JP2003180361A - s-DREBRIN A - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an isoform of a protein drebrin A, a DNA encoding the protein, a cell in which the DNA or protein is transduced and further a method for screening a prophylactic or a therapeutic agent for diseases developed with acceleration of functions of the drebrin A or inhibition of functions of the drebrin A using the cell. <P>SOLUTION: This isoform of drebrin, s-drebrin A is identified and cloned. The s-drebrin A and its substance in which an amino acid is deleted, substituted or added and a DNA encoding the same are provided. A host cell comprises the DNA integrated therein and a monoclonal antibody or a polyclonal antibody specifically binds to the protein. An animal neurocyte in which the DNA or protein is transduced into competitively inhibit the functions of the drebrin A is provided. The method, etc., for screening a substance having prophylactic or therapeutic effects on diseases developed with acceleration of the functions of the drebrin A or inhibition of the functions of the drebrin A are provided. <P>COPYRIGHT: (C)2003,JPO

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to an isoform of the protein drebrin A which is localized in the dendrites of nerve cells, binds to actin filaments there, and functions as a regulator of morphology of neurite, and DNA encoding the same. , And animal nerve cells that competitively inhibit the function of drebrin A using such proteins and DNA, and further, prevention or treatment of diseases caused by the enhancement of the function of drebrin A or the inhibition of drebrin A function using the animal nerve cells The present invention relates to a screening method for substances having effects.

[0002]

BACKGROUND OF THE INVENTION Actin filaments are the major cytoskeletal component of postsynaptic neuronal dendrites (Proc.
Natl. Acad. Sci. USA79, 7590-7594, 1982; Cell m
ol. Neurobiol. 5, 271-284, 1985), plays an important role in the regulation of synaptic structure and plasticity (Brai).
n Res. Mol. Brain Res. 43, 246-250, 1996; Neuron 2
0, 847-854, 1998; Neurosci. Res. 40, 1-7, 2001).
Drebrin is a developmentally regulated actin-binding protein that was first detected in the chicken brain (J. Neu.
rochem. 44, 1210-1216, 1985, Dev. Brain Res. 29, 2
33-244, 1986; Mol. Brain Res. 4, 71-74, 1988; Neur
oreport 3, 109-112, 1992; J Biochem. (Tokyo) 117, 2
31-236, 1995; Brain Res. 464, 207-215, 1988, Brain
Res. Mol. Brain Res. 19, 101-114, 1993). Recently, in vitro studies (Exp. Cell Res. 215, 145-153, 199
4; Exp. Cell Res. 253, 673-680, 1999) found that drebrin is widely involved in actin cytoskeletal dynamics.

Drebrin binds to filamentous actin (F-actin) with a high affinity to produce fascin (J. Neuro
chem. 66, 980-988, 1996), α-actinin and tropomyosin (J. Biol. Chem. 269, 29928-29933, 199).
4) Competitively binds to F-actin stabilizing protein such as. Besides having the ability to bind F-actin, drebrin has also been shown to interact with profilin, which is an actin monomer-binding protein that stimulates actin polymerization (Biochem. Biophys. Res.
Commun. 243, 86-89, 1998). Drebrin can also form a stable complex with gelsolin, myosin, α-actinin, and F-actin in nerve cells (J. Neurosc
i. 16, 7161-7170, 1996). Overexpression of drebrin in fibroblasts causes remodeling of actin filaments (Neuroreport 3, 109-112, 1992, Exp.
Cell Res. 215, 145-153, 1994; .J. Biol. Chem. 269,
29928-29933, 1994; Exp. Cell Res. 253, 673-680, 1
999). Drebrin has recently been elucidated to be a member of the actin depolymerizing factor homology (ADF-H) family (FIG. 1D) (Mol. Biol. Cell 9, 1951-19).
59, 1998). The high-affinity actin-binding domain of drebrin is not localized in the ADF-H domain, but both domains are localized in the N-terminal region (Exp. Cell Re
s. 253, 673-680, 1999).

Drebrin is classified into an embryonic type (Drebrin E) and a mature type (Drebrin A) based on its expression pattern and primary structure (Mol. Brain Res. 4, 71-74, 198).
8; Brain Res. 464, 207-215, 1988; Neuroreport 3, 1
09-112, 1992; Brain Res. Mol. Brain Res. 19, 101-1
14, 1993). Three drebrin isoforms (E1, E2 and A) detected in chickens have been identified. Drebrin E1 and E2 are mainly expressed in developing neurons (J. Neurochem. 44, 1210-1216, 1985, De.
v. Brain Res. 29, 233-244, 1986; Brain Res. 464, 2
07-215, 1988; Mol. Brain Res. 4, 71-74, 1988). E
In E2 compared to 1, there was an additional 129-b in the mRNA.
There are p exons (called ins1). Drebrin A having another 138-bp insert (ins2) from this ins1
Is expressed in mature neurons (Neurosci. Res. Supp.
l. 13, S106-111, 1990; Brain Res. Mol. Brain Res.
19, 101-114, 1993).

To date, two isoforms have been identified and cloned in mammals (drebrin E).
And A) only (Neuroreport 3, 109-112, 1992;
Biochem. Biophys. Res. Commun. 196, 468-472, 1993;
J. Cell Sci. 113, 325-336, 2000). Expression of drebrin in mammals is also tissue-specifically regulated during development. Mammalian drebrin E, like chicken drebrin E1 and E2, is cell-type-specifically expressed mainly in the developing brain and several types of non-neuronal tissues (J. Neurosci. 16, 7161-7170, 1996; Brain. Res. D
ev. Brain Res. 111, 137-141, 1998; Eur. J. Cell Bio
l. 78, 767-778, 1999; J. Cell Sci. 113, 325-336, 20.
00). Expression of drebrin E in the stomach and kidney is found in acid-secreting cells in which drebrin E is specifically localized in the apical plasma membrane (J. Cell Sci. 113, 325-336, 2000).

On the other hand, drebrin A is expressed mainly in the postnatal developmental stage and in the adult brain (J. Neurosci. 16,
7161-7170, 1996; Brain Res. Dev. Brain Res. 111,
137-141, 1998; J. Cell Sci. 113, 325-336, 2000),
It is localized in postsynaptic nerve cell dendrites of mature neurons (J. Neurosci. 16, 7161-7170, 1996). Moreover, when drebrin A is overexpressed in neurons of the cultured cortex, morphological changes occur in dendrites (J. Neurosci. 19, 3918-.
3925, 1999). This suggests that drebrin A is deeply involved in synaptic structure-based plasticity (J. Neurosci. 19, 3918-39).
25, 1999). In the brain of Alzheimer's disease (AD) patients,
Interestingly, drebrin is dramatically reduced in hippocampal neuronal dendrites (J. Neurosci. Re
s. 43, 87-92, 1996). An 81% reduction in drebrin protein was observed in AD brains compared to healthy subjects of the same age group (J. Neuropathol. Exp. Neuro
l. 58, 637-643, 1999).

Based on the results of an immunohistochemical method using adult rat brain, the M2F6 monoclonal antibody (Dev. Brain Re) which recognizes the C-terminal epitope of drebrin.
s. 29, 233-244, 1986) that the labeling pattern resembles that of a polyclonal antibody against full-length drebrin (J. Neurosci. 1).
6, 7161-7170, 1996).

