JP2005021093A - New protein and dna encoding the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for utilizing a protein based on the physiological activity mentioned below and a DNA encoding the same through analyzing the base sequence of a cDNA clone included in a full-length cDNA library, and for a cDNA with new sequence as a whole length, specifying the physiological activity of a protein encoded by the cDNA. <P>SOLUTION: The protein is provided, being (a) a protein comprising a specific amino acid sequence, or (b) a protein comprising an amino acid sequence where one or several amino acids is(are) deleted, substituted and/or added in the above-mentioned specific amino acid sequence and having transcription-regulatory activity. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

【０１４８】
表１より、ｃ−ｎｔ２ｒｉ３００７８７８によりコードされるタンパク質は、ＴＢＰの標的配列：ＴＡＴＡＡＡＡ（配列番号８２）、ＳＲＹ等のコンセンサス標的配列：ＡＡＡＡＡＡＣＡＡＴＡＧＧＧ（配列番号８３）、ｍａｔ−Ｍｃの標的配列：ＴＣＡＴＴＧＴＴ（配列番号８４）に非常に高い親和性を示した。このことから、該タンパク質はＤＮＡ結合性を有し、神経系の発生・分化、軟骨形成、性分化などの異常に起因する疾患と関連する転写調節因子である可能性がある。また、該タンパク質は、Ｏｃｔ−２等のコンセンサス標的配列：ＴＡＴＴＴＧＣＡＴ（配列番号６７）、ＨＳＦ１（ｓｈｏｒｔ）等のコンセンサス標的配列：ＡＧＡＡＡＡＧＡＡＡＡＧＡＡＡ（配列番号７０）に高い親和性を示し、ＵＳＦの標的配列：ＧＴＣＡＣＧＴＧＧＴ（配列番号４０）、ＧＲの標的配列：ＧＧＴＡＣＡＡＡＡＴＧＴＴＣＴ（配列番号４４）、ＲＸＲ−αの標的配列：ＧＧＴＣＡＴＡＧＧＧＧＴ（配列番号４７）、ＰＰＡＲαの標的配列：ＣＴＡＧＧＧＣＡＡＡＧＧＴＣＡ（配列番号４８）、ｃ−Ｍｙｂ等のコンセンサス標的配列：ＡＡＣＧＧＧＣＣＣ（配列番号７１）、ｃ−Ｅｔｓ−１＿ｐ５４等のコンセンサス標的配列：ＧＡＣＡＧＧＡＡＧＴＧ（配列番号７２）、ＩＲＦ−１等のコンセンサス標的配列（配列番号７３）、ｐ５０の標的配列：ＧＧＧＧＡＣＴＴＴＣＣ（配列番号７４）、ＣＰ１Ａ等のコンセンサス標的配列：ＣＴＧＡＴＴＧＧＣＴＡＣＣ（配列番号８５）に親和性を示した。
【０１４９】
ｃ−ｂｒａｍｙ２０４３１０３によりコードされるタンパク質は、ＥＲの標的配列：ＡＡＧＧＧＡＡＡＡＴＧＡＣＣＣＣＣ（配列番号４６）、ＲＸＲ−αの標的配列：ＧＧＴＣＡＴＡＧＧＧＧＴ（配列番号４７）、ＰＰＡＲ−α：ＣＴＡＧＧＧＣＡＡＡＧＧＴＣＡ（配列番号４８）、Ｐａｘ−３等のコンセンサス標的配列：ＣＧＴＣＡＣＧＣＴＴＧＡ（配列番号６８）、Ｐａｘ−１の標的配列：ＣＣＧＴＴＣＣＧＣＴＣＴＡＧＡＴＡＴ（配列番号６９）およびＨＳＦ１等のコンセンサス標的配列：ＡＧＡＡＡＡＧＡＡＡＡＧＡＡＡ（配列番号７０）に親和性を示した。このことから該タンパク質はＤＮＡ結合性を有し、脂質代謝や薬物代謝、筋肉形成などの異常に起因する疾患、例えば生活習慣病などの疾患と関連する転写調節因子である可能性がある。
【０１５０】
ｃ−ｂｒｓｓｎ２０１３７５３によりコードされるタンパク質はＧＡＴＡ−１等のコンセンサス標的配列：ＣＣＡＧＡＴＡＡＧＧ（配列番号５２）、およびＡＲＥＡ／ＮＩＴ−２の標的配列：ＴＡＴＣＴＣ（配列番号５３）に非常に高い親和性を示した。このことから該タンパク質はＤＮＡ結合性を有し、造血幹細胞の分化、血球系細胞、神経系細胞、循環器、アポトーシス制御などの異常に起因する疾患と関連する転写調節因子である可能性がある。また、該タンパク質は、ＨＮＦ−１Ａの標的配列：ＧＧＴＴＡＡＴＧＡＴＴＡＡＣＣＡＣ（配列番号６４）に高い親和性を示し、ＰＰＡＲγの標的配列：ＧＧＴＣＡＡＡＧＧＴＣＡ（配列番号４９）、ＣＦ２−ＩＩの標的配列：ＧＴＡＴＡＴＡＴＡ（配列番号５８）、Ｅｖｉ−１の標的配列：ＡＧＡＴＡＡＧＡＴＡＡ（配列番号５９）、Ｎｋｘ−２．２等のコンセンサス標的配列：ＴＴＡＡＧＴＧＧＴＴ（配列番号６５）、Ｏｃｔ−１Ａ等のコンセンサス標的配列：ＡＴＧＣＡＡＡＴ（配列番号６６）、ＩＲＦ−１等のコンセンサス標的配列：ＧＡＡＡＡＧＣＧＡＡＡＣＣ（配列番号７３）、ｐ５０の標的配列：ＧＧＧＧＡＣＴＴＴＣＣ（配列番号７４）、ＴＢＰの標的配列：ＴＡＴＡＡＡＡ（配列番号８２）、ＳＲＹ等のコンセンサス標的配列：ＡＡＡＡＡＡＣＡＡＴＡＧＧＧ（配列番号８３）、ＡＭＬ１ａの標的配列：ＴＧＴＧＧＴ（配列番号８６）に親和性を示した。
【０１５１】
ｃ−ｂｒｓｓｎ２０１７５３０によりコードされるタンパク質はＳｐ１の標的配列：ＧＧＧＧＧＧＧＧＧＧ（配列番号５４）、Ｔｒａｍｔｒａｃｋ６９Ｋの標的配列：ＧＧＡＣＣＴＧＣ（配列番号６１）、ＨＯＸ９の標的配列：ＴＧＡＣＡＧＴＴＴＡＡＣＧＡ（配列番号６２）およびＣＤＰの標的配列：ＣＣＡＡＴＡＡＴＣＧＡＴ（配列番号６３）に高い親和性を示した。このことから該タンパク質はＤＮＡ結合性を有し、造血幹細胞の分化、アポトーシス制御、サイトカイン産生などの異常に起因する疾患、ウイルス性疾患、細菌性疾患、癌などの疾患と関連する転写調節因子である可能性がある。また、該タンパク質は、ＯＰＩ１等のコンセンサス標的配列：ＡＡＣＡＧＡＴＧＧＴ（配列番号３７）、ＨＥＮ−１の標的配列：ＧＧＧＧＣＧＣＡＧＣＴＧＣＧＧＣＣＣ（配列番号３８）、ＡｈＲ等のコンセンサス標的配列：ＧＧＧＧＡＴＴＧＣＧＴＧ（配列番号３９）、ｐ９１（ＳＴＡＴ１）等のコンセンサス標的配列：ＧＡＡＴＴＣＣＧＧＧＡＡＡＴＧＧ（配列番号７６）、ＳＴＡＴ２等のコンセンサス標的配列：ＴＴＴＣＣＣＧＧＧＡＡＡＴＧ（配列番号７７）、ｐ５３の標的配列：ＧＧＡＣＡＴＧＣＣＣＧＧＧＣＡＴＧＴＣ（配列番号７８）に親和性を示した。
【０１５２】
ｃ−ｈｃｈｏｎ２０００４７３によりコードされるタンパク質はＨＥＮ−１の標的配列：ＧＧＧＧＣＧＣＡＧＣＴＧＣＧＧＣＣＣ（配列番号３８）、ＡｈＲ等のコンセンサス標的配列：ＧＧＧＧＡＴＴＧＣＧＴＧ（配列番号３９）、ＵＳＦの標的配列：ＧＴＣＡＣＧＴＧＧＴ（配列番号４０）、ＮＦ−１Ａ１等のコンセンサス標的配列：ＣＴＧＴＧＧＧＧＴＴＴＧＧＣＡＣＧＧＧＧＣＣＡ（配列番号４１）、ＲＦ−Ｘ１の標的配列：ＧＧＴＡＡＣＡＴＡＧＣＡＡＣ（配列番号４２）、Ｓｐ１の標的配列：ＧＧＧＧＧＧＧＧＧＧ（配列番号５４）、Ｅｇｒ−１等のコンセンサス標的配列：ＴＧＣＧＴＧＧＧＣＧ（配列番号５６）、ｐ５０の標的配列：ＧＧＧＧＡＣＴＴＴＣＣ（配列番号７４）、ＮＦ−ＡＴｃの標的配列：ＡＧＧＡＡＡＡ（配列番号７５）、ｐ９１等のコンセンサス標的配列：ＧＡＡＴＴＣＣＧＧＧＡＡＡＴＧＧ（配列番号７６）、ＳＴＡＴ２等のコンセンサス標的配列：ＴＴＴＣＣＣＧＧＧＡＡＡＴＧ（配列番号７７）およびｐ５３の標的配列：ＧＧＡＣＡＴＧＣＣＣＧＧＧＣＡＴＧＴＣ（配列番号７８）に非常に高い親和性を示した。このことから該タンパク質はＤＮＡ結合性を有し、細胞周期、炎症、アポトーシス制御、免疫反応、薬物代謝、サイトカイン産生などの異常に起因する疾患、ウイルス性疾患、癌などの疾患と関連する転写調節因子である可能性がある。また、該タンパク質は、ＡＲＥＡ／ＮＩＴ−２の標的配列：ＴＡＴＣＴＣ（配列番号５３）に高い親和性を、Ｃ／ＥＢＰα等のコンセンサス標的配列：ＡＡＧＴＧＧＣＧＡＡＡＧＡＧＡＣＡ（配列番号３５）、ＩＲＦ−１等のコンセンサス標的配列：ＧＡＡＡＡＧＣＧＡＡＡＣＣ（配列番号７３）に親和性を示した。
【０１５３】
ｃ−ｔｅｓｔｉ４００４６２６によりコードされるタンパク質はＹＹ１の標的配列：ＣＧＧＣＣＡＴＣＴＴＧＧＣＴ（配列番号５５）、Ｅｇｒ−１等のコンセンサス標的配列：ＴＧＣＧＴＧＧＧＣＧ（配列番号５６）、Ｓｎａｉｌの標的配列：ＣＡＣＣＴＧＴＴＴＴＣＡ（配列番号５７）、ＣＦ２−ＩＩの標的配列：ＧＴＡＴＡＴＡＴＡ（配列番号５８）、ＩＲＦ−１，ＩＲＦ−２のコンセンサス標的配列：ＧＡＡＡＡＧＣＧＡＡＡＣＣ（配列番号７３）、ＮＦ−ＡＴｃ等のコンセンサス標的配列：ＡＧＧＡＡＡＡ（配列番号７５）、およびｐ９１等のコンセンサス標的配列：ＧＡＡＴＴＣＣＧＧＧＡＡＡＴＧＧ（配列番号７６）に親和性を示した。このことから該タンパク質はＤＮＡ結合性を有し、免疫反応、サイトカイン産生などの異常に起因する疾患と関連する転写調節因子である可能性がある。
【０１５４】
ｃ−ｔｒａｃｈ３０１４３１６によりコードされるタンパク質はＧＡＴＡ−１等のコンセンサス標的配列：ＣＣＡＧＡＴＡＡＧＧ（配列番号５２）、Ｎｋｘ−２．２等のコンセンサス標的配列：ＴＴＡＡＧＴＧＧＴＴ（配列番号６５）およびＯｃｔ−１Ａ等のコンセンサス標的配列：ＡＴＧＣＡＡＡＴ（配列番号６６）に高い親和性を示した。このことから該タンパク質はＤＮＡ結合性を有し、造血幹細胞の分化、血球系細胞、神経系細胞、循環器、アポトーシス制御などの異常に起因する疾患と関連する転写調節因子である可能性がある。また、該タンパク質は、ＶＢＰ等のコンセンサス標的配列：ＡＧＡＡＧＣＡＣＧＴＧＧ（配列番号３６）、ＵＳＦの標的配列：ＧＴＣＡＣＧＴＧＧＴ（配列番号４０），ＮＦ−１Ａ１等のコンセンサス標的配列：ＣＴＧＴＧＧＧＧＴＴＴＧＧＣＡＣＧＧＧＧＣＣＡ（配列番号４１）、ＰＰＡＲαの標的配列：ＣＴＡＧＧＧＣＡＡＡＧＧＴＣＡ（配列番号４８）、ＰＰＡＲγの標的配列：ＧＧＴＣＡＡＡＧＧＴＣＡ（配列番号４９）、Ｓｎａｉｌの標的配列：ＣＡＣＣＴＧＴＴＴＴＣＡ（配列番号５７）、ＣＤＰの標的配列：ＣＣＡＡＴＡＡＴＣＧＡＴ（配列番号６３）、ＨＳＦ１（ｓｈｏｒｔ）等のコンセンサス標的配列：ＡＧＡＡＡＡＧＡＡＡＡＧＡＡＡ（配列番号７０）、ｃ−Ｅｔｓ−１＿ｐ５４等のコンセンサス標的配列：ＧＡＣＡＧＧＡＡＧＴＧ（配列番号７２）、ＩＲＦ−１等のコンセンサス標的配列：ＧＡＡＡＡＧＣＧＡＡＡＣＣ（配列番号７３）、ｐ９１（ＳＴＡＴ１）等のコンセンサス標的配列：ＧＡＡＴＴＣＣＧＧＧＡＡＡＴＧＧ（配列番号７６）、ＳＴＡＴ２等のコンセンサス標的配列：ＴＴＴＣＣＣＧＧＧＡＡＡＴＧ（配列番号７７）、ＳＲＦの標的配列：ＣＣＡＴＡＴＡＴＧＧＡＣＡＴ（配列番号８０），Ｅ２の標的配列：ＡＡＣＣＡＡＡＡＡＣＧＧＴＡＡ（配列番号８１）に親和性を示した。
【０１５５】
ｃ−ｂｅａｓｔ２００１４４４によりコードされるタンパク質はＮＦ−１Ａ１等のコンセンサス標的配列：ＣＴＧＴＧＧＧＧＴＴＴＧＧＣＡＣＧＧＧＧＣＣＡ（配列番号４１）、ＲＦ−Ｘ１の標的配列：ＧＧＴＡＡＣＡＴＡＧＣＡＡＣ（配列番号４２）、ＹＹ１の標的配列：ＣＧＧＣＣＡＴＣＴＴＧＧＣＴ（配列番号５５）、Ｓｎａｉｌの標的配列：ＣＡＣＣＴＧＴＴＴＴＣＡ（配列番号５７）、Ｅｖｉ−１の標的配列：ＡＧＡＴＡＡＧＡＴＡＡ（配列番号５９）、Ｉｋａｒｏｓ等のコンセンサス標的配列：ＴＴＧＧＧＡＧＧ（配列番号６０）、ＨＮＦ−１Ａの標的配列：ＧＧＴＴＡＡＴＧＡＴＴＡＡＣＣＡＣ（配列番号６４）、Ｎｋｘ−２．２等のコンセンサス標的配列：ＴＴＡＡＧＴＧＧＴＴ（配列番号６５）、Ｏｃｔ−１Ａの等のコンセンサス標的配列：ＡＴＧＣＡＡＡＴ（配列番号６６）、Ｏｃｔ−２等のコンセンサス標的配列：ＴＡＴＴＴＧＣＡＴ（配列番号６７）、Ｐａｘ−３等のコンセンサス標的配列：ＣＧＴＣＡＣＧＣＴＴＧＡ（配列番号６８）、Ｐａｘ−１の標的配列：ＣＣＧＴＴＣＣＧＣＴＣＴＡＧＡＴＡＴ（配列番号６９）、ＮＦ−ＡＴｃ等のコンセンサス標的配列：ＡＧＧＡＡＡＡ（配列番号７５）、ＭＥＦ−２Ａの標的配列：ＣＴＣＴＡＡＡＡＡＴＡ（配列番号７９）、ＳＲＦの標的配列：ＣＣＡＴＡＴＡＴＧＧＡＣＡＴ（配列番号８０）およびＥ２の標的配列：ＡＡＣＣＡＡＡＡＡＣＧＧＴＡＡ（配列番号８１）に親和性を示した。このことから該タンパク質はＤＮＡ結合性を有し、造血細胞や肝細胞の増殖、免疫系細胞の発生・分化、脂質代謝、筋肉形成などの異常に起因する疾患、例えば生活習慣病などの疾患と関連する転写調節因子である可能性がある。
【０１５６】
実施例５ 転写調節活性の測定（疾患関連転写調節因子に対する調節作用活性の解析）
（１）無細胞タンパク質合成系を用いた本発明のタンパク質の調製
本発明のｃＤＮＡがコードするタンパク質につき、さらに疾患関連転写調節因子に対する調節作用活性を解析した。各クローンのｃＤＮＡがコードするタンパク質は、実施例４（１）の方法に準じて調製した。
【０１５７】
（２）解析用プレートの調製
転写調節因子の疾患関連転写調節因子に対する調節作用活性の測定系に用いるＤＮＡは、疾患関連転写調節因子が結合する塩基配列を有することを特徴とし、５’側にビオチン（Ｂｉｏと表記する）が付加した一本鎖ＤＮＡ（ｏｌｉｇｏ−Ａと記載する）とその相補的配列を有する一本鎖ＤＮＡ（ｏｌｉｇｏ−Ｂ）と記載するをアニーリングさせることにより、二本鎖化したＤＮＡを用いた。以下に用いたＤＮＡ配列を記載する。
【０１５８】
疾患関連転写調節因子ＰＰＡＲγが認識する配列として、配列番号８７と配列番号８８を含むビオチン化ＤＮＡをアニーリングさせた二本鎖ＤＮＡ
ｏｌｉｇｏ−Ａ；５’− Ｂｉｏ−ＧＧＡＡＣＴＡＧＧＴＣＡＡＡＧＧＴＣＡＴＣＣＣＣＴ−３’（配列番号８７）
ｏｌｉｇｏ−Ｂ；３’−ＣＣＴＴＧＡＴＣＣＡＧＴＴＴＣＣＡＧＴＡＧＧＧＧＡ−５’（配列番号８８）
【０１５９】
疾患関連転写調節因子ｐ５３が認識する配列として、配列番号８９と配列番号９０を含むビオチン化ＤＮＡをアニーリングさせた二本鎖ＤＮＡ
ｏｌｉｇｏ−Ａ；５’−Ｂｉｏ−ＣＴＴＧＧＡＣＡＴＧＣＣＣＧＧＧＣＡＴＧＴＣＣＣＴＣ−３’（配列番号８９）
ｏｌｉｇｏ−Ｂ；３’−ＧＡＡＣＣＴＧＴＡＣＧＧＧＣＣＣＧＴＡＣＡＧＧＧＡＧ−５’（配列番号９０）
【０１６０】
疾患関連転写調節因子ＮＦκＢが認識する配列として、配列番号９１と配列番号９２を含むビオチン化ＤＮＡをアニーリングさせた二本鎖ＤＮＡ
ｏｌｉｇｏ−Ａ；５’−Ｂｉｏ−ＡＧＴＴＧＡＧＧＧＧＡＣＴＴＴＣＣＣＡＧＧＣ−３’（配列番号９１）
ｏｌｉｇｏ−Ｂ；３’−ＴＣＡＡＣＴＣＣＣＣＴＧＡＡＡＧＧＧＴＣＣＧ−５’（配列番号９２）
【０１６１】
疾患関連転写調節因子ＡＰ−１が認識する配列として、配列番号９３と配列番号９４を含むビオチン化ＤＮＡをアニーリングさせた二本鎖ＤＮＡ
ｏｌｉｇｏ−Ａ；５’−Ｂｉｏ−ＣＧＣＴＴＧＡＴＧＡＧＴＣＡＧＣＣＧＧＡＡ−３’（配列番号９３）
ｏｌｉｇｏ−Ｂ；３’−ＧＣＧＡＡＣＴＡＣＴＣＡＧＴＣＧＧＣＣＴＴ−５’（配列番号９４）
【０１６２】
疾患関連転写調節因子ＨＩＦ−１が認識する配列として、配列番号９５と配列番号９６を含むビオチン化ＤＮＡをアニーリングさせた二本鎖ＤＮＡ
ｏｌｉｇｏ−Ａ；５’−Ｂｉｏ−ＧＡＴＣＧＣＣＣＴＡＣＧＴＧＣＴＧＴＣＴＣＡＧＡＴＣ−３’（配列番号９５）
ｏｌｉｇｏ−Ｂ；３’−ＣＴＡＧＣＧＧＧＡＴＧＣＡＣＧＡＣＡＧＡＧＴＣＴＡＧ−５’（配列番号９６）
【０１６３】
疾患関連転写調節因子ＣＲＥＢが認識する配列として、配列番号９７と配列番号９８を含むビオチン化ＤＮＡをアニーリングさせた二本鎖ＤＮＡ
ｏｌｉｇｏ−Ａ；５’−Ｂｉｏ−ＡＧＡＧＡＴＴＧＣＣＴＧＡＣＧＴＣＡＧＡＧＡＧＣＴＡＧ−３’（配列番号９７）
ｏｌｉｇｏ−Ｂ；３’−ＴＣＴＣＴＡＡＣＧＧＡＣＴＧＣＡＧＴＣＴＣＴＣＧＡＴＣ−５’（配列番号９８）
【０１６４】
上記によって作成した二本鎖ＤＮＡを固定する固相担体として、ＢｉｏＴｅｃｈｎｉｑｕｅｓ ，３２：１１６８−１１７７（２００２）の方法に準じてストレプトアビジンをコートした９６ウェルプレート｛ビオチン結合能２０ｎｇ／ウェル（８０ ｐｍｏｌ／ウェル）、ストレプトアビジンコートエリア３００μｌ｝またはＴｒａｎｓＡＭキット（ＡＣＴＩＶＥ ＭＯＴＩＦ社）もしくはＢＤ Ｍｅｒｃｕｒｙ ＴｒａｎｓＦａｃｔｏｒキットに添付のプレートを適宜使用した。
【０１６５】
（３）ＰＰＡＲγに対する調節作用活性の解析
解析試料としてはＰＰＡＲγを含むＰＭＡ処理ＴＨＰ−１細胞核抽出溶液（２．５ｍｇ／ｍｌ ＡＣＴＩＶＥ ＭＯＴＩＦ社）１μｌを９μｌの希釈溶液｛２０ｍＭ Ｈｅｐｅｓ（ｐＨ７．５）、４００ｍＭ ＮａＣｌ，２０％ ｇｌｙｃｅｒｏｌ、０．１ｍＭ ＥＤＴＡ、１０ｍＭ ＮａＦ、１０μＭ Ｎａ_２ＭｏＯ_４、１ｍＭ ＮａＶＯ_３、１０ｍＭ ｐＮＰＰ、１０ｍＭ ｂ−ｇｌｙｃｅｒｏｐｈｏｓｐｈａｔｅ、１ｍＭ ＤＴＴ｝と混合後、３５μｌの反応溶液｛１０ｍＭ Ｈｅｐｅｓ（ｐＨ７．５）、４％ ｇｌｙｃｅｒｏｌ、５０ｍＭ ＮａＣｌ、０．５ｍＭ ＥＤＴＡ、１ｍＭ ＭｇＣｌ_２、１０μｇ／ｍｌ Ｈｅｒｒｉｎｇ ｓｐｅｒｍ｝および実施例５（１）の方法によって調製した本発明のタンパク質を含むコファクター溶液５μｌを混合したものを用いた。陽性試料としてはＰＭＡ処理ＴＨＰ−１細胞核抽出溶液１μｌ，９μｌ希釈溶液，３５μｌ反応溶液および本発明のタンパク質を含まないコファクター溶液５μｌを混合したものを用いた。また陰性試料としては１０μｌ希釈溶液、３５μｌ反応溶液および本発明のタンパク質を含まないコファクター溶液５μｌを混合したもの、測定ブランク用試料としては１０μｌ希釈溶液および４０μｌ反応溶液を混合したものを用いた。各試料は混合後室温で３０分反応させた。次に実施例５（２）によって作成した、ＰＰＡＲγが認識する二本鎖ＤＮＡを結合させた９６ウェルプレートに対し、１ウェルあたり上記試料５０μｌずつを添加し室温で１時間反応させた。
【０１６６】
この後洗浄用緩衝液｛１０ｍＭ ｐｈｏｓｐｈａｔｅ ｂｕｆｆｅｒ（ｐＨ７．５）、５０ｍＭ ＮａＣｌ、０．１％ Ｔｗｅｅｎ ２０、２．７ｍＭ ＫＣｌ｝を用いてウェルを充分な洗浄を行い、抗体希釈液｛１０ｍＭ ｐｈｏｓｐｈａｔｅ ｂｕｆｆｅｒ（ｐＨ７．５）、５０ｍＭ ＮａＣｌ、２．７ｍＭ ＫＣｌ、１０ｍｇ／ｍｌ ＢＳＡ｝に溶解した抗ＰＰＡＲヤギ抗体（０．２μｇ／ｍｌ）を１００μｌずつウェルに添加し、さらに室温で１時間反応させた。この後、洗浄用緩衝液によって充分な洗浄を行ない、抗体希釈液によって１０００倍に希釈したＨＲＰ（西洋わさびペルオキシターゼ）標識抗ヤギＩｇＧ抗体を１００μｌずつウェルに添加し、さらに室温で１時間反応させた。洗浄用緩衝液によって充分な洗浄を行なった後、発色用基質（ＴＭＢ）を含む１％ＤＭＳＯ溶液を１００μｌずつウェルに添加し室温で反応させ発色を行なった。この後０．５Ｍ硫酸溶液を１００μｌずつウェルに添加して反応を止め、測定波長４５０ｎｍ，リファレンス波長６５５ｎｍで測定を行なった。解析結果は、陽性試料と陰性試料測定値の差に対する解析試料測定値と陽性試料測定値の差の割合を百分率（％）で表示した。すなわち｛（解析試料値―陽性試料値）／（陽性試料値―陰性試料値）｝×１００で表記した。その結果を、表２の「ＰＰＡＲγ評価」のカラムに示す。表２で、−Ａはこの割合が−５０％未満、−Ｂは−５０％以上−３０％未満、−Ｃは−３０％以上−１０％未満、＋／−Ｄは−１０％以上１０％未満、＋Ｃは１０％以上３０％未満、＋Ｂは３０％以上５０％未満、＋Ａは５０％以上、であることを示す。
【０１６７】
【表２】

【０１６８】
ｃ−ｎｔ２ｒｉ３００７８７８、ｃ−ｂｒｓｓｎ２０１７５３０、ｃ−ｈｃｈｏｎ２０００４７３、ｃ−ｂｅａｓｔ２００１４４４の４クローンによりコードされるタンパク質は、ＰＰＡＲが有する作用を増強または抑制する活性を有し、内分泌・糖・脂質代謝異常等による糖尿病や肥満等の生活習慣病、血管機能異常等による循環器系疾患、炎症、癌等に関連することが推測された。
【０１６９】
（４）ｐ５３に対する調節作用活性の解析
解析試料としては活性化ｐ５３を含むＨ_２Ｏ_２処理ＭＣＦ−７細胞核抽出溶液（２．５ｍｇ／ｍｌ ＡＣＴＩＶＥ ＭＯＴＩＦ社）１μｌを９μｌの希釈溶液｛２０ｍＭ Ｈｅｐｅｓ（ｐＨ７．５）、４００ｍＭ ＮａＣｌ、２０％ ｇｌｙｃｅｒｏｌ、０．１ｍＭ ＥＤＴＡ、１０ｍＭ ＮａＦ、１０μＭ Ｎａ_２ＭｏＯ_４、１ｍＭ ＮａＶＯ_３、１０ｍＭ ｐＮＰＰ、１０ｍＭ ｂ−ｇｌｙｃｅｒｏｐｈｏｓｐｈａｔｅ、１ｍＭ ＤＴＴ｝と混合後、３５μｌの反応溶液｛２０ｍＭ Ｈｅｐｅｓ（ｐＨ７．５）、１０％ ｇｌｙｃｅｒｏｌ、５ｍＭ ＫＣｌ、０．５ｍＭ ＥＤＴＡ、５ｍＭ ＭｇＣｌ_２、１ｍＭ ＤＴＴ、０．１７μｇ／ｍｌ ｐｏｌｙ［ｄ（Ｉ−Ｃ）］｝および実施例５（１）の方法によって調製した本発明のタンパク質を含むコファクター溶液５μｌを混合したものを用いた。陽性試料としてはＨ_２Ｏ_２処理ＭＣＦ−７細胞核抽出溶液１μｌ、９μｌ希釈溶液、３５μｌ反応溶液および本発明のタンパク質を含まないコファクター溶液５μｌを混合したものを用いた。また陰性試料としては１０μｌ希釈溶液、３５μｌ反応溶液および本発明のタンパク質を含まないコファクター溶液５μｌを混合したもの、測定ブランク用試料としては１０μｌ希釈溶液および４０μｌ反応溶液を混合したものを用いた。各試料は混合後室温で３０分反応させた。次に実施例５（２）によって作成した、ｐ５３が認識する二本鎖ＤＮＡを結合させた９６ウェルプレートに対し、１ウェルあたり上記試料５０μｌずつを添加し室温で１時間反応させた。この後、洗浄用緩衝液｛１０ｍＭ ｐｈｏｓｐｈａｔｅ ｂｕｆｆｅｒ（ｐＨ７．５）、５０ｍＭ ＮａＣｌ、０．１％ Ｔｗｅｅｎ ２０、２．７ｍＭ ＫＣｌ｝を用いてウェルを充分な洗浄を行い、抗体希釈液｛１０ｍＭ ｐｈｏｓｐｈａｔｅ ｂｕｆｆｅｒ（ｐＨ７．５）、５０ｍＭ ＮａＣｌ、２．７ｍＭ ＫＣｌ、１０ｍｇ／ｍｌ ＢＳＡ｝に溶解した抗ｐ５３ウサギ抗体（０．２μｇ／ｍｌ）を１００μｌずつウェルに添加し、さらに室温で１時間反応させた。
【０１７０】
この後洗浄用緩衝液で充分な洗浄を行ない、抗体希釈液で１０００倍に希釈したＨＲＰ（西洋わさびペルオキシターゼ）標識抗ウサギＩｇＧ抗体を１００μｌずつウェルに添加し、さらに室温で１時間反応させた。洗浄用緩衝液で充分な洗浄を行なった後、発色用基質（ＴＭＢ）を含む１％ＤＭＳＯ溶液を１００μｌずつウェルに添加し室温で反応させ発色を行なった。この後０．５Ｍ硫酸溶液を１００μｌずつウェルに添加して反応を止め、測定波長４５０ｎｍ、リファレンス波長６５５ｎｍで測定を行なった。解析結果は、陽性試料と陰性試料測定値の差に対する解析試料測定値と陽性試料測定値の差の割合を百分率（％）で表示した。すなわち｛（解析試料値―陽性試料値）／（陽性試料値―陰性試料値）｝×１００で表記した。その結果を、表２の「ｐ５３評価」のカラムに示す。表２で、−Ａはこの割合が−５０％未満、−Ｂは−５０％以上−３０％未満、−Ｃは−３０％以上−１０％未満、＋／−Ｄは−１０％以上１０％未満、＋Ｃは１０％以上３０％未満、＋Ｂは３０％以上５０％未満、＋Ａは５０％以上、であることを示す。
【０１７１】
ｃ−ｎｔ２ｒｉ３００７８７８、ｃ−ｂｒｓｓｎ２０１７５３０、ｃ−ｔｅｓｔｉ４００４６２６の３クローンによりコードされるタンパク質は、ｐ５３が有する作用を増強または抑制する活性を有し、癌等に関連することが推測された。
【０１７２】
（５）ＮＦκＢに対する調節作用活性の解析
解析試料としてはＮＦκＢを含むＴＮＦ−α処理ＨｅＬａ細胞核抽出溶液（２．５ｍｇ／ｍｌ ＡＣＴＩＶＥ ＭＯＴＩＦ社）１μｌを１９μｌの希釈溶液｛２０ｍＭ Ｈｅｐｅｓ（ｐＨ７．５）、３５０ｍＭ ＮａＣｌ、２０％ ｇｌｙｃｅｒｏｌ、１％ Ｉｇｅｐａｌ−ＣＡ６３０、１ｍＭ ＭｇＣｌ_２， ０．５ｍＭ ＥＤＴＡ、０．１ｍＭ ＥＧＴＡ、５ｍＭ ＤＴＴ｝と混合後、２５μｌの反応溶液｛４ｍＭ Ｈｅｐｅｓ（ｐＨ７．５）、８％ ｇｌｙｃｅｒｏｌ、１２０ｍＭ ＫＣｌ、１％ ＢＳＡ、２ｍＭ ＤＴＴ、１０μｇ／ｍｌ Ｈｅｒｒｉｎｇ ｓｐｅｒｍ｝および実施例５（１）の方法によって調製した本発明のタンパク質を含むコファクター溶液５μｌを混合したものを用いた。陽性試料としてはＴＮＦ−α処理ＨｅＬａ細胞核抽出溶液１μｌ，１９μｌ希釈溶液，２５μｌ反応溶液および本発明のタンパク質を含まないコファクター溶液５μｌを混合したものを用いた。また陰性試料としては２０μｌ希釈溶液，２５μｌ反応溶液および本発明のタンパク質を含まないコファクター溶液５μｌを混合したもの、測定ブランク用試料としては２０μｌ希釈溶液および３０μｌ反応溶液を混合したものを用いた。各試料は混合後室温で３０分反応させた。次に実施例５（２）によって作成した、ＮＦκＢが認識する二本鎖ＤＮＡを結合させた９６ウェルプレートに対し、１ウェルあたり上記試料５０μｌずつを添加し室温で１時間反応させた。
【０１７３】
この後洗浄用緩衝液｛１０ｍＭ ｐｈｏｓｐｈａｔｅ ｂｕｆｆｅｒ（ｐＨ７．５）、５０ｍＭ ＮａＣｌ、０．１％Ｔｗｅｅｎ ２０｝を用いてウェルを充分な洗浄を行い、抗体希釈液｛１０ｍＭ ｐｈｏｓｐｈａｔｅ ｂｕｆｆｅｒ（ｐＨ７．５）、５０ｍＭ ＮａＣｌ、０．１％ Ｔｗｅｅｎ２０｝に溶解した抗ＮＦκＢ抗体（０．２μｇ／ｍｌ）を１００μｌずつウェルに添加し、さらに室温で１時間反応させた。この後、洗浄用緩衝液で充分な洗浄を行ない、抗体希釈液１０００倍で希釈したＨＲＰ（西洋わさびペルオキシターゼ）標識抗ＩｇＧ抗体を１００μｌずつウェルに添加し、さらに室温で１時間反応させた。洗浄用緩衝液で充分な洗浄を行なった後、発色用基質（ＴＭＢ）を含む１％ＤＭＳＯ溶液を１００μｌずつウェルに添加し室温で反応させ発色を行なった。この後０．５Ｍ硫酸溶液を１００μｌずつウェルに添加して反応を止め、測定波長４５０ｎｍ，リファレンス波長６５５ｎｍで測定を行なった。解析結果は、陽性試料と陰性試料測定値の差に対する解析試料測定値と陽性試料測定値の差の割合を百分率（％）で表示した。すなわち｛（解析試料値―陽性試料値）／（陽性試料値―陰性試料値）｝×１００で表記した。その結果を、表２の「ＮＦκＢ評価」のカラムに示す。表２で、−Ａはこの割合が−５０％未満、−Ｂは−５０％以上−３０％未満、−Ｃは−３０％以上−１０％未満、＋／−Ｄは−１０％以上１０％未満、＋Ｃは１０％以上３０％未満、＋Ｂは３０％以上５０％未満、＋Ａは５０％以上、であることを示す。
【０１７４】
ｃ−ｔｅｓｔｉ４００４６２６、ｃ−ｔｒａｃｈ３０１４３１６、ｃ−ｂｅａｓｔ２００１４４４の３クローンによりコードされるタンパク質は、ＮＦκＢが有する作用を増強または抑制する活性を有し、関節リウマチや変形性関節炎などの免疫性疾患、炎症性疾患、癌等に関連することが推測された。
【０１７５】
（６）疾患関連転写調節因子ＡＰ−１に対する調節作用活性の解析
