JP2001136967A - STRUCTURE AND FUNCTION OF NEW BIOLOGICAL RHYTHM MARKER GENE rPer3 - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a new protein involved in the control of the mammalian in vivo circadian rhythm and a gene encoding the protein, and information concerning its function. SOLUTION: A protein having an amino acid sequence shown by sequence 1 and a human mammalian homologue thereof, genes encoding the protein and a homologue thereof, and DNA fragments thereof, a method for measuring the circadian rhythm with which the gene and an expression product thereof are assayed using the DNA fragment, and a kit for assaying them are provided.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a circadian rhythm (ci
The present invention relates to a protein involved in the molecular mechanism of rcadian rhythm and its gene, and more particularly, to a novel biological clock-related protein and its gene whose expression fluctuates in a time-specific manner in relation to the circadian rhythm.

[0002] The present invention further relates to a partial polynucleotide of the above biological clock-related gene, particularly to a probe and a primer useful for detecting or amplifying the above gene.

[0003]

2. Description of the Related Art A physiological cycle phenomenon of about 24 hours inherent in an organism is called a circadian rhythm, and has been studied by many physiologists for a long time regardless of basic or clinical. Such a mechanism of generating a cycle is called a biological clock (or a biological clock). In order to clarify the molecular mechanism, a search has been made for a group of substances related thereto.

[0004] Drosophila is the most advanced molecular genetic analysis in the biological clock field. The period gene (per gene) was found as its internal clock gene, and the PER protein and mRNA encoded by the gene both show a certain circadian cycle of high night and low day in the brain,
The circadian rhythm of Drosophila was found to be controlled by genes. Further, the per gene is expressed not only in the brain, which is thought to play a central role in the biological clock, but also in somatic cells other than the eyes and the nervous system,
Similar to the brain, diurnal variations in mRNA and protein expression have been observed.

[0005] The structure of the protein encoded by the per gene has also been elucidated, and the translation region has a protein binding region called a PAS domain and a cytoplasmic localization region (CLD: cytoLD).
It has a structure required to retain the PER protein, a production protein called a plasmic location domein, in the cytoplasm (“genes controlling the biological clock”, Nario Ishida, Hyundai Chemistry, May 1996, 23) -28; “Biological clock genes and rhythm oscillation mechanisms common to flies and humans”, Katsutaka Oishi, Naoo Ishida, BIO Clinica 14 (7) 1999, 74-8
0).

[0006] Since many of the genes involved in fundamental life phenomena are conserved across species, it has been previously speculated that homologs of the Drosophila per gene will exist across species. .

In recent years, a per gene homologue having homology to the Drosophila per gene has been disclosed in organisms other than Drosophila and yeast (Mol. Cell. Biol. 14, 5569-557 (199)
4)) and from Neurospora crassa.
Like genes, it has been reported to be involved in the cell division cycle and the sporulation circadian cycle.

In 1997, the mouse per gene (mPer1) was reported as the first mammalian homolog of the Drosophila per gene from two groups in Japan and the United States (Sun, ZSe).
t al. Cell 90: (1997) pp. 1003-1011; Tei, H. et al.
Nature 389: (1997) pp.512-516). This gene has a PAS domain and a CLD similarly to the Drosophila per gene, and both have high homology in primary structure. Then, in succession, the above mPer1 and Drosophila
genes with homology to per, mPer2, rPer2 (rat p
er gene) and mPer3 have been reported (Takumi, T. et al.).
et al. EMBO J. 17, (1998) pp.4753-4759; Sakamoto,
K et al. J. Biol. Chem. 273 (1998) pp. 27039-2704
2).

[0009] As a result of in situ hybridization, these mammalian per genes are expressed locally and time-specifically in the suprachiasmatic nucleus (SCN). The same expression fluctuation as shown below indicates that gene expression is governed by an endogenous biological clock that forms a circadian rhythm.
In addition, it has been reported that these genes are expressed in peripheral tissues other than SCN as in Drosophila per (Sakamoto, K et al. J. Biol. Chem.
273 (1998) pp. 27039-27042).

[0010] Recently, results have been obtained indicating that the PER protein, which is a product of the per gene, acts on its own gene to influence its expression. It is suggested that circadian rhythm in vivo may be controlled by the mechanism. However, the PER protein itself has DN
Because there is no A-binding domain,
It is considered that the expression of the per gene is regulated.

The PAS domain in the Drosophila PER protein is known as a structure involved in protein-protein binding, and a second biological clock-related protein, timeless (tim), and its gene, which bind through this region. Has been reported, and the involvement of the complex in the biological clock molecule mechanism has been focused on and studied.

In order to grasp the prior art and conventional problems concerning the above-described biological clock gene in living organisms, the following documents can be referred to, and the teaching contents thereof are cited as disclosures in the present specification. Genes Governing the Clock ”, Nario Ishida, Modern Chemistry, May 1996, pp. 23-28; (2)“ Thinking about the molecular mechanism of biological clocks from genes: Implications for the cell cycle ”, Nario Ishida, Cell Engineering, Vol.15, No.8
1124-1130 (1996); (3) "Biological clock genes and rhythm oscillation mechanisms common to flies and humans", Katsutaka Oishi, Nario Ishida, BIO Clinica
14 (7) 1999, pp. 74-80; (4) “Brain clock and peripheral clock: central control of the diurnal rhythm of peripheral tissues viewed from the rat period gene”, Nario Ishida, Biological Sciences, 50 (3 ): 175-181, 1999.

[0013] Thus, information such as the overall picture of the biological clock obtained from the basic research and the detailed mechanism thereof is not limited to the fields of the basic research, but also the maintenance of sleep and homeostasis, the medication interval, the optimal time zone of the drug effect, and the like. It is also a very important issue from various clinical viewpoints, such as those involving human biological rhythms.

[0014]

An object of the present invention is to provide the desired information required in the art to elucidate the molecular mechanism of circadian rhythm in mammals including humans. Specifically, an object of the present invention is to provide a novel protein involved in the regulation of the circadian rhythm in the body of mammals including humans, its gene, and information on its function.

As such a protein and its gene,
Genes that are expressed with a certain circadian rhythm in the brain, which mainly controls the function of the biological clock, and their expression products (proteins) are listed. Such a gene is a novel gene having, in its structure, a PAS domain that is a region important for protein-protein interaction, which is characteristic of a biological clock gene. Hereinafter, in the present invention, the gene is referred to as rPer3 gene, and the expression product thereof is referred to as rPER3 protein.

By controlling the expression of the mammalian per gene group, which is a biological clock gene, development of a jet lag remedy or a novel sleeping pill, and application to treatment of rhythm disorders are expected.

[0017]

Means for Solving the Problems The present inventors have been studying day and night to isolate and identify mammalian clock-related substances involved in the molecular mechanism of the circadian rhythm for the above purpose. A novel gene having a structure (sequence) having extremely high homology with the PAS domain of the Drosophila and mouse per genes has been found from the upper cross nucleus (SCN), and the gene has been identified in the rat brain and peripheral organs. It was confirmed that the protein was expressed with a constant circadian rhythm.

The present invention has been completed based on the discovery of a series of biological clock-related genes and their expression products involved in the molecular mechanism of the circadian rhythm of mammals.

That is, the present invention provides the following 1. ~ 4. RPER3 protein and its gene (rPer3 gene), and useful partial DNA of the gene. 1. The following protein (a) or (b): (a) a protein consisting of the amino acid sequence represented by SEQ ID NO: 1, (b) one or more amino acids in the amino acid sequence represented by SEQ ID NO: 1 are deleted, substituted or A protein comprising an added amino acid sequence, having a homology of 85% or more with the amino acid sequence represented by SEQ ID NO: 1 and having a function of regulating a circadian rhythm. 2. The above 1. A gene encoding the protein described. 3. A gene consisting of the following polynucleotide (a) or (b): (a) a polynucleotide having the base sequence shown in SEQ ID NO: 2, (b) a polynucleotide consisting of the base sequence shown in SEQ ID NO: 2 and stringent Polynucleotide that hybridizes under various conditions The above polynucleotide includes a polynucleotide having the nucleotide sequence of SEQ ID NO: 3. 4. The above 2. Or 3. Detection of the gene described in
Or, a polynucleotide used as a specific probe or a specific primer for amplification.

The polynucleotide includes a polynucleotide used as a probe or a primer for detecting and / or amplifying a biological clock-related gene contained in the nucleotide sequence represented by SEQ ID NO: 2. The invention also provides such probes and primers.

The present invention also provides the following 5. ~ 11. These are listed below. 5. The above 1. A gene expression product having the amino acid sequence of 1. 6. 4 above. Using the specific probe or the specific primer of 2. Or 3. Or the amount of the gene described in 4. Circadian rhythm measurement method for measuring the amount of the gene expression product described in 1. 7. 4 above. Using the specific probe or the specific primer of 2. Or 3. Or the amount of the gene described in 4. A circadian rhythm detection kit for detecting the amount of the gene expression product according to 1. 8. The above 2. Or 3. Or the gene of 4. The gene expression product described in 1) is cultured in a medium containing a test substance, and Or 3. Or the amount of the gene described in 4. A method for screening a substance (agonist / antagonist) that affects the circadian rhythm, which measures the amount of a gene expression product of the above. 9. The above 1. Or a protein having the amino acid sequence of 5. An antibody against the gene expression product according to 1. 10. The above 2. Or 3. Or the gene of 4. A circadian rhythm abnormality improving agent comprising an effective amount of the gene expression product described in 1 above and a pharmaceutical carrier. 11. 9 above. A circadian rhythm abnormality improving agent comprising an effective amount of the antibody described in 1) and a pharmaceutical carrier.

The abbreviations of amino acids, peptides, base sequences, nucleic acids and the like in this specification are based on IUPAC.
-IUB regulations [IUPAC-IUB Communication on Biologi
calNomenclature, Eur.J. Biochem., 138: 9 (198
4)], "Guidelines for preparation of specifications containing base sequences or amino acid sequences" (edited by the Patent Office) and conventional symbols in the relevant field.