[0008]

An object of the present invention is to isolate the isoform of drebrin A, which is a protein localized in dendrites of nerve cells, where it binds to actin filaments and functions as a regulator of morphology of protrusions. , And animal nerve cells that competitively inhibit the function of drebrin A using such proteins and DNA, and further diseases caused by the enhanced function of drebrin A or the inhibition of drebrin A function using the animal nerve cells Another object of the present invention is to provide a method for screening a substance having a preventive or therapeutic effect on

[0009]

[Means for Solving the Problems] The immunostaining pattern (Exp. Cell Res. 215, 145-153, 1994) of mature rats against DAS1, which is a polyclonal antibody against ins2 polypeptide which is an insertion sequence of drebrin A, is as described above. The pattern of the two antibodies was very different (unpublished data). Therefore, the present inventors hypothesized from the above data that another isoform (s) of drebrin having an ins2 domain may exist in the adult brain. In the present invention, this drebrin isoform was identified and cloned to obtain the drebrin isoform of the present invention. The drebrin isoform of the present invention was designated as s-drebrin A. This drebrin isoform is a novel trunc with an ins2 domain.
ated) body drebrin isoform.

Such s-drebrin A is 13
It is produced by alternative splicing from a single drebrin gene located on chromosome # 3. The expression of s-drebrin A is brain-specific and increases in parallel with drebrin A during brain maturation. s-Drebrin A
Was found to associate with the actin cytoskeleton and regulate the organization of actin filaments in fibroblasts, as a result of functional analysis. The novel isoform of drebrin, s-drebrin A of the present invention, is a protein consisting of the amino acid sequence shown in SEQ ID NO: 2, and the DNA encoding the protein is shown as 119th to 1222nd in SEQ ID NO: 1. Consists of a base sequence.

In the present invention, a protein consisting of an amino acid sequence shown in SEQ ID NO: 2 in which one or several amino acids are deleted, substituted or added and having an s-drebrin function; The amino acid sequence shown in No. 2 consists of an amino acid sequence in which one or several amino acids have been deleted, substituted or added, has an actin cytoskeleton regulatory function, and has a proline rich domain, Homer binding domain, S
DNA encoding a protein lacking a function due to H2 binding domain deletion; SEQ ID NO: 1 119 to 1222
DNA constituting the gene consisting of the nucleotide sequence shown in No.
D that hybridizes with a stringent condition and encodes a protein having an s-drebrin function
NA; a monoclonal antibody or a polyclonal antibody that specifically binds to the protein of the present invention; an expression vector incorporating the gene of the present invention; a host cell containing the expression vector; Animal nerve cells that have been introduced and expressed to competitively inhibit the function of drebrin A; Animal nerve cells that have introduced the protein of the present invention into animal nerve cells and competitively inhibit the function of drebrin A; Of a substance having a preventive or therapeutic effect on a disease caused by an increase in the function of drebrin A or an inhibition of the function of drebrin A, in which a test substance is administered to animal nerve cells of the animal to evaluate and measure the state of the increase in the function of drebrin A Method: Diseases caused by enhancement of drebrin A function or inhibition of drebrin A function obtained by the screening method Substance having a prophylactic or therapeutic effect are also included in the present invention.

That is, the present invention is a DNA encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 2.
(Claim 1) or the amino acid sequence shown in SEQ ID NO: 2, which comprises an amino acid sequence in which one or several amino acids are deleted, substituted or added, and has an actin cytoskeleton regulatory function and a proline-rich domain. , Hom
er-binding domain, DNA encoding a protein lacking function due to SH2-binding domain deletion (claim 2)
Or a DNA comprising a nucleotide sequence represented by SEQ ID NO: 119 to 1222 or a complementary sequence thereof or a sequence containing a part or all of these sequences (claim 3),
A DNA which hybridizes with the DNA constituting the gene according to claim 3 under stringent conditions and which encodes a protein having an s-drebrin function (claim 4), or an amino acid sequence represented by SEQ ID NO: 2. A protein (claim 5) or the amino acid sequence shown in SEQ ID NO: 2, which comprises an amino acid sequence in which one or several amino acids are deleted, substituted or added, and s-
It is composed of a protein having a drebrin function (claim 6).

The present invention also provides an antibody that specifically binds to the protein according to claim 5 or 6 (claim 7), and the antibody is a monoclonal antibody or a polyclonal antibody. An antibody (claim 8), an expression vector (claim 9) incorporating a DNA comprising the sequence according to any one of claims 1 to 4, and a host cell comprising the expression vector according to claim 9 (claim). Item 10),
DNA comprising the sequence according to any one of claims 1 to 4,
Introduced into and expressed in animal nerve cells, animal nerve cells characterized by competitively inhibiting the function of drebrin A (claim 11), or the protein according to claim 5 or 6 introduced into animal nerve cells Animal nerve cells characterized by competitively inhibiting the function of drebrin A (claim 12).
And animal nerve cells (claim 13), characterized in that the competitive inhibition of the function of drebrin A according to claim 11 or 12 is inhibition of signal transduction from a neurotransmitter receptor to an actin filament (claim 13). The animal nerve cell is a mouse brain cell, which is the animal nerve cell according to any one of claims 11 to 13 (claim 14).

Furthermore, the present invention is characterized in that the test substance is administered to the animal nerve cells according to any one of claims 11 to 14 to evaluate and measure the state of hyperactivity of drebrin A. A method for screening a substance having a preventive or therapeutic effect on a disease caused by hyperfunction or inhibition of drebrin A function (claim 15), or a disease caused by inhibition of drebrin A function is Alzheimer's disease (A).
D), a method for screening a substance having a preventive or therapeutic effect on a disease caused by inhibition of drebrin A function (claim 16), or claim 15 or 16
It comprises a substance having a preventive or therapeutic effect on a disease developed by enhancing the function of drebrin A or inhibiting the function of drebrin A, which is obtained by the screening method described in any one of (17).

[0015]

BEST MODE FOR CARRYING OUT THE INVENTION s-Drebrin A, which is an isoform of drebrin A of the present invention, has a sequence number of 2
It is a protein consisting of the amino acid sequence described in.
The DNA sequence encoding the protein of the present invention consists of the nucleotide sequences shown as 119th to 1222th in SEQ ID NO: 1 in the sequence listing. The protein of the present invention includes a protein consisting of the amino acid sequence shown in SEQ ID NO: 2 in which one or several amino acids are deleted, substituted or added, and having the s-drebrin function. In addition, DNA encoding the protein of the present invention
As the sequence, the amino acid sequence shown in SEQ ID NO: 2 is composed of an amino acid sequence in which one or several amino acids are deleted, substituted or added, and has an actin cytoskeleton regulatory function, and has a proline-rich domain, Home.
Also included is a DNA encoding a protein that has a loss of function due to deletion of r-binding domain and SH2-binding domain.

To obtain the protein of the present invention using the gene comprising the DNA sequence of the present invention, the DN of the present invention is used.
The gene consisting of the A sequence can be incorporated into a known vector to construct an expression vector, which can be introduced into a host cell for expression, and the produced protein can be isolated and purified to obtain the protein. As an expression system, the protein
Any expression system capable of expressing the peptide in the host cell may be used, and expression systems derived from chromosomes, episomes, mammals and viruses, for example,
Vectors derived from bacterial plasmids, yeast plasmids, papovaviruses such as SV40, vaccinia virus, adenovirus, fowlpox virus, pseudorabies virus, retrovirus derived, bacteriophage derived, transposon derived and combinations thereof, eg , Cosmids and phagemids, and those derived from genetic elements of bacteriophage. This expression system (expression vector) may include a control sequence that regulates not only expression but also expression.