解析試料としては活性化ＡＰ−１を含むＰＭＡおよびＩｎｏｍｙｃｉｎ処理ＷＩ−３８細胞核抽出溶液（２．５ｍｇ／ｍｌ ＡＣＴＩＶＥ ＭＯＴＩＦ社）１μｌを１９μｌの希釈溶液｛２０ｍＭ Ｈｅｐｅｓ（ｐＨ７．５）、４００ｍＭ ＮａＣｌ、２０％ ｇｌｙｃｅｒｏｌ、０．１ｍＭ ＥＤＴＡ、１０ｍＭ ＮａＦ、１０μＭ Ｎａ_２ＭｏＯ_４、１ｍＭ ＮａＶＯ_３、１０ｍＭ ｐＮＰＰ、１０ｍＭ ｂ−ｇｌｙｃｅｒｏｐｈｏｓｐｈａｔｅ，１ｍＭ ＤＴＴ｝と混合後、２５μｌの反応溶液｛１０ｍＭ Ｈｅｐｅｓ（ｐＨ７．５）、 １２％ ｇｌｙｃｅｒｏｌ、８ｍＭ ＮａＣｌ、０．２ｍＭ ＥＤＴＡ， ０．１％ ＢＳＡ、１ｍＭ ＤＴＴ、０．１７μｇ／ｍｌ ｐｏｌｙ［ｄ（Ｉ−Ｃ）］｝および実施例５（１）の方法によって調製した本発明のタンパク質を含むコファクター溶液５μｌを混合したものを用いた。陽性試料としては活性化ＡＰ−１を含むＰＭＡおよびＩｎｏｍｙｃｉｎ処理ＷＩ−３８細胞核抽出溶液１μｌ、１９μｌ希釈溶液、２５μｌ反応溶液および本発明のタンパク質を含まないコファクター溶液５μｌを混合したものを用いた。また陰性試料としては２０μｌ希釈溶液、２５μｌ反応溶液および本発明のタンパク質を含まないコファクター溶液５μｌを混合したもの、測定ブランク用試料としては２０μｌ希釈溶液および３０μｌ反応溶液を混合したものを用いた。各試料は混合後室温で３０分反応させた。次に実施例５（２）によって作成した、ＡＰ−１が認識する二本鎖ＤＮＡを結合させた９６ウェルプレートに対し、１ウェルあたり上記試料５０μｌずつを添加し室温で１時間反応させた。この後洗浄用緩衝液｛１０ｍＭ ｐｈｏｓｐｈａｔｅ ｂｕｆｆｅｒ（ｐＨ７．５）、５０ｍＭ ＮａＣｌ、０．１％ Ｔｗｅｅｎ ２０、２．７ｍＭ ＫＣｌ｝を用いてウェルを充分な洗浄を行い、抗体希釈液｛１０ｍＭ ｐｈｏｓｐｈａｔｅ ｂｕｆｆｅｒ（ｐＨ７．５）、５０ｍＭ ＮａＣｌ、２．７ｍＭ ＫＣｌ、１％ ＢＳＡ｝に溶解した抗リン酸化−ｃ−Ｊｕｎ抗体（０．４μｇ／ｍｌ）を１００μｌずつウェルに添加し、さらに室温で１時間反応させた。
【０１７６】
この後、洗浄用緩衝液で充分な洗浄を行ない、抗体希釈液で１０００倍に希釈したＨＲＰ（西洋わさびペルオキシターゼ）標識抗ＩｇＧ抗体を１００μｌずつウェルに添加し、さらに室温で１時間反応させた。洗浄用緩衝液で充分な洗浄を行なった後、発色用基質（ＴＭＢ）を含む１％ ＤＭＳＯ溶液を１００μｌずつウェルに添加し室温で反応させ発色を行なった。この後０．５Ｍ硫酸溶液を１００μｌずつウェルに添加して反応を止め、測定波長４５０ｎｍ，リファレンス波長６５５ｎｍで測定を行なった。解析結果は、陽性試料と陰性試料測定値の差に対する解析試料測定値と陽性試料測定値の差の割合を百分率（％）で表示した。すなわち｛（解析試料値―陽性試料値）／（陽性試料値―陰性試料値）｝×１００で表記した。その結果を、表２の「ＡＰ−１評価」のカラムに示す。表２で、−Ａはこの割合が−５０％未満、−Ｂは−５０％以上−３０％未満、−Ｃは−３０％以上−１０％未満、＋／−Ｄは−１０％以上１０％未満、＋Ｃは１０％以上３０％未満、＋Ｂは３０％以上５０％未満、＋Ａは５０％以上、であることを示す。
【０１７７】
ｃ−ｎｔ２ｒｉ３００７８７８、ｃ−ｈｃｈｏｎ２０００４７３、ｃ−ｔｅｓｔｉ４００４６２６、ｃ−ｔｒａｃｈ３０１４３１６の４クローンによりコードされるタンパク質は、ＡＰ−１が有する作用を増強または抑制する活性を有し、炎症性疾患や癌等に関連することが推測された。
【０１７８】
（７）疾患関連転写調節因子ＨＩＦ−１に対する調節作用活性の解析
解析試料としてはＨＩＦ−１を含むＣｏＣｌ_２処理Ｃｏｓ−７細胞核抽出溶液（２．５ｍｇ／ｍｌ ＡＣＴＩＶＥ ＭＯＴＩＦ社）１μｌを９μｌの希釈溶液｛２０ｍＭ Ｈｅｐｅｓ（ｐＨ７．５）、４００ｍＭ ＮａＣｌ、２０％ ｇｌｙｃｅｒｏｌ、０．１ｍＭ ＥＤＴＡ、１０ｍＭ ＮａＦ、１０μＭ Ｎａ_２ＭｏＯ_４、１ｍＭＮａＶＯ_３、１０ｍＭ ｐＮＰＰ、１０ｍＭ ｂ−ｇｌｙｃｅｒｏｐｈｏｓｐｈａｔｅ、１ｍＭ ＤＴＴ｝と混合後、３５μｌの反応溶液｛１０ｍＭ Ｈｅｐｅｓ（ｐＨ７．５）、５％ ｇｌｙｃｅｒｏｌ、５０ｍＭ ＮａＣｌ、１ｍＭ ＥＤＴＡ、１０ｍｇ／ｍｌ ＢＳＡ， １ｍＭ ＤＴＴ｝および実施例５（１）の方法によって調製した本発明のタンパク質を含むコファクター溶液５μｌを混合したものを用いた。陽性試料としてはＨＩＦ−１を含むＣｏＣｌ_２処理Ｃｏｓ−７細胞核抽出溶液１μｌ、９μｌ希釈溶液、３５μｌ反応溶液および本発明のタンパク質を含まないコファクター溶液５μｌを混合したものを用いた。また陰性試料としては１０μｌ希釈溶液，３５μｌ反応溶液および本発明のタンパク質を含まないコファクター溶液５μｌを混合したもの，測定ブランク用試料としては１０μｌ希釈溶液および４０μｌ反応溶液を混合したものを用いた。各試料は混合後室温で３０分反応させた。次に実施例５（２）によって作成した、ＨＩＦ−１が認識する二本鎖ＤＮＡを結合させた９６ウェルプレートに対し、１ウェルあたり上記試料５０μｌずつを添加し室温で１時間反応させた。この後、洗浄用緩衝液｛１０ｍＭ ｐｈｏｓｐｈａｔｅ ｂｕｆｆｅｒ（ｐＨ７．５）、５０ｍＭ ＮａＣｌ、０．１％ Ｔｗｅｅｎ ２０、２．７ｍＭ ＫＣｌ｝を用いてウェルを充分な洗浄を行い、抗体希釈液｛１０ｍＭ ｐｈｏｓｐｈａｔｅ ｂｕｆｆｅｒ（ｐＨ７．５）、５０ｍＭ ＮａＣｌ、２．７ｍＭ ＫＣｌ、１０ｍｇ／ｍｌ ＢＳＡ｝に溶解した抗ＨＩＦ−１マウス抗体（０．２５μｇ／ｍｌ）を１００μｌずつウェルに添加し、さらに室温で１時間反応させた。
【０１７９】
この後、洗浄用緩衝液で充分な洗浄を行ない、抗体希釈液で１０００倍に希釈したＨＲＰ（西洋わさびペルオキシターゼ）標識抗マウスＩｇＧ抗体を１００μｌずつウェルに添加し、さらに室温で１時間反応させた。洗浄用緩衝液で充分な洗浄を行なった後、発色用基質（ＴＭＢ）を含む１％ ＤＭＳＯ溶液を１００μｌずつウェルに添加し室温で反応させ発色を行なった。この後０．５Ｍ硫酸溶液を１００μｌずつウェルに添加して反応を止め、測定波長４５０ｎｍ、リファレンス波長６５５ｎｍで測定を行なった。解析結果は、陽性試料と陰性試料測定値の差に対する解析試料測定値と陽性試料測定値の差の割合を百分率（％）で表示した。すなわち｛（解析試料値―陽性試料値）／（陽性試料値―陰性試料値）｝×１００で表記した。その結果を、表２の「ＨＩＦ−１評価」のカラムに示す。表２で、−Ａはこの割合が−５０％未満、−Ｂは−５０％以上−３０％未満、−Ｃは−３０％以上−１０％未満、＋／−Ｄは−１０％以上１０％未満、＋Ｃは１０％以上３０％未満、＋Ｂは３０％以上５０％未満、＋Ａは５０％以上、であることを示す。
【０１８０】
ｃ−ｂｒｓｓｎ２０１７５３０、ｃ−ｔｅｓｔｉ４００４６２６、ｃ−ｔｒａｃｈ３０１４３１６の３クローンによりコードされるタンパク質は、ＨＩＦ−１が有する作用を増強または抑制する活性を有し、血管新生異常や細胞内酸化異常等に起因する、虚血性疾患、貧血、低酸素症、糖尿病性網膜症、循環器系疾患、癌等に関連することが推測された。
【０１８１】
（８）疾患関連転写調節因子ＣＲＥＢに対する調節作用活性の解析
解析試料としてはＣＲＥＢを含むＦｏｒｓｋｏｌｉｎ処理ＷＩ−３８細胞核抽出溶液（２．５ｍｇ／ｍｌ ＡＣＴＩＶＥ ＭＯＴＩＦ社）１μｌを１９μｌの希釈溶液｛２０ｍＭ Ｈｅｐｅｓ（ｐＨ７．５）、４００ｍＭ ＮａＣｌ、２０％ ｇｌｙｃｅｒｏｌ、０．１ｍＭ ＥＤＴＡ、１０ｍＭ ＮａＦ、１０μＭ Ｎａ_２ＭｏＯ_４、１ｍＭ ＮａＶＯ_３、１０ｍＭ ｐＮＰＰ、１０ｍＭ ｂ−ｇｌｙｃｅｒｏｐｈｏｓｐｈａｔｅ、１ｍＭ ＤＴＴ）と混合後、２５μｌの反応溶液（１０ｍＭＨｅｐｅｓ（ｐＨ７．５）、４％ ｇｌｙｃｅｒｏｌ、５０ｍＭ ＮａＣｌ、０．５ｍＭ ＥＤＴＡ、１ｍＭ ＭｇＣｌ_２、１％ ＢＳＡ、１ｍＭ ＤＴＴ、１０μｇ／ｍｌ Ｈｅｒｒｉｎｇ ｓｐｅｒｍ）および実施例５（１）の方法によって調製した本発明のタンパク質を含むコファクター溶液５μｌを混合したものを用いた。陽性試料としてはＦｏｒｓｋｏｌｉｎ処理ＷＩ−３８細胞核抽出溶液１μｌ，１９μｌ希釈溶液，２５μｌ反応溶液および本発明のタンパク質を含まないコファクター溶液５μｌを混合したものを用いた。また陰性試料としては２０μｌ希釈溶液，２５μｌ反応溶液および本発明のタンパク質を含まないコファクター溶液５μｌを混合したもの、測定ブランク用試料としては２０μｌ希釈溶液および３０μｌ反応溶液を混合したものを用いた。各試料は混合後室温で３０分反応させた。次に実施例５（２）によって作成した、ＣＲＥＢが認識する二本鎖ＤＮＡを結合させた９６ウェルプレートに対し、１ウェルあたり上記試料５０μｌずつを添加し室温で３時間反応させた。
【０１８２】
この後、洗浄用緩衝液｛１０ｍＭ ｐｈｏｓｐｈａｔｅ ｂｕｆｆｅｒ（ｐＨ７．５）、１５１ｍＭ ＮａＣｌ、０．１％ Ｔｗｅｅｎ ２０、２．７ｍＭ ＫＣｌ｝を用いてウェルを充分な洗浄を行い、抗体希釈液｛１０ｍＭ ｐｈｏｓｐｈａｔｅ ｂｕｆｆｅｒ（ｐＨ７．５）、１５１ｍＭ ＮａＣｌ、 ２．７ｍＭ ＫＣｌ、１％ ＢＳＡ｝に溶解した抗ＣＲＥＢ抗体（０．２μｇ／ｍｌ）を１００μｌずつウェルに添加し、さらに室温で１時間反応させた。この後、洗浄用緩衝液で充分な洗浄を行ない、抗体希釈液で１０００倍に希釈したＨＲＰ（西洋わさびペルオキシターゼ）標識抗ＩｇＧ抗体を１００μｌずつウェルに添加し、さらに室温で１時間反応させた。洗浄用緩衝液で充分な洗浄を行なった後、発色用基質（ＴＭＢ）を含む１％ ＤＭＳＯ溶液を１００μｌずつウェルに添加し室温で反応させて発色を行なった。この後０．５Ｍ硫酸溶液を１００μｌずつウェルに添加して反応を止め、測定波長４５０ｎｍ、リファレンス波長６５５ｎｍで測定を行なった。解析結果は、陽性試料と陰性試料測定値の差に対する解析試料測定値と陽性試料測定値の差の割合を百分率（％）で表示した。すなわち｛（解析試料値―陽性試料値）／（陽性試料値―陰性試料値）｝×１００で表記した。その結果を、表２の「ＣＲＥＢ評価」のカラムに示す。表２で、−Ａはこの割合が−５０％未満、−Ｂは−５０％以上−３０％未満、−Ｃは−３０％以上−１０％未満、＋／−Ｄは−１０％以上１０％未満、＋Ｃは１０％以上３０％未満、＋Ｂは３０％以上５０％未満、＋Ａは５０％以上、であることを示す。
【０１８３】
ｃ−ｔｅｓｔｉ４００４６２６、ｃ−ｔｒａｃｈ３０１４３１６の２クローンによりコードされるタンパク質は、ＣＲＥＢが有する作用を増強または抑制する活性を有し、記憶障害などの中枢系疾患、循環器系疾患、糖尿病等に関連することが推測された。
【０１８４】
実施例６ ヒト組織発現解析
本発明のヒトタンパク質をコードするｍＲＮＡの正常ヒトおよび疾患患者での組織発現変動を検討するために、定法（Ｈｉｇｕｃｈｉ Ｒ， ｅｔ ａｌ．， Ｂｉｏｔｅｃｈｎｏｌｏｇｙ， １１： １０２６−３０ （１９９３））に従い、ＰＣＲ法を用いた組織発現解析を行った。
【０１８５】
（１）ｃＤＮＡ合成
ヒトの９組織より抽出・精製された市販の各ｍＲＮＡ（バイオチェイン社製：正常脳（＃５１０００５）、正常海馬（＃５１００６８）、正常視床（＃５１００７７）、正常腎臓（＃５１００３０）、正常肝臓（＃５１００３１）、正常膵臓（＃５１００３８）、正常骨格筋（＃５１００４７）、正常脂肪（＃５１００１８）、正常脾臓（＃５１００４９）、およびヒト正常末梢血白血球の市販の全ＲＮＡ（ユニーテック社製）より精製されたｍＲＮＡから、オリゴｄＴをプライマーとした逆転写反応を行いそれぞれのｃＤＮＡを合成した。また、ヒトの１０組織より調製された市販の各ｃＤＮＡ（クロンテック社製：正常乳房および乳癌（＃ＨＰ１０２Ｂ）、正常結腸および結腸癌（＃ＨＰ１０２Ｃ）、正常肺および肺癌（＃ＨＰ１０４Ｌ）、正常胃および胃癌（＃ＨＰ１０１Ｓ）、バイオチェイン社製：正常前頭葉（＃５１００６８）、アルツハイマー病前頭葉（＃５５０９０１）を使用した。
【０１８６】
（２）ＰＣＲ法による定量
本発明のヒトタンパク質をコードしているｍＲＮＡ（ｃ−ｎｔ２ｒｉ３００７８７８、ｃ−ｆｅｂｒａ２００１６２０）の発現は、ライトサイクラー定量ＰＣＲ装置（ロシュ・ダイアグノスティクス社）とＬｉｇｈｔＣｙｃｌｅｒ−ＦａｓｔＳｔａｒｔ ＤＮＡマスターＳＹＢＲ Ｇｒｅｅｎ Ｉ試薬を用いて、製品に添付されているプロトコールに従い定量した。定量ＰＣＲに用いた合成ＤＮＡ配列を以下に示す。
ｃ−ｎｔ２ｒｉ３００７８７８
５’側プライマー：ＧＡＡＧＴＣＧＡＣＴＧＣＣＡＡＡＧＡＧＧ（配列番号９９）
３’側プライマー：ＡＴＴＴＣＣＣＧＴＣＡＡＡＧＧＡＧＧＴＴ（配列番号１００）
ｃ−ｆｅｂｒａ２００１６２０
５’側プライマー：ＧＣＡＴＣＣＡＴＧＴＴＧＴＴＴＴＡＡＴＴＡＡＧＴＧ（配列番号１０１）
３’側プライマー：ＧＴＧＣＡＡＡＧＧＴＧＧＡＡＣＴＣＴＣＡ（配列番号１０２）
【０１８７】
定量結果は、Ｇｌｙｃｅｒａｌｄｅｈｙｄｅ ３−ｐｈｏｓｐｈａｔｅ ｄｅｈｙｄｒｏｇｅｎａｓｅ（ＧＡＰＤＨ）を内部標準として、補正した。即ち、各組織での対象遺伝子の発現量（コピー数／μｌ）をＧＡＰＤＨの発現量（コピー数／μｌ）で除し、定数（１×１０^５）を乗して表示した。その結果を表３に示す。
【０１８８】
【表３】

【０１８９】
表３から明らかな通り、ｃ−ｎｔ２ｒｉ３００７８７８のｍＲＮＡは膵臓で強く発現し、脾臓、肝臓、および脳で発現し、アルツハイマー病患者前頭葉（ＡＤ前頭葉）で発現が増加した。
ｃ−ｆｅｂｒａ２００１６２０のｍＲＮＡは膵臓、脾臓、および肝臓で発現し、乳癌、肺癌で発現が増加した。
この結果より、上記クローンのｃＤＮＡおよび該ｃＤＮＡによってコードされるタンパク質は、癌やアルツハイマー病などの治療や診断に応用できる。また該ｃＤＮＡによってコードされるタンパク質は、上記のようなｍＲＮＡ発現の変動が見られる組織あるいはｍＲＮＡ発現量の多い組織に関わる疾患に関与している可能性がある。
【０１９０】
実施例７ 各ｃＤＮＡクローンの解析結果
（１）ｃ−ｎｔ２ｒｉ３００７８７８（配列番号１、１１）
ｃ−ｎｔ２ｒｉ３００７８７８（以下、これを「本ＤＮＡ」と称し、該ＤＮＡによりコードされるタンパク質を「本タンパク質」と称する）は、配列番号１に示すように、６１５６塩基から成り、そのうち塩基番号２２７から２２４５までがオープンリーディングフレーム（終止コドンを含む）である。オープンリーディングフレームから予測されるアミノ酸配列は、６７２アミノ酸残基から成る（配列番号１１）。
【０１９１】
配列番号１がコードするアミノ酸配列についてＢＬＡＳＴを用いて相同性検索を行ったところ、ＮＲＤＢタンパク質データベース（ＳＷＩＳＳ−ＰＲＯＴ、ＰＩＲ、ＴＲＥＭＢＬＥ、ＧＥＮＰＥＰＴ、ＰＤＢから作成された重複のないアミノ酸配列のデータベース） 中の、（ｉ）データベース登録記号Ｘ６０１５６、ｚｉｎｃ ｆｉｎｇｅｒ ｐｒｏｔｅｉｎ（ＺＦ７）（Ｈｕｍａｎ）がヒットした。その内容として、Ｘ６０１５６は７３８アミノ酸から成り、そのアミノ酸配列中のアミノ酸番号３〜７０５が、配列番号１１に記載のアミノ酸配列のアミノ酸番号１〜６７０と、ｅ−ｖａｌｕｅ：０．０、かつ７０３アミノ酸残基に亘り５８％の一致度でヒットした。また、（ｉｉ）データベース登録記号Ｑ１４５８７、ｚｉｎｃ ｆｉｎｇｅｒ ｐｒｏｔｅｉｎ ２６８ （ｈｕｍａｎ） がヒットした。その内容として、Ｑ１４５８７は９４７アミノ酸から成り、そのアミノ酸配列中のアミノ酸番号８１〜７７４が、配列番号１１に記載のアミノ酸配列のアミノ酸番号６〜６７０と、ｅ−ｖａｌｕｅ：０．０、かつ６９８アミノ酸残基に亘り５４％の一致度でヒットした。さらに（ｉｉｉ）データベース登録記号Ｂ３２８９１，ｆｉｎｇｅｒ ｐｒｏｔｅｉｎ ２， ｐｌａｃｅｎｔａｌ （Ｈｕｍａｎ）がヒットしていた。その内容として、Ｂ３２８９１は６５１アミノ酸から成り、そのアミノ酸配列中のアミノ酸番号２〜６１８が、配列番号１１に記載のアミノ酸配列のアミノ酸番号８５〜６７０とｅ−ｖａｌｕｅ：０．０、かつ６１７アミノ酸残基に亘り５８％の一致度でヒットした。これらの結果より配列番号１１に示したアミノ酸配列からなるタンパク質は、ＫＲＡＢ ｚｉｎｃ ｆｉｎｇｅｒ ｐｒｏｔｅｉｎであることが推測された。
上記（ｉ）のタンパク質は、データベース中の文献情報Ｎｕｃｌｅｉｃ Ａｃｉｄ Ｒｅｓ． １９（２０）：５６６１−７から、ｆｉｎｇｅｒ ｐｒｅｃｅｄｉｎｇ ｂｏｘ（ＦＰＢ；ＫＲＡＢと同義）をもつｚｉｎｃ ｆｉｎｇｅｒ ｐｒｏｔｅｉｎであることが、また上記（ｉｉ）のタンパク質は、ＤＮＡ Ｃｅｌｌ Ｂｉｏｌ．１４（２）：１２５−３６から、単球の分化・増殖の調節を行うことが、それぞれ明らかになった。
【０１９２】
また、配列番号１に示す塩基配列がコードするアミノ酸配列について、ＨＭＭＰＦＡＭによるタンパク質特徴検索を行ったところ、配列番号１１のアミノ酸番号２００〜２２２、２２８〜２５０、２５６〜２７８、２８４〜３０６、３１２〜３３４、３４０〜３６２、３６８〜３９０、３９６〜４１８、４２４〜４４６、４５２〜４７４、４８０〜５０２、５０８〜５３０、５３６〜５５８、５６４〜５８６、５９２〜６１４、６２０〜６４２、６４８〜６７０に示されるアミノ酸配列にＣ２Ｈ２ ｚｉｎｃ ｆｉｎｇｅｒの特徴を示す配列（Ｐｆａｍにｚｆ−Ｃ２Ｈ２としてエントリーされるアミノ酸配列）を見出した。
Ｃ２Ｈ２ ｔｙｐｅ ｚｉｎｃ ｆｉｎｇｅｒは、保存された２つのＣｙｓと２つの Ｈｉｓが亜鉛イオンに配位する構造モチーフをもっており、転写因子等多くのＤＮＡ結合タンパク質に見られる。
また、アミノ酸番号６〜４６に示されるアミノ酸配列にＫｒｕｐｐｅｌ−ａｓｓｏｃｉａｔｅｄ ｂｏｘの特徴を示す配列（ＰｆａｍにＫＲＡＢとしてエントリーされるアミノ酸配列）も見出された。Ｋｒｕｐｐｅｌ−ａｓｓｏｃｉａｔｅｄ ｂｏｘ（ＫＲＡＢ）は、Ｃ２Ｈ２タイプのＺｎフィンガータンパク質のおよそ１／３に見られ、Ｎ末端部分に存在し、転写の抑制にはたらき、Ｚｎフィンガードメインとともに細胞分化・発生に関与することが知られている。
【０１９３】
さらに、タンパク質の細胞内局在の予測プログラムであるＰＳＯＲＴＩＩ（Ｔｒｅｎｄｓ Ｂｉｏｃｈｅｍ．Ｍｏｌ．Ｂｉｏｌ． ５８ （４）： ４１７−４２４ （１９９６）） による解析を行ったところ、本タンパク質の細胞内での局在の確率は、核に１００．０％であることがわかった。よって本タンパク質は、核に局在することが予測された。
【０１９４】
ゲノム情報を得るため、ヒトｃＤＮＡをヒトゲノム配列へ写像するソフトウエアｓｉｍ４（Ｇｅｎｏｍｅ Ｒｅｓ．８， ９６７−９７４ （１９９８））を用い、本ＤＮＡのゲノムへの写像を試みたところ、第１２番染色体上に５個のエクソンがマッピングされた。
真核生物ゲノムのアノテーション情報データベースＥｎｓｅｍｂｌ中のＤｉｓｅａｓｅ Ｂｒｏｗｓｅｒ （ｈｔｔｐ：／／ｗｗｗ．ｅｎｓｅｍｂｌ．ｏｒｇ／Ｈｏｍｏ＿ｓａｐｉｅｎｓ／ｄｉｓｅａｓｅｖｉｅｗ）によって、写像された第１２番染色体上の位置（ｑ２４．３）を含むｑ２４にヌーナン症候群、ｑ２４に脊髄性筋萎縮症の原因遺伝子座が存在することがわかった。これらによって本ＤＮＡがこれらの疾患の原因遺伝子である可能性や本ＤＮＡの変異がこれらの診断に応用できる可能性が推測された。
【０１９５】
以上より配列番号１１に示すタンパク質はヒトの器官・組織などの発生・分化・形成、細胞周期や細胞増殖などに関わる機能を有する新規Ｚｎフィンガータンパク質であることが予測された。
【０１９６】
本タンパク質を小麦胚芽無細胞タンパク質合成系を用いて発現させたところ、実施例４においては、ＴＢＰの標的配列：ＴＡＴＡＡＡＡ（配列番号８２）、ＳＲＹ，Ｓｏｘ−５，Ｓｏｘ−９のコンセンサス標的配列：ＡＡＡＡＡＡＣＡＡＴＡＧＧＧ（配列番号８３）、ｍａｔ−Ｍｃの標的配列：ＴＣＡＴＴＧＴＴ（配列番号８４）に非常に高い親和性を示した。このことから本タンパク質はＤＮＡ結合能を有し、神経系の発生・分化、軟骨形成、性分化などの異常に起因する疾患との関連が考えられる。また、実施例５によって、ＰＰＡＲが有する作用を増強する活性を有し、内分泌・糖・脂質代謝異常等による糖尿病や肥満等の生活習慣病、血管機能異常等による循環器系疾患、炎症、癌等に関連することが推測された。また、ｐ５３が有する作用を抑制する活性を有し、癌等に関連することが推測された。さらに、ＡＰ−１が有する作用を抑制する活性を有し、炎症性疾患や癌等に関連することが推測された。
【０１９７】
本ＤＮＡの発現組織を検討するため、本ＤＮＡをクエリーとしてｄｂＥＳＴ（Ｎａｔｕｒｅ Ｇｅｎｅｔｉｃｓ ４， （４） ３３２−３ （１９９３））に対してＢＬＡＳＴ検索を行い、ｅ−ｖａｌｕｅ≦１０^−１０でヒットしたヒトＥＳＴが５個以上ある器官名または組織名を抽出したところ、ヒトの正常な眼、子宮、胎盤、腸、免疫系、胎児肝臓、胎児脾臓、癌化した皮膚、眼であった。尚、本ＤＮＡは未分化のＮＴ２細胞を含む神経系細胞由来のｃＤＮＡライブラリーからクローニングされた。
ＤＮＡ Ｃｅｌｌ Ｂｉｏｌ．１４（２）：１２５−３６では、冒頭のＢＬＡＳＴヒットの（ｉｉ）のタンパク質（ｚｉｎｃ ｆｉｎｇｅｒ ｐｒｏｔｅｉｎ ２６８）は、単芽球で発現することが述べられている。さらにＤＮＡ ８（６）： ３７７−８７ （１９８９） からは、上記（ｉｉｉ）のタンパク質（ｆｉｎｇｅｒ ｐｒｏｔｅｉｎ ２）は胎盤由来のｃＤＮＡライブラリーからクローニングされたことがわかっている。
また、本タンパク質は、実施例６より、膵臓で強く発現し、脾臓、肝臓、および脳で発現し、アルツハイマー病患者前頭葉で発現が増加することが示された。
【０１９８】
本タンパク質はこれらの組織や細胞に特有の機能や疾患、例えばメラノ−マ・リンパ腫・白血病・神経膠腫・神経芽細胞腫・子宮癌・大腸癌・小腸癌・皮膚癌・肝臓癌などの癌、アルツハイマー病・パーキンソン病・舞踏病などの神経変性疾患、肝炎・皮膚炎・骨軟骨炎・変形性関節炎などの炎症性疾患、免疫系疾患、糖尿病、肥満、循環器系疾患、アレルギー疾患、脾臓疾患、不妊、ヌーナン症候群、脊髄性筋萎縮症などに関連する可能性が推測でき、これらの疾患の診断薬や治療薬のターゲットとしての利用が考えられる。
【０１９９】
（２）ｃ−ｆｅｂｒａ２００１６２０（配列番号２、１２）
ｃ−ｆｅｂｒａ２００１６２０（以下、これを「本ＤＮＡ」と称し、該ＤＮＡによりコードされるタンパク質を「本タンパク質」と称する）は、配列番号２に示すように、２１４６塩基から成り、そのうち塩基番号２１４から１９４７までがオープンリーディングフレーム （終止コドンを含む） である。オープンリーディングフレームから予測されるアミノ酸配列は、５７７アミノ酸残基から成る （配列番号１２）。
【０２００】
配列番号２がコードするアミノ酸配列についてＢＬＡＳＴを用いて相同性検索を行ったところ、ＮＲＤＢタンパク質データベース （ＳＷＩＳＳ−ＰＲＯＴ、ＰＩＲ、ＴＲＥＭＢＬＥ、ＧＥＮＰＥＰＴ、ＰＤＢから作成された重複のないアミノ酸配列のデータベース） 中の、（ｉ）データベース登録記号ＡＦ３２５１９１、ＫＲＡＢ ｚｉｎｃ ｆｉｎｇｅｒ ｐｒｏｔｅｉｎ ＨＺＦ２６ （Ｈｕｍａｎ） がヒットした。その内容として、ＡＦ３２５１９１は４７０アミノ酸から成り、そのアミノ酸配列中のアミノ酸番号２〜４６８が、配列番号１２に記載のアミノ酸配列のアミノ酸番号１７〜４８３と、ｅ−ｖａｌｕｅ：０．０かつ４６７アミノ酸残基に亘り８４％の一致度でヒットした。また（ｉｉ）データベース登録記号ＡＢ０７５８４９、ＫＩＡＡ１９６９ ｐｒｏｔｅｉｎ （Ｈｕｍａｎ）がヒットした。その内容として、ＡＢ０７５８４９は５９５アミノ酸から成り、そのアミノ酸配列中のアミノ酸番号５１〜５８３が、配列番号１２に記載のアミノ酸配列のアミノ酸番号１６〜５４８と、ｅ−ｖａｌｕｅ：０．０かつ５３３アミノ酸残基に亘り６９％の一致度でヒットした。さらに（ｉｉｉ）データベース登録記号、Ｑ０３９２３、ｚｉｎｃ ｆｉｎｇｅｒ ｐｒｏｔｅｉｎ ８５ （Ｚｉｎｃ ｆｉｎｇｅｒ ｐｒｏｔｅｉｎ ＨＰＦ４ ； Ｈｕｍａｎ）がヒットした。その内容として、Ｑ０３９２３は５９５アミノ酸から成り、そのアミノ酸配列中のアミノ酸番号２〜５６８が、配列番号１２に記載のアミノ酸配列のアミノ酸番号１７〜５５５と、ｅ−ｖａｌｕｅ：０．０かつ５６８アミノ酸残基に亘り６５％の一致度でヒットした。
【０２０１】
また、配列番号１２に示すアミノ酸配列について、ＨＭＭＰＦＡＭによるタンパク質特徴検索を行ったところ配列番号１２のアミノ酸番号１８８〜２１０、２１６〜２４３、２４４〜２６６、２７２〜２９４、３００〜３２２、３２８〜３５０、３５６〜３７８、３８４〜４０６、４４０〜４６２、４６８〜４９０、４９６〜５１８、５２４〜５４６に示されるアミノ酸配列にｚｉｎｃ ｆｉｎｇｅｒ，Ｃ２Ｈ２ ｔｙｐｅ ｒｅｇｉｏｎの特徴を示す配列（Ｐｆａｍにｚｆ−Ｃ２Ｈ２としてエントリーされるアミノ酸配列）を見出した。 このドメインは、保存された２つのＣｙｓと２つのＨｉｓが亜鉛イオンに配位する構造モチーフをもっており、転写因子等多くの ＤＮＡ結合タンパク質に見られる。
さらに配列番号１２のアミノ酸番号１９〜５９に示されるアミノ酸配列にＫＲＡＢ ｂｏｘ ｒｅｇｉｏｎの特徴を示す配列（ＰｆａｍにＫＲＡＢとしてエントリーされるアミノ酸配列）を見出した。Ｋｒｕｐｐｅｌ−ａｓｓｏｃｉａｔｅｄ ｂｏｘ（ＫＲＡＢ）はＣ２Ｈ２タイプのＺｎフィンガータンパク質の約１／３に見られ、Ｎ末端部分に存在し、ＤＮＡとの結合の際には転写の抑制にはたらき、Ｚｎフィンガードメインとともに細胞分化・発生に関与することが知られている。
前述のＢＬＡＳＴでヒットした（ｉ）のタンパク質は、データベース中の文献情報（ＤＮＡ Ｃｅｌｌ． Ｂｉｏｌ． １４２（２）：１２５−１３６（１９９５））から単球の分化・増殖の調節に関わることが、上記（ｉｉ）のタンパク質は、データベース中の文献情報（ＤＮＡ Ｒｅｓ． ８（６）：３１９−３２７（２００１））から細胞のシグナル伝達、細胞骨格や細胞の運動性の機能が予測されることが、さらに上記（ｉｉｉ）のタンパク質は、データベース中の文献情報（ＤＮＡ Ｃｅｌｌ． Ｂｉｏｌ． １７（１１）：９３１−９４３（１９９８））からＫＲＡＢドメインが、転写を抑制するコファクターを構成するいくつかの核内タンパク質とｉｎ ｖｉｔｒｏで結合することがそれぞれ明らかとなった。
【０２０２】
更にタンパク質の細胞内局在の予測プログラムであるＰＳＯＲＴＩＩ（Ｔｒｅｎｄｓ Ｂｉｏｃｈｅｍ．Ｍｏｌ．Ｂｉｏｌ． ５８，（４） ４１７−４２４ （１９９６））による解析を行ったところ、本タンパク質の細胞内での局在の確率は核に１００．０％であり、本タンパク質は核に存在することが予測された。この結果は本タンパク質が細胞核内でＤＮＡと結合し、細胞の分化・増殖、細胞周期などに関与する遺伝子の転写を調節するタンパク質であることを示唆するものである。
【０２０３】
一方、ゲノム情報を得るため、ヒトｃＤＮＡをヒトゲノム配列へ写像するソフトウエアｓｉｍ４（Ｇｅｎｏｍｅ Ｒｅｓ．８， ９６７−９７４ （１９９８））を用い、本ＤＮＡのゲノムへの写像を試みたところ、第１９番染色体上に６個のエクソンがマッピングされた。真核生物ゲノムのアノテーション情報データベースＥｎｓｅｍｂｌ中のＤｉｓｅａｓｅ Ｂｒｏｗｓｅｒ （ｈｔｔｐ：／／ｗｗｗ．ｅｎｓｅｍｂｌ．ｏｒｇ／Ｈｏｍｏ＿ｓａｐｉｅｎｓ／ｄｉｓｅａｓｅｖｉｅｗ）によれば、写像された第１９番染色体上の位置（ｐ１３．１１）を含むｐ（短腕）に外骨腫症、ｐ１３に急性骨髄性白血病の原因遺伝子座が存在し、更に外胚葉性異形成症、高バリン血症、高ロイシン−イソロイシン血症の原因遺伝子座は第１９番染色体上に存在するが、その詳細位置は同定されていないことがわかった。これらによって本ＤＮＡがこれらの疾患の原因遺伝子である可能性や本ＤＮＡの変異がこれらの診断に応用できる可能性が推測された。
