JP4400183B2 - Mutant estrogen receptor α and its gene - Google Patents

Description

  The present invention relates to a mutant estrogen receptor α and a gene thereof.

  Various physiological effects of estrogen are triggered through the estrogen receptor. The estrogen receptor is present in estrogen target cells in tissues such as uterus, vagina, oviduct, liver, breast cancer, bone, and is a ligand-responsive transcriptional regulator. The estrogen receptor is activated upon binding to estrogen, binds to an estrogen response element on the chromosome, and regulates transcription of a target gene located downstream of the response element. Such abnormalities in the activation of estrogen and receptor are known to cause various diseases, and are substances that act on estrogen receptor and exhibit antiestrogenic activity (hereinafter referred to as antiestrogenic substances). Attempts have been made to use these as therapeutic agents for such diseases. For example, tamoxifen, an anti-estrogen substance, is used as a therapeutic agent for breast cancer.

GenBank Accession No.M12674 GenBank Accession No. M38651 GenBank Accession No. X61098 GenBank Accession No. Y00102 Metzger D et al., J. Biol. Chem., 270: 9535-9542 (1995) Berry M. et al., EMBO J., 9: 2811-2818 (1990)

  However, if any of the amino acids constituting the estrogen receptor is substituted and the reactivity to the antiestrogenic substance is different from usual, the expected effect is not improved in the treatment using the antiestrogenic substance as described above. There are concerns about the possibility of occurrence of cases and undesirable symptoms. Therefore, if amino acid substitutions related to reactivity to antiestrogens are clarified, it is possible to determine the effectiveness of treatment such as administration of antiestrogens before starting treatment by examining the presence or absence of such amino acid substitutions. Become. It is also possible to construct a test system for searching for a substance exhibiting a desired effect even for an estrogen receptor having such an amino acid substitution.

As a result of intensive studies under such circumstances, the present inventor has found that an amino acid at a specific position is substituted with an amino acid different from the amino acid of the wild-type estrogen receptor α. The present inventors have found an estrogen receptor α showing reactivity different from that of the estrogen receptor α, and a DNA encoding the receptor.
That is, the present invention
1) An estrogen receptor α (hereinafter referred to as the receptor of the present invention) having the following properties.
(A) Among the amino acids constituting the receptor, 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 in the alignment based on the homology of the amino acid sequence is a wild-type estrogen It is substituted with an amino acid different from the amino acid at the corresponding position in the amino acid sequence of receptor α, and the substitution of the amino acid imparts the following properties (b) and (c) to the receptor.
(B) When contacted with any type of anti-estrogen 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, the transcription control region containing the estrogen response element Transcription of genes under transcriptional control can be activated.
(C) When contacted with estrogen, transcription of a gene under the transcriptional control of a transcriptional control region containing an estrogen response element can be activated, and this activation activates transcription of the gene in (b). Is not substantially inhibited by the compound that can.
2) Estrogen receptor α having the following properties.
(A) Among the amino acids constituting the receptor, 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 in the alignment based on the homology of the amino acid sequence is a wild-type estrogen It is substituted with an amino acid different from the amino acid at the corresponding position in the amino acid sequence of receptor α, and the substitution of the amino acid imparts the following properties (b) and (c) to the receptor.
(B) When contacted with any type of anti-estrogen 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, the transcription control region containing the estrogen response element Transcription of genes under transcriptional control can be activated.
(C) When contacted with estrogen, transcription of a gene under the transcriptional control of a transcriptional control region containing an estrogen response element can be activated, and this activation activates transcription of the gene in (b). Is not substantially inhibited by the compound that can.
(D) In alignment based on amino acid sequence homology, the amino acid sequence represented by SEQ ID NO: 1 is located at the position corresponding to the amino acid represented by amino acid number 390 and the amino acid sequence represented by amino acid number 578 The amino acid is identical to the amino acid at the corresponding position in the amino acid sequence of wild type estrogen receptor α.
3) Estrogen receptor α having the following properties.
(A) Among the amino acids constituting the receptor, 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 in the alignment based on the homology of the amino acid sequence is a wild-type estrogen It is substituted with an amino acid different from the amino acid at the corresponding position in the amino acid sequence of receptor α, and the substitution of the amino acid imparts the following properties (b) and (c) to the receptor.
(B) When contacted with any type of anti-estrogen 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, the transcription control region containing the estrogen response element Transcription of genes under transcriptional control can be activated.
(C) When contacted with estrogen, transcription of a gene under the transcriptional control of a transcriptional control region containing an estrogen response element can be activated, and this activation activates transcription of the gene in (b). Is not substantially inhibited by the compound that can.
(D) in alignment based on amino acid sequence homology, an amino acid at a position corresponding to the amino acids represented by amino acid numbers 303, 390, 396, 415, 494, 531 and 578 of the amino acid sequence represented by SEQ ID NO: 1; It is identical to the amino acid at the corresponding position in the amino acid sequence of wild type estrogen receptor α.
4) An estrogen receptor α having an amino acid sequence having the following properties.
(A) Among the amino acids constituting the receptor, 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 in the alignment based on the homology of the amino acid sequence is a wild-type estrogen It is substituted with an amino acid different from the amino acid at the corresponding position of the amino acid sequence of receptor α, and the substitution of the amino acid imparts the following properties (b) and (c) to the receptor.
(B) When contacted with any type of anti-estrogen 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, the transcription control region containing the estrogen response element Transcription of genes under transcriptional control can be activated.
(C) When contacted with estrogen, transcription of a gene under the transcriptional control of a transcriptional control region containing an estrogen response element can be activated, and this activation activates transcription of the gene in (b). Is not substantially inhibited by compounds that can be inhibited by pure antiestrogen.
5) An estrogen receptor α having an amino acid sequence having the following properties.
(A) Among the amino acids constituting the receptor, 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 in the alignment based on the homology of the amino acid sequence is a wild-type estrogen It is substituted with an amino acid different from the amino acid at the corresponding position of the amino acid sequence of receptor α, and the substitution of the amino acid imparts the following properties (b) and (c) to the receptor.
(B) When contacted with any type of anti-estrogen 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, the transcription control region containing the estrogen response element Transcription of genes under transcriptional control can be activated.
(C) When contacted with estrogen, transcription of a gene under the transcriptional control of a transcriptional control region containing an estrogen response element can be activated, and this activation activates transcription of the gene in (b). Is not substantially inhibited by compounds that can be inhibited by pure antiestrogen.
(D) In alignment based on amino acid sequence homology, the amino acid sequence represented by SEQ ID NO: 1 is located at the position corresponding to the amino acid represented by amino acid number 390 and the amino acid sequence represented by amino acid number 578 The amino acid is identical to the amino acid at the corresponding position in the amino acid sequence of wild type estrogen receptor α.
6) An estrogen receptor α having an amino acid sequence having the following properties.
(A) Among the amino acids constituting the receptor, 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 in the alignment based on the homology of the amino acid sequence is a wild-type estrogen It is substituted with an amino acid different from the amino acid at the corresponding position of the amino acid sequence of receptor α, and the substitution of the amino acid imparts the following properties (b) and (c) to the receptor.
(B) When contacted with any type of anti-estrogen 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, the transcription control region containing the estrogen response element Transcription of genes under transcriptional control can be activated.
(C) When contacted with estrogen, transcription of a gene under the transcriptional control of a transcriptional control region containing an estrogen response element can be activated, and this activation activates transcription of the gene in (b). Is not substantially inhibited by compounds that can be inhibited by pure antiestrogen.
(D) in alignment based on amino acid sequence homology, an amino acid at a position corresponding to the amino acids represented by amino acid numbers 303, 390, 396, 415, 494, 531 and 578 of the amino acid sequence represented by SEQ ID NO: 1; It is identical to the amino acid at the corresponding position in the amino acid sequence of wild type estrogen receptor α.
7) The estrogen receptor α according to any one of 1 to 6 above, wherein the anti-estrogen substance described in (b) is tamoxifen, 4-hydroxy tamoxifen, or raloxifene.
8) The estrogen receptor α according to any one of 1 to 7 above, wherein 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 isoleucine.
9) The estrogen receptor α according to any one of 1 to 8 above, wherein the amino acid different from the amino acid at the corresponding position in the amino acid sequence of the wild-type estrogen receptor α is threonine.
10) The estrogen receptor α according to any one of 1 to 9 above, wherein the estrogen receptor α is an estrogen receptor α derived from a mammal.
11) An estrogen receptor α having the amino acid sequence represented by SEQ ID NO: 2.
12) An isolated DNA encoding the estrogen receptor α according to any one of 1 to 11 above (hereinafter referred to as the DNA of the present invention).
13) The DNA according to item 12 above, wherein the DNA is cDNA.
14) The DNA according to any one of items 12 and 13, wherein the promoter is operably linked.
15) A vector containing the DNA according to any one of 12 to 14 above (hereinafter referred to as the present invention vector).
16) A method for producing a vector, wherein the DNA according to any one of 12 to 14 above is incorporated into a vector capable of replicating in a host cell.
17) A transformant obtained by introducing the DNA according to any one of 12 to 14 or the vector according to 15 above into a host cell (hereinafter referred to as the transformant of the present invention).
18) The transformant according to 17 above, wherein the host cell is an animal cell.
19) The transformant according to any one of items 17 and 18, wherein the host cell is a mammalian cell.
20) A method for producing a transformant, which comprises introducing the DNA according to any one of 12 to 14 above or the vector according to 15 above into a host cell.
21) A method for producing estrogen receptor α, comprising culturing the transformant according to any one of items 17 to 19 to produce estrogen receptor α.
22) A ligand / receptor binding assay comprising a step of bringing the estrogen receptor α according to any one of 1 to 11 above into contact with a test substance.
23) In alignment based on amino acid sequence homology, an amino acid at a position corresponding to the amino acid represented by amino acid number 424 of the amino acid sequence represented by SEQ ID NO: 1 is located at a position corresponding to the amino acid sequence of wild-type estrogen receptor α. An estrogen receptor α substituted with an amino acid different from a certain amino acid is produced, a transformant in which a reporter gene under the transcriptional control of a transcriptional control region including an estrogen response element is introduced into a chromosome, and a test substance A method for evaluating the ability of a test substance to regulate estrogen receptor α activity (hereinafter, referred to as an evaluation method of the present invention), which comprises the step of contacting and measuring the expression level of the reporter gene in the transformant.
24) producing an estrogen receptor α having the following properties, contacting a transformant obtained by introducing a reporter gene under the transcriptional control of a transcriptional control region containing an estrogen responsive element into a chromosome with a test substance, A method for evaluating the ability of a test substance to regulate estrogen receptor α activity, comprising measuring the expression level of the reporter gene in a transformant.
(A) Among the amino acids constituting the receptor, 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 in the alignment based on the homology of the amino acid sequence is a wild-type estrogen It is substituted with an amino acid different from the amino acid at the corresponding position of the amino acid sequence of receptor α, and the substitution of the amino acid imparts the following properties (b) and (c) to the receptor.
(B) When contacted with any type of anti-estrogen 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, the transcription control region containing the estrogen response element Transcription of genes under transcriptional control can be activated.
(C) When contacted with estrogen, transcription of a gene under the transcriptional control of a transcriptional control region containing an estrogen response element can be activated, and this activation activates transcription of the gene in (b). Is not substantially inhibited by the compound that can.
25) A test substance is brought into contact with a transformant that produces an estrogen receptor α having the following properties, and into which a reporter gene under the transcriptional control of a transcriptional control region containing an estrogen responsive element is introduced into a chromosome: A method for evaluating the ability of a test substance to regulate estrogen receptor α activity, comprising measuring the expression level of the reporter gene in a transformant.
(A) Among the amino acids constituting the receptor, 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 in the alignment based on the homology of the amino acid sequence is a wild-type estrogen It is substituted with an amino acid different from the amino acid at the corresponding position of the amino acid sequence of receptor α, and the substitution of the amino acid imparts the following properties (b) and (c) to the receptor.
(B) When contacted with any type of anti-estrogen 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, the transcription control region containing the estrogen response element Transcription of genes under transcriptional control can be activated.
(C) When contacted with estrogen, transcription of a gene under the transcriptional control of a transcriptional control region containing an estrogen response element can be activated, and this activation activates transcription of the gene in (b). Is not substantially inhibited by the compound that can.
(D) In alignment based on amino acid sequence homology, the amino acid sequence represented by SEQ ID NO: 1 is located at the position corresponding to the amino acid represented by amino acid number 390 and the amino acid sequence represented by amino acid number 578 The amino acid is identical to the amino acid at the corresponding position in the amino acid sequence of wild type estrogen receptor α.
26) producing a estrogen receptor α having the following properties, bringing a test substance into contact with a transformant in which a reporter gene under the transcriptional control of a transcriptional control region containing an estrogen responsive element is introduced into a chromosome; A method for evaluating the ability of a test substance to regulate estrogen receptor α activity, comprising measuring the expression level of the reporter gene in a transformant.
(A) Among the amino acids constituting the receptor, 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 in the alignment based on the homology of the amino acid sequence is a wild-type estrogen It is substituted with an amino acid different from the amino acid at the corresponding position of the amino acid sequence of receptor α, and the substitution of the amino acid imparts the following properties (b) and (c) to the receptor.
(B) When contacted with any type of anti-estrogen 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, the transcription control region containing the estrogen response element Transcription of genes under transcriptional control can be activated.
(C) When contacted with estrogen, transcription of a gene under the transcriptional control of a transcriptional control region containing an estrogen response element can be activated, and this activation activates transcription of the gene in (b). Is not substantially inhibited by the compound that can.
(D) in alignment based on amino acid sequence homology, an amino acid at a position corresponding to the amino acids represented by amino acid numbers 303, 390, 396, 415, 494, 531 and 578 of the amino acid sequence represented by SEQ ID NO: 1; It is identical to the amino acid at the corresponding position in the amino acid sequence of wild type estrogen receptor α.
27) A test substance is brought into contact with a transformant which produces an estrogen receptor α having the following properties and into which a reporter gene under the transcriptional control of a transcriptional control region containing an estrogen responsive element is introduced into a chromosome: A method for evaluating the ability of a test substance to regulate estrogen receptor α activity, comprising measuring the expression level of the reporter gene in a transformant.
(A) Among the amino acids constituting the receptor, 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 in the alignment based on the homology of the amino acid sequence is a wild-type estrogen It is substituted with an amino acid different from the amino acid at the corresponding position of the amino acid sequence of receptor α, and the substitution of the amino acid imparts the following properties (b) and (c) to the receptor.
(B) When contacted with any type of anti-estrogen 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, the transcription control region containing the estrogen response element Transcription of genes under transcriptional control can be activated.
(C) When contacted with estrogen, transcription of a gene under the transcriptional control of a transcriptional control region containing an estrogen response element can be activated, and this activation activates transcription of the gene in (b). Is not substantially inhibited by compounds that can be inhibited by pure antiestrogen.
28) producing a estrogen receptor α having the following properties, bringing a test substance into contact with a transformant in which a reporter gene under the transcriptional control of a transcriptional control region containing an estrogen response element is introduced into a chromosome; A method for evaluating the ability of a test substance to regulate estrogen receptor α activity, comprising measuring the expression level of the reporter gene in a transformant.
(A) Among the amino acids constituting the receptor, 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 in the alignment based on the homology of the amino acid sequence is a wild-type estrogen It is substituted with an amino acid different from the amino acid at the corresponding position of the amino acid sequence of receptor α, and the substitution of the amino acid imparts the following properties (b) and (c) to the receptor.
(B) When contacted with any type of anti-estrogen 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, the transcription control region containing the estrogen response element Transcription of genes under transcriptional control can be activated.
(C) When contacted with estrogen, transcription of a gene under the transcriptional control of a transcriptional control region containing an estrogen response element can be activated, and this activation activates transcription of the gene in (b). Is not substantially inhibited by compounds that can be inhibited by pure antiestrogen.
(D) In alignment based on amino acid sequence homology, the amino acid sequence represented by SEQ ID NO: 1 is located at the position corresponding to the amino acid represented by amino acid number 390 and the amino acid sequence represented by amino acid number 578 The amino acid is identical to the amino acid at the corresponding position in the amino acid sequence of wild type estrogen receptor α.
29) A test substance is brought into contact with a transformant that produces an estrogen receptor α having the following properties, and into which a reporter gene under the transcriptional control of a transcriptional control region containing an estrogen responsive element is introduced into a chromosome: A method for evaluating the ability of a test substance to regulate estrogen receptor α activity, comprising measuring the expression level of the reporter gene in a transformant.
(A) Among the amino acids constituting the receptor, 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 in the alignment based on the homology of the amino acid sequence is a wild-type estrogen It is substituted with an amino acid different from the amino acid at the corresponding position of the amino acid sequence of receptor α, and the substitution of the amino acid imparts the following properties (b) and (c) to the receptor.
(B) When contacted with any type of anti-estrogen 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, the transcription control region containing the estrogen response element Transcription of genes under transcriptional control can be activated.
(C) When contacted with estrogen, transcription of a gene under the transcriptional control of a transcriptional control region containing an estrogen response element can be activated, and this activation activates transcription of the gene in (b). Is not substantially inhibited by compounds that can be inhibited by pure antiestrogen.
(D) in alignment based on amino acid sequence homology, an amino acid at a position corresponding to the amino acids represented by amino acid numbers 303, 390, 396, 415, 494, 531 and 578 of the amino acid sequence represented by SEQ ID NO: 1; It is identical to the amino acid at the corresponding position in the amino acid sequence of wild type estrogen receptor α.
30) The method according to any one of items 24 to 29, wherein the anti-estrogen substance described in (b) is tamoxifen, 4-hydroxy tamoxifen or raloxifene.
31) The method according to any one of 23 to 30 above, wherein 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 isoleucine.
32) The method according to any one of 23 to 31 above, wherein the amino acid different from the amino acid at the corresponding position in the amino acid sequence of wild-type estrogen receptor α is threonine.
33) The method according to any one of items 23 to 32, wherein the estrogen receptor α is an estrogen receptor α derived from a mammal.
34) A transformant produced by producing an estrogen receptor α having the amino acid sequence represented by SEQ ID NO: 2 and having a reporter gene under the transcriptional control of a transcriptional control region containing an estrogen response element introduced into a chromosome, a test substance, A method for evaluating the ability of a test substance to regulate estrogen receptor α activity, the method comprising the step of:
35) A screening method for an estrogen receptor α activity regulating substance, comprising a step of evaluating the activity regulating ability of a test substance against estrogen receptor α produced by a transformant by the method according to any one of 23 to 34 above.
36) In the nucleic acid in the sample, the amino acid constituting the estrogen receptor α, and in the alignment based on the homology of the amino acid sequence, 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. Estrogen receptor α genotype comprising a step of examining whether a base sequence encoding an amino acid is substituted with a base sequence encoding an amino acid different from the amino acid at the corresponding position of the wild type estrogen receptor α Judgment method.
37) A nucleic acid in a sample, which is an amino acid constituting the estrogen receptor α, in an alignment based on amino acid sequence homology, at a position corresponding to the amino acid represented by amino acid number 424 of the amino acid sequence represented by SEQ ID NO: 1. Determine the estrogen receptor α genotype by investigating whether the base sequence encoding an amino acid has been replaced with a base sequence encoding an amino acid different from the amino acid at the corresponding position of the wild-type estrogen receptor α amino acid sequence A method for determining the effectiveness of treatment with an estrogen receptor α activity modulator.
38) In the nucleic acid in the sample, the amino acid constituting the estrogen receptor α, and in the alignment based on the homology of the amino acid sequence, 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. Determine the estrogen receptor α genotype by investigating whether the base sequence encoding an amino acid has been replaced with a base sequence encoding an amino acid different from the amino acid at the corresponding position of the wild-type estrogen receptor α amino acid sequence A method for determining the effectiveness of treatment with an anti-estrogen substance, comprising the step of:
39) Amplifying a nucleic acid encoding a region containing an amino acid at a position corresponding to the amino acid represented by amino acid number 424 of the amino acid sequence represented by SEQ ID NO: 1 using the nucleic acid in the sample as a template, and the base of the amplified nucleic acid 31. The method according to any one of items 29 and 30, which comprises a step of determining a sequence.
40) Using the nucleic acid in the sample as a template, a nucleic acid encoding a region containing an amino acid at a position corresponding to the amino acid represented by amino acid number 424 of the amino acid sequence represented by SEQ ID NO: 1 is amplified, and the amplified nucleic acid is electrically 37. The method according to 37 or 38 above, which comprises a step of performing electrophoresis and measuring the mobility, and examining whether the mobility of the nucleic acid encoding the region of wild-type estrogen receptor α is different from the mobility of the amplified nucleic acid. The method according to any one.
41) a probe comprising a base sequence encoding a region containing an amino acid at a position corresponding to the amino acid represented by amino acid 424 of the amino acid sequence represented by SEQ ID NO: 1 among the amino acid sequence of wild-type estrogen receptor α, and a sample 39. The method according to any one of 37 or 38 above, which comprises the step of examining whether or not the nucleic acid therein hybridizes.
42) Using the nucleic acid in the sample as a template, a nucleic acid encoding a region containing an amino acid at a position corresponding to amino acid number 424 of the amino acid sequence represented by SEQ ID NO: 1 is amplified, and the amplified nucleic acid is digested with a restriction enzyme. 39. The method according to any of 37 or 38 above, further comprising the step of examining the presence or absence of the restriction enzyme recognition sequence.
Is to provide.