The present invention also relates to a host cell containing an expression system (expression vector) capable of expressing the protein / peptide of the present invention. This protein
The gene encoding the peptide is introduced into the host cell by Da
vis et al. (BASIC METHODS IN MOLECULAR BIOLOGY, 1986)
And Sambrook et al. (MOLECULAR CLONING: A LABORATORY MA
NUAL, 2nd Ed., Cold Spring Harbor Laboratory Pres
S., Cold Spring Harbor, NY, 1989), methods described in many standard laboratory manuals such as calcium phosphate transfection, DEAE-dextran mediated transfection, transvection.
(transvection), microinjection, cationic lipid-mediated transfection, electroporation, transduction, scrape loading (scrape loa)
ding), ballistic introduction, infection, etc. Then, as the host cell, Escherichia coli, Streptomyces, Bacillus subtilis, Streptococcus, bacterial prokaryotic cells such as Staphylococcus, yeast, fungal cells such as Aspergillus, Drosophila S2, insect cells such as Spodoptera Sf9, L cells, CHO cells,
COS cells, NIH3T3 cells, HeLa cells, C12
7 cells, BALB / c3T3 cells (including mutant strains deficient in dihydrofolate reductase, thymidine kinase, etc.), BHK21 cells, HEK293 cells, Bowes
Animal cells such as malignant melanoma cells, plant cells and the like can be mentioned.

In order to recover and purify the protein of the present invention from cell culture, any suitable known method can be used. For example, ammonium sulfate or ethanol precipitation,
Known methods including acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxyapatite chromatography and lectin chromatography,
High performance liquid chromatography is preferably used. Particularly, as a column used for affinity chromatography, for example, a column to which an antibody against the protein / peptide of the present invention is bound, or an ordinary peptide tag added to the protein / peptide of the present invention has an affinity for this peptide tag. The protein / peptide of the present invention can be obtained by using a column to which a substance is bound.

In the present invention, the DNA of the present invention is introduced into an animal nerve cell and expressed therein to prepare an animal nerve cell in which the function of drebrin A is competitively inhibited, and the drebrin is used using the animal nerve cell. It can be used for screening a substance having a preventive or therapeutic effect on a disease caused by the enhancement of A function or inhibition of drebrin A function. Further, the protein of the present invention is introduced into animal nerve cells to produce animal nerve cells that competitively inhibit the function of drebrin A, and the animal nerve cells are used to enhance the function of drebrin A or inhibit the function of drebrin A. It can be used for screening a substance having a preventive or therapeutic effect on the disease that develops. The screening of the substance can be carried out by administering a test substance to the animal nerve cells of the present invention and evaluating / measuring the state of enhancement of drebrin A function.

[0020]

[Examples] The following are drebrin isoforms,
The present invention will be described more specifically with reference to Examples with respect to the identification, structure, function and the like of s-drebrin A of the present invention, but the scope of the present invention is not limited to these exemplifications.

(Identification of Novel Truncated Drebrin Isoform) In order to clone a novel drebrin isoform (s) having an ins2 domain, first, total RNA extracted from the brain and kidney of a mature mouse was expressed in
RT-using F1 and R1 primers surrounding s2
It was analyzed by the PCR method (FIG. 1A). Three bands were obtained from brain RNA, but only one band was obtained from kidney RNA (FIG. 1B). Sequence analysis revealed that the upper band was a novel cDNA fragment, which had an ins2 sequence and an additional 319-bp insert when compared to drebrin A cDNA (FIG. 1B). Next, R2 which is an insertion site-specific primer was prepared (see FIG.
A), 5'portion of the novel cDNA was cloned by RT-PCR using F2 and R2 primers (Fig. 1C). From the sequence of the novel cDNA, it was found that the cDNA encodes a truncated form of drebrin A and further two amino acids. This putative protein has an ADF-H domain, an actin-binding domain, and an ins2 domain, and its calculated molecular weight was 4.2 kDa, which was almost half the molecular weight of drebrin A (7.7 kDa). Since the primary structure of this protein was consistent with the N-terminal half of drebrin A, and its expression pattern was similar to drebrin A (see below), the novel protein was named s-drebrin A.

(Genre structure and splicing mutant of drebrin gene) In order to elucidate the gene structure of the novel drebrin, a genome live prepared from TT2 cells (F1 mouse obtained by mating CBA strain and B57BL / 6 strain mouse) The rally was screened using the rat drebrin cDNA as a probe to obtain 3 types of positive phage clones. The genomic organization of the mouse drebrin gene excluding intron 13 and exon 14 was considered by first sequencing the three phage clones (FIG. 2). In order to examine the comprehensive genomic organization of the mouse drebrin gene, a genomic DNA fragment of TT2 cells was cloned by PCR using F5 and R5 primers (Fig. 2) and its sequence was examined. From such sequence data, the mouse drebrin gene is composed of 14 exons (FIG. 2, upper row), and s-drebrin A is produced by alternative splicing of a precursor mRNA of one drebrin gene (FIG. 2 and Table 1). ) It became clear. s-Drebrin A has exons 11A, B and C, drebrin A has exons A and C but no exon B, and drebrin E has exons C but not exons A and B (Fig. 2). All splicing sites of the mouse drebrin gene conformed to the GT-AG paradigm (Table 1).

[0023]

[Table 1]

(Brain-specific isoform, s-drebrin A) The expression pattern was investigated as a way to understand the functional role of s-drebrin A. First, s-drebrin A-specific transcripts in various adult mouse tissues were detected by RT-PCR using F2 and R2 primers that specifically amplify s-drebrin A mRNA (FIG. 2). As shown in FIG. 3A, drebrin A2 is expressed in mature brain but not in non-neuronal mature tissue. On the other hand, RT using β-actin-specific primer
-From PCR, β-actin was detected in all RNA samples. From these results, it was found that s-drebrin A is a brain-specific isoform. Next, changes in the expression of s-drebrin A in the developing brain were examined. The results of RT-PCR (reverse transcription PCR) using F1 and R1 primers capable of amplifying the three drebrin isoforms (FIG. 1A, 2) showed that the expression profile of s-drebrin A was similar to that of drebrin A. Was found (Fig. 3B, upper panel). s-
Drebrin A and drebrin A are rarely expressed in embryonic brain, but expression was upregulated in the postnatal brain.
On the other hand, drebrin E was highly expressed in the embryonic brain, and postnatal expression was gradually downregulated. The relative amounts of s-drebrin A and drebrin E were reversed between 9 days and 4 weeks after birth (Fig. 3B). S-Drebrin A in the postnatal brain
The increase in mRNA was confirmed by RT-PCR using F2 and R2 primers (FIG. 3B, lower panel). From these results, it is considered that there is a correlation between the expression of s-drebrin A and the maturation of the brain.

(Regulation of actin cytoskeleton of s-drebrin A) In order to elucidate the functional role of s-drebrin A,
-PGFP-SDA encoding GFP fused with drebrin A (GFP-SDA) was transfected into CHO cells, and its actin cytoskeletal structure was changed to GFP or GFP.
-Compare with cytoskeletal structure of CHO cells expressing drebrin A fusion protein (GFP-DA). By highly expressing GFP-SDA in cells,
The actin cytoskeleton composition changed dramatically (Fig. 4, upper panel). Such GFP-SDA activity was similar to GFP-DA activity (FIG. 4, middle panel). On the other hand, when only GFP was overexpressed, the cell shape did not change at all (FIG. 4, lower panel). Next, in order to clarify the association between s-drebrin A and the actin cytoskeleton, the colocalization of s-drebrin A and actin filaments in transfected cells was considered. Most of the actin filaments stained with Rhodamine-Phalloidin are G
Although it was co-localized with FP-SDA and GFP-DA (FIG. 4, upper and middle panels), it was not co-localized with GFP (FIG. 4, lower panel). Similar results were obtained from experiments using COS cells. s-Drebrin A
It was revealed from the above observation results that it has the ability to regulate cell morphogenesis by interacting with the actin cytoskeleton.

