WO2004046352A1

WO2004046352A1 - MUTANT ESTROGEN RECEPTOR-α AND GENE THEREOF

Info

Publication number: WO2004046352A1
Application number: PCT/JP2003/014494
Authority: WO
Inventors: Ko Fujimori
Original assignee: Sumitomo Chemical Company, Limited
Priority date: 2002-11-15
Filing date: 2003-11-14
Publication date: 2004-06-03
Also published as: AU2003280793A1

Abstract

It is intended to provide a mutant estrogen receptor-α having the following characteristics, its gene, and so on: (a) in an alignment based on the amino acid sequence homology in the amino acids constituting the receptor, one or more amino acids at the positions corresponding to the amino acids represented by the amino acid numbers 404, 405 and 424 in the amino acid sequence represented by SEQ ID NO:1 being substituted into amino acid(s) different from the corresponding amino acid(s) in the amino acid sequence of the wild type estrogen receptor-α, and the amino acid substitution(s) imparting the following characteristics (b) and (c) to the receptor; (b) upon contact with an anti-estrogen substance which inhibits the function of the transcriptional activation region AF2 of the wild type estrogen receptor-α but not inhibiting the function of the transcriptional activation region AF1 thereof, being capable of activating the transcription of a gene under the transcriptional regulation by the transcriptional regulation region containing an estrogen response sequence; and (c) upon contact with estrogen, being capable of activating the transcription of a gene under the transcriptional regulation by the transcriptional regulation region containing an estrogen response sequence and the activation not being substantially inhibited by a compound capable of activating the transcription of the gene in the above (b).

Description

Specification

Mutant estrogen receptor α and its gene

The present invention relates to a mutant estrogen receptor α and its gene. Background art

Various physiological effects of estrogen are triggered through estrogen receptors. Estrogen receptors are present on estrogen target cells in tissues such as the uterus, vagina, oviduct, liver, breast cancer and bone, and are ligand-responsive transcription factors. The estrogen receptor is activated when bound to estrogen, binds to an estrogen response element on the chromosome, and regulates the transcription of a target gene downstream of the response element. It is known that such abnormalities in estrogen and receptor activation may cause various diseases, and substances that act on estrogen receptors and exhibit antiestrogenic activity (hereinafter referred to as antiestrogenic substances) Attempts have been made to use) as a remedy for such diseases. For example, tamoxifen, an antiestrogen, has been used as a treatment for breast cancer.

However, if one of the amino acids constituting the estrogen receptor is substituted and the reactivity to the antiestrogenic substance is different from the usual, the expected effect in the treatment using the antiestrogenic substance as described above will be obtained. It is feared that there is a possibility that it will not go up or may cause undesired symptoms. Therefore, if amino acid substitutions related to the response to antiestrogenic substances become clear, it will be possible to determine the effectiveness of treatment such as administration of antiestrogenic substances before starting treatment by examining the presence or absence of such amino acid substitutions. Become. In addition, it is possible to construct a test system for searching for a substance exhibiting a desired effect even for an estrogen receptor having a powerful amino acid substitution. Disclosure of the invention

Under such circumstances, the present inventors have conducted intensive studies and found that the amino acid at a specific position is The wild-type estrogen receptor, which has been substituted with an amino acid different from the amino acid of type estrogen receptor _α , has different reactivity to certain antiestrogenic substances, and DNA encoding the receptor. Thus, the present invention has been achieved.

That is, the present invention

1. Estrogen receptor having the following properties (hereinafter sometimes referred to as the receptor of the present invention):

(a) Among the amino acids constituting the receptor, in the alignment based on the homology of the amino acid sequence, the amino acid represented by amino acid number 404, 405 or 424 of the amino acid sequence represented by SEQ ID NO: 1 One or more of the amino acids at positions corresponding to the amino acid sequence of the wild-type estrogen receptor c has been replaced with an amino acid different from the amino acid at the corresponding position in the amino acid sequence of the wild-type estrogen receptor c. Imparting the properties of (b) and (c) to the receptor;

(b) Transcriptional activation region of wild-type estrogen receptor α Transcriptional activation region that contains an estrogen response element when contacted with any of the antiestrogenic substances that suppresses the function of AF2 but does not suppress the function of AF1 Can activate the transcription of a gene that is under the transcriptional control of

( _c ) contacting with an estrogen can activate the transcription of a gene under the transcriptional control of a transcription control region containing an estrogen response element, and the activation activates the transcription of the gene in the above (b). Not substantially inhibited by compounds that can

2. The estrogen receptor _{α according to the} above item 1, wherein in (b) and (c), the gene under the transcriptional control of a transcription control region containing an estrogen response element is a gene introduced into a chromosome of a cell;

3. The estrogen receptor α according to the above 1, wherein the activation in (c) is also an activation inhibited by a pure antiestrogen;

4. The estrogen receptor α according to 1 above, wherein the antiestrogenic substance according to (b) is tamoxifen, 4-hydroxytamoxifen or raloxifene;

5. In an alignment based on amino acid sequence homology, represented by SEQ ID NO: 1. The amino acid at the position corresponding to the amino acid represented by amino acid No. 390 and the amino acid at the position corresponding to the amino acid represented by amino acid No. 578 of the amino acid sequence, and the amino acid at the position corresponding to the amino acid sequence of the wild-type estrogen receptor The estrogen receptor-α according to the above 1, which is the same as a certain amino acid;

6. In an alignment based on the homology of amino acid sequences, this corresponds to the amino acid represented by amino acid number 303, 309, 390, 396, 396, 415, 494, 531, or 578 of the amino acid sequence represented by SEQ ID NO: 1. The estrogen receptor c according to item 1, wherein any of the amino acids at the corresponding positions is the same as the amino acid at the corresponding position in the amino acid sequence of the wild-type estrogen receptor α;

7. Amino acid at the position corresponding to the amino acid sequence of wild-type estrogen receptor α At the position corresponding to the amino acid represented by amino acid number 404, it is phenylalanine and at the position corresponding to the amino acid represented by amino acid number 405 Is estrogen receptor α according to item 1, wherein alanine is isoleucine at a position corresponding to the amino acid represented by amino acid number 424;

8. The amino acid that differs from the amino acid at the corresponding position in the amino acid sequence of wild-type estrogen receptor α is leucine at the position corresponding to the amino acid represented by amino acid number 404, and the amino acid represented by amino acid number 405 The estrogen receptor α according to the above item 1, which is valine at a position corresponding to the amino acid and threonine at a position corresponding to the amino acid represented by amino acid number 424;

9. The estrogen receptor α according to item 1, wherein the estrogen receptor ct is an estrogen receptor α derived from a mammal;

10. An estrogen receptor having an amino acid sequence represented by SEQ ID NO: 2 (hereinafter sometimes referred to as receptor F404L of the present invention);

1 1. An estrogen receptor having the amino acid sequence represented by SEQ ID NO: 28 (hereinafter sometimes referred to as receptor A405V of the present invention);

1 2. An estrogen receptor having the amino acid sequence represented by SEQ ID NO: 33 (hereinafter sometimes referred to as the receptor {424} of the present invention);

1 3. The isolated DNA encoding estrogen receptor α described in 1 above (hereinafter referred to as Below, it may be described as the present invention DNA. );

14. An isolated DNA encoding an estrogen receptor having the amino acid sequence set forth in SEQ ID NO: 2;

1 5. An isolated DNA encoding an estrogen receptor having the amino acid sequence set forth in SEQ ID NO: 28;

1 6. An isolated DNA encoding an estrogen receptor having the amino acid sequence of SEQ ID NO: 33;

1 7. The DNA according to 13 above, wherein the DNA is cDNA;

1 8. The DNA according to the above 13 which is operably linked with a promoter; 1 9. The vector containing the DNA according to the above 13 (hereinafter sometimes referred to as the vector of the present invention). ;

20. A method for producing a vector, which comprises incorporating the DNA described in the item 13 into a vector capable of replicating in a host cell;

21. A transformant obtained by introducing the DNA according to 13 above into a host cell;

22. The transformant according to the above item 21, wherein the host cell is an animal cell;

23. The transformant according to the above item 21, wherein the host cell is a mammalian cell;

24. A method for producing a transformant, which comprises introducing the DNA according to item 13 into a host cell;

25. A method for producing an estrogen receptor, which comprises culturing the transformant according to item 21 to produce estrogen receptor α;

26. (1) contacting the test substance with the estrogen receptor 1α according to the preceding item 1, to which the labeled ligand is bound; and

(2) The state of binding between the estrogen receptor α and the test substance is monitored by monitoring the amount of free labeled ligand or bound labeled ligand generated by competition between the labeled ligand and the test substance. A ligand / receptor binding assay, comprising a step of indirectly confirming the activity by performing

27. A method for evaluating the ability of a test substance to regulate estrogen receptor α activity, comprising the steps of: (1) producing the estrogen receptor α according to item 1 above; Contacting a test substance with a transformant in which a reporter gene under the transcriptional control of a transcription control region containing a sequence is introduced into a chromosome;

(2) measuring the expression level of the reporter gene contained in the transformant or an index value having a correlation with the expression level; and

(3) a step of evaluating the estrogen receptor activity regulating ability of the substance based on the measured expression level or an index value having a correlation with the level.

28. The activity of a test substance for estrogen receptor a produced by a transformant according to the method described in 27 above; A method of screening for an estrogen receptor α activity modulator, which comprises the step of evaluating a regulatory ability;

29. In the nucleic acid in the sample, the amino acids constituting estrogen receptor α, and the amino acid sequence of amino acid sequence represented by SEQ ID NO: 1 in the alignment based on the homology of the amino acid sequence. Has the nucleotide sequence encoding the amino acid at the position corresponding to the amino acid shown in 24 been replaced with a nucleotide sequence encoding an amino acid different from the amino acid at the corresponding position in the amino acid sequence of wild-type estrogen receptor _α ? A method for determining the gene type of estrogen receptor a, which comprises the step of determining whether or not the gene is estrogen receptor a;

30. In the nucleic acid in the sample, the amino acids constituting estrogen receptor α, and the amino acid sequence of amino acid sequence 404, 405, or 4 of the amino acid sequence represented by SEQ ID NO: 1 in an alignment based on amino acid sequence homology. Has the nucleotide sequence encoding the amino acid at the position corresponding to the amino acid shown in 4 been replaced with a nucleotide sequence encoding an amino acid different from the amino acid at the corresponding position in the amino acid sequence of wild-type estrogen receptor α? A method for determining the efficacy of treatment with an estrogen receptor activity modulator comprising the step of determining the gene type of estrogen receptor α by examining the presence or absence of estrogen receptor α;

31. In the nucleic acid in the sample, the amino acids that constitute estrogen receptor α, and the amino acid sequence of amino acid sequence represented by SEQ ID NO: 1 in the alignment based on the homology of the amino acid sequence. Equivalent to the amino acid shown in 24 The estrogen receptor is determined by examining whether the nucleotide sequence encoding the amino acid at the position corresponding to the estrogen receptor has been replaced with a base sequence encoding an amino acid different from the amino acid at the corresponding position in the amino acid sequence of the wild-type estrogen receptor _α. a method for determining the efficacy of treatment with an anti-estrogenic substance, including the step of determining the genotype of c; 32. The nucleic acid in the sample is designated as type II, and the amino acid number 40 of the amino acid sequence represented by SEQ ID NO: 1 The preceding item 30 including a step of amplifying a nucleic acid encoding a region containing an amino acid at a position corresponding to the amino acid represented by 4, 405 or 424 and determining the base sequence of the amplified nucleic acid Or the method according to any of 31;

33. Using the nucleic acid in the sample as type III, a region containing an amino acid at a position corresponding to the amino acid No. 404, 405 or 424 of the amino acid sequence shown in SEQ ID NO: 1 The encoding nucleic acid is amplified, the mobility of the amplified nucleic acid is measured by electrophoresis, and the mobility of the nucleic acid encoding the relevant region of wild-type estrogen receptor α and the mobility of the amplified nucleic acid are determined. 30. The method according to any one of the above items 30 or 31, comprising a step of checking whether or not they are different;

34. In the amino acid sequence of the wild-type estrogen receptor α, the amino acid at the position corresponding to the amino acid No. 404, 405 or 424 of the amino acid sequence represented by SEQ ID NO: 1 The method according to any one of the above items 30 or 31, comprising a step of examining the efficiency of hybridization between a probe consisting of a nucleotide sequence encoding a region containing the probe and a nucleic acid in the sample;

35. Nucleic acid encoding a region containing an amino acid at a position corresponding to amino acid number 404, 405 or 424 of the amino acid sequence represented by SEQ ID NO: 1, with the nucleic acid in the sample as type III The method according to any of the above items 30 or 31, comprising a step of amplifying the nucleic acid and digesting the amplified nucleic acid with a restriction enzyme to examine the presence or absence of a recognition sequence of the restriction enzyme;

And so on. BRIEF DESCRIPTION OF THE FIGURES

1, by the reporter mediation Si using a transformant reporter one coater gene is introduced into a chromosome of a host cell, human wild-type estrogen receptor _α or invention Les Fig. 3 shows the results of measuring the activation ability of 4-hydroxytamoxifen to sceptor F404L (indicated as "mutant F404LJ" in the figure). The value of luciferase activity in each test group was shown, assuming that the value of luciferase activity was 100%.

FIG. 2 shows that a reporter gene using a transformant in which a reporter gene was introduced into the chromosome of a host cell was used to report human wild-type estrogen receptor α or the receptor of the present invention, F404L (shown as `` mutant F404L '' in the figure). FIG. 9 shows the results of measuring the activation ability of raloxifene against). The luciferase activity in each of the test plots was shown assuming that the value of the luciferase activity in the control plot to which only the solvent was added (indicated as “control” in the figure) was 100%.

FIG. 3 shows that the reporter gene using a transformant in which a reporter gene has been introduced into the chromosome of a host cell is used to report human wild-type estrogen receptor α or the present receptor F404L (shown as “mutant F404LJ” in the figure). It is a figure which shows the result of having measured the activation ability of ZM189514 with respect to the luciferase activity value in the control group (only shown as "control" in a figure) which added only the solvent to 100%. The luciferase activity in the test plot was corrected.

FIG. 4 shows that the reporter gene using a transformant in which a reporter gene was introduced into the chromosome of a host cell was used to report on the human wild-type estrogen receptor α or the receptor F404L of the present invention (shown as `` mutant F404L '' in the figure). FIG. 4 shows the results of measuring the anti-estrogenic activity of 4-hydroxytamoxifen with respect to). The value of luciferase activity in each test group was shown assuming that the value of luciferase activity in the test group to which only E2 of {ρ} was added (indicated as “control” in the figure) was 100%.

FIG. 5 shows that the reporter atssii using a transformant in which a reporter gene was introduced into the chromosome of a host cell was used to report on the human wild-type estrogen receptor α or the receptor of the present invention, F404L (in FIG. FIG. 6 shows the results of measuring the anti-estrogenic activity of raloxifene against The value of luciferase activity in each test group was shown assuming that the value of luciferase activity in the test group to which only 100 ρΜ2 was added (indicated as “control” in the figure) was 100%. FIG. 6 shows the results of reporter assay using a transformant in which a reporter gene has been introduced into the chromosome of a host cell, using a human wild-type estrogen receptor α or a receptor of the present invention, F404L (in FIG. FIG. 4 shows the results of measuring the anti-estrogenic activity of ZM189154 against the following. The value of luciferase activity in each test group was shown assuming that the luciferase activity value in the test group supplemented with only 100 pM Ε2 (indicated as “control” in the figure) was 100%.

FIG. 7 shows the results of reporter assay using a transformant in which a reporter gene has been introduced into the chromosome of a host cell, by using the human wild-type estrogen receptor α or the receptor A405V of the present invention (shown as “mutant A405VJ” in the figure). The figure shows the results obtained by measuring the activation ability of 4-hydroxytamoxifen with respect to the luciferase activity in the control group to which only the solvent was added (indicated as “control” in the figure), assuming 100%. The values of luciferase activity in each test plot were shown.

FIG. 8 shows the results obtained by using a transformant in which a reporter gene has been introduced into the chromosome of a host cell, using a human wild-type estrogen receptor α or the receptor A405V of the present invention (mutation in the figure). FIG. 10 shows the results of measuring the activation ability of raloxifene with respect to “type A405V”). The value of luciferase activity in the control group to which only the solvent was added (indicated as “control” in the figure) was taken as 100%, and the luciferase activity in each test group was shown.

FIG. 9 shows that the reporter gene using a transformant in which the reporter gene was introduced into the chromosome of the host cell was used to report on the human wild-type estrogen receptor α or the receptor A405V of the present invention (shown as `` mutant A405V '' in the figure). FIG. 9 is a view showing the results of measuring the activation ability of ZM18914 with respect to). The value of luciferase activity in each of the test groups was shown assuming that the value of luciferase activity in the control group to which only the solvent was added (indicated as “control” in the figure) was 100%.

FIG. 10 shows the results of reporter assay using a transformant in which a reporter gene has been introduced into the chromosome of a host cell. 5 is a graph showing the results of measuring the anti-estrogenic activity of 4-hydroxy tamoxifen with respect to. The value of luciferase activity in each test group was shown assuming that the value of luciferase activity in each test group (shown as “control” in the figure) was 100%.

