US20040110171A1

US20040110171A1 - Method of examining ability to control nerve cell plasticity

Info

Publication number: US20040110171A1
Application number: US10/451,923
Authority: US
Inventors: Norishia Ohe
Original assignee: Sumitomo Chemical Co Ltd
Current assignee: Sumitomo Chemical Co Ltd
Priority date: 2000-12-27
Filing date: 2001-12-17
Publication date: 2004-06-10
Also published as: CA2433495A1; US7129065B2; WO2002053735A1; WO2002053729A1; ATE511539T1; US20060216705A1; EP1354951A1; ATE393168T1; US20040248247A1; US7541189B2; EP1354946A4; CA2433492A1; EP1354889B1; CA2433501C; EP1354889A1; CA2433501A1; EP1354946B1; WO2002053736A1; EP1354951B1; DE60133774D1

Abstract

The present invention provides a method for assaying an ability to control the neurocyte plasticity which is dependent on a transcription regulatory factor comprising any of the amino acid sequences selected for example from the amino acid sequence group consisting of the amino acid sequences represented by SEQ ID Nos.1 to 3 comprising the steps:

(1) a first step for bringing a test substance into contact with a mammalian cell expressing the transcription regulatory factor; and,

(2) a second step, after the first step, for measuring the expression level of a marker protein gene present on the transcription regulatory factor-dependent neurocyte plasticising pathway in the mammalian cell or an index value correlating with the level; and,

(3) a step for evaluating the ability possessed by the test substance based on the expression level or the index value correlating with the level measured in the second step, and the like.

Description

TECHNICAL FIELD

The present invention relates to a method for assaying an ability to control a neurocyte plasticity which is dependent on a transcription regulatory factor.

BACKGROUND ART

A cranial nervous function is based on a neural circuit consisting of various neurocytes. Such a complicated and precise network is formed as a result of a correct induction of a neuroaxon into a target cell to effect a synaptic binding with a correct target cell. This induction/binding process (hereinafter referred to as an axonal guidance) involves, as a matter of course, a neuroaxonal extension control (promotion, suppression, attraction, repelling and the like).

Recent studies suggested that a reduction in the cognitive ability in response to an ordinary aging is due to a neural dysfunction rather than due to a loss of neurocytes or synapses. Such a neural dysfunction is considered to be a disturbed mechanism of the neurocyte plasticity due to a difficulty in maintaining a normal condition of the structural plasticity (remodelling; hereinafter sometimes referred to as a neurocyte plasticity) of two protrusions possessed by a neurocyte (dendrite and neuroaxon) for some reason. Similarly, the disturbed mechanism of the neurocyte plasticity is considered recently to be one of the pathogenic factors in diseases such as a mental retardation and Alzheimer-related cognition insufficiency.

Based on the understandings mentioned above, it is possible to improve diseases such as an aging-related cognition insufficiency, mental retardation and Alzheimer-related cognition insufficiency by avoiding the disturbance of the neurocyte plasticity mechanism via an appropriate control of the neurocyte plasticity.

Accordingly, it has been desired to develop a method for assaying an ability to control the neurocyte plasticity, which is essential in searching for a substance employed for controlling the neurocyte plasticity in mammalian cells.

DISCLOSURE OF THE INVENTION

Now we discovered that a specific protein (i.e., a transcription regulatory factor) exists, which controls the expression of a marker protein gene present on a neurocyte plasticising pathway such as an Eph A receptor which is a tyrosine kinase-type cell membrane receptor believed to control the neurocyte plasticity and a controlling factor referred to as a Rho GDP dissociation inhibitor (or Rho GTPase inhibitor), and established the invention by means of utilizing such a protein.

Thus, the present invention provides:

1. a method for assaying (hereinafter sometimes referred to as an assay method of the present invention) an ability to control the neurocyte plasticity which is dependent on a transcription regulatory factor comprising any of the following amino acid sequences (hereinafter sometimes referred to as present amino acid sequences), comprising the steps:

(3) a step for evaluating the ability possessed by the test substance based on the expression level or the index value correlating with the level measured in the second step, wherein the group of the amino acid sequences being:

(a) the amino acid sequence represented by any of SEQ ID Nos.1 to 3,

(b) the amino acid sequence of a protein comprising an amino acid sequence exhibiting an amino acid identity of 90% or more to the amino acid sequence represented by any of SEQ ID Nos.1 to 3 and also having a transcription regulation ability,

(c) the amino acid sequence of a protein comprising an amino acid sequence encoded by a DNA which hybridizes under a stringent condition with a DNA consisting of the nucleotide sequence represented by the nucleotide numbers 102 to 2507 in the nucleotide sequence represented by SEQ ID No.4 and also having a transcription regulation ability,

(d) the amino acid sequence of a protein comprising an amino acid sequence encoded by a DNA which hybridizes under a stringent condition with a DNA consisting of the nucleotide sequence represented by the nucleotide numbers 51 to 2456 in the nucleotide sequence represented by SEQ ID No.5 and also having a transcription regulation ability,

(e) the amino acid sequence a protein comprising an amino acid sequence encoded by a DNA which hybridizes under a stringent condition with a DNA consisting of the nucleotide sequence represented by the nucleotide numbers 35 to 2440 in the nucleotide sequence represented by SEQ ID No.6 and also having a transcription regulation ability;

2. a method for assaying an ability to control the neurocyte plasticity which is dependent on a transcription regulatory factor comprising a present amino acid sequence, comprising the steps:

(1) a first step for bringing a test substance into contact with a transformed mammalian cell obtainable by introducing a gene comprising a nucleotide sequence encoding the present amino acid sequence; and,

(2) a second step, after the first step, for measuring the expression level of a marker protein gene present on the transcription regulatory factor-dependent neurocyte plasticising pathway in the transformed mammalian cell or an index value correlating with the level; and,

(3) a step for evaluating the ability possessed by the test substance based on the expression level or the index value correlating with the level measured in the second step;

3. an assay method according to the above 1 or 2, wherein the marker protein gene is an Eph A receptor gene or an Rho GDP dissociation inhibitor gene;

4. an assay method according to the above 1 or 2, wherein the marker protein genes are an Eph A receptor gene and an Rho GDP dissociation inhibitor gene;

5. an assay method according to the above 1 or 2, wherein the ability possessed by the test substance is evaluated based on the difference obtained by comparing the marker protein gene expression level or an index value correlating with the level in the groups employing two or more different substances independently as test substances;

6. an assay method according to the above 1 or 2, wherein at least one of the two or more different substance is a substance which does not have the ability;

7. a method for searching for a substance having an ability to control the neurocyte plasticity which is dependent on a transcription regulatory factor comprising a present amino acid sequence, wherein a substance having the ability is selected on the basis of the ability evaluated by an assay method according to the above 1 or 2 (hereinafter sometimes referred to as a searching method of the present invention);

8. a neurocyte plasticity regulator comprising as an active ingredient a substance selected by a searching method according to the above 7 or a pharmaceutically acceptable salt thereof and obtained by formulating the active ingredient into a pharmaceutically acceptable carrier (hereinafter sometimes referred to as a neurocyte plasticity regulator of the present invention);

9. a use of a gene comprising the nucleotide sequence encoding a present amino acid sequence for controlling neurocyte plasticity in a mammalian cell;

10. a use of a gene comprising the nucleotide sequence encoding a present amino acid sequence as an exogenous gene for promoting the expression of a marker protein gene present on the neurocyte plasticising pathway which is dependent on a transcription regulatory factor comprising a present amino acid sequence in a mammalian cell by means of providing the exogenous gene in a position enabling the expression in the cell; and the like.

BEST MODE FOR CARRYING OUT THE INVENTION

The invention is further detailed below.

As used herein, a “transcription regulatory factor comprising any of the following amino acid sequences (i.e., present amino acid sequences)” means a protein having a basic helix-loop-helix (hereinafter referred to as bHLH) motif and a PAS domain (Per-Arnt-Sim homology domain) involved in the neurocyte plasticity, which binds to a DNA by forming a heterodimer whereby acting as a transcription regulatory factor. Hereinafter it may be referred to as a present transcription regulatory factor. Detailed descriptions are made later.

As used herein, a “neurocyte plasticity which is dependent on a transcription regulatory factor comprising any of the following amino acid sequences (i.e., a present amino acid sequences)” means a neurocyte plasticity in which the transcription regulatory factor comprising the present amino acid sequence (i.e., a present transcription regulatory factor) is involved and which is believed to occur as a result of a series of the cascade reactions starting from the present transcription regulatory factor. Such a series of the cascade reactions (i.e., metabolic process or signal transmission) is referred to as a “the (i.e., present amino acid sequence-carrying) transcription regulatory factor-dependent neurocyte plasticising pathway” in the present invention, and a protein capable of being utilized as a measure of the neurocyte plasticity in the invention among the proteins present on the pathway as being subjected to the expression regulation is referred to as a “marker protein present on the (i.e., present amino acid sequence-carrying) transcription regulatory factor-dependent neurocyte plasticising pathway”. Those exemplified typically are an Eph A receptor, Rho GDP dissociation inhibitor (or Rho GTPase inhibitor) and the like.

In this context, the Eph A receptor is a tyrosine kinase type cell membrane receptor which is known widely to be related with the development of a brain, and several recent studies indicated the evidences of the involvement of the Eph A in the synapse plasticity, learning and memory, it is also implemented that the Eph A is involved in the potentiation of a memory, and its electrophysiological study results (LTP: long term potentiation of a synapse transmission efficiency) and behavior observation study results suggested that the activation of the Eph A is associated with the change in the cognition leading to the improvement in the memory in an adult brain. Also since the synapse plasticity is critical for the memory and the mnemonic data accumulation (as a basis of the mechanism), the activation of Eph A is considered to be associated with a neurocyte plasticising-promoting effect.

On the other hand, a Rho is an intracellular signal transmitter, which is a protein known widely to be involved in the cell growth control, cell adhesion formation regulation, actin cytoskeleton structure formation and the like. In order to control the activation of the Rho, a control factor (inhibitory protein) referred to as a Rho GDP dissociation inhibitor (or Rho GTPase inhibitor) is present. This forms a complex with a GDP-binding type of the Rho to prevent the Rho from forming a GTP-binding type (activated form of Rho), whereby inhibiting the activation of the Rho (Rho GTPase activity). Thus, the Rho GDP dissociation inhibitor is considered to regulate the neurocyte plasticity by means of controlling the GTPase activity of the Rho.

As used herein, a “transcription regulatory factor (i.e., a present transcription regulatory factor) comprising any of the following amino acid sequences (i.e., a present amino acid sequences)” is a protein consisting of any amino acid sequence included in the amino acid group consisting of (a) the amino acid sequence represented by any of SEQ ID Nos.1 to 3, (b) the amino acid sequence of a protein comprising an amino acid sequence exhibiting an amino acid identity of 90% or more to the amino acid sequence represented by any of SEQ ID Nos.1 to 3 and also having a transcription regulation ability, (c) the amino acid sequence of a protein comprising an amino acid sequence encoded by a DNA which hybridizes under a stringent condition with a DNA consisting of the nucleotide sequence represented by the nucleotide numbers 102 to 2507 in the nucleotide sequence represented by SEQ ID No.4 and also having a transcription regulation ability, (d) the amino acid sequence of a protein comprising an amino acid sequence encoded by a DNA which hybridizes under a stringent condition with a DNA consisting of the nucleotide sequence represented by the nucleotide numbers 51 to 2456 in the nucleotide sequence represented by SEQ ID No.5 and also having a transcription regulation ability and (e) the amino acid sequence of a protein comprising an amino acid sequence encoded by a DNA which hybridizes under a stringent condition with a DNA consisting of the nucleotide sequence represented by the nucleotide numbers 35 to 2440 in the nucleotide sequence represented by SEQ ID No.6 and also having a transcription regulation ability. Such a protein has a molecular weight preferably of about 80,000 to 100,000 as being measured by an SDS-PAGE.

A present transcription regulatory factor includes a protein comprising the amino acid sequence represented by any of SEQ ID Nos.1 to 3(i.e., a present amino acid sequences) (wherein the protein comprising the present amino acid sequence represented by SEQ ID No.1 is a human-derived present transcription regulatory factors which may sometimes be designated as hNXF; the protein comprising the present amino acid sequence represented by SEQ ID No.2 is a mouse-derived present transcription regulatory factor, which may sometimes be designated as mNXF; the protein comprising the present amino acid sequence represented by SEQ ID No.3 is a rat-derived present transcription regulatory factor, which may sometimes be designated as rNXF), a protein comprising an amino acid sequence exhibiting an amino acid identity of 90% or more to the amino acid sequence represented by any of SEQ ID Nos.1 to 3 and also having a transcription regulation ability, a protein comprising an amino acid sequence encoded by a DNA which hybridizes under a stringent condition with a DNA consisting of the nucleotide sequence represented by the nucleotide numbers 102 to 2507 in the nucleotide sequence represented by SEQ ID No.4 and also having a transcription regulation ability, a protein comprising an amino acid sequence encoded by a DNA which hybridizes under a stringent condition with a DNA consisting of the nucleotide sequence represented by the nucleotide numbers 51 to 2456 in the nucleotide sequence represented by SEQ ID No.5 and also having a transcription regulation ability and a protein comprising an amino acid sequence encoded by a DNA which hybridizes under a stringent condition with a DNA consisting of the nucleotide sequence represented by the nucleotide numbers 35 to 2440 in the nucleotide sequence represented by SEQ ID No.6 and also having a transcription regulation ability.

The difference from the amino acid sequence represented by any of SEQ ID Nos.1 to 3 observed in the amino acid sequence of a present transcription regulatory factor may for example be a variation such as the deletion, substitution, modification and addition of amino acids. Such a variation includes a variation which can artificially be introduced by means of a site-directed mutagenesis method or a mutagenic treatment as well as a polymorphic variation which occurs naturally such as a difference in an amino acid sequence resulting from the difference by the animal line, individual, organ and tissue.

In the invention, the “amino acid identity” means an identity and a homology in the amino acid sequence between two proteins. The “amino acid identity” described above can be determined by comparing two amino acid sequence which are aligned optimally over the entire range of a reference amino acid. A reference protein here may have an addition or deletion (for example, a gap) in the optimal alignment of the two amino acid sequences. Such an amino acid identity can be calculated for example by producing an alignment utilizing a Clustal W algorism [Nucleic Acid Res., 22 (22): 4673-4680 (1994)] using a Vector NTI. The amino acid identity can be investigated also by using a sequence analysis software, typically Vector NTI, GENETYX-MAC or any other analytical tools provide DNA public database. Such a public database can generally be available for example in the following URL: http://www.ddbj.nig.ac.jp.

A preferred amino acid identity in the invention may for example be 90% or higher.

A “DNA which hybridizes under a stringent condition” described above may for example be a DNA capable of maintaining a hybrid, which was formed previously as a DNA-DNA hybrid by a hybridization at 65° C. at a high ion concentration [for example using 6×SSC (900 mM sodium chloride, 90 mM sodium citrate)], even after washing for 30 minutes at 65° C. at a low ion concentration [for example using 0.1×SSC (15 mM sodium chloride, 1.5 mM sodium citrate)]. The transcription regulation ability of a present transcription regulatory factor can be evaluated based for example on an assay using a reporter gene described below.

A gene comprising the nucleotide sequence encoding the amino acid sequence of a present transcription regulatory factor (hereinafter referred to as a present transcription regulatory factor gene) may be obtained for example from a tissue of an animal such as human, mouse, rat and the like in accordance with a genetic engineering method described for example in J. Sambrook, E. F. Frisch, T. Maniatis, Molecular Cloning, 2nd Edition, Cold Spring Harbor Laboratory (1989).

Typically, a total RNA derived from a tissue of an animal such as human, mouse and rat is first prepared. For example, a brain tissue is pulverized in a solution containing a protein denaturant such as guanidine hydrochloride or guanidine thiocyanate, and then the pulverized material is treated with phenol, chloroform and the like, to denature the protein. The denatured protein is removed for example by a centrifugation to obtain a supernatant, from which the total RNA is extracted by a guanidine hydrochloride/phenol method, SDS-phenol method, guanidine thiocyanate/CsCl method and the like. A commercially available kit based on the methods described above may for example be ISOGEN (NIPPON GENE). The resultant total RNA is used as a template and an oligo dT primer is annealed to a poly A sequence of the RNA, whereby synthesizing a single-stranded cDNA using a reverse transcriptase. Then, the synthesized single-stranded cDNA is used as a template together with a primer which is an RNA obtained by inserting a nick and a gap into the RNA chain using an E. coli RnaseH, whereby synthesizing a double-stranded cDNA using an E. coli DNA polymerase I. Subsequently, the both ends of the synthesized double-stranded cDNA is made blunt using a T4 DNA polymerase. The double-stranded cDNA having both blunt ends is purified and recovered by means of a standard procedure such as a phenol-chloroform extraction and ethanol precipitation. A commercially available kit based on the methods described above may for example be a cDNA synthesis system plus (Amarsham Pharmacia Biotech) or a TimeSaver cDNA synthesis kit (Amarsham Pharmacia Biotech). Then the resultant double-stranded cDNA is ligated to a vector such as a plasmid pUC118 or phage λgt10 using a ligase to prepare a cDNA library. As such a cDNA library, a commercially available cDNA library (GIBCO-BPL or Clontech) may also be employed.

Alternatively, a genomic DNA may be prepared from a tissue sample of an animal such as human, mouse and rat in accordance with a standard method described for example in J. Sambrook, E. F. Frisch, T. Maniatis, Molecular Cloning, 2nd Edition, Cold Spring Harbor Laboratory (1989), or M. Muramatsu, “Labomanual genetic engineering” (Maruzen, 1988). For example, when the sample is a hair, 2 or 3 hairs are washed with a sterilized water and then with ethanol, cut into 2 to 3 mm pieces, which are combined with 200 μl of a BCL-Buffer [10 mM Tris-HCl (pH7.5), 5 mM MgCl ₂, 0.32 sucrose, 1 Triton X-100] followed by a Proteinase K at the final concentration of 100 μl/ml and SDS at the final concentration of 0.5 (w/v). The mixture thus obtained is incubated at 70° C. for 1 hour, and then subjected to a phenol/chloroform extraction to obtain a genomic DNA. When the sample is a peripheral blood, the sample is treated using a DNA-Extraction kit (Stratagene) and the like to obtain a genomic DNA. The resultant genomic DNA is ligated to a vector such as a λgt10 using a ligase to obtain a genomic DNA library. As such a genomic DNA library, a commercially available genomic DNA library (Stratagene) may also be employed.

From a cDNA library or genomic DNA library obtained as described above, a present transcription regulatory factor gene can be obtained for example by a polymerase chain reaction (hereinafter abbreviated as PCR) using as a primer an oligonucleotide comprising a partial nucleotide sequence of the nucleotide sequence represented by SEQ ID No.4, 5, 6 or 54 or the nucleotide sequence complementary to said partial nucleotide sequence or by a hybridization method using as a probe a DNA comprising the nucleotide sequence represented by SEQ ID No.4, 5, 6 or 54 or a partial nucleotide sequence of said partial nucleotide sequence.

A primer employed in a PCR may for example be an oligonucleotide having a length of about 10 nucleotides to about 50 nucleotides which is an oligonucleotide comprising a nucleotide sequence selected from a 5′ non-translation region of the nucleotide sequence represented by SEQ ID No.4, 5, 6 or 54 and which is an oligonucleotide comprising the nucleotide sequence complementary to a nucleotide sequence selected from a 3′ non-translation region of the nucleotide sequence represented by SEQ ID No.4, 5, 6 or 54. Typically, the forward primer may for example be the oligonucleotide consisting of the nucleotide sequence represented by SEQ ID NO.7 and the oligonucleotide consisting of the nucleotide sequence represented by SEQ ID NO.8. The reverse primer may for example be the oligonucleotide consisting of the nucleotide sequence represented by SEQ ID NO.9 and the oligonucleotide consisting of the nucleotide sequence represented by SEQ ID NO.10. An example of the PCR condition involves an incubation in 50 μl of a reaction solution containing 5 μl of a 10-fold diluted buffer for a LA-Taq polymerase (Takara), 5 μl of a 2.5 mM dNTP mixture (each 2.5 mM dATP, dGTP, dCTP and dTTP) (the final concentration of each of dATP, dGTP, dCTP and dTTP is 0.25 mM), each 0.25 to 1.25 μl of 20 μM primers (final concentration of 0.1 to 0.5 μM), 0.1 to 0.5 μg of a template cDNA and 1.25 units of a LA-Taq polymerase (Takara) for 1 minutes at 95° C. followed by 3 minutes at 68° C. in a single cycle, the cycle being repeated 35 times.

A probe employed in a hybridization method may for example be the DNA consisting of the nucleotide sequence represented by the nucleotide numbers 102 to 2507 in the nucleotide sequence represented by SEQ ID No.4, a DNA consisting of the nucleotide sequence represented by the nucleotide numbers 51 to 2456 in the nucleotide sequence represented by SEQ ID No.5, a DNA consisting of the nucleotide sequence represented by the nucleotide numbers 35 to 2440 in the nucleotide sequence represented by SEQ ID No.6, a DNA consisting of the nucleotide sequence represented by the nucleotide numbers 1419 to 6164 in the nucleotide sequence represented by SEQ ID No.54 and the like. An example of the hybridization condition involves an incubation at 65° C. in the presence of 6×SSC (0.9M sodium chloride, 0.9M sodium citrate), 5× Denhart's solution (0.1 (w/v) ficoll 400, 0.1 (w/v) polyvinyl pyrrolidone), 0.1 (w/v) BSA), 0.5 (w/v) SDS and 100 μg/ml denatured salmon sperm DNA followed by an incubation at room temperature for 15 minutes in the presence of 1×SSC (0.005M sodium chloride, 0.015M sodium citrate) and 0.5 (w/v) SDS; followed by an incubation at 68° C. for 30 minutes in the presence of 0.1×SSC (0.015M sodium chloride, 0.0015M sodium citrate) and 0.5 (w/v) SDS. Alternatively, an incubation at 65° C. in the presence of 5×SSC, 50 mM HEPES, pH7.0, 10× Denhart's solution and 20 μg/ml denatured salmon sperm DNA followed by an incubation at room temperature for 30 minutes in 2×SSC, followed by an incubation at 65° C. for 40 minutes in 0.1×SSC, which is repeated twice, may also be exemplified.

A present transcription regulatory factor gene can be prepared also by performing a chemical synthesis of a nucleic acid in accordance with a standard method such as a phosphite triester method (Hunkapiller, M. et al., Nature, 310, 105, 1984) based on the nucleotide sequence represented by SEQ ID NO.4, 5, 6 or 54.

A present transcription regulatory factor gene thus obtained can be cloned into a vector in accordance with a genetic engineering method described in J. Sambrook, E. F. Frisch, T. Maniatis, Molecular Cloning, 2nd Edition, Cold Spring Harbor Laboratory (1989). Typically, the cloning can for example be performed using a TA cloning kit (Invitrogen) or a commercially available plasmid vector such as pBluescriptII (Stratagene).

The nucleotide sequence of a resultant inventive DNA can be identified by a Maxam Gilbert method (described for example in Maxam, A. M. & W. Glibert, Proc. Natl. Acad. Sci. USA, 74, 560, 1997) or a Sanger method (described for example in Sanger, F. & A. R. Coulson, J. Mol. Biol., 94, 441, 1975, Sanger, F. & Nicklen and A. R. Coulson., Proc. Natl. Acad. Sci. USA, 74, 5463, 1997).

A typical example of a present transcription regulatory factor gene may for example be the DNA consisting of the nucleotide sequence represented by the nucleotide numbers 102 to 2507 in the nucleotide sequence represented by SEQ ID No.4, a DNA consisting of the nucleotide sequence represented by the nucleotide numbers 51 to 2456 in the nucleotide sequence represented by SEQ ID No.5, a DNA consisting of the nucleotide sequence represented by the nucleotide numbers 35 to 2440 in the nucleotide sequence represented by SEQ ID No.6, a DNA consisting of the nucleotide sequence represented by the nucleotide numbers 1419 to 6164 in the nucleotide sequence represented by SEQ ID No.54 and the like.

A present transcription regulatory factor gene is, as described above and below, can be utilized also for promoting the expression of a marker protein gene present on a present transcription regulatory factor-dependent neurocyte plasticising pathway in a mamalian cell by means of providing the DNA in the mammalian cell as an exogenous gene in a position enabling the expression in the cell.

A present transcription regulatory factor gene vector can be constructed by integrating a present transcription regulatory factor gene, in accordance with a standard genetic engineering method, into a vector capable of being utilized in a host cell to which said gene is introduced (hereinafter referred to as a basic vector), such as a vector which contains a gene information capable of being replicated in the host cell, which can independently be proliferated, which can be isolated and purified from the host cell and which has a detectable marker.

A basic vector which can be employed for constructing A present transcription regulatory factor gene vector may for example be a plasmid pUC119 (Takara) or phagimid pBluescriptII (Stratagene) when using a coliform as a host cell. When using a budding yeast as a host cell, then plasmids pGBT9, pGAD242, pACT2 (Clontech) may be exemplified. When using a mammalian cell as a host cell, a vector containing an autonomous replication origin derived from a virus such as pRc/RSV, pRc/CMV (Invitrogen), bovine papilloma virus plasmid pBV (Amarsham Pharmacia Biotech) or EB virus plasmid pCEP4 (Invitrogen) and a virus such as a vaccinia virus may be exemplified, while an insect virus such as a baculovirus may be exemplified when using a insect cell as a host cell.

In order to integrate a present transcription regulatory factor gene into a virus such as a baculovirus or vaccinia virus, a transfer vector containing a nucleotide sequence homologous to the genome of a virus to be employed can be used. Such a transfer vector is typically a plasmid available from Pharmingen such as pVL1372, pVL1393 (Smith, G. E., Summers M. E. et al., Mol. Cell Biol., 3, 2156-2165 (1983) and pSFB5 (Funahashi, S. et al., J. Virol., 65, 5584-5588 (1991). When a present transcription regulatory factor gene is inserted into a transfer vector described above and the transfer vector and the genome of a virus are introduced into a host cell simultaneously, a homologous recombination occurs between the transfer vector and the genome of the virus, whereby obtaining a virus into whose genome the present transcription regulatory factor gene is integrated. The genome of a virus may be the genome for example of Baculovirus, Adenovirus, Vacciniavirus and the like.