  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 estrogen exhibits a reactivity different from that of wild-type estrogen receptor α with respect to certain anti-estrogens. Receptor α, DNA encoding the receptor, method for evaluating estrogen receptor α activity-modulating ability of a test substance using 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 genotype determination method for determining the presence or absence of amino acid substitution at a specific position, and determination of the effectiveness of treatment with an estrogen receptor α activity modulator comprising the step of determining the estrogen receptor α genotype by examining the presence or absence of the amino acid substitution Methods can be provided It becomes ability.

Hereinafter, the present invention will be described in detail.
In the present invention,
“Alignment based on amino acid sequence homology” of estrogen receptor α refers to the amino acid sequences aligned so that the amino acid sequences of estrogen receptor α derived from various organisms are identical or highly similar. Means table. Specific examples include alignment of amino acid sequences of estrogen receptor α derived from human, mouse, rat and the like as shown in Table 1.

表１において、hERa.TXTは、ヒト由来のエストロゲンレセプターαのアミノ酸配列（GenBank Accession No.M12674記載のアミノ酸配列のアミノ末端から400番目のアミノ酸バリンがグリシンに置換されたアミノ酸配列；配列表に配列番号１で示す。）、mER.TXTは、マウス由来のエストロゲンレセプターαのアミノ酸配列（GenBank Accession No. M38651）、ratER(X6).TXTは、ラット由来のエストロゲンレセプターαのアミノ酸配列（GenBank Accession No. X61098）、ratER(Y0).TXTは、ラット由来のエストロゲンレセプターαのアミノ酸配列（GenBank Accession No. Y00102）を、それぞれアミノ酸の１文字表記で示す。当該アライメントは、市販のソフトウェアであるGenetyx-Win SV/R ver. 4.0（ソフトウエア開発株式会社）を使用して作成された。＊は、配列番号１で示されるアミノ酸配列のアミノ酸番号４２４のアミノ酸に相当する位置にあるアミノ酸を示す。

In Table 1, hERa.TXT is the amino acid sequence of human-derived estrogen receptor α (amino acid sequence in which the 400th amino acid valine from the amino terminus of the amino acid sequence described in GenBank Accession No. M12674 is substituted with glycine; MER.TXT is the amino acid sequence of mouse-derived estrogen receptor α (GenBank Accession No. M38651), and ratER (X6) .TXT is the amino acid sequence of rat-derived estrogen receptor α (GenBank Accession No. X61098), ratER (Y0) .TXT represents the amino acid sequence of the rat-derived estrogen receptor α (GenBank Accession No. Y00102), respectively, with one letter code for the amino acid. The alignment was created by 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 424 of the amino acid sequence represented by SEQ ID NO: 1.

  “In the alignment based on amino acid sequence homology, 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” refers to the estrogen receptor α derived from various organisms as described above. In the alignment of the amino acid sequences, when the amino acid sequence is shown side by side with the amino acid sequence shown by SEQ ID NO: 1, it means an amino acid at the same position as the amino acid shown by amino acid number 424 of the amino acid sequence shown by SEQ ID NO: 1. Specifically, for example, “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 in the alignment based on the homology of the amino acid sequence” refers to human-derived estrogen receptor α Isoleucine which is the 424th amino acid from the amino terminus of the amino acid sequence (shown in SEQ ID NO: 1), isoleucine which is the 428th amino acid from the amino terminus of the mouse-derived estrogen receptor α amino acid sequence (GenBank Accession No. M38651), Isoleucine, which is the 429th amino acid from the amino terminus of the rat-derived estrogen receptor α (GenBank Accession No. X61098), and 429th from the amino terminus of the rat-derived estrogen receptor α (GenBank Accession No. Y00102). The amino Isoleucine, and the like can be given is.