[0023]

BEST MODE FOR CARRYING OUT THE INVENTION (1) rPER3 protein The rPER3 protein of the present invention is a Drosophila PE
A novel circadian clock-related protein characterized by having a region homologous to the PAS domain of the R protein and the PAS domain of the mouse PER protein.

The rPER3 protein is characterized by having a cytoplasmic localization region (CLD) and a nuclear localization signal (NLD) in addition to a PAS domain (see FIG. 1). Its expression is time-specific as shown in the experimental examples described later, and from these facts, it is considered to be a protein involved in the molecular mechanism of circadian rhythm.

A specific example of such a protein is a rat-derived protein having the amino acid sequence represented by SEQ ID NO: 1. However, the rRER of the present invention
The three proteins are not limited to a protein having such a specific amino acid sequence.
Alternatively, it may have an amino acid sequence in which a plurality of amino acids have been deleted, substituted or added. That is, the present invention widely encompasses a mammalian homolog corresponding to the rat-derived rPER3 protein having the amino acid sequence represented by SEQ ID NO: 1.

Here, the degree of "deletion, substitution or addition of amino acids" and their positions are determined by the fact that the modified protein has the same function as the protein consisting of the amino acid sequence represented by SEQ ID NO: 1, that is, the circadian cycle. There is no particular limitation as long as the substance has a rhythm regulating function. The function of regulating the circadian rhythm is to change / increase or decrease the binding, responsiveness, or expression level of the rPER3 protein, rPER3 gene, and rPER3 gene expression product, thereby changing the body temperature and blood pressure inherent in the organism, It refers to the function of regulating abnormalities in the activity rhythm that occur in a living body in about a 24-hour cycle, such as changes in the secretion of endocrine substances and changes in sleep and wakefulness.

The rRR3 protein is specifically a protein having a PAS domain equivalent region, a CLD equivalent region, and an NLD equivalent region, and is about 85% or more, preferably 90% or more, of the amino acid sequence represented by SEQ ID NO: 1. And more preferably those having a homology of 93% or more. The proteins of the present invention include mouse-derived mPER1 protein, mPER2 protein and mPER2 protein.
Those having the same amino acid sequence as the PER3 protein are not included. The protein of the present invention is preferably 78-85%, preferably 7%, of the mPER3 protein in the amino acid sequence.
Having a homology of 8 to 84.4%, mPER1
Those having 30 to 35%, preferably 30 to 34.8% homology with the protein, or those having 30 to 34% homology with the mPER2 protein can be exemplified.

It should be noted that these amino acid sequence alterations (mutations) and the like may occur in nature, for example, due to mutations or post-translational modifications, but are based on naturally-occurring genes (eg, specific examples of the invention). It can also be modified artificially. The present invention encompasses all modified genes having the above-mentioned properties, irrespective of the cause and means of such modification / mutation.

As the above-mentioned artificial means, for example, site-specific mutagenesis [Methods in Enzym
ology, 154: 350, 367-382 (1987); id. 100: 468 (198
3); Nucleic Acids Res., 12: 9441 (1984); Lecture on Sequent Chemistry Experiment 1 "Genetic Research Method II", edited by The Japanese Biochemical Society, p105
(1986)] and chemical synthesis means such as the phosphate triester method and the phosphate amidite method [J. Am. Che.
m. Soc., 89: 4801 (1967); 91: 3350 (1969); Scien
ce, 150: 178 (1968); Tetrahedron Lett., 22: 1859 (1
981); ibid .: 24: 245 (1983)] and methods of combining them. (2) rPer3 gene The present invention is a gene encoding the above protein.

As one embodiment of the rPer3 gene of the present invention, a gene consisting of a polynucleotide having the nucleotide sequence of SEQ ID NO: 2 can be exemplified. However, this base sequence is an example of one combination of codons indicating each amino acid residue in the above amino acid sequence (SEQ ID NO: 1), and thus the gene of the present invention is not limited to these, Of course, it is also possible to have a base sequence in which the codons are selected in combination. 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 [Ncleic Acids Res., 9:43 (19
81)].

Further, the rPer3 gene of the present invention is not limited to a polynucleotide comprising the specific nucleotide sequence shown in SEQ ID NO: 2, but may be any one having a certain homology with the nucleotide sequence. One or more nucleotides may be deleted, substituted or added in the sequence. Such genes include those that hybridize under stringent conditions to the DNA consisting of the nucleotide sequence of SEQ ID NO: 2 and are not eliminated therefrom even by washing under certain conditions. For example,
A polynucleotide having the nucleotide sequence of SEQ ID NO: 2,
× SSC (standard saline citrate; 1 × SSC = 0.15M
NaCl, 0.015 M sodium citrate) at 65 ° C. overnight or in 4 × SSC containing 50% formamide at 37 ° C. overnight, and then hybridize at 55 ° C. in 2 × SSC.
Examples are polynucleotides having a nucleotide sequence that is not eliminated from the DNA even under washing conditions at 30 ° C. for 30 minutes. The length of the polynucleotide is not particularly limited as long as it hybridizes under such conditions. Such genes include the 5 'end of the nucleotide sequence of SEQ ID NO: 2 and / or
Alternatively, a gene having a (poly) nucleotide added to the 3 ′ end, specifically, a gene having the base sequence shown in SEQ ID NO: 3 is included.

The rPer3 gene of the present invention is represented, for example, in SEQ ID NO: 2 as a base sequence of a single-stranded DNA, but is not limited to the single-stranded polynucleotide, and is complementary to the base sequence of the polynucleotide. It naturally includes a polynucleotide comprising a simple nucleotide sequence, and a component containing both of them.
It is not limited to such DNA.

The gene of the present invention can be easily produced and obtained by general genetic engineering techniques on the basis of sequence information on specific examples [Molecular Clonin
g 2dEd, Cold Spring Harbor Lab. Press (1989); see Seismic Chemistry Laboratory Lecture “Gene Research Methods I, II and III”, edited by The Biochemical Society of Japan (1986), etc.). Specifically, a cDNA library is prepared from a suitable source expressing the gene of the present invention according to a conventional method, and the desired clone is cloned from the library using an appropriate probe or antibody specific to the gene of the present invention. [Proc. Natl. Aca
d. Sci., USA., 78: 6613 (1981); Science, 222: 778.
(1983) etc.].

In the above, the origin of cDNA is as follows:
Examples include various cells and tissues expressing the gene of the present invention, and cultured cells derived therefrom, and the total RNA therefrom.
, MRNA isolation and purification, cDNA acquisition and cloning thereof can all be carried out according to conventional methods. still,
cDNA libraries are commercially available, and in the present invention, such cDNA libraries, for example, various cDNA libraries commercially available from Clontech Lab. Inc. can also be used.

The method for screening the gene of the present invention from a cDNA library is not particularly limited, either, and any conventional method can be used. Specifically, for example, cDNA
For selecting a corresponding cDNA clone by immunoscreening using a specific antibody of a protein produced by the method, plaque hybridization using a probe that selectively binds to a target DNA sequence, colony hybridization, or a combination thereof Etc. can be exemplified.

As the probe used herein, DNAs chemically synthesized based on information on the nucleotide sequence of the gene of the present invention can be generally exemplified. Of course, the gene of the present invention itself or a fragment thereof which has already been obtained can of course be used. Etc. can be used well.

Further, sense primers and antisense primers set based on the nucleotide sequence information of the gene of the present invention can also be used as screening probes.

In obtaining the gene of the present invention, PCR
DNA / RN by the method [Science, 230: 1350 (1985)]
The A amplification method can also 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 (1994)), particularly 5′-race (5'-
RACE) method [Proc. Natl. Acad. Sci., USA., 8: 899
8 (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 amplified DNA / RNA fragment can be isolated and purified according to a conventional method as described above, for example, by gel electrophoresis.

The gene of the present invention or various DNs obtained above
The A fragment can be obtained by a conventional method, for example, the dideoxy method [Proc. Natl.
ad.Sci., USA., 74: 5463 (1977)) and the Maxam-Gilbert method (Method in Enzymology, 65: 499 (1980)), and the like, and conveniently using a commercially available sequence kit or the like. The sequence can be determined.

According to the use of the gene of the present invention, the rPER3 protein, which is the gene product, can be easily and stably produced in large quantities by using general genetic engineering techniques.

That is, the present invention provides a vector (expression vector) containing the rPer3 gene of the present invention, a host cell transformed with the vector, and a method for producing a desired rPER3 protein by culturing the host cell. Is also provided.

The production method can be carried out by a conventional gene recombination technique [Science, 224: 1431 (1984); Biochem. Biophys.
s. Comm., 130: 692 (1985); Proc. Natl. Acad. Sci.,
USA., 80: 5990 (1983) and the above-cited references].

As the host cell, either prokaryote or eukaryote can be used. For example, prokaryote hosts widely include commonly used ones such as Escherichia coli and Bacillus subtilis. Can be exemplified by Escherichia coli, especially those contained in the Escherichia coli K12 strain. In addition, eukaryotic host cells include vertebrates, yeast and other cells, and as the former,
For example, COS cells [Cell, 23: 175 (1
981)) and Chinese hamster ovary cells and their dihydrofolate reductase-deficient strains (Proc. Natl. Acad. Sc.
i., USA., 77: 4216 (1980)]. As the latter, yeast cells of the genus Saccharomyces and the like are preferably used, but not limited thereto.

In the case of using a vertebrate cell as a host, the expression vector usually has a promoter, an RNA splice site, a polyadenylation site, a transcription termination sequence, etc., located upstream of the gene of the present invention to be expressed. Which may optionally have a replication origin. Specific examples of the expression vector include, for example, pS having the early promoter of SV40.
V2 dhfr [Mol. Cell. Biol., 1: 854 (1981)] and the like. Further, as a specific example of an expression vector when a yeast cell is used as a host, for example, pAM82 [Proc.
Acad. Sci., USA., 80: 1 (1983)].

When a prokaryotic cell is used as a host, a promoter and an SD (Shine & Sand) are used upstream of the gene so that the gene of the present invention can be expressed in the vector using a vector that can be replicated in the host cell.・ Dargano)
An expression plasmid to which a base sequence and an initiation codon (for example, ATG) necessary for initiation of protein synthesis are added can be suitably used. As the above-mentioned vector, pBR322 and its improved vector are often used in general, but are not limited thereto, and various vectors can be used. There is no particular limitation on the promoter, for example, tryptophan (trp)
Promoter, lpp promoter, lac promoter,
Any of the PL / PR promoter and the like can be suitably used.