【０２０４】
以上より配列番号１２に示すタンパク質はヒトの器官・組織の発生・分化・形成、細胞周期や細胞増殖に関わる機能を有する新規Ｚｎフィンガータンパク質であることが予測された。
【０２０５】
本ＤＮＡの発現組織を検討するため、本ＤＮＡをクエリーとしてｄｂＥＳＴ（Ｎａｔｕｒｅ Ｇｅｎｅｔｉｃｓ ４， （４） ３３２−３ （１９９３））に対してＢＬＡＳＴ検索を行い、ｅ−ｖａｌｕｅ≦１０^−５０でヒットしたヒトＥＳＴが５個以上ある器官名または組織名を抽出したところ、正常な精巣・眼・脳・胃、癌化した精巣・子宮・脳・肺であった。尚、本ＤＮＡは胎児脳組織由来のｃＤＮＡライブラリーからクローニングされた。
更にＢＬＡＳＴのヒット上位３位について発現組織に関する文献情報を調べたところ、ＤＮＡ Ｃｅｌｌ． Ｂｉｏｌ． １４２（２）：１２５−１３６（１９９５）に、ＫＲＡＢ ｚｉｎｃ ｆｉｎｇｅｒ ｐｒｏｔｅｉｎ ＨＺＦ２６ （Ｈｕｍａｎ）はヒト単芽球において発現することが、ＤＮＡ Ｒｅｓ． ８（６）：３１９−３２７（２００１）に、ＫＩＡＡ１９６９ ｐｒｏｔｅｉｎ （Ｈｕｍａｎ）が脳・脊髄・胎児脳・胎児肝臓などで発現することが、さらにＤＮＡ Ｃｅｌｌ． Ｂｉｏｌ． １７（１１）：９３１−９４３（１９９８）に、ｚｉｎｃ ｆｉｎｇｅｒ ｐｒｏｔｅｉｎ ８５（Ｈｕｍａｎ）は精巣上皮腫組織に高発現することが、それぞれ述べられていた。
また、本タンパク質は、実施例６より、膵臓、脾臓、および肝臓で発現し、乳癌、肺癌で発現が増加することが示された。
【０２０６】
本タンパク質はこれらの組織や細胞に特有の機能や疾患、例えば精巣癌・メラノーマ・脳腫瘍・神経膠腫・神経芽細胞腫・胃癌・子宮癌・肺癌・肝臓癌・乳癌などの癌、アルツハイマー病・パーキンソン病・舞踏病などの神経変性疾患、肺炎・肝炎などの炎症性疾患、免疫系疾患、アレルギー疾患、不妊、高バリン血症、高ロイシン−イソロイシン血症、外胚葉性異形成症、外骨腫症などに関連する可能性が推測でき、これらの疾患の診断薬や治療薬のターゲットとしての利用が考えられる。
【０２０７】
（３）ｃ−ｂｒａｍｙ２０４３１０３（配列番号３、１３）
ｃ−ｂｒａｍｙ２０４３１０３（以下、これを「本ＤＮＡ」と称し、該ＤＮＡによりコードされるタンパク質を「本タンパク質」と称する）は、配列番号３に示すように、３０４９塩基から成り、そのうち塩基番号２８１から２２８４までがオープンリーディングフレーム （終止コドンを含む） である。オープンリーディングフレームから予測されるアミノ酸配列は、６６７アミノ酸残基からなる （配列番号１３）。
【０２０８】
配列番号１３のアミノ酸配列についてＢＬＡＳＴを用いて相同性検索を行ったところ、ＮＲＤＢタンパク質データベース （ＳＷＩＳＳ−ＰＲＯＴ、ＰＩＲ、ＴＲＥＭＢＬＥ、ＧＥＮＰＥＰＴ、ＰＤＢから作成された重複のないアミノ酸配列のデータベース） 中の、データベース登録記号Ｑ９ＵＪＷ８、Ｚｉｎｃ ｆｉｎｇｅｒ ｐｒｏｔｅｉｎ １８０ （Ｈｕｍａｎ）がヒットした。その内容としてＱ９ＵＪＷ８は６９２アミノ酸から成り、そのアミノ酸配列中の１〜６９２が、配列番号１３に記載のアミノ酸配列のアミノ酸番号１〜６６７と、ｅ−ｖａｌｕｅ：０．０、かつ６９２アミノ酸残基に亙り９６％の一致度でヒットした。
ＢＬＡＳＴ検索のアライメントから本タンパク質は、Ｑ９ＵＪＷ８のアミノ酸番号４５〜６９の２５残基に該当するアミノ酸を欠失しており、更に本ＤＮＡはＱ９ＵＪＷ８に該当する塩基配列ＡＦ１９２９１３の塩基番号３９８〜４７２に該当する７５塩基（本ＤＮＡの塩基番号４１２と４１３の間）を欠失していることもわかった。
【０２０９】
また配列番号１３のアミノ酸配列について、ＨＭＭＰＦＡＭによるタンパク質特徴検索を行ったところ、アミノ酸番号３２８〜３５０、３５６〜３７８、３８４〜４０６、４１２〜４３４、４４０〜４６２、４６８〜４９０、４９６〜５１８、５２４〜５４６、５５２〜５７４、５８０〜６０２、６０８〜６３０、６３６〜６５８に示されるアミノ酸配列にＺｉｎｃ Ｆｉｎｇｅｒ，Ｃ２Ｈ２ ｔｙｐｅ ｄｏｍａｉｎの特徴を示す配列（Ｐｆａｍにｚｆ−Ｃ２Ｈ２としてエントリーされるアミノ酸配列）を見出した。このドメインは、保存された２つのＣｙｓと２つのＨｉｓが亜鉛イオンに配位する構造モチーフをもっており、転写因子等多くの ＤＮＡ結合タンパク質に見られる。
さらに配列番号１３のアミノ酸番号４７〜８７に示されるアミノ酸配列にＫＲＡＢ ｂｏｘ ｒｅｇｉｏｎの特徴を示す配列（ＰｆａｍにＫＲＡＢとしてエントリーされるアミノ酸配列）を見出した。Ｋｒｕｐｐｅｌ−ａｓｓｏｃｉａｔｅｄ ｂｏｘ（ＫＲＡＢ）はＣ２Ｈ２タイプのＺｎフィンガータンパク質の約１／３に見られ、Ｎ末端部分に存在し、ＤＮＡとの結合の際には転写の抑制にはたらき、Ｚｎフィンガードメインとともに細胞分化・発生に関与することが知られている。
【０２１０】
ＨＭＭＰＦＡＭによるタンパク質特徴検索では更に本タンパク質のアミノ酸番号２５〜８０はＲｅｍｏｒｉｎ＿Ｎ ｒｅｇｉｏｎ（Ｒｅｍｏｒｉｎ，Ｎ−ｔｅｒｍｉｎａｌ ｒｅｇｉｏｎ）であり、この領域はＱ９ＵＪＷ８では見られない。Ｃ末端側でｃｏｉｌｅｄ−ｃｏｉｌが形成されるとき、このドメインはＮ末端側のプロリンに富んだ領域として存在し、ＤＮＡのような高分子と相互作用すると考えられている。Ｐｆａｍで、Ｃ末端側でＤＮＡ結合能を示すＺｎフィンガードメインが検出されているが、この領域がＱ９ＵＪＷ８とは異なるＤＮＡ結合能や相互作用、転写活性の調節を示す可能性が考えられる。
【０２１１】
本ＤＮＡおよびＡＦ１９２９１３ｃＤＮＡのエクソン構造を明らかにするために、ｃＤＮＡ配列をゲノム配列へ写像するソフトウエアｓｉｍ４（Ｇｅｎｏｍｅ Ｒｅｓ． ８， ９６７−７４ （１９９８））を用い、ゲノム配列へのアライメントを行ったところ、本ＤＮＡ配列はヒト第１９番染色体上の４個のエクソンにマッピングされ、ＡＦ１９２９１３配列は５個のエクソンにマッピングされることがわかった。本ＤＮＡの第１、第２、第３、第４エクソンはＡＦ１９２９１３と一致するが、本ＤＮＡはＡＦ１９２９１３の第３エクソンを１個欠失している。この違いは前述のＢＬＡＳＴ検索により本ＤＮＡの欠失部分（ＡＦ１９２９１３の塩基番号３９８〜４７２）に由来する。
これらの違いにより、ＤＮＡとの結合や相互作用、発現組織・発現量などに違いが生じる可能性がある。以上から、配列番号１３のタンパク質はＱ９ＵＪＷ８、Ｚｉｎｃ ｆｉｎｇｅｒ ｐｒｏｔｅｉｎ １８０ （Ｈｕｍａｎ）のスプライシングバリアントである。
【０２１２】
タンパク質の細胞内局在の予測プログラムであるＰＳＯＲＴ ＩＩ （Ｔｒｅｎｄｓ Ｂｉｏｃｈｅｍ． Ｓｃｉ． ２４， （１） ３４−６ （１９９９）） による解析を行ったところ、本タンパク質の細胞内での局在の確率は核９５．７％、ミトコンドリア４．３％であった。核への局在の確率が高いことから、本タンパク質は細胞核内でＤＮＡと結合し、細胞の分化・増殖、細胞周期などに関与する遺伝子の転写を調節するタンパク質であることが考えられる。
【０２１３】
真核生物ゲノムのアノテーション情報データベースＥｎｓｅｍｂｌ中のＤｉｓｅａｓｅ Ｂｒｏｗｓｅｒ （ｈｔｔｐ：／／ｗｗｗ．ｅｎｓｅｍｂｌ．ｏｒｇ／Ｈｏｍｏ＿ｓａｐｉｅｎｓ／ｄｉｓｅａｓｅｖｉｅｗ）によると、本ＤＮＡが写像された第１９番染色体上の位置（ｑ１３．３１）を包含するｑ１３．２−１３．３に進行性遺伝性心ブロック、３−ｍｅｔｈｙｌｇｌｕｔａｃｏｎｉｃａｃｉｄｕｒｉａの原因遺伝子座が、ｑ１３に常染色体優性難聴、ダイアモンド‐ブラックファン貧血、口蓋裂の原因遺伝子座が、ｑに遺伝性小児痙攣の原因遺伝子座が存在することがわかり、更に外胚葉性異形成症、高バリン血症、高ロイシン‐イソロイシン血症が第１９番染色体上に存在するが、その詳細位置は同定されていないことが明らかになった。以上より、本ＤＮＡがこれらの疾患の原因遺伝子である可能性や、スプライシングバリアントを含む本ＤＮＡの変異がこれらの疾患の診断に応用できる可能性が予想された。
【０２１４】
本タンパク質を小麦胚芽無細胞タンパク質合成系を用いて発現させたところ、実施例４においては、ＥＲの標的配列：ＡＡＧＧＧＡＡＡＡＴＧＡＣＣＣＣＣ（配列番号４６）、ＲＸＲ−αの標的配列：ＧＧＴＣＡＴＡＧＧＧＧＴ（配列番号４７）、ＰＰＡＲ−α：ＣＴＡＧＧＧＣＡＡＡＧＧＴＣＡ（配列番号４８）、Ｐａｘ−３等のコンセンサス標的配列：ＣＧＴＣＡＣＧＣＴＴＧＡ（配列番号６８）、Ｐａｘ−１の標的配列：ＣＣＧＴＴＣＣＧＣＴＣＴＡＧＡＴＡＴ（配列番号６９）およびＨＳＦ１等のコンセンサス標的配列：ＡＧＡＡＡＡＧＡＡＡＡＧＡＡＡ（配列番号７０）に親和性を示した。このことから本タンパク質はＤＮＡとの結合能を有し、脂質代謝や薬物代謝、筋肉形成などの異常に起因する疾患、例えば生活習慣病などの疾患との関連が考えられる。
【０２１５】
本ＤＮＡの発現組織を検討するため、本ＤＮＡをクエリーとしてｄｂＥＳＴ（Ｎａｔｕｒｅ Ｇｅｎｅｔｉｃｓ ４， （４） ３３２−３ （１９９３））に対してＢＬＡＳＴ検索を行い、ｅ−ｖａｌｕｅ≦１０^−１０でヒットしたヒトＥＳＴで、４個以上のヒットが見られる器官名または組織名を抽出したところ、ヒトの正常な肺・腸・脾臓、癌化した免疫系・リンパ系・子宮・眼・皮膚であった。尚、本ＤＮＡは扁桃核由来のｃＤＮＡライブラリーからクローニングされた。
【０２１６】
本タンパク質はこれらの組織や細胞に特有の機能や疾患、例えば肺癌・大腸癌・小腸癌・白血病・リンパ腫・子宮癌などの癌、うつ病、不安神経症、統合失調症などの中枢疾患、肺炎などの炎症性疾患、免疫系疾患、アレルギー疾患、高脂血症、動脈硬化、脾腫、口蓋裂、進行性遺伝性心ブロック、３−ｍｅｔｈｙｌｇｌｕｔａｃｏｎｉｃａｃｉｄｕｒｉａ、難聴、貧血、小児痙攣、高バリン血症、高ロイシン‐イソロイシン血症、筋形成不全に関連する可能性が推測でき、これらの疾患の診断薬や治療薬のターゲットとしての利用が考えられる。
【０２１７】
（４）ｃ−ｂｒｓｓｎ２０１３７５３（配列番号４、１４）
ｃ−ｂｒｓｓｎ２０１３７５３（以下、これを「本ＤＮＡ」と称し、該ＤＮＡによりコードされるタンパク質を「本タンパク質」と称する）は、配列番号４に示すように、２３１２塩基から成り、そのうち塩基番号１３８から１０１９までがオープンリーディングフレーム （終止コドンを含む） である。オープンリーディングフレームから予測されるアミノ酸配列は、２９３アミノ酸残基からなる （配列番号１４）。
【０２１８】
配列番号１４のアミノ酸配列についてＢＬＡＳＴを用いて相同性検索を行ったところ、ＮＲＤＢタンパク質データベース （ＳＷＩＳＳ−ＰＲＯＴ、ＰＩＲ、ＴＲＥＭＢＬＥ、ＧＥＮＰＥＰＴ、ＰＤＢから作成された重複のないアミノ酸配列のデータベース） 中の、（ｉ）データベース登録記号Ｑ６２９８１、Ｚｉｎｃ ｆｉｎｇｅｒ ｐｒｏｔｅｉｎ ＯＺＦ（Ｒａｔ）がヒットした。その内容として、Ｑ６２９８１は４０７アミノ酸から成り、そのアミノ酸配列中の１１６〜４０７が、配列番号１４に記載のアミノ酸配列のアミノ酸番号１〜２９３と、ｅ−ｖａｌｕｅ：５×１０^−１７６かつ２９３アミノ酸残基に亙り９５％の一致度でヒットした。
また（ｉｉ）データベース登録記号Ｑ６２５１３、Ｚｉｎｃ ｆｉｎｇｅｒ ｐｒｏｔｅｉｎ ＯＺＦ（Ｍｏｕｓｅ）がヒットした。その内容として、Ｑ６２９８１は４０７アミノ酸から成り、そのアミノ酸配列中の１１６〜４０７が、配列番号１４に記載のアミノ酸配列のアミノ酸番号１〜２９３と、ｅ−ｖａｌｕｅ：５×１０^−１７６かつ２９３アミノ酸残基に亙り９５％の一致度でヒットした。
更に（ｉｉｉ）データベース登録記号Ｑ１５０７２、Ｚｉｎｃ ｆｉｎｇｅｒ ｐｒｏｔｅｉｎ ＯＺＦ（Ｈｕｍａｎ）がヒットした。その内容として、Ｑ１５０７２は２９２アミノ酸から成り、そのアミノ酸配列中の４〜２９２が、配列番号１４に記載のアミノ酸配列のアミノ酸番号５〜２９３と、ｅ−ｖａｌｕｅ：５×１０^−１５１かつ２８９アミノ酸残基に亙り８１％の一致度でヒットした。
【０２１９】
また配列番号１４のアミノ酸配列について、ＨＭＭＰＦＡＭによるタンパク質特徴検索を行ったところ、アミノ酸番号１７〜３９、４５〜６７、７３〜９５、１０１〜１２３、１２９〜１５１、１５７〜１７９、１８５〜２０７、２１３〜２３５、２４１〜２６３、２６９〜２９１に示されるアミノ酸配列にＺｉｎｃ Ｆｉｎｇｅｒ，Ｃ２Ｈ２ ｔｙｐｅ ｄｏｍａｉｎの特徴を示す配列（Ｐｆａｍにｚｆ−Ｃ２Ｈ２としてエントリーされるアミノ酸配列）を見出した。このドメインは、保存された２つのＣｙｓと２つのＨｉｓが亜鉛イオンに配位する構造モチーフをもっており、転写因子等多くのＤＮＡ結合タンパク質に見られる。
またアミノ酸番号１５４〜１９４からＸＰＡ ｐｒｏｔｅｉｎ Ｎ−ｔｅｒｍｉｎａｌの特徴を示す配列（ＰｆａｍにＸＰＡ＿Ｎとしてエントリーされるアミノ酸配列）を抽出した。ＸＰＡ ｐｒｏｔｅｉｎは紫外線によって皮膚癌を高率に誘発する常染色体劣性遺伝病の色素性強皮症（ＸＰ）のパスウェイに関与するタンパク質であり、損傷したＤＮＡへの結合能をもち、ＤＮＡの除去修復の機能を担っている。
ＸＰＡ ｐｒｏｔｅｉｎ Ｎ−ｔｅｒｍｉｎａｌドメインは本タンパク質には存在するが、上記ヒット配列のうちヒトの配列であるＱ１５０７２には見られない。よって本タンパク質はＱ１５０７２が持たないＤＮＡ修復に関する機能を有する可能性が推測できる。
【０２２０】
本ＤＮＡおよびヒトのヒット配列であるＡＪ０１１８０６（Ｑ１５０７２に該当するＤＮＡ配列）ｃＤＮＡのエクソン構造を明らかにするために、ｃＤＮＡ配列をゲノム配列へ写像するソフトウエアｓｉｍ４（Ｇｅｎｏｍｅ Ｒｅｓ． ８， ９６７−７４ （１９９８））を用い、ゲノム配列へのアライメントを行ったところ、本ＤＮＡ配列はヒト第１９番染色体上の１個のエクソンにマッピングされ、ＡＪ０１１８０６配列は１個のエクソンにマッピングされることがわかった。尚、両者のマッピング位置は重複する部分は全く無い。
また本ＤＮＡはヒト由来のクローンであるが、本タンパク質ともっとも相同性の高い配列がラットの配列である。よって配列番号１４のタンパク質はラットやマウスのＺｉｎｃ ｆｉｎｇｅｒ ｐｒｏｔｅｉｎ ＯＺＦのオーソログであり、新規のヒトｃＤＮＡである可能性が高い。
【０２２１】
タンパク質の細胞内局在の予測プログラムであるＰＳＯＲＴ ＩＩ （Ｔｒｅｎｄｓ Ｂｉｏｃｈｅｍ． Ｓｃｉ． ２４， （１） ３４−６ （１９９９）） による解析を行ったところ、本タンパク質の細胞内での局在の確率は核９５．７％、ミトコンドリア４．３％であった。核への局在の確率が高いことから、本タンパク質は細胞核内でＤＮＡと結合し、細胞の分化・増殖、細胞周期などに関与する遺伝子の転写を調節するタンパク質であることが考えられる。
【０２２２】
真核生物ゲノムのアノテーション情報データベースＥｎｓｅｍｂｌ中のＤｉｓｅａｓｅ Ｂｒｏｗｓｅｒ （ｈｔｔｐ：／／ｗｗｗ．ｅｎｓｅｍｂｌ．ｏｒｇ／Ｈｏｍｏ＿ｓａｐｉｅｎｓ／ｄｉｓｅａｓｅｖｉｅｗ）によると、本ＤＮＡが写像された第１９番染色体上の位置（ｑ１３．１２）を包含するｑ１３．１にシスチン尿症、嚢胞性骨形成異常、先天性ネフローゼ、ｑ１３に常染色体優性難聴、ダイアモンド‐ブラックファン貧血、口蓋裂の原因遺伝子座が、ｑに遺伝性小児痙攣の原因遺伝子座が存在することがわかった。更に外胚葉性異形成症、高バリン血症・高ロイシン‐イソロイシン血症が第１９番染色体上に存在するが、その詳細位置は同定されていないことが明らかになった。
以上より、本ＤＮＡがこれらの疾患の原因遺伝子である可能性や、スプライシングバリアントを含む本ＤＮＡの変異がこれらの疾患の診断に応用できる可能性が予想された。
【０２２３】
本タンパク質を小麦胚芽無細胞タンパク質合成系を用いて発現させたところ、実施例４においては、ＧＡＴＡ−１等のコンセンサス標的配列：ＣＣＡＧＡＴＡＡＧＧ（配列番号５２）、およびＡＲＥＡ／ＮＩＴ−２の標的配列：ＴＡＴＣＴＣ（配列番号５３）に非常に高い親和性を示した。このことから本タンパク質はＤＮＡ結合能を有し、造血幹細胞の分化、血球系細胞、神経系細胞、循環器、アポトーシス制御などの異常に起因する疾患との関連が考えられる。
【０２２４】
本ＤＮＡの発現組織を検討するため、本ＤＮＡをクエリーとしてｄｂＥＳＴ（Ｎａｔｕｒｅ Ｇｅｎｅｔｉｃｓ ４， （４） ３３２−３ （１９９３））に対してＢＬＡＳＴ検索を行い、ｅ−ｖａｌｕｅ≦１０^−１０でヒットしたヒトＥＳＴで、４個以上のヒットが見られる器官名または組織名を抽出したところ、ヒトの正常な眼・脳・腸・肺・胎盤、癌化した精巣・子宮・眼・皮膚であった。尚、本ＤＮＡは黒質由来のｃＤＮＡライブラリーからクローニングされた。
Ｅｕｒ．Ｊ．Ｂｉｏｃｈｅｍ．２３６（３）：９９１−５（１９９６）によれば、ヒトのＯＺＦは乳腺上皮細胞で発現しており、腫瘍細胞で過剰発現が見られ、またＩｎｔ．Ｊ．Ｃａｎｃｅｒ ８０（３）：３６９−７２（１９９９）では、膵臓癌で高レベルに発現しており、発癌や腫瘍の発達への関与が推測される。
【０２２５】
以上より、本タンパク質はこれらの組織や細胞に特有の機能や疾患、例えばメラノーマ・脳腫瘍・大腸癌・小腸癌・肺癌・子宮癌・皮膚癌・精巣癌などの癌、パーキンソン病・舞踏病などの神経変性疾患、統合失調症などの中枢疾患、肺炎・皮膚炎などの炎症性疾患、免疫系疾患、アレルギー疾患、不妊症、シスチン尿症、嚢胞性骨形成異常、難聴、貧血、造血障害、口蓋裂、小児痙攣に関連する可能性が推測でき、これらの疾患の診断薬や治療薬のターゲットとしての利用が考えられる。
【０２２６】
（５）ｃ−ｂｒｓｓｎ２０１７５３０（配列番号５、１５）
ｃ−ｂｒｓｓｎ２０１１１９２（以下、これを「本ＤＮＡ」と称し、該ＤＮＡによりコードされるタンパク質を「本タンパク質」と称する）は、配列番号５に示すように、３８３９塩基から成り、そのうち塩基番号１５８２から２６８５までがオープンリーディングフレーム （終止コドンを含む） である。オープンリーディングフレームから予測されるアミノ酸配列は、３６７アミノ酸残基からなる （配列番号１５）。
【０２２７】
配列番号１５のアミノ酸配列についてＢＬＡＳＴを用いて相同性検索を行ったところ、ＮＲＤＢタンパク質データベース （ＳＷＩＳＳ−ＰＲＯＴ、ＰＩＲ、ＴＲＥＭＢＬＥ、ＧＥＮＰＥＰＴ、ＰＤＢから作成された重複のないアミノ酸配列のデータベース） 中の、データベース登録記号ＡＦ３２６２０６、ＺＮＦ１４０ − ｌｉｋｅ ｔｒａｎｓｃｒｉｐｔｉｏｎ ｆａｃｔｏｒ （Ｈｕｍａｎ）がヒットした。その内容としてＡＦ３２６２０６は３９９アミノ酸から成り、そのアミノ酸配列中の３３〜３９９が、配列番号１５に記載のアミノ酸配列のアミノ酸番号１〜３６７が、ｅ−ｖａｌｕｅ：０．０かつ３６７アミノ酸残基に亙り９９％の一致度でヒットした。
ＢＬＡＳＴ検索のアライメントから本タンパク質はＡＦ３２６２０６よりＮ末端が３２残基短く、ＡＦ３２６２０６のアミノ酸番号１３６（本タンパク質のアミノ酸番号１０４に該当）にグルタミン酸からグリシンの１残基置換が存在することがわかった。また本ＤＮＡはＡＦ３２６２０６と５’末端が１４９０塩基異なるが、翻訳開始点が塩基番号１５８２のため、ＡＦ３２６２０６との違いはＡＦ３２６２０６の翻訳開始点の塩基番号３３８から４３３までになり、これがアミノ酸のＮ末端の違いに反映している。またＡＦ３２６２０６の塩基番号７４３に該当する塩基（本ＤＮＡの塩基番号１８９１）がアデニンからグアニンに置換しており、これがアミノ酸の置換の由来である。
【０２２８】
また配列番号１５のアミノ酸配列について、ＨＭＭＰＦＡＭによるタンパク質特徴検索を行ったところ、アミノ酸番号１６９〜１９１、１９７〜２１９、２２５〜２４７、２５３〜２７５、２８１〜３０３、３０９〜３３１、３３７〜３５９に示されるアミノ酸配列にＺｉｎｃ Ｆｉｎｇｅｒ，Ｃ２Ｈ２ ｔｙｐｅ ｄｏｍａｉｎの特徴を示す配列（Ｐｆａｍにｚｆ−Ｃ２Ｈ２としてエントリーされるアミノ酸配列）を見出した。このドメインは、保存された２つのＣｙｓと２つのＨｉｓが亜鉛イオンに配位する構造モチーフをもっており、転写因子等多くのＤＮＡ結合タンパク質に見られる。
またＡＦ３２６２０６にはアミノ酸番号４〜４４にＫＲＡＢ ｂｏｘ ｒｅｇｉｏｎが存在するが、本タンパク質には見られない。このドメインはＤＮＡとの結合の際には転写の抑制にはたらき、Ｚｎフィンガードメインとともに細胞分化・発生に関与することが知られているので、本タンパク質はＡＦ３２６２０６とは異なる転写活性を示し、細胞分化に何らかの影響を及ぼすことが予測される。
【０２２９】
本ＤＮＡおよびＡＦ３２６２０６ｃＤＮＡのエクソン構造を明らかにするために、ｃＤＮＡ配列をゲノム配列へ写像するソフトウエアｓｉｍ４（Ｇｅｎｏｍｅ Ｒｅｓ． ８， ９６７−７４ （１９９８））を用い、ゲノム配列へのアライメントを行ったところ、本ＤＮＡ配列はヒト第１９番染色体上の３個のエクソンにマッピングされ、ＡＦ３２６２０６配列は５個のエクソン（ｐｏｌｙ Ａを除く）にマッピングされることがわかった。本ＤＮＡの第２、第３エクソンはＡＦ１９２９１３の第４、第５エクソンとそれぞれほぼ一致するが、本ＤＮＡの第１エクソンはＡＦ１９２９１３の第３エクソンより上流側に長い。また本ＤＮＡにはＡＦ１９２９１３の第１、第２エクソンは存在しない。ＡＦ１９２９１３の翻訳開始位置は塩基番号３３７であることから、両者は翻訳開始位置が異なり、これがアミノ酸配列上のＮ末端の違いの原因になっている。
これらの違いにより、ＤＮＡとの結合や相互作用、発現組織・発現量などに違いが生じる可能性がある。以上から、配列番号１５のタンパク質はＡＦ３２６２０６、Ｚｉｎｃ ｆｉｎｇｅｒ ｐｒｏｔｅｉｎ １４０−ｌｉｋｅ ｔｒａｎｓｃｒｉｐｔｉｏｎ ｆａｃｔｏｒ （Ｈｕｍａｎ）のスプライシングバリアントである。
【０２３０】
タンパク質の細胞内局在の予測プログラムであるＰＳＯＲＴ ＩＩ （Ｔｒｅｎｄｓ Ｂｉｏｃｈｅｍ． Ｓｃｉ． ２４， （１） ３４−６ （１９９９）） による解析を行ったところ、本タンパク質の細胞内での局在の確率は核に１００．０％であった。よって本タンパク質は細胞核内でＤＮＡと結合し、細胞の分化・増殖、細胞周期などに関与する遺伝子の転写を調節するタンパク質であることが考えられる。
【０２３１】
真核生物ゲノムのアノテーション情報データベースＥｎｓｅｍｂｌ中のＤｉｓｅａｓｅ Ｂｒｏｗｓｅｒ （ｈｔｔｐ：／／ｗｗｗ．ｅｎｓｅｍｂｌ．ｏｒｇ／Ｈｏｍｏ＿ｓａｐｉｅｎｓ／ｄｉｓｅａｓｅｖｉｅｗ）によると、本ＤＮＡが写像された第１９番染色体上の位置（ｑ１３．１１）を包含するｑ１３．１にシスチン尿症、先天性ネフローゼ、嚢胞性骨形成異常の原因遺伝子座が、ｑ１３に常染色体優性難聴、ダイアモンド‐ブラックファン貧血、口蓋裂の原因遺伝子座が、ｑに遺伝性小児痙攣の原因遺伝子座が存在することがわかり、更に外胚葉性異形成症、高バリン血症、高ロイシン‐イソロイシン血症が第１９番染色体上に存在するが、その詳細位置は同定されていないことが明らかになった。以上より、本ＤＮＡがこれらの疾患の原因遺伝子である可能性や、スプライシングバリアントを含む本ＤＮＡの変異がこれらの疾患の診断に応用できる可能性が予想された。
【０２３２】
本タンパク質を小麦胚芽無細胞タンパク質合成系を用いて発現させたところ、実施例４においては、Ｓｐ１の標的配列：ＧＧＧＧＧＧＧＧＧＧ（配列番号５４）、Ｔｒａｍｔｒａｃｋ６９Ｋの標的配列：ＧＧＡＣＣＴＧＣ（配列番号６１）、ＨＯＸ９の標的配列：ＴＧＡＣＡＧＴＴＴＡＡＣＧＡ（配列番号６２）およびＣＤＰの標的配列：ＣＣＡＡＴＡＡＴＣＧＡＴ（配列番号６３）に高い親和性を示した。このことから本タンパク質はＤＮＡ結合能を有し、造血幹細胞の分化、アポトーシス制御、サイトカイン産生などの異常に起因する疾患、ウイルス性疾患、細菌性疾患、癌などの疾患との関連が考えられる。また、実施例５によって、ＰＰＡＲが有する作用を強く抑制する活性を有し、内分泌・糖・脂質代謝異常等による糖尿病や肥満等の生活習慣病、血管機能異常等による循環器系疾患、炎症、癌等に関連することが推測された。また、ｐ５３が有する作用を抑制する活性を有し、癌等に関連することが推測された。さらに、ＨＩＦ−１が有する作用を増強する活性を有し、血管新生異常や細胞内酸化異常等に起因する、虚血性疾患、貧血、低酸素症、糖尿病性網膜症、循環器系疾患、癌等に関連することが推測された。
【０２３３】
本ＤＮＡの発現組織を検討するため、本ＤＮＡをクエリーとしてｄｂＥＳＴ（Ｎａｔｕｒｅ Ｇｅｎｅｔｉｃｓ ４， （４） ３３２−３ （１９９３））に対してＢＬＡＳＴ検索を行い、ｅ−ｖａｌｕｅ≦１０^−５０でヒットしたヒトＥＳＴで、５個以上のヒットが見られる器官名または組織名を抽出したところ、ヒトの正常な脳・眼・腎臓・胎盤、癌化した子宮・皮膚・眼・精巣であった。尚、本ＤＮＡは黒質由来のｃＤＮＡライブラリーからクローニングされた。
【０２３４】
本タンパク質はこれらの組織や細胞に特有の機能や疾患、例えば脳腫瘍・メラノーマ・腎臓癌・子宮癌・皮膚癌・精巣癌・白血病などの癌、パーキンソン病・舞踏病などの神経変性疾患、統合失調症などの中枢疾患、腎炎・皮膚炎などの炎症性疾患、免疫系疾患、アレルギー疾患、糖尿病、肥満、虚血性疾患、循環器系疾患、低酸素症、糖尿病性網膜症、シスチン尿症、ネフローゼ、難聴、貧血、口蓋裂、小児痙攣、外胚葉性異形成症、高バリン血症、高ロイシン‐イソロイシン血症に関連する可能性が推測でき、これらの疾患の診断薬や治療薬のターゲットとしての利用が考えられる。
【０２３５】
（６）ｃ−ｈｃｈｏｎ２０００４７３（配列番号６、１６）
ｃ−ｈｃｈｏｎ２０００４７３（以下、これを「本ＤＮＡ」と称し、該ＤＮＡによりコードされるタンパク質を「本タンパク質」と称する）は、配列番号６に示すように、３５８８塩基から成り、そのうち塩基番号３５２から２２７１までがオープンリーディングフレーム （終止コドンを含む） である。オープンリーディングフレームから予測されるアミノ酸配列は、６３９アミノ酸残基からなる （配列番号１６）。
【０２３６】
配列番号１６のアミノ酸配列についてＢＬＡＳＴを用いて相同性検索を行ったところ、ＮＲＤＢタンパク質データベース （ＳＷＩＳＳ−ＰＲＯＴ、ＰＩＲ、ＴＲＥＭＢＬＥ、ＧＥＮＰＥＰＴ、ＰＤＢから作成された重複のないアミノ酸配列のデータベース）ではゲノム配列以外に高いスコアを示すもは見当たらなかった。しかし商用の遺伝子配列特許データベースであるＧＥＮＥＳＥＱ（Ｄｅｒｗｅｎｔ社製）中の、アミノ酸配列データベースａａｇｅｎｅｓｅｑでは登録記号ＡＡＵ１５９４０およびＷＯ０１／５５３２２号公報にＳｅｑ ＩＤ ８９３として記載のアミノ酸配列、Ｈｕｍａｎ ｎｏｖｅｌ ｓｅｃｒｅｔｅｄ ｐｒｏｔｅｉｎがヒットした。その内容としてＡＡＵ１５９４０は３６１アミノ酸から成り、そのアミノ酸配列中の４〜３６１が、配列番号１６に記載のアミノ酸配列のアミノ酸番号２８２〜６３９と、ｅ−ｖａｌｕｅ：０．０かつ３５８アミノ酸残基に亙り１００．０％の一致度でヒットした。