  “Wild-type estrogen receptor α” means an estrogen receptor α consisting of the amino acid sequence found most frequently in nature in the amino acid sequence of the receptor derived from the same species of organism. For example, human-derived wild-type estrogen receptor α is an estrogen receptor consisting of the amino acid sequence represented by SEQ ID NO: 1 (the amino acid sequence in which the 400th amino acid valine is replaced by glycine from the amino terminus of the amino acid sequence described in GenBank Accession No. M12674) α can be mentioned.

  Whether it is possible to “activate transcription of a gene under the transcriptional control of a transcriptional control region containing an estrogen responsive element” is determined by, for example, using a reporter gene linked downstream of the transcriptional control region containing an estrogen responsive element. It can be used and evaluated by performing a test method such as a reporter assay described later.

  “Estrogen receptor α transcription activation region AF1” and “Estrogen receptor α transcription activation region AF2” are partial regions of estrogen receptor α, which are involved in the transcription activation ability of estrogen receptor α. [Metzger D et al., J. Biol. Chem., 270: 9535-9542 (1995), etc.]. “Anti-estrogens of the type that suppress the function of the transcriptional activation region AF2 of wild-type estrogen receptor α but do not suppress the function of the transcriptional activation region AF1”, for example, Berry M. et al., EMBO J., 9 : The characteristics can be evaluated by conducting a reporter assay according to the description of 2811-2818 (1990). Specifically, for example, it can be prepared according to the description of primary culture cells of chicken embryo fibroblast [Solomon. J. J, Tissue Cult. Assoc. Manual, 1: 7-11 (1975)]. ], A wild type estrogen receptor α gene is expressed in a cell having a strong activation ability of the transcription activation region AF1, and a reporter gene linked downstream of the transcription control region containing an estrogen response element is introduced. When a test substance is brought into contact with the obtained cells (hereinafter referred to as AF1 activity evaluation cells) and a reporter assay is performed as described below, “the function of the transcription activation region AF2 of wild-type estrogen receptor α is suppressed. The type of anti-estrogen substance that does not suppress the function of the transcription activation region AF1 activates transcription and increases the expression level of a reporter gene. On the other hand, a wild-type estrogen receptor α gene that lacks the region encoding the transcriptional activation region AF1 is expressed in cells such as primary cultured cells of chicken embryo fibroblast, and is ligated downstream of the transcriptional control region including the estrogen response element. Introduce a reporter gene. When a test substance is brought into contact with the obtained cells (hereinafter referred to as AF2 activity evaluation cells) and a reporter assay is performed as described below, “the function of the transcription activation region AF2 of wild-type estrogen receptor α is suppressed. However, an anti-estrogen substance that 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 “antiestrogens of the type that suppress the function of the transcriptional activation region AF2 of wild-type estrogen receptor α but do not suppress the function of the transcriptional activation region AF1” having such characteristics include tamoxifen, 4- Hydroxy tamoxifen, raloxifene and the like can be mentioned.

  “Pure anti-estrogen” means a substance that exhibits substantially no estrogen activity including partial agonist activity such as that possessed by tamoxifen, and such substance is a cell for AF1 activity evaluation as described above or AF2 activity evaluation When the reporter assay is performed using any of the cells for transcription, transcriptional activation is not induced and the expression level of the reporter gene is not changed. Specific examples of “pure antiestrogens” include ICI182780 [Wakeling, AE et al., Cancer Res., 512: 3867-3873 (1991)], ZM189154 [Dukes, M. et al., J. Endocrinol., 141 : 335-341 (1994)].

The receptor of the present invention is “the amino acid in the position corresponding to the amino acid represented by amino acid number 424 of the amino acid sequence represented by SEQ ID NO: 1 in the alignment based on the homology of the amino acid sequence among the amino acids constituting the receptor, Since it is substituted with an amino acid different from the amino acid at the corresponding position in the amino acid sequence of the wild-type estrogen receptor α ”, it has the following properties (e) and (f).
(e) Contact with any type of anti-estrogen such as tamoxifen, 4-hydroxy tamoxifen, raloxifene, etc. that suppresses the function of the transcriptional activation region AF2 of wild-type estrogen receptor α but does not suppress the function of the transcriptional activation region AF1 Then, transcription of the gene under the transcriptional control of the transcriptional regulatory region containing the estrogen response element can be activated.
(f) When contacted with estrogen, transcription of a gene linked downstream of a transcriptional control region containing an estrogen response element can be activated, and the activation activates transcription of the gene in (e) above. Is not substantially inhibited by the compound that can.
Specific examples of the substitution of amino acids as described above include substitution of isoleucine at a position corresponding to the amino acid represented by amino acid number 424 of the amino acid sequence represented by SEQ ID NO: 1 with threonine.
As the receptor of the present invention, a receptor derived from animals such as mammals such as humans, monkeys, rabbits, rats, and mice is preferable, and specific examples thereof include estrogen receptor α having the amino acid sequence shown by SEQ ID NO: 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, or produced by introducing mutation into DNA isolated from nature, for example, by site-directed mutagenesis or mutation treatment. It may also be a prepared DNA.
The DNA of the present invention can be prepared, for example, from cDNA encoding wild-type estrogen receptor α as follows.
In order to obtain cDNA encoding wild-type estrogen receptor α, first, animals such as mammals such as humans, monkeys, rabbits, rats, and mice based on gene databases such as GenBank and nucleotide sequences described in literatures. A primer for amplifying a DNA encoding the wild type estrogen receptor α derived therefrom by polymerase chain reaction (hereinafter referred to as PCR) is designed and synthesized. Specifically, for example, in order to obtain cDNA encoding human-derived wild-type estrogen receptor α, a primer designed based on the base sequence described in GenBank Accession No. M12674, for example, SEQ ID NO: 3 Chemical synthesis of a forward primer consisting of the base sequence shown in FIG. 5 and a reverse primer consisting of the base sequence shown in SEQ ID NO: 4.
On the other hand, for example, it is described in Sambrook, J., and Zrussell, DW; Molecular Cloning 3rd Edition, Cold Spring Harbor Laboratory (2001), etc. from tissues of mammals such as humans, monkeys, rabbits, rats and mice. RNA is extracted according to the genetic engineering method of, and single-stranded cDNA is synthesized. Specifically, first, total RNA is prepared from animal tissues such as mammals such as humans, monkeys, rabbits, rats, and mice. For example, tissue such as liver is pulverized in a solution containing a protein denaturant such as guanidine hydrochloride or guanidine thiocyanate, and the protein is denatured by adding phenol, chloroform or the like to the pulverized product. After removing the denatured protein by centrifugation or the like, total RNA is extracted from the collected supernatant fraction by a method such as guanidine hydrochloride / phenol method, SDS-phenol method, guanidine thiocyanate / cesium chloride-ultracentrifugation method. Examples of commercially available kits based on these methods include ISOGEN (manufactured by Nippon Gene) and TRIZOL reagent (manufactured by Invitrogen). Next, using the obtained total RNA as a template, oligo dT primer was bound to the polyA sequence of mRNA, reverse transcriptase such as RnaseH-Superscript II Reverse Transcriptase (Invitrogen) and attached buffer and oligo dT primer. To inactivate the reverse transcriptase by heating at 42 ° C. for 1 hour and then heating at 99 ° C. for 5 minutes. Examples of commercially available kits based on these methods include cDNA synthesis system plus (Amersham Biotech) and TimeSaver cDNA synthesis kit (Amersham Biotech).
Next, using the single-stranded cDNA synthesized as described above as a template, each of the above primers was added to 200 nM. For example, LA Taq DNA polymerase (manufactured by Takara Shuzo) and the enzyme were attached. PCR is performed using the prepared buffer. As such PCR, for example, a commercially available PCR thermal cycler such as PCR System 9700 (manufactured by Applied Biosystems) is used for about 35 cycles at 95 ° C. for 1 minute and then at 68 ° C. for 3 minutes. Here, in place of the cDNA synthesized as described above, commercially available cDNAs derived from various animals such as Clonetech quick clone cDNA may be used. A part of the obtained reaction solution is subjected to gel electrophoresis using agarose (Agarose L: Nippon Gene). After confirming the presence of a band made of DNA encoding wild-type estrogen receptor α having a size expected from a known sequence on the electrophoresed gel, the DNA of the band portion is recovered from the gel. For the base sequence of the recovered DNA, a sample for direct sequencing is prepared using the recovered DNA and a commercially available fluorescent sequencing reagent such as Dye Terminator Sequence Kit FS (manufactured by Applied Biosystems), and Applied Biosystems It can be confirmed that the DNA recovered by base sequence analysis using an automated DNA sequencer such as Model 3700 manufactured by the company is the target DNA. The DNA encoding the wild-type estrogen receptor α thus obtained is cloned into a vector that can replicate in E. coli and the like. Specifically, for example, about 1 μg of the recovered DNA is blunted with a DNA Blunting Kit (manufactured by Takara Shuzo Co., Ltd.) or the like, and then reacted with T4 polynucleotide kinase to phosphorylate the end. After the DNA is treated with phenol, it is purified by ethanol precipitation, and the purified DNA is introduced into a vector that can be replicated in Escherichia coli and the like, thereby obtaining a vector carrying the DNA encoding wild-type estrogen receptor α. .
Next, a DNA of a vector carrying the DNA encoding the wild-type estrogen receptor α thus prepared is prepared. Using the prepared DNA as a template, Sambrook, J., and Russell, DW; In accordance with the site-directed mutagenesis method described in the 3rd edition, Cold Spring Harbor Laboratory (2001), etc., a base substitution for performing the desired amino acid substitution is introduced into DNA encoding wild-type estrogen receptor α. . Specific examples include QuickChange Site-Directed Mutagenesis Kit manufactured by Stratagene. Using two types of primers designed and synthesized to perform the desired base substitution, a site-specific mutation is introduced according to the description of the kit manual. As a primer for substituting codon ATC encoding isoleucine, the 424th amino acid from the amino terminus of human-derived wild-type estrogen receptor α, with codon ACC encoding threonine, for example, the base sequence represented by SEQ ID NO: 5 And a reverse primer consisting of the base sequence shown in SEQ ID NO: 6 can be used.
By determining the base sequence of the obtained DNA, 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 that can replicate autonomously. The vector of the present invention can be constructed by incorporating into a vector having a detectable marker (hereinafter referred to as a basic vector) in accordance with a normal genetic engineering technique.
Specific examples of basic vectors that can be used to construct the vector of the present invention include plasmid pUC19 (manufactured by Takara Shuzo), phagemid pBluescript II (Stratagene), etc. be able to. When budding yeast is used as a host cell, plasmids pGBT9, pGAD404, pACT2 (Clontech) and the like can be mentioned. When mammalian cells are used as host cells, plasmids such as pRc / RSV and pRc / CMV (Invitrogen), viruses such as bovine papillomavirus plasmid pBPV (Amersham Biotech), and EB virus plasmid pCEP4 (Invitrogen) Examples include vectors containing the origin of autonomous replication (ori) derived from, viruses such as vaccinia virus, and insect cells such as baculoviruses when insect cells are used as host cells.
When the vector of the present invention is constructed using a vector containing an autonomous origin of replication, such as the above-mentioned yeast plasmid pACT2, bovine papilloma virus plasmid pBPV, Epstein-Barr virus plasmid pCEP4, etc., the vector is introduced into the host cell. It is retained in the cell as an episome.
In order to incorporate the DNA of the present invention into a virus such as baculovirus or vaccinia virus, a transfer vector containing a base sequence homologous to the genome of the virus to be used can be used. Specific examples of such transfer vectors include plasmids such as pVL1392, pVL1393 (Invitrogen), pSFB5 (Pharmingen). When the DNA of the present invention is inserted into a 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 is present on the genome. Recombinant virus incorporated into can be obtained. As the viral genome, genomes such as baculovirus, adenovirus, and vaccinia virus can be used.
More specifically, for example, when the DNA of the present invention is incorporated into a baculovirus, the DNA of the transfer vector and the baculovirus genomic DNA (BaculoGold; Pharmingen) is introduced into insect cell strain Sf21 (available from ATCC) by the calcium phosphate method or the like, and the cells are cultured. When the Baculogold DNA is used, only the virus particles containing the viral DNA into which the DNA of the present invention is inserted are released into the culture medium of the host cell. Such recombinant virus particles are recovered from the culture solution and deproteinized with phenol or the like to obtain virus DNA containing the DNA of the present invention. Furthermore, the recombinant virus particles can be increased by introducing the DNA of the virus into a host cell having the ability to form 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 also be directly integrated into a relatively small genome such as murine leukemia retrovirus without using a transfer vector. For example, the viral vector-DC (X) (Eli Gilboa et al., BioTechniques, 4: 504-512 (1986)) or the like may be incorporated into the cloning site on the vector. When the recombinant virus constructed in this way is introduced into a packaging cell such as Ampli-GPE (J. Virol., 66: 3755 (1992)), it contains the viral DNA inserted with the DNA of the present invention. Virus particles can be obtained.