FIG. 11 shows the results of reporter assay using a transformant in which a reporter gene has been introduced into the chromosome of a host cell, using human wild-type estrogen receptor c or receptor A405V of the present invention (in the figure, mutant A405V FIG. 7 shows the results of measuring the antiestrogenic activity of raloxifene against raloxifene. The value of luciferase activity in each test group was shown assuming that the value of luciferase activity in the test group to which only 100 pM E2 was added (indicated as “control” in the figure) was 100%.

FIG. 12 shows the results of reporter assay using a transformant in which a reporter gene has been introduced into the chromosome of a host cell, to obtain human wild-type estrogen receptor α or receptor A405V of the present invention (shown as `` mutant A405V '' in the figure). FIG. 4 shows the results of measuring the anti-estrogenic activity of ZM189154 against). The value of luciferase activity in each test group was shown assuming that the luciferase activity value in the test group supplemented with only 100 pM E2 (indicated as “control” in the figure) was 100%.

FIG. 13 shows the results of reporter assay using a transformant in which a reporter gene has been introduced into the chromosome of a host cell, to obtain human wild-type estrogen receptor α or the receptor {424} of the present invention (indicated as “mutant I424TJ” in the figure). Fig. 4 shows the results of measuring the activation ability of 4-hydroxytamoxifen with respect to luciferase activity in a control group to which only a solvent was added (indicated as "control" in the figure), assuming 100%. The values of luciferase activity in each test plot were shown.

FIG. 14 shows the results of reporter assay using a transformant in which a reporter gene has been introduced into the chromosome of a host cell, using human wild-type estrogen receptor α or the receptor of the present invention {424} (shown as `` mutant Ι424Τ '' in the figure). FIG. 6 shows the results of measuring the activation ability of raloxifene with respect to). The value of luciferase activity in each test group was shown assuming that the value of luciferase activity in the control group to which only the solvent was added (indicated as “control” in the figure) was 100%.

FIG. 15 shows that the reporter gene using the transformant in which the reporter gene was introduced into the chromosome of the host cell was used to obtain the human wild-type estrogen receptor or the present invention. It is a figure which shows the result of having measured the activation ability of ZM1891554 with respect to receptor _j424T (it shows as "mutant type I424TJ" in the figure). The value of luciferase activity in) was taken as 100%, and the change in luciferase activity in each test group was shown.

FIG. 16 shows the results of reporter assay using a transformant in which a reporter gene has been introduced into the chromosome of a host cell, to obtain human wild-type estrogen receptor α or the receptor of the present invention {424} (indicated as “mutant I424TJ” in the figure). The figure shows the results of measuring the antiestrogenic activity of 4-hydroxytamoxifen with respect to luciferase activity in the test group to which only ΜρΜ of Ε2 was added (indicated as “control” in the figure). The value of luciferase activity in each test group was shown as 100%.

FIG. 17 shows the results of reporter assay using a transformant in which a reporter gene has been introduced into the chromosome of a host cell, to obtain human wild-type estrogen receptor α or the present receptor {424} (indicated as “mutant I424TJ” in the figure). Fig. 3 shows the results of measuring the antiestrogenic activity of raloxifene against luciferase, where the value of luciferase activity in the test group to which only 100 pM Ε2 was added (indicated as “control” in the figure) was 100%, The value of luciferase activity was shown.

FIG. 18 shows the results of reporter assay using a transformant in which a reporter gene has been introduced into the chromosome of a host cell, using human wild-type estrogen receptor α or the receptor {424} of the present invention (indicated as “mutant I424TJ” in the figure). The figure shows the results of measuring the anti-estrogenic activity of ZM189154 against the luciferase activity in the test group to which only 100 pM of Ε2 was added (indicated as “control” in the figure). The values of luciferase activity in each test plot were shown.

FIG. 19 is a view showing alignment of amino acid sequences of estrogen receptor α derived from human, mouse or rat. * Indicates an amino acid at a position corresponding to the amino acid of amino acid number 404 in the amino acid sequence represented by SEQ ID NO: 1. BEST MODE FOR CARRYING OUT THE INVENTION

Hereinafter, the present invention will be described in detail. In the present invention,

"Alignment based on amino acid sequence homology" of estrogen receptor alpha refers to the alignment of amino acid sequences of estrogen receptor alpha derived from various organisms so that the amino acid sequences are identical or highly similar. Means table. Alignments based on amino acid sequence homology include, for example, FASTA [Pearson & Lipman, Proc. Natl. Acad. Sci. USA, 4, 2444-2448 (1988)], BLAST [Altschul et al., Journal of Molecular Biology, 215, 403-410 (1990)] and CLUSTAL W [Thompson, Higgins & Gibson, Nucleic Acid Research, 22, 4673-4680 (1994a)] and the like. The above program is, for example, the DNA Data Bank of Japan [National Institute of Genetics Life Information. International DNA Data Bank operated within the Center for Information Biology and DNA Data Bank of Japan (CIB / DDBJ)] Is generally available on the homepage (http: 〃www.ddbj.nig.ac.jp). The alignment was performed using a commercially available sequence analysis software, for example, GENETYX-WIN (manufactured by Software Development Co., Ltd.) using the Lipman-Pearson method [Lipman, DJ and Pearson, WR, Science, 227, 1435-1441, (1985)].

Specific examples of the "alignment based on amino acid sequence homology" of estrogen receptor include alignment of amino acid sequence of estrogen receptor _α derived from human, mouse or rat as shown in FIG. . In FIG. 19, hERa. TXT is the amino acid sequence of human-derived estrogen receptor α (an amino acid sequence in which the amino acid at position 400 from the amino terminal of the amino acid sequence described in GenBank Accession No. M12674 is substituted with glycine; The sequence listing shows SEQ ID NO: 1.), mER.TXT is a mouse-derived estrogen receptor α amino acid sequence (GenBank Accession No. M38651), ratER (X6) .TXT is a rat-derived estrogen receptor The amino acid sequences IJ (GenBank Accession No. X61098) and ratER (Y0) .TXT indicate the amino acid sequence of rat-derived estrogen receptor α (GenBank Accession No. Y00102) in one-letter amino acid notation. The alignment was made using commercially available software Genetyx-Win SV / R ver. 4.0 (Software Development Co., Ltd.). * Indicates an amino acid at a position corresponding to the amino acid of amino acid number 404 in the amino acid sequence represented by SEQ ID NO: 1.

“In an alignment based on the homology of the amino acid sequence, the amino acid at a position corresponding to the amino acid represented by amino acid number 404 in the amino acid sequence represented by SEQ ID NO: 1” includes various amino acids as described above. In the alignment of the amino acid sequence of the estrogen receptor α derived from the organism of the present invention, when described alongside the amino acid sequence of SEQ ID NO: 1, the amino acid represented by the amino acid sequence of the amino acid sequence of SEQ ID NO: 1 An amino acid is located at the same position as an acid. Specifically, for example, in the alignment based on the homology of amino acid sequences, the amino acid at the position corresponding to the amino acid represented by amino acid number 404 in the amino acid sequence represented by SEQ ID NO: 1 is human Amino acid sequence of the estrogen receptor _α derived from the amino acid sequence (shown in SEQ ID NO: 1), the amino acid sequence of feniralanine, which is the amino acid at the 404th position from the amino terminal, and the amino acid sequence of the estrogen receptor 1α derived from the mouse (GenBank Accession No. M38651) Phenylalanine, which is the 408th amino acid from the amino terminal, phenylalanine, which is the 409th amino acid from the amino acid sequence of the rat estrogen receptor α amino acid sequence (GenBank Accession No. X61098), rat From the amino terminal of the amino acid sequence of estrogen receptor α (GenBank Accession No. Y00102) Mention may be made of Hue two Ruaranin such as a eyes of the amino acid.

“In an alignment based on amino acid sequence homology, an amino acid at a position corresponding to the amino acid shown by amino acid No. 405 of the amino acid sequence shown by SEQ ID No. 1” refers to various organisms as described above. In the alignment of the amino acid sequence of the derived estrogen receptor α, when described alongside the amino acid sequence represented by SEQ ID NO: 1, the amino acid sequence represented by SEQ ID NO: 1 has the same position as the amino acid represented by amino acid number 405. Comes with the amino acids. Specifically, for example, in the alignment based on the homology of the amino acid sequence, the amino acid at the position corresponding to the amino acid No. 405 of the amino acid sequence represented by SEQ ID NO: 1 405 from the amino end of the amino acid sequence of estrogen receptor _α (shown in SEQ ID NO: 1) Alanine, the amino acid sequence of the estrogen receptor _α from the mouse (GenBank Accession No. 38651), the alanine amino acid at the 409th amino acid from the amino terminal, and the amino acid sequence of the estrogen receptor from the rat (GenBank Accession No. 38651) No. X61098), alanine, the amino acid at the 410th amino acid from the amino terminal, rat-derived estrogen receptor α, amino acid sequence at the 410th amino acid from the amino acid sequence (GenBank Accession No. Y00102), alanine, etc. Can be mentioned.

"In an alignment based on the homology of the amino acid sequence, the amino acid at a position corresponding to the amino acid shown by amino acid No. 424 of the amino acid sequence shown by SEQ ID NO: 1" includes various amino acids as described above. In the alignment of the amino acid sequence of the estrogen receptor ct derived from the organism, when described alongside the amino acid sequence represented by SEQ ID NO: 1, the amino acid sequence represented by SEQ ID NO: 1 is located at the same position as the amino acid represented by amino acid number 424. Means the amino acid that comes Specifically, for example, in the alignment based on the homology of the amino acid sequence, the amino acid at the position corresponding to the amino acid represented by amino acid number 424 in the amino acid sequence represented by SEQ ID NO: 1 is derived from human. Isoleucine, the 4th and 4th amino acid from the amino acid sequence of the estrogen receptor (shown in SEQ ID NO: 1), and the amino acid sequence of the amino acid sequence of estrogen receptor α from mouse (GenBank Accession No. M38651) Amino acid sequence of isoleucine, amino acid at position 428, isoleucine at amino acid position 429 from amino acid sequence of amino acid sequence of rat estrogen receptor α (GenBank Accession No. X61098), amino acid sequence of estrogen receptor α, rat (GenBank

And isoleucine which is the 429th amino acid from the amino terminal of Accession No. Y00102).

The term “wild-type estrogen receptor _α ” means an estrogen receptor _α consisting of an amino acid sequence naturally and most frequently found in the amino acid sequence of the receptor derived from the same species of organism. For example, as a human-derived wild-type estrogen receptor α, the amino acid sequence represented by SEQ ID NO: 1 (the amino acid valine at position 400 from the amino terminal of the amino acid sequence described in GenBank Accession No. M12674 was replaced with glycine) A A is _c es Bok Rogun response Rooster can be given estrogen receptor one α himself歹IJ (estrogen responsive element) from acid sequence), included in the transcription control region of the target genes regulated transcription by S. Bok Rokenrese Puta When a complex of estrogen and an estrogen receptor, for example, recognizes and binds to the specific base sequence, transcription of a target gene downstream of the sequence is regulated. Specific examples of such an estrogen response element include, for example, the base sequence of the 5 ′ upstream region of the vitellogenin gene of African alfalga (Cell., 57, 1139-1146). In addition, a consensus sequence of an estrogen response element [5′-AGGTCAnnnTGACCTT-3,; n represents A, G, C or T. ] One or more times. In order to obtain a sufficient transcription control ability, it is preferable that the consensus sequence as described above is usually tandemly linked in about 2 to 5 times. DNΑ having a strong nucleotide sequence can be prepared by chemical synthesis or amplification and cloning by PCR or the like according to a conventional method.

“Estrogen receptor transcription activation region AF1” and “estrogen receptor α transcription activation region AF2” are a part of estrogen receptor α, respectively, and Are involved

[Metzger D et al., J. Biol. Chem., 270: 9535-9542 (1995) and the like]. `` An anti-estrogenic substance that suppresses the function of the transcription activation region AF2 of the wild-type estrogen receptor α but does not suppress the function of the transcription activation region AF1 '' is described, for example, in Berry M. et al., ΕΜΒΟJ. , 9: 2811-2818 (1990), etc., the characteristics of which can be evaluated by performing a reporter assay. Specifically, for example, it can be prepared according to the description in primary culture cells of chicken embryo fibroblast [Solomon. JJ, Tissue Cult. Assoc. Manual, 1: 7-11 (1975) and the like. Expression of wild-type estrogen receptor gene in cells with strong ability to activate AF1 and transfection of a reporter gene linked downstream of the transcription control region containing estrogen response element . A test substance is brought into contact with the obtained cells (hereinafter, referred to as cells for AF1 activity evaluation), and a reporter is used as described below. When the assay is performed, a type of anti-estrogens that suppresses the function of the transcription-activating region AF2 of the wild-type estrogens receptor α, but does not suppress the function of the transcription-activating region AF1, activates transcription and activates the reporter. Increase the expression level of the gene. On the other hand, cells such as primary culture cells of chicken embryo fibroblast express the wild-type estrogen receptor ct gene lacking the transcriptional activation region AF1 coding region, and are linked downstream of the transcription control region containing the estrogen response element. Introduce a new reporter gene. When a test substance is brought into contact with the obtained cells (hereinafter referred to as cells for evaluating AF2 activity) and a reporter assay is performed as described below, the “function of the transcription activation region AF2 of wild-type estrogen receptor _α ” is obtained. A type of antiestrogens that suppresses transcription but does not suppress the function of the transcriptional activation region AF1 does not induce transcriptional activation and does not change the expression level of the reporter gene. Specific examples of `` an anti-estrogen substance of the type that suppresses the function of the transcriptionally active region AF2 of the wild-type estrogens receptor α but does not inhibit the function of the transcriptionally activated region AF1 '' having such properties Examples include tamoxifen, 4-hydroxytamoxifen, raloxifene and the like.

"Pure antiestrogen" refers to a substance that exhibits substantially no estrogen activity including partial agonist activity, such as that of tamoxifen, and such a substance can be used as a cell for assessing AF1 activity or AF2 activity as described above. When the reporter assay is performed using any of the cells for evaluation, transcriptional activation is not induced and the expression level of the reporter gene is not changed. Specific examples of “pure anti-estrogens” include ICI182780

[Wakeling, AE et al., Cancer Res., 512: 3867-3873 (1991)], ZM189154 [Dukes, M. et al., J. Endocrinol., 141: 335-341 (1994)] and the like. be able to. The receptor of the present invention is characterized by the fact that, among the amino acids constituting the receptor, the amino acid sequence of the amino acid sequence represented by SEQ ID NO: 1 in the alignment based on the homology of the amino acid sequence. One or more of the amino acids at the positions corresponding to the amino acids represented by are replaced with amino acids different from the amino acids at the corresponding positions in the amino acid sequence of the wild-type estrogen receptor. '' (B) And (c).

(b) Any of the types of antiestrogens such as tamoxifen, 4-hydroxytamoxifen, and raloxifene, which suppress the function of the transcription activation region AF2 but do not suppress the function of the transcription activation region AF1 of the wild-type estrogen receptor. When contacted with this, transcription of a gene under the transcriptional control of a transcription control region containing an estrogens response element can be activated.

(c) upon contact with estrogen, can activate the transcription of a gene linked downstream of a transcription control region containing an estrogen response element, and the activation activates the transcription of the gene in (b) above. And is not substantially inhibited by compounds that can.

The ability to activate transcription of a gene that is under the transcriptional control of a transcription control region containing an estrogen response element can be determined, for example, by determining whether a reporter gene linked downstream of the transcription control region containing an estrogen response element Can be evaluated by performing a test method such as a reporter assay described below. As the receptor of the present invention

In (b) and (c) above, examples of the receptor capable of activating transcription of a “gene under the transcriptional control of a transcription control region containing an estrogen response sequence” introduced into the chromosome of a cell are given. be able to. In addition, the activation of transcription of the “gene under the transcriptional control of a transcription control region containing an estrogen response sequence” in the above (c) is preferably an activation inhibited by a pure anti-estrogen.

Examples of the amino acid substitution as described above include, for example, substitution of phenylalanine with leucine at a position corresponding to the amino acid represented by amino acid number 404 in the amino acid sequence represented by SEQ ID NO: 1, sequence Substitution of alanine for valine at a position corresponding to the amino acid shown by amino acid No. 405 in the amino acid sequence shown by No. 1; Substitution of isoleucine at the corresponding position with threonine can be mentioned. On the other hand, in the receptor of the present invention, "in the alignment based on the homology of the amino acid sequences, the amino acid numbers 303, 309, 390, 396, 415, 4 of the amino acid sequence represented by SEQ ID NO: 1 Any amino acid at a position corresponding to the amino acid represented by 94, 531, or 5778 corresponds to the amino acid sequence of the wild-type estrogen receptor α. May be the same as the amino acid at a certain position.

The receptor of the present invention is preferably a receptor derived from an animal such as a mammal such as a human, a monkey, a rabbit, a rat, and a mouse. More specific examples of the receptor of the present invention include an estrogen receptor α having an amino acid sequence represented by SEQ ID NO: 2, an estrogen receptor α having an amino acid sequence represented by SEQ ID NO: 28, and an amino acid represented by SEQ ID NO: 33. Estrogen receptor having a sequence can be mentioned. The DNA of the present invention is an isolated DNA encoding the receptor of the present invention as described above. The DNA of the present invention may be DNA isolated from nature, for example, by introducing a mutation into DNA isolated from nature by site-directed mutagenesis, mutagenesis, or the like. It may be the created DN Α.