More specifically, a present transcription regulatory factor gene is integrated for example into a baculovirus by inserting the present transcription regulatory factor gene into a multiple cloning site of a transfer vector such as pVL1393 or pBL1392 followed by introducing the DNA of said transfer vector and a baculovirus genomic DNA (Baculogold; Pharmingen) into an insect cell line Sf21 (available from ATCC) for example by a calcium phosphate method followed by incubating the resulting cell. A virus particle containing the genome of the virus into which the present transcription regulatory factor gene has been inserted is recovered from the culture medium for example by a centrifugation, and then made free from proteins using phenol and the like, whereby obtaining the genome of the virus containing the present transcription regulatory factor gene. Subsequently, the genome of said virus is introduced into a host cell having a virus particle forming ability such as an insect cell line Sf21 for example by a calcium phosphate method and the resultant cell is incubated, whereby proliferating the virus particle containing the present transcription regulatory factor gene.

On the other hand, a relatively small genome such as that of a mouse leukemia retrovirus can directly be integrated with a present transcription regulatory factor gene without using any transfer vector. For example, a virus vector DC(X) (Eli Gilboa et al., BioTechniques, 4, 504-512 (1986)) is integrated with a present transcription regulatory factor gene on its cloning site. The resultant virus vector into which the present transcription regulatory factor gene has been integrated is introduced into a packaging cell such as an Ampli-GPE (J. Virol., 66, 3755 (1992)), whereby obtaining a virus particle containing the genome of the virus into which the present transcription regulatory factor gene has been inserted.

A promoter capable of functioning in a host-cell is operably connected to the upstream of a present transcription regulatory factor gene and then integrated into a basic vector such as those described above, whereby constructing an present transcription regulatory factor gene vector capable of allowing the present transcription regulatory factor gene to be expressed in the host cell. The expression “operably connected” means that a promoter and a present transcription regulatory factor gene are bound to each other in a condition which allows the present transcription regulatory factor gene is expressed under the control of the promoter in a host cell into which the present transcription regulatory factor gene is to be introduced. A promoter capable of functioning in a host cell may for example be a DNA exhibiting a promoter activity in a host cell into which it is to be introduced. Those which may be exemplified when the host cell is a coliform cell are E. coli lactose operon promoter (lacP), tryptophan operon promoter (trpP), arginine operon promoter (argP), galactose operon promoter (galP), tac promoter, T7 promoter, T3 promoter, λ phage promoter (λ-pL, λ-pR) and the like, while those which may be exemplified when the host cell is an animal cell or fission yeast are Rous sarcoma virus (RSV) promoter, cytomegalovirus (CMV) promoter, simian virus (SV40) early or late promoter, mouse mammary tumor virus (MMTV) promoter and the like. Those which may be exemplified when the host cell is a budding yeast are an ADH1 promoter and the like (the ADH1 promoter can be prepared by a standard genetic engineering method for example from an yeast expression vector pAAH5 comprising an ADH1 promoter and terminator [available from Washington Research Foundation, Ammerer et al., Method in Enzymology, 101 part (p.192-201)]; the ADH1 promoter is encompassed in the U.S. patent application Ser. No. 299,733 by Washington Research Foundation, and should be used industrially or commercially in United States only after obtaining the approval from the claimant).

When a basic vector which initially possesses a promoter capable of functioning in a host cell is employed, a present transcription regulatory factor gene may be inserted to the downstream of said promoter so that the vector-possessed promoter and the present transcription regulatory factor gene are operably connected to each other. For example, each of the plasmids such as pRc/RSV and pRc/CMV described above is provided with a cloning site downstream of a promoter capable of functioning in an animal cell, and by inserting a present transcription regulatory factor gene into said cloning site followed by a introduction into an animal cell, the present transcription regulatory factor gene can be expressed. Since any of these plasmids has previously been integrated with a SV40 autonomous replication origin, the introduction of said plasmid into a host cell which has been transformed with an SV40 genome from which an ori is deleted, such as a COS cell, leads to an extremely increased number of the intracellular plasmid copies, resulting in a high expression of the present transcription regulatory factor gene which has been integrated into said plasmid. Also since the plasmid pACT2 for yeast described above possesses an ADH1 promoter, an present transcription regulatory factor gene vector capable of allowing a present transcription regulatory factor gene to be expressed highly in a budding yeast such as CG1945 (Clontech) can be constructed by inserting the present transcription regulatory factor gene into the downstream of the ADH1 promoter of said plasmid or a derivative thereof.

By introducing a constructed present transcription regulatory factor gene vector into a host cell, a transformant can be obtained. A method for introducing a present transcription regulatory factor gene vector into a host cell may be a standard introducing method suitable for the host cell. For example, when E. coli is employed as a host cell, a standard method such as a calcium chloride method or electroporation described for example in J. Sambrook, E. F. Frisch, T. Maniatis, Molecular Cloning, 2nd Edition, Cold Spring Harbor Laboratory (1989) may be employed. When a mammalian cell or insect cell is employed as a host cell, the introduction into a cell described above can be effected in accordance with a general gene introduction method such as a calcium phosphate method, DEAE dextran method, electroporation, lipofection and the like. When an yeast is employed as a host cell, the introduction can be effected for example by means of an Yeast transformation kit (Clontech) based on a lithium method.

When a virus is employed as a vector, the genome of the virus can be introduced into a host cell by a standard gene introduction method described above, or a virus particle containing the genome of the virus into which a present transcription regulatory factor gene has been inserted is infected to a host cell, whereby introducing the genome of said virus into the host cell.

In order to screen for the transformant, a marker gene is introduced into a host cell simultaneously with a present transcription regulatory factor gene vector and the cell is cultured in a manner suitable to the nature of the marker gene. For example, when the marker gene is a gene which impart the host cell with a resistance to a lethally active screening drug, then the cell into which the present transcription regulatory factor gene vector has been introduced is cultured in a medium supplemented with said drug. The combination of such a drug resistance imparting gene and a screening drug may for example be the combination of a neomycin resistance imparting gene with neomycin, the combination of a hygromycin resistance imparting gene with hygromycin, and the combination of blasticidin S resistance imparting gene and blasticidin S. When the marker gene is a gene which compensates the auxotrophic nature of the host cell, then a minimum medium free from the relevant nutrition is used to culture the cell into which the present transcription regulatory factor gene vector has been introduced.

In order to obtain a transformant generated as a result of the introduction of a present transcription regulatory factor gene into a chromosome of a host cell, a present transcription regulatory factor gene vector and a marker gene-carrying vector are made linear by a digestion with restriction enzymes, and then introduced as described above into a host cell, which is cultured usually for several weeks to screen for an intended transformant on the basis of the expression of the introduced marker gene. Alternatively, it is also possible to screen for a transformant generated as a result of the introduction of a present transcription regulatory factor gene into a chromosome of a host cell by introducing a present transcription regulatory factor gene vector comprising as a marker gene a gene providing a resistance to a screening drug describe above into a host cell as described above, subculturing this cell for several weeks in a medium supplemented with the screening drug, and then incubating a selected drug resistance clone surviving as a colony in a pure culture manner. In order to verify that the introduced present transcription regulatory factor gene has surely been integrated into the chromosome of the host cell, a standard genetic engineering method may be employed to prepare the genomic DNA of the cell, from which the presence of the present transcription regulatory factor gene is detected by a PCR using as a primer an oligonucleotide comprising a partial nucleotide sequence of the introduced present transcription regulatory factor gene or by a southern hybridization method using as a probe the introduced present transcription regulatory factor gene. Since such a transformant can be stored frozen and can be made viable upon any need of use, it allows the step for producing the transformant at every time of the experiment to be omitted, and allows the experiment to be conducted using a transformant whose characteristics and the handling condition for which are well established.

By culturing a transformant obtained as described above, a present transcription regulatory factor can be produced.

For example, when a transformant described above is a microorganism, this transformant can be cultured using any culture medium containing carbon sources, nitrogen sources, organic salts and inorganic salts, as appropriate, used in an ordinary culture of an ordinary microorganism. The culture can be conducted in accordance with a usual procedure for an ordinary microorganism, such as a solid culture, liquid culture (rotary shaking culture, reciprocal shaking culture, Jar Fermenter, tank culture and the like). The culture temperature and the pH of the medium may appropriately be selected from the range enabling the growth of the microorganisms, and the culture is conducted usually at a temperature of about 15° C. to about 40° C. at a pH of about 6 to about 8. The culture time period is usually about 1 day to about 5 days, although it may vary depending on various culture conditions. When an expression vector comprising a promoter of a temperature shift type or an induction type such as an IPTG induction type, the induction time is preferably within 1 day, usually several hours.

When a transformant described above is an animal cell such as an insect cell, then the transformant can be cultured using a culture medium employed in an ordinary culture of an ordinary cell when such a transformant was prepared using a screening drug, then the culture is conducted preferably in the presence of the relevant drug. In the case of a mammalian cell, the culture is conducted for example in a DMEM medium supplemented with FBS at the final concentration of 10% (v/v) (NISSUI and the like) at 37° C. in the presence of 5% CO ₂with replacing the culture medium with a fresh medium every several days. When the culture became confluent, a PBS solution supplemented with trypsin for example at a concentration of about 0.25 (w/v) is added to disperse the culture into individual cells, which are subjected to a several-fold dilution and then inoculated to new dishes where they are further cultured. Similarly in the case of an insect cell, an insect cell culture medium such as a Grace's medium containing 10% (v/v) FBS and 2% (w/v) Yeastlate is employed to conduct the culture at a temperature of 25° C. to 35° C. In this case, a cell which tends to be peeled off from a dish easily such as a Sf21 cell can be dispersed by pipetting instead of using a trypsin solution, whereby continuing the subculture. In the case of a transformant containing a vector of a virus such as a baculovirus, the culture time period is preferably shorter than the time period allowing a cytoplasm effect to be evident to cause the cell death, for example up to 72 hours after the virus infection.

A present transcription regulatory factor produced by a transformant described above can be recovered appropriately by a combination of ordinary isolation and purification methods, and a fraction containing the present transcription regulatory factor can be obtained by collecting the transformant cells by a centrifugation after completion of the culture, suspending the collected cells in an ordinary buffer solution, pelletizing the cells for example using Polytron, ultrasonic treatment, Dounce homogenizer and the like, and then centrifuging the pelletized cell fluid to recover the supernatant. A further purified present transcription regulatory factor can be recovered by subjecting the supernatant fraction described above to various chromatographic procedures such as ion exchange chromatography, hydrophobic interaction chromatography, gel filtration chromatography, affinity chromatography and the like. When a present transcription regulatory factor or a polypeptide comprising its partial amino acid sequence is produced as a fusion protein with GST, the purification can be accomplished by an affinity chromatography using a glutathione sepharose (Amersham Pharmacia).

A present transcription regulatory factor thus produced can be employed as an immune antigen for producing an antibody which recognizes a present transcription regulatory factor or a polypeptide comprising its partial amino acid sequence, and can also be employed in an assay for screening for a substance which binds to the present transcription regulatory factor.

Using a present transcription regulatory factor produced as described above as an immune antigen, an animal such as mouse, rabbit, chicken and the like is immunized in accordance with an immunological procedure described in Frederick M. Ausubel et al., Short Protocols in Molecular Biology 3nd Edition, John Wiley & Sons, Inc, whereby producing an antibody which recognizes a present transcription regulatory factor or a polypeptide comprising its partial amino acid sequence. More typically and in one example, a present transcription regulatory factor as an antigen is mixed with a complete Freunds adjuvant to form an emulsion. The resultant emulsion is administered subcutaneously to a rabbit. After about 4 weeks, an antigen emulsified in an incomplete Freunds adjuvant is administered. If necessary, a similar administration is further conducted every two weeks. The blood is sampled to obtain a serum fraction, the antibody titre of which against the present transcription regulatory factor is then verified. The resultant serum fraction having the antibody titre which recognizes the present transcription regulatory factor or a polypeptide comprising its partial amino acid sequence is fractionated in accordance for example with an ordinary ammonium sulfate sedimentation method, whereby obtaining an IgG which recognizes the present transcription regulatory factor or a polypeptide comprising its partial amino acid sequence.

Alternatively, a polypeptide comprising a partial amino acid sequence of a present transcription regulatory factor is synthesized chemically and administered as an immune antigen to an animal, whereby producing an antibody which recognizes the present transcription regulatory factor or a polypeptide comprising its partial amino acid sequence. As the amino acid sequence of a polypeptide employed as an immune antigen, an amino acid sequence which has as a low homology as possible with the amino acid sequences of other proteins and which has many differences from the amino acid sequence of a present transcription regulatory factor possessed by an animal species to be immunized is selected for example from the amino acid sequences represented by SEQ ID Nos.1 to 3. A polypeptide having a length of 10 amino acids to 15 amino acids consisting of the selected amino acid sequence is synthesized chemically by a standard method and crosslinked for example with a carrier protein such as Limulus plyhemus hemocyanin using MBS and the like and then used to immunize an animal such as a rabbit as described above.

As a result, an antibody recognizing a polypeptide comprising a present transcription regulatory factor or a partial amino acid sequence thereof can be produced.

In order to assay an ability to control the neurocyte plasticity which is dependent on a present transcription regulatory factor thus produced (hereinafter sometimes referred to as a present neurocyte plasticity-controlling ability), an assay method (i.e., an assay method of the present invention) comprising the steps: (1) a first step for bringing a test substance into contact with a mammalian cell expressing a present transcription regulatory factor; and, (2) a second step, after the first step, for measuring the expression level of a marker protein gene present on the transcription regulatory factor-dependent neurocyte plasticising pathway in the mammalian cell or an index value correlating with the level; and a step for evaluating the ability possessed by the test substance based on the expression level (measured in the second step) or the index value correlating with the level can be employed. In this steps, a difference is investigated by comparing the marker protein gene expression level or an index value (first measured value, second measured value) correlating with the level in the groups employing two or more different substances independently as test substances. Based on the difference thus obtained (difference between the first measured value and the second measured value), the present neurocyte plasticity-controlling ability possessed by the test substance described above is evaluated, whereby accomplishing the assay of the ability. Based on the present neurocyte plasticity-controlling ability thus evaluated, the substance is identified as a substance having the present neurocyte plasticity-controlling ability.

In the method described above, it is possible to use at least one of the two or more different substance described above as a substance which does not have a present neurocyte plasticity-controlling ability whereby evaluating the present neurocyte plasticity-controlling ability possessed by any of the other test substances, and it is also possible to use the present neurocyte plasticity-controlling ability possessed by at least one of the two or more different substance described above as a standard for evaluating the present neurocyte plasticity-controlling ability of any of the other test substances.

Thus, a present transcription regulatory factor can be utilized in a method for effectively analyzing the expression level of a marker protein gene present on the transcription regulatory factor-dependent neurocyte plasticising pathway or an index value correlating with the level which is essential for assaying a present neurocyte plasticity-controlling ability possessed by a substance.

A mammalian cell employed in an assay method of the present invention may be a cell isolated from a tissue, or a cell forming a population having identical functions and morphology, or a cell present in the body of a mammal. It is also possible to use an extracted system of the cell mentioned above. The origin of the cell may for example be a mammal, typically human, monkey, cattle, rabbit, mouse, rat, hamster and the like.

In a first step of an assay method of the present invention, the concentration of a test substance to be brought into contact with a mammalian cell which expresses a present transcription regulatory factor may usually be about 0.1 μM to about 100 μM, preferably 1 μM to 50 μM. The time period during which the mammalian cell is in contact with the test substance is usually 10 minutes to 2 days, preferably several hours to 1 days.

The environment in which a test substance is brought into contact with a mammalian cell mentioned above is preferably an environment allowing the viable activity of the mammalian cell to be maintained, for example an environment in which an energy source for the mammalian cell is coexisting. Typically, it is convenient to perform a first step in a culture medium.

It is also possible to perform a first step of an assay method of the present invention for example by bringing a test substance into contact with a transformed mammalian cell (hereinafter sometimes referred to as a present transformed mammalian cell) obtainable by introducing a gene comprising a nucleotide sequence encoding the present amino acid sequence into the mammalian cell.

In this step, the concentration of a test substance to be brought into contact with a present transformed mammalian cell may usually be about 0.1 μM o about 100 μM, preferably 1 μM to 50 M. The time period during which the transformed mammalian cell is in contact with the test substance is usually 10 minutes to 2 days.

A present transformed mammalian cell can be prepared as follows.

A present transcription regulatory factor gene is inserted using an ordinary genetic engineering technology into a vector capable of being used in a mammalian cell into which the present transcription regulatory factor gene is introduced in such a manner it is connected to a promoter in an expressible form to form a plasmid. The promoter employed here may be one which is functional in the mammalian cell into which the present transcription regulatory factor gene is introduced, such as an SV40 virus promoter, cytomegalovirus promoter (CMV promoter), Raus sarcoma virus promoter (RSV promoter), β actin gene promoter and the like. It is also possible to use a commercially available vector containing any of these promoters upstream of the multiple cloning site.

Then, the plasmid is introduced into a mammalian cell. A method for such a introduction into the mammalian cell may for example be a calcium phosphate method, electroinduction, DEAE dextran method, micelle formation and the like. The calcium phosphate method may be a method described in Grimm, S. et al., Proc. Natl. Acad. Sci. USA, 93, 10923-10927, the electroinduciton and DEAE dextran method may for example be those described in Ting, A. T. et al. EMBO J., 15, 6189-6196, and the micelle formation may for example be a method described in Hawkins, C. J. et al., Proc. Natl. Acad. Sci. USA, 93, 13786-13790. When a micelle formation is employed, a commercially available reagent such as Lipofectamine (Gibco) or Fugene (Boehringer) may be utilized.

A mammalian cell which has been introduced with a plasmid described above is cultured in a medium providing a screening condition suitable to a screening marker gene for example by utilizing the screening marker gene which has previously been contained in vector, whereby screening for the transformed mammalian cell. It is also possible to screen further continuously to obtain the transformed mammalian cell which now became a stable transformed mammalian cell into whose chromosome the present transcription regulatory factor gene has been introduced. In order to verify that the introduced present transcription regulatory factor gene has been integrated into a chromosome present in the mammalian cell, the genomic DNA of the cell may be produced in accordance with an ordinary genetic engineering technology and then the presence of the present transcription regulatory factor gene in the genomic DNA may be detected and identified by means of a PCR employing a DNA comprising a partial nucleotide sequence of the present transcription regulatory factor gene as a primer, or by a southern hybridization method employing a DNA comprising a partial nucleotide sequence of the present transcription regulatory factor gene as a probe.

A present transformed mammalian cell may be prepared also from a transformed non-human animal tissue described below by an ordinary procedure.

In an assay method of the present invention, a method for measuring the expression level of a marker protein gene present on a present transcription regulatory factor-dependent neurocyte plasticising pathway or an index value correlating with the level may for example be:

(1) a method for measuring the level of the marker protein described above for example by a radioimmunoassay (RIA) employing an antibody which recognizes specifically the marker protein, an enzyme linked immunosorbent assay (ELISA), western blotting and the like;

(2) a method for measuring the level, in the mammalian cell, of an mRNA of a marker protein as an index value correlating with the marker protein;

(3) a method for measuring the level of a marker protein using a DNA array or DNA chip onto which an oligonucleotide capable of being hybridized with the marker gene to enable the measurement of the level of an mRNA of the marker protein has been immobilized.

The quantification of an mRNA in the procedure (2) described above may employ an ordinary method such as an RT-PCR or northern hybridization (for example see, J. Sambrook, E. F. Frisch, T. Maniatis, Molecular Cloning, 2nd Edition, Cold Spring Harbor Laboratory Press).

Based on the difference obtained by comparing the marker protein gene expression level or an index value correlating with the level in the groups employing two or more different substances independently as test substances which has been measured by an assay method of the present invention as described above, the present neurocyte plasticity-controlling ability of a substance mentioned above is evaluated. Thus, the present neurocyte plasticity-controlling ability possessed by a test substance can be assayed.

In an assay method of the present invention, it is possible to use at least one of the two or more different substance described above as a substance which does not have a present neurocyte plasticity-controlling ability (for example, solvent, test system solution serving as a background) whereby evaluating the present neurocyte plasticity-controlling ability possessed by any of the other test substances, and it is also possible to use the present neurocyte plasticity-controlling ability possessed by at least one of the two or more different substance described above as a standard for evaluating the present neurocyte plasticity-controlling ability of any of the other test substances.

It is also possible to compare the expression level of a marker protein gene relevant to a present transcription regulatory factor or an index value correlating with the level (hereinafter referred to as a measured value 1) with the expression level of the marker protein gene or the index value correlating with the level observed in a mammalian cell without any contact between the present transcription regulatory factor and a test substance (hereinafter referred to as a measured value 2), whereby evaluating the present neurocyte plasticity-controlling ability of the test substance. In such a case, the present neurocyte plasticity-controlling ability may be determined as a % control using the measured values described above in accordance with the following equation.

% Control=[(Measured value 1−Measured value 2)/Measured value 2]×100

In order to search for a substance having a present neurocyte plasticity-controlling ability, a substance having the present neurocyte plasticity-controlling ability may be selected based on the present neurocyte plasticity-controlling ability evaluated by an assay method of the present invention(a searching method of the present invention).

For example, a test substance whose absolute value of a % control indicating a present neurocyte plasticity-controlling ability is a statistically significant value, preferably 30% or higher, more preferably 50% or higher, is selected as a substance having the present neurocyte plasticity-controlling ability. When the % control is a positive value, the substance is selected as one having a promoting ability, while the substance is selected as one having an inhibiting ability when the % control is a negative value.

Such a substance may be any substance such as a low molecular weight compound, protein (including antibody) or peptide, as long as it has a present neurocyte plasticity-controlling ability.

A substance searched for by a searching method of the present invention has a present neurocyte plasticity-controlling ability, and may be used as an active ingredient of a neurocyte plasticity regulator (for example, therapeutic agent against cognitive ability insufficiency and mental retardation).

A neurocyte plasticity regulator containing as an active ingredient a substance selected by a searching method of the present invention or a pharmaceutically acceptable salt thereof (thus, an inventive neurocyte plasticity regulator) can be administered at an effective dose orally or parenterally to a mammalian animal such as human. For example, the inventive neurocyte plasticity regulator when administered orally can be given in an ordinary form such as a tablet, capsule, syrup and suspension. When the inventive neurocyte plasticity regulator given parenterally, it can be administered in an ordinary liquid form such as a solution, emulsion and suspension. A method for administering the inventive neurocyte plasticity regulator in a form described above parenterally may for example be an injection or a rectal administration of a suppository.

Such a suitable dosage form can be produced by incorporating a substance selected by a searching method of the present invention or a pharmaceutically acceptable salt thereof into a pharmaceutically acceptable ordinary carrier, excipient, binder, stabilizer, diluent and the like. When employing an injection formulation, it may be possible to add acceptable buffering agents, solubilizing aids, osmotic agents and the like.

The dosage may vary depending on the age and the sex of the mammalian subject, degree of the disease, type of the arteriosclerotic condition exacerbation factor secretion inhibitor, dosage form and the like, the oral dose is usually about 1 mg to about 2 g as an active ingredient per day in an adult, preferably about 5 mg to about 1 g, while the injection may be given at about 0.1 mg to about 500 mg as an active ingredient in an adult. Such a daily dose may be given all at once or in several portions.

A disease indicated for an inventive neurocyte plasticity regulator may for example be a senile cognition insufficiency, mental retardation and Alzheimer-related cognition insufficiency.

The present invention also provides a utilization of a present transcription regulatory factor gene for promoting the expression of a marker protein gene present on a present transcription regulatory factor-dependent neurocyte plasticising pathway in a mammalian cell by means of providing the present transcription regulatory factor gene in the mammalian cell as an exogenous gene in a position enabling the expression in the cell.

Such a mammalian cell may for example be a cell derived from a mammal such as human, monkey, mouse, rat, hamster and the like. Such a cell may be a cell which constitutes a population having identical functions and morphologies, or a cell present in the body of said mammalian animal.

Accordingly, when the mammalian animal is a human, a range from a human receiving a so called gene therapy to a cell line employed in various experiments is contemplated, while when the mammalian animal is a non-human animal then a range from a non-human animal receiving a so called gene therapy to an animal model or a cell line employed in various experiments is contemplated. In the latter case, a preferred species is rat, mouse and the like.

Moreover, a case in which a mammalian cell is a cell in the body of a mammalian animal which can be diagnosed to suffer from a disease accompanied with a mental retardation or from Alzheimer's disease can be exemplified as a more typical case.

A method for preparing a DNA encoding a present transcription regulatory factor may be prepared in accordance with a method equivalent to that described above.

Using such a DNA thus prepared, a transformant is prepared as described below, whereby obtaining a transformant in which said DNA is provided in a position which enables its expression in the mammalian cell.

In a present invention described above, the phrase “provided in a position enabling the expression” means that a DNA molecule is placed in the position adjacent to a DNA sequence directing the transcription and the translation of its nucleotide sequence (i.g., promoting the production of a present transcription regulatory factor or its RNA molecule).

The expression level of the present transcription regulatory factor gene may be any level which is sufficient to promote the expression of a marker protein gene when compared with a cell into which no present transcription regulatory factor gene has been introduced. In such a case, the present transcription regulatory factor gene may be a DNA encoding the entire or a part of the present transcription regulatory factor.

In a present invention described above, it is also possible to promote a marker protein gene by preparing a transformant in which a present transcription regulatory factor gene is integrated into a genome.

In an expression promoting method described above, a gene construct employed for introducing a present transcription regulatory factor gene into a mammalian cell (hereinafter sometimes referred to as a present gene construct) and a method for accomplishing a gene import may employ a virus vector having an affinity to the mammalian cell to which said DNA is to be introduced, such as a retrovirus vector, adenovirus vector, adeno-associated virus vector or others. For example, known vectors described in Miller, Human Gene Therapy 15 to 14, 1990; Friedman, Science 244:1275 to 1281, 1989; Eglitis and Anderson, BioTechniques 6:608 to 614, 1988; Tolstoshev and Anderson, Current opinion in Biotechnology 1;55 to 61, 1990; Sharp, The Lancet 337:1277 to 1278, 1991: Cornetta et al, Nucleic Acid Research and Molecular Biology 36:311 to 322, 1987; Anderson, Science 22-:401 to 409, 1984; Moen, Blood Cells 17:407 to 416, 1991; Miller et al. Biotechniques 7:980 to 990, 1989; Le Gai La Salle et al. Science 259:988 to 990, 1993; and Johnson) Chest 107;77S to 83S, 1995 and the like may be exemplified. The retroviruses described for example in Rosenberg et al, N. Engl. J. Med 323:370, 1990; Anderson et al., U.S. Pat. No. 5,399,346 have extensively developed, and have already been introduced into a clinical stage. For example, when said cell is an animal cell, those which may be exemplified are an SV40 virus promoter, cytomegalovirus promoter (CMV promoter), Rous sarcoma virus promoter (RSV promoter, β actin gene promoter, aP2 gene promoter and the like. It is also possible to use a commercially available vector containing any of these promoters upstream of the multiple cloning site.