ＢＬＡＳＴ検索のアライメントから本タンパク質はＡＡＵ１５９４０とＮ末端が２８１残基異なることがわかる。ＡＡＵ１５９４０は本タンパク質とは３残基異なっている。また本ＤＮＡではＡＡＳ２５９２７（ＡＡＵ１５９４０に該当するＤＮＡ配列データベースｎａｇｅｎｅｓｅｑ中のＤＮＡ配列）と５’末端が１１９３塩基異なり、またＡＡＳ２５９２７は本ＤＮＡと８塩基異なり、これらがアミノ酸配列のＮ末端の違いの原因になっている。
【０２３７】
また配列番号１６のアミノ酸配列について、ＨＭＭＰＦＡＭによるタンパク質特徴検索を行ったところ、アミノ酸番号２３４〜２５６、２６２〜２８４、２９０〜３１２、３１８〜３４０、３４６〜３６８、３７４〜３９６、４０２〜４２４、４３０〜４５２、４５８〜４８０、４８６〜５０８、５１４〜５３６、５４２〜５６４、５７０〜５９２、５９８〜６２０に示されるアミノ酸配列にＺｉｎｃ Ｆｉｎｇｅｒ，Ｃ２Ｈ２ ｔｙｐｅ ｄｏｍａｉｎの特徴を示す配列（Ｐｆａｍにｚｆ−Ｃ２Ｈ２としてエントリーされるアミノ酸配列）を見出した。このドメインは、保存された２つのＣｙｓと２つのＨｉｓが亜鉛イオンに配位する構造モチーフをもっており、転写因子等多くの ＤＮＡ結合タンパク質に見られる。
さらに配列番号１６のアミノ酸番号２７〜６７に示されるアミノ酸配列にＫＲＡＢ ｂｏｘ ｒｅｇｉｏｎの特徴を示す配列（ＰｆａｍにＫＲＡＢとしてエントリーされるアミノ酸配列）を見出した。Ｋｒｕｐｐｅｌ−ａｓｓｏｃｉａｔｅｄ ｂｏｘ（ＫＲＡＢ）はＣ２Ｈ２タイプのＺｎフィンガータンパク質の約１／３に見られ、Ｎ末端部分に存在し、ＤＮＡとの結合の際には転写の抑制にはたらき、Ｚｎフィンガードメインとともに細胞分化・発生に関与することが知られている。
ＫＲＡＢ ｂｏｘ ｒｅｇｉｏｎは本タンパク質には存在するが、ＡＡＵ１５９４０には見られない。よって本タンパク質はＡＡＵ１５９４０とは異なる転写活性を示し、細胞分化に何らかの影響を及ぼすことが考えられる。
【０２３８】
本ＤＮＡおよびＡＡＳ２５９２７ｃＤＮＡのエクソン構造を明らかにするために、ｃＤＮＡ配列をゲノム配列へ写像するソフトウエアｓｉｍ４（Ｇｅｎｏｍｅ Ｒｅｓ． ８， ９６７−７４ （１９９８））を用い、ゲノム配列へのアライメントを行ったところ、本ＤＮＡ配列はヒトＸ染色体上の７個のエクソンにマッピングされ、ＡＡＳ２５９２７配列は１個のエクソン（ｐｏｌｙ Ａを除く）にマッピングされることがわかった。本ＤＮＡの第７エクソンはＡＡＳ２５９２７の１エクソンより長く、これを包含するが、本ＤＮＡの第１から第６エクソンはＡＡＳ２５９２７には存在しない。この中でも第５、第６エクソンはＫＲＡＢドメインに該当するため、ＡＡＵ１５９４０はＫＲＡＢドメインを欠くというＨＭＭＰＦＡＭの結果と一致する。これらの違いにより、ＤＮＡとの結合や相互作用、発現組織・発現量などに違いが生じる可能性がある。以上から、配列番号１６のタンパク質は、ＷＯ０１／５５３２２号公報にＳｅｑ ＩＤ ８９３として記載のアミノ酸配列、Ｈｕｍａｎ ｎｏｖｅｌ ｓｅｃｒｅｔｅｄ ｐｒｏｔｅｉｎのスプライシングバリアントである。
【０２３９】
タンパク質の細胞内局在の予測プログラムであるＰＳＯＲＴ ＩＩ （Ｔｒｅｎｄｓ Ｂｉｏｃｈｅｍ． Ｓｃｉ． ２４， （１） ３４−６ （１９９９）） による解析を行ったところ、本タンパク質の細胞内での局在の確率は核に９５．７％、ミトコンドリア４．３％であった。よって本タンパク質は細胞核内でＤＮＡと結合し、細胞の分化・増殖、細胞周期などに関与する遺伝子の転写を調節するタンパク質であることが考えられる。
【０２４０】
真核生物ゲノムのアノテーション情報データベースＥｎｓｅｍｂｌ中のＤｉｓｅａｓｅ Ｂｒｏｗｓｅｒ （ｈｔｔｐ：／／ｗｗｗ．ｅｎｓｅｍｂｌ．ｏｒｇ／Ｈｏｍｏ＿ｓａｐｉｅｎｓ／ｄｉｓｅａｓｅｖｉｅｗ）によると、本ＤＮＡが写像されたＸ染色体上の位置（ｐ１１．２３）を包含するｐ１１−２３に精神遅滞、ｐ１１．４−１１．２に脆弱Ｘ症候群、ｐ１１．３−１１．２に関節拘縮症（脊髄性筋萎縮症）、ｐ１１．４−１１．２３にアルビノ（眼）、ｐ１１．４−１１．２１に弱視、ｐ１１．２３に夜盲症、ｐ１１．２に乳頭状腎細胞癌、ｐ１１にインスリン依存性糖尿病、ｐに先天性白内障の原因遺伝子座が存在することがわかった。以上より、本ＤＮＡがこれらの疾患の原因遺伝子である可能性や、スプライシングバリアントを含む本ＤＮＡの変異がこれらの疾患の診断に応用できる可能性が予想された。
【０２４１】
本タンパク質を小麦胚芽無細胞タンパク質合成系を用いて発現させたところ、実施例４においては、ＨＥＮ−１の標的配列：ＧＧＧＧＣＧＣＡＧＣＴＧＣＧＧＣＣＣ（配列番号３８）、ＡｈＲ等のコンセンサス標的配列：ＧＧＧＧＡＴＴＧＣＧＴＧ（配列番号３９）、ＵＳＦの標的配列：ＧＴＣＡＣＧＴＧＧＴ（配列番号４０）、ＮＦ−１Ａ１等のコンセンサス標的配列：ＣＴＧＴＧＧＧＧＴＴＴＧＧＣＡＣＧＧＧＧＣＣＡ（配列番号４１）、ＲＦ−Ｘ１の標的配列：ＧＧＴＡＡＣＡＴＡＧＣＡＡＣ（配列番号４２）、Ｓｐ１の標的配列：ＧＧＧＧＧＧＧＧＧＧ（配列番号５４）、Ｅｇｒ−１の標的配列：ＴＧＣＧＴＧＧＧＣＧ（配列番号５６）、ｐ５０の標的配列：ＧＧＧＧＡＣＴＴＴＣＣ（配列番号７４）、ＮＦ−ＡＴｃ等のコンセンサス標的配列：ＡＧＧＡＡＡＡ（配列番号７５）、ｐ９１等のコンセンサス標的配列：ＧＡＡＴＴＣＣＧＧＧＡＡＡＴＧＧ（配列番号７６）、ＳＴＡＴ２等のコンセンサス標的配列：ＴＴＴＣＣＣＧＧＧＡＡＡＴＧ（配列番号７７）およびｐ５３の標的配列：ＧＧＡＣＡＴＧＣＣＣＧＧＧＣＡＴＧＴＣ（配列番号７８）に非常に高い親和性を示した。このことから本タンパク質はＤＮＡ結合能を有し、細胞周期、炎症、アポトーシス制御、免疫反応、薬物代謝、サイトカイン産生などの異常に起因する疾患、ウイルス性疾患、癌などの疾患との関連が考えられる。また、実施例５によって、ＰＰＡＲが有する作用を増強する活性を有し、内分泌・糖・脂質代謝異常等による糖尿病や肥満等の生活習慣病、血管機能異常等による循環器系疾患、炎症、癌等に関連することが推測された。また、ＡＰ−１が有する作用を抑制する活性を有し、炎症性疾患や癌等に関連することが推測された。
【０２４２】
本ＤＮＡの発現組織を検討するため、本ＤＮＡをクエリーとしてｄｂＥＳＴ（Ｎａｔｕｒｅ Ｇｅｎｅｔｉｃｓ ４， （４） ３３２−３ （１９９３））に対してＢＬＡＳＴ検索を行い、ｅ−ｖａｌｕｅ≦１０^−１０でヒットしたヒトＥＳＴで、５個以上のヒットが見られる器官名または組織名を抽出したところ、ヒトの正常な脳・胎盤・胃・乳腺、癌化した眼・皮膚・肺・子宮であった。尚、本ＤＮＡは正常軟骨初代培養細胞由来のｃＤＮＡライブラリーからクローニングされた。
【０２４３】
本タンパク質はこれらの組織や細胞に特有の機能や疾患、例えば、白血病・リンパ腫・脳腫瘍・胃癌・乳癌・メラノーマ・皮膚癌・肺癌・子宮癌などの癌、肺炎・皮膚炎などの炎症性疾患、免疫系疾患、アレルギー疾患、肥満、循環器系疾患、不妊症、精神遅滞、脆弱Ｘ症候群、関節拘縮症、アルビノ、弱視、夜盲症、腎癌、糖尿病、変形関節症に関連する可能性が推測でき、本タンパク質はこれらの疾患の診断薬や治療薬のターゲットとしての利用が考えられる。
【０２４４】
（７）ｃ−ｔｅｓｔｉ４００４６２６（配列番号７、１７）
ｃ−ｔｅｓｔｉ４００４６２６（以下、これを「本ＤＮＡ」と称し、該ＤＮＡによりコードされるタンパク質を「本タンパク質」と称する）は、配列番号７に示すように、４０５３塩基から成り、そのうち塩基番号２０９７から３８８１までがオープンリーディングフレーム （終止コドンを含む） である。オープンリーディングフレームから予測されるアミノ酸配列は、５９４アミノ酸残基からなる （配列番号１７）。
【０２４５】
配列番号１７のアミノ酸配列についてＢＬＡＳＴを用いて相同性検索を行ったところ、ＮＲＤＢタンパク質データベース （ＳＷＩＳＳ−ＰＲＯＴ、ＰＩＲ、ＴＲＥＭＢＬＥ、ＧＥＮＰＥＰＴ、ＰＤＢから作成された重複のないアミノ酸配列のデータベース）のデータベース登録記号Ｐ５２７３６、Ｚｉｎｃ ｆｉｎｇｅｒ ｐｒｏｔｅｉｎ １３３ （Ｈｕｍａｎ）がヒットした。
その内容としてＰ５２７３６は６５４アミノ酸から成り、そのアミノ酸配列中の７３〜６５４が、配列番号１７に記載のアミノ酸配列のアミノ酸番号１３〜５９４と、ｅ−ｖａｌｕｅ：０．０かつ５８４アミノ酸残基に亙り９９％の一致度でヒットした。
ＢＬＡＳＴ検索のアライメントから本タンパク質はＰ５２７３６とＮ末端が１２残基異なり、Ｐ５２７３６は本タンパク質とＮ末端が７２残基異なることがわかる。また本タンパク質のアミノ酸番号１２１、４２８（Ｐ５２７３６のアミノ酸番号１８１、４８８に該当）においてはロイシンからフェニルアラニン、ヒスチジンからアルギニンの置換がそれぞれ存在する。また本ＤＮＡではアライメント上ではＵ０９３６６（Ｐ５２７３６に該当する塩基配列）と５’末端が２１３１塩基異なるが、翻訳開始点は本ＤＮＡは塩基番号２０９７なので、５’末端の違いは３５塩基であり、これがアミノ酸配列のＮ末端の違いの原因になっている。また本ＤＮＡの塩基番号２２８２、２４５７（Ｕ０９３６６の塩基番号８１１、９８６に該当）にアデニンからグアニン、シトシンからチミンへの１塩基置換がそれぞれ存在しており、これがアミノ酸置換の原因になっている。
【０２４６】
また配列番号１７のアミノ酸配列について、ＨＭＭＰＦＡＭによるタンパク質特徴検索を行ったところ、アミノ酸番号１５４〜１７６、１８２〜２０４、２１０〜２３２、２３８〜２６０、２６６〜２８８、２９４〜３１６、３２２〜３４４、３５０〜３７２、３７８〜４００、４０６〜４２８、４３４〜４５６、４６２〜４８４、４９０〜５１２、５１８〜５４０、５４６〜５７１に示されるアミノ酸配列にＺｉｎｃ Ｆｉｎｇｅｒ，Ｃ２Ｈ２ ｔｙｐｅ ｄｏｍａｉｎの特徴を示す配列（Ｐｆａｍにｚｆ−Ｃ２Ｈ２としてエントリーされるアミノ酸配列）を見出した。このドメインは、保存された２つのＣｙｓと２つのＨｉｓが亜鉛イオンに配位する構造モチーフをもっており、転写因子等多くのＤＮＡ結合タンパク質に見られる。
尚、Ｐ５２７３６にはアミノ酸番号１〜４１にＫＲＡＢ ｂｏｘ ｒｅｇｉｏｎが存在するが本タンパク質には見当たらない。
よって本タンパク質はＰ５２７３６とは異なる転写活性を示し、細胞分化に何らかの影響を及ぼす可能性が考えられる。
【０２４７】
本ＤＮＡおよびＵ０９３６６ｃＤＮＡのエクソン構造を明らかにするために、ｃＤＮＡ配列をゲノム配列へ写像するソフトウエアｓｉｍ４（Ｇｅｎｏｍｅ Ｒｅｓ． ８， ９６７−７４ （１９９８））を用い、ゲノム配列へのアライメントを行ったところ、本ＤＮＡ配列はヒト第２０番染色体上の２個のエクソンにマッピングされ、Ｕ０９３６６配列は６個のエクソン（ｐｏｌｙ Ａを除く）にマッピングされることがわかった。本ＤＮＡの第２エクソンはＵ０９３６６の第６エクソンとほぼ一致するが、本ＤＮＡの第１エクソンはＵ０９３６６には存在せず、位置としてはＵ０９３６６の第５と第６エクソンの間に存在する。Ｕ０９３６６の第４、第５エクソンはＫＲＡＢドメインに該当し、ＦＥＢＳ Ｌｅｔｔ． ３６９（２−３）：１５３−７にもＺｉｎｃ ｆｉｎｇｅｒ １３３はＫＲＡＢ ｂｏｘのＡとＢの両方のサブドメインをもっていることが述べられているので、両ＤＮＡは構造上の違いのみならず、機能的にも差異が生じることが考えられる。
これらの違いにより、ＤＮＡとの結合や相互作用、発現組織・発現量などに違いが生じる可能性がある。以上から、配列番号１７のタンパク質は、Ｐ５２７３６、Ｚｉｎｃ ｆｉｎｇｅｒ ｐｒｏｔｅｉｎ １３３ （Ｈｕｍａｎ）のスプライシングバリアントである。
【０２４８】
タンパク質の細胞内局在の予測プログラムであるＰＳＯＲＴ ＩＩ （Ｔｒｅｎｄｓ Ｂｉｏｃｈｅｍ． Ｓｃｉ． ２４， （１） ３４−６ （１９９９）） による解析を行ったところ、本タンパク質の細胞内での局在の確率は核に１００．０％であった。よって本タンパク質は細胞核内でＤＮＡと結合し、細胞の分化・増殖、細胞周期などに関与する遺伝子の転写を調節するタンパク質であることが考えられる。
【０２４９】
真核生物ゲノムのアノテーション情報データベースＥｎｓｅｍｂｌ中のＤｉｓｅａｓｅ Ｂｒｏｗｓｅｒ （ｈｔｔｐ：／／ｗｗｗ．ｅｎｓｅｍｂｌ．ｏｒｇ／Ｈｏｍｏ＿ｓａｐｉｅｎｓ／ｄｉｓｅａｓｅｖｉｅｗ）によると、本ＤＮＡが写像された第２０番染色体上の位置（ｐ１１．２３）を包含するｐ１１．２に遺伝性角膜ジストロフィーの原因遺伝子座が存在することがわかった。以上より、本ＤＮＡがこの疾患の原因遺伝子である可能性や、スプライシングバリアントを含む本ＤＮＡの変異がこれらの疾患の診断薬に応用できる可能性が予想された。
【０２５０】
本タンパク質を小麦胚芽無細胞タンパク質合成系を用いて発現させたところ、実施例４においては、ＹＹ１の標的配列：ＣＧＧＣＣＡＴＣＴＴＧＧＣＴ（配列番号５５）、Ｅｇｒ−１の標的配列：ＴＧＣＧＴＧＧＧＣＧ（配列番号５６）、Ｓｎａｉｌの標的配列：ＣＡＣＣＴＧＴＴＴＴＣＡ（配列番号５７）、ＣＦ２−ＩＩの標的配列：ＧＴＡＴＡＴＡＴＡ（配列番号５８）、ＩＲＦ−１，ＩＲＦ−２のコンセンサス標的配列：ＧＡＡＡＡＧＣＧＡＡＡＣＣ（配列番号７３）、ＮＦ−ＡＴｃ等のコンセンサス標的配列：ＡＧＧＡＡＡＡ（配列番号７５）、およびｐ９１等のコンセンサス標的配列：ＧＡＡＴＴＣＣＧＧＧＡＡＡＴＧＧ（配列番号７６）に親和性を示した。このことから本タンパク質はＤＮＡとの結合能を有し、免疫反応、サイトカイン産生などの異常に起因する疾患との関連が考えられる。また、実施例５によって、ｐ５３が有する作用を抑制する活性を有し、癌等に関連することが推測された。また、ＮＦκＢが有する作用を抑制する活性を有し、関節リウマチや変形性関節炎などの免疫性疾患、炎症性疾患、癌等に関連することが推測された。また、ＡＰ−１が有する作用を抑制する活性を有し、炎症性疾患や癌等に関連することが推測された。また、ＨＩＦ−１が有する作用を増強する活性を有し、血管新生異常や細胞内酸化異常等に起因する、虚血性疾患、貧血、低酸素症、糖尿病性網膜症、循環器系疾患、癌等に関連することが推測された。さらに、ＣＲＥＢが有する作用をやや強く増強する活性を有し、記憶障害などの中枢系疾患、循環器系疾患、糖尿病等に関連することが推測された。
【０２５１】
本ＤＮＡの発現組織を検討するため、本ＤＮＡをクエリーとしてｄｂＥＳＴ（Ｎａｔｕｒｅ Ｇｅｎｅｔｉｃｓ ４， （４） ３３２−３ （１９９３））に対してＢＬＡＳＴ検索を行い、ｅ−ｖａｌｕｅ≦１０^−５０でヒットしたヒトＥＳＴで、４個以上のヒットが見られる器官名または組織名を抽出したところ、ヒトの正常な骨・肝臓・膵臓・胎児心臓・脳・眼・胎児肝臓、癌化した乳腺・前立腺であった。尚、本ＤＮＡは精巣由来のｃＤＮＡライブラリーからクローニングされた。
【０２５２】
本タンパク質はこれらの組織や細胞に特有の機能や疾患、例えば、骨髄腫・肝臓癌・膵臓癌・脳腫瘍・メラノーマ・乳癌・前立腺癌・精巣癌などの癌、肺炎・膵炎・肝炎・変形性関節炎などの炎症性疾患、関節リウマチなどの免疫系疾患、アレルギー疾患、記憶障害などの中枢系疾患、糖尿病、虚血性疾患、貧血、低酸素症、糖尿病性網膜症、循環器系疾患、不妊症、角膜ジストロフィーに関連する可能性が推測でき、これらの疾患の診断薬や治療薬のターゲットとしての利用が考えられる。
【０２５３】
（８）ｃ−ｔｒａｃｈ３０１４３１６（配列番号８、１８）
ｃ−ｔｒａｃｈ３０１４３１６（以下、これを「本ＤＮＡ」と称し、該ＤＮＡによりコードされるタンパク質を「本タンパク質」と称する）は、配列番号８に示すように、４４５５塩基から成り、そのうち塩基番号１０３８から１９３７までがオープンリーディングフレーム （終止コドンを含む） である。オープンリーディングフレームから予測されるアミノ酸配列は、２９９アミノ酸残基からなる （配列番号１８）。
【０２５４】
配列番号１８のアミノ酸配列についてＢＬＡＳＴを用いて相同性検索を行ったところ、ＮＲＤＢタンパク質データベース （ＳＷＩＳＳ−ＰＲＯＴ、ＰＩＲ、ＴＲＥＭＢＬＥ、ＧＥＮＰＥＰＴ、ＰＤＢから作成された重複のないアミノ酸配列のデータベース）のデータベース登録記号ＡＫ０７４１１６、ＦＬＪ００１８７ ｐｒｏｔｅｉｎ （Ｈｕｍａｎ）がヒットした。
その内容としてＡＫ０７４１１６は５６３アミノ酸から成り、そのアミノ酸配列中の２６７〜５６３が、配列番号１８に記載のアミノ酸配列のアミノ酸番号３〜２９９と、ｅ−ｖａｌｕｅ：５×１０^−１７９かつ２９７アミノ酸残基に亙り１００．０％の一致度でヒットした。
ＢＬＡＳＴ検索のアライメントから本タンパク質はＡＫ０７４１１６とＮ末端が２残基異なり、ＡＫ０７４１１６は本タンパク質とＮ末端が２６６残基異なることがわかる。また本ＤＮＡではアライメント上ではＡＫ０７４１１６と５’末端が１０４３塩基異なるが、翻訳開始点は本ＤＮＡは塩基番号１０３８なので、５’末端の違いは６塩基であり、これがアミノ酸配列のＮ末端の違いの原因になっている。ＡＫ０７４１１６では５’末端は９９９塩基異なるが、翻訳開始点は塩基番号２０２であり、これらによってアミノ酸配列のＮ末端の違いが生じている。
【０２５５】
また配列番号１８のアミノ酸配列について、ＨＭＭＰＦＡＭによるタンパク質特徴検索を行ったところ、アミノ酸番号１１３〜１３５、１４１〜１６３、１６９〜１９１、１９７〜２１９、２２５〜２４７、２５３〜２７５に示されるアミノ酸配列にＺｉｎｃ Ｆｉｎｇｅｒ，Ｃ２Ｈ２ ｔｙｐｅ ｄｏｍａｉｎの特徴を示す配列（Ｐｆａｍにｚｆ−Ｃ２Ｈ２としてエントリーされるアミノ酸配列）を見出した。このドメインは、保存された２つのＣｙｓと２つのＨｉｓが亜鉛イオンに配位する構造モチーフをもっており、転写因子等多くのＤＮＡ結合タンパク質に見られる。
尚、ＡＫ０７４１１６はアミノ酸番号５０〜１４３からＳＣＡＮ ｄｏｍａｉｎ（ＰｆａｍにＳＣＡＮとしてエントリーされるアミノ酸配列）を、アミノ酸番号２２７〜２６７からＫＲＡＢ ｂｏｘ ｒｅｇｉｏｎ（ＰｆａｍにＫＲＡＢとしてエントリーされるアミノ酸配列）をそれぞれ抽出したが、これらのドメインは本タンパク質には存在しない。
ＳＣＡＮ ｄｏｍａｉｎは一部のＣ２Ｈ２タイプのＺｎフィンガータンパク質のＮ末端に見られる保存されたモチーフで、オリゴマー形成の調節と転写制御の役割をもつことが知られている。またＫｒｕｐｐｅｌ−ａｓｓｏｃｉａｔｅｄ ｂｏｘ（ＫＲＡＢ）はＣ２Ｈ２タイプのＺｎフィンガータンパク質の約１／３に見られ、Ｎ末端部分に存在し、ＤＮＡとの結合の際には転写の抑制にはたらき、Ｚｎフィンガードメインとともに細胞分化・発生に関与することが知られている。
よって本タンパク質はＡＫ０７４１１６とは異なる転写活性を示して細胞分化に影響したり、オリゴマー形成において異なる作用を有する可能性が考えられる。
【０２５６】
本ＤＮＡおよびＡＫ０７４１１６ｃＤＮＡのエクソン構造を明らかにするために、ｃＤＮＡ配列をゲノム配列へ写像するソフトウエアｓｉｍ４（Ｇｅｎｏｍｅ Ｒｅｓ． ８， ９６７−７４ （１９９８））を用い、ゲノム配列へのアライメントを行ったところ、本ＤＮＡ配列はヒト第７番染色体上の１個のエクソンにマッピングされ、ＡＫ０７４１１６配列は６個のエクソンにマッピングされることがわかった。本ＤＮＡの１エクソンはＡＫ０７４１１６の第６エクソンを包含し、これより長い。本ＤＮＡには存在しないＡＫ０７４１１６の第１から第５エクソンのうち、第４、第５エクソンはＫＲＡＢドメインに該当するため、両者の構造上の違いのみならず、機能的にも差異が生じることが考えられる。
これらの違いにより、ＤＮＡとの結合や相互作用、発現組織・発現量などに違いが生じる可能性がある。以上から、配列番号１８のタンパク質は、ＡＫ０７４１１６、ＦＬＪ００１８７ ｐｒｏｔｅｉｎ （Ｈｕｍａｎ）のスプライシングバリアントである。
【０２５７】
タンパク質の細胞内局在の予測プログラムであるＰＳＯＲＴ ＩＩ （Ｔｒｅｎｄｓ Ｂｉｏｃｈｅｍ． Ｓｃｉ． ２４， （１） ３４−６ （１９９９）） による解析を行ったところ、本タンパク質の細胞内での局在の確率は核に９５．７％、ミトコンドリア４．３％であった。よって本タンパク質は細胞核内でＤＮＡと結合し、細胞の分化・増殖、細胞周期などに関与する遺伝子の転写を調節するタンパク質であることが考えられる。
【０２５８】
真核生物ゲノムのアノテーション情報データベースＥｎｓｅｍｂｌ中のＤｉｓｅａｓｅ Ｂｒｏｗｓｅｒ （ｈｔｔｐ：／／ｗｗｗ．ｅｎｓｅｍｂｌ．ｏｒｇ／Ｈｏｍｏ＿ｓａｐｉｅｎｓ／ｄｉｓｅａｓｅｖｉｅｗ）によると、本ＤＮＡが写像された第７番染色体上の位置（ｑ２２．１）を包含するｑ２２−３１．１に先天性下痢症、大腸癌、ｑ２１．３−２２に出血性素因、血栓形成傾向の原因遺伝子座が、ｑ２１−２２に悪性高熱症感受性、ｑに自閉症感受性の遺伝子座がそれぞれ存在することがわかった。以上より、本ＤＮＡがこの疾患の原因遺伝子である可能性や、スプライシングバリアントを含む本ＤＮＡの変異がこれらの疾患の診断薬に応用できる可能性が予想された。
【０２５９】
本タンパク質を小麦胚芽無細胞タンパク質合成系を用いて発現させたところ、実施例４においては、ＧＡＴＡ−１等のコンセンサス標的配列：ＣＣＡＧＡＴＡＡＧＧ（配列番号５２）、Ｎｋｘ−２．２等のコンセンサス標的配列：ＴＴＡＡＧＴＧ配列番号６５）およびＯｃｔ−１Ａ等のコンセンサス標的配列：ＡＴＧＣＡＡＡＴ（配列番号６６）に高い親和性を示した。このことから本タンパク質はＤＮＡとの結合能を有し、造血幹細胞の分化、血球系細胞、神経系細胞、循環器、アポトーシス制御などの異常に起因する疾患との関連が考えられる。また、実施例５によって、ＮＦκＢが有する作用を増強する活性を有し、関節リウマチや変形性関節炎などの免疫性疾患、炎症性疾患、癌等に関連することが推測された。また、ＡＰ−１が有する作用を抑制する活性を有し、炎症性疾患や癌等に関連することが推測された。また、ＨＩＦ−１が有する作用をやや強く増強する活性を有し、血管新生異常や細胞内酸化異常等に起因する、虚血性疾患、貧血、低酸素症、糖尿病性網膜症、循環器系疾患、癌等に関連することが推測された。さらに、ＣＲＥＢが有する作用を増強する活性を有し、記憶障害などの中枢系疾患、循環器系疾患、糖尿病等に関連することが推測された。
【０２６０】
本ＤＮＡの発現組織を検討するため、本ＤＮＡをクエリーとしてｄｂＥＳＴ（Ｎａｔｕｒｅ Ｇｅｎｅｔｉｃｓ ４， （４） ３３２−３ （１９９３））に対してＢＬＡＳＴ検索を行い、ｅ−ｖａｌｕｅ≦１０^−６５でヒットしたヒトＥＳＴで、５個以上のヒットが見られる器官名または組織名を抽出したところ、ヒトの正常な骨・骨格筋・眼・膵臓・肺・腸・腎臓・血液・脳・胎盤・肝臓・胎児脳・胎児脾臓、癌化した皮膚・眼・肺・腸・乳腺・子宮・脳・膵臓・精巣であった。尚、本ＤＮＡは気管由来のｃＤＮＡライブラリーからクローニングされた。
【０２６１】
本タンパク質はこれらの組織や細胞に特有の機能や疾患、例えば、骨髄腫・肺癌・大腸癌・小腸癌・腎臓癌・白血病・リンパ腫・脳腫瘍・肝臓癌・皮膚癌・メラノ−マ・乳癌子宮癌・膵臓癌・精巣癌などの癌、肺炎・膵炎・腎炎・肝炎・気管支炎・変形性関節炎などの炎症性疾患、関節リウマチなどの免疫系疾患、気管支喘息などのアレルギー疾患、糖尿病、虚血性疾患、貧血、低酸素症、循環器系疾患、記憶障害などの中枢系疾患、筋ジストロフィー・多発性筋炎などの骨格筋疾患、不妊症、脾臓疾患、先天性下痢症、出血性素因、血栓形成傾向、悪性高熱症、自閉症に関連する可能性が推測でき、これらの疾患の診断薬や治療薬のターゲットとしての利用が考えられる。
【０２６２】
（９）ｃ−ｂｒｈｉｐ３００９１８１（配列番号９、１９）
ｃ−ｂｒｈｉｐ３００９１８１（以下、これを「本ＤＮＡ」と称し、該ＤＮＡによりコードされるタンパク質を「本タンパク質」と称する）は、配列番号９に示すように、４０１０塩基から成り、そのうち塩基番号１１４０から３５０６までがオープンリーディングフレーム （終止コドンを含む） である。オープンリーディングフレームから予測されるアミノ酸配列は、７８８アミノ酸残基からなる （配列番号１９）。
【０２６３】
配列番号１９のアミノ酸配列についてＢＬＡＳＴを用いて相同性検索を行ったところ、ＮＲＤＢタンパク質データベース （ＳＷＩＳＳ−ＰＲＯＴ、ＰＩＲ、ＴＲＥＭＢＬＥ、ＧＥＮＰＥＰＴ、ＰＤＢから作成された重複のないアミノ酸配列のデータベース）のデータベース登録記号Ｐ１９８３８、Ｎｕｃｌｅａｒ ｆａｃｔｏｒ ＮＦκＢ ｐ１０５ ｓｕｂｕｎｉｔ （Ｈｕｍａｎ）がヒットした。
その内容としてＰ１９８３８は９６８アミノ酸から成り、そのアミノ酸配列中の１９０〜９６８が、配列番号１９に記載のアミノ酸配列のアミノ酸番号１０〜７８８と、ｅ−ｖａｌｕｅ：０．０、かつ７７９アミノ酸残基に亙り１００％の一致度でヒットした。
ＮＦκＢは発癌や炎症、アポトーシスなどに関与する遺伝子発現を調節する疾患関連転写調節因子の１つとして知られている。
ＢＬＡＳＴ検索のアライメントから本タンパク質はＰ１９８３８とＮ末端が９残基異なり、Ｐ１９８３８は本タンパク質と１８９残基異なる。また本ＤＮＡではアライメント上ではＭ５８６０３（Ｐ１９８３８に該当するＤＮＡ配列）と５’末端が１１６５塩基異なり、Ｍ５８６０３は本ＤＮＡと９６３塩基異なる。本ＤＮＡは塩基番号１１４０が、Ｍ５８６０３は塩基番号３９８が、それぞれ翻訳開始点であり、これらのことにより、アミノ酸配列のＮ末端の違いが生じている。
【０２６４】
配列番号１９のアミノ酸配列について、ＨＭＭＰＦＡＭによるタンパク質特徴検索を行ったところ、アミノ酸番号７０〜１７０に示されるアミノ酸配列にＩＰＴ／ＴＩＧ ｄｏｍａｉｎの特徴を示す配列（ＰｆａｍにＴＩＧとしてエントリーされるアミノ酸配列）を見出した。このドメインは、イムノグロブリン様のフォールドをもち、ＤＮＡ結合能をもった細胞内の転写因子だけでなく、細胞表面の受容体にも見られる。
またアミノ酸番号６３７〜７１２にＤｅａｔｈ ｄｏｍａｉｎ（Ｐｆａｍにｄｅａｔｈとしてエントリーされるアミノ酸配列）を見出した。このドメインは、Ｃ末端に存在し、ＦＡＳやＴＮＦなどの細胞表面の受容体の多量体形成を調節して受容体の細胞質部分と相互作用し、アポトーシスを誘導するシグナルを伝達することが知られている。
更にアミノ酸番号３６２〜３９４、４０１〜４３３、４３４〜４６６、４７０〜５０２、５０４〜５３７、５３８〜５７０にＡｎｋｙｒｉｎ ｒｅｐｅａｔ（Ｐｆａｍにａｎｋとしてエントリーされるアミノ酸配列）を見出した。これは、タンパク質−タンパク質相互作用ドメインとしての機能をもち、Ａｎｋｙｒｉｎのアイソフォームには、Ｃ末端にＤｅａｔｈ ｄｏｍａｉｎをもつスプライスバリアントが存在することが知られている。
更に本タンパク質のアミノ酸番号２〜６２にＲｅｌ ｈｏｍｏｌｏｇｙ ｄｏｍａｉｎ ｒｅｇｉｏｎ（ＰｆａｍにＲＨＤとしてエントリーされるアミノ酸配列）の存在が認められた。このドメインは構造的に異なるドメインから成り、Ｎ末端側のドメインはｐ５３にも類似の構造が見出されるドメインであり、Ｃ末端側のドメインはＤＮＡに結合するイムノグロブリン様のフォールドをもつ。
このＲｅｌ ｈｏｍｏｌｏｇｙ ｄｏｍａｉｎについてはＰ１９８３８のアミノ酸番号４１〜２４２にも存在するが、比較すると本タンパク質のドメインのほうが短く、Ｒｅｌ ｈｏｍｏｌｏｇｙ ｄｏｍａｉｎのＮ末端側が欠損しているのがわかる。