A book capable of expressing the DNA of the present invention in a host cell by binding a promoter capable of functioning in the host cell in a functional form upstream of the DNA of the present invention and incorporating it into a basic vector as described above. Invention vectors can be constructed. Here, “to bind in a functional manner” means to bind the promoter and the DNA of the present invention so that the DNA of the present invention is expressed under the control of the promoter in the host cell into which the DNA of the present invention is introduced. It means that The promoter used exhibits promoter activity in the host cell to be transformed. For example, when the host cell is E. coli, the Lactose operon promoter (lacP) of E. coli, the promoter of tryptophan operon (trpP) ), Arginine operon promoter (argP), galactose operon promoter (galP), tac promoter, T7 promoter, T3 promoter, λ phage promoter (λ-pL, λ-pR), etc. In the case of animal cells or fission yeast, for example, rous sarcoma virus (RSV) promoter, cytomegalovirus (CMV) promoter, simian virus (SV40) early or late promoter, mouse papilloma virus (MMTV) promoter, etc. Can be mentioned. When the host cell is budding yeast, an alcohol dehydrogenase (ADH) 1 gene promoter and the like can be mentioned.
In addition, when a basic vector having a promoter that functions in a host cell in advance is used, the DNA of the present invention is placed downstream of the promoter so that the promoter of the vector and the DNA of the present invention are operably linked. Insert it. For example, the aforementioned plasmids pRc / RSV, pRc / CMV, etc. are provided with a cloning site downstream of a promoter that can function in animal cells, and if the DNA of the present invention is inserted into the cloning site and introduced into animal cells, The DNA of the present invention can be expressed. Since these plasmids have SV40 autonomous replication origin (ori) incorporated in advance, introduction of the plasmid into cultured cells transformed with the SV40 genome of ori (-), such as COS cells, The number of copies of the plasmid is greatly increased, and as a result, the DNA of the present invention incorporated in the plasmid can be expressed in a large amount. The yeast plasmid pACT2 has an ADH1 promoter. If the DNA of the present invention is inserted downstream of the ADH1 promoter of the plasmid or a derivative thereof, the DNA of the present invention is budding, for example, CG1945 strain (manufactured by Clontech). A vector of the present invention that can be expressed in large quantities in yeast can be constructed.

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 normal method for introducing it according to the host cell to be transformed can be applied. For example, when E. coli, a microorganism, is used as a host cell, the calcium chloride method described in Molecular Cloning 3rd Edition (Sambrook, J., and Russell, DW, Cold Spring Harbor, 2001), etc. A normal method such as a poration method can be used. In addition, when a mammalian cell or insect animal cell is used as a host cell, it is introduced into the cell by a general gene transfer method such as a calcium phosphate method, a DEAE dextran method, an electroporation method, or a lipofection method. Can do. When yeast is used as a host cell, it can be introduced using, for example, 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 a host cell by a general gene transfer method as described above, or by infecting a host cell with a recombinant virus particle containing the viral DNA. Viral DNA can be introduced into host cells.

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 selection marker gene is simultaneously introduced into the host cell, and the obtained cell is introduced into the selected selection cell. The culture is preferably performed under conditions according to the nature of the marker gene. For example, when the selection marker gene is a gene that confers drug resistance to a selection drug exhibiting lethal activity on the host cell, the host cell introduced with the vector of the present invention is cultured using a medium to which the drug is added. do it. Examples of combinations of a drug resistance-conferring gene and a selective drug include, for example, a combination of a neomycin resistance-conferring gene and neomycin, a combination of a hygromycin resistance-conferring gene and hygromycin, and a blasticidin S resistance-conferring gene and blasticidin S. Combinations can be mentioned. When the selection marker gene is a gene that complements the auxotrophy of the host cell, the host cell into which the vector of the present invention has been introduced may be cultured using a minimal medium that does not contain the nutrient. 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 is introduced into the chromosome of the 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 converted into the above-mentioned method. Then, the cells are introduced into the host cells, and the cells are usually cultured for several weeks, and the target transformant is selected using the expression of the selection marker gene encoded by the introduced vector of the present invention as an index. For example, the vector of the present invention having a gene for imparting resistance to a selection drug as described above as a selection marker gene is introduced into a host cell by the above-described method, and the cell is subcultured for several weeks or more in a medium to which the selection drug is added. The transformant of the present invention in which the DNA of the present invention is introduced into the chromosome of the host cell can be selected by subculturing and purifying the selected drug-resistant clone that survived in a colony form. Since the transformant can be cryopreserved and can be used after sleeping, the transformant can be prepared for each experiment as compared with the strain into which the DNA of the present invention was transiently introduced. In addition, the test can be carried out using transformants whose properties and handling conditions have been confirmed in advance.

The receptor of the present invention can be produced by culturing the transformant of the present invention obtained as described above.
For example, when the transformant of the present invention is a microorganism, the transformant is cultured using various media appropriately containing a carbon source and a nitrogen source, organic or inorganic salts, etc., used for normal culture in general microorganisms. Is done. 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 Fermenter culture, tank culture, etc.) are possible. is there. The culture temperature can be appropriately changed within the range in which the microorganism grows. For example, the culture temperature is generally about 15 ° C. to about 40 ° C. and the culture medium is pH 6.0 to 8.0. The culture time varies depending on the culture conditions, but is usually about 1 to 5 days. When an expression vector having an inducible promoter such as a temperature shift type or an isopropyl β-D-galactopyranoside (IPTG) inducible type is used, the induction time is preferably within one day, usually several hours.
In addition, when the transformant is an animal cell such as a mammal or an insect, the transformant can be cultured using a medium used for normal culture in general cultured cells. When the transformant is produced using a selective drug, it is desirable to culture in the presence of the selective drug. In the case of mammalian cells, for example, using Dulbecco's modified eagle (DMEM) medium (Nissui Pharmaceutical Co., Ltd.) supplemented with fetal bovine serum (FBS) to a final concentration of 10%, The culture may be performed while changing to a new culture solution every few days under the condition of% CO ₂ presence. When the cells grow to confluence, for example, a trypsin / PBS solution of about 0.25% (v / v) is added to disperse them into individual cells, diluted several times, seeded in a new petri dish, and cultured. Similarly, in the case of insect cells, culture is performed at a culture temperature of 25 ° C. to 35 ° C. using a culture solution for insect cells such as Grace's medium containing 10% (v / v) FBS and 2% (w / v) Yeastlate. do it. At this time, in the case of cells that are easily detached from the petri dish such as Sf21 cells, subculture can be performed by dispersing by pipetting or the like without using a trypsin solution. In the case of a transformant containing a viral vector such as baculovirus, the culture time is preferably set to 72 hours after the virus infection, for example, before the cytoplasmic effect appears and the cells die.
Recovery of the receptor protein of the present invention produced by the transformant of the present invention may be performed by appropriately combining conventional isolation and purification methods. For example, after completion of the culture, the cells of the transformant are centrifuged and the like. The collected cells are suspended in a normal buffer such as 20 mM HEPES (pH 7.0), 1 mM EDTA, 1 mM DTT, 0.5 mM PMSF, and then suspended with polytron, sonication, Dounce homogenizer, etc. The fraction containing the estrogen receptor can be obtained by crushing, ultracentrifugating the disrupted solution at 100,000 xg for several tens of minutes to about 1 hour, and collecting the supernatant fraction. Furthermore, the purified estrogen receptor can also be recovered by subjecting the supernatant fraction to various types of chromatography such as ion exchange, hydrophobicity, gel filtration, and affinity. In this case, the fraction containing the receptor of the present invention can be identified by a DNA binding assay using an oligonucleotide having a length of about 15 to 200 bp including an estrogen response element, that is, a base sequence to which the estrogen receptor binds, as a probe. .
The receptor of the present invention thus produced can be used, for example, in a ligand / receptor binding assay for evaluating the binding ability or binding 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 and quantifying the binding amount, as well as analyzing the binding specificity and binding force. For example, when a test substance is allowed to coexist where a labeled ligand (hereinafter referred to as labeled ligand) is bound to the receptor of the present invention recovered from the transformant of the present invention as described above, the test substance According to the competition between the ligand and the labeled ligand, the labeled ligand is released from the receptor in accordance with the affinity for both receptors, and the amount of the labeled ligand bound to the receptor is decreased, so that the amount of the label bound to the receptor is decreased. Therefore, the ability of the test substance to bind to the receptor can be indirectly determined by monitoring the labeling amount of the free labeled ligand or the labeling amount of the bound labeled ligand.
As the labeled ligand, for example, tritium-labeled 17β-estradiol (hereinafter referred to as E2) or the like can be used. Separation of the labeled ligand between bound and free forms can be performed by a hydroxyapatite method, a 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 place where the labeled ligand is bound to the receptor of the present invention, which corresponds to a system in which the concentration of the test substance is zero, and the bound labeled ligand obtained from this system. The amount of labeling indicates the total binding amount of the labeled ligand to the estrogen receptor. Another system is where the labeled ligand is bound to the receptor of the present invention, for example, so that unlabeled E2 is sufficiently saturated (e.g., 10 μM) so that the receptor cannot be sufficiently bound. The amount of the labeled labeled ligand obtained from this system is determined as 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 to the receptor of the present invention is a value obtained by subtracting this non-specific binding amount from the total binding amount. The third system is a system in which the test substance is added to a place where the labeled ligand is bound to the receptor of the present invention so that the test substance has a final concentration of 10 μM (this concentration is arbitrarily changed depending on the purpose). When the test substance has the ability to bind to the receptor of the present invention, the labeling amount of the bound labeled ligand obtained from this system is the amount to the receptor of the present invention when the test substance concentration determined as described above is zero. It becomes smaller than the specific binding amount of the labeled ligand. By conducting the ligand / receptor binding assay in this manner, the binding ability of the test substance to the receptor of the present invention can be examined. When the test substance contains a plurality of substances, the affinity for the receptor of the present invention is included therein. It is also possible to check whether the indicated substance 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 addition concentration of the test substance in the third system, the assay is performed in the same manner to determine the labeling amount of the bound labeled ligand. taking measurement. Based on the measured value, the amount of bound and free ligand in each assay is calculated, and, for example, by performing Scatchard analysis, the binding affinity, binding specificity, binding capacity between the test substance and the receptor of the present invention Etc. 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 estrogen receptor α activity-modulating ability include agonist activity and antagonist activity for estrogen receptor α, and more specifically include estrogen-like activity and anti-estrogenic activity.
The “reporter gene under the transcriptional control of a transcriptional control region containing an estrogen response element” used in the evaluation method of the present invention specifically refers to, for example, the transcriptional control region of Xenopus vitellogenin gene containing an estrogen response element A reporter gene in which a DNA encoding a reporter protein is linked downstream, or a reporter gene in which a base sequence necessary for initiation of transcription and a DNA encoding the reporter protein are linked downstream of an estrogen response element, etc. It is a gene used to monitor the transcriptional regulation ability of estrogen receptor in host cells. The “DNA encoding the reporter protein” used for the preparation of such a reporter gene includes DNA encoding luciferase, DNA encoding secretory alkaline phosphatase, DNA encoding β-galactosidase, chloramphenicol acetyltransferase. A DNA encoding a reporter protein that can be used, such as a DNA encoding a growth hormone and a DNA encoding a growth hormone, and having relatively high stability in a host cell is preferable.
In order to obtain a transformant used in the evaluation method of the present invention, for example, the DNA of the present invention and the reporter gene described above are used, for example, an estrogen receptor non-endogenous host cell, specifically, for example, HeLa cell, CV-1 It is introduced into cells, Hepa1 cells, NIH3T3 cells, HepG2 cells, COS1 cells, BF-2 cells, CHH-1 cells, etc. to produce transformants. Here, the DNA of the present invention may be, for example, linked to a promoter that can function in a host cell in a functional manner, incorporated into a basic vector, and then introduced into the cell as described above. The above reporter gene may also be used by being incorporated 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 selection marker gene are simultaneously introduced into a host cell, and the transformant is expressed using the expression of the selection marker gene as an index. By selecting, a transformant in which the reporter gene and the DNA of the present invention are introduced into the host cell chromosome is obtained. Since the transformant can be cryopreserved and can be used after sleeping, if necessary, once obtained, these genes are introduced into host cells each time a new assay is performed. Since it is not necessary to obtain a transformant and the performance of the transformant can be kept constant, the present invention is useful, for example, when carrying out a large-scale screening using an automated robot.
While the transformant produced as described above is cultured, for example, for 1 to several days, a test substance is added to the medium and brought into contact with the transformant, and the expression level of the reporter gene in the transformant Measure. When the receptor of the present invention produced by the transformant is activated by the binding of a test substance, transcription of the reporter gene is promoted, and the reporter protein encoded by the reporter 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 transformed cell is measured. Specifically, for example, when luciferase is used as the reporter protein, when luciferin, which is a luciferase substrate, is added to the crude cell extract, light is emitted with 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 hence the expression level of the luciferase reporter gene can be known. Similarly, the expression level of the reporter gene under the condition where the test substance is not contacted with the transformant is measured, and the expression level is compared with the expression level of the reporter gene under the condition where the test substance is contacted. If the expression level of the reporter gene under the condition where the test substance is contacted is higher than the expression level of the reporter gene under the condition where the test substance is not contacted with the transformant, the test substance is an agonist for the receptor of the present invention. It can be evaluated that it has activity, that is, has the ability to activate the receptor. In addition, for example, the expression level of the reporter gene can be determined in the same manner as described above under the conditions in which an estrogen such as E2 is brought into contact with the transformant and in the condition in which the estrogen and the test substance are simultaneously contacted. taking measurement. If the expression level of the reporter gene under the condition where the estrogen and the test substance are brought into contact with each other is lower than the expression level of the reporter gene under the condition where the estrogen is brought into contact with the transformant, the test substance is used as the receptor of the present invention. It can be evaluated as having an antagonist activity against, ie, an anti-estrogen activity against the receptor. Thus, for example, a reporter assay is performed using a transformant in which a reporter gene under the transcriptional control of a transcriptional control region containing an estrogen response element and the DNA of the present invention are introduced into the chromosome of a host cell. By evaluating the antiestrogenic activity of a substance, a substance exhibiting antiestrogenic activity against the receptor of the present invention can be selected.
According to the evaluation method of the present invention as described above, for example, tamoxifen, raloxifene, and the like are anti-estrogen substances of the type that suppress the function of the transcription activation region AF2 of wild-type estrogen receptor α but not the function of AF1. Thus, a substance that exhibits agonist activity and does not exhibit antiestrogenic activity for the receptor of the present invention can be found. On the other hand, since a substance that does not exhibit agonist activity with respect to the receptor of the present invention and exhibits antiestrogenic activity can be selected by such an evaluation method, screening of a test substance based on the evaluation method is performed with estrogen activity. Is useful for the development of pure antiestrogens that exhibit virtually no