Said DNA may be placed under the control of a promoter which allows a present transcription regulatory factor gene to be expressed constitutively. Such a DNA may also be placed under the control of a promoter which regulates the expression of a present transcription regulatory factor gene via an environmental stimulation. For example, said DNA may be expressed using a tissue-specific or cell type-specific promoter or a promoter which is activated by a chemical signal or exogenous signal such as a drug or by the introduction of a drug.

It is also possible to employ an non-viral technique. Those which may be exemplified are a lipofection described in Felgner et al., Proc. Natl. Acad. Sci. USA 84:7413, 1987; Ono et al., Neurosci. Lett. 117:259, 1990; Brigham et al., Am. J. Ned. Sci. 298:278, 1989; Staubinger et al., Meth. Enz. 101:512, 1983, asialoorosomucoid-polylysine conjugation (Wu et al., J. Biol. Chem. 263:14621, 1988, lipofection described in Wu et al., J. Biol. Chem. 264:16985, 1989 and the like, microinjection described in Wolff et al., Science 247:1465, 1990 and the like, calcium phosphate method, DEAE dextran method, electroporation, protoplast fusion method, liposome method and the like.

While in any of the technologies described above a present gene construct is applied (for example by an infusion) to the site where an underexpression of a maker protein gene described above is expected, it may be applied to a tissue near the site where an event such as an underexpression of a maker protein gene described above is expected or to a vessel supplying to the cell assumed to undergo an underexpression of a maker protein gene described above.

In a present gene construct, the expression of a present transcription regulatory factor gene (cDNA) can be directed by an appropriate promoter (for example, a promoter of human cytomegalovirus (CMV), simian virus 40 (SV40) or metallothionein and the like), and may also be regulated by an appropriate mammalian animal regulatory factor. For example, a present transcription regulatory factor gene can be expressed if necessary using an enhancer known to direct predominantly to the expression of the DNA in a neurocyte. Such an enhancer may be any enhancer whose expression is characterized to be specific to a tissue or cell. When a clone of a present transcription regulatory factor gene (genomic DNA) is employed as a gene construct [for example, a clone of a present transcription regulatory factor gene (genomic DNA) isolated by the hybridization with a present transcription regulatory factor gene (cDNA) described above], the regulation can be accomplished also via a cognate regulatory sequence, if necessary together with a regulatory sequence derived from an heterologous source containing any promoter or regulatory element described above.

When an expression promoting method described above is applied as a method for a gene therapy, it can be used by a direct administration of the present transcription regulatory factor gene into a cell. While the gene which may be employed may be any gene which has been produced or isolated by a standard method, a most convenient production can be accomplished by an in vivo transcription employing the present transcription regulatory factor gene under the control of a highly efficient promoter (for example, human cytomegalovirus promoter). The administration of the present transcription regulatory factor gene can be conducted by any of the direct nucleic acid administration methods described above.

An expression promoting method described above can be applied also as a gene therapy method in which a normal gene is implanted into a diseased cell of a patient. In this method, the normal present transcription regulatory factor gene is transfected into the cell which is exogenous or endogenous to the patient and which can be cultured. Then, the transfected cell is infused serologically into a target tissue.

Ideally, the production of a present transcription regulatory factor by all technologies for the gene therapy gives an intracellular level of the present transcription regulatory factor which is at least equal to a normal intracellular level of the present transcription regulatory factor in a non-diseased cell.

As an example, a present invention described above in the case where the mammalian animal is a transformed mouse is detailed below.

A method for introducing a present transcription regulatory factor gene in the production of a transformed mouse may for example be a microinjection method, a method employing a retrovirus, a method employing an embryonic stem cell (ES cell) and the like. Among those listed above, the microinjection method is employed most frequently. The microinjection method employs a micromanipulator to infuse a solution containing the relevant DNA into the pronucleus of a fertilized ovum under the observation by a microscope.

First, a present transcription regulatory factor gene is infused into a fertilized ovum. In this step, it is preferably to remove the vector region employed for isolating this DNA as much as possible, to remove an AU-rich region contributing to the instabilization of a mRNA and to make the DNA linear for the purpose of integrating the DNA into a chromosome at a high probability, it is also preferable to insert an intron previously into the DNA, and such an intron may for example be a β-globin intron and the like.

A fertilized ovum is obtained from a mouse of a line suitable for the purpose. An inbred C57BL/6 mouse or C3H mouse, a cross line of the C57BL/6 mouse with another line (such as (C57BL/6×DBA/2) F1), a non-inbred line ICR mouse may be exemplified. The fertilized ovum is obtained by mating a female mouse whose superovulation is induced by intraperitoneal administration of both of a pregnant mare's serum gonadotropin and chorionic gonadotropin with a male mouse followed by isolating the ovum from this female mouse. The isolated fertilized ovum is placed in a culture drop, which is maintained in a CO ₂gas incubator, whereby enabling the storage until the infusion of the relevant DNA.

The infusion of the DNA is conducted under the observation with an inverted microscope fitted with a micromanipulator. A fertilized ovum employed is preferably one in a developmental stage of the time when the male pronucleus becomes larger than the female pronucleus through the time-when the both pronuclei are fused with each other. First, the fertilized ovum is fixed, and a DNA solution containing the relevant DNA is infused into the male pronucleus of the fertilized ovum. This DNA solution can be prepared as a complex if necessary. A substance used for forming a complex may for example be a liposome, calcium phosphate, retrovirus and the like. The infusion of the DNA solution is evident from the swelling of the male pronucleus. The amount of the DNA infused may for example be an amount containing about 200 to about 3,000 copies of the relevant DNA.

A fertilized ovum into which a present transcription regulatory factor gene has been infused is then cultured as described above until it becomes a blastocyst, which is then implanted into the uterus of a surrogate mother. Preferably, the ovum is implanted into the oviduct of the surrogate mother immediately after the infusion of the DNA. The surrogate mother is preferably a female mouse in a pseudo-pregnant female mouse after mating with a male mouse whose seminal duct has been ligated. Typically, the relevant female mouse is excised at the back skin and muscle near the kidneys to take the ovaries, oviducts and uterus out, and the ovarian membrane is opened to search for the oviduct opening. Then a surviving fertilized ovum after infusing the relevant DNA is imported from the oviduct opening, and then the ovaries, oviducts and uterus are returned into the abdominal cavity, and then the muscle coats are sutured and the skin is clipped. After about 20 days, a neonate is born.

A part of the somatic tissue of the neonate thus obtained, such as a part of the tail, is cut out as a sample, from which DNAs are extracted and subjected for example to a southern blotting, whereby identifying the relevant DNA. As described above, it can be verified that the relevant DNA has been introduced into a non-human animal. Otherwise, a PCR may also be employed for identification.

While a present transcription regulatory factor gene as an active ingredient of a present transcription regulatory factor gene therapy agent may be prepared as described above, it can be employed in the form of a recombinant vector or recombinant virus containing the relevant DNA. Such a form may for example be a virus vector such as a retrovirus vector, adenovirus vector, adeno-associated virus vector, herpes simplex virus vector, SV40 vector, polyoma virus vector, papilloma virus vector, picornavirus vector and vaccinia virus vector and the like. When an adenovirus vector is employed, an AdEasy Kit produced by QUANTUM is employed to integrate a present transcription regulatory factor gene into a multiple cloning site of a Transfer Vector, and the resultant recombinant vector is made linear, and then transformed into a coliform microorganism together with a pAdEasy vector, and a homologous recombinant DNA is integrated into a human 293A cell, whereby producing a recombinant virus containing the present transcription regulatory factor gene, which is then recovered and used.

It is also possible to use a non-viral vector such as a plasmid DNA comprising a human cytomegalovirus promoter region. Similarly to a case where a present transcription regulatory factor gene is infused directly into a fibrotic tissue site, a use of a plasmid DNA is extremely beneficial in a system where the present transcription regulatory factor gene is delivered locally using a non-viral vector. By employing a method in which a cell once taken out of a body is introduced with an expression vector and then returned to the body, i.e., an ex vivo method, all of the known introduction methods can be utilized. For example, a non-viral vector can be introduced by means of a) direct infusion, b) liposome-mediated introduction, c) cell transfection by calcium phosphate method, electroporation and DEAE-dextran method, d) polybrene-mediated delivery, e) protoplast fusion, f) microinjection, g) introduction using polylysine and the like.

A present transcription regulatory factor gene therapy agent can be given at an effective dose parenterally to a mammalian animal such as a human. For example, a parenteral administration can be accomplished for example by an injection (subcutaneous, intravenosu) as described above. A suitable dosage form described above can be produced by incorporating a present transcription regulatory factor gene (including vector form, virus form, plasmid form of the present transcription regulatory factor gene) into a pharmaceutically acceptable carrier such as an aqueous solvent, non-aqueous solvent, buffering agent, solubilizing aid, osmotic agent, stabilizer and the like. If necessary, auxiliary agents such as a preservative, suspending agent, emulsifier and the like may also be added.

While the dose may vary depending on the age, sex, body weight of a mammalian animal to be treated, the type of a present fat accumulation inhibitor, and the dosage form, it is usually an amount of an active ingredient which gives an intracellular level of a present transcription regulatory factor which is equal to a level allowing the present transcription regulatory factor to act effectively in the cell of the patient. The daily dose described above may be given all at once or in portions.

EXAMPLES

The present invention is further described in the following Examples, which are not intended to restrict the invention. [0127]

Example 1

(Preparation of pGEM-mNXF as Vector Containing Present Transcription Regulatory Factor Gene) [0128]
Polynucleotides consisting of the nucleotide-sequences represented by SEQ ID NO.7 (aagcacggag gaggaagccg ccggtgcgtc gggac) and 8 (ggagagcggc tccacgtctt gatgacaata tgcca) were synthesized using a DNA synthesizer (Applied Biosystems Model 394). As a template, 10 ng of a mouse Brain cDNA library (#10655-017, Gibco BRL) was employed together with the polynucleotides described above as primers whereby effecting a PCR. In this PCR, each 10 pmol of the polynucleotide described above was added to 50 μl of the reaction solution, and an LA-Taq polymerase (Takara) and a buffer attached to the kit containing this enzyme were employed. The PCR reaction solution was incubated using a PCR system 9700 (Applied Biosystems) and subjected to 35 cycles, each cycle consisting of an incubation for 1 minutes at 95° C. followed by 3 minutes at 68° C. [0129]
Then, the entire volume of the PCR reaction solution was subjected to an agarose gel electrophoresis (agarose L, Nippon Gene). After identifying a single band of an about 2.5 kb DNA, this DNA was recovered. A part of the DNA recovered was used together with a dye terminator sequence kit FS (Applied Biosystems) to prepare a direct sequencing sample, which was subjected to a direct nucleotide sequencing using an autosequencer (Applied Biosystems, Model 3700). [0130]
Subsequently, the DNA (about 1 μg) which was recovered as described above was mixed with a pGEM T easy vector (Promega) (10 ng), and combined with a T4 DNA ligase to effect a reaction, whereby obtaining a pGEM-mNXF. The nucleotide sequence of the resultant pGEM-mNXF was determined using an ABI Model 3700 autosequencer by a dye terminator method. The determined nucleotide sequence was compared with the nucleotide sequence obtained by the direct sequencing described above, and it was confirmed that the nucleotide sequence in the translation region exhibited a complete agreement. [0131]

Example 2

(Preparation of pRC/RSV-mNXFsense and pRC/RSV-mNXFantisense) [0132]
Subsequently, a plasmid for expressing a full length present transcription regulatory factor in a mammalian cell (hereinafter sometimes referred to as a present expression plasmid) was prepared as described below. [0133]
First, the direction of the insertion fragment in relation with the multiple cloning site of the pGEM-mNXF obtained in EXAMPLE 1 was in such a construction that the Sp6 promoter of a commercial pGEM vector was positioned upstream of the initiation codon. Then this pGEM-mNXF (1 μg) was employed as a template together with the oligonucleotide primers represented by SEQ ID Nos.11 and 12 (primer pair: forward primer 5′-gggcgctgcagcccagccaccatgtaccgatccaccaaggg-3′, reverse primer 5′-aatctcggcgttgatctggt-3′) to effect a PCR using a KODplus polymerase (TOYOBO), whereby a partial DNA fragment of the present transcription regulatory factor gene into which a Kozac sequence (5′CCAGCCACC-3′) immediately before the initiation codon of the present transcription regulatory factor gene and a PstI restriction enzyme site upstream thereof had been introduced. [0134]
The PCR conditions employed 35 cycles, each cycle involving an incubation at 95° C. for 1 minute followed by 55° C. for 30 seconds followed by 72° C. for 1 minute. [0135]
The amplified DNA fragment thus obtained was cleaved with PstI and BssHII, and subjected to a low melting point agarose gel electrophoresis (NusieveGTG agarose; FMCbio), whereby accomplishing the purification and recovery. The DNA fragment thus purified and recovered was used as an insert fragment as described below. Then, a vector GEM-mNXF which had been cleaved with PstI and BssHII and then SAP-treated was subjected to a low melting point agarose gel electrophoresis (Agarose L, Nippon Gene) to recover a DNA fragment. The recovered DNA fragment (0.1 μg) was ligated with the insert fragment (0.5 μg) described above using a T4 Ligase to obtain a pGEM-mNXF kozac into which a Kozac sequence (5′CCAGCCACC-3′) had been introduced immediately before the initiation codon of the present protein gene derived from a mouse. The nucleotide sequence of the insert fragment was verified to be correct using a DNA sequencer (Model 3700; PE biosystems). [0136]
Then, this pGEM-mNXF kozac was cleaved simultaneously with 3 enzymes PstI, NotI and ScaI, and then subjected to a low melting point agarose electrophoresis to recover an about 2.5 kbp of an mNXF kozac PstI-NotI DNA fragment. The recovered DNA fragment was imparted with a blunt end using a T4 polymerase and then used as an insert fragment. After cleaving an RSV promoter-carrying pRC/RSV (Invitorgen) was cleaved with HindIII, imparted with a blunt end using a T4 polymerase, and subjected to a BAP treatment, whereby obtaining a vector. This vector (0.1 μg) was ligated with the insert fragment (0.5 μg) described above using a T4 Ligase to obtain (a) a pRC/RSV-mNXFsense which is a plasmid expressing the sense strand of the mNXF kozac under the control of the RSV promoter and (2) a pRC/RVS-mNXFantisense which is a plasmid expressing the antisense strand of the mNXF kozac under the control of the RSV promoter. Whether the prepared plasmid was the desired plasmid or not was checked by investigating the nucleotide sequence of the margin between the vector and the inserted fragment. [0137]

Example 3

(Promotion of EphA1 by Present Transcription Regulatory Factor) [0138]
First, about 1×10[0139] ⁷cells of an SK-N-MC cell (ATCC No.HTB10; purchased from DAINIPPON SEIYAKU) were cultured in a 10% FBS-supplemented DMEM medium (NISSUI SEIYAKU) at 37° C. in the presence of 5% CO₂in a petri dish (Falcon) whose diameter was about 10 cm. On the next day, the cultured cells were dispersed by a trypsin treatment, washed twice with an FBS-free DMEM medium, and then dispersed again in an FBS-free DMEM medium at the cell density of 1×10⁷. 0.4 ml of this cell dispersion was combined with each 10 μg of the plasmids prepared in EXAMPLE 2 ((a) pRC/RSV-mNXFsense or (b) pRC/RSV-mNXFantisense), and the mixture was transferred into an electroporation cuvette, where a transfection was conducted by an electroporation method employing a Gene pulser (BIORAD) under the conditions involving 200V and 950 μF. After the transfection, the culture medium was replaced with a 10% FBS-supplemented DMEM, and then further cultured in a 10-cm petri dish for about 24 hours. After the culture, the cells from five dishes were employed as a starting material to be subjected to a purification of DNA-free total RNA using an Atlas Pure Total RNA Labeling system (K1038-1; Clonetech) which is a commercial RNA purifying and radiolabelling kit. The RNA yield was 23 μg when using the plasmid (a) (i.e., pRC/RSV-mNXFsense) and 26 μg when using the plasmid (b) (i.e., pRC/RSV-mNXFantisense). Then, the commercial kit described above and the RNA obtained were employed as a starting material for radiolabeling each RNA with [α-P³²)-dATP (Amersham Pharmacia) using specific primers and reverse transcriptases contained in the commercial kit. Then the radiolabeled RNA (hereinafter referred to as a probe) was purified using the commercial kit described above, and the purified RNA was adjusted at 1.3×10⁵DPM, and then used in the hybridization reaction described below. The hybridization reaction was conducted using a commercial kit of a nylon membrane having various genes blotted thereon (Atlas cDNA Expression array-Neurobiology; 7736-1, Clontech) together with the hybridization buffers attached thereto. The hybridization employed a 18-hour reaction of (a) a nylon membrane corresponding to the probe derived from a pRC-RSV-mNXFsense-introduced cell and (b) a nylon membrane corresponding to the probe derived from a pRC-RSV-mNXFantisense-introduced cell each in an identical incubator under an identical condition. After the reaction, the nylon membrane was washed with 2×SSC, 1% SDS buffer (68° C., 30 minutes). This procedure was repeated 4 times, and then a further washing was made with 0.1×SSC, 0.5% SDS buffer (68° C., 30 minutes). Each nylon membrane was wrapped with a plastic film, exposed to an IP plate (FUJI FILM) for 7 days, and subjected to an image analyzer (BASstation; FUJI FILM) to quantify and compare the intensity of a probe hybridization signal corresponding to each of various genes on the nylon membrane.
As a result, the hybridization signal to the EphA1 gene on (a) the nylon membrane corresponding to the probe derived from a pRC-RSV-mNXFsense-introduced cell was significantly more intense than that on (b) the nylon membrane corresponding to the probe derived from a pRC-RSV-mNXFantisense-introduced cell. Thus, the present transcription regulatory factor was verified to have an ability of promoting the expression of the EphA1. [0140]

Example 4

(Inhibition of Rho GDP Dissociation Inhibitor by Present Transcription Regulatory Factor) [0141]
First, about 1×10[0142] ⁷cells of an SK-N-MC cell (ATCC No.HTB10; purchased from DAINIPPON SEIYAKU) were cultured in a 10% FBS-supplemented DMEM medium (NISSUI SEIYAKU) at 37° C. in the presence of 5% CO₂in a petri dish (Falcon) whose diameter was about 10 cm. On the next day, the cultured cells were dispersed by a trypsin treatment, washed twice with an FBS-free DMEM medium, and then dispersed again in an FBS-free DMEM medium at the cell density of 1×10⁷. 0.4 ml of this cell dispersion was combined with each 10 μg of the plasmids prepared in EXAMPLE 2 ((a) pRC/RSV-mNXFsense or (b)pRC/RSV-mNXFantisense), and the mixture was transferred into an electroporation cuvette, where a transfection was conducted by an electroporation method employing a Gene pulser (BIORAD) under the conditions involving 200V and 950 μF. After the transfection, the culture medium was replaced with a 10% FBS-supplemented DMEM, and then further cultured in a 10-cm petri dish for about 24 hours. After the culture, the cells from five dishes were employed as a starting material to be subjected to a purification of DNA-free total RNA using an Atlas Pure Total RNA Labeling system (K1038-1; Clonetech) which is a commercial RNA purifying and radiolabelling kit. The RNA yield was 23 μg when using the plasmid (a) (i.e., pRC/RSV-mNXFsense) and 26 μg when using the plasmid (b) (i.e., pRC/RSV-mNXFantisense). Then, the commercial kit described above and the RNA obtained were employed as a starting material for radiolabeling each RNA with [α-P³²]-dATP (Amersham Pharmacia) using specific primers and reverse transcriptases contained in the commercial kit. Then the radiolabeled RNA (hereinafter referred to as a probe) was purified using the commercial kit described above, and the purified RNA was adjusted at 1.3×10⁵DPM, and then used in the hybridization reaction described below. The hybridization reaction was conducted using a commercial kit of a nylon membrane having various genes blotted thereon (Atlas cDNA Expression array-Neurobiology; 7736-1, Clontech) together with the hybridization buffers attached thereto the hybridization employed a 18-hour reaction of (a) a nylon membrane corresponding to the probe derived from a pRC-RSV-mNXFsense-introduced cell and (b) a nylon membrane corresponding to the probe derived from a pRC-RSV-mNXFantisense-introduced cell each in an identical incubator under an identical condition. After the reaction, the nylon membrane was washed with 2×SSC, 1% SDS buffer (68° C., 30 minutes). This procedure was repeated 4 times, and then a further washing was made with 0.1×SSC, 0.5% SDS buffer (68° C., 30 minutes). Each nylon membrane was wrapped with a plastic film, exposed to an IP plate (FUJI FILM) for 7 days, and subjected to an image analyzer (BASstation; FUJI FILM) to quantify and compare the intensity of a probe hybridization signal corresponding to each of various genes on the nylon membrane.
As a result, the hybridization signal to the Rho GDP dissociation inhibitor gene on (a) the nylon membrane corresponding to the probe derived from a pRC-RSV-mNXFsense-introduced cell was significantly less intense than that on (b) the nylon membrane corresponding to the probe derived from a pRC-RSV-mNXFantisense-introduced cell. Thus, the present transcription regulatory factor was verified to have an ability of inhibiting the expression of the Rho GDP dissociation inhibitor. [0143]

Example 5

(Assay Method of the Present Invention) [0144]
The pRC/RSV-mNXFsense-introduced cells prepared in EXAMPLE 2 are cultured in a 10% FBS-supplemented DMEM medium (NISSUI SEIYAKU) at 37° C. in the presence of 5% CO[0145] ₂in a petri dish (Falcon) whose diameter is about 10 cm. On the next day, the cultured cells are dispersed by a trypsin treatment, washed twice with an FBS-free DMEM medium, and then dispersed again in the DMEM medium.
Subsequently, the dispersed cells are inoculated at 10[0146] ⁶cells/well in a 6-well plate to which (a) a culture medium supplemented only with DMSO or (b) a culture medium supplemented with a test substance dissolved at a varying concentration in DMSO has previously been added. The cells in this plate are cultured at 37° C. for about 24 hours, and then the culture medium is discarded from the plate, and the cells are washed with PBS (−), and then the total RNA is extracted from each well using Isogen (NIPPON GENE).
The total RNA thus extracted is employed as a starting material to produce a single-stranded cDNA using an oligodT (Amersham Pharmacia) and a Reverse transcriptase (Superscript II, Gibco etc.). Then this single-stranded cDNA is employed as a template to perform a PCR using a LA-Taq (Takara) together with a combination of the forward primers (SEQ ID Nos.13 to 17) and the reverse primers (SEQ ID Nos.18 to 22) listed below. [0147]

EXAMPLES OF FORWARD PRIMERS


	5′-agtgaacagcaacagatact-3′	(SEQ ID Nos. 13)

	5′-gcttcccgcgtccacgtgga-3′	(SEQ ID Nos. 14)

	5′-gatgtgggcattcagctccg-3′	(SEQ ID Nos. 15)

	5′-cagagcttcaccattcgaga-3′	(SEQ ID Nos. 16)

	5′-gtggccctggtgtctgtccg-3′	(SEQ ID Nos. 17)

EXAMPLES OF REVERSE PRIMERS


	5′-ctggcccgcgcgtggggcaa-3′	(SEQ ID Nos. 18)

	5′-acacatgcttcgccactgcc-3′	(SEQ ID Nos. 19)

	5′-taatggccgctctcacaggt-3′	(SEQ ID Nos. 20)

	5′-agctgagtccctgaggcaga-3′	(SEQ ID Nos. 21)

	5′-tgtgctgtgccctgacactg-3′	(SEQ ID Nos. 22)

The PCR employs 30 cycles, each cycle being performed at 95° C. for 1 minute followed by 68° C. for 1 minute. The amount of the mRNA of the EphA 1 present in the total RNA is thus calculated and the amount of the mRNA of the EphA 1 in the total RNA extracted from the cells cultured in the culture medium supplemented only with DMSO is compared with the amount of the mRNA of the EphA 1 in the RNA extracted from the cells cultured in the culture medium supplemented with any of various test substances, thereby assaying and evaluating the ability to control the neurocyte plasticity dependent on the present transcription regulatory factor possessed by the test substance. Based on the results, desired substances are selected. [0150]

EXAMPLES OF FORWARD PRIMERS


	5′-ttgcagcggagaacgaggag-3′	(SEQ ID Nos. 23)

	5′-gatgagcactcggtcaacta-3′	(SEQ ID Nos. 24)

	5′-gcatccaggagatccaggag-3′	(SEQ ID Nos. 25)

	5′-ctggacaaggacgacgagag-3′	(SEQ ID Nos. 26)

	5′-cctgcgaaagtacaaggagg-3′	(SEQ ID Nos. 27)

EXAMPLES OF REVERSE PRIMERS


	5′-cctaccatgtagtcagtctt-3′	(SEQ ID Nos. 28)

	5′-ggtcaggaactcgtactcct-3′	(SEQ ID Nos. 29)

	5′-ttgggtgcctcctccacggg-3′	(SEQ ID Nos. 30)

	5′-gcgggacttgatgctgtagc-3′	(SEQ ID Nos. 31)

	5′-gtcttgtcgtcgtctgtgaa-3′	(SEQ ID Nos. 32)