(Mapping of Mouse Drebrin Gene (DbnI) to Chromosome 13) Between C57BL / 6JEi and SPRET / Ei strains, useful for determining the chromosomal location of the mouse drebrin gene by backcross analysis. Restriction fragment length polymorphism (RFLP)
Was identified by long-PCR (Genomics 39, 419-4
21, 1997). As shown in FIG. 5A, a long-PCR product containing exons 2 to 8 of the mouse drebrin gene (4.9
As a result of digesting 8 kb) with Pst I, it became clear that RFLP was present in the parent strain. Next, C57BL / 6
F4 and R4 primers were generated that were able to amplify both JEi and SPRET / Ei alleles more efficiently (FIG. 5A). By Pst I digestion of the 510 bp PCR product,
360bp and 207bp from C57BL / 6JEi strain
, And three fragments of 255, 207 and 105 bp were generated from the SPRET / Ei strain, respectively (Fig. 5). After isolation of these polymorphic fragments, Jackson
The genomic DNA of 94 BSS backcross animals was analyzed. The segregation pattern of drebrin RFLP in the backcross was compared to the segregation pattern of another marker already typed in the same backcross. The mouse drebrin gene (DbnI) is located in the central part of chromosome 13 and contains D13Mit13, D13Mit249,
It was found to be co-separated with Il9 and Pitx1 (FIG. 5).

(Evaluation) The present inventors have identified a novel drebrin isoform, s-drebrin A, and have elucidated the genomic organization and chromosomal location of such mouse drebrin gene. In order to elucidate the function of s-drebrin A, its organic and developmental expression patterns are first examined,
Next, its F-actin remodeling activity in fibroblasts was examined. The nucleotide sequence of s-drebrin A cDNA cloned from mature brain by RT-PCR (FIG. 1) was identical to the sequence of drebrin A except for the 319 bp insert within the sequence. Such an insert corresponds to exon 11B located between exons 11A and 11C (FIG. 2). Since the termination codon is contained in frame at nucleotide 7 of the internal sequence insertion portion (see FIG. 2).
Exon 11B), the translation product of s-drebrin A mRNA contained ins2 and the N-terminal part of drebrin A which also contains two amino acids of cysteine and arginine. Assuming that both amino acid sequences are highly conserved (85% concordance), exon 11A of the mouse drebrin gene corresponds to ins2 of chicken drebrin A (Neurosci. Res. Suppl. 13, S106-111, 1990). ; Brai
Brain Res. 19, 101-114, 1993). As a result of examining the sequences of three types of drebrin cDNA and the genomic organization of the mouse drebrin gene, exons 11A, B,
It was found that the insertion and deletion of C and C give rise to an allogeneic mouse drebrin (FIG. 2).

Analyzing the expression pattern of s-drebrin A is an important first step in elucidating its functional role. Since an antibody that specifically recognizes s-drebrin A has not been found yet, RT-PCR was performed to examine the mRNA expression profile of s-drebrin A. Cells expressing s-drebrin A in the brain have not been identified to date, but s-drebrin A
Was found to be a brain-specific isoform, based on the results of our experiments. Based on this finding, the present invention has been completed by focusing on s-drebrin A in the brain. To clarify the difference in the roles of s-drebrin A and two other drebrin isoforms on brain development and function, changes in expression of each drebrin isoform in the developing brain were examined by RT-PCR. Drebrin E expression was transiently dominant in early brain development. On the other hand, s-drebrin A and drebrin A
Was up-regulated during postnatal brain development and was the dominant form in the adult brain. The present inventors have already reported that drebrin A expression is associated with synapse formation (Brain Res. Dev. Brain Res. 111, 137-141, 199).
8), as a result of evaluating the expression level of synaptophysin,
Synaptic vesicle membrane proteins were involved in synapse formation (Cell 41, 1017-1028, 1985; J. Neurochem. 47, 13
02-1304, 1986; Brain Res. 375, 37-48, 1986). s-
Similar expression patterns of drebrin A and drebrin A indicate that s-drebrin A expression is also associated with synapse formation.

Actin filaments are the major cytoskeletal components for neuronal dendrites (Proc. Natl. Ac
ad. Sci. USA79, 7590-7594 ,, 1982; Cell mol. Neu
robiol. 5, 271-284, 1985), the actin cytoskeleton plays an important role in regulating the structure and function of processes. Drebrin A localizes to dendrites, where it binds to actin filaments and functions as a regulator of morphology (J. Neurosci. 19, 3918-3925, 1999). Drebrin has been shown to exhibit actin binding and actin remodeling in fibroblasts (Neur
oreport 3, 109-112, 1992; Exp. Cell Res. 215, 145-
153, 1994; J. Biol. Chem. 269, 29928-29933, 1994;
Exp. Cell Res. 253, 673-680, 1999). To examine whether s-drebrin A can associate with and regulate the actin cytoskeleton, GF in CHO and COS cells
P-SDA was overexpressed. As in the case of GFP-DA, GFP-SDA fluorescence showed co-localization with actin filaments stained with rhodamine-phalloidin, and the cell morphology was changed by remarkably overexpressing GFP-SDA. (Fig. 4). The above results show that the actin-binding domain and the actin remodeling domain coincide with each other and both are localized in the central region of drebrin A (Exp. Cell Res. 2
53, 673-680, 1999). Further, the above results suggest that although half of the C-terminal region of drebrin A is deleted in s-drebrin A, the ability to associate with F-actin and regulate the actin cytoskeleton is still satisfactory. ing.

Various genes such as the erbB family and Homer protein regulate their own functions by expressing conical and short isoforms (Mol. Sell. Biol. 12, 883-893, 199
2; Nature 386, 284-288, 1997). In dendrites, long-form Homer protein is coiled on the C-terminal side
Form homodimers via the -coil (CC) domain. The dimeric Homer protein is a metabotropic glutamate receptor (mGluR) via the N-terminal region of the dimer.
And inositol triphosphate (IP3) receptor are bound to produce IP3 and C from the intracellular pool.
Form a useful signal complex that releases a ²⁺ (Neur
on 21, 717-726, 1998; Neuron 21, 707-716, 1998).
Homer 1a, which is a short form of Homer lacking the CC domain, is a long form of Home
The hypothesis of competing with the er protein and degrading the signal complex has been reported (Curr. Opin. Neurobio
l., 10, 370-374, 2000). s-drebrin A has an actin-binding domain, but a proline-rich domain,
There is no Homer binding domain or SH2 binding domain (FIG. 1D). From the above, it is considered that one of the physiological significances of s-drebrin A expression is that the function of drebrin A, which is associated with the C-terminal region of drebrin A, is negatively regulated by the expression of s-drebrin A. . However, whether or not s-drebrin A is localized in dendrites needs to be considered from now on. It is also important to measure the expression level of s-drebrin in such a structure.

The present inventor has already reported that drebrin is dramatically reduced in AD-affected brain (J. N.
eurosci. Res. 43, 87-92, 1996; J. Neuropathol. Ex
p. Neurol. 58, 637-643, 1999). AD is a complex genetic disease involving several genes, and multiple candidates as genetic risk factors have been mapped to human chromosomes 5, 9 and 10 (Hum. Mol. Genet. 8, 237). -245, 1
999). The human drebrin gene (DbnI) has been mapped on chromosome 5 by spot blot hybridization using a flow-sorted chromosome (Biochem. Biophys. Res. Commu.
n. 196, 468-472, 1993). In view of these data, it was decided to determine the location of the mouse drebrin gene on the chromosome and examine the neighborhood relationship with other mapped mouse genes. Using the Jackson BSS panel, the mouse drebrin gene (DbnI) was identified as D13Mit13, D13.
Co-separated with Mit249, Il9, and Pitx1 and
It was elucidated that it maps to the central part of chromosome 3 (Fig. 5). As a result, useful information was obtained in identifying a candidate gene for AD.

