of C. elegans was isolated as follows.

That is, mRNA extracted from human fetal brain was purchased from Clonetech and used as a starting material. The above m
CDNA was synthesized from RNA and inserted into the vector λZAPII (Stratagene) to construct a cDNA library (Otsuka GEN Research Institute,
Otsuka Pharmaceutical Co., Ltd.). In vivo excision method (invi
vo excision: Short, JM, et al., Nucleic Acids R
es., 16 , 7583-7600 (1988)), an E. coli colony containing a human gene was formed on an agar medium, the colony was randomly picked up, and an E. coli clone containing the human gene was registered in a 96-well microplate. The registered clone was stored at -80 ° C.

Next, each of the registered clones was replaced with 1.5 ml of L
Culture in B medium all day and night, automatic plasmid extractor PI-
DNA was extracted and purified using 100 (manufactured by Kurabo Industries, Ltd.). The contaminated RNA of Escherichia coli was decomposed and removed by RNase treatment. Finally, dissolve in 30 μl and add 2 μl
For l, the size and amount of DNA were roughly checked by minigel. 7 μl thereof was used for a sequencing reaction, and the remaining 21 μl was stored at 4 ° C. as plasmid DNA.

Subsequently, the dideoxy terminator method of Sanger et al. Using T3, T7 or synthetic oligonucleotide primers (Sanger, F., et al., Proc. Natl. A
cad. Sci., USA, 74 , 5463-5467 (1977)) or a cycle sequence method (Carothers, AM, et al., B) which is a method in which the PCR method is added to the dideoxy terminator method.
io. Techniques, 7 , 494-499 (1989)). These methods use a small amount of plasmid DNA (approximately 0.1-
0.5 μg) as a template (template) to carry out an elongation reaction to specifically stop four kinds of bases.

As a sequence primer, FITC was used.
(Fluorescein isothiocyanate) Fluorescein-labeled one was used and reacted for about 25 cycles with Taq polymerase. For the fluorescently labeled DNA fragment, a sequence of about 400 bases from the 5 ′ end of the cDNA was determined by an automatic DNA sequencer, ALF ^™ DNA sequencer (Pharmacia).

[0045] The 3 'untranslated region corresponds to the heterogeneity of each gene (he
In some cases, 3′-sequencing was also performed due to high terogeneity and suitable for distinguishing individual genes.

The vast amount of base sequence information obtained by the DNA sequencer is converted into a 64-bit computer DEC34.
00 and transferred to a computer for homology analysis. The homology analysis was performed using FAWG of UWGCG.
Programs (Pearson, WRand Lipman, DJ, Proc.N
Atl. Acad. Sci., USA., 85 , 2444-2448 (1988)) and a database (GenBank, EMBL) search.

The above analysis method for the human fetal brain cDNA library is described in Fujiwara et al. [Fujiwara, t., Et al., DN
A Res., 2 , 107-111 (1991)].

The human fetal brain cDN constructed as described above
Approximately 5040 ESTs (expressed sequence tags: partial DNA sequences of expressed gene fragments) randomly selected from the A library were sequenced.

In the sequence search of Gene Bank and EMBL by the FASTA program, a clone named GEN-086B12 was found to contain the cosmid C16, a gene encoding CE01498, a ring finger protein of nematodes.
It was found to show structural similarity with C10.7.

In order to clarify the full-length sequence of the GEN-086B12 clone, DNA sequencing using T7 DNA polymerase and synthetic primers was performed, and a vector (pBluescript vector: manufactured by Stratagene) was used as a template (template). Stratage
ne)), using the double-stranded DNA inserted in (1) and a synthetic oligonucleotide as a primer, the nucleotide sequence of the cDNA containing the entire coding region was determined by the dideoxy chain termination method of Sanger et al. And
It was compared with the DNA sequences of several other related genes.

SEQ ID NO: 3 encodes the nucleic acid sequence of the GEN-086B12 clone (cDNA), SEQ ID NO: 2 encodes the nucleic acid sequence of the coding region of the clone, and SEQ ID NO: 1 encodes the nucleic acid sequence. 1 shows the predicted amino acid sequence.