The amount of the mRNA of the Rho GDP dissociation inhibitor present in the total RNA is thus calculated and the amount of the mRNA of the Rho GDP dissociation inhibitor in the total RNA extracted from the cells cultured in the culture medium supplemented only with DMSO is compared with the amount of the mRNA of the Rho GDP dissociation inhibitor in the total RNA extracted from the cells cultured in the culture medium supplemented with any of various test substances, whereby assaying and evaluating the ability to control the neurocyte plasticity dependent on the present transcription regulatory factor possessed by the test substance. Based on the results, desired substances are selected. [0153]
Industrial Applicability [0154]
According to the invention, a method for assaying an ability to control a neurocyte plasticity which is essential for searching for a substance used for controlling the neurocyte plasticity in a mammalian cell can for example be provided. [0155]
Free Text in Sequence Listing [0156]
SEQ ID No.7 [0157]
Designed oligonucleotide primer for PCR [0158]
SEQ ID No.8 [0159]
Designed oligonucleotide primer for PCR [0160]
SEQ ID No.9 [0161]
Designed oligonucleotide primer for PCR [0162]
SEQ ID No.10 [0163]
Designed oligonucleotide primer for PCR [0164]
SEQ ID No.11 [0165]
Designed oligonucleotide primer for PCR [0166]
SEQ ID No.12 [0167]
Designed oligonucleotide primer for PCR [0168]
SEQ ID No.13 [0169]
Designed oligonucleotide primer for PCR [0170]
SEQ ID No.14 [0171]
Designed oligonucleotide primer for PCR [0172]
SEQ ID No.15 [0173]
Designed oligonucleotide primer for PCR [0174]
SEQ ID No.16 [0175]
Designed oligonucleotide primer for PCR [0176]
SEQ ID No.17 [0177]
Designed oligonucleotide primer for PCR [0178]
SEQ ID No.18 [0179]
Designed oligonucleotide primer for PCR [0180]
SEQ ID No.19 [0181]
Designed oligonucleotide primer for PCR [0182]
SEQ ID No.20 [0183]
Designed oligonucleotide primer for PCR [0184]
SEQ ID No.21 [0185]
Designed oligonucleotide primer for PCR [0186]
SEQ ID No.22 [0187]
Designed oligonucleotide primer for PCR [0188]
SEQ ID No.23 [0189]
Designed oligonucleotide primer for PCR [0190]
SEQ ID No.24 [0191]
Designed oligonucleotide primer for PCR [0192]
SEQ ID No.25 [0193]
Designed oligonucleotide primer for PCR [0194]
SEQ ID No.26 [0195]
Designed oligonucleotide primer for PCR [0196]
SEQ ID No.27 [0197]
Designed oligonucleotide primer for PCR [0198]
SEQ ID No.28 [0199]
Designed oligonucleotide primer for PCR [0200]
SEQ ID No.29 [0201]
Designed oligonucleotide primer for PCR [0202]
SEQ ID No.30 [0203]
Designed oligonucleotide primer for PCR [0204]
SEQ ID No.31 [0205]
Designed oligonucleotide primer for PCR [0206]
SEQ ID No.32 [0207]
Designed oligonucleotide primer for PCR [0208]
1 34 1 802 PRT Homo sapiens 1 Met Tyr Arg Ser Thr Lys Gly Ala Ser Lys Ala Arg Arg Asp Gln Ile 1 5 10 15 Asn Ala Glu Ile Arg Asn Leu Lys Glu Leu Leu Pro Leu Ala Glu Ala 20 25 30 Asp Lys Val Arg Leu Ser Tyr Leu His Ile Met Ser Leu Ala Cys Ile 35 40 45 Tyr Thr Arg Lys Gly Val Phe Phe Ala Gly Gly Thr Pro Leu Ala Gly 50 55 60 Pro Thr Gly Leu Leu Ser Ala Gln Glu Leu Glu Asp Ile Val Ala Ala 65 70 75 80 Leu Pro Gly Phe Leu Leu Val Phe Thr Ala Glu Gly Lys Leu Leu Tyr 85 90 95 Leu Ser Glu Ser Val Ser Glu His Leu Gly His Ser Met Val Asp Leu 100 105 110 Val Ala Gln Gly Asp Ser Ile Tyr Asp Ile Ile Asp Pro Ala Asp His 115 120 125 Leu Thr Val Arg Gln Gln Leu Thr Leu Pro Ser Ala Leu Asp Thr Asp 130 135 140 Arg Leu Phe Arg Cys Arg Phe Asn Thr Ser Lys Ser Leu Arg Arg Gln 145 150 155 160 Ser Ala Gly Asn Lys Leu Val Leu Ile Arg Gly Arg Phe His Ala His 165 170 175 Pro Pro Gly Ala Tyr Trp Ala Gly Asn Pro Val Phe Thr Ala Phe Cys 180 185 190 Ala Pro Leu Glu Pro Arg Pro Arg Pro Gly Pro Gly Pro Gly Pro Gly 195 200 205 Pro Ala Ser Leu Phe Leu Ala Met Phe Gln Ser Arg His Ala Lys Asp 210 215 220 Leu Ala Leu Leu Asp Ile Ser Glu Ser Val Leu Ile Tyr Leu Gly Phe 225 230 235 240 Glu Arg Ser Glu Leu Leu Cys Lys Ser Trp Tyr Gly Leu Leu His Pro 245 250 255 Glu Asp Leu Ala His Ala Ser Ala Gln His Tyr Arg Leu Leu Ala Glu 260 265 270 Ser Gly Asp Ile Gln Ala Glu Met Val Val Arg Leu Gln Ala Lys Thr 275 280 285 Gly Gly Trp Ala Trp Ile Tyr Cys Leu Leu Tyr Ser Glu Gly Pro Glu 290 295 300 Gly Pro Ile Thr Ala Asn Asn Tyr Pro Ile Ser Asp Met Glu Ala Trp 305 310 315 320 Ser Leu Arg Gln Gln Leu Asn Ser Glu Asp Thr Gln Ala Ala Tyr Val 325 330 335 Leu Gly Thr Pro Thr Met Leu Pro Ser Phe Pro Glu Asn Ile Leu Ser 340 345 350 Gln Glu Glu Cys Ser Ser Thr Asn Pro Leu Phe Thr Ala Ala Leu Gly 355 360 365 Ala Pro Arg Ser Thr Ser Phe Pro Ser Ala Pro Glu Leu Ser Val Val 370 375 380 Ser Ala Ser Glu Glu Leu Pro Arg Pro Ser Lys Glu Leu Asp Phe Ser 385 390 395 400 Tyr Leu Thr Phe Pro Ser Gly Pro Glu Pro Ser Leu Gln Ala Glu Leu 405 410 415 Ser Lys Asp Leu Val Cys Thr Pro Pro Tyr Thr Pro His Gln Pro Gly 420 425 430 Gly Cys Ala Phe Leu Phe Ser Leu His Glu Pro Phe Gln Thr His Leu 435 440 445 Pro Thr Pro Ser Ser Thr Leu Gln Glu Gln Leu Thr Pro Ser Thr Ala 450 455 460 Thr Phe Ser Asp Gln Leu Thr Pro Ser Ser Ala Thr Phe Pro Asp Pro 465 470 475 480 Leu Thr Ser Pro Leu Gln Gly Gln Leu Thr Glu Thr Ser Val Arg Ser 485 490 495 Tyr Glu Asp Gln Leu Thr Pro Cys Thr Ser Thr Phe Pro Asp Gln Leu 500 505 510 Leu Pro Ser Thr Ala Thr Phe Pro Glu Pro Leu Gly Ser Pro Ala His 515 520 525 Glu Gln Leu Thr Pro Pro Ser Thr Ala Phe Gln Ala His Leu Asp Ser 530 535 540 Pro Ser Gln Thr Phe Pro Glu Gln Leu Ser Pro Asn Pro Thr Lys Thr 545 550 555 560 Tyr Phe Ala Gln Glu Gly Cys Ser Phe Leu Tyr Glu Lys Leu Pro Pro 565 570 575 Ser Pro Ser Ser Pro Gly Asn Gly Asp Cys Thr Leu Leu Ala Leu Ala 580 585 590 Gln Leu Arg Gly Pro Leu Ser Val Asp Val Pro Leu Val Pro Glu Gly 595 600 605 Leu Leu Thr Pro Glu Ala Ser Pro Val Lys Gln Ser Phe Phe His Tyr 610 615 620 Ser Glu Lys Glu Gln Asn Glu Ile Asp Arg Leu Ile Gln Gln Ile Ser 625 630 635 640 Gln Leu Ala Gln Gly Met Asp Arg Pro Phe Ser Ala Glu Ala Gly Thr 645 650 655 Gly Gly Leu Glu Pro Leu Gly Gly Leu Glu Pro Leu Asp Ser Asn Leu 660 665 670 Ser Leu Ser Gly Ala Gly Pro Pro Val Leu Ser Leu Asp Leu Lys Pro 675 680 685 Trp Lys Cys Gln Glu Leu Asp Phe Leu Ala Asp Pro Asp Asn Met Phe 690 695 700 Leu Glu Glu Thr Pro Val Glu Asp Ile Phe Met Asp Leu Ser Thr Pro 705 710 715 720 Asp Pro Ser Glu Glu Trp Gly Ser Gly Asp Pro Glu Ala Glu Gly Pro 725 730 735 Gly Gly Ala Pro Ser Pro Cys Asn Asn Leu Ser Pro Glu Asp His Ser 740 745 750 Phe Leu Glu Asp Leu Ala Thr Tyr Glu Thr Ala Phe Glu Thr Gly Val 755 760 765 Ser Ala Phe Pro Tyr Asp Gly Phe Thr Asp Glu Leu His Gln Leu Gln 770 775 780 Ser Gln Val Gln Asp Ser Phe His Glu Asp Gly Ser Gly Gly Glu Pro 785 790 795 800 Thr Phe 2 802 PRT Mus musculus 2 Met Tyr Arg Ser Thr Lys Gly Ala Ser Lys Ala Arg Arg Asp Gln Ile 1 5 10 15 Asn Ala Glu Ile Arg Asn Leu Lys Glu Leu Leu Pro Leu Ala Glu Ala 20 25 30 Asp Lys Val Arg Leu Ser Tyr Leu His Ile Met Ser Leu Ala Cys Ile 35 40 45 Tyr Thr Arg Lys Gly Val Phe Phe Ala Gly Gly Thr Pro Leu Ala Gly 50 55 60 Pro Thr Gly Leu Leu Ser Ala Gln Glu Leu Glu Asp Ile Val Ala Ala 65 70 75 80 Leu Pro Gly Phe Leu Leu Val Phe Thr Ala Glu Gly Lys Leu Leu Tyr 85 90 95 Leu Ser Glu Ser Val Ser Glu His Leu Gly His Ser Met Val Asp Leu 100 105 110 Val Ala Gln Gly Asp Ser Ile Tyr Asp Ile Ile Asp Pro Ala Asp His 115 120 125 Leu Thr Val Arg Gln Gln Leu Thr Met Pro Ser Ala Leu Asp Ala Asp 130 135 140 Arg Leu Phe Arg Cys Arg Phe Asn Thr Ser Lys Ser Leu Arg Arg Gln 145 150 155 160 Ser Ser Gly Asn Lys Leu Val Leu Ile Arg Gly Arg Phe His Ala His 165 170 175 Pro Pro Gly Ala Tyr Trp Ala Gly Asn Pro Val Phe Thr Ala Phe Cys 180 185 190 Ala Pro Leu Glu Pro Arg Pro Arg Pro Gly Pro Gly Pro Gly Pro Gly 195 200 205 Pro Gly Pro Ala Ser Leu Phe Leu Ala Met Phe Gln Ser Arg His Ala 210 215 220 Lys Asp Leu Ala Leu Leu Asp Val Ser Glu Ser Val Leu Ile Tyr Leu 225 230 235 240 Gly Phe Glu Arg Ser Glu Leu Leu Cys Lys Ser Trp Tyr Gly Leu Leu 245 250 255 His Pro Glu Asp Leu Ala Gln Ala Ser Ser Gln His Tyr Arg Leu Leu 260 265 270 Ala Glu Ser Gly Asp Ile Gln Ala Glu Met Val Val Arg Leu Gln Ala 275 280 285 Lys His Gly Gly Trp Thr Trp Ile Tyr Cys Met Leu Tyr Ser Glu Gly 290 295 300 Pro Glu Gly Pro Phe Thr Ala Asn Asn Tyr Pro Ile Ser Asp Thr Glu 305 310 315 320 Ala Trp Ser Leu Arg Gln Gln Leu Asn Ser Glu Asp Thr Gln Ala Ala 325 330 335 Tyr Val Leu Gly Thr Pro Ala Val Leu Pro Ser Phe Ser Glu Asn Val 340 345 350 Phe Ser Gln Glu Gln Cys Ser Asn Pro Leu Phe Thr Pro Ser Leu Gly 355 360 365 Thr Pro Arg Ser Ala Ser Phe Pro Arg Ala Pro Glu Leu Gly Val Ile 370 375 380 Ser Thr Pro Glu Glu Leu Pro Gln Pro Ser Lys Glu Leu Asp Phe Ser 385 390 395 400 Tyr Leu Pro Phe Pro Ala Arg Pro Glu Pro Ser Leu Gln Ala Asp Leu 405 410 415 Ser Lys Asp Leu Val Cys Thr Pro Pro Tyr Thr Pro His Gln Pro Gly 420 425 430 Gly Cys Ala Phe Leu Phe Ser Leu His Glu Pro Phe Gln Thr His Leu 435 440 445 Pro Pro Pro Ser Ser Ser Leu Gln Glu Gln Leu Thr Pro Ser Thr Val 450 455 460 Thr Phe Ser Glu Gln Leu Thr Pro Ser Ser Ala Thr Phe Pro Asp Pro 465 470 475 480 Leu Thr Ser Ser Leu Gln Gly Gln Leu Thr Glu Ser Ser Ala Arg Ser 485 490 495 Phe Glu Asp Gln Leu Thr Pro Cys Thr Ser Ser Phe Pro Asp Gln Leu 500 505 510 Leu Pro Ser Thr Ala Thr Phe Pro Glu Pro Leu Gly Ser Pro Ala His 515 520 525 Glu Gln Leu Thr Pro Pro Ser Thr Ala Phe Gln Ala His Leu Asn Ser 530 535 540 Pro Ser Gln Thr Phe Pro Glu Gln Leu Ser Pro Asn Pro Thr Lys Thr 545 550 555 560 Tyr Phe Ala Gln Glu Gly Cys Ser Phe Leu Tyr Glu Lys Leu Pro Pro 565 570 575 Ser Pro Ser Ser Pro Gly Asn Gly Asp Cys Thr Leu Leu Ala Leu Ala 580 585 590 Gln Leu Arg Gly Pro Leu Ser Val Asp Val Pro Leu Val Pro Glu Gly 595 600 605 Leu Leu Thr Pro Glu Ala Ser Pro Val Lys Gln Ser Phe Phe His Tyr 610 615 620 Thr Glu Lys Glu Gln Asn Glu Ile Asp Arg Leu Ile Gln Gln Ile Ser 625 630 635 640 Gln Leu Ala Gln Gly Val Asp Arg Pro Phe Ser Ala Glu Ala Gly Thr 645 650 655 Gly Gly Leu Glu Pro Leu Gly Gly Leu Glu Pro Leu Asn Pro Asn Leu 660 665 670 Ser Leu Ser Gly Ala Gly Pro Pro Val Leu Ser Leu Asp Leu Lys Pro 675 680 685 Trp Lys Cys Gln Glu Leu Asp Phe Leu Val Asp Pro Asp Asn Leu Phe 690 695 700 Leu Glu Glu Thr Pro Val Glu Asp Ile Phe Met Asp Leu Ser Thr Pro 705 710 715 720 Asp Pro Asn Gly Glu Trp Gly Ser Gly Asp Pro Glu Ala Glu Val Pro 725 730 735 Gly Gly Thr Leu Ser Pro Cys Asn Asn Leu Ser Pro Glu Asp His Ser 740 745 750 Phe Leu Glu Asp Leu Ala Thr Tyr Glu Thr Ala Phe Glu Thr Gly Val 755 760 765 Ser Thr Phe Pro Tyr Glu Gly Phe Ala Asp Glu Leu His Gln Leu Gln 770 775 780 Ser Gln Val Gln Asp Ser Phe His Glu Asp Gly Ser Gly Gly Glu Pro 785 790 795 800 Thr Phe 3 802 PRT Rattus norvegicus 3 Met Tyr Arg Ser Thr Lys Gly Ala Ser Lys Ala Arg Arg Asp Gln Ile 1 5 10 15 Asn Ala Glu Ile Arg Asn Leu Lys Glu Leu Leu Pro Leu Ala Glu Ala 20 25 30 Asp Lys Val Arg Leu Ser Tyr Leu His Ile Met Ser Leu Ala Cys Ile 35 40 45 Tyr Thr Arg Lys Gly Val Phe Phe Ala Gly Gly Thr Pro Leu Ala Gly 50 55 60 Pro Thr Gly Leu Leu Ser Ala Gln Glu Leu Glu Asp Ile Val Ala Ala 65 70 75 80 Leu Pro Gly Phe Leu Leu Val Phe Thr Ala Glu Gly Lys Leu Leu Tyr 85 90 95 Leu Ser Glu Ser Val Ser Glu His Leu Gly His Ser Met Val Asp Leu 100 105 110 Val Ala Gln Gly Asp Ser Ile Tyr Asp Ile Ile Asp Pro Ala Asp His 115 120 125 Leu Thr Val Arg Gln Gln Leu Thr Met Pro Ser Ala Leu Asp Ala Asp 130 135 140 Arg Leu Phe Arg Cys Arg Phe Asn Thr Ser Lys Ser Leu Arg Arg Gln 145 150 155 160 Ser Ala Gly Asn Lys Leu Val Leu Ile Arg Gly Arg Phe His Ala His 165 170 175 Pro Pro Gly Ala Tyr Trp Ala Gly Asn Pro Val Phe Thr Ala Phe Cys 180 185 190 Ala Pro Leu Glu Pro Arg Pro Arg Pro Gly Pro Gly Pro Gly Pro Gly 195 200 205 Pro Gly Pro Ala Ser Leu Phe Leu Ala Met Phe Gln Ser Arg His Ala 210 215 220 Lys Asp Leu Ala Leu Leu Asp Ile Ser Glu Ser Val Leu Ile Tyr Leu 225 230 235 240 Gly Phe Glu Arg Ser Glu Leu Leu Cys Lys Ser Trp Tyr Gly Leu Leu 245 250 255 His Pro Glu Asp Leu Ala His Ala Ser Ser Gln His Tyr Arg Leu Leu 260 265 270 Ala Glu Asn Gly Asp Ile Gln Ala Glu Met Val Val Arg Leu Gln Ala 275 280 285 Lys His Gly Gly Trp Thr Trp Ile Tyr Cys Met Leu Tyr Ser Asp Gly 290 295 300 Pro Glu Gly Pro Ile Thr Ala Asn Asn Tyr Pro Ile Ser Asp Thr Glu 305 310 315 320 Ala Trp Ser Leu Arg Gln Gln Leu Asn Ser Glu Asn Thr Gln Ala Ala 325 330 335 Tyr Val Leu Gly Thr Pro Ala Val Leu Pro Ser Phe Ser Glu Asn Val 340 345 350 Phe Ser Gln Glu His Cys Ser Asn Pro Leu Phe Thr Pro Ala Leu Gly 355 360 365 Thr Pro Arg Ser Ala Ser Phe Pro Arg Ala Pro Glu Leu Gly Val Ile 370 375 380 Ser Thr Ser Glu Glu Leu Ala Gln Pro Ser Lys Glu Leu Asp Phe Ser 385 390 395 400 Tyr Leu Pro Phe Pro Ala Arg Pro Glu Pro Ser Leu Gln Ala Asp Leu 405 410 415 Ser Lys Asp Leu Val Cys Thr Pro Pro Tyr Thr Pro His Gln Pro Gly 420 425 430 Gly Cys Ala Phe Leu Phe Ser Leu His Glu Pro Phe Gln Thr His Leu 435 440 445 Pro Pro Pro Ser Ser Ser Leu Gln Glu Gln Leu Thr Pro Ser Thr Val 450 455 460 Thr Phe Ser Glu Gln Leu Thr Pro Ser Ser Ala Thr Phe Pro Asp Pro 465 470 475 480 Leu Thr Ser Ser Leu Gln Gly Gln Leu Thr Glu Ser Ser Ala Arg Ser 485 490 495 Phe Glu Glu Gln Leu Thr Pro Cys Thr Ser Thr Phe Pro Asp Gln Leu 500 505 510 Leu Pro Ser Thr Ala Thr Phe Pro Glu Pro Leu Gly Ser Pro Thr His 515 520 525 Glu Gln Leu Thr Pro Pro Ser Thr Ala Phe Gln Ala His Leu Asn Ser 530 535 540 Pro Ser Gln Thr Phe Pro Glu Gln Leu Ser Pro Asn Pro Thr Lys Thr 545 550 555 560 Tyr Phe Ala Gln Glu Gly Cys Ser Phe Leu Tyr Glu Lys Leu Pro Pro 565 570 575 Ser Pro Ser Ser Pro Gly Asn Gly Asp Cys Thr Leu Leu Ala Leu Ala 580 585 590 Gln Leu Arg Gly Pro Leu Ser Val Asp Val Pro Leu Val Pro Glu Gly 595 600 605 Leu Leu Thr Pro Glu Ala Ser Pro Val Lys Gln Ser Phe Phe His Tyr 610 615 620 Thr Glu Lys Glu Gln Asn Glu Ile Asp Arg Leu Ile Gln Gln Ile Ser 625 630 635 640 Gln Leu Ala Gln Gly Met Asp Arg Pro Phe Ser Ala Glu Ala Gly Thr 645 650 655 Gly Gly Leu Glu Pro Leu Gly Gly Leu Glu Pro Leu Asn Pro Asn Leu 660 665 670 Ser Leu Ser Gly Ala Gly Pro Pro Val Leu Ser Leu Asp Leu Lys Pro 675 680 685 Trp Lys Cys Gln Glu Leu Asp Phe Leu Val Asp Pro Asp Asn Leu Phe 690 695 700 Leu Glu Glu Thr Pro Val Glu Asp Ile Phe Met Asp Leu Ser Thr Pro 705 710 715 720 Asp Pro Asn Gly Glu Trp Gly Ser Gly Asp Pro Glu Ala Glu Val Pro 725 730 735 Gly Gly Thr Leu Ser Pro Cys Asn Asn Leu Ser Pro Glu Asp His Ser 740 745 750 Phe Leu Glu Asp Leu Ala Thr Tyr Glu Thr Ala Phe Glu Thr Gly Val 755 760 765 Ser Thr Phe Pro Tyr Glu Gly Phe Ala Asp Glu Leu His Gln Leu Gln 770 775 780 Ser Gln Val Gln Asp Ser Phe His Glu Asp Gly Ser Gly Gly Glu Pro 785 790 795 800 Thr Phe 4 3252 DNA Homo sapiens CDS (102)..(2507) 4 tgagcgagag acggggaagc acggaggagg aagccgccgg tgcgtcggga cgggagcgca 60 ggtgctcggg cacccgagct ggagctccgc agccgccggt c atg tac cgc tcc acc 116 Met Tyr Arg Ser Thr 1 5 aag ggc gcc tcc aag gcg cgc cgg gac cag atc aac gcc gag atc cgg 164 Lys Gly Ala Ser Lys Ala Arg Arg Asp Gln Ile Asn Ala Glu Ile Arg 10 15 20 aac ctc aag gag ctg ctg ccg ctg gcc gaa gcg gac aag gtc cgg ctg 212 Asn Leu Lys Glu Leu Leu Pro Leu Ala Glu Ala Asp Lys Val Arg Leu 25 30 35 tcc tac ctg cac atc atg agc ctc gcc tgc atc tac act cgc aag ggc 260 Ser Tyr Leu His Ile Met Ser Leu Ala Cys Ile Tyr Thr Arg Lys Gly 40 45 50 gtc ttc ttc gct ggt ggc act cct ctg gcg ggc ccc acg ggg ctt ctc 308 Val Phe Phe Ala Gly Gly Thr Pro Leu Ala Gly Pro Thr Gly Leu Leu 55 60 65 tca gct caa gag ctt gag gac atc gta gcg gca cta ccc ggc ttt ctg 356 Ser Ala Gln Glu Leu Glu Asp Ile Val Ala Ala Leu Pro Gly Phe Leu 70 75 80 85 ctt gtg ttc aca gcc gag ggg aaa ttg ctc tac ctg tct gag agt gtg 404 Leu Val Phe Thr Ala Glu Gly Lys Leu Leu Tyr Leu Ser Glu Ser Val 90 95 100 agc gag cat ctg ggc cac tcc atg gtg gac ctg gtt gcc cag ggt gac 452 Ser Glu His Leu Gly His Ser Met Val Asp Leu Val Ala Gln Gly Asp 105 110 115 agc atc tac gac atc att gac cca gct gac cac ctc act gtg cgc cag 500 Ser Ile Tyr Asp Ile Ile Asp Pro Ala Asp His Leu Thr Val Arg Gln 120 125 130 caa ctc acc ctg ccc tct gcc ctg gac act gat cgc ctc ttc cgc tgc 548 Gln Leu Thr Leu Pro Ser Ala Leu Asp Thr Asp Arg Leu Phe Arg Cys 135 140 145 cgc ttc aac acc tcc aag tcc ctc agg cgc cag agt gca ggc aac aaa 596 Arg Phe Asn Thr Ser Lys Ser Leu Arg Arg Gln Ser Ala Gly Asn Lys 150 155 160 165 ctc gtg ctt att cga ggc cga ttc cat gct cac cca cct gga gcc tac 644 Leu Val Leu Ile Arg Gly Arg Phe His Ala His Pro Pro Gly Ala Tyr 170 175 180 tgg gca gga aat ccc gtg ttc aca gct ttc tgt gcc cct ctg gag ccg 692 Trp Ala Gly Asn Pro Val Phe Thr Ala Phe Cys Ala Pro Leu Glu Pro 185 190 195 aga ccc cgc cca ggt cct ggc cct ggc cct ggc cct gcc tcg ctc ttc 740 Arg Pro Arg Pro Gly Pro Gly Pro Gly Pro Gly Pro Ala Ser Leu Phe 200 205 210 ctg gcc atg ttc cag agc cgc cat gct aaa gac ctg gct cta ctg gac 788 Leu Ala Met Phe Gln Ser Arg His Ala Lys Asp Leu Ala Leu Leu Asp 215 220 225 atc tcc gag agt gtc cta atc tac ctg ggc ttt gag cgc agt gaa ctg 836 Ile Ser Glu Ser Val Leu Ile Tyr Leu Gly Phe Glu Arg Ser Glu Leu 230 235 240 245 ctt tgt aaa tca tgg tat gga ctg ctg cac ccc gag gac ctg gcc cac 884 Leu Cys Lys Ser Trp Tyr Gly Leu Leu His Pro Glu Asp Leu Ala His 250 255 260 gct tct gct caa cac tac cgc ctg ttg gct gag agt gga gat att cag 932 Ala Ser Ala Gln His Tyr Arg Leu Leu Ala Glu Ser Gly Asp Ile Gln 265 270 275 gca gag atg gtg gtg agg cta cag gcc aag act gga ggc tgg gca tgg 980 Ala Glu Met Val Val Arg Leu Gln Ala Lys Thr Gly Gly Trp Ala Trp 280 285 290 att tac tgc ctg tta tac tca gaa ggt cca gag gga ccc att act gcc 1028 Ile Tyr Cys Leu Leu Tyr Ser Glu Gly Pro Glu Gly Pro Ile Thr Ala 295 300 305 aat aac tac cca atc agt gac atg gaa gcc tgg agc ctc cgc cag cag 1076 Asn Asn Tyr Pro Ile Ser Asp Met Glu Ala Trp Ser Leu Arg Gln Gln 310 315 320 325 ttg aac tct gaa gac acc cag gca gct tat gtc ctg ggc act ccg acc 1124 Leu Asn Ser Glu Asp Thr Gln Ala Ala Tyr Val Leu Gly Thr Pro Thr 330 335 340 atg ctg ccc tca ttc cct gaa aac att ctt tcc cag gaa gag tgc tcc 1172 Met Leu Pro Ser Phe Pro Glu Asn Ile Leu Ser Gln Glu Glu Cys Ser 345 350 355 agc act aac cca ctc ttc acc gca gca ctg ggg gct ccc aga agc acc 1220 Ser Thr Asn Pro Leu Phe Thr Ala Ala Leu Gly Ala Pro Arg Ser Thr 360 365 370 agc ttc ccc agt gct cct gaa ctg agt gtt gtc tct gca tca gaa gag 1268 Ser Phe Pro Ser Ala Pro Glu Leu Ser Val Val Ser Ala Ser Glu Glu 375 380 385 ctt ccc cga ccc tcc aaa gaa ctg gac ttc agt tac ctg aca ttc cct 1316 Leu Pro Arg Pro Ser Lys Glu Leu Asp Phe Ser Tyr Leu Thr Phe Pro 390 395 400 405 tct ggg cct gag cct tct ctc caa gca gaa cta agc aag gat ctt gtg 1364 Ser Gly Pro Glu Pro Ser Leu Gln Ala Glu Leu Ser Lys Asp Leu Val 410 415 420 tgc act cca cct tac acg ccc cat cag cca gga ggc tgt gcc ttc ctc 1412 Cys Thr Pro Pro Tyr Thr Pro His Gln Pro Gly Gly Cys Ala Phe Leu 425 430 435 ttc agc ctc cat gag ccc ttc cag acc cat ttg ccc acc cca tcc agc 1460 Phe Ser Leu His Glu Pro Phe Gln Thr His Leu Pro Thr Pro Ser Ser 440 445 450 act ctt caa gaa cag ctg act cca agc act gcg acc ttc tct gat cag 1508 Thr Leu Gln Glu Gln Leu Thr Pro Ser Thr Ala Thr Phe Ser Asp Gln 455 460 465 ttg acg ccc agc agt gca acc ttc cca gat cca cta act agc cca ctg 1556 Leu Thr Pro Ser Ser Ala Thr Phe Pro Asp Pro Leu Thr Ser Pro Leu 470 475 480 485 caa ggc cag ttg act gaa acc tcg gtc aga agc tat gaa gac cag ttg 1604 Gln Gly Gln Leu Thr Glu Thr Ser Val Arg Ser Tyr Glu Asp Gln Leu 490 495 500 act ccc tgc acc tcc acc ttc cca gac cag ctg ctt ccc agc aca gcc 1652 Thr Pro Cys Thr Ser Thr Phe Pro Asp Gln Leu Leu Pro Ser Thr Ala 505 510 515 acc ttc cca gag cct ctg ggc agc cct gcc cat gaa cag ctg act cct 1700 Thr Phe Pro Glu Pro Leu Gly Ser Pro Ala His Glu Gln Leu Thr Pro 520 525 530 ccc agc aca gca ttc caa gca cac ctg gac agc ccc agc caa acc ttc 1748 Pro Ser Thr Ala Phe Gln Ala His Leu Asp Ser Pro Ser Gln Thr Phe 535 540 545 cca gag caa ctg agc ccc aac cct acc aag act tac ttt gcc cag gag 1796 Pro Glu Gln Leu Ser Pro Asn Pro Thr Lys Thr Tyr Phe Ala Gln Glu 550 555 560 565 gga tgc agt ttt ctc tat gag aag ttg ccc cca agt cct agc agc cct 1844 Gly Cys Ser Phe Leu Tyr Glu Lys Leu Pro Pro Ser Pro Ser Ser Pro 570 575 580 ggt aat ggg gac tgc acg ctc ttg gcc cta gcc cag ctc cgg ggc ccc 1892 Gly Asn Gly Asp Cys Thr Leu Leu Ala Leu Ala Gln Leu Arg Gly Pro 585 590 595 ctc tct gtg gat gtc ccc ctg gtg ccc gaa ggc ctg ctc aca cct gag 1940 Leu Ser Val Asp Val Pro Leu Val Pro Glu Gly Leu Leu Thr Pro Glu 600 605 610 gcc tct cca gtc aag cag agt ttc ttc cac tac tct gaa aag gag cag 1988 Ala Ser Pro Val Lys Gln Ser Phe Phe His Tyr Ser Glu Lys Glu Gln 615 620 625 aat gag ata gac cgt ctc atc cag cag att agc caa ttg gct cag ggc 2036 Asn Glu Ile Asp Arg Leu Ile Gln Gln Ile Ser Gln Leu Ala Gln Gly 630 635 640 645 atg gac aga ccc ttc tca gct gag gct ggc act ggc gga cta gag cca 2084 Met Asp Arg Pro Phe Ser Ala Glu Ala Gly Thr Gly Gly Leu Glu Pro 650 655 660 ctt gga gga ctg gag ccc ctg gac tcc aac ctg tcc ctg tca ggg gca 2132 Leu Gly Gly Leu Glu Pro Leu Asp Ser Asn Leu Ser Leu Ser Gly Ala 665 670 675 ggc ccc cct gtg ctc agc ctg gac ctg aaa ccc tgg aaa tgc cag gag 2180 Gly Pro Pro Val Leu Ser Leu Asp Leu Lys Pro Trp Lys Cys Gln Glu 680 685 690 ctg gac ttc ctg gct gac cct gat aac atg ttc ctg gaa gag acg ccc 2228 Leu Asp Phe Leu Ala Asp Pro Asp Asn Met Phe Leu Glu Glu Thr Pro 695 700 705 gtg gaa gac atc ttc atg gat ctc tct acc cca gat ccc agt gag gaa 2276 Val Glu Asp Ile Phe Met Asp Leu Ser Thr Pro Asp Pro Ser Glu Glu 710 715 720 725 tgg ggc tca ggg gat cct gag gca gag ggc cca gga ggg gcc cca tcg 2324 Trp Gly Ser Gly Asp Pro Glu Ala Glu Gly Pro Gly Gly Ala Pro Ser 730 735 740 cct tgc aac aac ctg tcc cca gaa gac cac agc ttc ctg gag gac ctg 2372 Pro Cys Asn Asn Leu Ser Pro Glu Asp His Ser Phe Leu Glu Asp Leu 745 750 755 gcc aca tat gaa acc gcc ttt gag aca ggt gtc tca gca ttc ccc tat 2420 Ala Thr Tyr Glu Thr Ala Phe Glu Thr Gly Val Ser Ala Phe Pro Tyr 760 765 770 gat ggg ttt act gat gag ttg cat caa ctc cag agc caa gtt caa gac 2468 Asp Gly Phe Thr Asp Glu Leu His Gln Leu Gln Ser Gln Val Gln Asp 775 780 785 agc ttc cat gaa gat gga agt gga ggg gaa cca acg ttt tgaataagtc 2517 Ser Phe His Glu Asp Gly Ser Gly Gly Glu Pro Thr Phe 790 795 800 tgtgacttaa cgtcgtcaag tatggcatat tgtcatcaag acgtggagcc gctctccacc 2577 cccccgggac tgttgggggg attctgaggg ccagaggggg atatatatga ttccccaggc 2637 cctgcaggat tttggggggg gggaggtggg agggcaaggg aggggagctt ctttttaaaa 2697 tcaagagact tcgagcgatc ccagtttcca tttcaatctg tattcactcg tagtgagttt 2757 ccttgaatgg gatttcaagc ggagaatggg ggagtctcac ttccccgccg ccttgcccca 2817 ttggcctggg ccagttctcc actcctaggg gccaagccac ccctagcctt ggtgggggaa 2877 aggcagggcc cacccgggcc agcccgtgcc ctgaggggct cttgacaccc acgtagaatt 2937 ctctacacac cagtaacggg atttcaattc cgatggactc tgccgccctg gcggcccttc 2997 ctgtgacttt tgcgccccgc gcctggggtg gggggtgcga aaaaacgcta cgttcctttc 3057 cgatggagga aggcagacct gccgtcacac gtgtgcttgc acgagtgcgt gtacctggtg 3117 cgggactcac ccggccgcca gactgcctgg gcctgcccaa atggccacct cggtggtgct 3177 gcggtgactt tgtagccaac tttataataa agtccagttt gcctttttgg taaaaaaaaa 3237 aaaaaaaaaa aaaaa 3252 5 3087 DNA Mus musculus CDS (51)..