ＮＦκＢは通常細胞質でＩκＢと複合体を形成して不活性だが、炎症などにより生じた活性酸素によりＩκＢが遊離し、ＮＦκＢは核に移行して遺伝子の転写を誘導する。また細胞外からのＴＮＦやＩＬ−１からの刺激はプロテインキナーゼカスケードによってＮＦκＢ・ＩκＢ複合体に伝えられ、複合体をリン酸化して活性化することが知られている。
本タンパク質はその配列の特徴より、以上のＮｕｃｌｅａｒ ｆａｃｔｏｒ ＮＦκＢのパスウェイにおいてＰ１９８３８とは異なる転写活性や機能を示す可能性が考えられる。
【０２６５】
本ＤＮＡおよびＭ５８６０３ｃＤＮＡのエクソン構造を明らかにするために、ｃＤＮＡ配列をゲノム配列へ写像するソフトウエアｓｉｍ４（Ｇｅｎｏｍｅ Ｒｅｓ． ８， ９６７−７４ （１９９８））を用い、ゲノム配列へのアライメントを行ったところ、本ＤＮＡ配列はヒト第４番染色体上の１７個のエクソンにマッピングされ、Ｍ５８６０３配列は２４個のエクソンにマッピングされることがわかった。本ＤＮＡの全てのエクソンはＭ５８６０３の第８から第２４エクソンとほぼ一致するが、Ｍ５８６０３の第１から第７エクソンは本ＤＮＡには存在しない。
これらの違いにより、ＤＮＡとの結合や相互作用、発現組織・発現量などに違いが生じる可能性がある。以上から、配列番号１９のタンパク質はＰ１９８３８、Ｎｕｃｌｅａｒ ｆａｃｔｏｒ ＮＦκＢ ｐ１０５ ｓｕｂｕｎｉｔ （Ｈｕｍａｎ）のスプライシングバリアントである。
【０２６６】
タンパク質の細胞内局在の予測プログラムであるＰＳＯＲＴ ＩＩ （Ｔｒｅｎｄｓ Ｂｉｏｃｈｅｍ． Ｓｃｉ． ２４， （１） ３４−６ （１９９９）） による解析を行ったところ、本タンパク質の細胞内での局在の確率は核３９．１％、ミトコンドリア２１．７％、細胞質１７．４％、ペルオキシソーム８．７％、空胞４．３％、分泌系小胞４．３％、小胞体４．３％であった。
ＮＦκＢは細胞質から核へ移行して種々の転写を活性化するため、この分布は本タンパク質がＮＦκＢのスプライシングバリアントである可能性を示唆するものである。
【０２６７】
真核生物ゲノムのアノテーション情報データベースＥｎｓｅｍｂｌ中のＤｉｓｅａｓｅ Ｂｒｏｗｓｅｒ （ｈｔｔｐ：／／ｗｗｗ．ｅｎｓｅｍｂｌ．ｏｒｇ／Ｈｏｍｏ＿ｓａｐｉｅｎｓ／ｄｉｓｅａｓｅｖｉｅｗ）によると、本ＤＮＡが写像された第４番染色体上の位置（ｑ２４）を包含するｑに乾癬、象牙質異形成症の原因遺伝子座がそれぞれ存在することが、更に位置は特定されていないが、新生児好中球減少症の原因遺伝子座も第４番染色体上に存在することがわかった。以上より、本ＤＮＡがこの疾患の原因遺伝子である可能性や、スプライシングバリアントを含む本ＤＮＡの変異がこれらの疾患の診断に応用できる可能性が予想された。
【０２６８】
本ＤＮＡの発現組織を検討するため、本ＤＮＡをクエリーとしてｄｂＥＳＴ（Ｎａｔｕｒｅ Ｇｅｎｅｔｉｃｓ ４， （４） ３３２−３ （１９９３））に対してＢＬＡＳＴ検索を行い、ｅ−ｖａｌｕｅ≦１０^−５０でヒットしたヒトＥＳＴで、５個以上のヒットが見られる組織を抽出したところ、ヒトの正常な胎盤・肺・腸・骨・免疫系・リンパ系・脾臓・胎児肝臓・胎児眼、癌化した免疫系・腸・肺であった。尚、本ＤＮＡは海馬由来のｃＤＮＡライブラリーからクローニングされた。またＰｉｇｍｅｎｔ Ｃｅｌｌ Ｒｅｓ．１４（６）：４５６−６５ （２００１） には、ｐ１０５はヒトの正常なメラニン細胞よりも転移性メラノーマ細胞中のほうが高度に発現していることが述べられている。
【０２６９】
本タンパク質はこれらの組織や細胞に特有の機能や疾患、例えば、肺癌・大腸癌・小腸癌・骨髄腫・白血病・リンパ腫・肝臓癌・メラノ−マなどの癌、アルツハイマー病・パーキンソン病・舞踏病などの神経変性疾患、記憶障害、肺炎・肝炎などの炎症性疾患、免疫系疾患、アレルギー疾患、脾臓疾患、不妊症、乾癬、象牙質異形成症、好中球減少症に関連する可能性が推測でき、これらの疾患の診断薬や治療薬のターゲットとしての利用が考えられる。
【０２７０】
（１０）ｃ−ｂｅａｓｔ２００１４４４（配列番号１０、２０）
ｃ−ｂｅａｓｔ２００１４４４（以下、これを「本ＤＮＡ」と称し、該ＤＮＡによりコードされるタンパク質を「本タンパク質」と称する）は、配列番号１０に示すように、２７０９塩基から成り、そのうち塩基番号４６９から１４３７までがオープンリーディングフレーム （終止コドンを含む） である。オープンリーディングフレームから予測されるアミノ酸配列は、３２２アミノ酸残基からなる （配列番号２０）。
【０２７１】
配列番号２０のアミノ酸配列についてＢＬＡＳＴを用いて相同性検索を行ったところ、ＮＲＤＢタンパク質データベース （ＳＷＩＳＳ−ＰＲＯＴ、ＰＩＲ、ＴＲＥＭＢＬＥ、ＧＥＮＰＥＰＴ、ＰＤＢから作成された重複のないアミノ酸配列のデータベース）のデータベース登録記号ＡＪ００９９３６、Ｎｕｃｌｅａｒ ｈｏｒｍｏｎｅ ｒｅｃｅｐｔｏｒ ＰＲＲ１−Ｃ（Ｈｕｍａｎ）がヒットした。
その内容としてＡＪ００９９３６は３７９アミノ酸から成り、そのアミノ酸配列中の５８〜３７９が、配列番号２０に記載のアミノ酸配列のアミノ酸番号１〜３２２と、ｅ−ｖａｌｕｅ：０．０、かつ３２２アミノ酸残基に亙り１００％の一致度でヒットした。
Ｐｒｅｇｎａｎｅ Ｘ ｒｅｃｅｐｔｏｒ（ＰＸＲ）はＲｅｔｉｎｏｉｄ Ｘｒｅｃｅｐｔｏｒ（ＲＸＲ）とヘテロ二量体を作り、薬物代謝に関わるＣＹＰ２、ＣＹＰ３などのチトクロームＰ４５０、その他生体異物の代謝に関わるトランスポーターの発現を誘導することが知られている。チトクロームＰ４５０は多くの医薬品の代謝のみならず薬効や副作用の原因となる薬物相互作用にも関与するため、ＰＸＲの転写活性の変化をみることは重要である。またＣＹＰ３Ａはステロイドホルモンや胆汁酸の代謝にも関わっており、ＰＸＲが発現を調節するチトクロームＰ４５０が生体異物の代謝に広く関わっていることがうかがえる。
ＢＬＡＳＴ検索のアライメントから本タンパク質はＡＪ００９９３６よりＮ末端が５７残基短い。また本ＤＮＡはアライメント上ではＡＪ００９９３６と５’末端が４６３塩基異なり、ＡＪ００９９３６は本ＤＮＡと２１７０塩基異なる。本ＤＮＡは塩基番号４６９が、Ｍ５８６０３は塩基番号１７７１が、それぞれ翻訳開始点であり、これらのことにより、アミノ酸配列のＮ末端の違いが生じている。
【０２７２】
配列番号２０のアミノ酸配列について、ＨＭＭＰＦＡＭによるタンパク質特徴検索を行ったところ、アミノ酸番号１３６〜３１７に示されるアミノ酸配列にＬｉｇａｎｄ−ｂｉｎｄｉｎｇ ｄｏｍａｉｎ ｏｆ ｎｕｃｌｅａｒ ｈｏｒｍｏｎｅの特徴を示す配列（Ｐｆａｍにｈｏｒｍｏｎｅ＿ｒｅｃとしてエントリーされるアミノ酸配列）を見出した。このドメインは、核内受容体中でαへリックス構造の疎水性のポケットを形成して、ステロイドホルモンなどのリガンドと高親和的に結合することが知られており、この結合により標的遺伝子の転写活性が活性化または抑制され、発現量が調節される。
またＡＪ００９９３６からはこの他アミノ酸番号６２〜１３８に、Ｚｉｎｃ ｆｉｎｇｅｒ， Ｃ４ ｔｙｐｅ（ｔｗｏ ｄｏｍａｉｎｓ） （Ｐｆａｍにｚｆ−Ｃ４としてエントリーされるアミノ酸配列）も抽出された。このドメインは、保存されたシステインに富むＤＮＡ結合領域をもち、真核細胞のステロイドホルモン、活性型ビタミンＤ_３、レチノイン酸、甲状腺ホルモンなどの核内受容体ファミリーに見られる。
【０２７３】
タンパク質の機能の類似性によりドメイン構造やファミリーを分類したアミノ酸パターンのデータベースであり、機能的に重要な部位を検索可能なＰＲＯＳＩＴＥ（Ｎｕｃｌｅｉｃ Ａｃｉｄｓ Ｒｅｓ． ３０， （１） ２３５−８． （２００２））によれば、ＡＪ００９９３６のアミノ酸番号６４〜９１にはＳＴＥＲＯＩＤ＿ＦＩＮＧＥＲ ｒｅｇｉｏｎも存在する。この部分は核内のホルモン受容体のＤＮＡ結合領域である。
これら２つのドメインや領域は本タンパク質には存在しないため、本タンパク質はＡＪ００９９３６とは核内でのホルモンとの結合やＤＮＡへの結合の状態が異なったり、そのために転写活性も変化してＣＹＰ３の発現が抑制される可能性が考えられる。
【０２７４】
本ＤＮＡおよびＡＪ００９９３６ｃＤＮＡのエクソン構造を明らかにするために、ｃＤＮＡ配列をゲノム配列へ写像するソフトウエアｓｉｍ４（Ｇｅｎｏｍｅ Ｒｅｓ． ８， ９６７−７４ （１９９８））を用い、ゲノム配列へのアライメントを行ったところ、本ＤＮＡ配列はヒト第３番染色体上の６個のエクソンにマッピングされ、ＡＪ００９９３６配列は１０個のエクソンにマッピングされることがわかった。本ＤＮＡの全てのエクソンはＡＪ００９９３６の第４から第９エクソンとほぼ一致するが、ＡＪ００９９３６の第１から第３、第１０エクソンは本ＤＮＡには存在しない。
これらの違いにより、ＤＮＡとの結合や相互作用、発現組織・発現量などに違いが生じる可能性がある。以上から、配列番号２０のタンパク質はＡＪ００９９３６、Ｎｕｃｌｅａｒ ｈｏｒｍｏｎｅ ｒｅｃｅｐｔｏｒ ＰＲＲ１−Ｃ（Ｈｕｍａｎ）のスプライシングバリアントである。
【０２７５】
タンパク質の細胞内局在の予測プログラムであるＰＳＯＲＴ ＩＩ （Ｔｒｅｎｄｓ Ｂｉｏｃｈｅｍ． Ｓｃｉ． ２４， （１） ３４−６ （１９９９）） による解析を行ったところ、本タンパク質の細胞内での局在の確率は細胞質５６．５％、核３０．４％、ミトコンドリア８．７％、ゴルジ体４．３％であった。ＡＪ００９９３６の局在の確率は核８２．６％、細胞質８．７％、ミトコンドリア８．７％で、本タンパク質は細胞質への局在の確率が高い。よって本タンパク質はＡＪ００９９３６とは局在が異なる可能性がある。
【０２７６】
真核生物ゲノムのアノテーション情報データベースＥｎｓｅｍｂｌ中のＤｉｓｅａｓｅ Ｂｒｏｗｓｅｒ （ｈｔｔｐ：／／ｗｗｗ．ｅｎｓｅｍｂｌ．ｏｒｇ／Ｈｏｍｏ＿ｓａｐｉｅｎｓ／ｄｉｓｅａｓｅｖｉｅｗ）によると、本ＤＮＡが写像された第３番染色体上の位置（ｑ１３．３１）を包含するｑ１３−２１にＣｈｒｃｏｔ−Ｍａｒｉｅ−Ｔｏｏｔｈ病、ｑ１３に家族性副甲状腺機能低下症、家族性振戦、ｑに筋強直性ジストロフィーの原因遺伝子座がそれぞれ存在することがわかった。以上より、本ＤＮＡがこの疾患の原因遺伝子である可能性や、スプライシングバリアントを含む本ＤＮＡの変異がこれらの疾患の診断に応用できる可能性が予想された。
【０２７７】
本タンパク質を小麦胚芽無細胞タンパク質合成系を用いて発現させたところ、実施例４においては、ＮＦ−１Ａ１等のコンセンサス標的配列：ＣＴＧＴＧＧＧＧＴＴＴＧＧＣＡＣＧＧＧＧＣＣＡ（配列番号４１）、ＲＦ−Ｘ１の標的配列：ＧＧＴＡＡＣＡＴＡＧＣＡＡＣ（配列番号４２）、ＹＹ１の標的配列：ＣＧＧＣＣＡＴＣＴＴＧＧＣＴ（配列番号５５）、Ｓｎａｉｌの標的配列：ＣＡＣＣＴＧＴＴＴＴＣＡ（配列番号５７）、Ｅｖｉ−１の標的配列：ＡＧＡＴＡＡＧＡＴＡＡ（配列番号５９）、Ｉｋａｒｏｓ等のコンセンサス標的配列：ＴＴＧＧＧＡＧＧ（配列番号６０）、ＨＮＦ−１Ａの標的配列：ＧＧＴＴＡＡＴＧＡＴＴＡＡＣＣＡＣ（配列番号６４）、Ｎｋｘ−２．２等のコンセンサス標的配列：ＴＴＡＡＧＴＧＧＴＴ（配列番号６５）、Ｏｃｔ−１Ａの等のコンセンサス標的配列：ＡＴＧＣＡＡＡＴ（配列番号６６）、Ｏｃｔ−２等のコンセンサス標的配列：ＴＡＴＴＴＧＣＡＴ（配列番号６７）、Ｐａｘ−３等のコンセンサス標的配列：ＣＧＴＣＡＣＧＣＴＴＧＡ（配列番号６８）、Ｐａｘ−１の標的配列：ＣＣＧＴＴＣＣＧＣＴＣＴＡＧＡＴＡＴ（配列番号６９）、ＮＦ−ＡＴｃ等のコンセンサス標的配列：ＡＧＧＡＡＡＡ（配列番号７５）、ＭＥＦ−２Ａの標的配列：ＣＴＣＴＡＡＡＡＡＴＡ（配列番号７９）、ＳＲＦの標的配列：ＣＣＡＴＡＴＡＴＧＧＡＣＡＴ（配列番号８０）およびＥ２の標的配列：ＡＡＣＣＡＡＡＡＡＣＧＧＴＡＡ（配列番号８１）に親和性を示した。このことから本タンパク質はＤＮＡ結合能を有し、造血細胞や肝細胞の増殖、免疫系細胞の発生・分化、脂質代謝、筋肉形成などの異常に起因する疾患、例えば生活習慣病などの疾患との関連が考えられる。また、実施例５によって、ＰＰＡＲが有する作用をやや強く抑制する活性を有し、内分泌・糖・脂質代謝異常等による糖尿病や肥満等の生活習慣病、血管機能異常等による循環器系疾患、炎症、癌等に関連することが推測された。また、ＮＦκＢが有する作用を抑制する活性を有し、関節リウマチや変形性関節炎などの免疫性疾患、炎症性疾患、癌等に関連することが推測された。
【０２７８】
本ＤＮＡの発現組織を検討するため、本ＤＮＡをクエリーとしてｄｂＥＳＴ（Ｎａｔｕｒｅ Ｇｅｎｅｔｉｃｓ ４， （４） ３３２−３ （１９９３））に対してＢＬＡＳＴ検索を行い、ｅ−ｖａｌｕｅ≦１０^−０４でヒットしたヒトＥＳＴで、ヒットが存在する組織を抽出したところ、ヒトの正常な胎児肝臓・胎児脾臓・肝臓・胃・腸、癌化した免疫系であった。尚、本ＤＮＡは乳房由来のｃＤＮＡライブラリーからクローニングされた。
【０２７９】
本タンパク質はこれらの組織や細胞に特有の機能や疾患、例えば、肝臓癌・胃癌・大腸癌・小腸癌・白血病・リンパ腫などの癌、変形性関節炎などの炎症性疾患、関節リウマチなどの免疫系疾患、アレルギー疾患、糖尿病、肥満、循環器系疾患、脾臓疾患、不妊症、家族性副甲状腺機能低下症、家族性振戦、筋強直性ジストロフィーに関連する可能性が推測でき、これらの疾患の診断薬や治療薬のターゲットとしての利用が考えられる。
【０２８０】
【発明の効果】
本発明のタンパク質およびそれをコードするＤＮＡは転写調節活性等を有することから、該タンパク質あるいはそれをコードするＤＮＡを用いて該活性を調節する物質をスクリーニングすることができ、該タンパク質が関連する疾患等に作用し得る医薬の開発に有用である。
【０２８１】
【配列表】

[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a novel protein, a DNA encoding the protein, a full-length cDNA encoding the protein, a recombinant vector having the DNA, an oligonucleotide consisting of a partial sequence of the DNA, and a gene-transferred cell into which the DNA has been introduced , And an antibody that specifically binds to the protein.
[0002]
[Prior art]
Currently, the elucidation and analysis of genome sequences of various organisms are underway on a global level. The entire genome sequence has already been determined in about hundreds of prokaryotic microorganisms, lower eukaryotic budding yeast, and nematodes that are multicellular eukaryotes. The human genome, which is said to have 3 billion base pairs, was published in February 2001 as a draft of its base sequence. In April 2003, the complete sequence was decoded and published. The purpose of elucidating the genome sequence is to understand the complex life phenomenon as a network of interactions and functions of all genes or between genes, proteins, cells and even individuals. Elucidating biological phenomena from genome information of various species is not only important as a research subject in the academic field, but also how to develop the research results obtained there for industrial application In that sense, its social significance is also great.
[0003]
However, simply determining the genome sequence does not reveal the functions of all genes. For example, in yeast, only about half of the approximately 6,000 genes deduced from the genome sequence could estimate their functions. On the other hand, it is said that there are about 100,000 kinds of proteins in humans. Therefore, establishment of a “high-throughput gene function analysis system” for rapidly and efficiently elucidating the functions of a huge amount of new genes revealed from genome sequences is strongly desired.
[0004]
In eukaryotic genome sequences, a single gene is often divided into multiple exons by introns. Therefore, there are many problems in accurately predicting the structure of the protein encoded there from only the genomic sequence information. On the other hand, in cDNA prepared from mRNA from which introns have been removed, the information on the amino acid sequence of the protein can be obtained as a single piece of continuous sequence information, so that the primary structure can be easily clarified. In human cDNA research, more than 5 million EST (Expressed Sequence Tag) data has been published in public databases.
[0005]
Such information is utilized from various angles such as elucidation of human gene structure, prediction of exon region in genome sequence, and estimation of its expression profile. However, since most of these human EST information is concentrated in the vicinity of the 3 ′ end of the cDNA, the information in the vicinity of the 5 ′ end of the mRNA is particularly insufficient. In addition, more than 40,000 cDNAs have been revealed as a result of analysis conducted at research institutions around the world (Helix Research Institute, Kazusa DNA Research Institute, University of Tokyo Medical Research Institute, German Cancer Research Center, MGC Project, etc.). It seems that it covers the majority of gene loci, which are said to be several thousand in number, but the percentage of cDNA obtained as a full-length clone is about 80%, duplication and splicing Considering that the variants are included, it is considered that there are many cDNAs that have not yet been obtained.
[0006]
If full-length cDNA can be obtained, the mRNA transcription start point on the genome sequence can be estimated from the 5 ′ end sequence, and the factors involved in the stability of mRNA contained in the sequence and the expression control at the translation stage can be analyzed. Is possible. In addition, since atg, which is the translation start point, is included on the 5 'side, the protein can be translated into the correct frame. Therefore, by applying an appropriate gene expression system, it is possible to produce a large amount of the protein encoded by the cDNA or to analyze the biological activity by expressing the protein. Thus, analysis of full-length cDNA provides important information that complements genomic sequence analysis. In addition, a full-length cDNA clone that can be expressed is extremely important for empirical analysis of the function of the gene and for application to industrial applications.
[0007]
In addition, even in the case of a protein encoded by the same genome, when the mRNA encoding the same is transcribed, an isomer (hereinafter referred to as “splicing”) in which a part of the exons in the genome is inserted and deleted and combined. Sometimes referred to as "variant mRNA"). In fact, a plurality of types of similar proteins (hereinafter sometimes referred to as “splicing variants”) produced by translating these mRNAs have been confirmed in vivo. Splicing variants are expressed in a tissue-specific, developmental stage-specific or disease-specific manner and are considered to have different functions.
[0008]
For example, 10 types of exon-deleted splice variant mRNAs have been identified from various human tissues in Estrogen receptor β. Nine of them are expressed in normal breast tissue, and among them, the expression of mRNA lacking exons of Nos. 5 and 6 has been shown to be significantly reduced in most cancer tissues. Yes. In addition, it has been clarified that mRNA lacking the exon No. 5 significantly changes its expression level according to the degree of cancer progression (see, for example, Non-Patent Document 1). Thus, the expression level of the splice variant may show a significant correlation with the disease state.
[0009]
Such splicing variant mRNA or cDNA is also a clone that is difficult to obtain from a conventional cDNA library or EST and is highly likely to be obtained from a full-length cDNA library including a transcription start site.