As the expression vector for the gene of the present invention, an ordinary fusion protein expression vector can also be preferably used.
Specific examples of the vector include pGEX (Promega) for expression as a fusion protein with glutathione-S-transferase (GST).

The desired recombinant DNA (expression vector)
There is no particular limitation on the method of introducing and transforming the host into a host cell, and various general methods can be employed. The obtained transformant can also be cultured according to a conventional method, whereby the target rPER3 protein encoded by the gene of the present invention is expressed and produced, and the transformant is intracellularly, extracellularly or on the cell membrane of the transformant. Is accumulated or secreted.

As the medium used for the cultivation, various types of media commonly used depending on the host cells used can be appropriately selected and used, and the cultivation can be carried out under conditions suitable for the growth of the host cells.

The thus obtained recombinant protein (rPER
Protein) can be separated and purified according to various separation operations utilizing its physical properties, chemical properties, etc., if desired [“Biochemical Data Book II”, pp. 1175-1259,
1st edition, 1st printing, June 23, 1980, published by Tokyo Chemical Co., Ltd .; Biochemistry, 25 (25): 8274 (1986); Eur. J. Bio
Chem., 163: 313 (1987), etc.]. As the method,
Specifically, for example, normal reconstitution treatment, treatment with a protein precipitant (salting out method), centrifugation, osmotic shock method, ultrasonic crushing, ultrafiltration, molecular sieve chromatography (gel filtration), adsorption chromatography, Various liquid chromatography such as ion exchange chromatography, affinity chromatography, high performance liquid chromatography (HPLC), dialysis, combinations thereof and the like can be mentioned. Particularly preferable method is the specific antibody for rPER3 protein of the present invention. An example is affinity chromatography using a bound column.

Thus, the present invention also provides a novel rPER3 protein itself obtained, for example, as described above.

The rPER3 protein can also be used as an immunizing antigen for preparing a specific antibody of the protein.
The component used as an antigen here can be, for example, a protein or a fragment thereof produced in large amounts according to the above-described genetic engineering techniques. By using these antigens, a desired antiserum (polyclonal antibody) and monoclonal antibody can be used. An antibody can be obtained.
The method for producing the antibody itself is well understood by those skilled in the art, and these methods can be followed in the present invention [Sequence Chemistry Laboratory Course “Immunobiochemical Research Method”, edited by The Biochemical Society of Japan (1986) ) Etc.].

For example, a normal animal such as a rabbit, a guinea pig, a rat, a mouse or a chicken can be arbitrarily selected as an immunized animal used for obtaining an antiserum. It can be implemented according to the law.

In addition, a monoclonal antibody can be obtained by a conventional method by preparing a fusion cell of a plasma cell (immunocyte) and a plasmacytoma cell of an animal immunized with the above-mentioned immunizing antigen, and producing a clone producing the desired antibody therefrom. And culturing the clone. Immunized animals are generally selected in consideration of compatibility with plasmacytoma cells used for cell fusion, and mice and rats are usually advantageously used. Immunization is the same as in the case of the above antiserum, and can be carried out in combination with a usual adjuvant if desired.

The plasmacytoma cells used for the fusion are not particularly limited. For example, p3 (p3 / x63-Ag8) [Natu
re, 256: 495-497 (1975)], p3-U1 [Current Top
icsin Microbiology and Immunology, 81: 1-7 (197
8)], NS-1 [Eur. J. Immunol., 6: 511-519 (197
6)], MPC-11 [Cell, 8: 405-415 (1976)], S
All kinds of myeloma cells such as P2 / 0 [Nature, 276: 269-271 (1978)] and R210 in rats [Nature, 277: 131-133 (1979)] and cells derived therefrom are used. it can.

The fusion between the above immune cells and plasmacytoma cells can be carried out in the presence of a conventional fusion promoter such as polyethylene glycol (PEG) or Sendai virus (HVJ) according to a known method. Separation of the desired hybridoma can also be performed [Meth. In Enzym
ol., 73: 3 (1981);

Further, the search for the antibody-producing strain of interest and the monocloning are also performed by a conventional method. For example, the search for the antibody-producing strain is carried out by the ELISA method using the antigen of the present invention [Meth. In Enzymol. 70: 419-439 (1980)], plaque method, spot method, agglutination method, octeroney (Oucht)
erlony) method, radioimmunoassay and the like, and can be carried out according to various methods generally used for detection of antibodies.

The antibody of the present invention can be collected from the thus obtained hybridoma by culturing the hybridoma by a conventional method to obtain a culture supernatant thereof, or by administering the hybridoma to a mammal compatible with the hybridoma and growing it. It is carried out by a method for obtaining ascites or the like. The former method is
It is suitable for obtaining high-purity antibodies, and the latter method is suitable for mass production of antibodies. The antibody thus obtained can be further purified by ordinary means such as salting out, gel filtration, and affinity chromatography.

The antibody thus obtained is the rPE of the present invention.
Characterized by having binding to the R3 protein,
This can be advantageously used for the above-mentioned purification of rPER3 protein and its measurement or identification by immunological techniques.

The present invention also provides such a novel antibody.

Further, based on the sequence information of the gene of the present invention revealed by the present invention, for example, by utilizing a part or all of the nucleotide sequence of the gene, the present invention can be used in individuals or various tissues. A gene or expression of the gene can be qualitatively or quantitatively detected,
The detection makes it possible to measure the circadian rhythm of the living body. Therefore, the present invention also relates to a method for measuring the circadian rhythm of a living body using the gene of the present invention.

Detection of the above gene or its expression can be carried out according to a conventional method. For example, RT-PCR [Reve
rse transcribed-Polymerase chain reaction; ES Ka
wasaki, et al., Amplification of RNA.In PCR Proto
col, A Guide to methods and applications, Academic
Press, Inc., SanDiego, 21-27 (1991)]
Amplification and Northern blot analysis (Molecular Cloning, Col
d Spring Harbor Lab. (1989)], in situ RT-PCR
[Nucl. Acids Res., 21: 3159-3166 (1993)] and in si
tu Measurement of them at the cell level such as hybridization, NASBA method [Nucleic acid sequence-based am
plification, Nature, 350: 91-92 (1991)] and various other methods.

In the case where the RT-PCR method is employed, the primers used are not particularly limited as long as they are specific to the gene of the present invention and can specifically amplify only the gene of the present invention. Thus, the arrangement can be appropriately set. Usually, it can have a partial sequence of the order of 20 to 30 nucleotides.

As described above, the present invention relates to the present invention.
It also provides a DNA fragment used as a specific primer and / or a specific probe for detection and / or amplification of the PER3 gene.

The method for measuring a circadian rhythm according to the present invention is as follows.
By using a reagent kit for detecting the rPER3 gene in a sample, it can be easily carried out.

Therefore, the present invention provides a reagent kit for detecting rPER3, which comprises the above rPER3 DNA fragment.

The reagent kit comprises a DNA fragment that hybridizes to at least a part or all of the nucleotide sequence shown in SEQ ID NO: 2 or its complementary nucleotide sequence (provided that the D
If the NA sequence does not completely match a known sequence such as mPER3) as an essential component, as other components, a labeling agent, a reagent essential for PCR (for example, Ta)
qDNA polymerase, deoxynucleotide triphosphates, primers, etc.).

Examples of the labeling agent include a chemically modified substance such as a radioisotope or a fluorescent substance, and the DNA fragment itself may be conjugated with the labeling agent in advance. Further, the reagent kit may contain a suitable reaction diluent, a standard antibody, a buffer, a detergent, a reaction stop solution, etc., for the benefit of performing the measurement.

The present invention also provides a method for diagnosing a circadian rhythm abnormality using the above-described measurement method, a diagnostic agent and a diagnostic kit used in the method.

Further, by using the method described above, the rPER3 sequence obtained from the test sample is directly or indirectly sequenced, thereby obtaining a new rPER3 having a high homology with wild-type rPER3. Related genes related to genes can be found.

Accordingly, the present invention also provides a method for screening for a related gene related to the human rPER3 gene in a test sample by such measurement and sequencing of rPER3 DNA in the test sample.

The present invention can also provide an rPER3 binding protein present in the cytoplasmic fraction or cells having rPER3 binding activity to the rPER3 polypeptide. The rPER3 binding protein can be obtained by labeling rPER3 in a biomaterial sample containing a cell membrane fraction.
This is accomplished by conjugating the protein, extracting, isolating and purifying the rPER3 binding reaction, and identifying the amino acid sequence of the isolate. Acquisition and sequencing of the rPER3 binding protein is accomplished by those skilled in the art. Can be easily achieved.

The present invention also provides a method for screening a compound (rPER3-binding protein reactant: a compound is a low-molecular compound, a high-molecular compound,
It can be used for screening proteins (polypeptides), protein (polypeptide) partial fragments, antigens, antibodies, etc.). Preferably, an rPER3 binding protein is used. RP used for such screening test
The ER3 binding protein or fragment thereof may be free in solution attached to a solid support or carried to the cell surface.

As an example of drug screening, for example, prokaryotic or eukaryotic host cells stably transformed with a recombinant polypeptide expressing the rPER3 polypeptide or a fragment thereof can be used, preferably in a competitive binding assay. Can be used. Such cells, either in free or fixed form, can also be used in standard binding assays. More specifically, the formation of a complex between the rPER3 binding protein or fragment thereof and the substance to be tested is measured, and the rPER3 binding protein or fragment thereof and rPER
Compounds can be screened by detecting the extent to which the formation of the complex between the three polypeptides or fragments thereof is inhibited by the agent being tested.

Thus, the present invention provides for contacting such a substance with an rPER3-binding protein or a fragment thereof by methods known in the art, and then presenting a complex between the substance and the rPER3-binding protein or a fragment thereof. Or rP
It is possible to provide a drug screening method characterized by measuring the presence of a complex between an ER3 binding protein or a fragment thereof and a ligand.