(Materials and methods used in the above experiment) (Primers) The primers used for PCR are shown in FIG.
A, 2, and 5A. The sequence is shown below. F1, 5'-GGCTGCCATCATTGCCCAGCGGCCTG ATAA-3 '(SEQ ID NO: 3 P1) R1, 5'-CAGGGAGGCCTCAGCACCTGAGGGTGGTGT-3' (SEQ ID NO: 4: P2) F2, 5'-ACTCGAGGCATGGCCGGCGTCAGCTTCAGCG -3 '(SEQ ID NO: 5 P3) R2 , 5'-AGGGATCCTTACCCCACCCTGCCGAGGCCT -3 '(SEQ ID NO: 6 P4) F3, 5'-ATCTCCAGGGCTTGCTGCAGGTGAGGTGTG -3' (SEQ ID NO: 7: P5) R3, 5'-GGCACCCAGCACTTGGGATGCAGTGTCAGG -3 '(SEQ ID NO: 8: P6) F3. '-TATTGCACCGCCTGCGCCTTCGGGAGGATG--3' (SEQ ID NO: 9: P7) R4, 5'-CCTGAGCCTGGCGTCCAGAGCCTTCTTCCG (SEQ ID NO: 10: P8) F5, 5'-CCTCAACACTTCAGGCTGAACCCAGGGTGC-3 '(SEQ ID NO: 11: P9) R5, AG5CTAG, 5'- 3 '(SEQ ID NO: 12: P10) actin F, 5'-GGACTCCTATGTGGGTGACGAGGCCCAGAG -3' (SEQ ID NO: 13: P11) actin R, 5'-GGGCCGGACTCATCGTACTCCTGCTTGCTG -3 '(SEQ ID NO: 14: P12)

(RT-PCR and cDNA cloning) Platinum T was used for both cDNA synthesis and PCR amplification.
Superscript One-St by aq System (Gibco BRL)
It was performed in a single tube using ep RT-PCR.
Perform cDNA synthesis at 52 ° C for 30 minutes, denature at 94 ° C for 2 minutes, then at 94 ° C for 20 seconds, 72 ° C (or 70 ° C).
The RT-PCR program was carried out in which the amplification cycle was repeated 35 times, once for 30 seconds at 72 ° C and for 1.5 minutes at 72 ° C. Total RNA (3 μg) extracted from several types of tissues
Drebrin-specific primers F1-R1 or F2
Subjected to RT-PCR analysis using -R2 (Figures 1 and 2). RT amplified using F1 and R1 primers
-The PCR product was digested with Pvu II and then pBluescript
It was inserted into the EcoR V site of the II SK vector (pBSK) (Toyobo). RT amplified using F2 and R2 primers
-The PCR product was inserted into the XhoI-BamHI site of pBSK. R
A mouse β-actin specific primer pair was used as a positive control for T-PCR.

(Structural analysis of drebrin gene) EMBL3
As a probe for screening a genomic DNA library of TT2 (mouse embryonic stem cell line) inserted into a vector (Anal. Biochem. 214, 70-76, 1993), rat drebrin cDNA (Neuroreport 3, 109-112, 19
92) was used. Of the 10 ⁶ independent phage plaques analyzed, 3 independent clones were positive. After restriction mapping, such positive clones were subcloned into pBSK. The PCR product containing the region from exon 13 to exon 14 of the drebrin gene (FIG. 2)
Was amplified from TT2 cell genomic DNA and inserted into pBSK. ABI PRISM 377 DNA Sequencer (PE Applied Bios
ABI PRISM Dye Terminator Cycle
Using the Sequencing Ready Reaction kit (Perkin-Elmer), sequencing of all drebrin genome fragments was carried out bidirectionally and completely. GENETYX software (Software
Development) was used to analyze the sequence data.

(Expression vector) pEGFP-C1 vector (Clon
(manufactured by tech Co., Ltd.), a mouse cDNA encoding full-length s-drebrin A is inserted in-frame into the Xho I-BamH I site to express GFP-tag labeled s-drebrin A.
The pGFP-SDA vector was constructed. The GFP-DA expression vector has already been prepared by the present inventors (J. Neuro
sci. 19, 3918-3925, 1999).

(Cell culture and cytochemistry) Ham's F12 medium supplemented with 10% heat-inactivated fetal bovine serum (FBS) and 1
In each of DMEM medium supplemented with 0% FBS,
CHO and COS cells were retained. 24 of these cells
-Transfer to the well plate and use TransFast transfection
The expression vector was transfected with eagent (Promega). Two days later, such transfected cells were fixed with 4% paraformaldehyde in phosphate buffered saline (PBS) and 0.1% Triton X-100.
After treatment with, the cells were blocked in PBS containing 3% bovine serum albumin (BSA) for 30 minutes and incubated in the presence of rhodamine-labeled phalloidin (Molecular Probes) to label actin filaments. Fluorescein isothiocyanate (FITC) was added to GFP or rhodamine.
Or it observed using the Rhodamine filter set.

(Chromosome mapping) The Jackson Laboratory interspecific backcross written as Jackson BSS
panel (C57BL / 6JEi X SPRET / Ei) F ₁ X SPRET / Ei (Mam
m. Genome 5, 253-274, 1994) was used to determine the chromosomal location of the mouse drebrin gene. C57BL / 6
4.9 for both JEi and SPRET / Ei genomic DNA
Long-PCR for amplifying 8 kb fragment (3 minutes at 95 ° C,
Next, 95 ° C for 35 seconds, 69.5 ° C for 30 seconds, 72 ° C for 5 seconds.
Min, and 30 cycles of 10 minutes at 72 ° C.), the drebrin-specific primers F3 and R3 were used (FIG. 5). This long-PCR product was digested with various restriction enzymes such as Pst I to give C57BL / 6J.
We have found a restriction fragment length polymorphism (RFLP) that exists between the Ei and SPRET / Ei drebrin alleles. Ja
ckson BSS genomic DNA of 94 backcross animals
PCR analysis using F4 and R4 primers (95
3 minutes at ℃, then 35 seconds at 95 ℃, 20 seconds at 71.5 ℃,
30 cycles of 72 ° C for 30 seconds and 72 ° C for 2 minutes
(Fig. 5). The above PCR product (567 bpD
NA) was digested with Pst I and separated by 2% agarose gel electrophoresis. The Jackson Laboratory Mapping
Send the separation pattern of drebrin RFLP to Resource,
It was compared with the segregation pattern of markers that were typed in the same back cloth so far.

(Description of Figures) FIG. (A) Drebrin isoforms and a schematic diagram showing an RT-PCR primer set used for identifying s-drebrin A. The shaded box indicates the s-drebrin A-specific exon. i2 represents the drebrin A-specific exon ins2. Forward (F
1 and F2) primer and reverse (R1 and R2)
2) Primers are indicated by arrowheads. SDA: s-drebrin A, DA: drebrin A, DE: drebrin E. (B) It is a figure showing a state of detecting drebrin isoforms by RT-PCR using F1 and R1 primers. RT-PCR was performed on the total RNA extracted from the brain (B / 11W) and the kidney (K / 11W) of 11-week-old mice.
I went to the analysis. cDs obtained by amplifying mRNAs of s-drebrin A, drebrin A, and drebrin E, respectively
The NA fragments were designated SDA, DA, and DE, respectively. (C) is a diagram showing that s-drebrin A is amplified by RT-PCR using F2 and R2 primers. S-Drebrin A amplified from the brain of 11-week-old mice
The cDNA fragment was designated as SDA. (D) Schematic diagram of the domain structure of mouse drebrin isoforms. ADF-H: actin depolymerizing factor homology domain, AB: actin binding domain, i2:
ins2 domain, P: proline rich domain, H: Ho
mer binding domain, S: src homology 2-binding domain. The nucleotide sequence of mouse s-drebrin A cDNA is registered in DDBJ / EMBL / GenBank databases as Accession No. AB064321.