[0010]
In particular, a transcription factor is a protein having affinity for DNA involved in a step of synthesizing mRNA from DNA called transcription reaction or gene expression, and a large number of factors are known. The transcription reaction is an important reaction that regulates the synthesis amount of a protein that is a gene product, and controls the amount of protein involved in cell function and structure. This transcription reaction is caused by a group of transcription factors. For example, RNA polymerase binds to DNA having a specific base sequence of a transcription control region called a promoter composed of a TATA box and a transcription initiation site, and incorporates RNA polymerase. In this way, a transcription complex in which various transcription factors are assembled is formed and mRNA synthesis is started. A transcription reaction that depends on the promoter has no gene specificity and is called basic transcription, and a transcription factor that forms a transcription complex with RNA polymerase and RNA polymerase and works near the RNA polymerase is called a basic transcription factor. . Apart from this basic transcription, cells have a mechanism for raising and lowering the level of basic transcription and regulating time-specific or tissue-specific expression in response to changes in the physiological environment and external stimuli. Such a DNA having a specific base sequence of a transcriptional regulatory region that controls the basic transcription is also called an enhancer or a silencer, and usually exists upstream of the promoter and binds to this region (gene-specific transcription). Regulators, also called gene expression regulators or gene expression regulator proteins) interact with basic transcription factors either directly or through another transcriptional regulatory cofactor (also called mediator, cofactor or mediator) Regulation is performed (hereinafter, such basic transcriptional regulatory activity is referred to as “transcriptional regulatory activity”, and transcriptional regulatory factors and transcriptional regulatory cofactors may be collectively referred to as “transcriptional regulatory factors”). That is, transcription factors are classified into two categories: basic transcription factors and transcription regulatory factors. Basic transcription factors are not gene-specific and few in number, but a great many transcriptional regulators are known, and the number is expected to reach the order of 1000 when similar factors and families are included. The binding sites are also diverse.
[0011]
Modulating transcriptional responses in response to intracellular or intercellular stimuli is often desirable, for example, when such regulation is necessary for the development of an organism or for the survival of an organism against changes in the biological environment. The However, diseases can be caused by inappropriate activation or termination of transcription reactions. For example, evidence has been reported suggesting that estrogen-mediated gene expression is involved in several types of breast or ovarian cancer. In addition, various transcriptional regulatory factor genes have been discovered from the chromosomal translocation site associated with leukemia, and many of them encode fusion chimeric molecules of transcriptional regulatory factors. It has been suggested that the abnormality may be deeply involved in leukemia (see, for example, Non-Patent Document 2). Thus, among the transcriptional regulatory factors, there are transcriptional regulatory factors that regulate the transcription of various genes involved in diseases (hereinafter sometimes referred to as “disease-related transcriptional regulatory factors”). For example, PPAR, p53, NFκB, AP-1, HIF-1, CREB and the like are known as disease-related transcriptional regulatory factors.
[0012]
In addition, protein kinases are known to be involved in the control of various biological phenomena by regulating intracellular signal transduction systems (phosphorylation cascades) through protein phosphorylation, and their relationship with diseases has been elucidated. Although many genes are present (see, for example, Non-Patent Document 3), it is also known that a group of transcriptional regulatory factors that are phosphorylated and activated in this phosphorylation cascade are involved. It has been suggested that factors may be involved in diseases as well as protein kinases (see, for example, Non-Patent Document 4).
[0013]
However, it is estimated that there are about 1,000 different transcriptional regulators in the human body, and many transcriptional regulator genes are still left uncloned. Therefore, it is significant to provide a novel full-length cDNA of a transcriptional regulatory factor containing a splicing variant that has not been separated in humans. In addition, transcriptional regulatory factors can be expected to be useful as target molecules for treatment and to the proteins themselves as pharmaceuticals. Therefore, it is significant to clarify the full length of cDNA encoding these proteins.
[0014]
[Non-Patent Document 1] Poola I et al. , J. et al. Steroid Biochem. Mol. Biol. 82: 169-179 (2002).
[Non Patent Literature 2] Look, A .; T.A. , Science, 278: 1059-1064 (1997).
[Non-Patent Document 3] Hunter, T .; Cell, 50: 823-829 (1987).
[Non-Patent Document 4] Ninomiya-Tsuji, J. et al. et al. , Nature 398: 252-256 (1999)
[0015]
[Problems to be solved by the invention]
The present invention analyzes the nucleotide sequence of a cDNA clone contained in a full-length cDNA library, and among these, for a cDNA having a novel sequence as a full length including a splicing variant, the physiological activity of the protein encoded by this is identified. The purpose is to propose a protein based on activity and a method of using the DNA encoding the protein.
[0016]
[Means for Solving the Problems]
The inventors have used the oligo-capping method (Maruyama, K., et al., Gene, 138: 171-174 (1994); Suzuki, Y. et al., Gene, 200: 149-156 (1997)). When a database was searched based on the homology of the base sequence of the cDNA clone for a cDNA having a novel sequence containing a splicing variant obtained by using the sequence, a sequence specific to a protein having transcriptional regulatory activity was found in the sequence, The proteins encoded by these cDNAs were identified as transcriptional regulators. The present invention has been accomplished based on these findings.
[0017]
That is, according to the present invention, the following inventions 1 to 15 are provided.
1. The following protein (a) or (b):
(A) a protein comprising the amino acid sequence of any one of SEQ ID NOS: 11 to 20,
(B) A protein comprising an amino acid sequence in which one or several amino acids are deleted, substituted and / or added in the amino acid sequence of any one of SEQ ID NOs: 11 to 20, and having transcriptional regulatory activity.
2. A DNA encoding the protein according to item 1.
3. A full-length cDNA encoding the protein according to item 1.
4). DNA of any of the following (a) or (b);
(A) DNA having the base sequence according to any one of SEQ ID NOs: 1 to 10,
(B) a protein having a base sequence in which one or several bases are deleted, substituted and / or added in the base sequence described in any of SEQ ID NOs: 1 to 10 and having transcriptional regulatory activity DNA to do.
5. 5. A recombinant vector comprising the DNA according to any one of 2 to 4 above.
6). 7. A gene-transferred cell into which the DNA according to any one of 2 to 4 or the recombinant vector according to 5 above is introduced, or an individual comprising the cell.
7). The protein according to item 1, which is produced by the cell according to item 6.
8). A sense oligonucleotide having the same sequence as 5 to 100 consecutive bases in the base sequence of the DNA according to any one of 2 to 4 above, an antisense oligonucleotide having a sequence complementary to the sense oligonucleotide, and the An oligonucleotide selected from the group consisting of oligonucleotide derivatives of sense or antisense oligonucleotides.
9. 8. An antibody or a partial fragment thereof that specifically binds to the protein according to item 1 or 7.
10. 10. The antibody according to 9 above, wherein the antibody is a monoclonal antibody.
11. 11. The antibody according to item 10 above, wherein the monoclonal antibody has an action of neutralizing the transcriptional regulatory activity of the protein according to item 1 or 7.
12 8. A screening method for a substance that regulates the activity of the protein, which comprises contacting the test substance with the protein according to item 1 or 7, and measuring a change in activity of the protein by the test substance.
13. 7. A screening method for a substance that regulates the expression of DNA, comprising contacting the test cell with the gene-transferred cell according to item 6 and detecting a change in the expression level of the DNA introduced into the cell.
14 Preserving at least one or more amino acid sequence information selected from the amino acid sequence of the protein according to item 1 and / or at least one or more nucleotide sequence information selected from the DNA nucleotide sequence according to any one of items 2 to 4 above Computer readable recording medium.
15. A carrier on which the protein according to item 1 and / or the DNA according to any one of items 2 to 4 are bound.
[0018]
DETAILED DESCRIPTION OF THE INVENTION
Hereinafter, the present invention will be described in more detail.
(1) Acquisition of full-length cDNA and analysis of nucleotide sequence
The DNA of the present invention is a protein comprising the amino acid sequence described in any of SEQ ID NOs: 11 to 20, or one or several amino acids in the amino acid sequence described in SEQ ID NOs: 11 to 20 (here, several A protein having an amino acid sequence containing substitution, deletion and / or addition of amino acid residues and having transcriptional regulatory activity (for example, 5 or less, preferably 3 or less, more preferably 2 or less) Any code can be used as long as it can be coded. Specifically, it may be only the translation region encoding the amino acid sequence, or may include the full length of the cDNA.
[0019]
Specifically, examples of the DNA containing the full length of cDNA include DNA comprising the base sequence described in any one of SEQ ID NOs: 1 to 10. The translation region includes base numbers 227 to 2245 of SEQ ID NO: 1, base numbers 214 to 1947 of SEQ ID NO: 2, base numbers 281 to 2284 of SEQ ID NO: 3, base numbers 138 to 1019 of SEQ ID NO: 4, and SEQ ID NO: 5 base numbers 1582 to 2685, base numbers 352 to 2271 of SEQ ID NO: 6, base numbers 2097 to 3881 of SEQ ID NO: 7, base numbers 1038 to 1937 of SEQ ID NO: 8, base numbers 1140 to 3506 of SEQ ID NO: 9, The thing which has a sequence shown by 10 base numbers 469-1437 is mentioned. Furthermore, the DNA of the present invention includes not only the full length of the cDNA described above, but also those containing the translation region and the 3 ′ and / or 5 ′ end adjacent to the translation region at least necessary for expression. .
[0020]
The DNA of the present invention may be obtained by any method as long as it can be obtained. Specifically, for example, it can be obtained by the method described below. First, mRNA is prepared from human tissue or cultured cells by a commonly used method known per se. Next, cDNA is obtained by the oligo cap method (Maruyama, K., et al., Gene, 138: 171-174 (1994)) using this mRNA as a template. Specifically, 5 'cap is removed from the obtained mRNA with acid pyrophosphatase, and then the synthetic oligonucleotide is ligated using RNA ligase to the exposed 5'-terminal phosphate group as a target. Here, for RNA molecules that do not have a cap structure at the 5 ′ end, in order to prevent the oligonucleotide from binding, the phosphate group present at the 5 ′ end in advance is not removed from the 5 ′ cap but at the 5 ′ end. It is effective to remove the phosphoric acid group using phosphatase having the activity of removing only the phosphate group. Using this RNA molecule as a template, reverse transcription is performed with reverse transcriptase using an oligo dT primer as a 3'-side primer, and then the RNA strand is decomposed and removed.
[0021]
Furthermore, using the obtained single-stranded DNA as a template, a polynucleotide having a sequence complementary to the synthetic oligonucleotide is a 5 ′ primer, and a primer specific to the 3 ′ end (such as oligo dT) is used for the polymerase chain reaction. By performing (PCR), a full-length cDNA library can be prepared. Here, the 5 'primer and the 3' primer are not complementary to the total length of the synthetic oligonucleotide and the reverse transcription primer, and it is preferable to use sequences shifted by 3 to 10b on the 3 'side. The primer chain length is usually 15 to 100 bases, preferably 15 to 30 bases. However, when the length of the cDNA to be amplified is long, the length is preferably 25 to 35 bases. and Accurate PCR (LA PCR: Kenji Hayashi, experimental medicine separate volume, latest technology of PCR, Yodosha; Cheng, S. et al., Nature 369: 684-685 (1994)) is preferably used.
[0022]
The cDNA thus obtained is cloned by inserting it into an appropriate cloning vector. As the vector used here, a protein expression vector which can introduce the obtained cDNA clone into a cell and express the protein encoded by the cDNA is preferably used. Specifically, for example, when the host is a mammalian cell or the like, pME18SFL3 (Genbank AB009864) or the like is preferable, and when E. coli is used, pET3, pET11 (Stratagene), pGEX (Amersham Pharmacia Biotech), etc. In the case of yeast, pESP-I expression vector (Stratagene) is used, and in the case of insect cells, BacPAK6 (Clontech) is used. When the host is an animal cell, examples include ZAP Express (Stratagene), pSVK3 (Amersham Pharmacia Biotech), and the like.
[0023]
The cDNA library thus obtained is subjected to base sequence analysis by a commonly used method known per se. The DNA of the present invention is analyzed for the nucleotide sequence of the 5 ′ end or 3 ′ end of the obtained cDNA, and this is analyzed by NCBI (National Center of Biotechnology Information; http://www.ncbi.nlm.nih.gov/). Databases such as Genbank, EMBL, DDBJ, dbEST, etc. operated by BLAST (Basic local alignment search tool; Altschul, SF, et al., J. Mol. Biol., 215, 403-410 (1990) ) Was used, and when a completely identical sequence was not found for the entire length, it was decided to be subjected to the following analysis as new.
[0024]
Examples of the DNA having such a full-length cDNA base sequence include those having the base sequences described in SEQ ID NOs: 1 to 10. The translation region includes base numbers 227 to 2245 of SEQ ID NO: 1, base numbers 214 to 1947 of SEQ ID NO: 2, base numbers 281 to 2284 of SEQ ID NO: 3, base numbers 138 to 1019 of SEQ ID NO: 4, and SEQ ID NO: 5 base numbers 1582 to 2685, base numbers 352 to 2271 of SEQ ID NO: 6, base numbers 2097 to 3881 of SEQ ID NO: 7, base numbers 1038 to 1937 of SEQ ID NO: 8, base numbers 1140 to 3506 of SEQ ID NO: 9, The thing which has a sequence shown by 10 base numbers 469-1437 is mentioned.
[0025]
A novel nucleotide sequence as a full length of the cDNA thus obtained is obtained by using BLAST homology search (homology search) and HMMER (sequence analysis method using hidden Markov model; Eddy, SR, Bioinformatics 14: 755-763 (1998). The function of the protein encoded by the base sequence can be estimated by performing a protein feature search (profile search: http://pfam.wustl.edu) or the like using HMMPFAM, which is one of the functional groups of ()).
[0026]
In the homology search by BLAST, the function of the clone to be analyzed can be estimated from various annotation information attached to hit sequences with sufficiently significant homology obtained as a result of the search. Here, a sufficiently significant hit sequence means that the identity between the functional domain portion of the registered sequence and the corresponding portion of the DNA of the present invention is e-value (the query sequence is accidentally found in the database). 10 as the expected value)^-4The following are shown, or 30% or more.
[0027]
For example, if many of the top functional domain sequences have been confirmed to function as a transcriptional regulator, the clone to be analyzed that is similar in sequence also has the same function, that is, transcriptional regulatory activity. The prediction that it will be.
[0028]
In HMMPFAM, analysis is performed by a method for identifying whether the amino acid sequence encoded by the base sequence to be analyzed has the characteristics of the amino acid sequence of an entry in a database in which protein profiles called Pfam are accumulated. Profiles are extracted from a series of proteins having the same characteristics, and even if the function cannot be clarified by comparison over the entire length of one sequence to one sequence, if the characteristic region is present in the sequence, this can be found and the function can be predicted. In this way, the cDNA predicted that the protein encoded by it has transcriptional regulatory activity can be confirmed for its transcriptional regulatory activity by biochemical experiments described below.
[0029]
Specific examples of clones that are novel as full-length cDNAs described above include those having the base sequence shown in any one of SEQ ID NOs: 1 to 10. Moreover, the amino acid sequences encoded by these base sequences include those shown in any of SEQ ID NOS: 11 to 20.
[0030]
The DNA of the present invention thus obtained, whose base sequence is determined, and whose function is estimated has the base sequence described in any of SEQ ID NOs: 1 to 10 above, or the base sequence shown above as its translation region Not only those but also one or several bases in these base sequences (here, “several” means, for example, 15 or less, preferably 9 or less, more preferably 6 or less) are deleted. In addition, DNA encoding a protein having a substituted and / or added base sequence and having transcriptional regulatory activity is also included. As described above, these DNAs comprise an amino acid sequence in which one or several amino acid sequences are deleted, substituted and / or added in the amino acid sequence of SEQ ID NO: 11 to 20, and have transcriptional regulatory activity. Those that code for proteins are included.
[0031]
Furthermore, the DNA of the present invention may be obtained by the above-described method or may be synthesized. The substitution of the DNA base sequence can be easily performed with a commercially available kit such as a site-directed mutagenesis kit (Takara Shuzo) or a quick change site-directed mutagenesis kit (Stratagene).
[0032]
(2) Protein encoded by novel cDNA
The translation region of the protein encoded by the DNA of the present invention is, for example, converted into an amino acid using three types of reading frames for the base sequence of the DNA, and the range encoding the longest polypeptide is translated into the protein of the present invention. The amino acid sequence can be deduced as a region. Examples of such an amino acid sequence include those described in any of SEQ ID NOs: 11 to 20. In addition, the protein of the present invention is not limited to the amino acid sequence described above, and is composed of an amino acid sequence in which one or several amino acids are substituted, deleted, and / or added in the amino acid sequence, and has transcriptional regulatory activity. The thing which has is included.
[0033]
As a method for obtaining the protein of the present invention, the method of transcription / translation of the DNA of the present invention described in (1) by an appropriate method is preferably used. Specifically, an appropriate expression vector or a recombinant vector inserted into an appropriate vector together with an appropriate promoter is prepared, and an appropriate host microorganism is transformed with this recombinant vector or introduced into an appropriate cultured cell. And can be obtained by purifying it.
[0034]
In addition, the protein of the present invention includes a protein added with a tag inserted into a vector designed so that an appropriate tag is fused to the N-terminus or C-terminus. Specific examples of the tag include glutathione-S-transferase, polyhistidine, and Flag.
[0035]
The protein produced by the transformant can be modified by incorporating amino acids substituted or modified with heavy atoms during protein synthesis. In addition, the protein can be made into a modified protein by applying an appropriate protein-modifying enzyme before or after purification, by arbitrarily modifying the protein, or by partially removing the polypeptide. For example, terminal modification such as N-terminal acetylation, C-terminal amidation, glycosylation, lipid addition, acylation, methylation, sulfonation, carboxylation, hydroxylation, phosphorylation, ADP-ribosylation, etc. It is not necessarily limited to these. These modified proteins are also included in the scope of the present invention as long as they have the transcriptional regulatory activity described above.
[0036]
In addition, the protein produced by the transformant may be modified as desired by applying an appropriate protein-modifying enzyme before or after purification, or by partially removing the polypeptide. Can do. These modified proteins are also included in the scope of the present invention as long as they have the transcriptional regulatory activity described above.
[0037]
When the protein of the present invention is produced, the vector used for the production of the recombinant vector containing the DNA of the present invention is not particularly limited as long as the DNA is expressed in the transformant. Plasmid vector, phage Any of vectors may be used. Of these, usually a commercially available protein expression vector into which an expression control region DNA such as a promoter suitable for a host into which the DNA is introduced has already been inserted is used. Specific examples of such a protein expression vector include pET3, pET11 (manufactured by Stratagene), pGEX (manufactured by Amersham Pharmacia Biotech) when the host is Escherichia coli, and pESP when yeast is used. -I expression vector (Stratagene) and the like, and in the case of insect cells, BacPAK6 (Clontech) and the like are used. When the host is an animal cell, ZAP Express (Stratagene) or pSVK3 (Amersham Pharmacia Biotech) can be used. When the host is a mammalian cell, pME18SFL3 (Genbank AB009864) is preferred.
[0038]
When using a vector into which an expression control region is not inserted, it is necessary to insert at least a promoter as the expression control region. Here, the promoter possessed by the host microorganism or the cultured cell can be used as the promoter, but is not limited to this. Specifically, for example, when the host is Escherichia coli, T3, T7, tac, lac A promoter or the like can be used, and in the case of yeast, nmt1 promoter, Gal1 promoter and the like can be used. In the case of insect cells, a polyhedrin promoter or the like can be used. When the host is an animal cell, SV40 promoter, CMV promoter, etc. are preferably used.
[0039]
In addition, when a host capable of functioning a promoter derived from a mammal is used, a promoter specific to the gene of the present invention can also be used. Insertion of the DNA of the present invention into these vectors may be carried out by linking the DNA or a DNA fragment containing the DNA with the amino acid sequence of the protein encoded by the gene DNA downstream of the promoter in the vector.
[0040]
The recombinant vector thus prepared can be transformed into a host to be described later by a method known per se to prepare a DNA transductant. Specific examples of methods for introducing the vector into the host include heat shock (J. Mol. Biol., 53: 154 (1970)), calcium phosphate method (Science, 221: 551 (1983)), DEAE dextran method ( Science, 215: 166 (1982)), in vitro packaging method (Proc. Natl. Acad. Sci. USA, 72: 581 (1975)), viral vector method (Cell, 37: 1053 (1984)), and electric pulse (Chu. Et al., Nuc. Acids Res., 15: 1331 (1987)) and the like.
[0041]
The host for preparing the DNA transductant is not particularly limited as long as the DNA of the present invention is expressed in the body. For example, Escherichia coli, yeast, baculovirus (arthropod polyhedrosis virus) -insect cell, or Examples include animal cells. Specifically, BL21, XL-2Blue (manufactured by Stratagene) etc. are used in Escherichia coli, SP-Q01 (manufactured by Stratagene), etc. in yeast, and AcNPV (J. Biol. Chem., 263: 7406 (1988) in baculovirus. )) And its host, Sf-9 cells (J. Biol. Chem., 263: 7406 (1988)) and the like. Examples of animal cells include mouse fibroblast C127 (J. Viol., 26: 291 (1978)) and Chinese hamster ovary cell CHO cells (Proc. Natl. Acad. Sci. USA, 77: 4216 (1980)). Among them, from the expression level and ease of screening, COS-7 cells derived from African green monkey kidney (ATCC CRL1651: American type culture collection preservation cells), HEK293 cells derived from human fetal kidney (ATCC CRL1573) or human cervical cancer HeLa cells (ATCC CCL-2) are used.
[0042]
In addition to the expression method using the protein expression vector as described above, a homologous recombination technique (Vertes, A. A. et al., Which directly inserts the DNA fragment of the present invention linked with a promoter into the chromosome of the host microorganism. Biosci. Biotechnol. Biochem., 57: 2036 (1993)), or transposon and insertion sequence (Vertes, AA et al., Molecular Microbiol., 11: 739 (1994)) etc. It can also be produced.
[0043]
The culture obtained above is obtained by collecting cells or cells by a method such as centrifugation, suspending the cells or cells in an appropriate buffer, and using a suitable known per se method such as ultrasound, lysozyme, and / or freeze-thawing. After disruption by the method, a crude protein purification solution can be obtained by centrifugation, filtration or the like, and further purified by combining appropriate purification methods.
[0044]
Thus, the protein of the present invention is obtained. In addition to the expression method using the protein expression recombinant vector described above, the protein of the present invention is obtained by inducing protein expression by subjecting the DNA of the present invention obtained in (1) above to a cell-free transcription translation system. be able to. The cell-free transcription / translation system used in the present invention is a system including all elements necessary for transcription from DNA to mRNA and translation from mRNA to protein, and the DNA is encoded by adding DNA thereto. It refers to any system in which the protein being synthesized is synthesized. Specific examples of the cell-free transcription / translation system include transcription / translation systems prepared on the basis of extracts from eukaryotic cells and bacterial cells, or a part thereof. Specific examples of the cell-free protein synthesis system include known cell extracts such as Escherichia coli, plant seed germs, and rabbit reticulocytes. These may be commercially available, or a method known per se, specifically, Escherichia coli extract can be obtained from Pratt, J. et al. M.M. et al. Transcription and Translation, Hames, 179-209, B .; D. & Higgins, S .; J. et al. , Eds, IRL Press, Oxford (1984). Examples of commercially available cell-free protein synthesis systems or cell extracts include those derived from E. coli. E. coli S30 extract system (manufactured by Promega) and RTS500 Rapid Translation System (manufactured by Roche) and the like are those derived from rabbit reticulocytes.^TM(Manufactured by TOYOBO).
[0045]
Separation and purification of the protein of the present invention from the transcription-translation product of the obtained cell-free transcription / translation system can be performed by a commonly used method known per se. Specifically, for example, a DNA region encoding an epitope peptide, polyhistidine peptide, glutathione-S-transferase (GST), maltose binding protein, etc. is introduced into the DNA to be transcribed and translated as described above. The protein can be purified by utilizing affinity with a substance having affinity for the protein.
[0046]
Expression of the target protein is detected by SDS-polyacrylamide gel electrophoresis or the like, and stained with Coomassie Brilliant Blue (manufactured by Sigma) or detected by an antibody that specifically binds to the protein of the present invention described later. It can be confirmed by the method to do. In general, it is known that the expressed protein is cleaved (processing) by a proteolytic enzyme present in the living body. Of course, even if the protein of the present invention is a partial fragment of an amino acid sequence that has been cleaved, it may be included in the protein of the present invention as long as it has transcriptional regulatory activity.
[0047]
(3) Confirmation of the activity of the protein of the present invention
It can be confirmed that the protein of the present invention is produced as a recombinant protein as described in (2) above, and has the activity estimated in (1) by analyzing it. Furthermore, it can also analyze by the combination with the antibody etc. as described in the following (4).
[0048]
Examples of the transcriptional regulatory activity of the protein of the present invention include a function of regulating gene expression by changing the DNA structure. It can be confirmed that the protein of the present invention has transcriptional regulatory activity using a conventional method known per se. For example, as one method for analyzing transcriptional regulatory activity, a method for analyzing an interaction with another protein or DNA is known. For the analysis of the interaction, a conventional method known per se can be used. Specific examples include a yeast two-hybrid method, a fluorescence depolarization method, a surface plasmon method, a phage display method, and a ribosomal display method. Can be mentioned.
[0049]
More specifically, in the case of DNA binding activity which is one of the interaction activities with the DNA of the protein of the present invention, for example, an appropriate double-stranded DNA is contacted with the recombinant protein, and the recombinant protein This can be confirmed by measuring the binding property to the DNA strand. Specific methods include the methods described below.
[0050]
As the reaction solution, neutral to weakly basic buffer solution containing 60 mM potassium chloride, 1 mM dithiothreitol, 10% glycerol, 1 μg poly (dI-dC), for example, 20 mM Tris-HCl or HEPES buffer (pH 7-8) The reaction is started by adding double-stranded DNA of an appropriate length and the recombinant protein of the present invention. After the reaction under a certain condition, the double-stranded DNA binding activity of the protein is judged by detecting the conjugate of the recombinant protein and DNA.
[0051]
The binding between the recombinant protein and DNA can be detected using a method for detecting the size of the molecule, a method for detecting the difference in charge, a method for measuring the affinity between the two, and the like.
Examples of the method for detecting the size of the molecule include measurement by molecular sieve chromatography. The above reaction solution is added to a column for molecular sieve chromatography equilibrated with physiological saline, 0.1 M phosphate buffer (pH 7.4) or 0.1 M HEPES (pH 7.5), and developed in the same solution. To do. Compared with the elution position of each of the recombinant protein and DNA, the binding substance of both is eluted on the higher molecular weight side.
[0052]
Examples of the method for detecting the difference in charge include electrophoresis methods including ion exchange chromatography, gel electrophoresis, and capillary electrophoresis. In gel electrophoresis, a difference in charge and a difference in molecular size appear as a difference in mobility. The reaction mixture is electrophoresed in a polyacrylamide gel, and the gel after the electrophoresis is stained with silver, Coomassie brilliant blue, or fluorescently stained, and the conjugate has a different mobility compared to the recombinant protein alone. Confirmed as a band. In the case of these two methods, it is possible to detect with higher sensitivity as a difference in the elution position and mobility of DNA by fluorescently labeling one end of DNA.
[0053]
Examples of methods for measuring the affinity between the two include affinity chromatography and surface plasmon resonance. In these methods, DNA is immobilized on a carrier, a recombinant protein is brought into contact therewith, and the amount of binding is measured to determine the strength of activity. In affinity chromatography, the bound recombinant protein is eluted with a high-concentration salt solution, a denaturing agent, free DNA, etc., and the amount of the eluted protein is measured. In the surface plasmon resonance method, the amount of protein bound to immobilized DNA can be measured by surface plasmon resonance, and the strength of affinity can be measured at the bound protein concentration.
[0054]
In the case of a transcriptional regulatory factor, DNA of a gene transcriptional regulatory region such as a promoter or enhancer that is recognized and targeted by a known transcriptional regulatory factor (hereinafter sometimes referred to as “target transcriptional regulatory region”) or a portion thereof A method for analyzing biological activity by measuring the binding ability to fragments is known (Japanese Patent Application Laid-Open No. 2001-314190). The target transcriptional regulatory region DNA or a partial fragment thereof used for the analysis includes not only those having a known target transcriptional regulatory region base sequence (hereinafter sometimes referred to as “target sequence”) but also known targets. The base sequence of the transcriptional regulatory region was designed by database analysis and classification, and the common base sequence of the target transcriptional regulatory region recognized by transcriptional regulatory factors in common (hereinafter this may be referred to as the “consensus target sequence”) ) May be used.
[0055]
Known transcriptional regulatory factors include, for example, v-jun, c-jun, junB, junD, dJRA, c-fos, fosB1, fosB2, Fra-1, LRF-1, v-maf, mafG, NF-E2 p45. , ANF-E2, fNF-E2, Nrf short form, GCN4, yAP-1, CREB-2, ATF-3, CRE-BP1, CRE-BP3, ATF-a, CREB-341, CREB-327, CREM, dCREB2 , DCREB2-b, dCREB2-c, dCREB2-d, dCREB2-q, dCREB2-r, dCREB2-s, C / EBPα, C / EBPβ, p34C / EBPβ, CHOP-10, VBP, Hlf, CPRF-2, EmBP -1b, EmBP-1b, GBF1, GBF2, GBF3, CP F-1, TAF-1, HBP-1a, GBF9, GBF1, GBF12, CPRF-3, TGA1a, TGA1b, O2, STE4, OPI1, E2A, E47, ITF-2 / SEF2-1B, SEF-1A, MyoD, p42Tal-1, HEN-1, AhR, Arnt, USF, NF-1A1, NF-1A1.1, NF-1A6, NF-1B1, NF-1B1, NF-1B2, NF-1C2 / CTF-2, CTF- 4, CTF-6, RF-X1, AP2αA / AP-2α1, AP2α2, AP2α3, AP2α4, AP2αB, AP2β, AP2γ, GR, AR, ER, RXR-α, PPARα, PPARγ, COUP-TF1, HNF-4α1, HNF-4α2, CF1, GATA-1, GATA-2, GATA-3, GATA-4, AREA / NIT-2, Sp1, YY1, Egr-1, Egr-2, Egr-3, Snail, CF2-II, Evi-1, Ikaros, MZF-1, Tramtrack69K, HOX9, CDP, HNF-1A, Nkx-2 .2, Nkx-2.5, TTF-1, Oct-1A, Oct-1B, Oct-1C, Oct-2, Oct-2.1 / Oct-2B, Pax-3, Pax-6, Pax-1 , HSF1 (short), HSF2, dHSF, fungalHSF, c-Myb, A-Myb, v-Myb, P (long), P (short), C1 (long), C1 (short), c-Ets-1_p54, Ets-1_δiV / VII, Ets-2, Elk-1, SAP-1, SAP-1b, Erg-1, p55erg, Fli-1b, 4TF1-60 / GABP-α, E74A, IRF-1, IRF-2, p50, NF-ATc, NF-Atp, p91, p84, STAT2, STAT3, STAT4, STAT5A, STAT5B, STAT6, p53, MEF-2A, SRF, E2, TBP, SRY, Sox-5, Sox-9, mat-Mc, CP1A, CP1B, CBF-C, AML1a, etc. are known, and the nucleotide sequences of the target transcriptional regulatory regions of these transcriptional regulatory factors (Takaaki Tamura, 2 others, “Bio Science Experimental Medicine Separate Volume New Term Library Transcription Factor”, 2nd edition, Yodosha Co., Ltd., December 1999).
[0056]
The binding between the transcriptional regulatory factor and the target transcriptional regulatory region DNA or a partial fragment thereof can be detected using a measurement system known per se. For example, a test sample containing a transcriptional regulatory factor is added to a plate on which DNA of a target transcriptional regulatory region or a partial fragment thereof is immobilized, and the direct binding of both is performed using the SPR (Surface Plasmon Resonance, Surface Plasmon Resonance) method. Can be detected. In that case, DNA of a target transcriptional regulatory region recognized by the transcriptional regulatory factor as described above or a partial fragment thereof is immobilized on a sensor chip. The annealed DNA or its partial fragment is immobilized. Alternatively, the binding of both can be detected using a transcriptional regulatory factor or a target transcriptional regulatory region DNA or a fragment thereof fluorescently labeled.
[0057]
The test sample for analysis containing the protein encoded by the DNA of the present invention can be prepared by a method for transcription / translation of the DNA of the present invention by an appropriate method as described above. Specifically, for example, the protein of the present invention can be obtained by inducing protein expression by subjecting the DNA of the present invention to a cell-free transcription / translation system.
[0058]
The protein encoded by the DNA of the present invention (SEQ ID NO: 11 to 20) is a target sequence or consensus target sequence (SEQ ID NO: 34 to SEQ ID NO: 86) designed by database analysis of the base sequence of a known target transcriptional regulatory region. It had binding activity to at least one DNA or partial fragment thereof.
[0059]
Furthermore, in the case of a transcriptional regulatory factor, as described above, the transcriptional regulatory factor is identified by analyzing the action on a disease-related transcriptional regulatory factor such as PPAR, p53, NFκB, AP-1, HIF-1, and CREB. It is possible to analyze whether the disease is directly or indirectly involved.