(2456) 5 aggatcgcag gtgctcggga gccggagctg gagctccaca gccggcagtc atg tac 56 Met Tyr 1 cga tcc acc aag ggc gcc tcc aag gcg cgc cgc gac cag atc aac gcc 104 Arg Ser Thr Lys Gly Ala Ser Lys Ala Arg Arg Asp Gln Ile Asn Ala 5 10 15 gag att cgg aac ctc aag gag ctg ctg ccg ttg gct gaa gcg gac aag 152 Glu Ile Arg Asn Leu Lys Glu Leu Leu Pro Leu Ala Glu Ala Asp Lys 20 25 30 gtc cgg ctg tcc tac ctg cac atc atg agt ctt gcc tgc atc tac act 200 Val Arg Leu Ser Tyr Leu His Ile Met Ser Leu Ala Cys Ile Tyr Thr 35 40 45 50 cgc aag ggt gtc ttc ttt gct gga ggc act cct ttg gct ggc ccc acc 248 Arg Lys Gly Val Phe Phe Ala Gly Gly Thr Pro Leu Ala Gly Pro Thr 55 60 65 ggg ctt ctc tct gct caa gag ctt gaa gac att gtg gca gca cta cct 296 Gly Leu Leu Ser Ala Gln Glu Leu Glu Asp Ile Val Ala Ala Leu Pro 70 75 80 gga ttt ctc ctt gta ttc aca gct gag ggg aag ttg cta tac ctg tcg 344 Gly Phe Leu Leu Val Phe Thr Ala Glu Gly Lys Leu Leu Tyr Leu Ser 85 90 95 gag agt gtg agc gag cat ctg ggc cac tct atg gtg gac ctg gtt gcc 392 Glu Ser Val Ser Glu His Leu Gly His Ser Met Val Asp Leu Val Ala 100 105 110 cag ggc gac agt atc tac gat atc att gac cct gct gac cat ctc act 440 Gln Gly Asp Ser Ile Tyr Asp Ile Ile Asp Pro Ala Asp His Leu Thr 115 120 125 130 gtg cgc cag cag ctc acc atg ccc tct gct ctg gat gct gat cgc ctt 488 Val Arg Gln Gln Leu Thr Met Pro Ser Ala Leu Asp Ala Asp Arg Leu 135 140 145 ttc cgt tgt cga ttc aac acc tcc aag tcc ctc cgg cgc cag agt tca 536 Phe Arg Cys Arg Phe Asn Thr Ser Lys Ser Leu Arg Arg Gln Ser Ser 150 155 160 gga aac aaa ctg gtg ctt att cga ggt cga ttc cat gct cac cca cct 584 Gly Asn Lys Leu Val Leu Ile Arg Gly Arg Phe His Ala His Pro Pro 165 170 175 ggg gcc tac tgg gca gga aac cct gtg ttc acc gct ttc tgc gcc cca 632 Gly Ala Tyr Trp Ala Gly Asn Pro Val Phe Thr Ala Phe Cys Ala Pro 180 185 190 ctg gag cca aga ccc cgc cct ggc ccc ggc cct ggc cct ggc cct ggt 680 Leu Glu Pro Arg Pro Arg Pro Gly Pro Gly Pro Gly Pro Gly Pro Gly 195 200 205 210 cct gct tct ctc ttc ctg gcc atg ttc cag agc cgg cat gct aag gac 728 Pro Ala Ser Leu Phe Leu Ala Met Phe Gln Ser Arg His Ala Lys Asp 215 220 225 cta gcc cta ctg gac gtt tct gaa agt gtc cta atc tac ctg ggc ttt 776 Leu Ala Leu Leu Asp Val Ser Glu Ser Val Leu Ile Tyr Leu Gly Phe 230 235 240 gag cgc agc gaa ctg ctc tgt aaa tca tgg tat gga ctg cta cac ccc 824 Glu Arg Ser Glu Leu Leu Cys Lys Ser Trp Tyr Gly Leu Leu His Pro 245 250 255 gag gac ctg gcc caa gct tct tct caa cac tac cgc ctg ttg gct gaa 872 Glu Asp Leu Ala Gln Ala Ser Ser Gln His Tyr Arg Leu Leu Ala Glu 260 265 270 agt gga gat att cag gct gaa atg gtg gtg aga ctt caa gcc aag cat 920 Ser Gly Asp Ile Gln Ala Glu Met Val Val Arg Leu Gln Ala Lys His 275 280 285 290 gga ggc tgg aca tgg att tac tgc atg cta tac tca gaa ggt cca gaa 968 Gly Gly Trp Thr Trp Ile Tyr Cys Met Leu Tyr Ser Glu Gly Pro Glu 295 300 305 ggc cct ttt act gcc aat aac tac cct atc agt gac acg gaa gcc tgg 1016 Gly Pro Phe Thr Ala Asn Asn Tyr Pro Ile Ser Asp Thr Glu Ala Trp 310 315 320 agc ctc cgc cag cag cta aac tct gaa gac acc cag gca gcc tat gtc 1064 Ser Leu Arg Gln Gln Leu Asn Ser Glu Asp Thr Gln Ala Ala Tyr Val 325 330 335 cta gga acc cca gct gtg cta ccc tca ttc tct gag aat gtc ttc tcc 1112 Leu Gly Thr Pro Ala Val Leu Pro Ser Phe Ser Glu Asn Val Phe Ser 340 345 350 cag gag caa tgc tct aat cca ctc ttt aca cca tcc ctg ggg act cct 1160 Gln Glu Gln Cys Ser Asn Pro Leu Phe Thr Pro Ser Leu Gly Thr Pro 355 360 365 370 aga agt gcc agc ttc ccc agg gct cct gaa cta ggt gtg atc tca aca 1208 Arg Ser Ala Ser Phe Pro Arg Ala Pro Glu Leu Gly Val Ile Ser Thr 375 380 385 cca gaa gag ctt ccc caa ccc tcc aaa gag ctg gac ttc agt tac ctg 1256 Pro Glu Glu Leu Pro Gln Pro Ser Lys Glu Leu Asp Phe Ser Tyr Leu 390 395 400 cca ttc cct gct agg cct gag cct tcc ctc caa gca gac ctg agc aag 1304 Pro Phe Pro Ala Arg Pro Glu Pro Ser Leu Gln Ala Asp Leu Ser Lys 405 410 415 gat ttg gtg tgt act cca cct tac aca ccc cac cag cca gga ggc tgt 1352 Asp Leu Val Cys Thr Pro Pro Tyr Thr Pro His Gln Pro Gly Gly Cys 420 425 430 gcc ttc ctc ttc agc ctc cat gaa ccc ttc cag act cac ttg ccc cct 1400 Ala Phe Leu Phe Ser Leu His Glu Pro Phe Gln Thr His Leu Pro Pro 435 440 445 450 ccg tcc agc tct ctc caa gaa cag ctg aca cca agt aca gtg act ttc 1448 Pro Ser Ser Ser Leu Gln Glu Gln Leu Thr Pro Ser Thr Val Thr Phe 455 460 465 tct gaa cag ttg aca ccc agc agt gct acc ttc cca gac cca cta acc 1496 Ser Glu Gln Leu Thr Pro Ser Ser Ala Thr Phe Pro Asp Pro Leu Thr 470 475 480 agt tca cta caa gga cag ttg aca gaa agc tca gcc aga agc ttt gaa 1544 Ser Ser Leu Gln Gly Gln Leu Thr Glu Ser Ser Ala Arg Ser Phe Glu 485 490 495 gac cag ttg act cca tgc acc tct tcc ttc cct gac cag cta ctt ccc 1592 Asp Gln Leu Thr Pro Cys Thr Ser Ser Phe Pro Asp Gln Leu Leu Pro 500 505 510 agc act gcc aca ttc cca gag cct ctg ggc agc ccc gcc cat gag cag 1640 Ser Thr Ala Thr Phe Pro Glu Pro Leu Gly Ser Pro Ala His Glu Gln 515 520 525 530 ctg act cct ccc agc aca gca ttc cag gct cat ctg aac agc ccc agc 1688 Leu Thr Pro Pro Ser Thr Ala Phe Gln Ala His Leu Asn Ser Pro Ser 535 540 545 caa acc ttc cca gag caa ctg agc ccc aat cct acc aag act tac ttc 1736 Gln Thr Phe Pro Glu Gln Leu Ser Pro Asn Pro Thr Lys Thr Tyr Phe 550 555 560 gcc cag gag gga tgc agt ttt ctc tat gag aag ttg ccc cca agt cct 1784 Ala Gln Glu Gly Cys Ser Phe Leu Tyr Glu Lys Leu Pro Pro Ser Pro 565 570 575 agc agc cct ggt aat ggg gac tgt aca ctc ctg gcc cta gct cag ctc 1832 Ser Ser Pro Gly Asn Gly Asp Cys Thr Leu Leu Ala Leu Ala Gln Leu 580 585 590 cgg ggc ccc ctc tct gtg gat gtc ccc ctg gtg ccc gaa ggc ctg ctc 1880 Arg Gly Pro Leu Ser Val Asp Val Pro Leu Val Pro Glu Gly Leu Leu 595 600 605 610 aca cct gag gcc tct cca gtc aag caa agt ttc ttc cac tac aca gag 1928 Thr Pro Glu Ala Ser Pro Val Lys Gln Ser Phe Phe His Tyr Thr Glu 615 620 625 aaa gag caa aat gag ata gat cgt ctc att cag cag atc agc cag ttg 1976 Lys Glu Gln Asn Glu Ile Asp Arg Leu Ile Gln Gln Ile Ser Gln Leu 630 635 640 gct cag ggc gtg gac agg ccc ttc tca gct gag gct ggc act ggg ggg 2024 Ala Gln Gly Val Asp Arg Pro Phe Ser Ala Glu Ala Gly Thr Gly Gly 645 650 655 ctg gag cca ctt gga ggg ctg gag ccc ctg aac cct aac ctg tcc ctg 2072 Leu Glu Pro Leu Gly Gly Leu Glu Pro Leu Asn Pro Asn Leu Ser Leu 660 665 670 tca ggg gct gga ccc cct gtg ctt agc ctg gat ctt aaa ccc tgg aaa 2120 Ser Gly Ala Gly Pro Pro Val Leu Ser Leu Asp Leu Lys Pro Trp Lys 675 680 685 690 tgc cag gag ctg gac ttc ctg gtt gac cct gat aat tta ttc ctg gaa 2168 Cys Gln Glu Leu Asp Phe Leu Val Asp Pro Asp Asn Leu Phe Leu Glu 695 700 705 gag acg cca gtg gaa gac atc ttc atg gat ctt tct act cca gac ccc 2216 Glu Thr Pro Val Glu Asp Ile Phe Met Asp Leu Ser Thr Pro Asp Pro 710 715 720 aat ggg gaa tgg ggt tca ggg gat cct gag gca gag gtc cca gga ggg 2264 Asn Gly Glu Trp Gly Ser Gly Asp Pro Glu Ala Glu Val Pro Gly Gly 725 730 735 acc ctg tca cct tgc aac aac ctg tcc cca gaa gat cac agc ttc ctg 2312 Thr Leu Ser Pro Cys Asn Asn Leu Ser Pro Glu Asp His Ser Phe Leu 740 745 750 gag gac ttg gcc acc tat gaa acc gcc ttt gag aca ggt gtc tca aca 2360 Glu Asp Leu Ala Thr Tyr Glu Thr Ala Phe Glu Thr Gly Val Ser Thr 755 760 765 770 ttc ccc tac gaa ggg ttt gct gat gag ttg cat caa ctc cag agc caa 2408 Phe Pro Tyr Glu Gly Phe Ala Asp Glu Leu His Gln Leu Gln Ser Gln 775 780 785 gtt caa gac agc ttc cat gaa gat gga agt gga ggg gaa cca acg ttt 2456 Val Gln Asp Ser Phe His Glu Asp Gly Ser Gly Gly Glu Pro Thr Phe 790 795 800 tgaataagtc tgtgacttaa cgtcttcaag tatggcatat tgtcatcaag acgtggagcc 2516 gctctccacc cccccgggac tgttgggggg attctggggg ccagaggggg atatatctga 2576 ttctccaggc cctgaaggat ttagggggga ggtgggaggg taagggaggg gagcaacttt 2636 ttaaaatcaa gagacttcga gcgatcccag tttccatttc aatctgtatt cactcgtagt 2696 gagtttcctt gaatggattt caagcggaga atgggggagt ctcacttcct caccgcgctg 2756 ccccatgggc ctgggccagt tctccactcc taggggcaaa gccacccctg ggctttggtg 2816 ggggaaaggc atggcccacc tggggctagc ctgtgccccg aggggctctt gacacccacg 2876 tagaattctc tacaaaccag taacgggatt tcaattccga cggactctgc cgccctggcg 2936 gctcttcctg tgacttttgc gccccgcgcc tggggtgggg ggcgcgaaga gacgctacat 2996 tcctttccga tggaggaagg cagatctgcc gtcacacgtg tgcttgcacg agtgcgtgta 3056 cctggtgcgg gactcacccg gccgccagac c 3087 6 2459 DNA Rattus norvegicus CDS (35)..(2440) 6 gggagccgga gctggagctc cacggccggc agtc atg tac cga tcc acc aag ggc 55 Met Tyr Arg Ser Thr Lys Gly 1 5 gcc tcc aag gcg cgc cgc gac cag atc aac gcc gag att cgg aac ctc 103 Ala Ser Lys Ala Arg Arg Asp Gln Ile Asn Ala Glu Ile Arg Asn Leu 10 15 20 aag gaa ctg ctg ccg ttg gct gaa gcg gac aag gtc cgg ctg tcc tac 151 Lys Glu Leu Leu Pro Leu Ala Glu Ala Asp Lys Val Arg Leu Ser Tyr 25 30 35 ctg cac atc atg agt ctt gcc tgc atc tac act cgc aag ggt gtc ttc 199 Leu His Ile Met Ser Leu Ala Cys Ile Tyr Thr Arg Lys Gly Val Phe 40 45 50 55 ttt gct gga ggc act cct ttg gct ggc ccc acg ggg ctt ctc tct gct 247 Phe Ala Gly Gly Thr Pro Leu Ala Gly Pro Thr Gly Leu Leu Ser Ala 60 65 70 caa gag ctt gaa gac ata gtg gca gca cta cct gga ttt cta ctt gtg 295 Gln Glu Leu Glu Asp Ile Val Ala Ala Leu Pro Gly Phe Leu Leu Val 75 80 85 ttc aca gct gag ggg aag ttg cta tac ctg tcg gag agt gtg agc gag 343 Phe Thr Ala Glu Gly Lys Leu Leu Tyr Leu Ser Glu Ser Val Ser Glu 90 95 100 cat ctg ggc cat tct atg gtg gat ctg gtt gcc cag ggt gac agt att 391 His Leu Gly His Ser Met Val Asp Leu Val Ala Gln Gly Asp Ser Ile 105 110 115 tac gac atc att gac cct gct gac cat ctc act gtg cgc cag cag ctc 439 Tyr Asp Ile Ile Asp Pro Ala Asp His Leu Thr Val Arg Gln Gln Leu 120 125 130 135 acc atg ccc tct gct ctg gat gct gat cgc ctt ttc cgt tgt cga ttt 487 Thr Met Pro Ser Ala Leu Asp Ala Asp Arg Leu Phe Arg Cys Arg Phe 140 145 150 aac aca tcc aag tcc ctc cgg cgc cag agt gca ggc aac aaa ctg gtg 535 Asn Thr Ser Lys Ser Leu Arg Arg Gln Ser Ala Gly Asn Lys Leu Val 155 160 165 ctt att cga ggt cga ttc cat gct cac cca cct ggg gcc tac tgg gca 583 Leu Ile Arg Gly Arg Phe His Ala His Pro Pro Gly Ala Tyr Trp Ala 170 175 180 gga aac ccc gtg ttc aca gct ttc tgt gcc cca ctg gag cca aga ccc 631 Gly Asn Pro Val Phe Thr Ala Phe Cys Ala Pro Leu Glu Pro Arg Pro 185 190 195 cgt ccc ggc cct ggc cct ggc cct ggc cct ggt cct gcc tct ctc ttc 679 Arg Pro Gly Pro Gly Pro Gly Pro Gly Pro Gly Pro Ala Ser Leu Phe 200 205 210 215 ctg gcc atg ttc cag agc cgg cat gct aag gac cta gcc cta ctg gac 727 Leu Ala Met Phe Gln Ser Arg His Ala Lys Asp Leu Ala Leu Leu Asp 220 225 230 att tct gaa agt gtc cta atc tac ctg ggc ttt gag cgc agc gaa ctg 775 Ile Ser Glu Ser Val Leu Ile Tyr Leu Gly Phe Glu Arg Ser Glu Leu 235 240 245 ctc tgt aaa tca tgg tat gga ctg cta cac ccc gag gac ctg gcc cac 823 Leu Cys Lys Ser Trp Tyr Gly Leu Leu His Pro Glu Asp Leu Ala His 250 255 260 gct tct tct caa cac tac cgc ctg ttg gct gaa aat gga gat att cag 871 Ala Ser Ser Gln His Tyr Arg Leu Leu Ala Glu Asn Gly Asp Ile Gln 265 270 275 gct gaa atg gtg gtg aga ctt caa gcc aag cat gga ggc tgg aca tgg 919 Ala Glu Met Val Val Arg Leu Gln Ala Lys His Gly Gly Trp Thr Trp 280 285 290 295 att tac tgc atg cta tac tcg gat ggt cca gaa ggc cct att act gcc 967 Ile Tyr Cys Met Leu Tyr Ser Asp Gly Pro Glu Gly Pro Ile Thr Ala 300 305 310 aat aac tac cct atc agt gac acg gaa gcc tgg agt ctt cgc cag cag 1015 Asn Asn Tyr Pro Ile Ser Asp Thr Glu Ala Trp Ser Leu Arg Gln Gln 315 320 325 cta aac tct gaa aac acc cag gca gcc tat gtc cta gga acc cca gct 1063 Leu Asn Ser Glu Asn Thr Gln Ala Ala Tyr Val Leu Gly Thr Pro Ala 330 335 340 gtg cta ccc tca ttc tct gag aat gtc ttc tcc cag gag cac tgc tct 1111 Val Leu Pro Ser Phe Ser Glu Asn Val Phe Ser Gln Glu His Cys Ser 345 350 355 aat cca ctc ttt aca cca gcc ctg ggg act cct aga agt gcc agc ttc 1159 Asn Pro Leu Phe Thr Pro Ala Leu Gly Thr Pro Arg Ser Ala Ser Phe 360 365 370 375 ccc agg gcc cct gaa cta ggt gtg atc tca aca tca gaa gag ctt gcc 1207 Pro Arg Ala Pro Glu Leu Gly Val Ile Ser Thr Ser Glu Glu Leu Ala 380 385 390 caa ccc tcc aaa gaa ctg gac ttc agt tac ctg cca ttc cct gca agg 1255 Gln Pro Ser Lys Glu Leu Asp Phe Ser Tyr Leu Pro Phe Pro Ala Arg 395 400 405 cct gag cct tcc ctc caa gca gac ttg agc aag gat ttg gtg tgt act 1303 Pro Glu Pro Ser Leu Gln Ala Asp Leu Ser Lys Asp Leu Val Cys Thr 410 415 420 cca cct tac aca ccc cac cag cca gga ggc tgc gcc ttc ctc ttc agc 1351 Pro Pro Tyr Thr Pro His Gln Pro Gly Gly Cys Ala Phe Leu Phe Ser 425 430 435 ctc cat gaa ccc ttc cag act cac ttg ccc cct cca tcc agc tct ctc 1399 Leu His Glu Pro Phe Gln Thr His Leu Pro Pro Pro Ser Ser Ser Leu 440 445 450 455 caa gaa cag ctg acg cca agc acg gtg act ttc tct gaa cag ttg aca 1447 Gln Glu Gln Leu Thr Pro Ser Thr Val Thr Phe Ser Glu Gln Leu Thr 460 465 470 cca agc agt gca acc ttc cca gat cca cta acc agt tca cta caa gga 1495 Pro Ser Ser Ala Thr Phe Pro Asp Pro Leu Thr Ser Ser Leu Gln Gly 475 480 485 cag ttg act gaa agc tca gcc aga agc ttt gaa gaa caa ttg act ccg 1543 Gln Leu Thr Glu Ser Ser Ala Arg Ser Phe Glu Glu Gln Leu Thr Pro 490 495 500 tgc acc tct acc ttc cct gac cag ctg ctt ccc agc act gcc acg ttc 1591 Cys Thr Ser Thr Phe Pro Asp Gln Leu Leu Pro Ser Thr Ala Thr Phe 505 510 515 cca gaa cct ctg ggt agc ccc acc cat gag cag ctg act cct ccc agc 1639 Pro Glu Pro Leu Gly Ser Pro Thr His Glu Gln Leu Thr Pro Pro Ser 520 525 530 535 aca gca ttc caa gca cat ctg aac agt cct agc caa acc ttc cca gag 1687 Thr Ala Phe Gln Ala His Leu Asn Ser Pro Ser Gln Thr Phe Pro Glu 540 545 550 caa ctg agc cct aat cct acc aag act tac ttc gcc cag gag gga tgc 1735 Gln Leu Ser Pro Asn Pro Thr Lys Thr Tyr Phe Ala Gln Glu Gly Cys 555 560 565 agt ttt ctc tat gag aag ttg ccc cca agt cct agc agc cct ggt aat 1783 Ser Phe Leu Tyr Glu Lys Leu Pro Pro Ser Pro Ser Ser Pro Gly Asn 570 575 580 ggg gac tgt aca ctc ttg gcc cta gct caa ctc cgg ggt ccc ctc tct 1831 Gly Asp Cys Thr Leu Leu Ala Leu Ala Gln Leu Arg Gly Pro Leu Ser 585 590 595 gtg gac gtc ccc ctg gtg cct gaa ggc ctg ctc aca cct gag gcc tct 1879 Val Asp Val Pro Leu Val Pro Glu Gly Leu Leu Thr Pro Glu Ala Ser 600 605 610 615 cca gtc aag caa agt ttc ttc cac tat aca gag aaa gag cag aat gag 1927 Pro Val Lys Gln Ser Phe Phe His Tyr Thr Glu Lys Glu Gln Asn Glu 620 625 630 ata gat cgt ctc atc cag cag atc agc cag ttg gct cag ggc atg gac 1975 Ile Asp Arg Leu Ile Gln Gln Ile Ser Gln Leu Ala Gln Gly Met Asp 635 640 645 agg ccc ttc tca gct gag gct ggc act ggg ggg ctg gag cca ctt gga 2023 Arg Pro Phe Ser Ala Glu Ala Gly Thr Gly Gly Leu Glu Pro Leu Gly 650 655 660 ggg ctg gag ccc ctg aac ccc aac ctg tcc ctg tca ggg gct gga ccc 2071 Gly Leu Glu Pro Leu Asn Pro Asn Leu Ser Leu Ser Gly Ala Gly Pro 665 670 675 cct gtg ctt agc ctg gat ctt aaa ccc tgg aaa tgc cag gag ctg gac 2119 Pro Val Leu Ser Leu Asp Leu Lys Pro Trp Lys Cys Gln Glu Leu Asp 680 685 690 695 ttc ttg gtt gac cct gat aat tta ttc ctg gaa gag acg cca gtg gaa 2167 Phe Leu Val Asp Pro Asp Asn Leu Phe Leu Glu Glu Thr Pro Val Glu 700 705 710 gac atc ttc atg gat ctt tct act cca gac ccc aat ggg gaa tgg ggt 2215 Asp Ile Phe Met Asp Leu Ser Thr Pro Asp Pro Asn Gly Glu Trp Gly 715 720 725 tca ggg gat cct gag gca gag gtc cca gga ggg acc ctg tca cct tgc 2263 Ser Gly Asp Pro Glu Ala Glu Val Pro Gly Gly Thr Leu Ser Pro Cys 730 735 740 aac aac ctg tcc cca gaa gat cac agc ttc ctg gag gac ttg gcc acc 2311 Asn Asn Leu Ser Pro Glu Asp His Ser Phe Leu Glu Asp Leu Ala Thr 745 750 755 tat gaa acc gcc ttt gag aca ggt gtc tca aca ttc ccc tat gaa ggg 2359 Tyr Glu Thr Ala Phe Glu Thr Gly Val Ser Thr Phe Pro Tyr Glu Gly 760 765 770 775 ttt gct gat gag ttg cat caa ctc cag agc caa gtt caa gac agc ttc 2407 Phe Ala Asp Glu Leu His Gln Leu Gln Ser Gln Val Gln Asp Ser Phe 780 785 790 cat gaa gat gga agt gga ggg gaa cca acg ttt tgaataagtc tgtgactta 2459 His Glu Asp Gly Ser Gly Gly Glu Pro Thr Phe 795 800 7 35 DNA Artificial Sequence Description of Artificial Sequence PCR primer 7 aagcacggag gaggaagccg ccggtgcgtc gggac 35 8 35 DNA Artificial Sequence Description of Artificial Sequence PCR primer 8 acgggagcgc aggtgctcgg gcacccgagc tggag 35 9 35 DNA Artificial Sequence Description of Artificial Sequence PCR primer 9 ggagagcggc tccacgtctt gatgacaata tgcca 35 10 35 DNA Artificial Sequence Description of Artificial Sequence PCR primer 10 ccacgtcttg atgacaatat gccatacttg acgac 35 11 41 DNA Artificial Sequence Description of Artificial Sequence PCR primer 11 gggcgctgca gcccagccac catgtaccga tccaccaagg g 41 12 20 DNA Artificial Sequence Description of Artificial Sequence PCR primer 12 aatctcggcg ttgatctggt 20 13 20 DNA Artificial Sequence Description of Artificial Sequence PCR primer 13 agtgaacagc aacagatact 20 14 20 DNA Artificial Sequence Description of Artificial Sequence PCR primer 14 gcttcccgcg tccacgtgga 20 15 20 DNA Artificial Sequence Description of Artificial Sequence PCR primer 15 gatgtgggca ttcagctccg 20 16 20 DNA Artificial Sequence Description of Artificial Sequence PCR primer 16 cagagcttca ccattcgaga 20 17 20 DNA Artificial Sequence Description of Artificial Sequence PCR primer 17 gtggccctgg tgtctgtccg 20 18 20 DNA Artificial Sequence Description of Artificial Sequence PCR primer 18 ctggcccgcg cgtggggcaa 20 19 20 DNA Artificial Sequence Description of Artificial Sequence PCR primer 19 acacatgctt cgccactgcc 20 20 20 DNA Artificial Sequence Description of Artificial Sequence PCR primer 20 taatggccgc tctcacaggt 20 21 20 DNA Artificial Sequence Description of Artificial Sequence PCR primer 21 agctgagtcc ctgaggcaga 20 22 20 DNA Artificial Sequence Description of Artificial Sequence PCR primer 22 tgtgctgtgc cctgacactg 20 23 20 DNA Artificial Sequence Description of Artificial Sequence PCR primer 23 ttgcagcgga gaacgaggag 20 24 20 DNA Artificial Sequence Description of Artificial Sequence PCR primer 24 gatgagcact cggtcaacta 20 25 20 DNA Artificial Sequence Description of Artificial Sequence PCR primer 25 gcatccagga gatccaggag 20 26 20 DNA Artificial Sequence Description of Artificial Sequence PCR primer 26 ctggacaagg acgacgagag 20 27 20 DNA Artificial Sequence Description of Artificial Sequence PCR primer 27 cctgcgaaag tacaaggagg 20 28 20 DNA Artificial Sequence Description of Artificial Sequence PCR primer 28 cctaccatgt agtcagtctt 20 29 20 DNA Artificial Sequence Description of Artificial Sequence PCR primer 29 ggtcaggaac tcgtactcct 20 30 20 DNA Artificial Sequence Description of Artificial Sequence PCR primer 30 ttgggtgcct cctccacggg 20 31 20 DNA Artificial Sequence Description of Artificial Sequence PCR primer 31 gcgggacttg atgctgtagc 20 32 20 DNA Artificial Sequence Description of Artificial Sequence PCR primer 32 gtcttgtcgt cgtctgtgaa 20 33 7408 DNA Mus musculus CDS (1419)..(1593) intron (1594)..(2347) CDS (2348)..(2499) intron (2500)..(2673) CDS (2674)..(2776) intron (2677)..(2886) CDS (2887)..(3160) intron (3161)..(3347) CDS (3348)..(3457) intron (3458)..(3784) CDS (3785)..(3920) intron (3921)..(4050) CDS (4051)..(5480) intron (5481)..(6138) CDS (6139)..