In the above nucleic acid sequence, the start signal sequence is 67
It was found at the position of the nucleic acid residue at position −69, which was assumed to be the translation initiation codon. The putative stop codon is 607-
It was found at the position of the 609th nucleic acid residue.

The cDNA was 1074 nucleotides long and contained a 540 base pair open reading frame encoding a predicted protein of 180 amino acids.

By homology search using the FASTA program, this gene was found to be cosmid C16, a gene encoding CE01498, a ring finger protein of nematodes.
C10.7 [Wilson, R., et al., Nature, 368 , 32-38.
(1994)], and the homology of the nucleotide sequence was calculated to be 53%.

At the amino acid sequence level, the predicted expression product of the gene of the present invention showed 51% homology with the ring finger protein of the nematode.

(2) Northern blot analysis The expression of RNF5 mRNA in normal human tissues was determined by the random oligonucleotide priming method.
Evaluation was performed by Northern blot using a labeled human cDNA clone as a probe.

The Northern blot analysis was carried out according to the product usage, using a human MTN blot (Human Multiple Tissue Noth).
ern blot; Clonetech, La Joll
a), California, USA).

That is, the clone GEN-086B12
Is amplified by PCR, and the PCR amplification product is [ ³² P]
Probes were labeled with dCTP (random primed DNA labeling kit, Boehringer Mannheim).

After prehybridizing the blot for 4 hours,
50% formamide / 5 × SSC / 10 overnight at 42 ° C.
× Denhardz solution / 2% SDS solution (100 μg / ml
(Containing denatured salmon sperm DNA). After washing twice with 2 × SSC / 0.1% SDS at room temperature, then 65% with 0.2 × SSC / 0.1% SDS.
Washed twice at 10 ° C. for 10 minutes. Filter at -70 ° C
It was exposed to X-ray film (manufactured by Kodak) for one day below.

As a result of the exposure for 36 hours, all organs (liver, heart, placenta, pancreas, skeletal muscle, prostate, kidney, ovary, brain, lung, spleen, thymus, testis, small intestine) of the 16 adult human tissues tested , Colon and peripheral lymphocytes), and a transcript of about 1.2 kb was detected.

(3) FISH, Chromosome Mapping by Radiation Hybrid Method FISH for chromosome alignment can be performed by a known method (Takahas
hi E., et al., Hum. Genet., 86 , 14-16 (1990)) using 0.5 μg of each cosmid DNA as a probe. FISH is Provia 100 film (Fuji, ISO100) or CCD camera system (Applied Imaging, SightVision)
Caught by

As a result, 100 typical R-bands
The signal examined in (pre) metaphase cells was localized to chromosome 6 band 6p21.31. Therefore, the localization site of the human RNF5 gene could be identified as 6p21.31.

According to the present invention, a novel human RNF5 gene is provided, and the use of the gene enables detection of the expression of the gene in various tissues and production of human RNF5 protein by genetic engineering. Thus, it becomes possible to elucidate canceration caused by expressed proteins, DNA repair, rearrangement of immunoglobulin and T cell receptor genes, and to elucidate pathological conditions, such as cancer and the like, elucidate, diagnose, treat, etc. Conceivable.

[0064]

[Sequence list]

(1) GENERAL INFORMATION: (i) APPLICANT: Ostuka Pharmaceutical Co., Ltd. (ii) TITLE OF INVENTION: human RNF5 gene (iii) NUMBER OF SEQUENCES: 3 (iv) COMPUTER READABLE FORM: (A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS / MS-DOS (D) SOFTWARE: PatentIn Release # 1.0, Version # 1.30 (vi) CURRENT APPLICATION DATA: (A) APPLICATION NUMBER : (B) FILING REFERENCE: 29C7JP (C) FILING DATE: 24-DECEMBER-1997