FIG. 2. It is a figure which shows the genomic organization of mouse drebrin gene, and the coding region of three kinds of drebrin isoforms. A schematic diagram of the exon and intron structures of the mouse drebrin locus is shown at the top and the coding regions for the three drebrin isoforms are shown below. Exons corresponding to the open reading frame are represented by black boxes, and exons corresponding to the untranslated region are represented by white boxes. Exon 11A,
B and C are contained in s-drebrin A mRNA. The in-frame termination codon is located at nucleotide 7 of exon 11-B. The nucleotide sequence of mouse drebrin gene is DDBJ / EMBL / GenBank database.
s is registered as accession No. AB041033.

FIG. s-Drebrin A is a brain-specific isoform whose expression in the brain is upregulated postnatally. (A) Total RNA extracted from a predetermined tissue of a mature mouse
Was subjected to RT-PCR analysis using F2 and R2 primers. A PCR product amplified from s-drebrin A mRNA was detected in the brain but not in non-neural tissue (upper panel). Actin cDNA was amplified by RT-PCR using an actin-specific primer, and used as a positive control for RT-PCR (lower panel). (B) Total RNA extracted from the brain (B) and kidney (K) of mouse 17-day embryos (E17), 9-day-old (P9) and 4-week-old (4W) mice, respectively, was F1-R1 (upper panel). , And F2-R2 primer (lower panel). The PCR-amplified cDNAs prepared from the mRNAs of s-drebrin A, drebrin A, and drebrin E were designated as SDA, DA, and DE, respectively.

FIG. It is a figure which shows the association property of GFP-SDA and an actin cytoskeleton by overexpressing GFP-SDA, and the morphological change of a fibroblast. Expression vectors pGFP-SDA (a, b and c), pGFP-DA (d, e)
And f), or CHO cells transfected with pEGFP-C1 (g, h and i) were stained with rhodamine-phalloidin to label actin filaments. Such G
The FP tag-labeled protein and actin filament were labeled with FITC (a, d and g), and rhodamine (b,
Visualization was performed using each filter set of e and h). Superimpose the images of GFP and Rhodamine
Co-localization of P-SDA and actin filaments was observed (c, f and i).

FIG. Mouse drebrin gene (Dbn
It is a figure which shows a mode that gene mapping of I) was carried out on the 13th chromosome. (A) Restriction fragment length polymorphism (RF) present between C57BL / 6JEi and SPRET / Ei drebrin alleles
It is a figure which shows LP. Long-PCR product containing exons 2-8 of mouse drebrin gene (4.98 kb)
Was digested with Pst I to identify RFLPs. PCR amplification product (567b) obtained from 94 Jackson BSS backcross animals by the above Pst I-RFLP.
p) was analyzed and its genotype was determined. (B) is a diagram showing a haplotype of Jackson BSS back cross showing a part of a locus linked to DbnI in chromosome 13. Each locus is listed in order from the gene closest to the centromere. The black box represents the C57BL / 6JEi allele,
White boxes represent SPRET / Ei alleles. The bottom number is the number of offspring inheriting each genotype. The recombination rate (R) occurring between loci having a close relationship is shown on the right side of the figure, and the standard error (SE) for each R is also shown.

(C) is a linkage map of mouse chromosome 13 constructed from the halotype distributions in (B). In this linkage map, the centromere is on top. The A3cM scale bar is shown on the right side of the figure. The loci that map to the same position are shown in alphabetical order. The drebrin gene (DbnI) is a mouse 13
Mapped to the central part of the chromosome. The symbol for the mouse drebrin locus, DbnI, is Mouse Nomencla
It has been approved by the ture committee.

[0044]

INDUSTRIAL APPLICABILITY In the present invention, the protein drebrin A which is localized in the dendrites of nerve cells, binds to actin filaments there, and functions as a regulator of protrusion morphology
Isoforms, s-drebrin A, and DNA encoding the same, and animal nerve cells that competitively inhibit the function of drebrin A using such proteins and DNA, and further the function of drebrin A using the animal nerve cells The present invention provides a method for screening a substance having a preventive or therapeutic effect on a disease caused by the enhancement of erythrocyte or inhibition of drebrin A function. By using the gene or protein of the present invention, it becomes possible to regulate nerve synapse function,
It can be applied to the treatment of nerve diseases and nerve damage. In addition, the protein of the present invention, s-drebrin A, is drebrin A.
Competitively inhibits the function of. Functional suppression and enhancement of drebrin A protein are closely related to maturation of nerve cells / circuits and synaptic plasticity. Therefore, the function inhibitor of drebrin A is useful in developing a diagnostic agent or therapeutic agent for brain diseases.

[0045]