Furthermore, by measuring the rPER3 binding protein activity, such substances can inhibit the rPER3 binding protein, thus regulating the circadian rhythm of rPER3 as defined above,
For example, whether it is possible to control a cyclic rhythm that moves forward or backward the cycle of the circadian rhythm, or rPER3m
It is determined whether the expression level of RNA can be regulated. In such competitive binding assays, more specifically, rPE
The R3 binding protein or a fragment thereof is labeled. Free rPE
The R3 binding protein or fragment thereof is separated from that present in the protein: protein complex, and the amount of free (uncomplexed) label depends on the binding of the agent to be tested to the rPER3 binding protein or the rPER3 binding protein: It is a measure of the inhibition of rPER3 protein binding. A small peptide (peptide mimetic) of the rPER3 protein was analyzed in this way and rPE3
Those having R3 binding protein inhibitory activity can be measured.

In the present invention, another method for drug screening is a screening method for a compound having an appropriate binding affinity for an rPER3-binding protein. The peptide test compound is synthesized on a solid support, such as the surface of a plastic pin or other material, and the peptide
React with ER3 binding protein and wash. Subsequently, a method for detecting the reaction-bound rPER3 binding protein using a known method can also be exemplified (PCT Patent Publication No. WO84-035).
No. 64). The purified rPER3 binding protein can be directly coated on a plate used in the drug screening technique described above. However, the rPER3 binding protein can be immobilized on a solid phase by capturing the antibody with a non-neutralizing antibody against the polypeptide. The present invention is further directed to the use of a competitive drug screening assay, wherein a test compound competes with a neutralizing antibody capable of specifically binding to the rPER3 binding protein for binding to the rPER3 binding protein or a fragment thereof. The competition by the antibodies makes it possible to detect the presence of any peptide having one or more antigenic determinants of the rPER3 binding protein.

Further, with respect to drug screening, additional methods include the use of host eukaryotic cell lines or cells containing a non-functional rPER3 gene. After growing the host cell line or cell in the presence of the drug compound for a period of time, the rate of growth of the host cell is measured to allow the compound to, for example, modulate the circadian rhythm, e.g., advance or cycle the circadian rhythm. It can control the cyclic rhythm of regression, or can bind to or dissociate from the protein that regulates the expression level of rPER3 mRNA to regulate the transfer of the binding protein in blood and tissues, See if you can adjust it. As one means of measuring the growth rate, it is possible to measure the biological activity of the rPER3 binding protein.

Also according to the present invention, to develop a more active or stable form of a rPER3 polypeptide derivative or, for example, an agent that enhances or interferes with the function of an rPER3 polypeptide in vivo, It is possible to make biologically active polypeptides or structural analogs of interest with which they interact, such as rPER3 agonists, rPER3 antagonists, rPER3 inhibitors and the like. The structural analog may be, for example, a three-dimensional structure of a complex of rPER3 and another protein by X-ray crystallography, computer modeling, or
It can be determined by a combination of these methods. Information on the structure of a structural analog can also be obtained by protein modeling based on the structure of a homologous protein.

The more active or more stable form of r
As a method for obtaining a PER3 polypeptide derivative, analysis can be performed by, for example, alanine scanning. The method involves substituting Ala for an amino acid residue and measuring each of the amino acid residues of the peptide in a manner that measures its effect on the activity of the peptide, thus determining regions important for the activity and stability of the peptide. Is the way. By this method, more active or stable rPER3 polypeptide derivatives can be designed.

It is also possible to isolate the target-specific antibody selected by the functional assay and then analyze its crystal structure. In principle, this approach allows the pharmacoma (pharmac
ore). By generating anti-idiotypic antibodies to functional pharmacologically active antibodies, it is possible to identify and isolate peptides from a bank of chemically or biologically generated peptides. Therefore, the selected peptide is also expected to act as a pharmacore.

Thus, a drug having improved rPER3 activity or stability or an action as an inhibitor, agonist, antagonist or the like of rPER3 activity can be designed and developed.

In view of the above, the present invention provides a circadian rhythm abnormality improving agent having an effective amount of the above-mentioned rPER3 gene, its expression product or antibody of the present invention. The circadian rhythm abnormality ameliorating agent can contain a pharmaceutically acceptable pharmaceutical carrier or additive, as long as the effects of the present invention are not impaired. In addition, diseases associated with the circadian rhythm include advanced sleep phase syndrome, delayed sleep phase syndrome, irregular sleep-wake disorders, jet lag, and the like (WO99-14324), and the circadian cycle of the present invention. It is considered that a rhythm ameliorating agent is effective for treating such diseases.

[0084]

According to the present invention, there is provided a novel mammal-derived biological clock-related protein responsible for the molecular mechanism of the circadian rhythm, and a novel gene encoding the protein. These applications will enable the elucidation of the circadian rhythm and the molecular mechanism of the biological clock in mammals.

The rPer3 gene of the present invention is expressed daily in the suprachiasmatic nucleus (SCN), which is the center of the circadian clock. It seems that it is also possible to control and control the circadian rhythm.

Thus, development of a jet lag remedy or a novel sleeping pill that does not go against the biological rhythm is expected. Recently, it has been revealed that certain types of manic depression and school refusal are also rhythm disorders. Therefore, SCN
It is hoped that controlling the expression of many circadian genes within them will alleviate or treat the symptoms of these diseases.

As shown in the experimental examples, the rPER3 of the present invention
The mRNA shows expression with a constant circadian rhythm, high at night and low during the day, not only in the hypothalamus SCN but also in peripheral organs such as the cerebellum, cerebrum and eyes. Therefore, the rPER3 protein of the present invention or the protein having a partial amino acid thereof, the rPer3 gene or the polynucleotide having a partial base sequence thereof, based on this property, can be used as a time-specific marker in the brain, which is the center controlling the biological clock. It seems to work. Therefore, it is useful as a marker of the circadian rhythm.

[0088]

The present invention will now be described in further detail with reference to examples. Example 1 Acquisition of rPer3 gene From cDNA library of rat hypothalamic suprachiasmatic nucleus (SCN), (1) Preparation of cDNA library of rat hypothalamus suprachiasmatic nucleus (SCN) Rat suprachiasmatic nucleus (SCN) The cDNA library (3.3 × 10 ⁷ cDNA) was prepared as follows. First, rat suprachiasmatic nucleus cells were dissolved in a guanidine thiocyanate solution, layered on a CsCl solution, and total RNA was collected as a precipitate by ultracentrifugation and purified by ordinary phenol / chloroform and ethanol treatment. Poly (A) by oligo dT cellulose chromatography
+ RNA was isolated, cDNA was purified by reverse transcriptase, double-stranded by incubation with E. coli DNA polymerase I and RNaseH, and an EcoRI linker was ligated to its end. This cDNA is converted to λ
gt10 into the EcoRI site, λ in vitro
Packaging was performed using a packaging kit (manufactured by Amersham) to prepare a cDNA library. (2) Screening A clone homologous to the PAS region, which is a Drosophila protein binding region, was screened. The obtained clone was subjected to PCR according to a conventional method using a Gesmer in the PAS region. The amplified PCR product is 180
bp DNA fragment.

Using this DNA fragment, the rat SCN cDNA library prepared in (1) was screened. Clones that hybridize under appropriate conditions (rPe
r3 clone) and the DNA sequence was determined by a conventional method. The hybridization conditions that can be used here are 50-60 ° C., 1 × SSC for 30-50 minutes, preferably 50-60 ° C., 1 × SSC, 0.1% SSC.
DS, 40 minutes.

MRN corresponding to the rPER3 protein encoded by the cDNA of the obtained rPer3 clone
The presence of A in the normal rat SCN-derived poly (A) + RNA was confirmed by RT-PCR. Further, by the primer extension method, this cDNA is not a fragment but a full-length r.
It was confirmed that PER3 was encoded. It was also confirmed that this mRNA was actually translated into the expected protein in a cell-free translation system.

The nucleotide sequence of the obtained cDNA and the amino acid sequence encoded thereby are shown in SEQ ID NO: 3. As a result of sequencing, the rPER3 protein of the present invention showed PAS-A and 270 to 164 amino acid regions from the N-terminus.
The 319 amino acid region had the PAS-B PAS domain region, the 337-392 amino acid region had the cytoplasmic localization region (CLD), and the 761-777 amino acid region had the nuclear localization signal (NLD). . As described above, the rPER3 protein and the rPer3 gene of the present invention have a PAS domain which is a characteristic of a biological clock-related protein (gene), indicating that they are biological clock-related proteins and genes.

FIG. 1 schematically shows the structure of the rPER3 protein encoded by the gene. As you can see from this figure,
The protein has a PAS-A domain in the 130 to 164 amino acid region and a P
CL in AS-B domain, 337-392 amino acid region
NLS (nuclea) in the D region, 761-777 amino acid region
r localization signal). 2 to 4 show the homology between the amino acid sequence of the rPER3 protein of the present invention and the amino acid sequence of the mouse PER protein family (mPER1, mPER1, mPER1). Surround the common amino acids in black,
From this figure, it can be seen that the rPER3 protein of the present invention has a homology as high as 78.0 to 84.4% with the mPER3 protein. On the other hand, mPER1 protein and mPER2
For proteins, the homologies were 30.0-34.8% and 30.0-34.0%, respectively.

The above rPer3 clone was replaced with PBS.
SK^-PBSSK recombined into vector^-The JM109 bacteria
Introduced into the strain (Identification: rPer3 PBS
SK ^-/ JM109) on October 19, 1999
Industrial Technology Institute with an address at 1-3 1-3 Higashi, Tsukuba City, Castle Prefecture
Accession number FERM P-17 to Biotechnology Industrial Technology Research Institute
No. 614.

Example 2 rPER3 in rat SCN
Diurnal Variation of mRNA Expression Rats were cultured under each condition [bright and dark conditions (LD: 12-
12 hours), dark and dark (DD) and light and bright (L
L)]. The rats used in the experiment [Wistar rats (male: 10 weeks old, 300 to 350 g, CLEA Japan)]
At least one week before the experiment, under light and dark conditions (LD: 12-1)
2 hours).