In order to examine whether or not the estrogen receptor α produced in the cells of an individual animal such as a human is the receptor of the present invention, for example, it is examined whether or not the estrogen receptor α gene of the individual is the DNA of the present invention. Good.
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, from samples of cell tissues from which genomic DNA such as hair roots, peripheral blood, and oral epithelial cells can be extracted, Masamura Muramatsu, “Lab Manual Genetic Engineering” (Maruzen; 1988) and TAKARA PCR Technical news No. 2, Genomic DNA can be prepared according to the usual method described in Takara Shuzo (1991). Specifically, for example, when the sample is hair, one hair with a root is washed with sterilized water and ethanol, and then BCL-Buffer [10 mM Tris-Cl (pH 7.5), 5 mM MgCl ₂ , 0.32 M Sucrose, 1% (v / v) Triton X-100] 200 μl, and then add proteinase K to a final concentration of 100 μl / ml and SDS to a final concentration of 0.5% (w / v) and mix. . The mixture is incubated at 70 ° C. for 1 hour, and then extracted with phenol / chloroform to obtain genomic DNA. 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, RNA is prepared using, for example, TRIZOL reagent (manufactured by Invitrogen) while the sample is fresh, and the prepared RNA is used as a template. If cDNA is synthesized by reverse transcriptase, the synthesized cDNA can be used for testing instead of genomic DNA.

In order to examine whether or not the estrogen receptor α gene present in the genomic DNA or cDNA thus prepared is the DNA of the present invention, for example, the estrogen receptor α encoded by the estrogen receptor α gene is constructed. A nucleotide sequence encoding an amino acid at a position corresponding to the amino acid represented by amino acid number 424 of the amino acid sequence represented by SEQ ID NO: 1 in an alignment based on amino acid sequence homology, It is good to examine whether or not the amino acid sequence of α is substituted with a base sequence encoding an amino acid different from the amino acid at the position corresponding to the amino acid. 1 is the amino acid number 424 of the amino acid sequence represented by Nucleic acid encoding a region containing an amino acid "in the position corresponding to amino acids that are the nucleic acid in the sample was amplified as a template, and a method for determining the nucleotide sequence of the amplified nucleic acid.
A nucleic acid encoding a region containing “an amino acid at a position corresponding to the amino acid represented by amino acid number 424 of the amino acid sequence represented by SEQ ID NO: 1” can be amplified by PCR, for example.
As a primer that can be used in such PCR, the ability to amplify by PCR a nucleic acid encoding a region containing "an amino acid at a position corresponding to the amino acid represented by amino acid number 424 of the amino acid sequence represented by SEQ ID NO: 1" An oligonucleotide having a GC content of about 30% to about 70%, preferably about 35% to about 60%, and comprising 8 bases to 50 bases, preferably about 15 bases to about 40 bases. I can give you. More specifically, for example, an oligonucleotide having a base sequence represented by any of SEQ ID NOs: 7 to 11 can be used as the oligonucleotide for forward primer, and SEQ ID NOs: 12 to 16 can be used as the oligonucleotide for reverse primer. The oligonucleotide which consists of a base sequence shown by either of these can be mention | raise | lifted.
When PCR is performed using the above oligonucleotide as a primer, generally two types of oligonucleotides for forward and reverse are used in combination. These oligonucleotides can be chemically synthesized by, for example, the β-cyanoethyl phosphoramidite method or the thiophosphite method.
PCR can be performed according to the method described in, for example, Saiki, RK et al., Science, 230: 1350-1354 (1985). For example, prepare an amplification buffer containing oligonucleotides used as primers, DNA polymerase, 4 types of bases (dATP, dTTP, dGTP, dCTP), about 1.5 mM to about 3.0 mM magnesium chloride, and genomic DNA. . For example, a three-step amplification cycle is repeated using the prepared amplification buffer under the following conditions. First, as a denaturation step, for example, incubation is performed at about 90 ° C. to about 95 ° C., preferably about 94 ° C. to about 95 ° C., for 1 minute to about 5 minutes, preferably about 1 minute to about 2 minutes. Next, as a primer annealing step, for example, a temperature is maintained at about 30 ° C. to 70 ° C., preferably 40 ° C. to 60 ° C., for about 3 seconds to about 3 minutes, preferably about 5 seconds to about 2 minutes. Further, as an extension step by DNA polymerase, for example, a temperature is maintained at about 70 ° C. to about 75 ° C., preferably about 72 ° C. to about 74 ° C., for about 15 seconds to about 5 minutes, preferably about 30 seconds to about 4 minutes. Is called. This three-step amplification cycle is performed about 20 to about 50 times, preferably about 25 to about 40 times.
The nucleic acid amplified by PCR is recovered by subjecting it to agarose electrophoresis or the like, and the base sequence of the DNA can be confirmed by performing a direct sequence [BioTechniques, 7,494 (1989)] on the recovered nucleic acid. it can. The nucleotide sequence can be determined using the Maxam Gilbert method (for example, Maxam, AM and Gilbert, W., Proc. Natl. Acad. Sci. USA., 74; 560, 1977) or the Sanger method (for example, Sanger, F. and Coulson, AR, J. Mol. Biol., 94; 441, 1975., Sanger, F., Nicklen, S. and Coulson, AR, Proc. Natl. Acad. Sci. USA., 74; 5463, 1977) That's fine. When an automatic DNA sequencer such as Applied Biosystems model 3700 is used, a corresponding DNA sequence kit such as BigDye terminator cycle sequencing ready reaction kit manufactured by Applied Biosystems can be used.

An amino acid that constitutes estrogen receptor α in a nucleic acid in a sample and that is in a position corresponding to the amino acid represented by amino acid number 424 of the amino acid sequence represented by SEQ ID NO: 1 in the alignment based on the homology of the amino acid sequence As a method for investigating whether or not the nucleotide sequence coding for is substituted with a nucleotide sequence encoding an amino acid different from the amino acid at the corresponding position of the amino acid sequence of wild-type estrogen receptor α, for example, A nucleic acid encoding a region containing "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 amplified using the nucleic acid in the sample as a template, and the amplified nucleic acid is electrophoresed Thus, the mobility of the nucleic acid is measured, and the wild type estrogen receptor α is detected. And mobility of the nucleic acid encoding the region and mobility of the nucleic acids may be mentioned a method to check different or not.
For example, when examining the estrogen receptor α gene in a human sample, first, for example, the ends of the oligonucleotides shown in SEQ ID NO: 7 to SEQ ID NO: 16 are labeled with a fluorescent substance such as FITC, and this is used as a primer. PCR is performed as described above to amplify a nucleic acid encoding a region containing “an amino acid at a position corresponding to the amino acid represented by amino acid number 424 of the amino acid sequence represented by SEQ ID NO: 1”. In addition, the nucleic acid encoding the corresponding region of the wild type estrogen receptor α is amplified in the same manner.
The amplified nucleic acid is electrophoresed according to the SSCP (single strand conformation polymorphism) method described in Hum. Mutation, 2; 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 then subjected to non-denaturing polyacrylamide electrophoresis to separate the single strands. Examples of the buffer used for electrophoresis include tris-phosphate system (pH 7.5-8.0), tris-acetic acid system (pH 7.5-8.0), and tris-boric acid system (pH 7.5-8.3). May be tris-boric acid. If necessary, glycerol or the like is added to the gel. Examples of conditions for electrophoresis include conditions for electrophoresis at a constant power of 30 W to 40 W, room temperature (about 20 ° C. to about 25 ° C.) or 4 ° C. for 4 to 8 hours. Next, the fluorescence signal in the gel after electrophoresis is detected by a scanner capable of reading fluorescence, and is “at a position corresponding to the amino acid represented by amino acid number 424 of the amino acid sequence represented by SEQ ID NO: 1 derived from the sample. The mobility of the nucleic acid encoding the region containing “amino acid” and the nucleic acid encoding the region of wild-type estrogen receptor α are compared. When the mobility of these nucleic acids is different, the wild-type estrogen is added to the base sequence encoding the region containing “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”. It can be determined that a base sequence different from the base sequence encoding the region of receptor α is included. Furthermore, the nucleic acid contained in the gel containing nucleic acids having different mobilities is extracted into sterilized water, the nucleic acid is amplified by PCR using this as a template, and the amplified product is amplified by pGEM-T Easy vector ( After cloning into a vector such as Invitrogen), the nucleotide sequence of the cloned nucleic acid can be confirmed, and a nucleotide sequence different from the nucleotide sequence encoding wild-type estrogen receptor α can be identified.