The DNA of the present invention can be prepared, for example, from cDNA encoding the wild-type estrogen receptor ct as follows.

To obtain cDNA encoding the wild-type estrogen receptor, first, a mammal such as a human, monkey, monkey, rat, mouse, etc. should be Design and synthesize primers to amplify the DNA encoding wild-type estrogen receptor _α from the animal by polymerase chain reaction (hereinafter referred to as PCR). Specifically, for example, in order to obtain a cDNA encoding human-derived wild-type estrogen receptor _α , a primer designed based on the nucleotide sequence described in GenBank Accession No.M12674, for example, A formal primer consisting of the nucleotide sequence represented by SEQ ID NO: 3 and a reverse primer consisting of the nucleotide sequence represented by SEQ ID NO: 4 are chemically synthesized.

On the other hand, for example, Sambrook, J., and Zrussel, DW; Molecular Cloning 3rd edition, Cornole Red Spring RNA is extracted and single-stranded cDNA is synthesized according to the genetic engineering method described in Nover Laboratory (Cold Spring Harbor Laboratory) (2001). Specifically, first, total RNA is prepared from animal tissues of mammals such as humans, monkeys, egrets, rats and mice. For example, liver The tissue is ground in a solution containing a protein denaturing agent such as guanidine hydrochloride / guanidine thiocyanate, and the protein is denatured by adding phenol, croperform, etc. to the ground material. After removing denatured proteins by centrifugation or the like, total RNA is extracted from the collected supernatant fraction by methods such as guanidine hydrochloride Z-phenol method, SDS-phenol method, guanidine thiocyanoate cesium chloride-ultracentrifugation method, etc. I do. Commercially available kits based on these methods include, for example, IS0GEN (manufactured by Futtsubon Gene) and TRIZ0L reagent (manufactured by Invitrogen). Next, using the obtained total RNA as a 铸 type, an oligo dT primer was annealed to the poly A sequence of mRNA contained in the RNA, followed by a reverse transcription enzyme, followed by loss of the enzyme. Activate. For example, RnaseH-Superscript II Reverse Transcriptase (manufactured by Invitrogen), a buffer attached to the enzyme, oligo dT primer and total RNA are mixed, reacted at 42 ° C for 1 hour, and then heated at 99 ° C for 5 minutes. To Commercially available kits based on these methods include, for example, a cDNA synthesis system plus (Amersham Biotech) and a TimeSaver cDNA synthesis kit (Amersham Biotech).

Next, the single-stranded cDNA synthesized as described above is converted into a type II, and the above-mentioned primers are added to each at 200 nM. For example, LA Taq DNA polymerase (manufactured by Takara Shuzo) and the enzyme are added. Perform PCR using the buffer attached to. For example, using a commercially available thermal cycler for PCR such as PCR System9700 (manufactured by Applied Biosystems), the PCR is performed at 95 ° C. for 1 minute, and then at 68 ° C. for 3 minutes for about 35 cycles. Do. Here, instead of the cDNA synthesized as described above, commercially available cDNAs derived from various animals, such as Clontech Quick Clone cDNA, may be used. A part of the obtained reaction solution is subjected to gel electrophoresis using agarose (agarose L: Futaba Gene). After confirming the presence of the DNA encoding the wild-type estrogen receptor of the size expected from the known sequence on the electrophoresed gel, the DNA is recovered from the gel. The nucleotide sequence of the recovered DNA was determined by preparing a sample for direct sequence using the recovered DNA and a commercially available fluorescent sequence reagent such as Dye Terminator Sequence Kit FS (manufactured by Applied Biosystems). Using an automatic DNA sequencer such as model 3700 manufactured by Applied Biosystems It can be determined by performing a base sequence analysis, and as a result, it can be confirmed that the recovered DNA is the target DNA. The DNA encoding the wild-type estrogen receptor _α thus obtained is cloned into a vector capable of replicating in E. coli or the like. Specifically, for example, using about lg of the recovered DNA, its end is blunted with a DNA Blunting Kit (manufactured by Takara Shuzo) or the like, and then the end is phosphorylated by reacting with T4 polynucleotide kinase. After the DNA is treated with phenol, it is purified by the ethanol precipitation method. By introducing the purified DNA into a replicable vector in E. coli or the like, a vector having a DNA encoding a wild-type estrogen receptor can be obtained.

Next, DNA of a vector carrying DNΑ encoding wild-type estrogen receptor α thus prepared is prepared. The prepared DNA was converted into type III, and it was published in Sambrook, J., and Zrussel l, DW; Molecular Cloning 3rd edition, Cold Spring Harbor Laboratory (2001), etc. In accordance with the site-directed mutagenesis method and the like described, a base substitution for performing the desired amino acid substitution is introduced into DNA encoding wild-type estrogen receptor c. Commercially available kits that can be used for such a method include, for example, the QuickChange Site-Directed Mutagenesis Kit manufactured by Stratagene. Using two kinds of primers designed and synthesized for performing the desired base substitution, a site-specific mutation is introduced according to the description of the kit as described above. Primers for replacing codon TTT encoding phenylalanine, which is the fourth amino acid from the amino terminus of human-derived wild-type estrogen receptor c, with codon CTT encoding leucine include, for example, SEQ ID NO: A forward primer consisting of the base sequence represented by SEQ ID NO: 5 and a reverse primer consisting of the base sequence represented by SEQ ID NO: 6 can be used. Primers for replacing codon GCT encoding alanine, which is the amino acid at position 450 from the amino terminus of human-derived wild-type estrogen receptor α, with codon GTT encoding valine include, for example, SEQ ID NO: 29 And a reverse primer consisting of the base sequence represented by SEQ ID NO: 30. Human estrogen The primer for replacing the codon ATC encoding isoloisin, which is the 424th amino acid from the amino terminus of the receptor _α , with the codon ACC encoding threonine is, for example, a nucleotide sequence represented by SEQ ID NO: 34 And a reverse primer consisting of the nucleotide sequence represented by SEQ ID NO: 35.

By determining the nucleotide sequence of the obtained DN N, it can be confirmed that the intended base substitution has been performed. A vector that can be used in a host cell to be transformed with the DNA of the present invention thus obtained, for example, a vector that contains genetic information that can be replicated in the host cell and is autonomously replicable, The vector of the present invention can be constructed by incorporating it into a vector that can be isolated and purified and has a detectable marker (hereinafter, referred to as a basic vector) according to ordinary genetic engineering techniques.

As a basic vector that can be used for the construction of the vector of the present invention, specifically, when Escherichia coli which is a microorganism is used as a host cell, for example, plasmid pUC19 (manufactured by Takara Shuzo), phagemid pBluescript iKStratagene, etc. Can be mentioned. When budding yeast is used as the host cell, plasmids pGBT9, pGAD404, pACT2 (manufactured by Clontech) and the like can be mentioned. In addition, _P Rc / RSV is the case of a mammalian cell is used as the host cell, pRc / CMV (Invitrogen), and the like plasmid of, © Shea papilloma virus plus Mi de pBPV (§ Ma Siam Biotech), EB virus plus _Mi-de-P Vectors containing a virus-derived autonomous origin of replication (ori) such as CEP4 (Invitrogen), viruses such as vaccinia virus, and the like. When insect cells are used as host cells, insect viruses such as baculovirus. Can be mentioned.

Vectors containing autonomous replication origin, for example, and the above-mentioned yeast for plasmid _P ACT2, Ushipa pillow Ma virus plus mi de pBPV, Epusutain - incorporation of bar one Honoré virus plus Mi de pCEP4, etc. In the present invention DNA, resulting vector Can be retained intracellularly as an episome when introduced into a host cell.

Incorporating the DNA of the present invention into viruses such as baculovirus vaccinia virus For this purpose, a transfer vector containing a nucleotide sequence homologous to the genome of the virus to be used can be used. Specific examples of such transfer vectors include pVL1392, pVL1393 (Smith, GE, Summers MD et al .: ol. Cell. Biol., 3, 2156-2165 (1983)) (Invitrogen), Plasmids such as pSFB5 (Funahashi, S. et al .: J. Virol., 65, 5584-5588 (1991)) (Pharmingen) can be mentioned. When the DNA of the present invention is inserted into the transfer vector as described above, and the transfer vector and the viral genome are simultaneously introduced into a host cell, homologous recombination occurs between the transfer vector and the viral genome, and the DNA of the present invention becomes A recombinant virus integrated on the genome can be obtained. As the virus genome, genomes of baculovirus, adenovirus, vaccinia virus and the like can be used.

More specifically, for example, when the DNA of the present invention is incorporated into a baculovirus, the DNA of the present invention is inserted into a multiple access site of a transfer vector such as pVL1392 or pVL1393, and then the DNA of the transfer vector is used. Baculovirus genomic DNA [for example, BaculoGold (Pharmingin)] is introduced into insect cell strain Sf21 (available from ATCC) by a calcium phosphate method or the like, and the cells are cultured. When the Baculogold DNA is used, only the virus particles containing the virus DNA into which the DNA of the present invention has been inserted are released into the culture medium of the host cell. Viral DNA containing the DNA of the present invention can be obtained by collecting the recombinant virus particles from the culture solution and subjecting them to deproteinization with phenol or the like. Furthermore, the recombinant virus particles can be recovered by introducing the DNA of the virus into a host cell capable of forming virus particles, such as insect cell strain Sf21, by the calcium phosphate method or the like and culturing the cells. .

On the other hand, the DNA of the present invention can be directly integrated into a relatively small genome such as a mouse leukemia retrovirus without using a transfer vector. For example, virus vector-DC (X) (Eli Gilboa et al., BioTechniques, 4: 504-512 (1986)) may incorporate the DNA of the present invention into the clawing site on the vector. When the recombinant virus thus constructed is introduced into a packaging cell such as Ampli-GPE (J. Virol., 66: 3755 (1992)), the virus containing the DNA of the present invention is contained. Virus particles can be obtained. A promoter capable of functioning in a host cell is operably linked to the upstream of the DNA of the present invention, and this is integrated into the above-described basic vector, whereby a DNA capable of expressing the DNA of the present invention in the host cell can be obtained. Invention vectors can be constructed. Here, the term "functionally linked" means that the promoter and the DNA of the present invention are combined so that the DNA of the present invention is expressed under the control of a promoter in a host cell into which the DNA of the present invention is introduced. Means to combine. The promoter used exhibits a promoter activity in the host cell to be transformed. For example, when the host cell is Escherichia coli, the E. coli lactose operon promoter (lacP) and tryptophan operatin are used. raising etc. - of the promoter (trpP), promoter of arginine operon (argP), the promoter of the galactose operon _(g alP), tac promoter, T7 promoter, T3 promoter and foremost, promoter of example phage (pR tut -pL, e) When the host cell is a animal cell or fission yeast, for example, the Rous sarcoma virus (RSV) promoter, the cytomegalovirus (CMV) promoter, the simian virus (SV40) early or late promoter, Mouse papilloma virus (MMTV) promoter and the like can be mentioned. When the host cell is a budding yeast, examples thereof include an alcohol dehydrogenase (ADH) l gene promoter.

When a basic vector having a promoter that functions in a host cell is used in advance, the promoter is placed downstream of the promoter so that the DNA of the present invention and the DNA of the present invention can be operably linked to the promoter of the vector. The invention DNA may be inserted. For example, the aforementioned plasmid pRc / RSV, _P Rc / CMV and the like, under flow promoter operable in animal cells cloning sites is provided in the present invention DNA in the cloning sites揷入and into animal cells When introduced, the DNA of the present invention can be expressed. Since these plasmids contain the autonomous replication origin (ori) of SV40 in advance, when the plasmid is introduced into cultured cells transformed with the ori (-) SV40 genome, for example, COS cells, etc. On the other hand, the number of copies of the plasmid is greatly increased in the cell, and as a result, the DNA of the present invention incorporated in the plasmid can be expressed in a large amount. In addition, the above-mentioned yeast plasmid PACT2 has an ADH1 promoter, and the ADH1 promoter of the plasmid or a derivative thereof is used. If the DNA of the present invention is inserted downstream of the motor, the vector of the present invention can be constructed that allows the DNA of the present invention to be expressed in large amounts in budding yeast such as CG1945 strain (manufactured by Kuguchi Tech). The transformant of the present invention can be obtained by introducing the DNA of the present invention or the vector of the present invention into a host cell. As a method for introducing the DNA of the present invention or the vector of the present invention into a host cell, a usual method for introduction depending on the host cell to be transformed can be applied. For example, when a host cell is a microorganism, Escherichia coli, the calcium chloride method described in Molecular 'Cloning 3rd edition (Sambrook, J., and Russell, DW, Cold Spring' Harbor, 2001), etc. An ordinary method such as a deposition method can be used. When a mammalian cell or an insect cell is used as the host cell, for example, the cell may be prepared by a common gene transfer method such as the calcium phosphate method, the DEAE dextran method, the electroporation method, or the lipofection method. Can be introduced. When yeast is used as a host cell, it can be introduced using, for example, a Yeast Transformation Kit (manufactured by Clontech) based on the lithium chloride method.

When a virus is used as a vector, viral DNA can be introduced into host cells by a general gene transfer method as described above, and recombinant virus particles containing viral DNA can be transmitted to host cells. By doing so, the viral DNA can be introduced into the host cell. In order to select the transformant of the present invention, for example, when the DNA of the present invention or the vector of the present invention is introduced into a host cell, a selectable marker gene is simultaneously introduced into the host cell, and the obtained cell is subjected to the introduced selection. It is preferable to culture under conditions according to the properties of the marker gene. For example, when the selectable marker gene is a gene that confers drug resistance to a selective drug exhibiting lethal activity on host cells, the DNA of the present invention or the vector of the present invention is selected using a medium containing the drug. A host cell into which the marker gene has been introduced may be cultured. Examples of combinations of drug resistance imparting genes and selected drugs include neomycin resistance Examples of the combination include a combination of a given gene and neomycin, a combination of a hygromycin resistance imparting gene and hygromycin, and a combination of a blasticidin S resistance imparting gene and blasticidin S. When the selectable marker gene is a gene that complements the auxotrophy of the host cell, the host into which the DNA of the present invention or the vector of the present invention and the selectable marker gene have been introduced using a minimal medium not containing the nutrient. The cells may be cultured. Furthermore, when the vector of the present invention capable of expressing the DNA of the present invention in a host cell is introduced, a detection method based on estrogen binding activity can also be used.

In order to obtain the transformant of the present invention in which the DNA of the present invention has been introduced into the chromosome of a host cell, for example, the vector of the present invention is linearized by digestion with a restriction enzyme or the like, and then this is selected as a selection marker gene. At the same time, the cells may be introduced into host cells by the above-described method, and the cells are usually cultured for several weeks, and the transformant of interest may be selected using the expression of the introduced selectable marker gene as an index. For example, a gene conferring resistance to a selection drug as described above is introduced as a selection marker gene into a host cell together with the vector of the present invention by the method described above, and the cell is subcultured for several weeks or more in a medium to which the selection drug has been added. Then, the transformant of the present invention, in which the DNA of the present invention has been introduced into the chromosome of a host cell, can be selected by purifying and culturing the selected drug-resistant clone remaining in a colony. Since the transformant can be cryopreserved and awakened when necessary, it can be used to prepare a transformant for each experiment as compared with a strain into which the DNA of the present invention has been transiently introduced. This makes it possible to save time and labor, and to conduct tests using transformants whose properties and handling conditions have been confirmed in advance. By culturing the transformant of the present invention obtained as described above, the receptor of the present invention can be produced.

For example, when the transformant of the present invention is a microorganism, the transformant may be prepared by using various media appropriately containing a carbon source, a nitrogen source, organic or inorganic salts, etc., which are used for ordinary culture in a general microorganism. And cultured. Cultivation is carried out according to the usual method for general microorganisms, and solid culture, liquid culture (test tube shaking culture, reciprocating shaking culture, jar armamenter (Jar Fermenter) culture, tank culture, etc.). The culture temperature can be appropriately changed within a range in which the microorganism grows. For example, culture is generally performed in a culture temperature of about 15 ° C. to about 40 ° C. and a medium having a pH of about 6 to about 8. The culturing time varies depending on the culturing conditions, but is usually about 1 day to about 5 days. In the case of using an expression vector having an inducible promoter such as a temperature-shifted disopropyl β-D-galactopyranoside (IPTG) -inducible type, the induction time is desirably within one day, usually several hours.

When the transformant is an animal cell such as a mammal or an insect, the transformant can be cultured using a medium used for ordinary culture of general cultured cells. When the transformant is prepared using a selective drug, it is preferable to culture the transformant in the presence of the selective drug. For mammalian cells, use a Dulbecco's Modified Eagle (DMEM) medium (such as Nissui Pharmaceutical) supplemented with fetal bovine serum (FBS) to a final concentration of 10% at 37 ° C. 5% C0 may be cultured while exchanging the fresh medium every few days in conditions such as ₂ presence. When the cells have grown to confluence, add 0.25% (v / v) trypsin / PBS solution to disperse them into individual cells, dilute them several times, inoculate them into new dishes, and culture them. to continue. Similarly, in the case of insect cells, use a culture medium for insect cells such as Grace's medium containing 10% (v / v) FBS and 2% (w / v) yeast late at a culture temperature of 25 ° C. Culture may be performed at 35 ° C. At this time, when cells such as Sf21 cells are easily detached from a petri dish, subculture can be performed by dispersing the cells by pipetting or the like without using a trypsin solution. In the case of a transformant containing a virus vector such as baculovirus, the culturing time is preferably up to 72 hours after the virus infection, for example, before the cells are killed due to the cytoplasmic effect.