A predetermined time (5 hours) after lighting (subjective day)
5 hours and 11 hours or 2 hours and 8 hours, indicated on the bar in FIG. 5) and a predetermined time after lighting (subjective night) (17 hours and 23 hours or 14 hours and 20 hours, indicated on the bar in FIG. 5) ), Each rat was killed to remove the hypothalamus, and total mRNA was extracted from the suprachiasmatic nucleus (SCN) of the hypothalamus by the guanidine thiocyanate method. For each mRNA, the expression level of rPER3 mRNA was measured by Northern blotting using a probe designed from the nucleotide sequence of the rPer3 gene.

FIG. 5 shows the results. In the figure, the light period is indicated by a white bar, and the dark period is indicated by a black bar. The total mRNA used for each lane was 10 μg, and the washing conditions were 1 × SSC,
0.1% SDS, 50 ° C., 40 minutes was used.

As a result, two of 9.5 kb and 7.5 kb were obtained.
Revealed that there are major types of mRNA,
Furthermore, it turned out that these two types are expressed with a fixed diurnal rhythm of high night and low day.

Example 3 rPE in rat peripheral organs
Expression of R3 mRNA Rats were bred under light and dark conditions (LD: 12-12 hours), and after 6 hours of lighting (ZT6: subjective day) and 18 hours after lighting (ZT18: subjective night),
Total mRNA was extracted from the pineal gland, eye, brain, heart, lung, pancreas, liver and kidney of each rat according to a conventional method using guanidine thiocyanate, and the expression level of rPER3 mRNA was measured by Northern blotting. FIG. 6 shows the results.

[0099] With respect to the heart and lungs in particular, the expression level of rPER3 mRNA at each time point was measured more minutely. Specifically, under light and dark conditions (LD: 12
-12 hours), after lighting (subjecti
ve day) 2 hours, 5 hours, 8 hours and 11 hours, and 14 hours and 17 hours after lighting (subjective night)
At predetermined times of time, 20 hours and 23 hours, the heart and lungs are removed and total mRNA is extracted from these organs and
The expression level of PER3 mRNA was measured. FIG. 7 shows the results.

From these results, it was found that rPER3 mRNA was also expressed in peripheral organs as in SCN, and that all regions showed circadian expression of high night and low day. In particular, the oscillation of expression in the pancreas and spleen was remarkable.

As described above, as shown in Examples 2 and 3, rP
ER3 mRNA showed expression with a constant circadian rhythm, high at night and low during the day, not only in the hypothalamus SCN but also in the pineal gland, eye, brain, heart, lung, pancreas, liver and kidney. Thus, the rPER3 protein or its partial protein, the rPer3 gene or its partial gene of the present invention is useful as a time-specific marker (biological rhythm marker) in the brain, which is the center controlling the biological clock, based on this property. is there.

[0102] Further, since the rPer3 gene is expressed daily in the mammalian biological clock center (SCN), it is considered that the biological rhythm can be adjusted by controlling the expression of rPER3 with an antisense nucleic acid or a specific antibody.
Therefore, according to the present invention, development of a jet lag remedy or a novel sleeping pill that does not go against the biological rhythm is expected. Recently, it has been revealed that certain types of manic depression and school refusal are also rhythm disorders. Therefore, it is expected that the regulation of the expression of many circadian genes in SCN can be a therapeutic agent for these diseases.

[0103]