An amino acid that constitutes estrogen receptor α in a nucleic acid in a sample and that is in a position corresponding to the amino acid represented by amino acid number 424 of the amino acid sequence represented by SEQ ID NO: 1 in the alignment based on the homology of the amino acid sequence As a method for investigating whether or not the nucleotide sequence coding for is substituted with a nucleotide sequence encoding an amino acid different from the amino acid at the corresponding position of the amino acid sequence of wild-type estrogen receptor α, for example, Corresponding to the above-mentioned amino acid among the nucleic acid encoding the region containing "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" and the wild-type estrogen receptor α Salt encoding the region containing the amino acid at position It may be a method to check whether a probe comprising sequences hybridize.
Of the amino acid sequence of wild-type estrogen receptor α, a probe comprising a base sequence encoding a region containing an amino acid at a position corresponding to the amino acid represented by amino acid number 424 of the amino acid sequence represented by SEQ ID NO: 1 is used as such a base. An oligonucleotide having a sequence and a GC content of about 30% to about 60% can be mentioned. The number of bases constituting the oligonucleotide is preferably 15 or more and 40 or less so as to be suitable for detection by hybridization. Specifically, for example, an oligonucleotide having a base sequence selected from the following base sequence group can be raised. Among the amino acid sequences of human-derived wild-type estrogen receptor α, as a probe consisting of a base sequence encoding a region containing isoleucine at a position corresponding to the amino acid represented by amino acid number 424 of the amino acid sequence represented by SEQ ID NO: 1, Examples thereof include a probe comprising the base sequence represented by any of SEQ ID NOs: 17 to 21.
In general, when the number of bases constituting the oligonucleotide is about 100 or less, the oligonucleotide as described above can be chemically synthesized by, for example, the β-cyanoethyl phosphoramidite method or the thiophosphite method.
“Amino acid at a position corresponding to the amino acid represented by amino acid number 424 of the amino acid sequence represented by SEQ ID NO: 1” present in the genomic DNA or cDNA prepared from a sample of an animal individual such as a human as described above. The nucleic acid encoding the region to be included is amplified by PCR using, for example, the above-mentioned oligonucleotide as a primer, and then mixed with a probe having the base sequence shown by any of SEQ ID NOs: 17 to 21 for hybridization. Do. Hybridization may be performed by a conventional dot blot hybridization method or a mismatch detection method using an enzyme called Taq MutS, which is an enzyme that binds to a mismatch hybridization site [Biswas, I., and Hsieh, P., J. Biol. Chem. 271; 5040-5048 (1996) and Nippon gene information 1999 No. 125, described in Nippon Gene Co., Ltd.].
As a specific procedure of the dot blot hybridization method, for example, first, genomic DNA or cDNA prepared from a sample of an animal individual such as a human, or “amino acid of the amino acid sequence represented by SEQ ID NO: 1 amplified by PCR” A nucleic acid encoding a region containing the amino acid at the position corresponding to the amino acid represented by the number 424 is incubated at, for example, 90 ° C. to 100 ° C. for 3 to 5 minutes, and then a nylon filter [Hybond N ⁺ (Amersham Biotech The spotted filter is dried on a filter paper, and then irradiated with ultraviolet light to fix the nucleic acid to the filter. Next, the obtained DNA fixing filter and the above probe are hybridized by, for example, incubating at 40 to 50 ° C. for 10 to 20 hours, and a hybrid containing the probe is detected according to a normal method. When a radioactive probe labeled with a radioisotope such as ³² P is used as the probe, the hybrid containing the probe can be detected by exposing the hybridized filter to an X-ray film. When using a non-radioactive probe labeled with a biotinylated nucleotide, the hybrid containing the probe is enzymatically labeled with biotinylated alkaline phosphatase via streptavidin, and the probe is detected by detecting the coloration or luminescence of the substrate due to the enzymatic reaction. Hybrids containing can be detected. In addition, a non-radioactive probe directly labeled with an enzyme such as alkaline phosphatase or peroxidase via a spacer can also be used. When a hybrid containing a probe is not detected, the base of the probe used in the nucleic acid encoding the region containing “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” It can be determined that a base sequence different from the sequence is included.
When using a mismatch detection method using Taq MutS, 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. Using Taq MutS binding properties that can recognize and bind mismatched base pairs, gel shift assay using native polyacrylamide gel or dot blotting method on solid phase such as nylon filter and nitrocellulose filter Thus, mismatched base pairs can be detected. If a mismatch is detected, the nucleic acid encoding the region containing “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” and the base sequence of the probe used It can be determined that different base sequences are included.

An amino acid that constitutes estrogen receptor α in a nucleic acid in a sample and that is in a position corresponding to the amino acid represented by amino acid number 424 of the amino acid sequence represented by SEQ ID NO: 1 in the alignment based on the homology of the amino acid sequence As a method for investigating whether or not the nucleotide sequence coding for is substituted with a nucleotide sequence encoding an amino acid different from the amino acid at the corresponding position of the amino acid sequence of wild-type estrogen receptor α, for example, A nucleic acid that encodes a region containing "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 amplified from the gene in the sample, digested with a restriction enzyme, and gene mutation By detecting the disappearance or expression of the restriction enzyme site A method for examining the presence or absence of a restriction enzyme recognition sequence can also be mentioned.
For example, when examining the estrogen receptor α gene in a human sample, first, for example, PCR is performed as described above using the oligonucleotide having the nucleotide sequence represented by any of SEQ ID NOs: 7 to 16 as a primer, A nucleic acid encoding a region containing 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 amplified. In addition, the nucleic acid encoding the corresponding region of the wild type estrogen receptor α is amplified in the same manner. Both nucleic acids are cleaved according to a standard method using a restriction enzyme that recognizes a difference in sequence derived from a gene mutation in both amplified nucleic acids. If these nucleic acid fragments after restriction enzyme treatment are subjected to gel electrophoresis using agarose, polyacrylamide, etc., the base sequence that differs from the base sequence encoding wild-type estrogen receptor α due to the difference in the fragment pattern of both nucleic acid digests. The sequence can be specified.
The determination of the estrogen receptor α genotype in a sample derived from an animal individual such as a human that can be performed as described above is performed by determining the reactivity of the estrogen receptor α possessed by the individual to an estrogen activity regulating substance such as an anti-estrogen substance. It is very useful in predicting the effectiveness of treatment such as administration of the substance before the start of treatment. It is also useful for judging the continuity and effectiveness of treatment with an administered substance by determining the genotype of the receptor in a specific tissue during or after the administration of an estrogen activity modulator such as an anti-estrogen substance. is there.

  EXAMPLES Hereinafter, although an Example demonstrates this invention further in detail, this invention is not limited by 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 α was obtained. An oligonucleotide consisting of the base sequence shown in SEQ ID NO: 3 and an oligonucleotide consisting of the base sequence shown in SEQ ID NO: 4 were chemically synthesized. Using 10 ng of human liver-derived cDNA (quick clone cDNA # 7113-1; manufactured by Clontech) as a template, an oligonucleotide consisting of the base sequence shown in SEQ ID NO: 3 and an oligonucleotide consisting of the base sequence shown in SEQ ID NO: 4 10 pmol of each was added, and PCR was performed using LA Taq DNA polymerase (Takara Shuzo) and a buffer attached to the enzyme. In the PCR, the reaction solution was incubated for 35 cycles using PCR System 9700 (Applied Biosystems) at 95 ° C. for 1 minute, 68 ° C. for 3 minutes. Next, the total amount of the reaction solution was subjected to agarose gel electrophoresis using agarose (Agarose S: Nippon Gene). After confirming that about 1.8 kb of DNA was amplified, the DNA was recovered. Prepare a sample for direct sequencing using a part of the recovered DNA and Dye Terminator Sequence Kit FS (Applied Biosystems), and base it on an automatic DNA sequencer (Applied Biosystems, Model 3700). Sequence analysis was performed, and it was confirmed that the recovered DNA had a base sequence encoding the amino acid sequence represented by SEQ ID NO: 1.
Next, about 100 ng of the DNA obtained as described above was used as a template, and the oligonucleotide consisting of the base sequence shown by SEQ ID NO: 22 and the oligonucleotide consisting of the base sequence shown by SEQ ID NO: 4 PCR was performed under the same conditions as described above to amplify a DNA having a base sequence in which a base sequence encoding human wild-type estrogen receptor α was linked immediately after the Kozak consensus sequence. The obtained DNA was separated by low melting point agarose gel electrophoresis, and about 1.8 kb of DNA was recovered. About 1 μg of the mixture was blunted with a DNA Blunting Kit (Takara Shuzo Co., Ltd.), and reacted with T4 polynucleotide kinase to phosphorylate the end. The obtained DNA was treated with phenol and purified by an ethanol precipitation method, and the entire amount was used as an insert DNA for preparing the following expression plasmid. Plasmid pRc / RSV (manufactured by Invitrogen) was digested with restriction enzyme Hind III, and then DNA was collected by phenol treatment and ethanol precipitation. The recovered DNA is treated with a DNA Blunting Kit (Takara Shuzo) to smooth the ends, and subjected to agarose gel electrophoresis using a low melting point agarose (Nippon Gene; Agarose L). It was collected. Bacterial alkaline phosphatase (BAP) was added to about 100 ng of the recovered DNA and incubated at 65 ° C. for 1 hour, and then mixed with the total amount of the above insert DNA, and T4 ligase was added to perform a ligation reaction. Escherichia coli DH5α competent cell (manufactured by Toyobo Co., Ltd.) is transformed using the reaction solution, plasmid DNA is prepared from a colony exhibiting ampicillin resistance, and its base sequence is converted into an automatic DNA sequencer (Applied Biosystems, model 3700). ) To determine by the die terminator method. The obtained base sequence is compared with the base sequence obtained by the above-mentioned direct sequence, and a plasmid in which the base sequence of the translation region is confirmed to be completely matched is selected, and pRc / RSV-hERα Kozak is selected. I named it.

Example 2 (Preparation of expression plasmid for DNA encoding receptor of the present invention)
(1) Preparation of mutation-introduced plasmid Using the plasmid pRc / RSV-hERα Kozak prepared in Example 1 as a template, mutation using a synthetic oligonucleotide for base substitution and Quickchange Site-directed mutagenesis Kit (Stratagene) Was introduced. First, an upper-strand oligonucleotide consisting of the base sequence shown in SEQ ID NO: 5 and a lower-strand oligonucleotide consisting of the base sequence shown in SEQ ID NO: 6 were chemically synthesized and performed according to the description in the kit instructions. . PCR uses Stratagene's Pfu Turbo DNA polymerase, 200 μM of each 4 bases (dATP, dTTP, dGTP, dCTP) are added, and pRc / RSV is added in the dedicated buffer attached to the enzyme. -hERα Kozak was used as a template, and the conditions were 95 ° C, 30 seconds, 55 ° C, 1 minute, 68 ° C, 10 minutes. Next, a part of the PCR reaction solution was taken and digested with the restriction enzyme Dpn I (Stratagene) at 37 ° C. for 1 hour. Escherichia coli XLI-Blue competent cells (Stratagene) were transformed with the reaction solution. A codon encoding the isoleucine represented by amino acid number 424 in the amino acid sequence represented by SEQ ID NO: 1 by purifying the plasmid DNA retained from several colonies of Escherichia coli showing ampicillin resistance and analyzing the base sequences thereof. The introduction of a mutation in which (ATC) is replaced with a threonine-encoding codon (ACC) was confirmed. The plasmid in which the introduction of the mutation was confirmed was named pRc / RSV-hERαI424T Kozak.