[Sequence list]                                SEQUENCE LISTING <110> JAPAN SCIENCE AND TECNOLOGY CORPORATION <120> S-DrebrinA <130> A091P24 <140> <141> <160> 14 <170> PatentIn Ver. 2.1 <210> 1 <211> 2771 <212> DNA <213> Mus musculus <220> <221> CDS <222> (119) .. (1222) <400> 1 ctctcagcgc tgcagaggct ctgaggcggc ggcggcgact ccctcatccc ctccctccgg 60 tgtcggtcgg tccgcctgtg cgtgcgtctg tccgttcggc ctcggtccgg cccgcagc 118 atg gcc ggc gtc agc ttc agc ggc cac cgc ctg gag ctg ctg gcg gcg 166 Met Ala Gly Val Ser Phe Ser Gly His Arg Leu Glu Leu Leu Ala Ala   1 5 10 15 tac gag gag gtg atc cgg gag gag agc gca gcc gac tgg gct ctg tac 214 Tyr Glu Glu Val Ile Arg Glu Glu Ser Ala Ala Asp Trp Ala Leu Tyr              20 25 30 aca tac gag gat ggc tca gat gac ctc aag ctt gca gcg tca gga gaa 262 Thr Tyr Glu Asp Gly Ser Asp Asp Leu Lys Leu Ala Ala Ser Gly Glu          35 40 45 ggg ggc ttg cag gag ctt tcc ggc cac ttc gag aac cag aaa gtg atg 310 Gly Gly Leu Gln Glu Leu Ser Gly His Phe Glu Asn Gln Lys Val Met      50 55 60 tat ggt ttc tgc agc gtc aag gac tcc caa gct gcc ctg cca aca tat 358 Tyr Gly Phe Cys Ser Val Lys Asp Ser Gln Ala Ala Leu Pro Thr Tyr  65 70 75 80 gtg ctc atc aac tgg gtt ggt gag gat gtg cct gat gcc cga aaa tgt 406 Val Leu Ile Asn Trp Val Gly Glu Asp Val Pro Asp Ala Arg Lys Cys                  85 90 95 gct tgc gcc agt cat gtg gcc aag gtg gct gaa ttc ttc cag ggt gtt 454 Ala Cys Ala Ser His Val Ala Lys Val Ala Glu Phe Phe Gln Gly Val             100 105 110 gat gtc att gtg aat gcc agc agt gtg gaa gac atc gat gct ggt gcc 502 Asp Val Ile Val Asn Ala Ser Ser Val Glu Asp Ile Asp Ala Gly Ala         115 120 125 att ggg cag cgg ctc tcc aat gga ctg gca cgg ctc tcc agc cca gta 550 Ile Gly Gln Arg Leu Ser Asn Gly Leu Ala Arg Leu Ser Ser Pro Val     130 135 140 ttg cac cgc ctg cgc ctt cgg gag gat gaa aat gct gaa ccg gtg ggt 598 Leu His Arg Leu Arg Leu Arg Glu Asp Glu Asn Ala Glu Pro Val Gly 145 150 155 160 acc acc tac cag aag acg gat gca gca gtg gag atg aag cgg att aac 646 Thr Thr Tyr Gln Lys Thr Asp Ala Ala Val Glu Met Lys Arg Ile Asn                 165 170 175 cgt gag cag ttt tgg gag cag gcc aag aag gag gaa gag ctg cgg aag 694 Arg Glu Gln Phe Trp Glu Gln Ala Lys Lys Glu Glu Glu Leu Arg Lys             180 185 190 gag gag gag cgg aag aag gct ctg gac gcc agg ctc agg ttt gaa cag 742 Glu Glu Glu Arg Lys Lys Ala Leu Asp Ala Arg Leu Arg Phe Glu Gln         195 200 205 gaa cgg atg gag cag gag cgg cag gag cag gaa gaa cgt gag cgg cgc 790 Glu Arg Met Glu Gln Glu Arg Gln Glu Gln Glu Glu Arg Glu Arg Arg     210 215 220 tac cgg gag cgg gag cag cag att gag gag cac agg agg aaa cag cag 838 Tyr Arg Glu Arg Glu Gln Gln Ile Glu Glu His Arg Arg Lys Gln Gln 225 230 235 240 agt ctg gaa gct gaa gaa gcc aag agg agg tta aag gag cag tct atc 886 Ser Leu Glu Ala Glu Glu Ala Lys Arg Arg Leu Lys Glu Gln Ser Ile                 245 250 255 ttt ggt gac cag cgg gat gaa gag gaa gag tcc cag atg aag aag tcg 934 Phe Gly Asp Gln Arg Asp Glu Glu Glu Glu Ser Gln Met Lys Lys Ser             260 265 270 gag tca gag gtg gag gag gcg gct gcc atc att gcc cag cgg cct gat 982 Glu Ser Glu Val Glu Glu Ala Ala Ala Ile Ile Ala Gln Arg Pro Asp         275 280 285 aac cca cgg gag ttc ttc aga cag cag gaa cga gtg gca tcg gcc tct 1030 Asn Pro Arg Glu Phe Phe Arg Gln Gln Glu Arg Val Ala Ser Ala Ser     290 295 300 ggt ggc agc tgt gac gcg cct gcg cct gca ccc ttc aac cac cga cca 1078 Gly Gly Ser Cys Asp Ala Pro Ala Pro Ala Pro Phe Asn His Arg Pro 305 310 315 320 ggt cgt ccg tac tgc cct ttc ata aag gca tcg gac agt ggg cct tcc 1126 Gly Arg Pro Tyr Cys Pro Phe Ile Lys Ala Ser Asp Ser Gly Pro Ser                 325 330 335 tcc tcc tcc tct tcc tcc tct tcc cct cca cgg act ccc ttt ccc tat 1174 Ser Ser Ser Ser Ser Ser Ser Ser Pro Pro Arg Thr Pro Phe Pro Tyr             340 345 350 atc acc tgc cac cgc acc cca aac ctc tct tcc tcc ctc cca tgt agg 1222 Ile Thr Cys His Arg Thr Pro Asn Leu Ser Ser Ser Leu Pro Cys Arg         355 360 365 tagcagcccc aggcctcggc agggtggggt aaggagggcc ccgcttccca gcagccccct 1282 cgccctttct cccccagcct gactgccgac tgctggtgtt tgtccccaga gcaggtacta 1342 cctgcctctt gtccctggcc ctgtagggca ctggccccca gcggcctggt tcacagggtc 1402 tggagcagca atctggcccc tggggggcct ggggagctca cagcccccac ccacacttga 1462 gacacaaggt ggatagtcct gattccacct ggctcccact tcctcactca ctagcctggg 1522 catttgctcc gcaggcagcc acctggacag ccaccggagg atggcaccca ctcctattcc 1582 cacccggagc ccatctgatt ccagcacagc ctctaccccc atcgctgagc agatcgagag 1642 ggccctggat gaggtcacat cctcgcagcc tccacctcca cctccaccac ctccaccaac 1702 tcaagaggcc caggagacta ccccaagcct ggatgaagag ctcagcaagg aggccaaagt 1762 aacagcagct cctgaggtct gggctggctg tgcggcagag ccccctcagg cacaggaacc 1822 tcccctgttg caaagcagcc ccctggagga ctcgatgtgc acagaatctc cagagcaggc 1882 tgccctggct gcccctgcgg agcctgctgc ctctgtcacc tcagtagctg atgtccatgc 1942 agctgacacc attgagacca ccactgccac tactgacacc actattgcca acaacgtcac 2002 ccctgccgct gccagcctca ttgatctatg gcctggcaac ggggaagagg cctcaacact 2062 tcaggctgaa cccagggtgc ccacaccacc ctcaggtgct gaggcctccc tggcagaggt 2122 gcccctgctg aatgaggccg ctcaggagcc gctgccgccg gtaggcgaag gctgtgctaa 2182 ccttcttaat tttgatgagc tgccagaacc tccagccacc ttctgtgacc cagaggagga 2242 agtaggagaa acgctggctg cctcccaggt cctaactatg ccctcagctc tagaggaggt 2302 agatcaggtg ctggagcagg agctggagcc agaacctcac ctgctgacca atggagagac 2362 cactcaaaag gaggggaccc aggccagcga aggatacttc agtcagtcac aggaggaaga 2422 gttcgcccaa tcagaagagc catgtgcaaa ggttccgcct cctgtatttt acaacaagcc 2482 tccagaaatc gacatcacct gctgggatgc agacccagtt cctgaagagg aagagggctt 2542 cgagggtggt gattagtagc ggcgactgcc ccctggctgc cctcgccaag gctgcctacc 2602 tgcagtggcc tctggccagc cggcttgcag tgccagcatt agcagcagcc ccgcctggct 2662 cccactctgg attccggcac tggccgggga cctgtctgct tccttaccca cagggcctga 2722 cttttacagc ttttctcttc tttttaaaag ttgatagact tgtaaaaaa 2771 <210> 2 <211> 368 <212> PRT <213> Mus musculus <400> 2 Met Ala Gly Val Ser Phe Ser Gly His Arg Leu Glu Leu Leu Ala Ala   1 5 10 15 Tyr Glu Glu Val Ile Arg Glu Glu Ser Ala Ala Asp Trp Ala Leu Tyr              20 25 30 Thr Tyr Glu Asp Gly Ser Asp Asp Leu Lys Leu Ala Ala Ser Gly Glu          35 40 45 Gly Gly Leu Gln Glu Leu Ser Gly His Phe Glu Asn Gln Lys Val Met      50 55 60 Tyr Gly Phe Cys Ser Val Lys Asp Ser Gln Ala Ala Leu Pro Thr Tyr  65 70 75 80 Val Leu Ile Asn Trp Val Gly Glu Asp Val Pro Asp Ala Arg Lys Cys                  85 90 95 Ala Cys Ala Ser His Val Ala Lys Val Ala Glu Phe Phe Gln Gly Val             100 105 110 Asp Val Ile Val Asn Ala Ser Ser Val Glu Asp Ile Asp Ala Gly Ala         115 120 125 Ile Gly Gln Arg Leu Ser Asn Gly Leu Ala Arg Leu Ser Ser Pro Val     130 135 140 Leu His Arg Leu Arg Leu Arg Glu Asp Glu Asn Ala Glu Pro Val Gly 145 150 155 160 Thr Thr Tyr Gln Lys Thr Asp Ala Ala Val Glu Met Lys Arg Ile Asn                 165 170 175 Arg Glu Gln Phe Trp Glu Gln Ala Lys Lys Glu Glu Glu Leu Arg Lys             180 185 190 Glu Glu Glu Arg Lys Lys Ala Leu Asp Ala Arg Leu Arg Phe Glu Gln         195 200 205 Glu Arg Met Glu Gln Glu Arg Gln Glu Gln Glu Glu Arg Glu Arg Arg     210 215 220 Tyr Arg Glu Arg Glu Gln Gln Ile Glu Glu His Arg Arg Lys Gln Gln 225 230 235 240 Ser Leu Glu Ala Glu Glu Ala Lys Arg Arg Leu Lys Glu Gln Ser Ile                 245 250 255 Phe Gly Asp Gln Arg Asp Glu Glu Glu Glu Ser Gln Met Lys Lys Ser             260 265 270 Glu Ser Glu Val Glu Glu Ala Ala Ala Ile Ile Ala Gln Arg Pro Asp         275 280 285 Asn Pro Arg Glu Phe Phe Arg Gln Gln Glu Arg Val Ala Ser Ala Ser     290 295 300 Gly Gly Ser Cys Asp Ala Pro Ala Pro Ala Pro Phe Asn His Arg Pro 305 310 315 320 Gly Arg Pro Tyr Cys Pro Phe Ile Lys Ala Ser Asp Ser Gly Pro Ser                 325 330 335 Ser Ser Ser Ser Ser Ser Ser Ser Pro Pro Arg Thr Pro Phe Pro Tyr             340 345 350 Ile Thr Cys His Arg Thr Pro Asn Leu Ser Ser Ser Leu Pro Cys Arg         355 360 365 <210> 3 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: P1 <400> 3 ggctgccatc attgcccagc ggcctgataa 30 <210> 4 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: P2 <400> 4 cagggaggcc tcagcacctg agggtggtgt 30 <210> 5 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: P3 <400> 5 actcgaggca tggccggcgt cagcttcagc g 31 <210> 6 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: P4 <400> 6 agggatcctt accccaccct gccgaggcct 30 <210> 7 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: P5 <400> 7 atctccaggg cttgctgcag gtgaggtgtg 30 <210> 8 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: P6 <400> 8 ggcacccagc acttgggatg cagtgtcagg 30 <210> 9 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: P7 <400> 9 tattgcaccg cctgcgcctt cgggaggatg 30 <210> 10 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: P8 <400> 10 cctgagcctg gcgtccagag ccttcttccg 30 <210> 11 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: P9 <400> 11 cctcaacact tcaggctgaa cccagggtgc 30 <210> 12 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: P10 <400> 12 ctcgagaaaa gctgtaaaag tcaggccctg 30 <210> 13 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: P11 <400> 13 ggactcctat gtgggtgacg aggcccagag 30 <210> 14 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: P12 <400> 14 gggccggact catcgtactc ctgcttgctg 30