[Sequence List] <110> Otsuka Pharmaceutical Co. Ltd .; National Institute of Bioscience and Human-Technology; Norio Ishida <120> Structure of Novel Circadian Rhythm Marker Gene rPer3 and Function there <130> 24D9JP <160> 3 <210 > 1 <211> 1119 <212> PRT <213> Wister rat <220> <221> PAS A <222> (130) ... (164) <220> <221> PAS B <222> (270). .. (319) <220> <221> CLD <222> (337) ... (392) <220> <221> NLS <222> (761) ... (777) <400> 1 Met Asp Pro Cys Gly Asn Pro Ala Val Pro Gly Gly Asp Cys Pro Gln 5 10 15 Thr Arg Gly Pro Gly Leu Gln Gly Ser Ser Gly Gln Glu Gly Pro Leu 20 25 30 Gln Gly Ile Cys Val Asp Ser Ser His Ser Glu His Glu Asp Arg Asn 35 40 45 Arg Met Ser Glu Glu Leu Ile Met Val Val Gln Glu Met Lys Lys Tyr 50 55 60 Phe Pro Ala Glu Arg His Thr Lys Pro Ser Thr Leu Asp Ala Leu Asn 65 70 75 80 Tyr Ala Leu Arg Cys Val His Ser Val Gln Ala Asn Ser Glu Phe Phe 85 90 95 Gln Ser Leu Ser Pro Arg Gly Ala Arg Gln Ala Glu Ala Thr Val Tyr 100 105 110 Asn Leu Glu Glu Leu Thr Ser Leu Ala Ser Glu His Thr Ser L ys Asn 115 120 125 Thr Asp Thr Phe Val Ala Val Phe Ser Phe Leu Ser Gly Arg Leu Val 130 135 140 His Ile Ser Glu Gln Ala Ala Trp Ile Leu Asn Ser Lys Lys Gly Phe 145 150 155 160 Leu Lys Ser Leu His Phe Val Asp Leu Leu Ala Pro Arg Asp Val Arg 165 170 175 Val Phe Tyr Ala His Thr Ala Pro Thr Gln Leu Pro Phe Trp Asn Thr 180 185 190 Trp Thr Gln Arg Ala Ser Gln Tyr Glu Cys Ala Pro Val Lys Pro Phe 195 200 205 Phe Cys Arg Ile Cys Gly Gly Gly Asp Arg Glu Gln Lys Arg His Tyr 210 215 220 Ser Pro Phe Arg Ile Leu Pro Tyr Leu Val His Val His Ser Pro Ala 225 230 235 240 Gln Pro Glu Pro Glu Pro Cys Cys Leu Thr Leu Val Glu Lys Ile His 245 250 255 Ser Gly Tyr Glu Ala Pro Arg Ile Pro Val Asp Lys Arg Val Phe Thr 260 265 270 Thr Thr His Thr Pro Gly Cys Val Phe Leu Glu Val Asp Glu Arg Ala 275 280 285 Val Pro Leu Leu Gly Phe Leu Pro Gln Asp Leu Ile Gly Thr Ser Ile 290 295 300 Leu Thr Tyr Leu His Pro Glu Asp Arg Pro Leu Met Val Ala Val His 305 310 315 320 Gln Lys Val Leu Lys Tyr Val Gly His Pro Pro Phe Glu His S er Pro 325 330 335 Ile Arg Phe Cys Thr Gln Asn Gly Asp Tyr Val Ile Leu Asp Ser Ser 340 345 350 Trp Ser Ser Phe Val Asn Pro Trp Ser Arg Lys Val Ser Phe Ile Ile 355 360 365 Gly Arg His Lys Val Arg Thr Ser Pro Leu Asn Glu Asp Val Phe Ala 370 375 380 Thr Arg Ile Lys Lys Ala Thr Ser His Asp Glu Asp Ile Thr Glu Leu 385 390 395 400 Gln Glu Gln Ile His Arg Leu Leu Leu Gln Pro Val His Ala Ser Ala 405 410 415 Ser Ser Gly Tyr Gly Ser Leu Gly Ser Ser Gly Ser Gln Glu Gln His 420 425 430 Ile Ser Val Thr Ser Ser Ser Glu Ser Ser Gly His Cys Val Glu Glu 435 440 445 Ala Gln Gln Glu Gln Met Thr Leu Gln Gln Val Tyr Ala Ser Val Asn 450 455 460 Lys Ile Lys Asn Val Gly Gln Gln Leu Tyr Ile Glu Ser Met Ala Arg 465 470 475 480 Ser Ser Val Lys Pro Val Met Glu Thr Cys Thr Glu Pro Gln Gly Ser 485 490 495 Asp Glu Gln Lys Asp Phe Ser Ser Ser Gln Thr Leu Lys Asn Lys Ser 500 505 510 Thr Asp Thr Gly Ser Gly Gly Asp Leu Arg Pro Glu Gln His Ser Ser 515 520 525 Ser Tyr Gln Gln Met Asn Cys Ile Asp Ser Val Ile Arg Tyr Leu T hr 530 535 540 Ser Tyr Ser Phe Pro Ala Leu Lys Arg Lys Cys Ile Ser Cys Thr Asn 545 550 555 560 Thr Ser Ser Ser Ser Glu Glu Ala Lys Pro Asn Pro Glu Ala Asp Gly 565 570 575 Ser Leu Arg Asp Thr Glu Gln Leu Leu Asp Ile Pro Glu Gln Glu Thr 580 585 590 Thr Thr Pro Ser Ala Asp Ala Glu Gly Gly Val Ala Arg Thr Leu Ser 595 600 605 Thr Ala Ala Leu Ser Met Ala Ser Gly Val Ser Gln Cys Ser Cys Ser 610 615 620 Ser Thr Thr Asp His Val Pro Pro Leu Gln Ser Glu Ser Val Ala Gly 625 630 635 640 Ala Cys Glu Pro Trp Ala Leu Arg Thr Lys Ala His Val Thr Ala Glu 645 650 655 Gly Phe Lys Pro Val Gly Leu Thr Ala Ala Val Leu Ser Ala His Thr 660 665 670 Gln Lys Glu Glu Gln Asn Tyr Val Asp Arg Phe Arg Glu Lys Ile Leu 675 680 685 Thr Ser Pro Tyr Gly Cys Tyr Leu Gln Gln Glu Gly Arg Asn His Ala 690 695 700 Lys Tyr Ala Cys Val Val Gly Ala Gly Ala Thr Pro Lys His Ser Arg 705 710 715 720 720 Cys Ala Gly Ser Glu Arg Arg Lys His Lys Arg Lys Lys Leu Pro Thr 725 730 735 Pro Val Asp Ser Ser Ser Ser Ser Ala His Leu Cys Pro His Val A rg 740 745 750 Gly Leu Leu Pro Asp Val Gln His Trp Ser Ala Ser Val Thr Ser Pro 755 760 765 Cys Ala Thr Gly Leu Ala Leu Pro Ser Ala Leu Val Val Pro Asn Gln 770 775 780 Thr Pro Tyr Leu Leu Ser Ser Phe Pro Leu Gln Asp Met Ala Pro His 785 790 795 800 Gly Val Gly Asp Ser Ala Pro Trp Gly Ala Ala Ala Glu Cys Pro Pro 805 810 815 Leu Ser Ala Gly Pro His Pro Val Ser Thr Phe Pro Ser Ala Tyr Met 820 825 830 Gly Thr Phe Met Thr Val Leu Leu His Asn Ser Pro Leu Phe Pro Leu 835 840 845 Trp Pro Ala Ser Phe Ser Pro Tyr Pro Phe Leu Gly Ala Thr Gly Pro 850 855 860 Ser Gln Met Ala Pro Leu Val Pro Ala Met Ala Pro Asp Leu Glu Pro 865 870 875 880 Thr Pro Ser Asp His Gly Pro Arg Arg Val Glu Glu Asn Trp Glu Thr 885 890 895 His Ser Glu Glu Glu His Pro Phe Ile Ser Ser Arg Ser Ser Ser Pro 900 905 910 Leu Gln Leu Asn Leu Leu Gln Glu Glu Met Pro Ala Pro Ser Glu Tyr 915 920 925 Ala Asp Ala Leu Arg Arg Gly Ala Cys Pro Asp Ala Lys Gln Leu Cys 930 935 940 Val Thr Gly Asn Ser Gly Ser Arg Ser Pro Pro Cys Ala Thr Gly Glu 9 45 950 955 960 Leu Ala Thr Ala Ser Val Gln Gln Glu Ser Pro Ser Ala Ala Ala Ser 965 970 975 Gly Ser Ser Ala Ser Ser Val His Gly Ser Gly Ser Asp Tyr Thr Ser 980 985 990 Glu Val Ser Glu Asn Gly Gln Arg Ser Gln Asp Thr His Arg Asp Arg 995 1000 1005 Ala Phe Ser Gly Ala Ala Glu Glu Ser Ile Trp Arg Met Ile Glu Arg 1010 1015 1020 Thr Pro Gln Cys Val Leu Met Thr Tyr Gln Val Pro Glu Arg Gly Arg 1025 1030 1035 1040 Asp Thr Val Leu Arg Glu Asp Leu Glu Lys Leu His Ser Met Glu Arg 1045 1050 1055 Gln Arg Pro Gln Phe Ser Ser Ala Gln Lys Glu Glu Leu Ala Lys Val 1060 1065 1070 Arg Ser Trp Ile His Ser His Pro Ala Pro Glu Glu Arg Gln Leu Gln 1075 1080 1085 Arg Ala Met Ser Pro Val Lys Thr Glu Val Gln Leu Val Thr Leu Gln 1090 1095 1100 Arg Pro Val Asn Ser Val Gln Gln Lys Thr Pro Val Glu Gln Leu 1105 1110 1115 <211> 3357 <212 > DNA <213> Wister rat <220> <221> CDS <222> (1) ... (3357) <400> 2 atggatccct gtggaaaccc ggcagtgcct ggtggcgact gtccccaaac taggggaccg 60 gggctccaag ggtcgtctgg ccaggagggt cctctgcagg gcatttg cgt ggacagcagc 120 cacagtgaac atgaagaccg aaacagaatg tctgaagagc ttataatggt tgtccaagaa 180 atgaaaaagt atttcccagc cgagagacac actaaaccca gcaccctaga tgcccttaac 240 tatgccctac gctgtgtaca tagtgtgcaa gcaaacagtg aatttttcca gagtctcagt 300 ccacgtggag cacgtcaggc agaagcgact gtatataatc tcgaggagct gacctctctg 360 gcttctgaac atacttccaa gaacacagat accttcgtgg cagtgttttc gtttctgtct 420 ggaaggctgg tgcacatttc tgaacaggct gcttggatcc tgaattctaa gaaaggtttc 480 ctcaagagct tgcacttcgt cgacctgctt gcccctcggg acgtgagggt gttctacgct 540 cacaccgctc caactcaact ccctttctgg aacacctgga cccaaagagc ctcgcagtat 600 gaatgcgcac cagtgaagcc ctttttctgc agaatctgtg gcggtggaga cagggagcag 660 aagagacatt actccccatt ccggatcctc ccctacttgg ttcatgtaca cagccctgcc 720 cagccagagc cagagccttg ctgtctaaca ctggttgaaa agattcactc tggttatgaa 780 gctcctcgaa tccctgtaga taaaagagtt tttaccacaa cacacacgcc gggatgtgtg 840 tttcttgaag tagatgaaag agcagtgcct ttgctgggtt tcctacctca ggatctgatt 900 ggaacgtcga tcttaacgta cttgcaccca gaagatcggc ctctgatggt tgcagtacac 960 c aaaaagttt taaagtacgt gggccaccct ccatttgaac actcacccat aagattctgc 1020 acgcagaatg gagattatgt cattctggat tccagctggt ccagctttgt gaacccctgg 1080 agccggaagg tctccttcat cattggtcga cataaagtcc gaacgagccc gttaaatgaa 1140 gatgtttttg ccaccagaat aaaaaaggca accagtcatg acgaagacat aacagaatta 1200 caagaacaaa ttcacagact tctcttgcag ccggttcatg ccagtgcttc cagtggctac 1260 gggagcctgg gcagcagtgg ctcgcaggag cagcacatca gcgtcacctc ttccagtgag 1320 tcaagcggac actgtgtgga ggaagcccag caggagcaga tgaccctgca gcaggtctat 1380 gccagtgtaa acaaaattaa gaatgtgggc caacagctct acatcgagtc gatggccaga 1440 tcgtcggtga agccagtgat ggagacgtgc acggaaccgc agggtagtga tgagcagaag 1500 gacttttctt cctctcagac actgaaaaat aaaagcacag ataccggctc tggaggcgat 1560 ctgcggccag aacagcatag ctcgtcctat cagcagatga actgcatcga cagtgtcatc 1620 aggtacctga caagctacag tttcccagcc ttgaaaagaa agtgcatctc ctgcacaaac 1680 acgtcttcat cctcagagga agccaagcca aacccagagg cagacggcag cctgcgagac 1740 accgaacagc tcctggacat tccagaacag gaaacaacca caccatccgc agacgccgag 1800 ggaggtg ttg cacggaccct gtccactgcc gcactgagca tggcgtctgg cgtaagccag 1860 tgcagctgca gcagcaccac ggaccatgtc ccccccctac agtcagaaag tgttgccgga 1920 gcgtgtgagc cctgggccct gagaacgaag gctcacgtga ccgcagaagg attcaagccc 1980 gtggggctca cagcggccgt cctctccgcg cacacgcaga aggaagagca gaactatgtt 2040 gacaggttcc gggagaagat cctgacctcg ccctacggct gttatcttca gcaagaaggc 2100 aggaaccatg ccaagtacgc ctgcgtcgtc ggagcagggg ccacccctaa gcacagcaga 2160 tgtgctggaa gtgagagacg gaagcacaaa cggaagaagc tgccgacgcc tgtggacagc 2220 agcagctcca gtgcccacct ctgtccccat gtcagaggac tccttccaga tgtgcagcac 2280 tggagcgctt ccgttacctc tccctgtgct acaggcctag ctctcccctc agccctggtg 2340 gttcccaacc agaccccgta tctcctctcc tcttttcccc ttcaagacat ggcccctcac 2400 ggagtagggg actccgcacc ctggggtgct gcagccgaat gtccacctct gtccgctggt 2460 ccccaccctg tttccacgtt cccttctgct tacatgggta ctttcatgac cgtcctcctg 2520 cacaacagcc ctctctttcc tctgtggccg gcatcattct ctccatatcc attcctgggg 2580 gccacaggtc cttctcaaat ggcaccctta gtaccagcaa tggctccaga cctggaacca 2640 accccttcag ac cacggccc aaggagagtg gaggagaact gggagacaca cagcgaagaa 2700 gagcatccgt ttattagctc acggagcagt tcaccattgc agttaaattt actccaggaa 2760 gaaatgcctg caccatcaga gtatgcagat gcactgagaa gaggcgcctg cccagacgct 2820 aagcaactct gtgttacagg taacagtggc agtaggagcc ctccctgtgc cacaggtgag 2880 ctggccacag catcagtaca gcaagaatct ccatccgcag ctgcctcagg aagcagtgcc 2940 agcagtgtac acggcagtgg cagtgactac acttctgaag tctctgaaaa tggacagagg 3000 tcacaggata cacacagaga cagagccttt tccggggcgg ctgaagaatc tatctggaga 3060 atgatagaac ggacgccaca gtgtgttctc atgacatacc aggtgccgga gaggggtaga 3120 gacaccgtgc tgagggaaga cctggaaaaa cttcacagca tggagcggca gcggccccag 3180 ttctcttctg cgcagaagga ggagctggcc aaggtgcggt cctggatcca cagccaccct 3240 gcccctgagg aaagacagct ccaaagagct atgtcacctg tgaaaacaga ggttcagttg 3300 gtgacactgc agaggcctgt gaacagtgtc cagcagaaga caccagttga gcagctg 3357 <211> 3582 <212> PRT <213> Wister rat <220> < 221> CDS <222> (43) ... (3399) <400> 3 tgctgcaaac tgagagaagc aggctgaggg ctgccaggca gg 42 atg gat ccc tgt gga aac ccg gca gtg cct ggt ggc gac tgt ccc caa 90 Met Asp Pro Cys Gly Asn Pro Ala Val Pro Gly Gly Asp Cys Pro