(2) INFORMATION FOR SEQ ID NO: 1: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 180 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi ) SEQUENCE DESCRIPTION: SEQ ID NO: 1: Met Ala Ala Ala Glu Glu Glu Asp Gly Gly Pro Glu Gly Pro Asn Arg 1 5 10 15 Glu Arg Gly Gly Ala Gly Ala Thr Phe Glu Cys Asn Ile Cys Leu Glu 20 25 30 Thr Ala Arg Glu Ala Val Val Ser Val Cys Gly His Leu Tyr Cys Trp 35 40 45 Pro Cys Leu His Gln Trp Leu Glu Thr Arg Pro Glu Arg Gln Glu Cys 50 55 60 Pro Val Cys Lys Ala Gly Ile Ser Arg Glu Lys Val Val Pro Leu Tyr 65 70 75 80 Gly Arg Gly Ser Gln Lys Pro Gln Asp Pro Arg Leu Lys Thr Pro Pro 85 90 95 Arg Pro Gln Gly Gln Arg Pro Ala Pro Glu Ser Arg Gly Gly Ply Ghe 100 100 110 110 Pro Phe Gly Asp Thr Gly Gly Phe His Phe Ser Phe Gly Val Gly Ala 115 120 125 Phe Pro Phe Gly Phe Phe Thr Thr Val Phe Asn Ala His Glu Pro Phe 130 135 140 Arg Arg Gly Thr Gly Val Asp Leu Gly Gln Gly His Pro Ala Ser Ser 145 150 155 160 Trp Gln Asp Ser Leu Phe Leu Phe Leu Ala Ile Phe Phe Phe Phe Trp 165 170 175 Leu Leu Ser Ile 180

(2) INFORMATION FOR SEQ ID NO: 2: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 540 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2: ATGGCAGCAG CGGAGGAGGA GGACGGGGGC CCCGAAGGGC CAAATCGCGA GCGGGGCGGG 60 GCGGGCGCGA CCTTCGAATG TAATATATGT TTGGAGACTG CTCGGGAAGC TGTGGTCAGT 120 GTGTGTGGCC ACCTGTACTG TTGGCCATGT CTTCATCAGT GGCTGGAGAC ACGGCCAGAA 180 CGGCAAGAGT GTCCAGTATG TAAAGCTGGG ATCAGCAGAG AGAAGGTTGT CCCGCTTTAT 240 GGGCGAGGGA GCCAGAAGCC CCAGGATCCC AGATTAAAAA CTCCACCCCG CCCCCAGGGC 300 CAGAGACCAG CTCCGGAGAG CAGAGGGGGA TTCCAGCCAT TTGGTGATAC CGGGGGCTTC 360 CACTTCTCAT TTGGTGTTGG TGCTTTTCCC TTTGGCTTTT TCACCTCGT TTTCAATGCC 420 CATGAGCCTT TCCGCCTCGGTC TAGCGGTCTC GCGGTC TAGCGGTCTC GATCAGCTCGTCTCGGTCTC