[0060]
PPAR is a peroxisome proliferator-activated receptor that is a family of steroid hormone receptors, and includes endocrine / sugar / lipid metabolism associated with development of lifestyle-related diseases such as diabetes and obesity, It is known as a disease-related transcriptional regulator that regulates the expression of various target genes involved in the circulatory system such as vascular function and inflammation and carcinogenic mechanisms, and has been attracting attention as a target molecule for therapeutic agents for these diseases Yes. In this PPAR, a plurality of subtype genes are found, and there are mainly three types of α, γ, and δ (also called NUC I in humans, FAAR (fat acid-activated receptor) in mice, and PPARβ in frogs) in mammals. The subtype of is clear. α-form is mainly expressed in fat consuming organs such as liver, kidney and brown adipocytes, as well as in the myocardium and gastrointestinal tract, and is involved in fatty acid oxidation, ketone body formation, and apolipoprotein production. The β type is expressed in the brain, and the δ type is universally expressed with no tissue specificity, but its expression in colon cancer cells is remarkable, and its relationship with carcinogenesis has attracted attention. Several types of isoforms, such as γ1 type and γ2 type, are known as γ type, γ1 type is expressed in adipose tissue, immune system organs, adrenal gland, and small intestine, and γ2 type is specifically expressed in adipocytes It is considered that it plays an important role in the induction of adipocyte differentiation and fat synthesis. These PPARs form heterodimers with another transcriptional regulator, such as RXR (retinoid X receptor), and are known to bind to the response element PPRE (PPAR response element) upstream of the target gene to control transcription. It has been.
[0061]
p53 is the second tumor suppressor gene identified in 1989 following the RB gene. The p53 gene is present on chromosome 17p13.1 and its gene product is a nuclear protein with a molecular weight of 53 kD. At first, the p53 gene was thought to be an oncogene similar to myc. After that, the p53 protein has a wild type and a mutant type, and the wild type has a function of controlling the cell proliferation function. It became clear that it is a tumor suppressor gene with a similar function. The p53 protein controls several genes as transcriptional regulators. Among them, the most important one is considered to be the expression control of the p21 gene that inhibits the function of the cyclin / Cdk complex. The function of p53 itself is controlled by phosphorylation. From the viewpoint of suppression of canceration, the following two functions are important as the function of p53. The first is on the signaling pathway that causes apoptosis, a programmed cell death. The second function is to stop the cell cycle while the cell performs DNA repair. Through such functions, p53 stops the cell cycle of cells damaged by radiation or drugs, or removes them by causing apoptosis to prevent the cells that have undergone genetic changes from growing as cancer cells. Therefore, it has been attracting attention as a target molecule for therapeutic agents such as cancer.
[0062]
NFκB regulates gene expression of cytokines (eg, IL-1, IL-2, IL-6, IL-8, GM-CSF, TNF, etc.), chemokines, interferons, MHC molecules, growth factors, cell adhesion molecules, etc. In particular, it is known as a disease-related transcriptional regulator that plays an important role in the immune system. For example, it has attracted attention as a target molecule for therapeutic agents for immune diseases such as rheumatoid arthritis and osteoarthritis, inflammatory diseases, and cancer. Has been.
[0063]
AP-1 is a complex composed of Fos and Jun family of transcriptional regulators, and is known to be involved in cell proliferation and differentiation, particularly cell canceration and inflammatory diseases. It is attracting attention as a target molecule for therapeutic drugs such as cancer.
[0064]
HIF-1 is a gene transcriptional regulator that is activated in a hypoxic situation, and has been shown to be closely involved in the control of angiogenesis through the control of gene expression of vascular endothelial growth factor, such as abnormal angiogenesis. It is attracting attention as a target molecule for therapeutic agents for ischemic diseases, anemia, hypoxia, diabetic retinopathy, cardiovascular diseases, cancer, and the like caused by oxidative abnormalities and intracellular oxidation.
[0065]
CREB (Cyclic AMP Responses Element Binding Protein) is a disease-related disease that plays an important role in cell growth, differentiation, learning and memory processes, nervous system adaptation to drug addiction, and metabolic pathway control such as gluconeogenesis by insulin and glucagon. It is known as a transcriptional regulatory factor, and has attracted attention as a target molecule for therapeutic agents for central system diseases such as memory impairment, cardiovascular diseases, and diabetes.
[0066]
Specifically, the base sequence of the gene site recognized by each disease-related transcriptional regulatory factor is immobilized on a solid phase. In particular, DNA of a target transcriptional regulatory region recognized by a disease-related transcriptional regulatory factor or a partial fragment thereof is preferably used. Examples of the sequence of DNA to be immobilized or a partial fragment thereof include SEQ ID NO: 87 and SEQ ID NO: 88 for PPARγ analysis, SEQ ID NO: 89 and SEQ ID NO: 90 for p53 analysis, SEQ ID NO: 91 and SEQ ID NO: 92 for NFκB analysis, SEQ ID NO: 93 and SEQ ID NO: 94 are used for AP-1 analysis, SEQ ID NO: 95 and SEQ ID NO: 96 are used for HIF-1 analysis, and SEQ ID NO: 97 and SEQ ID NO: 98 are used for CREB analysis. is not. The annealed DNA or its partial fragment is immobilized. For example, a well plate is used as the solid phase, and it is immobilized directly or on, for example, a streptavidin-treated plate or a neutravidin-treated plate. It is more preferable to use biotin-labeled DNA or a partial fragment thereof. The addition amount of DNA or a partial fragment thereof is preferably 1 to 100 nM / 100 μl / well. The reaction is preferably carried out at 10 to 30 ° C. (room temperature) for about 30 minutes to 2 hours. After the completion of the reaction, unreacted substances are removed, and more preferably a protein blocking operation is performed.
[0067]
A test sample containing a transcriptional regulatory factor is added to the well plate prepared as described above together with each disease-related transcriptional regulatory factor, and the action of the transcriptional regulatory factor contained in the test sample is analyzed. If the transcriptional regulatory factor contained in the test sample binds to the disease-related transcriptional regulatory factor and enhances the affinity with the immobilized DNA, the conjugate of the disease-related transcriptional regulatory factor and the transcriptional regulatory factor even after reaction washing Remains in the well in a state of being bound to the immobilized DNA. For example, when measured by absorbance, the OD value is increased compared to the control, and the transcriptional regulatory factor is positive for the affinity between the disease-related transcriptional regulatory factor and the immobilized DNA. It can be estimated that it works. On the other hand, if the transcriptional regulatory factor contained in the test sample binds or does not bind to the disease-related transcriptional regulatory factor and reduces the affinity between the disease-related transcriptional regulatory factor and the immobilized DNA, reaction washing After that, the conjugate of the disease-related transcription regulatory factor and the transcriptional regulatory factor is removed from the well. For example, if measured by absorbance, the OD value is decreased compared to the control, and the transcriptional regulatory factor is the disease-related transcriptional regulatory factor and the immobilized DNA. It can be estimated that it negatively affects the affinity with.
[0068]
The test sample for analysis containing the protein encoded by the clone of the present invention can be prepared by a method for transcription / translation of the DNA of the present invention by an appropriate method as described above. Specifically, for example, the protein of the present invention can be obtained by inducing protein expression by subjecting the DNA of the present invention to a cell-free transcription / translation system.
[0069]
The disease-related transcriptional regulatory factor can be obtained from a cell extract or a cell nucleus extract containing the disease-related transcriptional regulatory factor. Further, as described above, it can be prepared by a method of transcription / translation of DNA encoding a disease-related transcriptional regulatory factor by an appropriate method. Specifically, for example, a cDNA encoding a disease-related transcriptional regulatory factor is used, and the full length or a part thereof is inserted into an appropriate expression vector, and this is inserted into a microorganism such as E. coli, insect cells, yeast, animal cells or animals. The disease-related transcriptional regulator, which is a recombinant protein, can be obtained from the culture supernatant or intracellular, tissue, or body fluid of these gene-introduced cells in which the disease-related transcriptional regulatory factor is expressed.
[0070]
The protein encoded by 4 clones of c-nt2ri3007878, c-brsn2017530, c-hchon20000473, and c-best2001444 has an activity to enhance or suppress the action of PPAR, and it may cause diabetes caused by abnormal endocrine / sugar / lipid metabolism, etc. It was speculated that it is related to lifestyle-related diseases such as obesity, circulatory system diseases due to abnormal vascular function, inflammation, cancer and the like.
[0071]
It was speculated that the protein encoded by the three clones c-nt2ri3007878, c-brsn2017530, and c-testi4004626 has an activity to enhance or suppress the action of p53 and is related to cancer and the like.
[0072]
Proteins encoded by the three clones c-testi 4004626, c-trach 3014316, and c-best 2001444 have an activity to enhance or suppress the action of NFκB, and are immune diseases such as rheumatoid arthritis and osteoarthritis, inflammatory diseases It was speculated to be related to cancer and the like.
[0073]
A protein encoded by 4 clones of c-nt2ri3007878, c-hchon20000473, c-testi4004626, c-trac3014316 has an activity to enhance or suppress the action of AP-1, and is related to inflammatory diseases and cancer It was speculated.
[0074]
The protein encoded by the three clones c-brssn2017530, c-testi4004626, c-trac3014316 has an activity to enhance or suppress the action of HIF-1, resulting from abnormal angiogenesis, abnormal intracellular oxidation, etc. It was speculated that it is related to ischemic disease, anemia, hypoxia, diabetic retinopathy, cardiovascular disease, cancer and the like.
[0075]
The protein encoded by two clones of c-testi 4004626 and c-trac 3014316 has an activity to enhance or suppress the action of CREB and is related to central diseases such as memory impairment, cardiovascular diseases, diabetes, etc. Was guessed.
[0076]
Such a system for analyzing transcriptional regulatory activity can also be used for evaluating agonists and antagonists of proteins having transcriptional regulatory activity of the present invention. The confirmation of the activity of the protein of the present invention is not limited to the method described above.
[0077]
(4) Functional analysis of the protein of the present invention
The proteins of the present invention having novel transcriptional activity including those identified as splicing variants thus obtained are analyzed by analyzing functions other than the transcriptional regulatory activity confirmed in (3) above. The novel utilization method is provided (a protein to be further analyzed for functions other than the transcriptional regulatory activity may be hereinafter referred to as “analyzed protein”). In particular, since the protein of the present invention includes a known protein splicing variant, it is important to identify how this variant functions differently from the known variant.
[0078]
Specific functional analysis methods include, for example, (i) a method for comparative analysis of expression states in each tissue, disease, or developmental stage, (ii) a method for analyzing interactions with other proteins and DNA, ( iii) a method of introducing into a suitable cell or individual and analyzing the phenotypic change; and (iv) a method of analyzing the phenotypic change by inhibiting the expression of the protein in a suitable cell or individual.
[0079]
In the method (i), the expression of the protein of the present invention can be analyzed at the mRNA level or the protein level. In the case of analyzing the expression level at the mRNA level, for example, in situ hybridization (Application to Developmental Biology & Medicine, Ed. by Harris, N. and Wilkins, U.S. 1990)), a hybridization method using a DNA chip, a quantitative PCR method and the like are used. Moreover, when analyzing at a protein level, the tissue staining method using the antibody specifically couple | bonded with the protein of this invention mentioned later, ELISA, a Western blot method etc. are mentioned. If the target protein to be analyzed is a splicing variant with a known variant, use a probe that is present only in the cDNA encoding the target protein and does not hybridize with the cDNA encoding the known variant. Is preferred. In the case of the quantitative PCR method, a method of selecting primers capable of producing amplified fragments having different lengths between the target variant and the known variants (Wong, Y., Neuroscience Let., 320: 141-145 (2002)), etc. Is mentioned. Also, when analyzing at the protein level, it is preferable to use an antibody that reacts only with the target protein and does not react with a known variant.
[0080]
In the method (ii), the function of the protein of the present invention can be analyzed by examining the presence or absence of interaction between the protein of the present invention and a known protein. As an analysis method of the interaction, a conventional method known per se can be used. Specifically, for example, yeast two-hybrid method, fluorescence depolarization method, surface plasmon method, phage display method, ribosomal display Law. Also in this method, when the protein to be analyzed is a splicing variant in which a known variant exists, a substance that interacts with the known variant in the same manner is analyzed, and a substance that specifically interacts with the target protein is identified. Is preferred.
[0081]
In the method (iii), the cells into which the cDNA of the present invention is introduced are not particularly limited, but human cultured cells and the like are particularly preferably used. Examples of the method for introducing DNA into cells include those described in (2) above. In addition, the phenotype of the introduced cell can be observed with a microscope, such as cell viability, cell growth rate, cell differentiation, neurite elongation when cells are nerve cells, and localization and migration of intracellular proteins. And those that can be analyzed by biochemical experiments such as changes in expression of specific proteins in cells. In the case of a splicing variant in which a known variant is present, these phenotypes are similarly introduced into the cell by the present DNA or a DNA encoding the known variant, and subjected to comparative analysis to obtain a phenotype related to the analysis target variant. Can be identified. Moreover, since it is known that the protein of the present invention has transcriptional regulatory activity, it is also preferable to analyze by paying attention to phenotypes and the like found in diseases associated with kinases.
[0082]
The method (iv) can be carried out efficiently by a method using an oligonucleotide described later or an RNA interference method. Also in this method, when the target protein to be analyzed is a splicing variant in which a known variant exists, the same analysis is performed on the known variant and other variants, and the target protein-specific is obtained by performing comparative analysis. Function can be identified.
[0083]
(5) Preparation of oligonucleotide and functional analysis using the oligonucleotide Using the DNA of the present invention obtained by the method described in the above (1) or a fragment thereof, by a conventional method using a DNA synthesizer or the like, Oligonucleotides such as antisense oligonucleotides and sense oligonucleotides having a partial sequence of DNA can be prepared. Examples of the oligonucleotide include DNA having the same sequence as 5 to 100 consecutive bases in the base sequence of the DNA or DNA having a sequence complementary to the DNA. Specific examples thereof include DNA having the same sequence as 5 to 100 consecutive bases in the base sequence represented by any of SEQ ID NOs: 1 to 10, or DNA having a sequence complementary to the DNA. Here, when the target protein is a splicing variant in which a known variant DNA exists, it is preferable to select a base sequence of a portion different from the known variant. When used as a sense primer and an antisense primer, the above oligonucleotides in which the melting temperature (Tm) and the number of bases of both do not change extremely are preferable. The length of the sequence is generally 5 to 100 bases, preferably 10 to 60 bases, and more preferably 15 to 50 bases.
[0084]
In addition, derivatives of these oligonucleotides can also be used as the oligonucleotide of the present invention. The oligonucleotide derivative includes an oligonucleotide derivative in which a phosphodiester bond in an oligonucleotide is converted to a phosphorothioate bond, and an oligonucleotide in which a phosphodiester bond in an oligonucleotide is converted to an N3′-P5 ′ phosphoramidate bond. Nucleotide derivatives, oligonucleotide derivatives in which ribose and phosphodiester bonds in oligonucleotides are converted to peptide nucleic acid bonds, oligonucleotide derivatives in which uracil in oligonucleotides is substituted with C-5 propynyl uracil, uracil in oligonucleotides Oligonucleotide derivative substituted with C-5 thiazole uracil, oligonucleotide derivative substituted with cytosine in oligonucleotide with C-5 propynylcytosine, oligonucleotide An oligonucleotide derivative in which the cytosine is replaced with a phenoxazine-modified cytosine, an oligonucleotide derivative in which the ribose in the oligonucleotide is replaced with 2′-O-propylribose, or 2 in the oligonucleotide Examples thereof include oligonucleotide derivatives substituted with '-methoxyethoxyribose.
[0085]
Moreover, the oligonucleotide of the present invention can be applied to the RNA interference method by preparing it as a double-stranded RNA. For example, the method described in (Elbashir, S., et al., Nature, 411: 494-498 (2001)) can be used for the production method of double-stranded RNA and the RNA interference method. . The double-stranded RNAs need not all be RNA. Specifically, those described in WO 02/10374 can be used as a part of which is DNA.
[0086]
The target gene in this RNA interference method (hereinafter sometimes referred to as “target gene”) may be any DNA as long as it is the DNA of the present invention. A double-stranded polynucleotide comprising RNA having a sequence substantially the same as at least a part of the base sequence of these DNAs (hereinafter sometimes referred to as “double-stranded polynucleotide”) refers to a target gene Among these nucleotide sequences, the nucleotide sequence is substantially the same as a sequence of 15 bp or more which may be any part. Here, “substantially identical” means having a homology of 80% or more with the sequence of the target gene.
[0087]
Further, when the protein to be analyzed is an insertion type or substitution type variant as compared with a known protein, the double-stranded polynucleotide sequence can be set at the insertion or substitution site. In addition, when the protein to be analyzed is a deletion variant of a known protein, it is possible to select a sequence that is specifically effective for the protein by setting the sequence across the deletion to a double-stranded polynucleotide sequence. it can. Furthermore, by comparing the base sequence of the DNA encoding each of the protein to be analyzed and the known protein, by selecting a base sequence specific to the DNA encoding the protein to be analyzed, Expression can be inhibited.
[0088]
The nucleotide chain length may be any length from 15 bp to the entire length of the open reading frame (ORF) of the target gene, but a length of about 15 to 500 bp is preferably used. However, in cells derived from mammals, the existence of a signal transduction system that is activated in response to a long double-stranded RNA of 30 bp or more is known. This is called an interferon reaction (Mareus, PI, et al., Interferon, 5: 115-180 (1983)). When the double-stranded RNA enters the cell, PKR (dsRNA-responsive) protein kinase: Bass, B. L., Nature, 411: 428-429 (2001)), the initiation of translation of many genes is non-specifically inhibited, and at the same time, 2′-5 ′ oligoadenylate synthetase (Bass, B. L., Nature, 411: 428-429 (2001)), activation of RNase L occurs, causing non-specific degradation of intracellular RNA. These non-specific reactions mask the specific reaction of the target gene. Therefore, when a mammal or a cell or tissue derived from the animal is used as the recipient, it is preferable to use a double-stranded polynucleotide of 15 to 30 bp, preferably 19 to 24 bp, more preferably 21 bp. The double-stranded polynucleotide need not be entirely double-stranded, and includes those in which the 5 'or 3' end partially protrudes. However, it is preferable to use a polynucleotide in which the 3 'end protrudes 2 bases. The double-stranded polynucleotide means a double-stranded polynucleotide having complementarity, but may be a self-annealed single-stranded polynucleotide having self-complementarity. Examples of the single-stranded polynucleotide having self-complementarity include those having an inverted repeat sequence.
[0089]
Although there is no restriction | limiting in particular as a preparation method of double stranded polynucleotide, It is preferable to use the chemical synthesis method known per se. In chemical synthesis, single-stranded polynucleotides having complementarity can be synthesized separately and associated with each other by an appropriate method to be double-stranded. Examples of the association method include a method in which the polynucleotide is mixed, heated to a temperature at which the double strands dissociate, and then gradually cooled. The associated double-stranded polynucleotide is confirmed using an agarose gel or the like, and the remaining single-stranded polynucleotide is removed by, for example, decomposing with an appropriate enzyme.
[0090]
The recipient to which the double-stranded polynucleotide thus prepared is introduced may be any recipient as long as the target gene can be transcribed into RNA or translated into protein in the cell. Specific examples include those belonging to plant, animal, protozoan, virus, bacteria, or fungal species. The plant may be monocotyledonous, dicotyledonous or gymnosperm, and the animal may be a vertebrate or invertebrate. Preferred microorganisms are those used in agriculture or industry and are pathogenic to plants or animals. Fungi include organisms in both mold and yeast forms. Examples of vertebrates include mammals including fish, cows, goats, pigs, sheep, hamsters, mice, rats, and humans, and invertebrates include nematodes and other reptiles, Drosophila melanogaster ( Drosophila), and other insects. Preferably, the cell is a vertebrate cell.
[0091]
An introducer means a cell, tissue or individual. Here, the cells may be germline or somatic, totipotent or multipotent, divided or undivided, parenchyma or epithelium, immortalized or transformed, and the like. The cell may be a gamete or embryo, and in the case of an embryo, it is a single-cell embryo or constitutive cell, or a cell from a multi-cell embryo and includes fetal tissue. Furthermore, it may be an undifferentiated cell such as a stem cell, or a differentiated cell such as from a cell of an organ or tissue including fetal tissue, or any other cell present in an organism. Differentiated cell types include adipocytes, fibroblasts, muscle cells, cardiomyocytes, endothelial cells, neurons, glia, blood cells, megakaryocytes, lymphocytes, macrophages, neutrophils, eosinophils, Examples include basophils, mast cells, leukocytes, granulocytes, keratinocytes, chondrocytes, osteoblasts, osteoclasts, hepatocytes and cells of endocrine or exocrine glands.
[0092]
As a method for introducing a double-stranded polynucleotide into a recipient, when the recipient is a cell or tissue, a calcium phosphate method, an electroporation method, a lipofection method, a virus infection, a double-stranded polynucleotide solution Soaking or transformation method is used. Examples of the method for introduction into the embryo include microinjection, electroporation, and virus infection. When the recipient is a plant, a method by injection or perfusion into the body cavity or stromal cells of the plant or spraying is used. In the case of an animal individual, it is introduced systemically by oral, topical, parenteral (including subcutaneous, intramuscular and intravenous administration), vaginal, rectal, nasal, ophthalmic, intraperitoneal administration, etc. The method, electroporation method, virus infection or the like is used. For methods for oral introduction, double-stranded polynucleotides can be mixed directly with the food of the organism. Furthermore, when introduced into an individual, it can be administered, for example, as an implanted long-term release preparation or by ingesting an introduced body into which a double-stranded polynucleotide has been introduced.
[0093]
The amount of the double-stranded polynucleotide to be introduced can be appropriately selected depending on the introduced body and the target gene, but it is preferable to introduce an amount sufficient to introduce at least one copy per cell. Specifically, for example, when the recipient is a cultured human cell and a double-stranded polynucleotide is introduced by the calcium phosphate method, 0.1 to 1000 nM is preferable.
[0094]
By suppressing the expression of the DNA of the present invention in the introduced body by RNA interference, the function of the protein encoded by the DNA of the present invention can be confirmed, or a new function can be analyzed.
[0095]
(6) An antibody that specifically binds to the protein of the present invention
As a method for preparing an antibody that specifically binds to the protein of the present invention, a commonly used known method can be used. A polypeptide used as an antigen also has a high antigenicity according to a known method and has an epitope (antigen determination). A sequence suitable as the group) can be selected and used. As an epitope selection method, for example, commercially available software such as Epitope Advisor (manufactured by Fujitsu Kyushu System Engineering Co., Ltd.) can be used. In addition, when the target protein is a splicing variant in which a known variant exists, it is specific to the target protein by using an antibody that reacts only with the target protein and does not react with a known or other variant. Function can be identified. As an epitope of such an antibody, for example, when there is an amino acid sequence in which the target protein is deleted compared to a known variant, an amino acid sequence (junction part) before and after the deleted part is preferable. In addition, when the target protein has an amino acid sequence to which a known variant N-terminal or C-terminal is added, it is also preferable to use the added amino acid sequence as an epitope. As a method other than the above, an antibody that reacts only with the target protein can be obtained by removing an antibody that reacts with a known or other variant from the polyclonal antibody obtained against the target protein. As a method for removal, affinity chromatography in which a known or other variant is immobilized as a ligand, or an immunoprecipitation method using a known or other variant is used.
[0096]
The polypeptide used as the antigen may be a synthetic peptide synthesized according to a known method, or the protein of the present invention itself. Polypeptides that serve as antigens can be prepared in an appropriate solution according to known methods and immunized to mammals such as rabbits, mice, rats, etc. In order to perform stable immunization or increase antibody titers It is preferable to immunize by using an antigen peptide as a conjugate with an appropriate carrier protein or adding an adjuvant or the like.
[0097]
There are no particular limitations on the route of administration of the antigen during immunization, and any route such as subcutaneous, intraperitoneal, intravenous, or intramuscular may be used. Specifically, for example, a method of inoculating BALB / c mice several times every several days to several weeks is used. The antigen intake is preferably about 0.3 to 0.5 mg / dose when the antigen is a polypeptide, but is appropriately adjusted depending on the type of polypeptide and the animal species to be immunized.
[0098]
After immunization, blood is collected on a trial basis as appropriate, and an increase in antibody titer is confirmed by a method such as octalony method, solid phase enzyme immunoassay (hereinafter sometimes referred to as “ELISA method”) or Western blotting, Blood is collected from animals with sufficiently elevated antibody titers. A polyclonal antibody can be obtained by performing an appropriate treatment used for preparing the antibody. Specific examples include a method of obtaining a purified antibody obtained by purifying antibody components from serum according to a known method. For purification of antibody components, methods such as salting out, ion exchange chromatography, affinity chromatography and the like can be used.
[0099]
A monoclonal antibody can also be produced by using a hybridoma fused with the spleen cells and myeloma cells of the animal according to a known method (Milstein, et al., Nature, 256: 495 (1975)). . A monoclonal antibody can be obtained, for example, by the following method.
[0100]
First, antibody-producing cells are obtained from an animal having an increased antibody titer by immunization with the antigen described above. The antibody-producing cells are plasma cells and lymphocytes that are precursor cells thereof, and these may be obtained from any individual, but are preferably obtained from spleen, lymph nodes, peripheral blood, and the like. The myeloma to be fused with these cells is generally a cell line obtained from a mouse, for example, an 8-azaguanine resistant mouse (BALB / c-derived etc.) myeloma cell line P3X63-Ag8.653 (ATCC: CRL). -1580), P3-NS1 / 1Ag4.1 (RIKEN Cell Bank: RCB0095) and the like are preferably used. For cell fusion, antibody-producing cells and myeloma cells are mixed at an appropriate ratio, and 50% polyethylene is added to an appropriate cell fusion medium such as RPMI 1640, Iskov modified Dulbecco medium (IMDM), Dulbecco modified Eagle medium (DMEM), or the like. It can be performed by using a solution in which glycol (PEG) is dissolved. It can also be carried out by the electrofusion method (Zimmermann, U. et al., Naturewissenschafften, 68: 577 (1981)).
[0101]
The hybridoma was prepared by using 5% CO 2 in a normal medium (HAT medium) containing an appropriate amount of hypoxanthine / aminopterin / thymidine (HAT) solution using the fact that the myeloma cell line used was an 8-azaguanine resistant strain.₂It can be selected by culturing at 37 ° C. for an appropriate time. This selection method can be appropriately selected depending on the myeloma cell line used. The antibody titer of the antibody produced by the selected hybridoma is analyzed by the method described above, the hybridoma producing the antibody having a high antibody titer is separated by a limiting dilution method, etc., and the isolated fused cells are cultured in an appropriate medium. A monoclonal antibody can be obtained by purifying the resulting culture supernatant by an appropriate method such as ammonium sulfate fractionation or affinity chromatography. For purification, a commercially available monoclonal antibody purification kit can also be used. Furthermore, ascites containing a large amount of the monoclonal antibody of the present invention can be obtained by growing the antibody-producing hybridoma obtained above in the abdominal cavity of an animal of the same strain as the immunized animal or a nude mouse.
[0102]
In addition, when a protein derived from human is obtained as the protein of the present invention, the above-described method is applied to a Severe combined immunology (SCID) mouse transplanted with human peripheral blood lymphocytes using such a polypeptide or a partial peptide thereof as an antigen. The human-type antibody can also be prepared by immunizing in the same manner as described above and preparing a hybridoma between the antibody-producing cells of the immunized animal and human myeloma cells (Mosier, D. E., et al., Nature). 335: 256-259 (1988); Duchosal, M. A., et al., Nature, 355: 258-262 (1992)).
[0103]
In addition, RNA is extracted from the obtained hybridoma producing the human antibody, the gene encoding the desired human antibody is cloned, this gene is inserted into an appropriate vector, and this is introduced into an appropriate host. By expressing it, it is possible to produce a human antibody in a larger amount. Here, an antibody having a low binding property to an antigen can also be obtained as an antibody having a higher binding property by using a known evolutionary engineering technique. A partial fragment such as a transient antibody can be prepared by cleaving the Fab portion and the Fc portion using, for example, papain, and collecting the Fab portion using an affinity column or the like.
[0104]
The antibody that specifically binds to the protein of the present invention thus obtained can also be used as a neutralizing antibody that inhibits the transcriptional regulatory activity and the like of the protein by specifically binding to the protein of the present invention. There is no particular limitation on the method for selecting the protein that inhibits the activity of the protein. For example, whether or not the function of the target protein in the introduced body is inhibited by bringing the antibody into contact with the DNA introduced body prepared in (2) above. And a method for analyzing the above.
[0105]
Such a neutralizing antibody can be used alone in the clinical application, but can also be used as a pharmaceutical composition by blending with a pharmaceutically acceptable carrier. The ratio of the active ingredient to the carrier at this time can vary between 1 and 90% by weight. In addition, such drugs can be administered in various forms, such as tablets, capsules, granules, powders, syrups or the like, or injections, drops, liposomes, Parenteral administration with a suppository or the like can be mentioned. Further, the dose can be appropriately selected depending on symptoms, age, body weight and the like.
[0106]
(7) Screening for molecules that modulate the activity of the protein of the present invention
By screening for a substance that specifically binds to the protein of the present invention and has an action of inhibiting, antagonizing or enhancing the function (activity) of the protein of the present invention, (Sometimes referred to as “regulators”).
[0107]
Any method may be used for this modulator screening method as long as it is capable of obtaining a substance that specifically binds to the protein of the present invention and has an action of inhibiting, antagonizing or enhancing the activity of the protein. For example, first, the protein of the present invention is contacted with a substance to be screened (hereinafter sometimes referred to as “test substance”) and selected with the binding property to the protein as an index. A method of selecting a test substance using the change in activity of the protein as an index can be used.
[0108]
The test substance may be any substance that can interact with the protein of the present invention and affect the activity of the protein. Specifically, for example, a peptide , Proteins, non-peptidic compounds, low molecular weight compounds, synthetic compounds, fermentation products, cell extracts, animal tissue extracts and the like. These substances may be novel substances or known substances. As a method for analyzing the interaction between the test substance and the protein of the present invention, a conventional method known per se can be used. Specifically, for example, the yeast two-hybrid method, the fluorescence depolarization method, the surface plasmon method , Phage display method, ribosomal display method, competition analysis method with the antibody described in (6) above, and the like. By such a method, a substance found to bind to the protein of the present invention is then analyzed by analyzing how the activity of the protein of the present invention is affected in the presence of the substance. Whether it is used as a modulator is identified. Here, when the target protein is a splicing variant in which a known variant exists, the effect is analyzed on a substance that binds only to the target protein and does not bind to a known or other variant, or a known Alternatively, the effect of the substance on the target protein can be analyzed by identifying whether or not other variants bind in the same manner, and analyzing the difference in the effects when binding. Further, by examining the distribution of the substance in an individual, the influence on the target protein and known or other variants can be analyzed.
[0109]
As a specific analysis method, for example, when analyzing a substance that regulates transcriptional regulatory activity, a protein serving as a substrate is also introduced into the DNA transductant described in (2) by the same method. The phosphorylation of the protein serving as a substrate in the presence / absence of the substance selected for this transductant is analyzed by a commonly used method known per se. Specifically, it can be performed using the method described in (3) above. If the transcriptional regulatory activity is increased compared to that in the absence of the substance, the substance may function as an activator of the transcriptional regulator, and if it is reduced or inhibited, the substance Can be identified as having the potential to function as inhibitors of transcriptional regulators.
[0110]
Here, when using the DNA or recombinant protein of the present invention used for screening a regulatory substance for the purpose of obtaining a pharmaceutically active ingredient, it is preferable to use the human homologous protein described above. Furthermore, the substance screened by the above method may be further selected as a pharmaceutical candidate by in vivo screening. The evaluation of the protein function regulator of the present invention is not limited to the method described above.
[0111]
Transcriptional regulators control pathway signaling related to cancer, signaling related to myocardial development, signaling to control sperm differentiation and motility, and control germline differentiation Signal transduction function on the pathway, signal transduction function on the pathway that controls cell differentiation, function to produce glycerol triphosphate, signal transduction function on the pathway that controls the development, differentiation, proliferation and survival of neurons, Alzheimer In the signal transmission function on the pathway that controls the pathogenesis of the disease, the signal transmission function of the pathway that controls the generation, differentiation, growth, proliferation, survival, regeneration, and cell function of various cells, etc. It is related to the regulation of gene expression by binding to DNA. Therefore, it can be a target for screening for therapeutic agents for various diseases related to these signal transductions. Compounds that can be identified by this screening method include anticancer agents, diabetes treatment agents, anti-inflammatory agents, neurodegenerative disease treatment agents, cardiac disease treatment agents, infertility treatment agents, regenerative tissue inducers, Alzheimer's disease treatment agents, obesity treatment agents, It can be used as a therapeutic agent for diabetes, cardiovascular disease, metabolic disorder, anorexia, bulimia and the like.
[0112]
The DNA encoding the protein of the present invention is a tissue such as brain (amygdala, hippocampus, substantia nigra, fetal brain), testis, trachea, adult breast, chondrocyte, nervous system cell including undifferentiated neural progenitor cells, etc. The protein of the present invention, which has been cloned from a cDNA library constructed from organ or cell-derived RNA, may have a specific function in the above tissues or organs. The protein function-regulating substance of the invention can be used as a therapeutic agent for diseases peculiar to the tissue or organ.
[0113]
In the clinical application, the above-mentioned active ingredient can be used alone for clinical application, but it can also be used as a pharmaceutical composition by blending with a pharmaceutically acceptable carrier. The ratio of the active ingredient to the carrier at this time can vary between 1 and 90% by weight. In addition, such drugs can be administered in various forms, such as tablets, capsules, granules, powders, syrups or the like, or injections, drops, liposomes, Parenteral administration with a suppository or the like can be mentioned. Further, the dose can be appropriately selected depending on symptoms, age, body weight and the like.