(6164) 33 aaggaaaaaa aaaaaaagaa aggtgtatgt tgtgcgctca ctcagtgaca agtgcacagg 60 cagaacgagg agccctggag ctaaagatgg agcaagaatt gaagggagat ggggagggga 120 ctctggcaga attaaagggt cttgggtagg tgcagcagcc actgagggca cagcagaccc 180 tggctacttg gcagcctccc cctcttcccg gctgaagcag tggggagagc tttctagagc 240 tgtgcggagg ccggtaggcc ccgcccgccg ctgccgccgc cgcagccgcc ggaggattcc 300 tgtcctaata tggagctggg attcccccgg ccccgccccc gccccccagc ccgccggaga 360 gactggggct cgcaagaggg cgggggaaca gctcgtcttg ggggctgaca agcgggaggg 420 gatcgtggga gaggttcaaa cacacatcca gatcctcacc aggccctggg tctttgcctc 480 agtttccccg acaggtggct aagatgaact aatagaggaa aaaggaatcc ctgcagatca 540 cagtggagat gtttgtgttc ctatggtgct aaggaaatat gatcaagacc gaattcaagt 600 ggtctcttct ccacaggacc accattccac cctatcctgg agatttagac cctcaggtca 660 gggcatggag gcgaagaagg aattatttat ttgaaactgg ctaagggact ttcagattga 720 atagacccca gaaaagaccc ccagttgtga cctagcccct ccccaaaagc caaggaaagt 780 ccctcatgta atttcgaccc ctgcctggca gatggcggca aattggcaga ggaccaagcc 840 ccttctcatc ctttgcctcc ttagaaaaat ctatgttgat agcagcctcg ctttgccttc 900 tacaaactct cttgttaagg gcttcagacc accctaatcc atgtactgtt cctcctccta 960 atagaatgta atggaaaacc tgggtccctg caccccattc ctggctctgc acagctcttg 1020 ccagccggcc ccctctgcac cctcccttct cccccttccc ccgccccctc cctctcccgt 1080 tcgacgtcac gggatgacgt cggaagtctg ggagggagga ggagcacccc ccctccccag 1140 ccagtggctc cctctgcagc ttgctttagc ccagcctccc gcctcccgct gccccccccg 1200 tctctaaaaa cgagcccccc acgcctgtca ggagctatat aaggcggatc gaggcaggcg 1260 aggggggcag cgctgccgag cggagcccag gagtggagcg agagcgagca agagcctgag 1320 cgaaaagacc gggaagcaag gaagaggaag cctccggtgc atcgggaaag gatcgcaggt 1380 gctcgggagc cggagctgga gctccacagc cggcagtc atg tac cga tcc acc aag 1436 Met Tyr Arg Ser Thr Lys 1 5 ggc gcc tcc aag gcg cgc cgc gac cag atc aac gcc gag att cgg aac 1484 Gly Ala Ser Lys Ala Arg Arg Asp Gln Ile Asn Ala Glu Ile Arg Asn 10 15 20 ctc aag gag ctg ctg ccg ttg gct gaa gcg gac aag gtc cgg ctg tcc 1532 Leu Lys Glu Leu Leu Pro Leu Ala Glu Ala Asp Lys Val Arg Leu Ser 25 30 35 tac ctg cac atc atg agt ctt gcc tgc atc tac act cgc aag ggt gtc 1580 Tyr Leu His Ile Met Ser Leu Ala Cys Ile Tyr Thr Arg Lys Gly Val 40 45 50 ttc ttt gct gga g gtgagcagct tgggctaccg gagaccagag ctgacgggga 1633 Phe Phe Ala Gly 55 ccagggatgg aggagctgag ggaatgtgct aaaactgccg cttgtctagc acagcgtgct 1693 ggaagcctgt ggagagaagg gacttgaggg gaccctggac ttcctacttt ttcttctgag 1753 ctccatctag actagcctaa acgatagtcc tagcactgga tttgtgtgag atagagcgct 1813 aaaacagaat ggtccaggct cccattgcct cagaggcact ccaggaatcc ggggagggta 1873 cggaaggaag cctggcaagc tacagggaaa gcctgcaaag gcaaagtatg aggaagagta 1933 gcttgtgcta gaaaatgctg agagggtctt tctatgcgct ctggagctgt tcgacgtcct 1993 gaagccatca ccctttctgg cgctgcccgc ggtgctgaaa tggccatagc cccttttcgc 2053 aggagctgtc cgcggtgctg aatcccagtc ctttcgggag agctctgtcc acagtgctga 2113 cagcagaggg ctgctgaggt tccggccagg cttggaagtc caggggctcc ctggctagat 2173 agttttagca acaggtctcc tggccaagat ccacaagcat agggtcaaca ggtgttggca 2233 gaaataggtc tatgggaatt tcctgtgtct tctccaagac tcaaaagatg ttctctttat 2293 ttctgtgttg tccctgattc ttatcctgac tcaccacatc ttctcaccct acag gc 2349 Gly act cct ttg gct ggc ccc acc ggg ctt ctc tct gct caa gag ctt gaa 2397 Thr Pro Leu Ala Gly Pro Thr Gly Leu Leu Ser Ala Gln Glu Leu Glu 60 65 70 75 gac att gtg gca gca cta cct gga ttt ctc ctt gta ttc aca gct gag 2445 Asp Ile Val Ala Ala Leu Pro Gly Phe Leu Leu Val Phe Thr Ala Glu 80 85 90 ggg aag ttg cta tac ctg tcg gag agt gtg agc gag cat ctg ggc cac 2493 Gly Lys Leu Leu Tyr Leu Ser Glu Ser Val Ser Glu His Leu Gly His 95 100 105 tct atg gtgagtacta aaagtccttg catctcaagt tggggtatat gtgagataaa 2549 Ser Met atgagcctct cactactgaa aacagagtta ttagaggcga gtgtggggga gtcttcccta 2609 agaaaaatca ttggttgcag ataggctctt gctgccttca ctaatgatca cttctccttt 2669 ctag gtg gac ctg gtt gcc cag ggc gac agt atc tac gat atc att gac 2718 Val Asp Leu Val Ala Gln Gly Asp Ser Ile Tyr Asp Ile Ile Asp 110 115 120 cct gct gac cat ctc act gtg cgc cag cag ctc acc atg ccc tct gct 2766 Pro Ala Asp His Leu Thr Val Arg Gln Gln Leu Thr Met Pro Ser Ala 125 130 135 140 ctg gat gct g gtaagaacct cctctcggtt cttcagttta ctcctctgct 2816 Leu Asp Ala gccctgccct aactatctac tctcctccaa tgcccaccct cttagtcagt ttttcctttt 2876 gctcacctag at cgc ctt ttc cgt tgt cga ttc aac acc tcc aag tcc ctc 2927 Asp Arg Leu Phe Arg Cys Arg Phe Asn Thr Ser Lys Ser Leu 145 150 155 cgg cgc cag agt tca gga aac aaa ctg gtg ctt att cga ggt cga ttc 2975 Arg Arg Gln Ser Ser Gly Asn Lys Leu Val Leu Ile Arg Gly Arg Phe 160 165 170 cat gct cac cca cct ggg gcc tac tgg gca gga aac cct gtg ttc acc 3023 His Ala His Pro Pro Gly Ala Tyr Trp Ala Gly Asn Pro Val Phe Thr 175 180 185 gct ttc tgc gcc cca ctg gag cca aga ccc cgc cct ggc ccc ggc cct 3071 Ala Phe Cys Ala Pro Leu Glu Pro Arg Pro Arg Pro Gly Pro Gly Pro 190 195 200 205 ggc cct ggc cct ggt cct gct tct ctc ttc ctg gcc atg ttc cag agc 3119 Gly Pro Gly Pro Gly Pro Ala Ser Leu Phe Leu Ala Met Phe Gln Ser 210 215 220 cgg cat gct aag gac cta gcc cta ctg gac gtt tct gaa ag gtaagcccaa 3170 Arg His Ala Lys Asp Leu Ala Leu Leu Asp Val Ser Glu Ser 225 230 235 agtgttcaaa ctccagtaag aagggaggcc agaaagaagg gaactttaga ttcgtgatct 3230 tagattcagg gcagggagga tggggcttaa gtgggcagag agcatgggag ggagtgaagt 3290 gcatgcattt tgagtaaggt aaacagaaag ctgacctcat catttccacc ttcccag t 3348 gtc cta atc tac ctg ggc ttt gag cgc agc gaa ctg ctc tgt aaa tca 3396 Val Leu Ile Tyr Leu Gly Phe Glu Arg Ser Glu Leu Leu Cys Lys Ser 240 245 250 tgg tat gga ctg cta cac ccc gag gac ctg gcc caa gct tct tct caa 3444 Trp Tyr Gly Leu Leu His Pro Glu Asp Leu Ala Gln Ala Ser Ser Gln 255 260 265 cac tac cgc ctg t gtgagtgtcc tgagaggccg tgcataacac aggaagctgg 3497 His Tyr Arg Leu 270 gagaaagcat gggagacagg ccagggactg gctgtggtcc aaactgatgt taaggagttt 3557 cggaggctac agagtgagct tgaggatgag aagtcaaggc aagaatagga cagagttaga 3617 aaacactgtg tgataaggtc aagtggggag cctagaggta caggttaggg tagttagaag 3677 agaatatgtc atggctccct caattcagtg tagaggtaag aaaggtgggt gtgtaggtgg 3737 tgttgattga tggaccttct aatccggtat tccttttttc tccccag tg gct gaa 3792 Leu Ala Glu agt gga gat att cag gct gaa atg gtg gtg aga ctt caa gcc aag cat 3840 Ser Gly Asp Ile Gln Ala Glu Met Val Val Arg Leu Gln Ala Lys His 275 280 285 290 gga ggc tgg aca tgg att tac tgc atg cta tac tca gaa ggt cca gaa 3888 Gly Gly Trp Thr Trp Ile Tyr Cys Met Leu Tyr Ser Glu Gly Pro Glu 295 300 305 ggc cct ttt act gcc aat aac tac cct atc ag gtaagctgta agatacaaga 3940 Gly Pro Phe Thr Ala Asn Asn Tyr Pro Ile Ser 310 315 tggcggagag gggaggggag ctgaggtcag catagaagaa atgcaacgaa gaaaactact 4000 ctggtaatgg acagcagacc cttacaagct gccacctctt ccctttccag t gac acg 4057 Asp Thr gaa gcc tgg agc ctc cgc cag cag cta aac tct gaa gac acc cag gca 4105 Glu Ala Trp Ser Leu Arg Gln Gln Leu Asn Ser Glu Asp Thr Gln Ala 320 325 330 335 gcc tat gtc cta gga acc cca gct gtg cta ccc tca ttc tct gag aat 4153 Ala Tyr Val Leu Gly Thr Pro Ala Val Leu Pro Ser Phe Ser Glu Asn 340 345 350 gtc ttc tcc cag gag caa tgc tct aat cca ctc ttt aca cca tcc ctg 4201 Val Phe Ser Gln Glu Gln Cys Ser Asn Pro Leu Phe Thr Pro Ser Leu 355 360 365 ggg act cct aga agt gcc agc ttc ccc agg gct cct gaa cta ggt gtg 4249 Gly Thr Pro Arg Ser Ala Ser Phe Pro Arg Ala Pro Glu Leu Gly Val 370 375 380 atc tca aca cca gaa gag ctt ccc caa ccc tcc aaa gag ctg gac ttc 4297 Ile Ser Thr Pro Glu Glu Leu Pro Gln Pro Ser Lys Glu Leu Asp Phe 385 390 395 agt tac ctg cca ttc cct gct agg cct gag cct tcc ctc caa gca gac 4345 Ser Tyr Leu Pro Phe Pro Ala Arg Pro Glu Pro Ser Leu Gln Ala Asp 400 405 410 415 ctg agc aag gat ttg gtg tgt act cca cct tac aca ccc cac cag cca 4393 Leu Ser Lys Asp Leu Val Cys Thr Pro Pro Tyr Thr Pro His Gln Pro 420 425 430 gga ggc tgt gcc ttc ctc ttc agc ctc cat gaa ccc ttc cag act cac 4441 Gly Gly Cys Ala Phe Leu Phe Ser Leu His Glu Pro Phe Gln Thr His 435 440 445 ttg ccc cct ccg tcc agc tct ctc caa gaa cag ctg aca cca agt aca 4489 Leu Pro Pro Pro Ser Ser Ser Leu Gln Glu Gln Leu Thr Pro Ser Thr 450 455 460 gtg act ttc tct gaa cag ttg aca ccc agc agt gct acc ttc cca gac 4537 Val Thr Phe Ser Glu Gln Leu Thr Pro Ser Ser Ala Thr Phe Pro Asp 465 470 475 cca cta acc agt tca cta caa gga cag ttg aca gaa agc tca gcc aga 4585 Pro Leu Thr Ser Ser Leu Gln Gly Gln Leu Thr Glu Ser Ser Ala Arg 480 485 490 495 agc ttt gaa gac cag ttg act cca tgc acc tct tcc ttc cct gac cag 4633 Ser Phe Glu Asp Gln Leu Thr Pro Cys Thr Ser Ser Phe Pro Asp Gln 500 505 510 cta ctt ccc agc act gcc aca ttc cca gag cct ctg ggc agc ccc gcc 4681 Leu Leu Pro Ser Thr Ala Thr Phe Pro Glu Pro Leu Gly Ser Pro Ala 515 520 525 cat gag cag ctg act cct ccc agc aca gca ttc cag gct cat ctg aac 4729 His Glu Gln Leu Thr Pro Pro Ser Thr Ala Phe Gln Ala His Leu Asn 530 535 540 agc ccc agc caa acc ttc cca gag caa ctg agc ccc aat cct acc aag 4777 Ser Pro Ser Gln Thr Phe Pro Glu Gln Leu Ser Pro Asn Pro Thr Lys 545 550 555 act tac ttc gcc cag gag gga tgc agt ttt ctc tat gag aag ttg ccc 4825 Thr Tyr Phe Ala Gln Glu Gly Cys Ser Phe Leu Tyr Glu Lys Leu Pro 560 565 570 575 cca agt cct agc agc cct ggt aat ggg gac tgt aca ctc ctg gcc cta 4873 Pro Ser Pro Ser Ser Pro Gly Asn Gly Asp Cys Thr Leu Leu Ala Leu 580 585 590 gct cag ctc cgg ggc ccc ctc tct gtg gat gtc ccc ctg gtg ccc gaa 4921 Ala Gln Leu Arg Gly Pro Leu Ser Val Asp Val Pro Leu Val Pro Glu 595 600 605 ggc ctg ctc aca cct gag gcc tct cca gtc aag caa agt ttc ttc cac 4969 Gly Leu Leu Thr Pro Glu Ala Ser Pro Val Lys Gln Ser Phe Phe His 610 615 620 tac aca gag aaa gag caa aat gag ata gat cgt ctc att cag cag atc 5017 Tyr Thr Glu Lys Glu Gln Asn Glu Ile Asp Arg Leu Ile Gln Gln Ile 625 630 635 agc cag ttg gct cag ggc gtg gac agg ccc ttc tca gct gag gct ggc 5065 Ser Gln Leu Ala Gln Gly Val Asp Arg Pro Phe Ser Ala Glu Ala Gly 640 645 650 655 act ggg ggg ctg gag cca ctt gga ggg ctg gag ccc ctg aac cct aac 5113 Thr Gly Gly Leu Glu Pro Leu Gly Gly Leu Glu Pro Leu Asn Pro Asn 660 665 670 ctg tcc ctg tca ggg gct gga ccc cct gtg ctt agc ctg gat ctt aaa 5161 Leu Ser Leu Ser Gly Ala Gly Pro Pro Val Leu Ser Leu Asp Leu Lys 675 680 685 ccc tgg aaa tgc cag gag ctg gac ttc ctg gtt gac cct gat aat tta 5209 Pro Trp Lys Cys Gln Glu Leu Asp Phe Leu Val Asp Pro Asp Asn Leu 690 695 700 ttc ctg gaa gag acg cca gtg gaa gac atc ttc atg gat ctt tct act 5257 Phe Leu Glu Glu Thr Pro Val Glu Asp Ile Phe Met Asp Leu Ser Thr 705 710 715 cca gac ccc aat ggg gaa tgg ggt tca ggg gat cct gag gca gag gtc 5305 Pro Asp Pro Asn Gly Glu Trp Gly Ser Gly Asp Pro Glu Ala Glu Val 720 725 730 735 cca gga ggg acc ctg tca cct tgc aac aac ctg tcc cca gaa gat cac 5353 Pro Gly Gly Thr Leu Ser Pro Cys Asn Asn Leu Ser Pro Glu Asp His 740 745 750 agc ttc ctg gag gac ttg gcc acc tat gaa acc gcc ttt gag aca ggt 5401 Ser Phe Leu Glu Asp Leu Ala Thr Tyr Glu Thr Ala Phe Glu Thr Gly 755 760 765 gtc tca aca ttc ccc tac gaa ggg ttt gct gat gag ttg cat caa ctc 5449 Val Ser Thr Phe Pro Tyr Glu Gly Phe Ala Asp Glu Leu His Gln Leu 770 775 780 cag agc caa gtt caa gac agc ttc cat gaa g gtaagtctag cctgaatgtc 5500 Gln Ser Gln Val Gln Asp Ser Phe His Glu 785 790 caagagccct gcccttctaa tcagacattg catagattgg gtgaatcagt ccccaactct 5560 gaaactctgt tttattaaga gaacaatatt acctcctact aagaagagta gtgaggtagg 5620 aataatacaa agctttgtgt gaaagatgag tagacctggt gggcggggga ggtgagctag 5680 aaaaacgcga tagacaatcc ctaggcaaaa gcttgaaagc ttctgagaga cctagaccag 5740 acaacaccgt cattttatag acaaaaataa tcaaggcccc agagttaaag aaactttaag 5800 tggcacaaaa attgatagaa gttgatgctt ccccctgaag gggacccaga gcaacaactg 5860 gttaaaatta ggagacagaa agaacaatgc caagccccta gctccaatct ggcggccttg 5920 tgctgtttgt ccaaagctgt ggccacagtt tccctccata tttgcatatt gcctcttatc 5980 tgctgacacc ctggggatca gttcatttgg ctaacacatt tgacgtccat agactatagc 6040 aatattgtac cactgcctga gcccaatgac gcttttactg aataagcttg actaacatac 6100 gcactttctc tcttctctct ctctctcttt ccccacag at gga agt gga ggg gaa 6155 Asp Gly Ser Gly Gly Glu 795 cca acg ttt tgaataagtc tgtgacttaa cgtcttcaag tatggcatat 6204 Pro Thr Phe 800 tgtcatcaag acgtggagcc gctctccacc cccccgggac tgttgggggg attctggggg 6264 ccagaggggg atatatctga ttctccaggc cctgaaggat ttagggggga ggtgggaggg 6324 taagggaggg gagcaacttt ttaaaatcaa gagacttcga gcgatcccag tttccatttc 6384 aatctgtatt cactcgtagt gagtttcctt gaatggattt caagcggaga atgggggagt 6444 ctcacttcct caccgcgctg ccccatgggc ctgggccagt tctccactcc taggggcaaa 6504 gccacccctg ggctttggtg ggggaaaggc atggcccacc tggggctagc ctgtgccccg 6564 aggggctctt gacacccacg tagaattctc tacaaaccag taacgggatt tcaattccga 6624 cggactctgc cgccctggcg gctcttcctg tgacttttgc gccccgcgcc tggggtgggg 6684 ggcgcgaaga gacgctacat tcctttccga tggaggaagg cagatctgcc gtcacacgtg 6744 tgcttgcacg agtgcgtgta cctggtgcgg gactcacccg gccgccagac cgcctaggct 6804 tgcccaggtg gccacctcgt ggtgctgcgg tgactttgta gccaacttta taataaagtc 6864 cagtttgcct ttttggtatc tctggtgtca tgcgctattg tgaaaaggga agggagggga 6924 agggagagat tgaggagccc agataggagg ctggggcagg agtcacaggt tagacctcct 6984 ctcagccctg gtatctctaa gtgagtttgt tcatatctcc atttgactct gcttggtcca 7044 cactgtgcta gaagactaag tacttgtcag aagcagacat tgcaccaaag acactggagt 7104 cttctctctg ccctgggttt atggtgtgat ggggaggaaa gagcctgggg ctgagcaagt 7164 ttgtcactgg tcttggatat gggtttaaag tttctggtca tttcctgcct ggtctttcag 7224 gatattgatt tcctcatgga ggcttagatt ttaaaaatca gaagctgaaa cctgttacgc 7284 ttgcgtaggg ctgttcagtt agcaaatacc caatccactg caataaattt ccacttcatt 7344 gggaaagcaa cccgataacg ggtgttcctc cagttacagg tgagaaacac atcaacccct 7404 cccc 7408 34 20775 DNA Mus musculus CDS (9594)..(9768) intron (9769)..(10522) CDS (10523)..(10674) intron (10675)..(10848) CDS (10849)..(10951) intron (10952)..(11061) CDS (11062)..(11335) intron (11336)..(11522) CDS (11523)..(11632) intron (11633)..(11959) CDS (11960)..(12095) intron (12096)..(12225) CDS (12226)..(13655) intron (13656)..(14313) CDS (14314)..(14339) 34 tctcctgatt tttaaagccc ctctgtcttt cctggccccg cttggcctcc ctgaagatgc 60 cctgccctct gcatacctag ggccaatagg agtgatgagc ccatgtcatg tctgctctgg 120 gattctaatg acccaatccc tacaccagac acacaaggca tggacatctg ctcacctgta 180 ggctccatgt cactgggtac acgcaggtga tattacagac aagtgtaaag cttcggtctg 240 tggtggcctg caggtttgtg tgtacctagg tagaagagga agtgaggagg caccagtcag 300 aagcaactct gagaaacagg agccagaatt taagctgggt aagaacatga aagatggcca 360 aggattgcaa ttgttggccc ctggagaaca cactgggact ggtcttggat gttctgttct 420 gtactggagg gatatgggat gcctgctgac acacaggaag ggtctgaacc cagaccctca 480 gggtcactag gtatgcgtac ctcagtttcc taaggctcat tgacttcttt gttcgtttat 540 tcggagaaca gcacctattc tggccacctc cataaggagg gtttcaggaa gcacccaggg 600 ctatgaaccc atcgagccac ttctgtctga ctgcattcaa aacgatagtt tccttaagac 660 aatggccact ccccgtgcat tctccaacac ttactccgtt ccttccgtgc ctatggcttt 720 gttctgagtg ttttgacaaa ttagttcacc tggctcttgt gtcagagctc taacacaaat 780 tgtattctcc tcttcacaac tctatttaat acactggtaa actgaggcat gagaggctgc 840 agtccttacc tcagcagtgt cacagtctgt aacagaggca agacctccct ccaggcccca 900 ccctcttgcc taccctgcct tggctctctt ccggtctcta tgcgaagatc ctatgtattc 960 agacccttct tttaattttt taatggcttt tttatttact ctgtgtgcat gcatgtgagt 1020 gagtgtgtgc cgtggtttat gtgtggcagt cagaggacat ctttcggctc tccttccacc 1080 atggaggtcc tggggattga agttaggctg tcaggcttgg cagcaagtgc ctttacctga 1140 ttaaccttgc tgcccacccc taaccccttc ttgctggctc ttccattcgg aataaggcaa 1200 accatgccct ttcagccttc ttttcaccga gaagaattat cttccttctt ttcatcttct 1260 caatttttcc tacaaatata cctggaatgc ttcgatcaga gctgatggca gacaaaggtg 1320 acagctccta cccaggggtc tccaatacaa gccagagaag acagcagctc attaatgaaa 1380 cgaagtgtaa aactgtcacc atcacaactg gcaacagaag cacagggaac cctgggacct 1440 acagctgggg atttgacccg atagagaaga ttttctggag tgatatttga gccaagctat 1500 tgtgaaaaat gaggatcagg tgcaaggaga ggcaaggggc gtgcatgtgt gcacggagct 1560 gcagaaccac aatggaagag ctgcctgtgg ctagaagaga gggacgggga ggaaggaggc 1620 agggtcgggg gttgggggga agatcaccag agtgcagcct gggagaaggc ttagggtggc 1680 tcctcacagt tcttgacatc cttgatgatg gaagctaagc ctggccactt cacctcatag 1740 gacagctcct gcagccatac ctgctgcgta aagaagcctc agctcccttc ccccacagca 1800 ccacctcatt cccatctaat taattgtttg cttttacctt ggctactgct actcaccaca 1860 ccttataaag ccatgagacc acgctcttgc taatctcatc ctccccaccc acccagcaca 1920 gccacgttgg tcagttggcc acttgactcc caagcaacgt ggcgcaaaca cacctcccta 1980 ggaaccccac tgccatatcc ctaggcttgg tcttccccat gttgcagcca ccgagcaccc 2040 cagatgcccc tttccagaca gcatctcatt cagatggctt cctcttaccc tgtggaagct 2100 ccatgatgtt aaagccaagc ttgtgcctct ccccgacccc cgccagtatc caccagagag 2160 gctggcctct ggcctcaatt catcccacag ccctgtgcag tgagcgtgac atccatcccc 2220 acggtccctg tgacagatgc tggcagtatg gcggccagcc tgaggtcccg tgtgggtggg 2280 caaggaatag catttgagaa gcagaggcag gagggtcaca agttcaaggt tatcctctgc 2340 tatatatgag gatgcatgcg attctttctc aaattttaga aaatgtgcat caaggaagag 2400 gcacaggtcg ggtgtgaggg cagagggggc aagctagtca cctctagaag atcagcaggg 2460 cagagttccc ttgctgagga aagtcagaca tgaacatgtg aggcagatct agagggcagg 2520 ggccacaccc tcggtttcta tcttcatgcc actgaggcac atggggtccc tggtctgaat 2580 tttatctctg gtccatgaat taattttcct ctcctccttg gagcagatgc ctccagtcag 2640 ccccatcctc aagccttgcc cagatacctt caattttctc atccaggttc cagtctctcc 2700 ctcctgccca caccatccct ccccgccctc acctctgctc agcccactcc cctggctctg 2760 accgtttcta tgcgtaggtg gcagcgtgta ccctcttcac aggagatttg ttgatttcat 2820 aaccataaat agataaaatg ttctgagtgc ttccatgagc gagtagaatt gagggagtga 2880 tcacacaatg aaaaggctgt aggagaagga aaacagcctg tggagacaca gctgaaaccg 2940 gcttggtgct gcacaaacca gcactacctg agggcgagct tgccgttgca taagaggtat 3000 accataaaca caggacttgg gactgccaga gaaccttctg gaaaacactt atgagactgc 3060 aagtctgtca attcaaagga acaatatatt aagaaaatct agatttaaaa tgaaaacaga 3120 acgtggaagc caacaaacat ggatttttaa ttctgagttc atctcatttt ggctgtgtga 3180 ctatgagcaa gtttctcatc ctctctggga aggtgtatat tcatctcttg ggtcagacta 3240 gagggaccaa tcatataata tgctcatttt ttcctctaag aaaaagtggt cttctcgatt 3300 acaacttaac ctccaatatg gaacaatttg tcttcccaaa acgcagtccc aagacactaa 3360 ggatggatag ggtaacctgc tttttcattg tcaagtagaa ctcatgttga catggaaatg 3420 ggttatgcac aatcattctt ggatggggag accatatatt catagttaca aagaaagcta 3480 gacattagga aggacttccc aagttcttct ccctaagatg ggtaaagaga gtagagagag 3540 gatgtgacag ctcagtttgt tctgagactg gagagctttc cagagagagg gaaagctatt 3600 tctttacctt ctgctctaag ggtggcagga ttttctgtca agggttaggt agtaggtgtt 3660 tgggcttggt gggagctcta gtcccttctg cacttaactc tgtgactgtg tgaagaaggg 3720 caccagccat aagtatgtga atggtctgaa tgtgtcccag taaaacttca ttaatgaaaa 3780 gaaacagcgg accagatttt attcggtgcc atagtgtata ggccccaatc tcgttctaca 3840 cggcaagaga acaagtttga atggggagga atgaaccatg cacagggcac tgccagagcc 3900 ctgctgtctg actttaagtc attgctcact tctgatctta acctcatcga ctatagaatg 3960 aacgtaataa tctcaatcat cagtcctgag acaatagcta agaggaaggt tagggtggct 4020 aggaaggctg tgtgtcaaag tgaaagaact gacgcctgca agttaacctc tgacctccac 4080 acacagacac catgacacat gtgtgttctc ttcatgtgac aaattaaaaa ttgcaaaata 4140 aaaagtgcct aagagatcag agtaagtctc tctctccctt tactccaccc ctttgagtgg 4200 cactgagtct agcagcacac gaggccacat ccttgtctgc tgcaggtgac ggtggccttc 4260 ttggatggag acaaatattt cattatagtt ggattcttgg tctgtctttc taacatgcgg 4320 tcctcagtga ccccatttct ggagcaagcc cagcacagga ggaaacgagg aatctctctt 4380 cctctccact gtccgggcat ttggcagggt gctagagttc atgtcaggga gcaacatggc 4440 cgcagtggct ggtgccagac cttgggagag gccttcaaga ctcaggctgg gatcagagtc 4500 aggaacagaa agctctgagt tctcccagaa cattcagctc tggtcccagc ttccctgggg 4560 tctccacgaa gcagccacag ctgtggtcca ctgggaacct gcagccccac ccacggcatc 4620 ataaagtgaa agttgtcctg ctcatctgct cagatgatct cggagtgctg catccttcag 4680 cactgattta tctcagaagc cctagcaagg gattccttta ttttctcatt ctgtccctct 4740 tcctcttccc ctccctctcc tttgcttcat ccttccttct cttcctcata ggcatacttg 4800 tgcagacaaa taccacatgt atgccgacag tcccccgtca catccttgct ccagtatttg 4860 agaaaaggag ccaggagtct ccatgatatt cttaagaatc aaaccctcca ttccaattcc 4920 tcaggaggtc ttcctcctgg acaatctctg aaaaagatgc accatttctc taatagggat 4980 tgaggggtga tgaccctcta gagccccaat aaagccatga agagaggagc agaggacttc 5040 atggtctgct cttgctataa aaaggccttt ttcgggaaaa aaataaagaa aggaatcagc 5100 caatcccttc acgatgccat cacctcttct tggtggtttt tcggggaagg agtgggtggg 5160 tttccatggc aacagatgcg agctctgctc agtaaagaag ggaccttgat attttttctc 5220 tctcattctc tcagttgtgt gtgtgcctgt gtgtatgtgc gtgctacaca tgcctatgcc 5280 cacaccagca atttttttag aactaaagaa agcccttcta tcagctcccc aaatatggag 5340 tgatagaaaa ccatgcactc ctgcaggcca gagaaggttc tggatggttc cagagaaggg 5400 tgctcctgtg aacttgtttt cctccattgc agagattgtg tgcagcagag aggcctttgc 5460 aaactgttag aggctaagag ttagaaaaaa ggatgtttgg tggagagagg ggaacaaagg 5520 atagatggtg caaaaaccaa cgaatggcgt cctagtgggc aaccaaaggt gcacggagtc 5580 tcaggaagca cagtcagcac aaaccaccta acgctgaaga aaaggctcaa ggcagactca 5640 tatatggaca caaacacaca gagaggtata aaagcaaata tattaggcaa aaccgcaaaa 5700 ctgcatacaa cacagaaagg cagagactta gagaaataaa acagacaaat aaaaacacag 5760 atgcaggtac tggcagatat gtagacacac aaggatgcag agcctatcat caaaacacag 5820 gcaaatagat acatggatgc agatagataa gtgtatccag acagataggt ttggatgcag 5880 acatagaaca tgcaacacag cctcattcaa gtgcacacac tcgtgtgcgc tcacacacac 5940 actccccttt cccccttcag ttgctcaagc ttcctatagc aggaaggcag atctgcaaat 6000 gctgcatgtt cacccagtaa gtttggctgt gaatatcttg taacccccac ctattgcttc 6060 tacacacaca cacacacaca cacacacaca cacacacaca cacacacacc ccaaagcccc 6120 tctcccaact ttgtccactt tcccataacc aaaggctgtg atacctcccc catctcaggc 6180 ttccaattct gttttgctgc tgctgctgcc gccgcctgcc gcttgggggg ggagagagtg 6240 gggtgactca gccaggccag gatgactcac actgacagta tttttagcag cggccaggag 6300 ctctctagcg tgccagccgc ccccctcctc ctgcttgcta tttcggaacc gtcactggtg 6360 atataaatag ctcttctccc acggcctgaa gctgctgcca ggctattttt ggttctgcac 6420 agttaaaaat agtttcatgg aggtgggagg caagaggagt gggagctggc ctagggagag 6480 gagacattgg gggcatacag agcttctcaa cttgaatcag agtagtcgaa ctaagatgat 6540 cccttcccta ccccctccct tgcccctttc tagaaccttc tccccttcca acgttcctta 6600 tccctagtcc atcctcctgg aaaaatccaa ggattcctcc cttgagccca attttctttc 6660 caagcttaac taattcctag aaccgaggag tcttgacagc cacacctgta aatagcccat 6720 atgtattctc aatgaggagg atgacagcat cgggatgcca ttctcatcta tcccgaggcc 6780 cagctcggct ttgatgtcac aggcaaacca cgaccattct gagtgggaag gcaacattca 6840 gcaccacgga cagcgacaac atcccccccc cccctccccc ttccaggtct gcttaaattg 6900 cttggagacc agctgtggac ccagcagaga gatgcaactt attgtggagg