Example 3 (Preparation of a plasmid containing a reporter gene and a selection marker gene)
An oligonucleotide consisting of a base sequence upstream of the Xenopus vitellogenin gene containing the estrogen response sequence (base sequence shown by SEQ ID NO: 23) and an oligonucleotide consisting of a base sequence complementary to the base sequence were synthesized with a DNA synthesizer. These were annealed to form double-stranded DNA (hereinafter referred to as ERE DNA), and then T4 ligase was allowed to act to bind ERE DNA to tandem, which was then allowed to act on T4 polynucleotide kinase. Both ends were phosphorylated.
Further, two oligonucleotides consisting of a base sequence in the vicinity of the TATA box of the mouse metallothionein I gene and a base sequence derived from the leader sequence, that is, an oligonucleotide consisting of the base sequence shown in SEQ ID NO: 24 and a base shown in SEQ ID NO: 25 An oligonucleotide consisting of the sequence was annealed to form a double-stranded DNA, and T4 polynucleotide kinase was allowed to act on the DNA to phosphorylate both ends thereof (the DNA is hereinafter referred to as TATA DNA). On the other hand, a plasmid pGL3 (Promega) containing a firefly luciferase gene was digested with restriction enzymes Bgl II and Hind III, and then bacterial-derived alkaline phosphatase (BAP) was added thereto and incubated at 65 ° C. for 1 hour. Next, the heat retaining solution was subjected to electrophoresis using a low melting point agarose (Agarose L; manufactured by Nippon Gene Co., Ltd.), and DNA was recovered from the gel in the band portion. About 100 ng of the DNA and 1 μg of the TATA DNA were mixed and ligated with T4 ligase to prepare a plasmid pGL3-TATA.
Next, pGL3-TATA was digested with the restriction enzyme Sma I, BAP was added, and the mixture was incubated at 65 ° C. for 1 hour. The incubated solution was subjected to low melting point agarose gel electrophoresis, and DNA was recovered from the band gel. About 100 ng of the DNA and about 1 μg of ERE DNA bound to the above tandem and phosphorylated at the end were reacted with T4 ligase, and then E. coli DH5α competent cell (manufactured by Toyobo Co., Ltd.) was used. ) Was transformed. The DNA of each plasmid was purified from several colonies of Escherichia coli showing ampicillin resistance, digested with restriction enzymes Kpn I and Xho I, and the digested solution was analyzed by agarose gel electrophoresis. A plasmid having a structure in which 5 copies of ERE DNA were introduced in tandem at the Sma I site of pGL3-TATA was selected and named plasmid pGL3-TATA-EREx5.
Next, plasmid pUCSV-BSD (Funakoshi Co., Ltd.) was digested with BamH I to prepare DNA encoding a blasticidin S deaminase gene expression cassette. The DNA was mixed with the DNA obtained by digesting the plasmid pGL3-TATA-EREx5 with BamH I and treated with BAP, and reacted with T4 ligase. Then, the reaction solution was used for E. coli DH5α competent cell. (Toyobo Co., Ltd.) was transformed. Plasmid DNA was prepared from Escherichia coli showing ampicillin resistance, each was digested with the restriction enzyme Bam HI, 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 assay)
The DNA of plasmid pGL3-TATA-EREx5-BSD prepared as described above was introduced into human-derived HeLa cells in a linear form to prepare stable transformed cells.
First, the DNA of plasmid pGL3-TATA-EREx5-BSD was digested with SalI.
On the other hand, about 5 × 10 ⁵ cells of HeLa cells were obtained using a DMEM medium (Nissui Pharmaceutical Co., Ltd.) containing 10% FBS at 37 ° C. in the presence of 5% CO _{2 and} a petri dish with a diameter of about 10 cm (Falcon). For 24 hours. The linearized plasmid pGL3-TATA-EREx5-BSD DNA was introduced into the cells by lipofection using lipofectamine (Invitrogen). The conditions of the lipofection method were as described in the manual attached to the lipofectamine, the treatment time was 5 hours, the total amount of the linearized plasmid DNA was 7 μg / dish, and the lipofectamine amount was 21 μl / dish. After the lipofection treatment, the medium was replaced with DMEM medium containing 10% FBS, and cultured for about 36 hours. Subsequently, the cells are detached from the petri dish by trypsin treatment and collected, transferred to a culture vessel containing a medium supplemented with blasticidin S having a final concentration of 16 μg / ml, and the medium is renewed every 3 to 4 days ( It was cultured for about 1 month while changing to Blasticidin S. The appearing cell colonies having a diameter of 1 mm to several mm were transferred to the 96-well view plate (Berthold) with a culture medium dispensed in advance, and further cultured. When the cells grew to such an extent that they occupied more than half of the bottom of the well (about 5 days after transplantation), the cells were detached by trypsin treatment and collected, and divided into two equal 96-well view plates. One plate was continuously subcultured and cultured to serve as a master plate. The remaining one was cultured for 2 days, the medium was removed from the wells, the cells adhering to the vessel wall were washed twice with PBS (-), and then PGC50 (Toyo Ink Co., Ltd.) diluted 5 times was added to the wells. 20 μl per well was added and left at room temperature for 30 minutes. The plates were respectively set on a luminometer LB96p (manufactured by Bertrud) equipped with an enzyme substrate automatic injector, and 50 μl of substrate solution PGL100 (manufactured by Toyo Ink) was automatically dispensed to measure luciferase activity. Of these, 10 cells showing high luciferase activity were selected and stored. Furthermore, each cell was expanded and cultured on a 10 cm plate. The wild-type estrogen receptor α expression plasmid prepared in Example 1 was introduced into these 10 types of cells by the lipofection method using Lipofectamine (manufactured by Invitrogen) according to the description in the attached manual. To the obtained cells, E2 dissolved in DMSO was added to a final concentration of 10 nM in the medium and cultured for 2 days, and luciferase activity was measured according to the method described above. The cells before introduction of the wild-type estrogen receptor α expression plasmid corresponding to the cells showing the highest activity induction in the system to which E2 was added were selected as stable transformed cells for reporter assay into which the reporter gene was introduced.

Example 5 (Reporter assay using stably transformed cells)
The stable transformed cells selected in Example 4 were seeded with about 2 × 10 ⁶ cells in a 10 cm plate, and added with charcoal dextran-treated FBS to 10% (hereinafter referred to as FBS-containing medium). E-MEM medium) was cultured for 1 day at 37 ° C. under 5% CO ₂ . 7 μg of human wild-type estrogen receptor α gene expression plasmid pRc / RSV-hERα Kozak or 7 μg of human mutant estrogen receptor α gene expression plasmid pRc / RSV-hERαI424T Kozak was introduced. After culturing at 37 ° C. for 16 hours, the medium was changed and further culturing was performed for 3 hours. Thereafter, the cells were collected, suspended in FBS-containing E-MEM medium, homogenized, and seeded in 96-well plates to which various concentrations of anti-estrogen-like compounds previously dissolved in DMSO were added (DMSO final concentration 0.1%). Similarly, the cells were seeded in a 96-well plate to which various concentrations of an anti-estrogen-like compound and 10 nM estradiol were simultaneously added (DMSO final concentration 0.1%). The 96-well plate on which the cells were seeded was cultured at 37 ° C. for about 40 hours, and then a 5-fold diluted cell lysis agent PGC50 (manufactured by Nippon Gene Co., Ltd.) was added at 50 μl / well. Cells were lysed by standing for minutes. 10 μl of the cell lysate prepared in this way is collected in a 96-well white sample plate (Berthold), and 50 μl / well of enzyme substrate solution PGL100 (Nippon Gene) at a luminometer LB96p (Berthold) with an automatic substrate injector. The amount of luminescence was immediately measured for 5 seconds.
The measurement results of the estrogen-like action of 4-hydroxy tamoxifen, raloxifene or ZM189154 on the wild-type estrogen receptor α or the receptor of the present invention are shown in FIGS.
Moreover, the measurement results of the anti-estrogen action of 4-hydroxy tamoxifen, raloxifene or ZM189154 on wild-type estrogen receptor α or the receptor of the present invention are shown in FIGS.

Example 6 (Production of oligonucleotide for amplification)
In order to detect a nucleic acid containing the nucleotide sequence of the mutation site of DNA encoding a human-derived mutant estrogen receptor, the site encoding the 424th threonine from the amino terminus of the human-derived wild-type estrogen receptor is within the nucleic acid amplification range. And an oligonucleotide having a GC content of 30% to 70% and a length of about 20 bases is designed. An oligonucleotide is prepared based on the designed base sequence.

Example 7 (Gene diagnosis using human tissue as material)
Add 100 mg of frozen human liver sample to 5 ml of [4 M guanidine thiocyanate, 0.1 M Tris-Cl (pH 7.5) 1% β-mercaptoethanol] and grind it with a Polytron homogenizer. This is layered on 25 ml of 5.7 M cesium chloride solution previously placed in an ultracentrifuge tube, and RNA is separated by density gradient ultracentrifugation at 90,000 × g for 24 hours. The RNA is recovered, rinsed with 70% ethanol, and air-dried at room temperature. This is dissolved in 10 μl of sterilized water and the concentration is measured. Using 1-5 μg of this RNA as a template, 1 μg of oligo dT primer (Amersham Biotech) as a primer for reverse transcription synthesis, reaction with Superscript II (Invitrogen) in attached buffer at 42 ° C. for 1 hour To synthesize cDNA. Using 1 / 50th of the cDNA solution thus obtained as a template, PCR reaction is carried out using the PCR oligonucleotides having the sequences shown in SEQ ID NO: 10 and SEQ ID NO: 16. PCR was performed using Stratagene's Pfu DNA polymerase, 200 µM of each of the four bases (dATP, dTTP, dGTP, dCTP) and the dedicated buffer attached to the enzyme at 94 ° C for 1 minute at 55 ° C. 35 cycles of 30 seconds at 72 ° C for 1 minute. DNA fragments obtained by the PCR method are separated by electric start in a gel containing 1% agarose (Agarose S, manufactured by Nippon Gene) and collected. Using this total amount as a template, a sample for direct sequencing is prepared with a dye terminator sequencing kit FS (manufactured by Applied Biosystems) using 5 pM of oligonucleotide having the sequence shown in SEQ ID NO: 11 as a sequencing primer. This is subjected to base sequence analysis using an automatic DNA sequencer (Applied Biosystems, model 3700) to determine the base sequence.

Example 8 (Extraction of genomic DNA contained in specimen)
Genomic DNA contained in the sample is prepared by the usual method described in TAKARA PCR Technical news No. 2. Takara Shuzo (1991). Specifically, one hair with a hair root is transferred to a plastic tube and BCL buffer [10 mM Tris-Cl (pH 7.5), 5 mM MgCl ₂ , 0.32 M sucrose, 1% (v / v) Triton 200 μl of X-100] is added, and a proteinase K solution with a final concentration of 100 μg / ml and SDS with a final concentration of 0.5% (w / v) are mixed. Incubate the mixture at 70 ° C for 1 hour, add an equal volume of phenol / chloroform, shake vigorously, and centrifuge (15,000 rpm, 5 minutes, 4 ° C). The aqueous layer is collected and phenol extracted once again. Add an equal amount of chloroform to the recovered aqueous layer, shake vigorously and collect the aqueous layer by centrifugation. Add 500 μl of 100% ethanol to this, incubate at -80 ° C. for 20 minutes, and then centrifuge. The obtained pellets are dried and then dissolved in sterilized water.
In addition, peripheral blood is used as a cell from which genomic DNA can be collected. 10 ml of blood is collected, and genomic DNA is extracted using a DNA Extraction kit (Stratagene) according to the instructions attached to the kit.

Example 9 (Analysis of base substitution by PCR-SSCP method)
First, one type of forward primer is selected from the oligonucleotide consisting of the base sequence shown in any of SEQ ID NOs: 7 to 11, and one type of reverse primer is selected from the oligonucleotide consisting of the base sequence shown in any of SEQ ID NOs: 12 to 16 And chemically synthesized with a DNA synthesizer. At this time, the 5 ′ end is modified with a fluorescent material FITC. Next, the estrogen receptor α gene fragment is amplified by a PCR method using 100 ng of genomic DNA obtained as described above as a template and 200 pM each of the oligonucleotides of the present invention modified with FITC as primers. The PCR method uses Ex Taq DNA polymerase (Takara Shuzo) as a thermostable polymerase, using 200 μM of each of the four types of bases (dATP, dTTP, dGTP, dCTP) and a dedicated buffer attached to the enzyme. 40 cycles of 94 ° C, 30 seconds, 55 ° C, 30 seconds, 74 ° C, 30 seconds. After the reaction, 1/20 of the amplification product obtained is heat-denatured in 95% formamide at 95 ° C. for 5 minutes and then rapidly cooled. Among them, 2.5 μl is electrophoresed in 180 mM Tris-borate buffer (pH 8.0) using 5% native polyacrylamide gel. Electrophoresis is performed at room temperature, constant power of 40 W, and 5 hours. After completion of the electrophoresis, the amplified nucleic acid fragment is detected by detecting the fluorescence signal in the gel with a fluorescence reading scanner. Compared with the mobility of the amplified product band in the wild-type estrogen receptor α gene that migrates side-by-side, the mobility of the amplified product in the mutant estrogen receptor α gene differs, so the presence or absence of mutations in the amplified sequence Can be tested.

Example 10 (Analysis of base substitution by base sequence analysis)
A portion of the gel at the position corresponding to the band of the mutant estrogen receptor α gene fragment detected in Example 9 is cut into 1 mm squares, and permeated in 400 μl of sterile water overnight to elute the DNA. After removing the gel and purification by ethanol precipitation, the DNA is dissolved in 50 μl of sterile water. Among them, 1 μl is used as a template, and PCR is performed using the oligonucleotides used in the PCR-SSCP to amplify the estrogen receptor α gene fragment. PCR is performed using Ex Taq DNA polymerase (Takara Shuzo Co., Ltd.) as a thermostable DNA polymerase in the attached buffer for 30 cycles of 94 ° C, 30 seconds, 55 ° C, 30 seconds, 74 ° C, 30 seconds. . After completion of the reaction, the amplified product obtained is confirmed by agarose gel electrophoresis, and then the amplified fragment is cloned into a TA cloning vector such as pGEM-T Easy vector (Promega). Using the cloned plasmid as a template, the base sequence is determined using a BigDye Terminator cycle sequence ready reaction kit (Applied Biosystems) and an automatic DNA sequencer (Applied Biosystems model 3700).