(2) INFORMATION FOR
SEQ ID NO: 3: (i) SEQUENCE CHARACTERIST
ICS: (A) LENGTH: 1074 base pai
rs (B) TYPE: nucleic acid (C) STRANDENDESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (c)
DNA) (ix) FEATURE: (A) NAME / KEY: CDS (B) LOCATION: 67. . 606 (xi) SEQUENCE DESCRIPTION: SEQ I
D NO: 3: TTGTTGGGGGC GTGTGTATGT GTATTTGGGGG GGA
CTGAAGG GTACGTGGGG CGAAACAAAA 60 CCGGCC ATG GCA GCA GCG GAG GAG GAG GAG G
AC GGG GGC CCC GAA GGG CCA 108 Met Ala Ala Ala Glu Glu Glu A
sp Gly Gly Pro Glu Gly Pro 15
10 AAT CGC GAG CGG GGC GGG GCG GGC GCG
ACC TTC GAA TGT AAT ATA TGT 156 Asn Arg Glu Arg Gly Gly Gly Ala Gly Ala
Thr Phe Glu Cys Asn Ile Cys 15 20
25 30 TTG GAG ACT GCT CGG GAA GCT GTG GTC
AGT GTG TGT GGC CAC CTG TAC 204 Leu Glu Thr Ala Arg Glu Ala Val Val
Ser Val Cys Gly His Leu Tyr 35
40 45 TGT TGG CCA TGT CTT CAT CAG TGG CTG
GAG ACA CGG CCA GAA CGG CAA 252 Cys Trp Pro Cys Leu His Gln Trp Leu
Glu Thr Arg Pro Glu Arg Gln 50 55
60 GAG TGT CCA GTA TGT AAA GCT GGG ATC
AGC AGA GAG AAG GTT GTC CCG 300 Glu Cys Pro Val Cys Lys Ala Gly Ile
Ser Arg Glu Lys Val Val Pro 65 70
75 CTT TAT GGG CGA GGG AGC CAG AAG CCC
CAG GAT CCC AGA TTA AAA ACT 348 Leu Tyr Gly Arg Gly Ser Gln Lys Pro
Gln Asp Pro Arg Leu Lys Thr 80 85
90 CCA CCC CGC CCC CAG GGC CAG AGA CCA
GCT CCG GAG AGC AGA GGG GGA 396 Pro Pro Arg Pro Gln Gly Gln Arg Pro
Ala Pro Glu Ser Arg Gly Gly 95 100
105 110 TTC CAG CCA TTT GGT GAT ACC GGG GGC
TTC CAC TTC TCA TTT GGT GTT 444 Phe Gln Pro Phe Gly Asp Thr Gly Gly
Phe His Phe Ser Phe Gly Val 115
120 125 GGT GCT TTT CCC TTT GGC TTT TTC ACC
ACC GTC TTC AAT GCC CAT GAG 492 Gly Ala Phe Pro Phe Gly Phe Phe Thr
Thr Val Phe Asn Ala His Glu 130 135
140 CCT TTC CGC CGG GGT ACA GGT GTG GAT
CTG GGA CAG GGT CAC CCA GCC 540 Pro Phe Arg Arg Gly Thr Gly Val Asp
Leu Gly Gln Gly His Pro Ala 145 150
155 TCC AGC TGG CAG GAT TCC CTC TTC CTG
TTT CTC GCC ATC TTC TTC TTT 588 Ser Ser Trp Gln Asp Ser Leu Phe Leu
Phe Leu Ala Ile Phe Phe Phe 160 165
170 TTT TGG CTG CTC AGT ATT TGAGCTATGT C
TGCTCCCTG CCCACCTCCA 636 Phe Trp Leu Leu Ser Ile
175 180
GCCAGAGAAG AATCAGTATT GAGGTCCCTG CTG
ACCCTTC CGTACTCCTG GACCCCCTTG 696 ACCCCTCTAT TTCGTGTTGGC TAAGGCCAGC CCT
GGACATT GTCCAGGAAG GCCTGGGGAG 756 GAGGAGTGAA GTTCTGTGCAT AGATGGGAGA GCC
TTCTGCT CAGAGGCTCA CTCAGTAACG 816 TTGTTTAATT CTCGCCCTG GGGAAGGAGG ATG
GATTGAG AGAATGTCTT TCTCCTCTCC 876 TAAGTCTTTG CTTTCCCTGA TTTCTTGATT TGA
TCTTCAA AGGTGGGCAA AGTTCCCTCT 936 GACTCTTCCC CCACTCCCCA TCTTACTGAT TTA
ATTTAAT TTTTCACTCC CCAGAGTCTA 996 ATATGGATCTC TGACTCTTAA GTGCTTCCGC CCC
CTCACTA CCTCCTTTAA TACAAAATTCA 1056 ATAAAAAAGGG TGAAATAT
1074

Claims (3)

[Claims] 1. Encoding a protein having the amino acid sequence represented by SEQ ID NO: 1 or a protein comprising an amino acid sequence in which one or more amino acids are deleted, substituted or added in the amino acid sequence, and having a binding activity to RNF5. A human RNF5 gene comprising a nucleotide sequence. 2. A human RNF5 gene comprising a nucleotide sequence represented by SEQ ID NO: 2 or a nucleotide sequence which hybridizes with the nucleotide sequence under stringent conditions. 3. The human RNF5 gene according to claim 2, which is a nucleotide sequence represented by SEQ ID NO: 3.