The receptor protein of the present invention produced by the transformant of the present invention may be appropriately collected by a combination of ordinary isolation and purification methods.For example, after culturing, the cells of the transformant are centrifuged or the like. The cells are collected, and the collected cells are suspended in a normal buffer, for example, a buffer composed of 20 mM HEPES (pH 7.0), 1 mM EDTA, 1 mM DTT, 0.5 mM PMSF. Processing, crushing with a Dounce homogenizer, etc., ultracentrifugation of the crushed liquid at 100,000 Xg for several tens of minutes to about 1 hour, and collection of the supernatant fraction to obtain a fraction containing estrogen receptor be able to. Further, the supernatant fraction is subjected to ion exchange. A more purified estrogen receptor can be recovered by subjecting it to various types of chromatography such as chromatography, hydrophobicity, gel filtration, and abundance. At this time, the fraction containing the receptor of the present invention is identified by a DNA binding assay using an estrogen response element, ie, an oligonucleotide having a length of about 15 bp to 200 bp containing the base sequence to which the estrogen receptor binds. Can also.

The receptor of the present invention thus produced can be used, for example, as a ligand 'receptor binding assay for evaluating the binding ability and amount of a test substance to the receptor of the present invention. The ligand-receptor binding assay using the receptor of the present invention is a test method capable of measuring the binding ability of a chemical substance to the receptor, quantifying the amount of binding, and analyzing the binding specificity and binding strength. For example, when a test substance is allowed to coexist with a labeled ligand (hereinafter referred to as a labeled ligand) bound to the receptor of the present invention recovered from the transformant of the present invention as described above, Due to the competition between the substance and the labeled ligand, the labeled ligand is released from the receptor according to the affinity of both for the receptor, and the amount of the labeled ligand bound to the receptor decreases, and therefore, the amount of the label bound to the receptor decreases. . Therefore, by monitoring the amount of the free labeled ligand or the amount of the bound labeled ligand, the ability of the test substance to bind to the receptor can be determined indirectly.

As the labeled ligand, for example, tritium-labeled 17 / 3-estradiol (hereinafter referred to as E2) or the like can be used. Separation of the bound and free forms of the labeled ligand can be performed by the hydroxyapatite method ゃ glycerol density gradient ultracentrifugation method or the like. The reaction system is roughly divided into three groups. One system is a group in which only the solvent is added to the site where the labeled ligand is bound to the receptor of the present invention, and corresponds to a system where the concentration of the test substance is zero. The labeling amount of the bound labeled ligand obtained from this system indicates the total binding amount of the labeled ligand to the estrogen receptor. In another system, the concentration of the labeled ligand bound to the receptor of the present invention, for example, such that unlabeled E2 sufficiently saturates the receptor to prevent the labeled ligand from binding (eg, 10 μm). Μ) It is a system added so that The amount of the labeled labeled ligand obtained from the second system is determined to be the amount of nonspecific binding of the labeled ligand to the receptor of the present invention. Therefore, the specific binding amount of the labeled ligand of the receptor of the present invention is a value obtained by subtracting this non-specific binding amount from the total binding amount. In the third system, the test substance was added to the receptor of the present invention where the labeled ligand was bound so that the test substance had a final concentration of, for example, ΙΟμΜ (this concentration may be arbitrarily changed depending on the purpose). System. If the test substance has the ability to bind to the receptor of the present invention, the amount of the labeled labeled ligand obtained from this system will be the same as that of the receptor of the present invention when the test substance concentration obtained as described above is zero. Is smaller than the specific binding amount of the labeled ligand. By performing ligand / receptor binding as described above, the binding ability of the test substance to the receptor of the present invention can be determined. When the test substance contains a plurality of substances, the test substance can be incorporated into the receptor of the present invention. It is also possible to check whether a substance exhibiting affinity exists. Further, in order to evaluate the binding ability of the test substance to the receptor of the present invention in more detail, for example, by changing the concentration of the test substance in the third system and performing the same assay, the amount of the Is measured. Based on the measured values, the amounts of bound and free ligands in each assay were calculated, for example, by performing Scatchard analysis to determine the binding affinity and specificity between the test substance and the receptor of the present invention. The coupling capacity and the like can be evaluated. The evaluation method of the present invention can be carried out, for example, by conducting a reporter assay of a test substance using the DNA of the present invention. Examples of the ability to regulate estrogen receptor α activity include agonist activity and antagonist activity for estrogen receptor _α , and more specifically, for example, estrogen-like activity and antiestrogenic activity.

The “reporter gene under the transcriptional control of a transcription control region containing an estrogen response element” used in the evaluation method of the present invention is, for example, specifically, A reporter gene in which DNA encoding a reporter protein is linked downstream of the transcription control region of the genin gene, or a base sequence and a reporter protein required for transcription initiation downstream of an estrogen response element This is a reporter gene linked to DNA which is used to monitor the ability of estrogen receptor to regulate transcription in host cells. DNAs encoding luciferase, DNA encoding secretory alkaline phosphatase, DNA encoding 3-galactosidase, and chloramphene used as DNAs encoding reporter proteins used for the production of such reporter genes. A DNA encoding a Nicol acetylenotransferase, a DNA encoding a growing honoremon, and the like can be used, and a DNA encoding a reporter protein having relatively high stability in host cells is preferable.

In order to obtain the transformant used in the evaluation method of the present invention, for example, the DNA of the present invention and the above-described reporter gene are combined with, for example, an estrogen receptor non-endogenous host cell, specifically, for example, a HeLa cell, CV -Transform 1 cells, Hepal cells, NIH3T3 cells, HepG2 cells, C0S1 cells, BF-2 cells, CHH-1 cells, etc. to prepare transformants. Here, the DNA of the present invention may be, for example, operably linked to a promoter operable in a host cell, incorporated into a basic vector, and introduced into the above-described cells. The above reporter gene may also be used by incorporating it into a basic vector. For example, a vector incorporating the DNA of the present invention, a vector incorporating the reporter gene, and a vector containing a selectable marker gene are simultaneously introduced into a host cell, and the transformant is expressed using the expression of the selectable marker gene as an index. By selecting, a transformant in which the reporter gene and the DNA of the present invention have been introduced into the chromosome of the host cell is obtained. Since the transformant can be cryopreserved and can be used after awakening as needed, once it is obtained, these genes are introduced into a host cell and newly renewed every time the assay is performed. Since it is not necessary to obtain a suitable transformant and the performance of the transformant can be kept constant, it is useful, for example, when performing a large-scale screening by an immobilized robot.

While culturing the transformant prepared as described above, for example, for about 1 to several days, a test substance is added to the medium and brought into contact with the transformant, and the reporter gene in the transformant is The expression level is measured. When the receptor of the present invention produced by the transformant is activated by binding of a test substance, transcription of a reporter gene is promoted, The reporter protein encoded by the gene is accumulated in the cells of the transformant or secreted into the medium. By measuring the amount of this protein, the expression level of the reporter gene per transformant cell is measured. Specifically, for example, when luciferase is used as the reporter protein, when luciferin which is a substrate of luciferase is added to the crude cell extract, light is emitted at an intensity proportional to the amount of luciferase in the cell extract. Therefore, by measuring the luminescence intensity with a measuring device such as a luminometer, the amount of luciferase and, consequently, the expression level of the luciferase reporter gene can be determined. Similarly, the expression level of the reporter gene is measured under the condition that the test substance is not brought into contact with the transformant, and the expression level is compared with the expression level of the reporter gene under the condition that the test substance is brought into contact. If the expression level of the reporter gene under the condition in which the test substance is contacted is higher than the expression level of the reporter gene under the condition in which the test substance is not contacted with the transformant, the test substance is It can be evaluated as having agonist activity, that is, having the ability to activate the receptor. Further, for example, the reporter gene may be prepared in the same manner as described above under the conditions in which the above-mentioned transformant is contacted with an estrogen such as E2, and the condition in which the estrogen and the test substance are simultaneously contacted. Is measured. If the expression level of the reporter gene under the condition of contacting the estrogen with the test substance is lower than the expression level of the reporter gene under the condition of contacting the estrogen with the transformant, Can be evaluated as having antagonist activity against the receptor of the present invention, that is, antiestrogenic activity against the receptor. In this manner, for example, a reporter assay is performed using a transformant in which a reporter gene under the transcriptional control of a transcription control region containing an estrogen response element and the DNA of the present invention are introduced into the chromosome of a host cell. By conducting the test and evaluating the anti-estrogenic activity of the test substance, a substance exhibiting anti-estrogenic activity with respect to the receptor of the present invention can be selected.

According to the evaluation method of the present invention as described above, for example, anti-estrogens such as tamoxifen and raloxifene, which suppress the function of the transcription activation region AF2 of the wild-type estrogen receptor, but do not suppress the function of AF1. To the receptor of the present invention Thus, a substance having agonist activity and no antiestrogenic activity can be found. Conversely, a substance that does not exhibit agonist activity with respect to the receptor of the present invention and that exhibits antiestrogenic activity can be selected by such an evaluation method. Therefore, screening of a test substance based on the evaluation method requires estrogen. Useful for the development of pure antiestrogens that show virtually no activity. To determine whether or not the estrogen receptor produced by cells of an animal individual such as a human is the receptor of the present invention, for example, whether or not the estrogen receptor α gene of the individual encodes the receptor of the present invention You should find out.

First, a sample is collected from an animal individual such as a human, and genomic DNA or cDNA is prepared from the sample. For example, Masataka Muramatsu, "Lab Manual Genetic Engineering" (Maruzen; 1988) and TAKARA PCR Technical news No. 2 from samples of cell tissues from which genomic DNA such as hair roots, peripheral blood, and oral epithelial cells can be extracted. Genomic DNA can be prepared according to the usual method described in Takara Shuzo (1991). Specifically, for example, if the sample is hair, wash one hair with hair follicles with sterile water and ethanol, then add BCL-Buffer

[10 mM Tris-Cl (pH 7.5), 5 mM MgCl ₂ , 0.32 M Sucrose, l% (v / v) Triton X-100] 200 μΐ was added, and proteinase K was added to a final concentration of 100 il / ml, SDS Are mixed with each other to a final concentration of 0.5% (w / v). After keeping this mixture at 70 ° C. for 1 hour, genomic DNA can be obtained by performing phenol / chloroform extraction. When the sample is peripheral blood, genomic DNA can be obtained by treating the sample with a DNA-Extraction Kit (Stratagene) or the like. When the sample is a tissue sample obtained by surgery or biopsy, while the sample is fresh, RNA is prepared using, for example, TRIZOL reagent (manufactured by Invitrogen), and the prepared RNA is analyzed. If cDNA is synthesized using reverse transcriptase as a template, the synthesized cDNA can be used for testing instead of genomic DNA. To examine whether the estrogen receptor _α gene present in the genomic DNA or cDNA prepared in this way encodes the receptor of the present invention, for example, For example, the amino acids constituting the estrogen receptor alpha encoded by the gene, which are based on the homology of the amino acid sequences, are identified by the amino acid numbers 404, 405 of the amino acid sequence represented by SEQ ID NO: 1. Or a nucleotide sequence encoding an amino acid at a position corresponding to the amino acid represented by 424 is different from the amino acid at a position corresponding to the amino acid in the amino acid sequence of the wild-type estrogen receptor. It is advisable to check whether or not the nucleotide sequence is replaced by the coding base sequence. As a method therefor, for example, a region containing the above-mentioned `` amino acid at a position corresponding to the amino acid number 404, 405 or 424 of the amino acid sequence represented by SEQ ID NO: 1 '' is encoded. A method of amplifying a nucleic acid to be amplified with the nucleic acid in the sample as type III and determining the base sequence of the amplified nucleic acid can be mentioned.

A nucleic acid encoding a region including "an amino acid at a position corresponding to the amino acid represented by amino acid No. 404, 405 or 424 of the amino acid sequence represented by SEQ ID NO: 1" may be amplified by, for example, PCR. Can be.

As a primer that can be used for such PCR, a region containing `` the amino acid at a position corresponding to the amino acid shown by amino acid No. 404, 405 or 424 of the amino acid sequence shown by SEQ ID NO: 1 '' Has the ability to amplify the nucleic acid encoding by PCR, has a GC content of about 30% to about 70%, preferably about 35% to about 60%, and 8 to 50 bases, preferably Can produce an oligonucleotide consisting of about 15 bases to about 40 bases. More specifically, for example, as the oligonucleotide for the forward primer, an oligonucleotide consisting of the nucleotide sequence represented by any one of SEQ ID NOs: 7 to 11 can be mentioned, and as the oligonucleotide for the reverse primer, SEQ ID NO: 1 An oligonucleotide having a base sequence represented by any one of 2 to 16 can be used.

When PCR is performed using the above-described oligonucleotides as primers, two types of oligonucleotides, one for forward and one for reverse, are generally used in combination. These oligonucleotides can be chemically synthesized, for example, by a 3-cyanoethylphosphoramidite method or a thiophosphite method.

PCR is described in, for example, Saiki, RK et al., Science, 230: 1350-1354 (1985). It can be performed according to the method. For example, an amplification buffer containing oligonucleotides used as primers, DNA polymerase, four types of bases (dATP, dTTP, dGTP, dCTP), about 1.5 mM to about 3.0 mM magnesium chloride, and genomic DNA Prepare solution. Using the prepared amplification buffer, for example, a three-step amplification cycle under the following conditions is repeated. First, as a denaturation step, for example, at about 90 ° C. to about 95 ° C., preferably about 94 ° C. to about 95 ° C., for about 1 minute to about 5 minutes, preferably about 1 minute to about 2 minutes Insulation is performed. Next, as a step of annealing the primer, for example, at about 30 to about 70 ° C, preferably about 40 ° C to about 60, for about 3 seconds to about 3 minutes, preferably for about 5 seconds to about 2 minutes. Insulation is performed. Further, as an extension step by DNA polymerase, for example, about 70 ° C. to about 75 ° C., preferably about 72 ° C. to about 74 ° C., for about 15 seconds to about 5 minutes, preferably for about 30 seconds to about 4 minutes Is kept warm. The amplification cycle consisting of three steps is carried out about 20 to about 50 times, preferably about 25 to about 40 times.

After the nucleic acid amplified by such PCR is subjected to agarose electrophoresis and collected, the nucleotide sequence of the DNA is confirmed by performing a direct sequence [BioTechniques, 7, 494 (1989)] on the collected nucleic acid. can do. The nucleotide sequence can be determined by the Maxam Gilbert method (eg, Maxam, A.M. and Gilbert, W., Proc. Natl. Acad. Sci. USA., 74; 560, 1977) or the Sanger method (eg, Sanger, F. and Coulson, AR, J. ol. Biol., 94; 441, 1975., Sanger, F., Nicklen, S. and Coulson, AR, Proc. Natl. Acad. Sci. USA., 74; 5463, 1977). You only need to analyze it. When using an automatic DNA sequencer such as model 3700 manufactured by Applied Biosystems, a corresponding DNA sequence kit, for example, BigDye terminator cycle sequencing ready reaction kit manufactured by AFP Systems Co., Ltd. can be used. In the nucleic acid in the sample, the amino acids constituting the estrogen receptor α, and the alignment based on the homology of the amino acid sequence, the amino acid sequence of the amino acid sequence represented by SEQ ID NO: 1 The nucleotide sequence encoding the amino acid at the position corresponding to the amino acid shown in 4 is different from the amino acid at the corresponding position in the amino acid sequence of wild-type estrogen receptor _α. As a method for checking whether or not the amino acid is substituted in the column, for example, the amino acid sequence represented by the amino acid sequence represented by the amino acid sequence represented by SEQ ID NO: 1 A nucleic acid encoding a region containing `` the amino acid at the corresponding position '' is amplified with the nucleic acid in the sample as type 、, and the amplified nucleic acid is subjected to electrophoresis to measure the mobility of the nucleic acid. A method of examining whether or not the mobility of the nucleic acid encoding the relevant region of the receptor α is different from the mobility of the nucleic acid may be mentioned.

For example, when examining the estrogen receptor c gene in a human sample, first, for example, the end of the oligonucleotide having the nucleotide sequence represented by any one of SEQ ID NOs: 7 to 16 is treated with a fluorescent substance such as FITC. After labeling, use this as a primer and perform PCR as described above, and confirm that `` at a position corresponding to the amino acid represented by amino acid number 404, 405 or 424 of the amino acid sequence represented by SEQ ID NO: 1. Amplify nucleic acids that encode regions containing "amino acids." In addition, a nucleic acid encoding the corresponding region of the wild-type estrogen receptor _α is amplified in the same manner.