Example 11 (Analysis of base substitution combining PCR and restriction enzyme)
PCR is performed as usual using genomic DNA or cDNA as a template and the mutation detection primers shown in SEQ ID NO: 26 and SEQ ID NO: 27 to amplify the human estrogen receptor α gene. In the above PCR method, Pfu DNA polymerase (Stratagene) is used as the thermostable DNA polymerase, and one cycle of 94 ° C, 1 minute, 55 ° C, 30 seconds, 72 ° C, 1 minute in the attached buffer. As 30 cycles. When the obtained DNA fragment of 100 base pairs in length is treated with the restriction enzyme Acc I, it is not digested when the wild-type estrogen receptor α is used as a template, but the 424th position from the amino terminus of the human-derived wild-type estrogen receptor α. When a mutant estrogen receptor α in which isoleucine is mutated to threonine is used as a template, a new sequence called GTAGAC is formed, so that Acc I digests it into DNA fragments of 75 and 25 base pairs. In this way, DNA encoding the estrogen receptor α having the mutation can be detected.

  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 estrogen exhibits a reactivity different from that of wild-type estrogen receptor α with respect to certain anti-estrogens. Receptor α, DNA encoding the receptor, method for evaluating estrogen receptor α activity-modulating ability of a test substance using 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 genotype determination method for determining the presence or absence of amino acid substitution at a specific position, and determination of the effectiveness of treatment with an estrogen receptor α activity modulator comprising the step of determining the estrogen receptor α genotype by examining the presence or absence of the amino acid substitution Methods can be provided It becomes ability.

Activation of 4-hydroxy tamoxifen to human wild-type estrogen receptor α or the receptor of the present invention (shown as “mutant” in the figure) by a reporter assay using a transformant in which a reporter gene is introduced into the host cell chromosome It is a figure which shows the result of having measured the performance. The value of luciferase activity in each test group was shown with the value of luciferase activity in the control group to which only the solvent was added (shown as “solvent control” in the figure) as 100%.

Measure the ability of raloxifene to activate human wild-type estrogen receptor α or the receptor of the present invention (shown as “mutant” in the figure) by reporter assay using a transformant in which the reporter gene is introduced into the host cell chromosome It is a figure which shows the result. The value of luciferase activity in each test group was shown with the value of luciferase activity in the control group to which only the solvent was added (shown as “solvent control” in the figure) as 100%.

Measure the ability of ZM189154 to activate human wild-type estrogen receptor α or the receptor of the present invention (shown as “mutant” in the figure) by reporter assay using a transformant in which the reporter gene is introduced into the host cell chromosome It is a figure which shows the result. The value of the luciferase activity in each test group was shown with the value of luciferase activity in the control group to which only the solvent was added (shown as “solvent control” in the figure) as 100%.

Anti-estrogens of 4-hydroxy tamoxifen against human wild-type estrogen receptor α or the receptor of the present invention (shown as “mutant” in the figure) by a reporter assay using a transformant in which a reporter gene is introduced into the host cell chromosome It is a figure which shows the result of having measured activity. The value of luciferase activity in each test group was shown with the value of luciferase activity in the test group to which only 100 pM E2 was added (shown as “solvent control” in the figure) as 100%.

Measure the antiestrogenic activity of raloxifene against human wild-type estrogen receptor α or the receptor of the present invention (shown as “mutant” in the figure) by reporter assay using a transformant in which the reporter gene is introduced into the host cell chromosome It is a figure which shows the result. The value of luciferase activity in each test group was shown with the value of luciferase activity in the test group to which only 100 pM E2 was added (shown as “solvent control” in the figure) as 100%.

Measures anti-estrogen activity of ZM189154 against human wild-type estrogen receptor α or the receptor of the present invention (shown as “mutant” in the figure) by reporter assay using a transformant in which a reporter gene is introduced into the host cell chromosome It is a figure which shows the result. The value of luciferase activity in each test group was shown with the value of luciferase activity in the test group to which only 100 pM E2 was added (shown as “solvent control” in the figure) as 100%.

SEQ ID NO: 3
Oligonucleotide primer designed for PCR SEQ ID NO: 4
Oligonucleotide primer designed for PCR SEQ ID NO: 5
Oligonucleotide primer designed for PCR SEQ ID NO: 6
Oligonucleotide primer designed for PCR SEQ ID NO: 7
Oligonucleotide primer designed for PCR SEQ ID NO: 8
Oligonucleotide primer designed for PCR SEQ ID NO: 9
Oligonucleotide primer designed for PCR SEQ ID NO: 10
Oligonucleotide primer designed for PCR SEQ ID NO: 11
Oligonucleotide primer designed for PCR SEQ ID NO: 12
Oligonucleotide primer designed for PCR SEQ ID NO: 13
Oligonucleotide primer designed for PCR SEQ ID NO: 14
Oligonucleotide primer designed for PCR SEQ ID NO: 15
Oligonucleotide primer designed for PCR SEQ ID NO: 16
Oligonucleotide primer designed for PCR SEQ ID NO: 17
Oligonucleotide probe SEQ ID NO: 18 designed for Southern hybridization
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 SEQ ID NO: 22 designed for Southern hybridization
Oligonucleotide primer designed for PCR SEQ ID NO: 23
Oligonucleotide designed to generate an estrogen response element SEQ ID NO: 24
Oligonucleotide designed to make promoter DNA SEQ ID NO: 25
Oligonucleotide designed to create promoter DNA SEQ ID NO: 26
Oligonucleotide primer designed for PCR SEQ ID NO: 27
Oligonucleotide primers designed for PCR

Claims (27)

Estrogen receptor α having the following properties.
(A) Among the amino acids constituting the receptor, 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 in the alignment based on the homology of the amino acid sequence is a wild-type estrogen It is substituted with an amino acid different from the amino acid at the corresponding position in the amino acid sequence of receptor α, and the substitution of the amino acid imparts the following properties (b) and (c) to the receptor.
Here, 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 isoleucine, and the amino acid at the position corresponding to the amino acid sequence of wild-type estrogen receptor α is A different amino acid is threonine.
(B) When contacted with any type of anti-estrogen 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, the transcription control region containing the estrogen response element Transcription of genes under transcriptional control can be activated.
(C) When contacted with estrogen, transcription of a gene under the transcriptional control of a transcriptional control region containing an estrogen response element can be activated, and this activation activates transcription of the gene in (b). Is not substantially inhibited by the compound that can. The estrogen receptor α according to claim 1, wherein in (b) and (c), the gene under the transcriptional control of the transcriptional control region containing an estrogen response element is a gene introduced into the chromosome of the cell. The estrogen receptor α according to claim 1 or 2, wherein the activation in (c) is also an activation inhibited by pure antiestrogen. The estrogen receptor α according to any one of claims 1 to 3, wherein the anti-estrogen substance according to (b) is tamoxifen, 4-hydroxy tamoxifen or raloxifene. 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 390 of the amino acid sequence represented by SEQ ID NO: 1 and the amino acid at the position corresponding to the amino acid represented by amino acid number 578 are: The estrogen receptor α according to any one of claims 1 to 4, which is the same as the amino acid at the corresponding position in the amino acid sequence of wild-type estrogen receptor α. The estrogen receptor α according to any one of claims 1 to 5 , wherein the estrogen receptor α is an estrogen receptor α derived from a mammal.   An estrogen receptor α having the amino acid sequence represented by SEQ ID NO: 2. An isolated DNA encoding the estrogen receptor α according to any one of claims 1 to 7 . The DNA according to claim 8 , wherein the DNA is cDNA. The DNA according to claim 8 or 9 , wherein the promoter is operably linked. A vector containing the DNA according to any one of claims 8 to 10 . A method for producing a vector, comprising incorporating the DNA according to claim 11 into a vector capable of replicating in a host cell. A transformant obtained by introducing the DNA according to any one of claims 8 to 10 or the vector according to claim 11 into a host cell. The transformant according to claim 13 , wherein the host cell is an animal cell. The transformant according to any one of claims 13 and 14 , wherein the host cell is a mammalian cell. A method for producing a transformant, wherein the DNA according to any one of claims 8 to 10 or the vector according to claim 11 is introduced into a host cell. A method for producing estrogen receptor α, comprising culturing the transformant according to any one of claims 13 to 15 to produce estrogen receptor α. (1) a step of contacting the estrogen receptor α and the test substance according to any one of claims 1 to 7 , wherein a labeled ligand is bound , and
(2) The binding state between the estrogen receptor α and the test substance is monitored by the amount of the free labeled ligand or the bound labeled ligand produced by competition between the labeled ligand and the test substance. A ligand-receptor binding assay comprising a step of confirming indirectly by It is a method for evaluating the estrogen receptor α activity regulating ability of a test substance,
(1) A transformant that produces the estrogen receptor α according to any one of claims 1 to 7 and has a reporter gene under the transcriptional control of a transcriptional control region containing an estrogen response element introduced into a chromosome; A step of contacting the test substance,
(2) measuring the expression level of the reporter gene possessed by the transformant, and
(3) A step of evaluating the ability of the substance to regulate estrogen receptor activity based on the measured expression level
Including methods. A method for screening an estrogen receptor α activity modulator comprising the step of evaluating the ability of a test substance to modulate the activity of an estrogen receptor α produced by a transformant by the method according to claim 19 . An amino acid that constitutes estrogen receptor α in a nucleic acid in a sample and that is in a position corresponding to the amino acid represented by amino acid number 424 of the amino acid sequence represented by SEQ ID NO: 1 in the alignment based on the homology of the amino acid sequence Determination of the estrogen receptor α genotype, which includes the step of examining whether the nucleotide sequence coding for is substituted with a nucleotide sequence encoding an amino acid different from the amino acid at the corresponding position of the amino acid sequence of wild-type estrogen receptor α Method.
Here, 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 isoleucine, and the amino acid at the position corresponding to the amino acid sequence of wild-type estrogen receptor α is A different amino acid is threonine. An amino acid that constitutes estrogen receptor α in a nucleic acid in a sample and that is in a position corresponding to the amino acid represented by amino acid number 424 of the amino acid sequence represented by SEQ ID NO: 1 in the alignment based on the homology of the amino acid sequence Determining the estrogen receptor α genotype by investigating whether the nucleotide sequence coding for is substituted with a nucleotide sequence encoding an amino acid different from the amino acid at the corresponding position of the amino acid sequence of wild-type estrogen receptor α For determining the effectiveness of treatment with an estrogen receptor α activity modulator comprising:
Here, 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 isoleucine, and the amino acid at the position corresponding to the amino acid sequence of wild-type estrogen receptor α is A different amino acid is threonine. An amino acid that constitutes estrogen receptor α in a nucleic acid in a sample and that is in a position corresponding to the amino acid represented by amino acid number 424 of the amino acid sequence represented by SEQ ID NO: 1 in the alignment based on the homology of the amino acid sequence Determining the estrogen receptor α genotype by investigating whether the nucleotide sequence coding for is substituted with a nucleotide sequence encoding an amino acid different from the amino acid at the corresponding position of the amino acid sequence of wild-type estrogen receptor α For determining the effectiveness of treatment with an anti-estrogen substance comprising
Here, 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 isoleucine, and the amino acid at the position corresponding to the amino acid sequence of wild-type estrogen receptor α is A different amino acid is threonine. Using the nucleic acid in the sample as a template, a nucleic acid encoding a region containing an amino acid at a position corresponding to the amino acid represented by amino acid number 424 of the amino acid sequence represented by SEQ ID NO: 1 is amplified, and the base sequence of the amplified nucleic acid is determined 24. A method according to any of claims 22 or 23 , comprising the step of determining. Using the nucleic acid in the sample as a template, a nucleic acid encoding a region containing an amino acid at a position corresponding to the amino acid represented by amino acid number 424 of the amino acid sequence represented by SEQ ID NO: 1 is amplified, and the amplified nucleic acid is electrophoresed its mobility is measured, one of the claim 22 or 23 and the mobility of the nucleic acids the amplified mobility of a nucleic acid encoding the region of the wild-type estrogen receptor α comprises a step to check different or not Te The method of crab. A probe comprising a base sequence encoding a region containing an amino acid at a position corresponding to the amino acid represented by amino acid number 424 of the amino acid sequence represented by SEQ ID NO: 1 among the amino acid sequences of wild-type estrogen receptor α, The method according to any one of claims 22 and 23 , comprising a step of examining whether or not the nucleic acid hybridizes. Using the nucleic acid in the sample as a template, a nucleic acid encoding a region containing an amino acid at a position corresponding to amino acid number 424 of the amino acid sequence represented by SEQ ID NO: 1 is amplified, the amplified nucleic acid is digested with a restriction enzyme, and The method according to any one of claims 22 and 23 , comprising a step of examining the presence or absence of a restriction enzyme recognition sequence.

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