[Brief description of drawings]

1] FIG. 1 of the present invention, (A) is a schematic diagram showing drebrin isoforms and an RT-PCR primer set used for identifying s-drebrin A, and (B) is F1. And RT-using R1 primer
The figure which shows the state which detects drebrin isoforms by PCR, (C) is a figure showing the mode that s-drebrin A is amplified by RT-PCR using F2 and R2 primers, (D) is a mouse drebrin It is a figure which shows the schematic of the domain structure of isoforms.

FIG. 2 shows the genomic organization of the mouse drebrin gene and the coding regions of three drebrin isoforms.

FIG. 3: Total RNA extracted from selected tissues of adult mice
FIG. 3 shows the results of RT-PCR analysis using F2 and R2 primers.

FIG. 4 G by overexpressing GFP-SDA
It is a figure which shows the association property of FP-SDA and an actin cytoskeleton, and the morphological change of a fibroblast.

FIG. 5: Mouse drebrin gene (DbnI)
It is a figure which shows a mode that gene mapping was carried out on the 13th chromosome, (A) is C57BL / 6JEi and SPRE.
It is a figure which shows the restriction fragment length polymorphism (RFLP) which exists between T / Ei drebrin alleles, (B) is Jackson BSS which shows the part with the locus linked to DbnI among 13 chromosomes. Back cloth haplotype,
(C) is a diagram showing a linkage map of mouse chromosome 13 constructed from the haplotype distribution in (B).

Front page continuation (51) Int.Cl. ⁷ identification code FI theme code (reference) C07K 16/18 C12N 1/15 4H045 C12N 1/15 1/19 1/19 C12Q 1/02 5/10 G01N 33/15 Z C12Q 1/02 33/50 Z G01N 33/15 C12N 15/00 ZNAA 33/50 5/00 A BF Term (reference) 2G045 AA40 BA13 BB03 BB20 CB01 CB21 DA12 DA13 DA14 DA36 FB03 4B024 AA11 BA43 BA80 CA04 DA06 EA04 GA11 HA12 HA15 4B063 QA18 QR59 QR69 QR80 QS24 QS28 4B065 AA91Y AB01 AC14 CA24 CA46 4C084 AA17 ZA012 ZB212 4H045 AA11 BA10 CA40 DA76 DA86 EA50 FA72 FA74

Claims (17)

[Claims] 1. A DNA encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 2. 2. The amino acid sequence represented by SEQ ID NO: 2 is composed of an amino acid sequence in which one or several amino acids are deleted, substituted or added, and has an actin cytoskeleton regulatory function, a proline rich domain, and Home.
A DNA encoding a protein that has a loss of function due to deletion of r-binding domain and SH2-binding domain. 3. A DNA comprising the nucleotide sequence shown in SEQ ID NO: 1 to 119 to 1222, its complementary sequence, or a sequence containing a part or all of these sequences. 4. A DNA constituting the gene according to claim 3.
D that hybridizes with a stringent condition and encodes a protein having an s-drebrin function
NA. 5. A protein comprising the amino acid sequence shown in SEQ ID NO: 2. 6. A protein consisting of the amino acid sequence shown in SEQ ID NO: 2 in which one or several amino acids are deleted, substituted or added, and having a s-drebrin function. 7. An antibody that specifically binds to the protein according to claim 5 or 6. 8. The antibody according to claim 7, wherein the antibody is a monoclonal antibody or a polyclonal antibody. 9. An expression vector incorporating a DNA comprising the sequence according to any one of claims 1 to 4. 10. A host cell comprising the expression vector of claim 9. 11. A DNA comprising the sequence according to any one of claims 1 to 4 is introduced into an animal nerve cell and expressed therein,
An animal neuron characterized by competitively inhibiting the function of drebrin A. 12. An animal nerve cell, which comprises introducing the protein according to claim 5 or 6 into an animal nerve cell and competitively inhibiting the function of drebrin A. 13. An animal nerve cell, wherein the competitive inhibition of the function of drebrin A according to claim 11 or 12 is inhibition of signal transduction from a neurotransmitter receptor to an actin filament. 14. The animal nerve cell according to claim 11, wherein the animal nerve cell is a mouse brain cell. 15. Enhancement of the function of drebrin A, which comprises administering a test substance to the animal nerve cells according to claim 11 and evaluating / measuring the state of enhancement of the function of drebrin A. Alternatively, a method for screening a substance having a preventive or therapeutic effect on a disease caused by inhibition of drebrin A function. 16. A method for screening a substance having a preventive or therapeutic effect on a disease caused by drebrin A function inhibition, wherein the disease caused by drebrin A function inhibition is Alzheimer's disease (AD). 17. A substance having a preventive or therapeutic effect on a disease caused by the enhanced function of drebrin A or the inhibition of drebrin A function, which is obtained by the screening method according to claim 15 or 16.