The amplified nucleic acid is subjected to electrophoresis according to, for example, the SSCP (single strand conformation polymorphism) method described in Hum. Mutation, 2; 338. Specifically, PCR is performed using a fluorescently labeled primer, and the amplified nucleic acid is denatured by heating to form a single strand, which is subjected to non-denaturing polyacrylamide electrophoresis to separate the single strands. Buffers used for electrophoresis include tris-phosphate (pH 7.5-8.0), tris-acetic acid (pH 7.5-8.0), and tris-boric acid (pH 7.0). 5-8.3) and the like, and preferably a tris-borate-based compound. If necessary, add glycerol to the gel. The conditions of the electric swimming include, for example, electrophoresis at a constant power of 30 W to 40 W, at room temperature (about 20 ° C. to about 25 ° C.) or at about 4 ° C. for about 4 hours to about 8 hours. it can. Next, the fluorescence signal in the gel after electrophoresis is detected by a scanner capable of reading fluorescence, and the amino acid number of the amino acid sequence represented by SEQ. The mobility of a nucleic acid encoding a region containing an amino acid at a position corresponding to the amino acid represented by 424 is compared with the mobility of a nucleic acid encoding the region of the wild-type estrogen receptor. When these nucleic acids have different mobilities, the amino acid sequence represented by the amino acid sequence represented by SEQ ID NO: 1 corresponds to the amino acid sequence represented by amino acid number 404, 405 or 424. It can be determined that the nucleotide sequence encoding the region containing the “amino acid at the position” contains a nucleotide sequence different from the nucleotide sequence encoding the region of the wild-type estrogen receptor α. Furthermore, the nucleic acid contained in the gel containing nucleic acids having different mobilities is extracted into sterilized water, the nucleic acid is converted into a 錄 form, the nucleic acid is amplified by PCR, and the amplified product is pGEM-T Easy After cloning into a vector such as a vector (manufactured by Invitrogen), the nucleotide sequence of the cleaved nucleic acid can be confirmed. Thus, a nucleotide sequence different from the nucleotide sequence encoding the wild-type estrogen receptor can be specified. In the nucleic acid in the sample, the amino acids constituting the estrogen receptor α, and in an alignment based on the homology of the amino acid sequence, the amino acid numbers of the amino acid sequence represented by SEQ ID NO: 1 A nucleotide sequence encoding an amino acid at a position corresponding to the amino acid represented by 5 or 4 24 is different from an amino acid at an amino acid position corresponding to the amino acid sequence of the wild-type estrogen receptor A method for examining whether or not the amino acid sequence has been substituted includes, for example, a probe consisting of a nucleotide sequence encoding a region containing an amino acid at a position corresponding to the amino acid in the amino acid sequence of wild-type estrogen receptor _α. And a method for examining the efficiency of hybridization with a nucleic acid in a sample.

In the amino acid sequence of the wild-type estrogen receptor, a region containing an amino acid at a position corresponding to the amino acid No. 404, 405 or 424 of the amino acid sequence shown in SEQ ID NO: 1 Examples of the probe consisting of the base sequence to encode include oligonucleotides consisting of a strong base sequence and having a GC content of about 30% or more and about 60% or less. The number of bases constituting the oligonucleotide is preferably about 15 or more and about 40 or less so as to be suitable for detection by hybridization. Specifically, in the amino acid sequence of human-derived wild-type estrogen receptor α, phenylalanine at a position corresponding to the amino acid shown by amino acid number 404 in the amino acid sequence shown by SEQ ID NO: 1 Examples of a probe consisting of a base sequence encoding a region including a nucleotide include a probe consisting of the base sequence represented by any one of SEQ ID NOS: 17 to 21. Human Of the amino acid sequence of the wild-type estrogen receptor α, which comprises an alanine-containing region at a position corresponding to the amino acid No. 405 of the amino acid sequence No. 1 shown in SEQ ID NO: 1. Examples of the probe include a probe having a base sequence represented by SEQ ID NO: 17, 18, 18, 20 or 21. Nucleotide sequence encoding a region containing isoleucine at a position corresponding to the amino acid shown by amino acid No. 424 in the amino acid sequence shown by SEQ ID No. 1 in the amino acid sequence of human-derived wild-type estrogen receptor α Examples of the probe consisting of: a probe consisting of the nucleotide sequence represented by any one of SEQ ID NOs: 38 to 42, and the like.

Generally, when the number of bases constituting the oligonucleotide is about 100 or less, the oligonucleotide as described above is chemically synthesized by, for example, the j3-cyanoethylphosphamidide method or the thiophosphite method. be able to.

A nucleic acid such as genomic DNA or cDNA prepared from a sample of an animal individual such as a human as described above is mixed with the above-described probe, and the mixture is usually hybridized under stringent conditions. I do. If necessary, the nucleic acid in the sample comprises, for example, an oligonucleotide consisting of the nucleotide sequence represented by any of SEQ ID NOs: 7 to 11 and a nucleotide sequence represented by any of the SEQ ID NOs: 12 to 16 By PCR using an oligonucleotide as a primer, the `` amino acid at a position corresponding to the amino acid No. 405, 405 or 424 of the amino acid sequence shown in SEQ ID NO: 1 '' was obtained. After amplifying the nucleic acid encoding the containing region, hybridization with the above-described probe can be performed.

As stringent conditions for hybridization, for example, prehybridization and hybridization may be performed using 6 × SSC (0.9 M NaCl, 0.09 M sodium citrate), 5 × Denhardt solution (0 l% (w / v) Ficoll 400, 0.1% (w / v) polyvinylpyrrolidone, 0.1% BSA), 0.5% (w / v) SDS and 100 g / ml denatured salmon Force in the presence of sperm DNA or in a DIG EASY Hyb solution (Boehringer Mannheim) containing lOOig / ml denatured salmon sperm DNA, and wash with 1X SSC (0.15M NaCl, 0.015M sodium citrate) ) And 0.5% SDS in the presence of 0.5X SSC (0.15M NaCl, 0.0015M sodium citrate) and 0.5% SDS. , Keep for 30 minutes The conditions for heating can be increased. The incubation temperature in the prehybridization, hybridization and washing steps can be varied according to the length and composition of the probe used, and is generally based on the same Tm value as the Tm value of the probe. Is also set to a slightly lower temperature. Specifically, for example, in hybridization, assuming base pairs when an interbase hydrogen bond is formed between a probe and a nucleic acid in a sample, one base pair of A and one base is assumed. Assuming 2 ° C, 4 ° C for each G and C base pair, sum the values of all base pairs that form hydrogen bonds and use this as the Tm value. For the pre-hybridization, hybridization, and washing steps, the temperature is selected to be the same as the Tm value calculated in this way or a temperature lower by about 2 ° C to 3 ° C. .

Specific procedures of the dot blot hybridization method include, for example, first, genomic DNA or cDNA prepared from a sample of an animal individual such as a human, or `` SEQ ID NO: 1 amplified by PCR. A nucleic acid encoding a region containing `` the amino acid at a position corresponding to the amino acid number 404, 405 or 424 of the amino acid sequence shown '', for example, at 90 ° C. to 100 ° C. After keeping it warm for 3 to 5 minutes, spot it on a nylon filter [Hybond N + (Amersham Biotech) etc.], dry the spotted filter on filter paper, and irradiate it with ultraviolet light. To the filter. Next, the obtained DNA-immobilized filter and the above-mentioned probe are hybridized by incubating, for example, at 40 ° C. to 50 ° C. for 10 to 20 hours, and the hybrid containing the probe is subjected to a conventional method. To detect. In the case of using a radioactive probe labeled with a radioactive isotope such as ^{3 2} P as a probe, it is possible to detect the hybrid comprising the probe by photosensitive filter after Haiburidi's the X-ray film. When using a non-radioactive probe labeled with a biotinylated nucleotide, the hybrid containing the probe is enzymatically labeled with streptavidin using a biotinylated alkaline phosphatase, etc., and the color or luminescence of the substrate due to the enzyme reaction is detected. Thus, a hybrid containing a probe can be detected. A non-radioactive probe directly labeled with an enzyme such as alkaline phosphatase or peroxidase via a spacer can also be used. No hybrid containing the probe is detected in the test sample or wild-type estrogen When the amount of the hybrid detected in the test sample is smaller than the amount of the hybrid detected in the nucleic acid containing the DNA encoding the receptor α, the nucleotide sequence of the probe used for the nucleic acid in the test sample is used. Can be determined to contain a different base sequence.

When using a mismatch detection method that uses an enzyme called Taq MutS, which is an enzyme that binds to the mismatch hybridization site, it is stable against heat (0 to 75 ° C) and maintains its activity even at high temperatures. Utilizes the binding characteristics of Taq MutS, which can recognize and bind to mismatched base pairs in DNA, and use gel shift assays using native polyacrylamide gels or solid phases such as nylon filters and nitrocellulose filters. The mismatched base pair can be detected by the dot blotting method described above. If a mismatch is detected, encode a region containing `` the amino acid at a position corresponding to the amino acid No. 404, 405 or 424 of the amino acid sequence shown by SEQ ID NO: 1 '' Can be determined to contain a base sequence different from the base sequence of the probe used. In the nucleic acid in the sample, the amino acids constituting the estrogen receptor c, and in an alignment based on the homology of the amino acid sequences, the amino acid sequence of the amino acid sequence represented by SEQ ID NO: 1 in the amino acid sequence of SEQ ID NO: 1; 4 The nucleotide sequence encoding the amino acid at the position corresponding to the amino acid shown in 4 is replaced with a nucleotide sequence encoding an amino acid different from the amino acid at the corresponding position in the amino acid sequence of the wild-type estrogen receptor. As a method for examining whether or not the amino acid is represented by the position corresponding to the amino acid represented by amino acid No. 404, 405 or 424 of the amino acid sequence represented by SEQ ID NO: 1 A nucleic acid encoding a region containing an amino acid in the sample is amplified from a gene in the sample, and the amplified nucleic acid is replaced with a base sequence to produce a recognition sequence for the nucleic acid. A method of examining the presence or absence of a recognition sequence for the restriction enzyme by extinguishing with a restriction enzyme that appears or disappears can also be used.

For example, when examining the estrogen receptor gene in a human sample, first, genomic DNA or cDNA prepared from the sample is referred to as type III, for example, SEQ ID NO: PCR was performed as described above using an oligonucleotide consisting of the nucleotide sequence represented by any one of 7 to 16 as a primer, and `` amino acid number 404, 405 or 42 of the amino acid sequence represented by SEQ ID NO: 1 '' A nucleic acid encoding a region containing an amino acid at a position corresponding to the amino acid indicated by 4 is amplified. In addition, a nucleic acid encoding a region corresponding to the wild-type estrogen receptor is similarly amplified. The amplified nucleic acid is digested with a restriction enzyme whose recognition sequence appears or disappears due to base substitution. When the obtained nucleic acid digest is subjected to gel electrophoresis using agarose, polyacrylamide, or the like, the nucleic acid digest from the test sample and the nucleotide sequence encoding the region corresponding to wild-type estrogen receptor α can be obtained. Based on the difference in the fragment pattern from the digested nucleic acid, it can be determined whether or not the nucleic acid in the sample has a nucleotide sequence different from the nucleotide sequence encoding the wild-type estrogen receptor.

The determination of the estrogen receptor _α genotype in a sample derived from an animal individual such as a human, which can be performed as described above, is based on the determination of the estrogen receptor _α of the individual with respect to estrogen activity modulators such as antiestrogenic substances. It is very useful in predicting reactivity and predicting the efficacy of treatment such as administration of the substance before starting treatment. In addition, by determining the genotype of the receptor in a specific tissue during or after administration of an estrogen activity modulator such as an anti-estrogen substance, it is also useful for determining the duration and efficacy of treatment with the administered substance. It is. Example

Hereinafter, the present invention will be described in more detail with reference to Examples, but the present invention is not limited to these Examples. Example 1 (Preparation of Expression Plasmid for DNA Encoding Human Wild-Type Estrogen Receptor α)

First, cDNA encoding human wild-type estrogen receptor was obtained. An oligonucleotide having the nucleotide sequence represented by SEQ ID NO: 3 and an oligonucleotide having the nucleotide sequence represented by SEQ ID NO: 4 were chemically synthesized. Human liver-derived cDNA ( Quick clone cDNA # 7113-l; manufactured by Clontech) 10 ng was made into type III, and 10 pmol of each of the oligonucleotide having the nucleotide sequence of SEQ ID NO: 3 and the oligonucleotide having the nucleotide sequence of SEQ ID NO: 4 were added. Then, PCR was performed using LA Taq DNA polymerase (Takara Shuzo) and a buffer attached to the enzyme. In the PCR, the reaction solution was maintained at a temperature of 95 ° C. for 1 minute, followed by 68 ° C. for 3 minutes for 35 cycles using PCR System 9700 (manufactured by Applied Biosystems). Next, the entire amount of the reaction solution was subjected to agarose gel electrophoresis using agarose (Agarose S: Futaba Gene). After confirming that about 1.8 kb of DNA was amplified, the DNA was recovered. A sample for the direct sequence is prepared using a part of the recovered DNA and a dye terminator-sequence kit FS (manufactured by Applied Biosystems), and the sample is prepared using an automatic DNA sequencer (manufactured by Applied Biosystems, Inc.). Model 3700) was used for nucleotide sequence analysis. As a result, it was confirmed that the recovered DNA had a nucleotide sequence encoding the amino acid sequence represented by SEQ ID NO: 1. Next, about 100 ng of the DNA obtained as described above was converted into a type II, and an oligonucleotide consisting of the base sequence shown in SEQ ID NO: 22 and an oligonucleotide consisting of the base sequence shown in SEQ ID NO: 4 were separated. PCR was performed under the same conditions as described above to amplify DNA having a nucleotide sequence in which a nucleotide sequence encoding human wild-type estrogen receptor was ligated immediately after the Kozak consensus sequence. The obtained DNA was separated by low melting point agarose gel electrophoresis, and a DNA of about 1.8 kb was recovered. Approximately l / g was treated with a DNA Blunting Kit (manufactured by Takara Shuzo Co., Ltd.) to blunt its ends, and then reacted with T4 polynucleotide kinase to phosphorylate the ends. . After the obtained DNA was treated with phenol, it was purified by an ethanol precipitation method, and the entire amount was used as the insert DNA for preparing the following expression plasmid. Plasmid pRc / RSV (manufactured by Invitrogen) was digested with a restriction enzyme HindIII, phenol-treated, and then precipitated with ethanol to recover DNA. Recovered DNA into DNA

The ends were blunted by treatment with a Blunting Kit (Takara Shuzo) and subjected to agarose gel electrophoresis using a low-melting point agarose (Nippon Gene; Agarose L), and DNA in a band portion was recovered from the gel. Bacterial alkaline to about 100 ng of recovered DNA After adding phosphatase (BAP) and incubating at 65 ° C for 1 hour, the mixture was mixed with the whole amount of the insert DNA, and T4 ligase was added to carry out a ligation reaction. The resulting reaction solution was used to transform E. coli DH5 recombinant cells (manufactured by Toyobo Co., Ltd.), plasmid DNA was prepared from ampicillin-resistant colonies, and its base sequence was automatically sequenced using an automatic DNA sequencer. It was determined by the Dye Terminator method using Applied Biosystems, Model 3700). The obtained nucleotide sequence is compared with the nucleotide sequence obtained by the above-mentioned direct sequence, and a plasmid having a confirmed that the nucleotide sequence of the translation region is completely identical is selected, and pRc / RSV- Named hERa Kozak. Example 2 (Preparation of Expression Plasmid of DNA Encoding Receptor of the Present Invention F404L) Plasmid pRc / RSV-hERa Kozak prepared in Example 1 was made into type III, and a synthetic oriconucleotide for base substitution and Quickchange Site- Mutations were introduced using a directed mutagenesis Kit (manufactured by Stratagene) according to the method described in the kit instructions. First, an oligonucleotide consisting of the base sequence represented by SEQ ID NO: 5 and an oligonucleotide consisting of the base sequence represented by SEQ ID NO: 6 were chemically synthesized. The extension reaction was carried out using pRc / RSV-hER a Kozak as a 铸 type, and using the two oligonucleotides as primers, Pfu Turbo DNA polymerase (Stratagene) and four types of bases (dATP, dTTP, 200 μM each). , dGTP, dCTP), and in a dedicated buffer attached to the enzyme, 95 ° C, 30 seconds, then 55 ° C, 1 minute, and further 68 ° C, 10 minutes as one cycle. The test was carried out under a cycle condition. Next, a part of this reaction solution is taken and the restriction enzyme Dpn I (

(Stratagene) at 37 ° C for 1 hour. Escherichia coli XLI-Blue competent cells (Stratagene) were transformed using the digested solution. From each of several colonies of E. coli that showed ampicillin resistance, the plasmid DNAs possessed by each were purified, and their nucleotide sequences were deciphered. In the amino acid sequence represented by SEQ ID NO: 1, it was confirmed that a mutation in which the codon (TTT) encoding phenylalanine represented by amino acid number 404 was replaced with a codon (CTT) encoding leucine was identified. The name was pRc / RSV-hERaF404L Kozak. Example 3 (Preparation of Plasmid Containing Reporter Gene and Selectable Marker Gene) Oligonucleotide consisting of a base sequence (base sequence represented by SEQ ID NO: 23) upstream of Afat Xenopus vitellogenin gene containing an estrogen response element and said base An oligonucleotide consisting of a nucleotide sequence complementary to the sequence was synthesized using a DNA synthesizer. These are annealed to form double-stranded DNA (the DNA is hereinafter referred to as ERE DNA), and then T4 ligase is actuated to bind the ERE DNA to tandem, which is then acted upon by T4 polynucleotide kinase. Both ends were phosphorylated.