agatatcaag 6960 aacgtctcct ggccagggct taaagcacct gtctgtgagg aagacagggc agagatgaac 7020 cccagagata gaatggttgt ctagcataca cagagccctg gtttcatcct cagctttggg 7080 aaactaatct agaaactcca tcttggattt gcatatggaa agagaatcca aaaaccaagg 7140 gaagagaatg gaacagggag tggtggtgtt gagtggggat atcagagtta ataaggatga 7200 aacatgccag agagaaatac atcctgaaga aaccatttct gtcacccata aggttggaaa 7260 cagtgtctta cagacacaca ccattttctc catctcagct ataccactgg ctggctacat 7320 ggttgtatat gtagatgctt tctatctgaa ctaaaattgt acaaaatatt aggatagggg 7380 ctctacaacc atgaacctct ccccgcccct ccccggcatt actagggagt gcactcaagt 7440 cttgagcatg atagaagtgt gaactcctac taagccatgg ccctggtcac caaagtaccc 7500 tcttcccata ccccctgctt ttcactccac gttgcctctc ttgctatcac ccctttccat 7560 gaagaacagg ggtttcttga ccacaaactt ttctccttgg tgtcaaagtt catctctaac 7620 tttctgcagc cagttctgtc cctctctccc aatttttttt tgttttttgt tctgtttgtt 7680 tgtgtgtttt tgttttttga gacagggttt ctctgtgtag ccttggctgt cctggaactc 7740 actttgtaga ccaggctggc ctcgaactca gaaatctgct tgcctctgcc tcccaagtgc 7800 tgggattaaa ggcgtgtgcc accacgcccg gcttccctca actttttaaa tggtcttgtt 7860 tttcaggctc taaaagtgct tttatatgtt cctactctaa atgaaatttt gggcaaaaag 7920 tttctctagt cctttgtgaa atggttgtgg gataaaaaaa gggctcccat accctgtgta 7980 gacagcaatc gcatgtaagt gacctgaaga aaggtgtgtg tgggggtgtg tgtctggagg 8040 ggtggggtga tgcaaaggcc acactacaaa gacaagcctg acatgacagg tagttaaacc 8100 aaaggtgcaa attagagggg tgggggtggg gggcgcccac aaagccgaga tagactgtcc 8160 aacgctcaat gaacgaagga aaaaaaaaaa aagaaaggtg tatgttgtgc gctcactcag 8220 tgacaagtgc acaggcagaa cgaggagccc tggagctaaa gatggagcaa gaattgaagg 8280 gagatgggga ggggactctg gcagaattaa agggtcttgg gtaggtgcag cagccactga 8340 gggcacagca gaccctggct acttggcagc ctccccctct tcccggctga agcagtgggg 8400 agagctttct agagctgtgc ggaggccggt aggccccgcc cgccgctgcc gccgccgcag 8460 ccgccggagg attcctgtcc taatatggag ctgggattcc cccggccccg cccccgcccc 8520 ccagcccgcc ggagagactg gggctcgcaa gagggcgggg gaacagctcg tcttgggggc 8580 tgacaagcgg gaggggatcg tgggagaggt tcaaacacac atccagatcc tcaccaggcc 8640 ctgggtcttt gcctcagttt ccccgacagg tggctaagat gaactaatag aggaaaaagg 8700 aatccctgca gatcacagtg gagatgtttg tgttcctatg gtgctaagga aatatgatca 8760 agaccgaatt caagtggtct cttctccaca ggaccaccat tccaccctat cctggagatt 8820 tagaccctca ggtcagggca tggaggcgaa gaaggaatta tttatttgaa actggctaag 8880 ggactttcag attgaataga ccccagaaaa gacccccagt tgtgacctag cccctcccca 8940 aaagccaagg aaagtccctc atgtaatttc gacccctgcc tggcagatgg cggcaaattg 9000 gcagaggacc aagccccttc tcatcctttg cctccttaga aaaatctatg ttgatagcag 9060 cctcgctttg ccttctacaa actctcttgt taagggcttc agaccaccct aatccatgta 9120 ctgttcctcc tcctaataga atgtaatgga aaacctgggt ccctgcaccc cattcctggc 9180 tctgcacagc tcttgccagc cggccccctc tgcaccctcc cttctccccc ttcccccgcc 9240 ccctccctct cccgttcgac gtcacgggat gacgtcggaa gtctgggagg gaggaggagc 9300 accccccctc cccagccagt ggctccctct gcagcttgct ttagcccagc ctcccgcctc 9360 ccgctgcccc ccccgtctct aaaaacgagc cccccacgcc tgtcaggagc tatataaggc 9420 ggatcgaggc aggcgagggg ggcagcgctg ccgagcggag cccaggagtg gagcgagagc 9480 gagcaagagc ctgagcgaaa agaccgggaa gcaaggaaga ggaagcctcc ggtgcatcgg 9540 gaaaggatcg caggtgctcg ggagccggag ctggagctcc acagccggca gtc atg 9596 Met 1 tac cga tcc acc aag ggc gcc tcc aag gcg cgc cgc gac cag atc aac 9644 Tyr Arg Ser Thr Lys Gly Ala Ser Lys Ala Arg Arg Asp Gln Ile Asn 5 10 15 gcc gag att cgg aac ctc aag gag ctg ctg ccg ttg gct gaa gcg gac 9692 Ala Glu Ile Arg Asn Leu Lys Glu Leu Leu Pro Leu Ala Glu Ala Asp 20 25 30 aag gtc cgg ctg tcc tac ctg cac atc atg agt ctt gcc tgc atc tac 9740 Lys Val Arg Leu Ser Tyr Leu His Ile Met Ser Leu Ala Cys Ile Tyr 35 40 45 act cgc aag ggt gtc ttc ttt gct gga g gtgagcagct tgggctaccg 9788 Thr Arg Lys Gly Val Phe Phe Ala Gly 50 55 gagaccagag ctgacgggga ccagggatgg aggagctgag ggaatgtgct aaaactgccg 9848 cttgtctagc acagcgtgct ggaagcctgt ggagagaagg gacttgaggg gaccctggac 9908 ttcctacttt ttcttctgag ctccatctag actagcctaa acgatagtcc tagcactgga 9968 tttgtgtgag atagagcgct aaaacagaat ggtccaggct cccattgcct cagaggcact 10028 ccaggaatcc ggggagggta cggaaggaag cctggcaagc tacagggaaa gcctgcaaag 10088 gcaaagtatg aggaagagta gcttgtgcta gaaaatgctg agagggtctt tctatgcgct 10148 ctggagctgt tcgacgtcct gaagccatca ccctttctgg cgctgcccgc ggtgctgaaa 10208 tggccatagc cccttttcgc aggagctgtc cgcggtgctg aatcccagtc ctttcgggag 10268 agctctgtcc acagtgctga cagcagaggg ctgctgaggt tccggccagg cttggaagtc 10328 caggggctcc ctggctagat agttttagca acaggtctcc tggccaagat ccacaagcat 10388 agggtcaaca ggtgttggca gaaataggtc tatgggaatt tcctgtgtct tctccaagac 10448 tcaaaagatg ttctctttat ttctgtgttg tccctgattc ttatcctgac tcaccacatc 10508 ttctcaccct acag gc act cct ttg gct ggc ccc acc ggg ctt ctc tct 10557 Gly Thr Pro Leu Ala Gly Pro Thr Gly Leu Leu Ser 60 65 70 gct caa gag ctt gaa gac att gtg gca gca cta cct gga ttt ctc ctt 10605 Ala Gln Glu Leu Glu Asp Ile Val Ala Ala Leu Pro Gly Phe Leu Leu 75 80 85 gta ttc aca gct gag ggg aag ttg cta tac ctg tcg gag agt gtg agc 10653 Val Phe Thr Ala Glu Gly Lys Leu Leu Tyr Leu Ser Glu Ser Val Ser 90 95 100 gag cat ctg ggc cac tct atg gtgagtacta aaagtccttg catctcaagt 10704 Glu His Leu Gly His Ser Met 105 tggggtatat gtgagataaa atgagcctct cactactgaa aacagagtta ttagaggcga 10764 gtgtggggga gtcttcccta agaaaaatca ttggttgcag ataggctctt gctgccttca 10824 ctaatgatca cttctccttt ctag gtg gac ctg gtt gcc cag ggc gac agt 10875 Val Asp Leu Val Ala Gln Gly Asp Ser 110 115 atc tac gat atc att gac cct gct gac cat ctc act gtg cgc cag cag 10923 Ile Tyr Asp Ile Ile Asp Pro Ala Asp His Leu Thr Val Arg Gln Gln 120 125 130 ctc acc atg ccc tct gct ctg gat gct g gtaagaacct cctctcggtt 10971 Leu Thr Met Pro Ser Ala Leu Asp Ala 135 140 cttcagttta ctcctctgct gccctgccct aactatctac tctcctccaa tgcccaccct 11031 cttagtcagt ttttcctttt gctcacctag at cgc ctt ttc cgt tgt cga ttc 11084 Asp Arg Leu Phe Arg Cys Arg Phe 145 150 aac acc tcc aag tcc ctc cgg cgc cag agt tca gga aac aaa ctg gtg 11132 Asn Thr Ser Lys Ser Leu Arg Arg Gln Ser Ser Gly Asn Lys Leu Val 155 160 165 ctt att cga ggt cga ttc cat gct cac cca cct ggg gcc tac tgg gca 11180 Leu Ile Arg Gly Arg Phe His Ala His Pro Pro Gly Ala Tyr Trp Ala 170 175 180 gga aac cct gtg ttc acc gct ttc tgc gcc cca ctg gag cca aga ccc 11228 Gly Asn Pro Val Phe Thr Ala Phe Cys Ala Pro Leu Glu Pro Arg Pro 185 190 195 cgc cct ggc ccc ggc cct ggc cct ggc cct ggt cct gct tct ctc ttc 11276 Arg Pro Gly Pro Gly Pro Gly Pro Gly Pro Gly Pro Ala Ser Leu Phe 200 205 210 215 ctg gcc atg ttc cag agc cgg cat gct aag gac cta gcc cta ctg gac 11324 Leu Ala Met Phe Gln Ser Arg His Ala Lys Asp Leu Ala Leu Leu Asp 220 225 230 gtt tct gaa ag gtaagcccaa agtgttcaaa ctccagtaag aagggaggcc 11375 Val Ser Glu Ser 235 agaaagaagg gaactttaga ttcgtgatct tagattcagg gcagggagga tggggcttaa 11435 gtgggcagag agcatgggag ggagtgaagt gcatgcattt tgagtaaggt aaacagaaag 11495 ctgacctcat catttccacc ttcccag t gtc cta atc tac ctg ggc ttt gag 11547 Val Leu Ile Tyr Leu Gly Phe Glu 240 cgc agc gaa ctg ctc tgt aaa tca tgg tat gga ctg cta cac ccc gag 11595 Arg Ser Glu Leu Leu Cys Lys Ser Trp Tyr Gly Leu Leu His Pro Glu 245 250 255 gac ctg gcc caa gct tct tct caa cac tac cgc ctg t gtgagtgtcc 11642 Asp Leu Ala Gln Ala Ser Ser Gln His Tyr Arg Leu 260 265 270 tgagaggccg tgcataacac aggaagctgg gagaaagcat gggagacagg ccagggactg 11702 gctgtggtcc aaactgatgt taaggagttt cggaggctac agagtgagct tgaggatgag 11762 aagtcaaggc aagaatagga cagagttaga aaacactgtg tgataaggtc aagtggggag 11822 cctagaggta caggttaggg tagttagaag agaatatgtc atggctccct caattcagtg 11882 tagaggtaag aaaggtgggt gtgtaggtgg tgttgattga tggaccttct aatccggtat 11942 tccttttttc tccccag tg gct gaa agt gga gat att cag gct gaa atg gtg 11994 Leu Ala Glu Ser Gly Asp Ile Gln Ala Glu Met Val 275 280 gtg aga ctt caa gcc aag cat gga ggc tgg aca tgg att tac tgc atg 12042 Val Arg Leu Gln Ala Lys His Gly Gly Trp Thr Trp Ile Tyr Cys Met 285 290 295 cta tac tca gaa ggt cca gaa ggc cct ttt act gcc aat aac tac cct 12090 Leu Tyr Ser Glu Gly Pro Glu Gly Pro Phe Thr Ala Asn Asn Tyr Pro 300 305 310 315 atc ag gtaagctgta agatacaaga tggcggagag gggaggggag ctgaggtcag 12145 Ile Ser catagaagaa atgcaacgaa gaaaactact ctggtaatgg acagcagacc cttacaagct 12205 gccacctctt ccctttccag t gac acg gaa gcc tgg agc ctc cgc cag cag 12256 Asp Thr Glu Ala Trp Ser Leu Arg Gln Gln 320 325 cta aac tct gaa gac acc cag gca gcc tat gtc cta gga acc cca gct 12304 Leu Asn Ser Glu Asp Thr Gln Ala Ala Tyr Val Leu Gly Thr Pro Ala 330 335 340 gtg cta ccc tca ttc tct gag aat gtc ttc tcc cag gag caa tgc tct 12352 Val Leu Pro Ser Phe Ser Glu Asn Val Phe Ser Gln Glu Gln Cys Ser 345 350 355 aat cca ctc ttt aca cca tcc ctg ggg act cct aga agt gcc agc ttc 12400 Asn Pro Leu Phe Thr Pro Ser Leu Gly Thr Pro Arg Ser Ala Ser Phe 360 365 370 375 ccc agg gct cct gaa cta ggt gtg atc tca aca cca gaa gag ctt ccc 12448 Pro Arg Ala Pro Glu Leu Gly Val Ile Ser Thr Pro Glu Glu Leu Pro 380 385 390 caa ccc tcc aaa gag ctg gac ttc agt tac ctg cca ttc cct gct agg 12496 Gln Pro Ser Lys Glu Leu Asp Phe Ser Tyr Leu Pro Phe Pro Ala Arg 395 400 405 cct gag cct tcc ctc caa gca gac ctg agc aag gat ttg gtg tgt act 12544 Pro Glu Pro Ser Leu Gln Ala Asp Leu Ser Lys Asp Leu Val Cys Thr 410 415 420 cca cct tac aca ccc cac cag cca gga ggc tgt gcc ttc ctc ttc agc 12592 Pro Pro Tyr Thr Pro His Gln Pro Gly Gly Cys Ala Phe Leu Phe Ser 425 430 435 ctc cat gaa ccc ttc cag act cac ttg ccc cct ccg tcc agc tct ctc 12640 Leu His Glu Pro Phe Gln Thr His Leu Pro Pro Pro Ser Ser Ser Leu 440 445 450 455 caa gaa cag ctg aca cca agt aca gtg act ttc tct gaa cag ttg aca 12688 Gln Glu Gln Leu Thr Pro Ser Thr Val Thr Phe Ser Glu Gln Leu Thr 460 465 470 ccc agc agt gct acc ttc cca gac cca cta acc agt tca cta caa gga 12736 Pro Ser Ser Ala Thr Phe Pro Asp Pro Leu Thr Ser Ser Leu Gln Gly 475 480 485 cag ttg aca gaa agc tca gcc aga agc ttt gaa gac cag ttg act cca 12784 Gln Leu Thr Glu Ser Ser Ala Arg Ser Phe Glu Asp Gln Leu Thr Pro 490 495 500 tgc acc tct tcc ttc cct gac cag cta ctt ccc agc act gcc aca ttc 12832 Cys Thr Ser Ser Phe Pro Asp Gln Leu Leu Pro Ser Thr Ala Thr Phe 505 510 515 cca gag cct ctg ggc agc ccc gcc cat gag cag ctg act cct ccc agc 12880 Pro Glu Pro Leu Gly Ser Pro Ala His Glu Gln Leu Thr Pro Pro Ser 520 525 530 535 aca gca ttc cag gct cat ctg aac agc ccc agc caa acc ttc cca gag 12928 Thr Ala Phe Gln Ala His Leu Asn Ser Pro Ser Gln Thr Phe Pro Glu 540 545 550 caa ctg agc ccc aat cct acc aag act tac ttc gcc cag gag gga tgc 12976 Gln Leu Ser Pro Asn Pro Thr Lys Thr Tyr Phe Ala Gln Glu Gly Cys 555 560 565 agt ttt ctc tat gag aag ttg ccc cca agt cct agc agc cct ggt aat 13024 Ser Phe Leu Tyr Glu Lys Leu Pro Pro Ser Pro Ser Ser Pro Gly Asn 570 575 580 ggg gac tgt aca ctc ctg gcc cta gct cag ctc cgg ggc ccc ctc tct 13072 Gly Asp Cys Thr Leu Leu Ala Leu Ala Gln Leu Arg Gly Pro Leu Ser 585 590 595 gtg gat gtc ccc ctg gtg ccc gaa ggc ctg ctc aca cct gag gcc tct 13120 Val Asp Val Pro Leu Val Pro Glu Gly Leu Leu Thr Pro Glu Ala Ser 600 605 610 615 cca gtc aag caa agt ttc ttc cac tac aca gag aaa gag caa aat gag 13168 Pro Val Lys Gln Ser Phe Phe His Tyr Thr Glu Lys Glu Gln Asn Glu 620 625 630 ata gat cgt ctc att cag cag atc agc cag ttg gct cag ggc gtg gac 13216 Ile Asp Arg Leu Ile Gln Gln Ile Ser Gln Leu Ala Gln Gly Val Asp 635 640 645 agg ccc ttc tca gct gag gct ggc act ggg ggg ctg gag cca ctt gga 13264 Arg Pro Phe Ser Ala Glu Ala Gly Thr Gly Gly Leu Glu Pro Leu Gly 650 655 660 ggg ctg gag ccc ctg aac cct aac ctg tcc ctg tca ggg gct gga ccc 13312 Gly Leu Glu Pro Leu Asn Pro Asn Leu Ser Leu Ser Gly Ala Gly Pro 665 670 675 cct gtg ctt agc ctg gat ctt aaa ccc tgg aaa tgc cag gag ctg gac 13360 Pro Val Leu Ser Leu Asp Leu Lys Pro Trp Lys Cys Gln Glu Leu Asp 680 685 690 695 ttc ctg gtt gac cct gat aat tta ttc ctg gaa gag acg cca gtg gaa 13408 Phe Leu Val Asp Pro Asp Asn Leu Phe Leu Glu Glu Thr Pro Val Glu 700 705 710 gac atc ttc atg gat ctt tct act cca gac ccc aat ggg gaa tgg ggt 13456 Asp Ile Phe Met Asp Leu Ser Thr Pro Asp Pro Asn Gly Glu Trp Gly 715 720 725 tca ggg gat cct gag gca gag gtc cca gga ggg acc ctg tca cct tgc 13504 Ser Gly Asp Pro Glu Ala Glu Val Pro Gly Gly Thr Leu Ser Pro Cys 730 735 740 aac aac ctg tcc cca gaa gat cac agc ttc ctg gag gac ttg gcc acc 13552 Asn Asn Leu Ser Pro Glu Asp His Ser Phe Leu Glu Asp Leu Ala Thr 745 750 755 tat gaa acc gcc ttt gag aca ggt gtc tca aca ttc ccc tac gaa ggg 13600 Tyr Glu Thr Ala Phe Glu Thr Gly Val Ser Thr Phe Pro Tyr Glu Gly 760 765 770 775 ttt gct gat gag ttg cat caa ctc cag agc caa gtt caa gac agc ttc 13648 Phe Ala Asp Glu Leu His Gln Leu Gln Ser Gln Val Gln Asp Ser Phe 780 785 790 cat gaa g gtaagtctag cctgaatgtc caagagccct gcccttctaa tcagacattg 13705 His Glu catagattgg gtgaatcagt ccccaactct gaaactctgt tttattaaga gaacaatatt 13765 acctcctact aagaagagta gtgaggtagg aataatacaa agctttgtgt gaaagatgag 13825 tagacctggt gggcggggga ggtgagctag aaaaacgcga tagacaatcc ctaggcaaaa 13885 gcttgaaagc ttctgagaga cctagaccag acaacaccgt cattttatag acaaaaataa 13945 tcaaggcccc agagttaaag aaactttaag tggcacaaaa attgatagaa gttgatgctt 14005 ccccctgaag gggacccaga gcaacaactg gttaaaatta ggagacagaa agaacaatgc 14065 caagccccta gctccaatct ggcggccttg tgctgtttgt ccaaagctgt ggccacagtt 14125 tccctccata tttgcatatt gcctcttatc tgctgacacc ctggggatca gttcatttgg 14185 ctaacacatt tgacgtccat agactatagc aatattgtac cactgcctga gcccaatgac 14245 gcttttactg aataagcttg actaacatac gcactttctc tcttctctct ctctctcttt 14305 ccccacag at gga agt gga ggg gaa cca acg ttt tgaataagtc tgtgacttaa 14359 Asp Gly Ser Gly Gly Glu Pro Thr Phe 795 800 cgtcttcaag tatggcatat tgtcatcaag acgtggagcc gctctccacc cccccgggac 14419 tgttgggggg attctggggg ccagaggggg atatatctga ttctccaggc cctgaaggat 14479 ttagggggga ggtgggaggg taagggaggg gagcaacttt ttaaaatcaa gagacttcga 14539 gcgatcccag tttccatttc aatctgtatt cactcgtagt gagtttcctt gaatggattt 14599 caagcggaga atgggggagt ctcacttcct caccgcgctg ccccatgggc ctgggccagt 14659 tctccactcc taggggcaaa gccacccctg ggctttggtg ggggaaaggc atggcccacc 14719 tggggctagc ctgtgccccg aggggctctt gacacccacg tagaattctc tacaaaccag 14779 taacgggatt tcaattccga cggactctgc cgccctggcg gctcttcctg tgacttttgc 14839 gccccgcgcc tggggtgggg ggcgcgaaga gacgctacat tcctttccga tggaggaagg 14899 cagatctgcc gtcacacgtg tgcttgcacg agtgcgtgta cctggtgcgg gactcacccg 14959 gccgccagac cgcctaggct tgcccaggtg gccacctcgt ggtgctgcgg tgactttgta 15019 gccaacttta taataaagtc cagtttgcct ttttggtatc tctggtgtca tgcgctattg 15079 tgaaaaggga agggagggga agggagagat tgaggagccc agataggagg ctggggcagg 15139 agtcacaggt tagacctcct ctcagccctg gtatctctaa gtgagtttgt tcatatctcc 15199 atttgactct gcttggtcca cactgtgcta gaagactaag tacttgtcag aagcagacat 15259 tgcaccaaag acactggagt cttctctctg ccctgggttt atggtgtgat ggggaggaaa 15319 gagcctgggg ctgagcaagt ttgtcactgg tcttggatat gggtttaaag tttctggtca 15379 tttcctgcct ggtctttcag gatattgatt tcctcatgga ggcttagatt ttaaaaatca 15439 gaagctgaaa cctgttacgc ttgcgtaggg ctgttcagtt agcaaatacc caatccactg 15499 caataaattt ccacttcatt gggaaagcaa cccgataacg ggtgttcctc cagttacagg 15559 tgagaaacac atcaacccct ccccaaatct ggggagctcc cagatctcaa tgccagcgaa 15619 taaccatcat agaccatctc accacagagc tgaggaccag tcactgggga ggaaatttca 15679 gaaaatggtg tttgactcta aactcgtagg ctcaacccca cagggtgtgg ttagtggagg 15739 acaaatgaaa gttaggtggt agaaggacct gacagatcca atcacgatcc caccttttgt 15799 atttggagtg cacctaaagc ccccacttcc tcacaggtca aaggagggca gcaatcaaga 15859 ggcagtgtca gaacaggaca agtctcttcc agctcacgaa gtgcagtgaa ggcttggtcg 15919 gtgcgacctc catttcagtg gtgacccgca gacttagaga aagccttgtc ctcaaggaga 15979 ggacaacaac tccaggctcc agtctttcca cagaagcaca ggggcacagc cttgaaaacc 16039 ctgtagcctc cactcatcct gaagcccagc tgtggagaca gacaggccct ttggagggtc 16099 cttccttcac tgtggagaca gacaggccct ttggagggtc cttccttcac tgtggagaca 16159 gacaggccct ttggagggtc cttccttcac tgtggagaca ggccctttgg atccttcctt 16219 cacagaaagg aaggatccac agggaccttt cccttctttg atgggtattt gggtggagcc 16279 aagaacttcc ctgtcactcc caagaggaac ctgtcttagc tcagttccct cctcagcaca 16339 gggacacgga gatggggaga tggataaagg tgctgggcca agcatgatgc tctgatttga 16399 tccttgatgg gaagagataa ctgacagttg tcctctgacg tgtaactgca ctccaggaca 16459 tgttacactc acatgtgcac acacacacac tacacacact acatacacat accatacaca 16519 tactatacac aatataccac acacacacat actatacaca cataccacat acactacaca 16579 cagtacacat gctacacata catacacaca ccacacacat ataccacaca caaacactct 16639 acacacacac actacacaca ctacatacat ataccacaca cacttaccac acatacagta 16699 tatacagtac atacatatgc cacacacaca taacacacac tcacacacac catatatact 16759 actaatagaa aataataaaa atttttaaat ggggtggatt taggaaatga aatttctgtg 16819 agaataaagg aaaggcttcc ttgatgtttg gtggtggctg gcaatagtgt atgctttctt 16879 tgtctttgtt tgttgtagtt tttttgttta ttttgctttt gatttttttt ttgttgttgt 16939 tgttacttgt ttgttgaaaa cctgcctctg cctcctaagc actgaattgt cttgggtggt 16999 ttttaaaaat taattaatgt tgaaatattt ttttcatttt tgagacaaga tttctttgtg 17059 tagccttagc tgtcttagaa ctagctctgt agactaagct ggccttaaac ttacagagat 17119 ctgcttgctt ctgcctcctg agtgctggga ttaaagtttt tagtttttaa aaaaatataa 17179 ttacagatat gcactgtctt tgcatcatgt cctcttgttt tgggcttatt tttgttgttg 17239 tggtggtgat aagtgatttt ttttgtttgt ttttgttttt agttttgttt ttcttcagct 17299 caggtcaatc tggagttcac tatgtagtcc aaggtggcca cagacttttg caaatccccc 17359 tgcctcagcc tcccaggtgc taggattaca gaagaaccag accaactggt cctgtgtgag 17419 gaaaataaag tagaagaggc aatactgcca cctgctggaa ggaaaagaag ctgcttcctt 17479 gctggctgct gaggcccttg cagctcagaa tatcttcacc ttagaatgga gagataaact 17539 gagtccctgg gagagaaaag gacttcagga tctgagagtg agtgatgttc tggaagcaga 17599 gtgcatgaga gaaggtgtct taatcattgt agtactgctg tgagaagaca ccatgaccaa 17659 ggtaacataa aataaagcat ttagttgggg acttgcttag agtttaaaag ggttgctcca 17719 tgaccagcag agcagggagc atgggagtat gcaggtagac acggcactgg agaagtacct 17779 gagagcttcc atctgatccc caagatagag gcagagagaa ccctcaaagc ccacaccccc 17839 tccaacaaca aacacctcct gatccttcct aaacagtcca ccaaatggag actaagcatt 17899 cagatatggg gaccattatc atccaaacca ctatggaagg ctcgagtctg gggaccagac 17959 agactgaacc caggagacca aggggatagc ttagtgggta aaggcgctag ctgccgagct 18019 tggagacgca agtccaatcc ctaggttctg tatggtggaa agaaacggga ttccagtaag 18079 tcaccccctg gccttcgcgc acaccatgat gctcatgccc acacacatac aaatccaaaa 18139 gaaagaccga acctaaggat ggttctgctg ttgtacattt ttcctgtaat agatcatcca 18199 tgacacttgc ctgagttctg ggaaaactga acaaacaaga tgggtggggc cagacagctg 18259 tgctctaact gggaacatca caagaggtaa gacagagcct gagtgctgaa ggcaagagct 18319 agggtatcgt gacagagtaa ccggggactg atttatagtg ccactttctg agaaggtgac 18379 actgagcttg ttagcaacag gtgacaacaa agaagagtcc aacctaaagg aagcatctgt 18439 aatgacatta aaacgggaga gtgtctgagc tgcttaagaa gtacacagga agtgggctga 18499 gacaagcagg agaggggctg gagagaaggt cgcccagtac ttctagacca caataaaaga 18559 tgtaggttgc attctggctg agcgtggtag tgcacacctg caattgcagc ctcaaaaggc 18619 cgagggtgga aaatcttgag ctcctggaca gcctgggctc catagaaaga aaagtctgca 18679 aacaacagca acaaaaaacc caaaccaaaa accaaagtgc tggtgtccta gtgagggttc 18739 ctattgctat gaggaaaaac aatgatcaaa aacaaactgc ggacgaaagg gtttgtttgc 18799 ctggcacttc cacatcacag tccatcattg aaggaatcca gaacaggaac gcaagcaagg 18859 caggaacctg gaggcaggag ccaatgaaga ggtcatgaag ggttgctgct tatggcttgc 18919 tccacatggc tttacagcct gctagatctc agcaccaaca gcctaccatg agcgtggccc 18979 tcctccatca atcactgatt aagaaaatgt cctacacagg aagggaggaa ggaagagaga 19039 gttaggagca tattggatgg ggatagtgac aggataagat gtagctacta gagtcttctg 19099 gtttagatgg tgaatctgcc agaatttgcc actgaaggat ttagatttag atttaacata 19159 acttacaaga ttagcattct agttgttgca cccagagact gagttaccat tgtttctgaa 19219 ctaagtttgt gtgctgtttt tcttcacgcg gtggctcgac tgggttcaag agagaaaggt 19279 acagcggcaa agcctgggtt tgccagatgc gcaccacaaa ggcagtgggg gtttgaacga 19339 tggggctagc acggcagtgg gaactcattg agccgggtgg agggattttg gagctccagg 19399 tcagagagtt tgctgagatg agaacaccag gctggagcca tgtggcctgc cggtaccttg 19459 gcataatgag ggaacttgct gttcttttta atatttccca caacaggtgg tgaaccagca 19519 tgttggggaa gaatccacta gaaatgtaag attatgccgg gcgtggtggt gctcgccttt 19579 aatcccagca ctcgggaggc agaggcaggc agatttctga gttcgaggcc agcctggtct 19639 acaaagtgag ttccaggaca gccagggcta cacagagaaa ccctgtctcg aaagacaaaa 19699 caaaacaaaa caaacaaaac aaaacaaaac gtatgatcat tagcctgaga gttagagttt 19759 tatttgtttg tttgtttgtt tgttatttaa aatgagtagc tgggtagtgc tgacacaagt 19819 catgtggacc caagcgtgga attgaaacaa agactgtaac tctgaggtcc cctgctgtgg 19879 gggctgcagg ctgttctgag tcaggagaag aaggatgaag ttgcctactt cttagggcag 19939 agatggattg aactgtgaat ttataaaatt ggtattattt gcttttagga aagatttata 19999 tctgggtttt gcctgaatca catggggatt ttcgcccact gttcagaatt aggataggaa 20059 aaaaatcagt ccctgactcc aggtagaaaa gacagtgatt atcgtctgct acaaacaggt 20119 atcaattaac tatgtctgtg gctccctgta gagagctcaa aagatggata ttataacagg 20179 tattaataaa attaatgtca cccaggcagt ggtggcacac gcctttaatc ccagcacttg 20239 ggaggcagag gcaggcggat ttctgagttc gaggccagcc tggtctacag agtgagttcc 20299 agcacagcca gggctacaca gagaaaccct atcttgaaaa aaaaattaaa taaaattaat 20359 gtctgtggcc ccagtgctga gcagatagac agtgtaacaa gatggctgct ctaggcagag 20419 agctgaacag gaagatggta tgaagatagt ttgctctaac acacctcaca ggatgctcaa 20479 atcctgtcta tgtgggctcc atgggaatct tttttttaat taggtatttt cctcatttac 20539 atttccaatg ctatcccaaa agtcccccat accctcctcc caacccccca accacccact 20599 cccacttttt ggccctggcg ttcccctgta ctggggcata taaagtttgc gtgtccaatg 20659 ggcctctctt tccagtgatg gctgactagg ccaccttttg atacatatgc agctagagtc 20719 aagagctccg gggtactggt tagttcataa tgttgttcca cctatagggt tgcaga 20775