Gln 5 10 15 act agg gga ccg ggg ctc caa ggg tcg tct ggc cag gag ggt cct ctg 138 Thr Arg Gly Pro Gly Leu Gln Gly Ser Ser Gly Gln Glu Gly Pro Leu 20 25 30 cag ggc att tgc gtg gac agc agc cac agt gaa cat gaa gac cga aac 186 Gln Gly Ile Cys Val Asp Ser Ser His Ser Glu His Glu Asp Arg Asn 35 40 45 aga atg tct gaa gag ctt ata atg gtt gtc caa gaa atg aaa aag tat 234 Arg Met Ser Glu Glu Leu Ile Met Val Val Gln Glu Met Lys Lys Tyr 50 55 60 ttc cca gcc gag aga cac act aaa ccc agc acc cta gat gcc ctt aac 282 Phe Pro Ala Glu Arg His Thr Lys Pro Ser Thr Leu Asp Ala Leu Asn 65 70 75 80 tat gcc cta cgc tgt gta cat agt gtg caa gca aac agt gaa ttt ttc 330 Tyr Ala Leu Arg Cys Val His Ser Val Gln Ala Asn Ser Glu Phe Phe 85 90 95 cag agt ctc agt cca cgt gga gca cgt cag gca gaa gcg act gta tat 378 Gln Ser Leu Ser Pro Arg Gly Ala Arg Gln Ala Glu Ala Thr Val Tyr 100 105 110 aat ctc gag gag ctg acc tct ctg gct tct g aa cat act tcc aag aac 426 Asn Leu Glu Glu Leu Thr Ser Leu Ala Ser Glu His Thr Ser Lys Asn 115 120 125 aca gat acc ttc gtg gca gtg ttt tcg ttt ctg tct gga agg ctg gtg 474 Thr Asp Thr Phe Val Ala Val Phe Ser Phe Leu Ser Gly Arg Leu Val 130 135 140 cac att tct gaa cag gct gct tgg atc ctg aat tct aag aaa ggt ttc 522 His Ile Ser Glu Gln Ala Ala Trp Ile Leu Asn Ser Lys Lys Gly Phe 145 150 155 160 ctc aag agc ttg cac ttc gtc gac ctg ctt gcc cct cgg gac gtg agg 570 Leu Lys Ser Leu His Phe Val Asp Leu Leu Ala Pro Arg Asp Val Arg 165 170 175 gtg ttc tac gct cac acc gct cca act caa ctc cct cttc acc 618 Val Phe Tyr Ala His Thr Ala Pro Thr Gln Leu Pro Phe Trp Asn Thr 180 185 190 tgg acc caa aga gcc tcg cag tat gaa tgc gca cca gtg aag ccc ttt 666 Trp Thr Gln Arg Ala Ser Gln Tyr Glu Cys Ala Pro Val Lys Pro Phe 195 200 205 ttc tgc aga atc tgt ggc ggt gga gac agg gag cag aag aga cat tac 714 Phe Cys Arg Ile Cys Gly Gly Gly Asly Arg Glu Gln Lys Arg His Tyr 210 215 220 tcc cca ttc cgg atc ctc ccc t ac ttg gtt cat gta cac agc cct gcc 762 Ser Pro Phe Arg Ile Leu Pro Tyr Leu Val His Val His Ser Pro Ala 225 230 235 240 cag cca gag cca gag cct tgc tgt cta aca ctg gtt gaa aag att cac 810 Gln Pro Glu Pro Glu Pro Cys Cys Leu Thr Leu Val Glu Lys Ile His 245 250 255 tct ggt tat gaa gct cct cga atc cct gta gat aaa aga gtt ttt acc 858 Ser Gly Tyr Glu Ala Pro Arg Ile Pro Val Asp Lys Arg Val Phe Thr 260 265 270 aca aca cac acg ccg gga tgt gtg ttt ctt gaa gta gat gaa aga gca 906 Thr Thr His Thr Pro Gly Cys Val Phe Leu Glu Val Asp Glu Arg Ala 275 280 285 gtg cct ttg ctg ggt ttc cta cg gat gat gga acg tcg atc 954 Val Pro Leu Leu Gly Phe Leu Pro Gln Asp Leu Ile Gly Thr Ser Ile 290 295 300 tta acg tac ttg cac cca gaa gat cgg cct ctg atg gtt gca gta cac 1002 Leu Thr Tyr Leu His Pro Glu Asp Arg Pro Leu Met Val Ala Val His 305 310 315 320 caa aaa gtt tta aag tac gtg ggc cac cct cca ttt gaa cac tca ccc 1050 Gln Lys Val Leu Lys Tyr Val Gly His Pro Pro Phe Glu His Ser Pro 325 330 335 ata aga ttc tgc acg cag aat gga gat tat gtc att ctg gat tcc agc 1098 Ile Arg Phe Cys Thr Gln Asn Gly Asp Tyr Val Ile Leu Asp Ser Ser 340 345 350 tgg tcc agc ttt gtg aac ccc tgg agc cgg aag gtc tcctt 146 Trp Ser Ser Phe Val Asn Pro Trp Ser Arg Lys Val Ser Phe Ile Ile 355 360 365 ggt cga cat aaa gtc cga acg agc ccg tta aat gaa gat gtt ttt gcc 1194 Gly Arg His Lys Val Arg Thr Ser Pro Leu Asn Glu Asp Val Phe Ala 370 375 380 acc aga ata aaa aag gca acc agt cat gac gaa gac ata aca gaa tta 1242 Thr Arg Ile Lys Lys Ala Thr Ser His Asp Glu Asp Ile Thr Glu Leu 385 390 395 400 400 caa gaa caa att cac aga ctt ctc ttg cag ccg gtt cat gcc agt gct 1290 Gln Glu Gln Ile His Arg Leu Leu Leu Gln Pro Val His Ala Ser Ala 405 410 415 tcc agt ggc tac ggg agc ctg ggc agc agt ggc tcg cag gag cag cac 1338 Ser Ser Gly Ser Leu Gly Ser Ser Gly Ser Gln Glu Gln His 420 425 430 atc agc gtc acc tct tcc agt gag tca agc gga cac tgt gtg gag gaa 1386 Ile Ser Val Thr Ser Ser Ser Glu Ser Ser Gly His Cys Val Glu Glu 435 440 445 gcc cag cag gag cag atg acc ctg cag cag gtc tat gcc agt gta aac 1434 Ala Gln Gln Glu Gln Met Thr Leu Gln Gln Val Tyr Ala Ser Val Asn 450 455 460 aaa att aag aat gtg ggc caa cag ctc tac atg tcg atg gcc aga 1482 Lys Ile Lys Asn Val Gly Gln Gln Leu Tyr Ile Glu Ser Met Ala Arg 465 470 475 480 tcg tcg gtg aag cca gtg atg gag acg tgc acg gaa ccg cag ggt agt 1530 Ser Ser Val Glus Pro M Thr Cys Thr Glu Pro Gln Gly Ser 485 490 495 gat gag cag aag gac ttt tct tcc tct cag aca ctg aaa aat aaa agc 1578 Asp Glu Gln Lys Asp Phe Ser Ser Ser Gln Thr Leu Lys Asn Lys Ser 500 505 510 aca gat acc ggc tct gga ggc gat ctg cgg cca gaa cag cat agc tcg 1626 Thr Asp Thr Gly Ser Gly Gly Asp Leu Arg Pro Glu Gln His Ser Ser 515 520 525 tcc tat cag cag atg aac tgc atc gac agt gtc atc agg tac74cg Ser Tyr Gln Gln Met Asn Cys Ile Asp Ser Val Ile Arg Tyr Leu Thr 530 535 540 agc tac agt ttc cca gcc ttg aaa aga aag tgc atc tcc tgc aca aac 1722 Ser Tyr Ser Phe Pro Ala Leu Lys Arg Lys Cys Ile S er Cys Thr Asn 545 550 555 560 acg tct tca tcc tca gag gaa gcc aag cca aac cca gag gca gac ggc 1770 Thr Ser Ser Ser Ser Glu Glu Ala Lys Pro Asn Pro Glu Ala Asp Gly 565 570 575 agc ctg cga gac acc gaa cag ctc ctg gac att cca gaa cag gaa aca 1818 Ser Leu Arg Asp Thr Glu Gln Leu Leu Asp Ile Pro Glu Gln Glu Thr 580 585 590 acc aca cca tcc gca gac gcc gag gga ggt gtt gca cgg acc ctg tcc 1866 Thr Pro Ser Ala Asp Ala Glu Gly Gly Val Ala Arg Thr Leu Ser 595 600 605 act gcc gca ctg agc atg gcg tct ggc gta agc cag tgc agc tgc agc 1914 Thr Ala Ala Leu Ser Met Ala Ser Gly Val Ser Gln Cys Ser Cys Ser 610 615 620 agc acc acg gac cat gtc ccc ccc cta cag tca gaa agt gtt gcc gga 1962 Ser Thr Thr Asp His Val Pro Pro Leu Gln Ser Glu Ser Val Ala Gly 625 630 635 640 640 gcg tgt gag ccc tgg gcc ctg aga acg aag gct cac gtg acc gca gaa 2010 Ala Cys Glu Pro Trp Ala Leu Arg Thr Lys Ala His Val Thr Ala Glu 645 650 655 gga ttc aag ccc gtg ggg ctc aca gcg gcc gtc ctc tcc gcg cac acg 2058 Gly Phe Lys Pro Val G Le u Thr Ala Ala Val Leu Ser Ala His Thr 660 665 670 cag aag gaa gag cag aac tat gtt gac agg ttc cgg gag aag atc ctg 2106 Gln Lys Glu Glu Gln Asn Tyr Val Asp Arg Phe Arg Glu Lys Ile Leu 675 680 685 acc tcg ccc tac ggc tgt tat ctt cag caa gaa ggc agg aac cat gcc 2154 Thr Ser Pro Tyr Gly Cys Tyr Leu Gln Gln Glu Gly Arg Asn His Ala 690 695 700 aag tac gcc tgc gtc gtc gga gca ggg gcc acc cct aag aga 2202 Lys Tyr Ala Cys Val Val Gly Ala Gly Ala Thr Pro Lys His Ser Arg 705 710 715 720 tgt gct gga agt gag aga cgg aag cac aaa cgg aag aag ctg ccg acg 2250 Cys Ala Gly Ser Glu Arg Arg Lys His Lys Arg Lys Lys Leu Pro Thr 725 730 735 cct gtg gac agc agc agc tcc agt gcc cac ctc tgt ccc cat gtc aga 2298 Pro Val Asp Ser Ser Ser Ser Ser Ala His Leu Cys Pro His Val Arg 740 745 750 gga ctc ctt cca gat gtg cag cac tgg agc gct tcc gtt acc tct ccc 2346 Gly Leu Leu Pro Asp Val Gln His Trp Ser Ala Ser Val Thr Ser Pro 755 760 765 tgt gct aca ggc cta gct ctc ccc tca gcc ctg gtg gtt ccc aac cag 2394 Cys Ala Thr Gly Leu Ala Leu Pro Ser Ala Leu Val Val Pro Asn Gln 770 775 780 acc ccg tat ctc ctc tcc tct ttt ccc ctt caa gac atg gcc cct cac 2442 Thr Pro Tyr Leu Leu Ser Ser Phe Pro Leu Gln Asp Met Ala Pro His 785 790 795 800 gga gta ggg gac tcc gca ccc tgg ggt gct gca gcc gaa tgt cca cct 2480 Gly Val Gly Asp Ser Ala Pro Trp Gly Ala Ala Ala Gla Cys Pro Pro 805 810 815 ctg tcc gct ggt ccc cac cct gtt tcc acg ttc cct tct gct tac atg 2538 Leu Ser Ala Gly Pro His Pro Val Ser Thr Phe Pro Ser Ala Tyr Met 820 825 830 ggt act ttc atg acc gtc ctc ctg cac aac agc cct ctc ttt cct ctg 2586 Gly Thr Phe Met Thr Val Leu Leu His Asn Ser Pro Leu Phe Pro Leu 835 840 845 tgg ccg gca tca ttc tct cca tat cca ttc ctg ggg gcc aca ggt cct 2634 Trp Pro Ala Ser Phe Ser Pro Tyr Pro Phe Leu Gly Ala Thr Gly Pro 850 855 860 tct caa atg gca ccc tta gta cca gca atg gct cca gac ctg gaa cca 2682 Ser Gln Met Ala Pro Leu Val Pro Ala Met Ala Pro Asp Leu Glu Pro 865 870 870 875 880 acc cct tca gac cac ggc cca agg aga gtg gag gag aac t gg gag aca 2730 Thr Pro Ser Asp His Gly Pro Arg Arg Val Glu Glu Asn Trp Glu Thr 885 890 895 cac agc gaa gaa gag cat ccg ttt att agc tca cgg agc agt tca cca 2778 His Ser Glu Glu Glu His Pro Phe Ile Ser Ser Arg Ser Ser Ser Pro 900 905 910 ttg cag tta aat tta ctc cag gaa gaa atg cct gca cca tca gag tat 2826 Leu Gln Leu Asn Leu Leu Gln Glu Glu Met Pro Ala Pro Ser Glu Tyr 915 920 925 925 gca gat gca ctg aga aga ggc gcc tgc cca gac gct aag caa ctc tgt 2874 Ala Asp Ala Leu Arg Arg Gly Ala Cys Pro Asp Ala Lys Gln Leu Cys 930 935 940 gtt aca ggt aac agt ggc agt agg ag agc cct ccc tgt gcc aca ggt Gly Asn Ser Gly Ser Arg Ser Pro Pro Cys Ala Thr Gly Glu 945 950 955 960 ctg gcc aca gca tca gta cag caa gaa tct cca tcc gca gct gcc tca 2970 Leu Ala Thr Ala Ser Val Gln Gln Glu Ser Pro Ser Ala Ala Ala Ser 965 970 975 gga agc agt gcc agc agt gta cac ggc agt ggc agt gac tac act tct 3018 Gly Ser Ser Ala Ser Ser Val His Gly Ser Gly Ser Asp Tyr Thr Ser 980 985 990 gaa gtc tct gaa aat gga cag agg tc a cag gat aca cac aga gac aga 3066 Glu Val Ser Glu Asn Gly Gln Arg Ser Gln Asp Thr His Arg Asp Arg 995 1000 1005 gcc ttt tcc ggg gcg gct gaa gaa tct atc tgg aga atg ata gaa cgg 3114 Ala Phe Ser Gly Ala Ala Glu Glu Ser Ile Trp Arg Met Ile Glu Arg 1010 1015 1020 acg cca cag tgt gtt ctc atg aca tac cag gtg ccg gag agg ggt aga 3162 Thr Pro Gln Cys Val Leu Met Thr Tyr Gln Val Pro Glu Arg Gly Arg 1025 1030 1035 1040 gac acc gtg ctg agg gaa gac ctg gaa aaa ctt cac agc atg gag cgg 3210 Asp Thr Val Leu Arg Glu Asp Leu Glu Lys Leu His Ser Met Glu Arg 1045 1050 1055 cag cgg ccc cag ttc tct tct gcg gag ag gcc aag gtg 3258 Gln Arg Pro Gln Phe Ser Ser Ala Gln Lys Glu Glu Leu Ala Lys Val 1060 1065 1070 cgg tcc tgg atc cac agc cac cct gcc cct gag gaa aga cag ctc caa 3306 Arg Ser Trp Ile His Ser His Pro Ala Pro Glu Glu Arg Gln Leu Gln 1075 1080 1085 aga gct atg tca cct gtg aaa aca gag gtt cag ttg gtg aca ctg cag 3354 Arg Ala Met Ser Pro Val Lys Thr Glu Val Gln Leu Val Thr Leu Gln 1090 1095 1100 agg cct gtg aac agt gtc cag cag aag aca cca gtt gag cag ctg 3399 Arg Pro Val Asn Ser Val Gln Gln Lys Thr Pro Val Glu Gln Leu 1105 1110 1115 tgaagatgtc ccaccctctc caggtcat ggtcatgca tg tcatgca tg tcatg tcatg tgttg tggcgctggg gatcaaaccc agggcctcgc ctccgccagg ctggcctcgt 3579 gcc 3582