In addition, two oligonucleotides consisting of the nucleotide sequence near the TATA box of the mouse metallothionein I gene and the nucleotide sequence derived from the leader sequence, that is, an oligonucleotide consisting of the nucleotide sequence represented by SEQ ID NO: 24 and SEQ ID NO: 25, An oligonucleotide having the base sequence shown was subjected to a double-stranded DNA reaction, and T4 polynucleotide kinase was acted thereon to phosphorylate both ends of the double-stranded DNA (the DNA is hereinafter referred to as TATA DNA). . On the other hand, plasmid PGL3 (promega) containing the firefly luciferase gene was digested with restriction enzymes Bgl II and Hind III, and bacterial alkaline phosphatase (BAP) was added thereto. Incubated for hours. Next, the heat-retained solution was subjected to electrophoresis using low-melting point agarose (Agarose L; manufactured by Futatsu Gene Co., Ltd.), and DNA was recovered from the gel at the band portion. Approximately 100 ng of the DNA was mixed with 1 / g of the TATA DNA and bound with T4 ligase to prepare plasmid pGL3-TATA.

Next, pGL3-TATA was digested with a restriction enzyme SmaI, BAP was added thereto, and the mixture was incubated at 65 ° C for 1 hour. The incubation solution was subjected to low-melting point agarose gel electrophoresis, and DNA was recovered from the gel in the band portion. After mixing about 100 ng of the DNA and about the ERE DNA bound to the tandem and phosphorylated at the end to react with T4 ligase, the reaction solution is used to form a DH5a competent cell of Escherichia coli (Toyobo Co., Ltd.). ) Was transformed. From several colonies of Escherichia coli showing ampicillin resistance, the DNA of each plasmid was purified, digested with restriction enzymes KpnI and XhoI, and the digested solution was analyzed by agarose gel electrophoresis. A plasmid having a structure in which five copies of ERE DNA were introduced in tandem at the SmaI site of pGL3-TATA was selected, and named plasmid pGL3-TATA_EREx5. Next, the plasmid pUCSV-BSD (Funakoshi) was digested with BamHI to prepare a DNA encoding the blasticidin S deaminase gene expression cassette. The DNA is mixed with the DNA obtained by digesting the plasmid pGL3-TATA-EREx5 with BamHI and treating with BAP, and reacting with T4 ligase. Tent cells (Toyobo) were transformed. Plasmid DNA was prepared from Escherichia coli showing ampicillin resistance, each was digested with the restriction enzyme BamHI, and the digested solution was analyzed by agarose gel electrophoresis. A plasmid having a structure in which the blasticidin S deaminase gene expression cassette was introduced into the BamHI site was selected and named plasmid pGL3-TATA_EREx5-BSD. Example 4 (Preparation of Stable Transformed Cells for Reporter Atsushi)

The DNA of the plasmid pGL3-TATA_EREx5-BSD prepared as described above was linearized and introduced into human-derived HeLa cells to prepare stable transformed cells.

First, DNA of plasmid pGL3-TATA-EREx5-BSD was digested with SalI.

On the other hand, the HeLa cell of approximately 5 x lO ⁵ cells, 10 ° /. The cells were cultured in a DMEM medium containing FBS (manufactured by Nissui Pharmaceutical Co., Ltd.) at 37 ° C. in the presence of 5% CO ₂ for 24 hours using a petri dish having a diameter of about 10 cm (manufactured by Falcon). The linearized plasmid pGL3-TATA-EREx5-BSD DNA was introduced into the cells by a lipofection method using lipofectamine (manufactured by Invitrogen). The conditions for the ribofecting method were as described in the manual attached to the ribofectamine, the processing time was 5 hours, the total amount of linearized plasmid DNA was TgZ Petri dish, and the amount of ribofectamine was 21/1 Petri dish. did. After ribofection, remove the medium at 10 ° /. The medium was replaced with a DMEM medium containing FBS and cultured for about 36 hours. Next, the cells are detached from the Petri dish by trypsinization and collected, transferred to a culture vessel containing a medium containing a final concentration of blasticidin S at a final concentration of ml, and the medium is cultivated every 3 to 4 days. The cells were cultured for about one month while exchanging them for Scidin S. A cell colony of several mm from the appearing cell was transferred to a 96-well view plate (manufactured by Berthold) into which the medium had been previously dispensed, and further cultured. Once the cells have grown to cover more than half of the bottom of the well (about 5 days after transplantation), Cells were detached and collected by trypsinization, divided into two equal parts, and seeded on two new 96-well view plates. Subculture and culturing were continued for one sheet as it was, and used as a master plate. After culturing the remaining one for 2 days, remove the medium from the wells, wash the cells adhering to the vessel wall twice with PBS (-), and dilute PGC50 (manufactured by Toyo Ink Co., Ltd.) diluted 5 times with the wells. 20 μl each and left at room temperature for 30 minutes. Each of the plates was set on a luminometer LB96p (manufactured by Berthold) with an automatic enzyme substrate injector, and 50 μl of a substrate solution PGL100 (manufactured by Toyo Ink) was automatically dispensed to measure luciferase activity. Among them, 10 cells showing high luciferase activity were selected and stored. Further, each of the cells was expanded and cultured on a 10 cm plate. The wild-type estogen receptor ct expression plasmid prepared in Example 1 was introduced into these 10 cells by a lipofection method using lipofectamine (manufactured by Invitrogen) according to the description of the attached manual. E2 dissolved in DMS0 was added to the obtained cells so that the final concentration in the medium was 10 nM, and the cells were cultured for 2 days, and luciferase activity was measured according to the method described above. Select the cells before the introduction of the wild-type estrogen receptor-expressing plasmid corresponding to the cells that showed the highest level of activity induction in the system to which E2 was added, as the stable transfected cells with the reporter gene introduced for the reporter assay. did. Example 5 (Reporter assay using stable transformed cells)

Approximately 2 × 10 ⁶ cells of the reporter stable transfected cells selected in Example 4 were seeded on a 10-cm plate, and E-MEM medium supplemented with 10% of charcoal dextran-treated FBS was added. (hereinafter referred to as FBS-containing E-MEM medium), the was one day culture at 5% C0 ₂ under 37 ° C. To the cell, Lvov We accordance with the flop port tocol using transfected § Min (Ir itrogen Inc.), 7 human wild-type estrogen receptor ig shed gene expression plasmid _P Rc / RSV_hER a Kozak or human mutant estrogen Receptor gene expression plasmid pRc / RSV-hERaF404L Kozak was introduced. After culturing at 37 ° C for 16 hours, the medium was replaced and the cells were further cultured for 3 hours. The cells were then collected, suspended and homogenized in E-MEM medium containing FBS, and seeded in a 96-well plate containing various concentrations of antiestrogenic compounds previously dissolved in DMS0 (DMS0 final concentration 0.1%). did. Similarly, various concentrations of anti-s The cells were seeded on a 96-well plate in which a trogen-like compound and 10 nM E2 were simultaneously added (DMS0 final concentration: 0.1%). The 96-well plate in which the cells have been seeded is cultured at 37 ° C for about 40 hours, and the cell lysing agent PGC50 (manufactured by Futtsubon Gene), diluted 5 times, is added at 50 / l Zwell at a time, and then shaken occasionally at room temperature. And left for 30 minutes to lyse the cells. 10 μl of the cell lysate prepared in this manner was collected into a 96-well white sample plate (Berthold), and the enzyme substrate solution was added at 50 μΐ / well using a luminometer LB96p (Berthold) with an automatic substrate injector. PGL100 (manufactured by Futtsubon Gene) was added, and the light emission was measured immediately for 5 seconds.

The results of measuring the estrogenic effect of 4-hydroxytamoxifen, raloxifene or ZM189154 on the wild-type estrogen receptor α or the receptor F404L of the present invention are shown in FIGS. 1 to 3, respectively.

4 to 6 show the results of measuring the antiestrogenic action of 4-hydroxytamoxifen, raloxifene or ZM189154 on wild-type estrogen receptor α or the receptor F404L of the present invention, respectively. Example 6 (Preparation of amplification oligonucleotide)

The nucleotide sequence S encoding the amino acid at the position corresponding to the amino acid No. 404 of the amino acid sequence represented by SEQ ID NO: 1, the amino acid at the corresponding position in the amino acid sequence of the wild-type estrogen receptor α In order to detect whether or not the amino acid sequence has been replaced with a nucleotide sequence encoding a different amino acid, amplification of the nucleic acid at the 404th phenylalanine-encoding site from the amino terminal of the human wild-type estrogen receptor is performed. Design oligonucleotides that fall within the range and have a GC content between 30% and 70% and a length of about 20 bases. An oligonucleotide is prepared based on the designed nucleotide sequence. (Hereinafter, the amino acid at the corresponding position of the base sequence forces the wild-type amino acid sequence of the estrogen receptor _α encoding the amino acid at the position corresponding to amino acid represented by amino acid numbers 4 0 4 of the amino acid sequence shown in SEQ ID NO: 1 May be referred to as a 404 mutation when the mutation is replaced by a nucleotide sequence encoding a different amino acid.) Example 7 (Analysis of 404 mutation using human tissue as material)

Add 100 mg of frozen human liver sample to 5 ml of [4 M guar-zine thiosinate, 0.1 M Tris-Cl (pH 7.5) 1% / 3_mercaptoethanol], and grind with a polytron homogenizer. . This is overlaid on 25 ml of a 5.7 M cesium chloride solution previously placed in an ultracentrifuge tube, and RNA is separated by performing a density gradient ultracentrifugation at 90,000 X g for 24 hours. Collect the RNA, rinse with 70% ethanol, and air-dry at room temperature. Dissolve this in sterile water 10 and measure the concentration. And the RNA 1 to 5 _§ to鎳型, oligo dT primer (Amersham Biotech) using l // g as a primer during reverse transcription synthesis, in the accompanying buffer by Superscript II (Invitrogen) The cDNA is synthesized by reacting at 42 ° C for 1 hour. One-fiftieth of the cDNA solution thus obtained was made into type III, and an oligonucleotide having the base sequence of SEQ ID NO: 10 and an oligonucleotide having the base sequence of SEQ ID NO: 16 were separated. Perform PCR using The PCR uses the Pfu DNA Polymerase (Stratagene Co., Ltd.), 200 _mu 4 kinds of each of the bases of Μ (dATP, dTTP, dGTP, dCTP) and in only buffer one attached to the enzyme, 94 ° C, 1 minute, then 55 ° C, 30 seconds, 72 ° C, 1 minute, 1 cycle, 35 cycles. Amplified DNA at 1 ° /. Separate by electrophoresis in a gel containing Agarose S (Agarose S, manufactured by Futaba Gene) and collect. Using this total amount as a type I, a sample for direct sequence was prepared using a dye terminator-sequence kit FS (manufactured by Applied Biosystems) using 5 pM of oligonucleotide having the nucleotide sequence of SEQ ID NO: 11 as a sequence primer. Prepare. This is subjected to nucleotide sequence analysis using an automatic DNA sequencer (Applied Biosystems, Model 3700) to determine the nucleotide sequence. In this manner, the nucleotide sequence encoding the amino acid at the position corresponding to the amino acid represented by amino acid number 404 in the amino acid sequence represented by SEQ ID NO: 1 is clarified. Example 8 (extraction of genomic DNA contained in specimen)

Transfer one hair with hair follicle to a plastic tube and add BCL buffer [10 mM 200 µl of Tris-Cl (pH 7.5), 5 mM MgCl ₂ , 0.32 M sucrose, l% (v / v) Triton X-100], and a final concentration of 100; g / ml proteinase. The solution and the final concentration of 0.5% (w / v) SDS are mixed respectively. Incubate the mixture at 70 ° C for 1 hour, add an equal volume of chloroform // shake the form, shake vigorously, and centrifuge (15,000 rpm, 5 minutes, 4 ° C). Collect the aqueous layer and perform another phenol extraction. Add an equal volume of black-mouthed form to the collected aqueous layer, shake vigorously, and collect the aqueous layer by centrifugation. This is 500 μl of 100 ° /. Add ethanol, incubate at -80 ° C for 20 minutes, and centrifuge. After drying the obtained pellet, dissolve it in sterile water.

In addition, peripheral blood is used as cells from which genomic DNA can be collected. Collect 10 ml of blood, and extract genomic DNA using a DNA Extraction kit (Stratagene) according to the instructions attached to the kit. Example 9 (Analysis of 404 mutation by PCR—SSCP method)

First, a forward primer is selected from an oligonucleotide consisting of the nucleotide sequence shown in any of SEQ ID NOs: 7 to 11, and a reverse primer is obtained from an oligonucleotide consisting of the nucleotide sequence shown in any of SEQ ID NOs: 12 to 16. And chemically synthesize it with a DNA synthesizer. At this time, the 5 'end is modified with the fluorescent substance FITC. Next, 100 ng of the genomic DNA obtained in Example 8 was converted into type II, and the DNA encoding the amino acid sequence of estrogen receptor α was subjected to PCR using 200 pM of each of the above-mentioned FITC-modified oligonucleotides as primers. Amplify. The PCR was performed using Ex Taq DNA polymerase (Takara Shuzo) and 200 bases (dATP, dTTP, dGTP, dCTP) and the dedicated buffer attached to the enzyme. Perform 40 cycles of 30 ° C, 30 seconds, then 55 ° C, 30 seconds, and 74 ° C, 30 seconds. After the reaction, incubate 1/20 of the resulting amplified product in 95% formamide at 95 ° C for 5 minutes, then quench. Of these, 2.5 1 is 5 ° /. Run on a native polyacrylamide gel and perform electrophoresis in 180 mM Tris-monophosphate buffer (pH 8.0). The electrophoresis conditions are room temperature, constant power of 40 W, and 5 hours. After the electrophoresis, the amplified nucleic acid fragment is detected by detecting the fluorescent signal in the gel with a fluorescence reading scanner. Wild-type estrogens running side by side Compared with the mobility of the band of the amplified product in DNΑ coding for the amino acid sequence of receptor _α, the amplified product in the DNA coding for the amino acid sequence of mutant estrogen receptor c has a different mobility. The presence or absence of can be detected. Example 10 (Analysis of 404 mutation by nucleotide sequence analysis)

A part of the gel at the position corresponding to the DNA band encoding the amino acid sequence of the mutant estrogen receptor c detected in Example 9 was cut into 1 mm squares, and permeated in 400 μl of sterile water. Elute the DNA. After removing the gel and purifying by ethanol precipitation, dissolve the DNA in 50 μl of sterile water. Among them, 1 μl is converted into type 、 and PCR is performed using the oligonucleotide used for PCR-SSCP in Example 9 to amplify DN D encoding the amino acid sequence of the estrogen receptor. PCR was performed using Ex Taq DNA Polymerase (Takara Shuzo) in a buffer attached to the enzyme at 94 ° C for 30 seconds, followed by 55 ° C for 30 seconds, and a further cycle at 74 ° C for 30 seconds. Perform 30 cycles. After completion of the reaction, the amplified DNA is confirmed by agarose gel electrophoresis, and cloned into pGEM-T Easy vector (promega). Using the resulting plasmid as a template, determine the nucleotide sequence using the BigDye Terminator cycle sequence ready reaction Kit (available from Applied Biosystems) and an automatic DNA sequencer (available from Applied Biosystems, model 3700). I do. Thus, the nucleotide sequence encoding the amino acid at the position corresponding to the amino acid represented by amino acid number 404 in the amino acid sequence represented by SEQ ID NO: 1 is clarified. Example 1 1 (Analysis of 404 mutation by combining PCR and restriction enzyme digestion)

PCR was performed using a primer having the nucleotide sequence of SEQ ID NO: 26 and a primer having the nucleotide sequence of SEQ ID NO: 27, using genomic DNA or cDNA as a rust type, to thereby obtain human estrogen receptor α. Promote the DNA encoding the amino acid sequence. The above PCR uses Pfu DNA polymerase (manufactured by Stratagene) in a buffer attached to the enzyme, at 94 ° C for 1 minute, then at 55 ° C for 30 seconds, and further at 72 ° C for 1 minute. For 30 cycles. The resulting 100 bp DNA is treated with the restriction enzyme NheI. The DNA encoding the amino acid sequence of wild-type estrogen receptor α is not digested. On the other hand, DNA encoding the amino acid sequence of the mutant estrogen receptor α in which the phenylalanine at the 4th position from the amino terminal of the human-derived wild-type estrogen receptor is mutated to leucine has the sequence GCTAGC. Digested with NheI to yield 75 bp DNA and 25 bp DNA. Thus, the DNA encoding the estrogen receptor α having the mutation can be detected. Example 1 2 (Preparation of Expression Plasmid of DNA Encoding Receptor A405V of the Present Invention) Plasmid pRc / RSV-hERa prepared in Example 1 was made into a type III, a synthetic oligonucleotide for base substitution and a Quickchange Site Using a -directed mutagenesis Kit (manufactured by Stratagene), a mutation was introduced according to the method described in the instruction manual of the kit. First, an oligonucleotide consisting of the base sequence shown by SEQ ID NO: 29 and an oligonucleotide consisting of the base sequence shown by SEQ ID NO: 30 were chemically synthesized. The extension reaction was carried out using pRc / RSV-hERa Kozak as type III, the two oligonucleotides as primers, Pfu Turbo DNA polymerase (Stratagene) and four types of 200 μM bases (dATP , dTTP, dGTP, dCTP), and in a dedicated buffer attached to the enzyme, 95 ° C, 30 seconds, then 55 ° C, 1 minute, further 68 ° C, 10 minutes incubation as one cycle. This was performed under the condition of performing 16 cycles. Next, a part of this reaction solution was taken and digested with a restriction enzyme Dpn I (Stratagene) at 37 ° C for 1 hour. Escherichia coli XLI-Blue competent cells (Stratagene) were transformed using the digested solution. From each of several colonies of Escherichia coli showing ampicillin resistance, the respective plasmid DNAs were purified and their nucleotide sequences were analyzed. Codon (GCT) encoding alanine represented by amino acid number 405 in the amino acid sequence represented by SEQ ID NO: 1 Plasmid confirmed to have a mutation substituted for valine-encoded codon (GTT) Was named pRc / RSV-hERaA405V Kozak. Example 13 (Reporter assay using stable transformed cells)