Claims

1. a method for assaying an ability to control the neurocyte plasticity which is dependent on a transcription regulatory factor comprising any of the following amino acid sequences, comprising the steps:

(3) a step for evaluating the ability possessed by the test substance based on the expression level or the index value correlating with the level measured in the second step,

wherein the group of the amino acid sequences being:

(a) the amino acid sequence represented by any of SEQ ID Nos.1 to 3,

(e) the amino acid sequence a protein comprising an amino acid sequence encoded by a DNA which hybridizes under a stringent condition with a DNA consisting of the nucleotide sequence represented by the nucleotide numbers 35 to 2440 in the nucleotide sequence represented by SEQ ID No.6 and also having a transcription regulation ability.

2. a method for assaying an ability to control the neurocyte plasticity which is dependent on a transcription regulatory factor comprising any of the following amino acid sequences, comprising the steps:

(1) a first step for bringing a test substance into contact with a transformed mammalian cell obtainable by introducing a gene comprising a nucleotide sequence encoding the amino acid sequence; and,

wherein the group of the amino acid sequences being:

(a) the amino acid sequence represented by any of SEQ ID Nos.1 to 3,

3. an assay method according to claim 1 or 2, wherein the marker protein gene is an Eph A receptor gene or an Rho GDP dissociation inhibitor gene.

4. an assay method according to claim 1 or 2,

wherein the marker protein genes are an Eph A receptor gene and an Rho GDP dissociation inhibitor gene.

5. an assay method according to claim 1 or 2,

wherein the ability possessed by the test substance is evaluated based on the difference obtained by comparing the marker protein gene expression level or an index value correlating with the level in the groups employing two or more different substances independently as test substances.

6. an assay method according to claim 1 or 2,

wherein at least one of the two or more different substance is a substance which does not have the ability.

7. a method for searching for a substance having an ability to control the neurocyte plasticity which is dependent on a transcription regulatory factor comprising any of the following amino acid sequences, wherein a substance having the ability is selected on the basis of the ability evaluated by an assay method according to claim 1 or 2,

wherein the group of the amino acid sequences being;

(a) the amino acid sequence represented by any of SEQ ID Nos.1 to 3,

8. a neurocyte plasticity regulator comprising as an active ingredient a substance selected by a searching method according to claim 7 or a pharmaceutically acceptable salt thereof and obtained by formulating the active ingredient into a pharmaceutically acceptable carrier.

9. a use of a gene comprising the nucleotide sequence encoding any of the following amino acid sequences for controlling neurocyte plasticity in a mammalian cell, wherein the group of the amino acid sequences being:

(a) the amino acid sequence represented by any of SEQ ID Nos.1 to 3,

10. a use of a gene comprising the nucleotide sequence encoding any of the following amino acid sequences as an exogenous gene for promoting the expression of a marker protein gene present on the neurocyte plasticising pathway which is dependent on a transcription regulatory factor comprising the amino acid sequence in a mammalian cell by means of providing the exogenous gene in a position enabling the expression in the cell,

wherein the group of the amino acid sequences being:

(a) the amino acid sequence represented by any of SEQ ID Nos.1 to 3,