[Brief description of the drawings]

FIG. 1 is a diagram schematically showing the structure of an rPER3 protein encoded by an rPer3 gene.

FIG. 2: Amino acid sequence of rat-derived rPER3 protein and mouse-derived mPER1 protein, mPER2 protein and mPER
FIG. 4 is a diagram showing homology with the amino acid sequence of PER3 protein. The amino acids enclosed in black in the figure are common parts.

FIG. 3. Amino acid sequence of rat-derived rPER3 protein and mouse-derived mPER1 protein, mPER2 protein and mPER
FIG. 4 is a diagram showing homology with the amino acid sequence of PER3 protein. The amino acids enclosed in black in the figure are common parts.

FIG. 4. Amino acid sequence of rat-derived rPER3 protein and mouse-derived mPER1 protein, mPER2 protein and mPER
FIG. 4 is a diagram showing homology with the amino acid sequence of PER3 protein. The amino acids enclosed in black in the figure are common parts.

FIG. 5. rPER in rat suprachiasmatic nucleus (SCN)
9 is a photograph replaced with a drawing showing the result of Experimental Example 1 in which the diurnal variation of 3 mRNA expression was examined using Northern blotting.

FIG. 6 is a photograph replaced with a drawing showing the results of Experimental Example 2 in which diurnal fluctuations in the expression of rPER mRNA in rat pineal gland, eye, brain, heart, lung, pancreas, liver and kidney were examined by Northern blotting. .

FIG. 7 is a photograph instead of a drawing, showing the results of Experimental Example 2 in which diurnal variations in the expression of rPER mRNA in rat heart and lung were examined by Northern blotting.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) A61P 43/00 C07K 16/18 4C085 C07K 14/47 C12Q 1/68 A 4H045 16/18 G01N 33/15 Z C12Q 1/68 33/50 Z G01N 33/15 C12P 21/08 33/50 C12N 15/00 ZNAA // C12P 21/08 A61K 37/02 (74) The above two agents 100065215 Patent Attorney Eiji Saegusa (72 ) Inventor Nario Ishida 1-3-1 Higashi, Tsukuba, Ibaraki Pref., Institute of Biotechnology, Industrial Technology Institute (72) Inventor Satoru Suzuki 1-3-1, Higashi, Tsukuba, Ibaraki Pref. 72) Inventor Mikio Kikuchi 4-29-14 Imaizumidai, Kamakura City, Kanagawa Prefecture F-term (reference) 2G045 AA34 AA35 AA40 DA12 DA13 DA14 DA36 FB01 FB03 4B 024 AA01 AA11 BA80 CA04 DA06 EA04 GA11 GA19 HA01 HA14 4B063 QA01 QA08 QA18 QQ02 QQ54 QR32. EA20 FA74

Claims (11)

[Claims] 1. A protein of the following (a) or (b): (a) a protein consisting of the amino acid sequence represented by SEQ ID NO: 1, and (b) one or more amino acids in the amino acid sequence represented by SEQ ID NO: 1. A protein consisting of a deleted, substituted or added amino acid sequence, having a homology of 85% or more with the amino acid sequence represented by SEQ ID NO: 1 and having a function of regulating a circadian rhythm. 2. A gene encoding the protein according to claim 1. 3. A gene consisting of the following polynucleotide (a) or (b): (a) a polynucleotide having the nucleotide sequence represented by SEQ ID NO: 2, (b) consisting of the nucleotide sequence represented by SEQ ID NO: 2 A polynucleotide that hybridizes to a polynucleotide under stringent conditions. 4. A polynucleotide used as a specific probe or a specific primer for detecting and / or amplifying the gene according to claim 2 or 3. A gene expression product having the amino acid sequence according to claim 1. 6. A circadian rhythm measuring method for measuring the amount of the gene according to claim 2 or the amount of the gene expression product according to claim 5 in a sample using the specific probe or the specific primer according to claim 4. . 7. A circadian rhythm detecting method for detecting the amount of the gene according to claim 2 or the amount of the gene expression product according to claim 5 in a sample using the specific probe or the specific primer according to claim 4. kit. 8. The gene according to claim 2 or 3 or the gene expression product according to claim 5 is cultured in a medium containing a test substance. A method for screening a substance (agonist / antagonist) that affects the circadian rhythm, wherein the amount or the amount of the gene expression product of claim 5 is measured. 9. An antibody against the protein having the amino acid sequence according to claim 1 or the gene expression product according to claim 5. 10. A circadian rhythm abnormality improving agent comprising an effective amount of the gene according to claim 2 or 3 or the gene expression product according to claim 5 and a pharmaceutical carrier. (11) A circadian rhythm abnormality improving agent comprising an effective amount of the antibody according to (9) and a pharmaceutical carrier.