Approximately 2 × 10 ⁶ cells of the stable transformant for the reporter assay selected in Example 4 were seeded on a 10-cm plate, and an E-MEM medium (10%) supplemented with charcoal dextran-treated FBS was added at 10%. In the following, the cells were cultured in an E-MEM medium containing FBS at 37 ° C. under 5% CO _{2 for} 1 day. According to the protocol, lipofectamine (manufactured by Invitrogen) was used to prepare the human wild-type estrogen receptor α gene expression plasmid pRc / RSV-hER a Kozak or 7 // g human mutation. Estrogen receptor α gene expression plasmid pRc / RSV-hERaA405V Kozak was introduced. After culturing at 37 ° C for 16 hours, the medium was replaced and the cells were further cultured for 3 hours. Thereafter, the cells were collected, suspended in an E-MEM medium containing FBS, homogenized, and supplemented with various concentrations of antiestrogen-like compound previously dissolved in DMS0 (DMS0 final concentration 0.1%) in a 96-well plate. Seeded. Similarly, the above cells were seeded on a 96-well plate to which various concentrations of an antiestrogenic compound and 10 nM of E2 were simultaneously added (final concentration of DMS0 0.1%). The 96-well plate in which the cells have been seeded is cultured at 37 ° C for about 40 hours. And left for 30 minutes to lyse the cells. In thus prepared cell lysate 10 1 by 96 well white sample plate was taken (manufactured by Berthold), luminometer with substrate automatic injector LB96P (manufactured by Berthold) 50; ul / w _e ll by enzyme The substrate solution PGL100 (manufactured by Futtsubon Gene) was added, and the emitted light was immediately measured for 5 seconds.

The measurement results of the estrogenic effect of 4-hydroxytamoxifen, raloxifene or ZM189154 on the wild-type estrogen receptor α or the receptor A405V of the present invention are shown in FIGS. 7 to 9, respectively.

In addition, the measurement results of the antiestrogenic action of 4-hydroxytamoxifen, raloxifene or ZM189154 on the wild-type estrogen receptor or the receptor A405V of the present invention are shown in FIGS. 10 to 12, respectively. Example 14 (Preparation of oligonucleotide for amplification)

The amino acid sequence of the amino acid sequence represented by SEQ ID NO: 1 Detects whether the nucleotide sequence encoding the amino acid at the corresponding position has been replaced with a nucleotide sequence encoding an amino acid different from the amino acid at the corresponding position in the amino acid sequence of the wild-type estrogen receptor 1α For this purpose, the region encoding the alanine at position 405 from the amino terminus of the human wild-type estrogen receptor is included in the range of nucleic acid amplification, and the GC content is 30% or more and 70% or less, and about 20 bases. Design oligonucleotides that have a length. Create oligonucleotides based on the designed base sequence. (Hereinafter, the nucleotide sequence encoding the amino acid at the position corresponding to the amino acid No. 405 of the amino acid sequence shown by SEQ ID No. 1 is A mutation substituted with a nucleotide sequence encoding an amino acid different from a certain amino acid may be referred to as a 405 mutation.) Example 15 (Analysis of 405 mutation using human tissue as material)

Add 100 mg of the frozen human liver sample to 5 ml of [4 M guanidine thiocyanate, 0.1 M Tris-Cl (pH 7.5) 1% 3-mercaptoethanol], and grind with a polytron homogenizer. This is layered on 25 ml of a 5.7 M cesium chloride solution previously placed in an ultracentrifuge tube, and RNA is isolated by performing density gradient ultracentrifugation at 90,000 X g for 24 hours. Collect the RNA, rinse with 70% ethanol, and air-dry at room temperature. Dissolve this in 10 xl of sterile water and measure the concentration. This l ~ 5 g of RNA was converted into type III, oligo dT primer (Amersham Biotech) l / zg was used as a primer for reverse transcription synthesis, and Superscript II (Invitrogen) was used in the attached buffer. The cDNA is synthesized by reacting at ° C for 1 hour. One-fiftieth of the cDNA solution thus obtained was made into type III, and an oligonucleotide having the base sequence of SEQ ID NO: 10 and an oligonucleotide having the base sequence of SEQ ID NO: 16 were separated. Perform PCR using The PCR was performed using Pfu DNA polymerase (manufactured by Stratagene) at 200 ° C. in a dedicated buffer attached to each of the four bases (dATP, dTTP, dGTP, dCTP) and the enzyme at 94 ° C. Perform 35 cycles of 1 minute, followed by 55 ° C, 30 seconds, and 72 ° C, 1 minute, 1 cycle. Amplified DNA at 1 ° /. Separation and recovery by electrophoresis in a gel containing Agarose S (Agarose S, manufactured by Futatsu Gene) . Using this total amount as a type I, a sample for direct sequence was prepared using a dye terminator-sequence kit FS (manufactured by Applied Biosystems) using 5 pM of oligonucleotide having the nucleotide sequence of SEQ ID NO: 11 as a sequence primer. Prepare. This is subjected to nucleotide sequence analysis using an automatic DNA sequencer (Applied Biosystems, Model 3700) to determine the nucleotide sequence. Thus, the nucleotide sequence encoding the amino acid at the position corresponding to the amino acid represented by amino acid number 405 in the amino acid sequence represented by SEQ ID NO: 1 is clarified. Example 16 (Analysis of 405 mutations by PCR-SSCP method)

First, one forward primer is derived from the oligonucleotide consisting of the nucleotide sequence shown in any of SEQ ID NOs: 7 to 11, and the reverse primer is derived from the oligonucleotide consisting of the nucleotide sequence shown in any of SEQ ID NOs: 12 to 16. And chemically synthesize it with a DNA synthesizer. At this time, the 5 'end is modified with the fluorescent substance FITC. Next, 100 ng of the genomic DNA obtained in Example 8 was converted into type II, and the DNA encoding the amino acid sequence of estrogen receptor α was subjected to PCR using 200 pM of each of the above-mentioned FITC-modified oligonucleotides as primers. Amplify. The PCR was performed using Ex Taq DNA polymerase (Takara Shuzo) and 200 bases (dATP, dTTP, dGTP, dCTP) and the dedicated buffer attached to the enzyme. Perform 40 cycles of 30 ° C, 30 seconds, then 55 ° C, 30 seconds, and 74 ° C, 30 seconds. After the reaction, incubate 1/20 of the resulting amplified product in 95% formamide at 95 ° C for 5 minutes, then quench. Among them, 2.5 μl is applied to a 5% native polyacrylamide gel, and electrophoresed in 180 mM Tris-borate buffer (H8.0). The electrophoresis conditions are room temperature, constant power of 40 W, and 5 hours. After the electrophoresis, the amplified nucleic acid fragment is detected by detecting the fluorescent signal in the gel with a fluorescence reading scanner. Compared with the band mobility of the amplified product in DNΑ encoding the amino acid sequence of wild-type estrogen receptor α, which is run side by side, the amplified product in the DNA encoding the amino acid sequence of the mutant estrogen receptor is Since the mobilities are different, the presence or absence of a mutation in the amplified sequence can be detected. Example 17 (Analysis of 405 mutations by nucleotide sequence analysis)

A part of the gel at a position corresponding to the band of DNΑ encoding the amino acid sequence of the mutant estrogen receptor α detected in Example 16 was cut into 1 mm squares, and permeated in 400 μΐ of sterile water. Elute the DNA. After removing the gel and purifying by ethanol precipitation, dissolve the DNA in 501 sterile water. Among them, Ιμΐ is changed to 铸, and PCR is performed using the oligonucleotide used in PCR-SSCP in Example 9 to amplify DNA encoding the amino acid sequence of estrogen receptor α. PCR was performed using Ex Taq DNA Polymerase (Takara Shuzo) in a buffer attached to the enzyme at 94 ° C for 30 seconds, followed by 55 ° C for 30 seconds, and a further cycle at 74 ° C for 30 seconds. Perform 30 cycles. After completion of the reaction, the amplified DNA is confirmed by agarose gel electrophoresis, and the DNA is cloned into pGEM-T Easy vector (promega). Using the obtained plasmid type II, the nucleotide sequence was analyzed using the BigDye Terminator cycle sequence ready reaction kit (manufactured by Nopphid Biosystems) and an automatic DNA sequencer (model 3700 manufactured by Applied Biosystems). decide. Thus, the nucleotide sequence encoding the amino acid at the position corresponding to the amino acid represented by amino acid number 405 in the amino acid sequence represented by SEQ ID NO: 1 is clarified. Example 18 (Analysis of 405 mutations combining PCR and restriction enzyme digestion)

PCR was performed using genomic DNA or cDNA as a type I primer having the nucleotide sequence of SEQ ID NO: 31 and a primer having the nucleotide sequence of SEQ ID NO: 32 to encode the amino acid sequence of human estrogen receptor α. Amplify DNA. The above PCR uses Pfu DNA polymerase (manufactured by Stratagene) in a buffer attached to the enzyme, at 94 ° C for 1 minute, then at 55 ° C for 30 seconds and then at 72 ° C for 1 minute for 1 cycle. Perform 30 cycles. 100 base pairs in length DNA obtained is treated with a restriction enzyme Bs _P T104 I. The DNA encoding the amino acid sequence of wild-type estrogen receptor α is not digested. On the other hand, a mutant estrogen in which the 405th araene from the amino terminal of human-derived wild-type estrogen receptor α is mutated to valine. DN DN, which encodes the amino acid sequence of receptor α, has the sequence TTCGAA, and is digested with BspT104I to produce 80 base pairs of DNA and 20 base pairs of DNA. Thus, the DNA encoding the estrogen receptor α having the mutation can be detected. Example 19 (Preparation of Expression Plasmid of DNA Encoding Receptor {424} of the Present Invention) Plasmid pRc / RSV-hERa produced in Example 1 was made into type III, and a synthetic oligonucleotate for base substitution was prepared. Mutations were introduced using the Quickchange Site-directed mutagenesis Kit (Stratagene) according to the method described in the kit instructions. First, an oligonucleotide consisting of the nucleotide sequence shown by SEQ ID NO: 34 and an oligonucleotide consisting of the nucleotide sequence shown by SEQ ID NO: 35 were chemically synthesized. The elongation reaction was performed using pFc / RSV-hERa Kozak as type III, the above two kinds of oligonucleotides as primers, Pfu Turbo DNA polymerase (Stratagene) and four kinds of bases (200 μM each). dATP, dTTP, dGTP, dCTP) [], and in a dedicated buffer attached to the enzyme, at 95 ° C for 30 seconds and then 55. C, 1 minute, 68 ° C for 10 minutes, and 10 minutes of incubation as one cycle. Next, a part of this reaction solution was taken and digested with a restriction enzyme Dpn I (Stratagene) at 37 ° C for 1 hour. Escherichia coli XLI-Blue competent cells (Stratagene) were transformed using the digested solution. From each of several colonies of Escherichia coli showing ampicillin resistance, plasmid DNAs possessed by each were purified, and their nucleotide sequences were analyzed. In the amino acid sequence represented by SEQ ID NO: 1, a plasmid in which a mutation in which the codon (ATC) encoding isoleucine represented by amino acid numbers 424 was replaced by a codon (ACC) encoding threonine was confirmed was confirmed.

It was named PRc / RSV-hERaI424T Kozak. Example 20 (Reporter assay using stable transformed cells)

Approximately 2 × 10 ⁶ cells of the reporter-assembly-stable transformed cells selected in Example 4 were seeded on a 10 era plate, and E-MEM supplemented with charcoal dextran-treated FBS so that the FBS became 10%. medium (hereinafter referred to as FBS-containing E-MEM medium), the daily at 5% C0 ₂ under 37 ° C Interculture was performed. The cells were treated with 7 g of human wild-type estrogen receptor α gene expression plasmid pRc / RSV-hER α Kozak or 7 μg of human wild-type estrogen receptor α gene using ribofectamine (Invitrogen) according to the protocol. A mutant pRc / RSV_hERaI424T Kozak was introduced. After culturing at 37 ° C. for 16 hours, the medium was replaced and the cells were further cultured for 3 hours. Thereafter, the cells were collected, suspended and homogenized in E-MEM medium containing FBS, and supplemented with various concentrations of antiestrogen-like compound previously dissolved in DMS0 (final concentration of DMS0 0.1%) in a 96-well plate. Seeded. Similarly, the above cells were seeded on a 96-well plate to which various concentrations of an anti-estrogens-like compound and 10 nM E2 were simultaneously added (DMS0 final concentration: 0.1%). The 96-well plate in which the cells have been seeded is cultured at 37 ° C for about 40 hours, and then the cell lysing agent PGC50 (manufactured by Etsubon Gene) diluted 5 times is calorificized by SO wlZwell at a time, and occasionally lightly shaken at room temperature. The cells were left for 30 minutes to lyse. The cell lysate prepared in this manner was collected in a 96-well white sample plate (Berthold) in a quantity of 10 1 each, and the enzyme substrate solution was fed at 50 / il / well using a luminometer LB96p (Berthold) with an automatic substrate injector. PGL100 (manufactured by Futtsubon Gene) was added, and the light emission was measured immediately for 5 seconds.

The measurement results of the estrogen-like effects of 4-hydroxytamoxifen, raloxifene or ZM189154 on the wild-type estrogen receptor _α or the receptor I424T of the present invention are shown in FIGS. 13 to 15, respectively.

In addition, the measurement results of the antiestrogenic action of 4-hydroxytamoxifen, raloxifene or ZM189154 on the wild-type estrogen receptor α or the receptor {424} of the present invention are shown in FIGS. 16 to 18, respectively. Example 21 (Preparation of oligonucleotide for amplification)

The nucleotide sequence encoding the amino acid at the position corresponding to the amino acid No. 424 of the amino acid sequence No. 4 of the amino acid sequence represented by SEQ ID No. 1. Was used to detect whether or not the nucleotide sequence encoding a different amino acid had been substituted, by inserting a site encoding isoleucine at the 424th position from the amino terminus of the human wild-type estrogen receptor. Design an oligonucleotide that is within the amplification range, has a GC content of 30% or more and 70% or less, and has a length of about 20 bases. An oligonucleotide is prepared based on the designed base sequence. (Hereinafter, the nucleotide sequence encoding the amino acid at the position corresponding to the amino acid No. 424 of the amino acid sequence represented by SEQ ID NO: 1 is located at the position corresponding to the amino acid sequence of the wild type estrogen receptor α A mutation substituted with a nucleotide sequence encoding an amino acid different from a certain amino acid may be referred to as a 424 mutation.) Example 22 (Analysis of 424 mutation using human tissue as material)

Add 100 mg of the frozen human liver sample to 5 ml of [4 M guanidine thiocyanate, 0.1 M Tris-Cl (pH 7.5) 1%] 3_mercaptoethanol, and grind with a polytron homogenizer. . This is overlaid on 25 ml of a 5.7 M cesium chloride solution previously placed in an ultracentrifuge tube, and RNA is separated by performing a density gradient ultracentrifugation at 90,000 X g for 24 hours. Collect the RNA, rinse with 70% ethanol, and air-dry at room temperature. Dissolve this in sterile water (ΙΟμΙ) and measure the concentration. Using this RNA l-5 / xg as type III, 1 μ オリゴ of oligo dT primer (Amersham Biotech) was used as a primer for reverse transcription synthesis, and Superscript II (Invitrogen) was used in the attached buffer. The cDNA is synthesized by reacting at 42 ° C for 1 hour. One-fiftieth of the cDNA solution thus obtained was made into type III, and an oligonucleotide having the base sequence of SEQ ID NO: 10 and an oligonucleotide having the base sequence of SEQ ID NO: 16 were separated. Perform PCR using The PCR was carried out using Pfu DNA polymerase (manufactured by Stratagene) in a buffer of 94 ° C. in 200 μL of each of the four types of bases (dATP, dTTP, dGTP, dCTP) and a dedicated buffer attached to the enzyme. C, 1 minute, then 55 ° C, 30 seconds, 72 ° C, 1 minute, 1 cycle, 35 cycles. The amplified DNA is separated by electrophoresis in a gel containing 1% agarose (Agaroses, manufactured by Futaba Gene) and collected. Using this whole amount as type III, a sample for direct sequence was prepared using a dye terminator-sequence kit FS (manufactured by Applied Biosystems) using 5 pM of oligonucleotide having the nucleotide sequence of SEQ ID NO: 11 as a sequence primer. I do. This is called an automated DNA sequencer (Applied Biosystems, The base sequence is determined by subjecting it to base sequence analysis using Model 3700). In this manner, the nucleotide sequence encoding the amino acid at the position corresponding to the amino acid represented by amino acid number 424 of the amino acid sequence represented by SEQ ID NO: 1 is clarified. Example 2 3 (Analysis of 424 mutations by PCR-SSCP method)

First, a forward primer is selected from an oligonucleotide consisting of the nucleotide sequence shown in any of SEQ ID NOs: 7 to 11, and a reverse primer is obtained from an oligonucleotide consisting of the nucleotide sequence shown in any of SEQ ID NOs: 12 to 16. And chemically synthesize it with a DNA synthesizer. At this time, the 5 'end is modified with the fluorescent substance FITC. Next, 100 ng of the genomic DNA obtained in Example 8 was transformed into a 铸 form, and the DNTC encoding the amino acid sequence of estrogen receptor α was subjected to PCR by using 200 pM of each of the above FITC-modified oligonucleotides as primers. To amplify. The PCR was performed using Ex Taq DNA polymerase (Takara Shuzo Co., Ltd.) and 200 μL of each of the four bases (dATP, dTTP, dGTP, dCTP) and the dedicated buffer attached to the enzyme. , 94 ° C, 30 seconds, then 55 ° C, 30 seconds, and further 40 cycles of 74 ° C, 30 seconds. After the reaction, 1/20 amount of the obtained amplified product was 95 ° /. After incubating in formamide at 95 ° C for 5 minutes, cool rapidly. Among them, 2.51 is applied to a 5% non-denaturing polyacrylamide gel, and electrophoresed in 180 mM Tris-borate buffer (pH 8.0). The electrophoresis conditions are room temperature, constant power of 40 W, and 5 hours. After the electrophoresis, the amplified nucleic acid fragment is detected by detecting the fluorescent signal in the gel with a fluorescence reading scanner. Compared to the mobility of the band of the amplified product in the DNA encoding the amino acid sequence of the wild-type estrogen receptor, which is run side by side, the mobility of the amplified product in the DNA encoding the amino acid sequence of the mutant estrogen receptor α has a lower mobility. Because of the difference, the presence or absence of the mutation in the amplified sequence can be detected. Example 24 (Analysis of 424 mutations by nucleotide sequence analysis)

A part of the gel at a position corresponding to the band of the DNA encoding the amino acid sequence of the mutant estrogen receptor α detected in Example 23 was cut into a square, Permeate in 400 μl of bacterial water to elute DNA. After removing the gel and purifying by ethanol precipitation, dissolve DNII in 501 sterile water. Among them, Ιμΐ is changed to 铸, and PCR is performed using the oligonucleotide used for PCR-SSCP in Example 9 to widen the DNA encoding the amino acid sequence of estrogen receptor α. PCR was performed using Ex Taq DNA Polymerase (Takara Shuzo) in a buffer attached to the enzyme, at 94 ° C for 30 seconds, followed by 55 ° C, 30 seconds, and a further cycle at 74 ° C, 30 seconds. Perform 30 cycles. After completion of the reaction, the amplified DNA is confirmed by agarose gel electrophoresis, and cloned into pGEM-T Easy vector (Promega). The obtained plasmid was converted into type III, and the base was prepared using a BigDye Terminator cycle sequence ready reaction kit (/ 7F Biosystems) and an automatic DNA sequencer (Applied Biosystems 3700). Determine the sequence. Thus, the nucleotide sequence encoding the amino acid at the position corresponding to the amino acid represented by amino acid number 424 in the amino acid sequence represented by SEQ ID NO: 1 is clarified. Example 25 (Analysis of 424 mutations combining PCR and restriction enzyme digestion)

PCR was performed using genomic DNA or cDNA as a type I primer having the nucleotide sequence of SEQ ID NO: 36 and a primer having the nucleotide sequence of SEQ ID NO: 37 to encode the amino acid sequence of human estrogen receptor α. Increase the DNA. The above PCR uses Pfu DNA polymerase (manufactured by Stratagene) in a buffer attached to the enzyme, at 94 ° C for 1 minute, then at 55 ° C for 30 seconds and then at 72 ° C for 1 minute for 1 cycle. Perform 30 cycles. The resulting 100 bp DNA is treated with the restriction enzyme AccI. The DNA encoding the amino acid sequence of wild-type estrogen receptor α is not digested. On the other hand, DNΑ, which encodes the amino acid sequence of mutant estrogen receptor α in which the isoleucine at position 424 from the amino terminal of human-derived wild-type estrogen receptor is mutated to threonine, has the sequence GTAGAC. Digestion with Acc I yields 75 base pairs of DNA and 25 base pairs of DNA. Thus, the DNA encoding the estrogen receptor α having the mutation can be detected. Industrial potential

According to the present invention, an amino acid at a specific position is substituted with an amino acid different from the amino acid of wild-type estrogen receptor α, and the amino acid at a specific position has a different reactivity to wild-type estrogen receptor α from wild-type estrogen receptor α. Using an estrogen receptor α, a DNA encoding the receptor, and an estrogen receptor α in which the amino acid at the specific position is substituted with an amino acid different from the amino acid of the wild-type receptor. Estrogen receptor _α activity regulation method comprising the steps of: estimating an estrogen receptor α genotype by examining the presence or absence of amino acid substitution at the specific position; and estimating estrogen receptor α genotype by examining the amino acid substitution. How to determine the effectiveness of treatment with a substance But it is possible to provide. Sequence listing free text

SEQ ID NO: 3

Oligonucleotide primers designed for PCR

SEQ ID NO: 4

Oligonucleotide primers designed for PCR

SEQ ID NO: 5

Oligonucleotide primers designed for PCR

SEQ ID NO: 6

Oligonucleotide primers designed for PCR

SEQ ID NO: 7

Oligonucleotide primers designed for PCR

SEQ ID NO: 8

Oligonucleotide primers designed for PCR

SEQ ID NO: 9

Oligonucleotide primers designed for PCR SEQ ID NO: 10

Oligonucleotide primers designed for PCR

SEQ ID NO: 1 1

Oligonucleotide primers designed for PCR

SEQ ID NO: 12

Oligonucleotide primers designed for PCR

SEQ ID NO: 13

Oligonucleotide primers designed for PCR

SEQ ID NO: 14

Oligonucleotide primers designed for PCR

SEQ ID NO: 15

Oligonucleotide primers designed for PCR

SEQ ID NO: 16

Oligonucleotide primers designed for PCR

SEQ ID NO: 17

Oligonucleotide probe designed for Southern hybridization SEQ ID NO: 18

Oligonucleotide probe designed for Southern hybridization SEQ ID NO: 19

Oligonucleotide probe designed for Southern hybridization SEQ ID NO: 20

Oligonucleotide probe designed for Southern hybridization SEQ ID NO: 21

Oligonucleotide probe designed for Southern hybridization SEQ ID NO: 22

Oligonucleotide primers designed for PCR

SEQ ID NO: 23

Oligonucleotides designed to create estrogen responsive elements SEQ ID NO: 24

Oligonucleotide designed to generate promoter DNA SEQ ID NO: 25

Oligonucleotide designed to generate promoter DNA SEQ ID NO: 26

Oligonucleotide primers designed for PCR

SEQ ID NO: 27

Oligonucleotide primers designed for PCR

SEQ ID NO: 29

Oligonucleotide primers designed for PCR

SEQ ID NO: 30

Oligonucleotide primers designed for PCR

SEQ ID NO: 31

Oligonucleotide primers designed for PCR

SEQ ID NO: 32

Oligonucleotide primers designed for PCR

SEQ ID NO: 34

Oligonucleotide primers designed for PCR

SEQ ID NO: 35

Oligonucleotide primers designed for PCR

SEQ ID NO: 36

Oligonucleotide primers designed for PCR

SEQ ID NO: 37

Oligonucleotide primers designed for PCR

SEQ ID NO: 38

Oligonucleotide probe designed for Southern hybridization SEQ ID NO: 39

Oligonucleotide probes designed for Southern hybridization SEQ ID NO: 40

Oligonucleotide probe designed for Southern hybridization SEQ ID NO: 41

Oligonucleotide probe designed for Southern hybridization SEQ ID NO: 42

Oligonucleotide probes designed for Southern hybridization

Claims

The scope of the claims

1. Estrogen receptor having the following properties:

(a) Among the amino acids constituting the receptor, in an alignment based on the homology of the amino acid sequence, the amino acid represented by amino acid number 404, 405 or 424 of the amino acid sequence represented by SEQ ID NO: 1 One or more of the amino acids at positions corresponding to the amino acid sequence has been substituted with an amino acid different from the amino acid at the corresponding position in the amino acid sequence of wild-type estrogen receptor α. Impart the properties of (b) and (c) to the receptor.

(B) contacting the one of the anti-estrogen agent types wildtype Est port plasminogen receptor _α inhibits the function of the transcriptional activation region AF2 of not suppressed transcriptional function of the active region AF1 Then, the estrogen ² emissions responsive element It can activate the transcription of a gene that is under the transcriptional control of the included transcriptional control region.

(c) contacting with an estrogen can activate transcription of a gene under the transcriptional control of a transcription control region containing an estrogen response element, and the activation activates the transcription of the gene in the above (b). And is not substantially inhibited by compounds that can.

2. The estrogen receptor α according to claim 1, wherein in (b) and (c), the gene under the transcriptional control of a transcription control region containing an estrogen response element is a gene introduced into a chromosome of a cell.

3. The estrogen receptor α according to claim 1, wherein the activation in (c) is also an activation inhibited by a pure antiestrogen.

4. The estrogen receptor α according to claim 1, wherein the antiestrogenic substance according to (b) is tamoxifen, 4-hydroxytamoxifen or raloxifene.

5. In an alignment based on amino acid sequence homology, the amino acid at the position corresponding to the amino acid shown by amino acid No. 390 in the amino acid sequence shown by SEQ ID NO: 1 2. The estrogen receptor-α according to claim 1, which is the same as the amino acid at the position corresponding to the amino acid sequence of the wild-type estrogen receptor-α.

6. In the alignment based on the homology of the amino acid sequences, the amino acid numbers 303, 309, 390, 396, 415, 494, 531 of the amino acid sequence represented by SEQ ID NO: 1 Or the estrogen receptor ct according to claim 1, wherein any of the amino acids at positions corresponding to the amino acid represented by 578 is the same as the amino acid at the corresponding position in the amino acid sequence of the wild-type estrogen receptor.

7. Amino acid at a position corresponding to the amino acid sequence of wild-type estrogen receptor α At a position corresponding to the amino acid represented by amino acid number 404, it is phenylalanine, and the amino acid represented by amino acid number 2. The estrogen receptor α according to claim 1, wherein alanine is present at a corresponding position, and isoleucine is present at a position corresponding to the amino acid represented by amino acid number 424.

8. The amino acid that differs from the amino acid at the corresponding position in the amino acid sequence of the wild-type estrogen receptor is leucine at the position corresponding to the amino acid indicated by amino acid number 404, and 2. The estrogen receptor α according to claim 1, wherein the position corresponding to the amino acid shown is valine, and the position corresponding to the amino acid shown by amino acid number 424 is threonine.

9. The estrogen receptor α power according to claim 1, which is an estrogen receptor α derived from a mammal.

10. An estrogen receptor α having the amino acid sequence represented by SEQ ID NO: 2.

11. An estrogen receptor α having the amino acid sequence represented by SEQ ID NO: 28.

1 2. An estrogen receptor having the amino acid sequence of SEQ ID NO: 33.

1 3. An isolated DNA encoding the estrogen receptor α according to claim 1.

14. An isolated DNA encoding an estrogen receptor ct having the amino acid sequence shown in SEQ ID NO: 2.

15. An isolated DNA encoding an estrogen receptor _α having the amino acid sequence represented by SEQ ID NO: 28.

16. An isolated DNA encoding an estrogen receptor having the amino acid sequence of SEQ ID NO: 33.

17. The DNA according to claim 13, wherein the DNA is cDNA.

18. The DNA according to claim 13, wherein the promoter is operably linked.

19. A vector containing the DNA according to claim 13.

20. A method for producing a vector, comprising incorporating the DNA according to claim 13 into a vector capable of replicating in a host cell.

21. A transformant obtained by introducing the DNA according to claim 13 into a host cell.

22. The transformant according to claim 21, wherein the host cell is an animal cell.

23. The transformant according to claim 21, wherein the host cell is a mammalian cell.

24. A method for producing a transformant, which comprises introducing the DNA according to claim 13 into a host cell.

25. A method for producing estrogen receptor α, comprising culturing the transformant according to claim 21 to produce estrogen receptor α.

26. (1) contacting the estrogen receptor of claim 1 with a test substance, wherein the labeled ligand is bound; and

(2) The binding state between the estrogen receptor α and the test substance is determined by measuring the amount of free labeled ligand or bound labeled ligand produced by competition between the labeled ligand and the test substance. A ligand 'receptor binding assay, comprising a step of indirectly confirming by monitoring.

27. A method for evaluating the ability of a test substance to regulate estrogen receptor α activity,

(1) a transformant which produces the estrogen receptor α according to claim 1 and has a reporter gene under the transcriptional control of a transcription control region containing an estrogen response sequence, which is introduced into a chromosome; and a test substance. Contacting with

(2) measuring the expression level of the reporter gene of the transformant or an index value having a correlation with the level, and

(3) a step of evaluating the ability of the substance to regulate estrogen receptor activity based on the measured expression level or an index value having a correlation with the level.

An evaluation method comprising:

28. A method for screening for an estrogen receptor α-activity modulator, which comprises the step of evaluating the activity-modulating ability of a test substance on an estrogen receptor produced by a transformant according to the method of claim 27.

29. In the nucleic acid in the sample, the amino acids constituting the estrogen receptor, and the alignment based on the homology of the amino acid sequence, the amino acid sequence of the amino acid sequence represented by SEQ ID NO: 1, 4, 4, 5, or 4 Has the nucleotide sequence encoding the amino acid at the position corresponding to the amino acid shown in 24 been replaced with a nucleotide sequence encoding an amino acid different from the amino acid at the corresponding position in the amino acid sequence of wild-type estrogen receptor _α ? A method for determining the gene type of estrogen receptor α, comprising the step of determining whether or not the gene is estrogen receptor α.

30. In the nucleic acid in the sample, the amino acids that constitute estrogen receptor α, and in the alignment based on the homology of the amino acid sequence, the amino acid number of the amino acid sequence represented by SEQ ID NO: 1 4 24 The nucleotide sequence encoding the amino acid at the position corresponding to the amino acid indicated by 4 4 Has the amino acid sequence of the wild-type estrogen receptor _α been substituted with a base sequence encoding an amino acid different from the amino acid at the corresponding position? A method for determining the efficacy of treatment with an estrogen receptor activity modulator comprising the step of determining the genotype of estrogen receptor α by examining the presence or absence of estrogen receptor α.

31. In the nucleic acid in the sample, the amino acid constituting estrogen receptor α, and the amino acid sequence of amino acid sequence represented by SEQ ID NO: 1 in the alignment based on the homology of the amino acid sequence. Has the nucleotide sequence encoding the amino acid at the position corresponding to the amino acid indicated by 24 been replaced with a nucleotide sequence encoding an amino acid different from the amino acid at the corresponding position in the amino acid sequence of wild-type estrogen receptor _α ? A method for judging the efficacy of treatment with an anti-estrogen substance, comprising the step of judging the gene type of estrogen receptor α by examining the presence or absence of the estrogen receptor α.

32. With the nucleic acid in the sample as type III, a region containing an amino acid at a position corresponding to the amino acid No. 404, 405 or 424 of the amino acid sequence shown in SEQ ID NO: 1 Amplifying the encoding nucleic acid and determining the base sequence of the amplified nucleic acid. 30. The method according to claim 30 or 31.

33. Using the nucleic acid in the sample as type III, a region containing an amino acid at a position corresponding to the amino acid No. 404, 405 or 424 of the amino acid sequence shown in SEQ ID NO: 1 The encoding nucleic acid is amplified, the mobility of the amplified nucleic acid is measured by electrophoresis, and the mobility of the nucleic acid encoding the relevant region of wild-type estrogen receptor α and the mobility of the amplified nucleic acid are determined. 32. The method according to claim 30, further comprising a step of checking whether or not they are different.

34. In the amino acid sequence of the wild-type estrogen receptor α, the amino acid at the position corresponding to the amino acid No. 404, 405 or 424 of the amino acid sequence represented by SEQ ID NO: 1 32. The method according to claim 30, further comprising a step of examining the efficiency of hybridization between a probe consisting of a nucleotide sequence encoding a region containing the nucleic acid and a nucleic acid in the sample.

35. Nucleic acid encoding a region containing an amino acid at a position corresponding to amino acid number 404, 405 or 424 of the amino acid sequence represented by SEQ ID NO: 1, with the nucleic acid in the sample as type III 32. The method according to claim 30, further comprising a step of amplifying the nucleic acid, and digesting the amplified nucleic acid with a restriction enzyme to examine the presence or absence of a recognition sequence of the restriction enzyme.