[0114]
(8) Screening for DNA expression regulators of the present invention
Examples of the screening method include a method of analyzing the expression level of the protein of the present invention or mRNA encoding the same in the presence of a test substance. Specifically, for example, cells expressing the protein of the present invention described in (2) are cultured in an appropriate medium containing a test substance, and the amount of the protein of the present invention expressed in the cells is determined by ELISA or the like. Or the amount of mRNA encoding the protein of the present invention in the cells can be analyzed by quantitative reverse transcription PCR, Northern blotting, or the like.
[0115]
As the test substance, those described in (7) can be used. If this analysis shows that the amount of protein or mRNA expressed in the cells cultured in the absence of the test substance increases compared to the amount of the test substance, the test substance becomes a substance that promotes the expression of the DNA of the present invention. In the case of decrease, it can be determined that this test substance can be used as a DNA expression inhibitor of the present invention.
[0116]
Such an expression regulating substance can be used alone in the clinical application, but can also be used as a pharmaceutical composition by blending with a pharmaceutically acceptable carrier. The ratio of the active ingredient to the carrier at this time can vary between 1 and 90% by weight. In addition, such drugs can be administered in various forms, such as tablets, capsules, granules, powders, syrups or the like, or injections, drops, liposomes, Parenteral administration with a suppository or the like can be mentioned. Further, the dose can be appropriately selected depending on symptoms, age, body weight and the like.
[0117]
(9) DNA-introduced animal of the present invention
The introduction DNA containing the DNA of the present invention described in (1) above is constructed, introduced into a fertilized egg of a mammal other than a human, and transplanted into a female individual uterus to generate it. Non-human mammals into which DNA has been introduced can be produced. More specifically, for example, a female individual is over-ovulated by hormone administration, then mated with a male, a fertilized egg is extracted from the oviduct on the first day after mating, and the introduced DNA is microinjected into the fertilized egg, etc. It introduces by the method of. Thereafter, after culturing by an appropriate method, the surviving fertilized eggs are transplanted into the uterus of a pseudo-pregnant female individual (temporary parent) to give birth. Whether or not the target DNA has been introduced into the newborn can be identified by performing Southern blot analysis of DNA extracted from the cells of the individual. Examples of mammals other than humans include mice, rats, guinea pigs, hamsters, rabbits, goats, pigs, dogs, cats and the like.
[0118]
The thus-obtained DNA-introduced animal of the present invention is mated and obtained offspring by subculture in a normal breeding environment while confirming that the introduced DNA is stably retained. be able to. Moreover, the offspring can be obtained by repeating in vitro fertilization and a line can be maintained.
[0119]
The non-human mammal into which the DNA of the present invention has been introduced can be used as an analysis of the function of the DNA of the present invention in vivo, or as a screening system for substances that regulate it.
[0120]
(10) Other uses of the protein of the present invention and DNA containing the base sequence encoding it
The protein of the present invention can be used as a carrier on which the protein is bound. Moreover, the base sequence encoding the protein of the present invention, for example, the DNA having the base sequence set forth in any of SEQ ID NOs: 1 to 10 or a partial fragment thereof can be used as a carrier on which they are bound on a substrate. Hereinafter, these may be referred to as “protein chip”, “DNA chip” or “DNA array” (DNA microarray and DNA macroarray). These protein chips, or DNA chips or arrays, may contain other proteins and DNAs in addition to the proteins and DNAs of the present invention. Here, when the target protein is a splicing variant in which a known variant exists, an amino acid sequence partial fragment specific to the target protein can be used for the protein chip, but has a three-dimensional structure different from other variants. Since there is a possibility, the full length can also be used. For the DNA array, it is preferable to select a sequence different from other variant DNAs among the DNA sequences encoding the target protein.
[0121]
In addition, as a substrate for binding proteins and DNA, a resin substrate such as a nylon membrane or a polypropylene membrane, a nitrocellulose membrane, a glass plate, a silicon plate, or the like is used. When performing using a substance etc., the glass plate, silicon plate, etc. which do not contain a fluorescent substance are used suitably. The protein or DNA can be easily bound to the substrate by a commonly used method known per se. These protein chips, DNA chips, or DNA arrays are also included in the scope of the present invention.
[0122]
The amino acid sequence of the protein of the present invention and the base sequence of DNA can also be used as sequence information. The base sequence of this DNA includes the base sequence of the corresponding RNA. That is, an amino acid sequence or base sequence database can be constructed by storing the obtained amino acid sequence or base sequence in an appropriate recording medium in a predetermined format readable by a computer. This database may include base sequences of other types of proteins and DNAs encoding the same. In the present invention, the database also means a computer system in which the above array is written on an appropriate recording medium and a search is performed according to a predetermined program. Examples of suitable recording media include magnetic media such as flexible disks, hard disks, and magnetic tapes, optical disks such as CD-ROM, MO, CD-R, CD-RW, DVD-R, and DVD-RAM, and semiconductor memory. Etc.
[0123]
【Example】
EXAMPLES Hereinafter, although an Example is given and this invention is demonstrated in detail, the scope of the present invention is not limited by these Examples. In addition, the result of having performed the following experiment about each acquired cDNA clone was put together in Example 7, and was described.
[0124]
Example 1 Preparation of cDNA library by oligo cap method
Human cultured cells (HC cells that are primary human cartilage primary cultured cells, manufactured by Toyobo: # T402K-05) and human cultured cells (teratocarcinoma cells derived from human fetal testis, nerves that can be differentiated into neurons by retinoic acid treatment NT2 cells, which are progenitor cells, manufactured by Stratagene: # 204101) were induced by retinoic acid treatment, cultured for 5 weeks, further treated with a growth inhibitor, and cultured for 2 weeks (Sambrook, J. et al). , Molecular Cloning 2nd ed., Cold Spring Harbor Laboratory Press, (1989)), and mRNA is extracted with oligo dT cellulose.⁺RNA was purified.
[0125]
Poly (A) obtained above⁺RNA, commercially available mRNA extracted as total RNA from human tissues (Clontech: testis (# 64027-1), fetal brain (# 64019-1), trachea (# 64091-1), made by Stratagene) : Adult breast (# 7335044)) and poly (A) from human tissues⁺Polynucleotides from total RNA in the whole brain are extracted into each commercially available mRNA extracted and purified as RNA (Clontech: amygdala (# 6574-1), substantia nigra (# 6580-1), hippocampus (# 65778-1)). (A)⁺Poly (A) prepared by removing RNA with oligo dT cellulose⁻From each RNA mixed with RNA, cDNA libraries were prepared by the oligo cap method (Maruyama, K., et al., Gene, 138: 171-174 (1994)). The relationship between the library name and its origin is shown below.
c-best: adult breast
c-bramy: amygdala
c-brhip: Hippocampus
c-brassn: Black quality
c-febra: fetal brain
c-hchon: Normal cartilage primary cultured cells (HC cells)
c-nt2ri: cells obtained by differentiating neural progenitor cells (NT2 cells) into neuron-like cells by retinoic acid treatment
c-testi: testis
c-trach: trachea
[0126]
First, the RNA was treated with BAP (Bacterial Alkaline Phosphatase) and TAP (Tobacco Acid Pyrophosphatase), and then an oligocap linker (SEQ ID NO: 21) was ligated using RNA ligase. First strand cDNA was synthesized by reverse transcription reaction using this RNA strand as a template and oligo dT primer (SEQ ID NO: 22), and then RNA strand was decomposed and removed (Suzuki et al., Protein Nucleic Acid Enzyme, 41: 603-607 ( 1996); Suzuki, Y. et al., Gene, 200: 149-156 (1997)). Subsequently, double-stranded cDNA was amplified by PCR (polymerase chain reaction) using the 5 ′ PCR primer (SEQ ID NO: 23) and the 3 ′ PCR primer (SEQ ID NO: 24), and the amplified DNA strand was cleaved with SfiI. .
[0127]
Next, the SfiI digested fragment obtained above was cloned into the DraIII site of pME18SFL3 (GenBank AB009864), which is an expression vector, to prepare a cDNA library. In the pME18SFL3 vector used above, the SRα promoter and the SV40 small t intron are incorporated upstream of the cloning site, and the SV40 poly (A) addition signal sequence is inserted downstream thereof. Since the cloning site of pME18SFL3 is an asymmetric DraIII site and a complementary SfiI site is added to the end of the cDNA fragment, the cloned cDNA fragment is unidirectional downstream of the SRα promoter. Inserted into. Therefore, in a clone containing a full-length cDNA, it is possible to express a gene transiently by directly introducing the obtained plasmid into a COS cell. That is, it can be analyzed very easily as a protein that is a gene product or as an experimental analysis of their biological activity.
[0128]
With respect to the plasmid DNA of the clones obtained from these, the nucleotide sequence at the 5 'end or 3' end of the cDNA was converted into a DNA sequencing reagent (Dye Terminator Cycle Sequencing FS Ready Reaction Kit, dRhodamine). Terminator Cycle Sequencing FS Ready Reaction Kit or BigDye Terminator Cycle Sequencing FS Ready Reaction Kit: Bios, sequenced by M did.
[0129]
Example 2 Evaluation of full length of 5 'end of clone from cDNA library prepared by oligocap method
The base sequence of the 5 ′ end of the human cDNA library prepared in Example 1 is compared with the sequence of known human mRNA in the public database, and all clones having the same 5 ′ end sequence are known in the public database. When the 5 ′ end extends longer than the mRNA sequence, or when the 5 ′ end is short but has a translation initiation codon, it is determined as “full length”, and when the translation initiation codon is not included, “non-full length” It was judged.
[0130]
Next, clones were evaluated using ESTiMateFL. ESTiMateFL is a method developed by Nishikawa and Ota et al. Of Helix Laboratories in order to select clones with high possibility of full-length cDNA by comparing with EST 5 'and 3' end sequences in public databases. is there. The 5 ′ end or 3 ′ end sequence of the cDNA clone analyzed in Example 1 is compared with the base sequence registered in the EST database, and extends to the 5 ′ side or 3 ′ side of the obtained cDNA clone sequence. When the EST was present, it was judged that the clone was “possibly not full length”. When the 5 'end is extended from the EST sequence in the public database, or even if the 5' end is short, the difference is within 50 bases for the sake of convenience, and when it is shorter than that, the non-full length is assumed. .
[0131]
Example 3 Analysis of nucleotide sequence and amino acid sequence of cDNA clone
According to BLAST (Basic local alignment search tool; Altschul, SF, et al., J. Mol. Biol., 215: 403-410 (1990)) for the entire nucleotide sequence of the cDNA clone analyzed in Example 2. Protein feature search by HMMPFAM which is one of the functional groups of homology search (homology search) and HMMER (sequence analysis method by hidden Markov model; Eddy, SR, Bioinformatics, 14: 755-763 (1998)) profile search: http://pfam.ustl.edu), and the function of the protein encoded by each cDNA clone was estimated. In addition, with regard to a clone presumed to be a splicing variant in which there is a known clone in which a part of the base sequence is completely identical, any exon deleted and spliced if the genome sequence can be analyzed It was analyzed.
[0132]
Example 4 Measurement of transcriptional regulatory activity (analysis of binding activity to target transcriptional regulatory region)
(1) Preparation of the protein of the present invention using a cell-free protein synthesis system
The cDNA clone presumed to have transcriptional regulatory activity in Example 3 was synthesized using a cell-free protein synthesis system, and whether or not the protein had transcriptional regulatory activity was determined by the following biochemical method. It was analyzed by experiment.
[0133]
PCR method using the ORF fragment of the cDNA clone presumed to have transcriptional regulatory activity in Example 3 using the following primers specific for each clone as 5'-side primers and the following common primers as 3'-side primers Obtained by.
[0134]
5 'primer
(A) c-nt2ri3007878: ATGATCCCAGTCACAGATACATCATTGAG (SEQ ID NO: 25)
(B) c-bramy2043103: ATGCCGCGCATGCGC (SEQ ID NO: 26)
(C) c-brsn2013753: ATGCCAACCATCATCAGACAC (SEQ ID NO: 27)
(D) c-brsn2017530: ATGGTCCCAAATTATGAGAACCCTGG (SEQ ID NO: 28)
(E) c-hcho20000473: ATGACACCAGCATCTGCCATC (SEQ ID NO: 29)
(F) c-testi 4004626: ATGATTTATCTCAGTGTGCATGGGTTG (SEQ ID NO: 30)
(G) c-trach 3014316: AGTTCAGGTGGGTGAGAACAGGG (SEQ ID NO: 31)
(H) c-best2001444: ATGTCCGACGGAGCCG (SEQ ID NO: 32)
3 'common primer
GGCCCTTATGGCCGGAGAAAAGGCGGACAGGTAT (SEQ ID NO: 33)
[0135]
A translation control region containing SP6 promoter-glutathione-S-transferase gene-PreScission Protease (manufactured by Amersham Pharmacia Biotech) -cleavage site-DNA cloning site (SmaI, SfiI) -poly (A) signal sequence vector (pEU) -Inserted into the cloning site of SS4).
[0136]
Using the plasmid DNA prepared above as a template, transcription was performed using SP6 RNA polymerase (Promega), and the resulting mRNA was extracted with phenol / chloroform and precipitated with ethanol, followed by Nick Column (Amersham Pharmacia Biotech). Purified by
[0137]
Protein synthesis using the mRNA purified as described above is described in JP-A No. 2002-204689 and Proc. Natl. Acad. Sci. USA, 99: 14652-14657 (2002). The translation solution (25 μl) used in the layered cell-free protein synthesis system includes Proc. Natl. Acad. Sci. USA, 97: 559-564 (2000), 6 μl of wheat germ extract and the above-mentioned mRNA (0.02 nmol) were added and used, the composition being 24 mM Hepes / KOH (pH 7.8), 1. 2 mM ATP, 0.25 mM GTP, 16 mM creatine phosphate, 10 μg creatine kinase, ribonuclease inhibitor (20 units), 2 mM DTT, 0.4 mM spermidine, 0.3 mM sac um sac met 5 μg wheat germ derived tRNA, 0.05% Nonidet P-40 and 0.005% NaN₃Consists of. The translation buffer was 31.3 mM HEPES / KOH (pH 7.8), 2.67 mM Mg (OAc).₂, 93 mM KOAc, 1.2 mM ATP, 0.257 mM GTP, 16 mM creatine phosphate, 2.1 mM DTT, 0.41 mM spermidine, 0.3 mM L-type amino acid (20 species), 1 μM E-64, 0.005% NaN₃0.05% NP-40. In cell-free protein synthesis by the multilayer method, 125 μl of translation buffer was first added to a 96-well plate, and the translation solution was slowly layered from the bottom into each hole containing the translation buffer. This was carried out by incubating for 16 hours while keeping the temperature in a incubator.
[0138]
(2) SPR measurement method using a sensor chip on which DNA having a target sequence or a consensus target sequence is immobilized
According to the description of BIAapplications handbook, chapter 4.4, the following 53 types of double-stranded DNA biotinylated were immobilized separately on the surface of the sensor chip. The sensor chip used was SA type (manufactured by Biacore), and BIACORE 3000 (manufactured by Biacore) was used for SPR measurement and analysis.
[0139]
The relationship between known transcriptional regulators and designed or consensus target sequences and the functions of these transcriptional regulators are as follows.
[1] v-jun, c-jun, junB, junD, dJRA, c-fos, fosB1, fosB2, Fra-1, LRF-1, v-maf, mafG, NF-E2 p45, aNF-E2, fNF- E2, Nrf short form, GCN4, yAP-1, CREB-2, ATF-3, CRE-BP1, CRE-BP3, ATF-a, CREB-341, CREB-327, CREM, dCREB2, dCREB2-b, dCREB2- consensus target sequence of c, dCREB2-d, dCREB2-q, dCREB2-r, dCREB2-s: TGATGACGT (SEQ ID NO: 34)
[2] Consensus target sequence of C / EBPα, C / EBPβ, p34C / EBPβ, and CHOP-10: AAGTGGCGAAAGAGACA (SEQ ID NO: 35)
The above transcriptional regulatory factors are known to be involved in, for example, induction of insulin resistant diabetes, intermittent ketonuria, and multidrug resistance.
[3] VBP, Hlf, CPRF-2, EmBP-1b, EmBP-1b, GBF1, GBF2, GBF3, CPRF-1, TAF-1, HBP-1a, GBF9, GBF1, GBF12, CPRF-3, TGA1a, TGA1b , O2, STE4 consensus target sequence: AGAAGCACGTGGG (SEQ ID NO: 36)
[4] Consensus target sequence of OPI1, E2A, E47, ITF-2 / SEF2-1B, SEF-1A, MyoD, p42Tal-1: AACAGATGGGT (SEQ ID NO: 37)
[5] Target sequence of HEN-1: GGGGCGCAGCTGGCGCCC (SEQ ID NO: 38)
[6] AhR, Arnt consensus target sequence: GGGGATTGCCGTG (SEQ ID NO: 39)
The above transcriptional regulatory factors are universally expressed in most cells and tissues in adults, and are known to be involved in, for example, induction of drug metabolizing enzymes by dioxins.
[7] USF target sequence: GTCACGTGGT (SEQ ID NO: 40)
[8] Consensus target sequence of NF-1A1, NF-1A1.1, NF-1A6, NF-1B1, NF-1B1, NF-1B2, NF-1C2 / CTF-2, CTF-4, CTF-6: CTGTGGGGGTTTGGCACGGGGCCCA (SEQ ID NO: 41)
The above transcriptional regulators are not only involved in transcription of housekeeping genes, but also include transcription of many cell-specific genes such as activation of transcription by TGF-β and suppression of glucose transporter gene expression by insulin. It has been shown to be involved in the promotion and suppression of It is also suggested that it may interact with oncogene function.
[9] RF-X1 target sequence: GGTAACATAGCAAC (SEQ ID NO: 42)
[10] Consensus target sequence of AP2αA / AP-2α1, AP2α2, AP2α3, AP2α4, AP2αB, AP2β, AP2γ: CGCCCCCCGGCG (SEQ ID NO: 43)
[0140]
[11] Target sequence of GR: GGTACAAAATGTTCT (SEQ ID NO: 44)
The above transcriptional regulatory factors include, for example, promotion of Glucocorticoid release, stress response,
Glucocorticoid requirement in fetus, pulmonary hypoplasia, angiogenesis related cancer, uterine maladaptation in early pregnancy, growth hormone deficiency, fetal intrauterine growth retardation, uterine hypoxia, osteogenesis related disease, vitamin D It is known to be involved in the induction of
[12] AR target sequence: AACATTAGTTTCT (SEQ ID NO: 45)
[13] Target sequence of ER: AAGGGGAAAATGACCCC (SEQ ID NO: 46)
[14] RXR-α target sequence: GGTCATAGGGGT (SEQ ID NO: 47)
The above transcriptional regulators include juvenile-onset diabetes, obesity, drug metabolism in the liver, cancer, low HDL / low apoprotein, atherosclerosis, diarrhea, dyspepsia, malnutrition, multiple sclerosis, RA, SLE, insulin dependence It is known to be involved in the induction of cardiovascular diseases such as diabetes mellitus, Crohn's disease, asthma, high HDL, low β lipoproteinosis, high α lipoproteinosis, amyloidosis, arteriosclerosis and the like.
[15] Target sequence of PPARα: CTAGGGCAAAGGTCA (SEQ ID NO: 48)
As mentioned above, the above transcriptional regulators are mainly expressed in fat consuming organs such as liver, kidney, brown adipocytes, and other myocardium and gastrointestinal tract, and are used for fatty acid oxidation, ketone body production, apolipoprotein production, etc. It regulates the expression of various target genes involved in cardiovascular and carcinogenic mechanisms such as endocrine, sugar and lipid metabolism related to the onset of lifestyle-related diseases such as diabetes and obesity, as well as vascular function and inflammation. Known as a disease-related transcriptional regulator
[16] Target sequence of PPARγ: GGTCAAAGGTCA (SEQ ID NO: 49)
As described above, the above transcriptional regulators are expressed in adipocytes, adipose tissue, immune system organs, adrenal glands, and small intestine, and are thought to play an important role in adipocyte differentiation induction and fat synthesis. .
[17] COUP-TF1, HNF-4α1, HNF-4α2 target sequences: TGAACTTTGA (SEQ ID NO: 50)
[18] Target sequence of CF1: GGGGTCACC (SEQ ID NO: 51)
[19] GATA-1, GATA-2, GATA-3, GATA-4 consensus target sequence: CCAGATAAGG (SEQ ID NO: 52)
The above transcriptional regulators are expressed in internal organs such as blood cells, nervous system, heart and intestine, and differentiation, formation, maintenance, proliferation and apoptosis of blood cells, leukemia, spermatogenesis, differentiation of nervous system cells And has been shown to be involved in formation, organogenesis, and the like.
[20] AREA / NIT-2 target sequence: TATCTC (SEQ ID NO: 53)
[0141]
[21] Sp1 target sequence: GGGGGGGGGG (SEQ ID NO: 54)
The above transcriptional regulators have been shown to be involved in transcription of oncogenes and viral genes as well as transcription of housekeeping genes
[22] YY1 target sequence: CGGCCCATCTGGCT (SEQ ID NO: 55)
[23] Consensus target sequence of Egr-1, Egr-2, Egr-3: TGCGTGGGCG (SEQ ID NO: 56)
[24] Target sequence of Snail: CACCTGTTTTCA (SEQ ID NO: 57)
[25] Target sequence of CF2-II: GTATATATA (SEQ ID NO: 58)
[26] Target sequence of Evi-1: AGATA AGATAA (SEQ ID NO: 59)
[27] Ikaros, MZF-1 consensus target sequence: TTGGGAGG (SEQ ID NO: 60)
[28] Target sequence of Tramtrack69K: GGACCTGC (SEQ ID NO: 61)
[29] Target sequence of HOX9: TGACAGTTTAACGA (SEQ ID NO: 62)
[30] CDP target sequence: CCAATAATCGAT (SEQ ID NO: 63)
[0142]
[31] Target sequence of HNF-1A: GGTTAATGATTAACCAC (SEQ ID NO: 64)
[32] Consensus target sequence of Nkx-2.2, Nkx-2.5, TTF-1: TTAAGTGTT (SEQ ID NO: 65)
[33] Consensus target sequence of Oct-1A, Oct-1B, Oct-1C: ATGCAAAT (SEQ ID NO: 66)
[34] Consensus target sequence of Oct-2, Oct-2.1 / Oct-2B: TATTTGCAT (SEQ ID NO: 67)
[35] Consensus target sequence of Pax-3, Pax-6: CGTCACGCTTTGA (SEQ ID NO: 68)
[36] Target sequence of Pax-1: CCGTTCCGCTCTAGATAT (SEQ ID NO: 69)
[37] Consensus target sequence of HSF1 (short), HSF2, dHSF, fungal HSF: AGAAAGAAAAAGAAA (SEQ ID NO: 70)
The above transcription regulatory factors are universally expressed in most cells and tissues, and are known to be involved in, for example, the heat shock response.
[38] Consensus target sequence of c-Myb, A-Myb, v-Myb, P (long), P (short), C1 (long), C1 (short): AACGGGCCC (SEQ ID NO: 71)
[39] c-Ets-1_p54, Ets-1_δiV / VII, Ets-2, Elk-1, SAP-1, SAP-1b, Erg-1, p55erg, Fli-1b, E4TF1-60 / GABP-α, E74A Consensus target sequence: GACAGGAAGTG (SEQ ID NO: 72)
The above transcriptional regulatory factors are known to be involved in the induction of cancer, circadian rhythm-related diseases and the like.
[40] IRF-1, IRF-2 consensus target sequence: GAAAAGCGAAACC (SEQ ID NO: 73)
[0143]
[41] p50 target sequence: GGGGATTTTCC (SEQ ID NO: 74)
[42] NF-ATc, NF-Atp consensus target sequence: AGGAAAA (SEQ ID NO: 75)
[43] p91 (STAT1), p84 consensus target sequence: GAATTCCGGGAAATG (SEQ ID NO: 76)
It is known that the above transcriptional regulatory factors are stimulated and activated by various interferons and are involved in protection against viral and bacterial infections.
[44] Consensus target sequence of STAT2, STAT3, STAT4, STAT5A, STAT5B, STAT6: TTTCCCGGGAAATG (SEQ ID NO: 77)
The above transcription regulatory factors are known to be stimulated and activated by cytokines such as various interferons and various interleukins, and are involved in cancer, autoimmune diseases and inflammation.
[45] Target sequence of p53: GGACATGCCCGGGGCATGTC (SEQ ID NO: 78)
As mentioned above, the above transcriptional regulators are known to be tumor suppressor genes, are involved in signal transduction that causes apoptosis, and stop the cell cycle while cells perform DNA repair. Because of its function, it has attracted attention as a target molecule for therapeutic agents such as cancer.
[46] Target sequence of MEF-2A: CTCTAAAAAATA (SEQ ID NO: 79)
[47] SRF target sequence: CCATATATGGACAT (SEQ ID NO: 80)
[48] E2 target sequence: AACCAAAAACGGTAA (SEQ ID NO: 81)
[49] TBP target sequence: TATAAA (SEQ ID NO: 82)
[50] Consensus target sequence of SRY, Sox-5, Sox-9: AAAAAACAATAGGGG (SEQ ID NO: 83)
The above transcriptional regulatory factors are known to be involved in the development / differentiation of nervous system development / differentiation, cartilage formation, sex differentiation and the like.
[51] mat-Mc target sequence: TCATTGTT (SEQ ID NO: 84)
[52] CP1A, CP1B, CBF-C consensus target sequence: CTGATGTGCACC (SEQ ID NO: 85)
[53] Target sequence of AML1a: TGTGGT (SEQ ID NO: 86)
[0144]
First, regarding the DNAs having the 53 types of target sequences or consensus target sequences (SEQ ID NO: 34 to SEQ ID NO: 86), DNAs having biotin added to the 5 ′ side and DNAs having complementary base sequences thereof are individually separated by a conventional method. In total, 53 types of double-stranded DNA were prepared. On the other hand, the sensor cell used for the analysis has one flow cell divided into four. The flow cell 1 was used as a control section without immobilizing anything, and the flow cells 2, 3 and 4 were each immobilized with three types of the double-stranded DNA prepared above. In order to make the DNA immobilization density of the sensor chip constant, the value (D) obtained by dividing the increase in SPR response value (ΔRU-DNA) by DNA immobilization by the DNA molecular weight (MW) is constant in each flow cell. ΔRU-DNA was regulated. The remaining DNA was immobilized in the same manner.
[0145]
After the sensor chip was prepared as described above, the binding activity analysis with the protein of the present invention encoded by the clone cDNA was performed. There have already been many reports on the binding activity analysis between DNA and protein by the SPR method. In this example, the measurement was performed with reference to the measurement conditions of Molecular Microbiology, 36 (3), 557-569 (2000). First, the flow cell 1-2-3-4 is set to a flow path connected in series. The running buffer is allowed to flow at a constant flow rate (5 μL / min) to stabilize the SPR measurement value, and then the baseline value (SPR-baseline) of each flow cell is measured. Next, the protein solution is flowed at the same flow rate to form a specific bond between the protein molecule and the DNA strand. After injecting for a certain time, the SPR response value (SPR-bound) of each flow cell was measured.
[0146]
(4) Analysis method of measurement results obtained by SPR method
By subtracting the baseline value from the SPR response value ([SPR-bound]-[SPR-baseline]), the true binding amount (B) was determined, and further, the standardized value (nB) was determined.
From the measurement results, the protein (SEQ ID NO: 11 to 20) encoded by the clone of the present invention is a total of 53 types of target sequences or consensus target sequences (SEQ ID NO: 34 to SEQ ID NO: 86) of [1] to [53]. It was found to have a binding activity for at least one of the DNAs having (Table 1). In Table 1, A indicates that nB is 100 or more, B indicates that nB is 50 or more and less than 100, C indicates that nB is 5 or more and less than 50, and D indicates that nB is 0 to less than 5.
[0147]
[Table 1]

[0148]
From Table 1, the protein encoded by c-nt2ri3007878 is the target sequence of TBP: TATAAA (SEQ ID NO: 82), consensus target sequence such as SRY: AAAAAACAATAGGG (SEQ ID NO: 83), mat-Mc target sequence: TCATTGTT (sequence) No. 84) showed a very high affinity. This indicates that the protein has DNA binding properties and may be a transcriptional regulator associated with diseases caused by abnormalities such as development / differentiation of the nervous system, cartilage formation, and sex differentiation. In addition, the protein exhibits high affinity for consensus target sequences such as Oct-2: TATTTGCAT (SEQ ID NO: 67), consensus target sequences such as HSF1 (short): AGAAAGAAAAAAAAA (SEQ ID NO: 70), and USF target sequence: GTCACGTGGT (SEQ ID NO: 40), GR target sequence: GGTACAAAATGTTCT (SEQ ID NO: 44), RXR-α target sequence: GGTCATAGGGGT (SEQ ID NO: 47), PPARα target sequence: CTAGGGCAAAGGTCA (SEQ ID NO: 48), c-Myb, etc. Consensus target sequences: AACGGGCCC (SEQ ID NO: 71), consensus target sequences such as c-Ets-1_p54: GACAGGAAGTG (SEQ ID NO: 72), consensus target sequences such as IRF-1 (SEQ ID NO: 7) 3) The p50 target sequence: GGGGATTTTCC (SEQ ID NO: 74), CP1A and other consensus target sequences: CTGATTGGCTACC (SEQ ID NO: 85) showed affinity.
[0149]
The proteins encoded by c-bramy2043103 are ER target sequence: AAGGGGAAAATGACCCC (SEQ ID NO: 46), RXR-α target sequence: GGTCATAGGGGT (SEQ ID NO: 47), PPAR-α: CTAGGGCAAAGGTCA (SEQ ID NO: 48), Pax-3 Consensus target sequences such as: CGTCACGCTTTGA (SEQ ID NO: 68), Pax-1 target sequence: CCGTCCCCTCTTAGATAT (SEQ ID NO: 69) and HSF1 and other consensus target sequences: AGAAAGAAAAAAAAA (SEQ ID NO: 70). This indicates that the protein has DNA-binding properties and may be a transcriptional regulator associated with diseases caused by abnormalities such as lipid metabolism, drug metabolism, and muscle formation, such as diseases such as lifestyle-related diseases.
[0150]
The protein encoded by c-brassn2013753 showed very high affinity to consensus target sequences such as GATA-1: CCAGATAAGG (SEQ ID NO: 52), and AREA / NIT-2 target sequence: TATCTC (SEQ ID NO: 53) . This indicates that the protein has DNA-binding properties and may be a transcriptional regulator associated with diseases caused by abnormalities such as hematopoietic stem cell differentiation, hematopoietic cells, neural cells, circulatory organs, and apoptosis control. . In addition, the protein shows high affinity for the target sequence of HNF-1A: GGTTAATGATTACCACC (SEQ ID NO: 64), the target sequence of PPARγ: GGTCAAAGGTCCA (SEQ ID NO: 49), and the target sequence of CF2-II: GTATATATA (SEQ ID NO: 58 ), Evi-1 target sequence: AGATAGATAAA (SEQ ID NO: 59), consensus target sequence such as Nkx-2.2: TTAAGTGGTT (SEQ ID NO: 65), consensus target sequence such as Oct-1A: ATGCAAAT (SEQ ID NO: 66), Consensus target sequences such as IRF-1: GAAAAGCGGAAACC (SEQ ID NO: 73), p50 target sequence: GGGACTTTCC (SEQ ID NO: 74), TBP target sequences: TATAAA (SEQ ID NO: 82), SRY, etc. Column: AAAAAACAATAGGG (SEQ ID NO: 83), the target sequence of AML1a: showed affinity TGTGGT (SEQ ID NO: 86).
[0151]
The protein encoded by c-brassn2017530 is Sp1 target sequence: GGGGGGGGGG (SEQ ID NO: 54), Tramtrack69K target sequence: GGACCCTGC (SEQ ID NO: 61), HOX9 target sequence: TGACAGTTTAACGA (SEQ ID NO: 62) and CDP target sequence: It showed high affinity for CCAATAATCGAT (SEQ ID NO: 63). This indicates that the protein has DNA-binding properties and is a transcriptional regulator associated with diseases such as hematopoietic stem cell differentiation, apoptosis control, cytokine production, and other diseases such as viral diseases, bacterial diseases, and cancers. There is a possibility. In addition, the protein includes consensus target sequences such as OPI1: AACAGATGGGT (SEQ ID NO: 37), HEN-1 target sequence: GGGGCGGCAGCTGCGGCCCC (SEQ ID NO: 38), AhR and other consensus target sequences: GGGGATTGCCGTG (SEQ ID NO: 39), p91 ( Consensus target sequences such as STAT1): GAATTCCGGGAAATG (SEQ ID NO: 76), consensus target sequences such as STAT2: TTTCCCGGGAAATG (SEQ ID NO: 77), and p53 target sequence: GGACATGCCCGGGGCATGGTC (SEQ ID NO: 78).
[0152]
The proteins encoded by c-hcho20000473 are HEN-1 target sequence: GGGGCGGCGCTGCGGCCC (SEQ ID NO: 38), consensus target sequence such as AhR: GGGGATTGCCGTG (SEQ ID NO: 39), USF target sequence: GTCACGTGGT (SEQ ID NO: 40), NF Consensus target sequences such as -1A1: CGTTGGGGTTTGGCACGGGGCCA (SEQ ID NO: 41), RF-X1 target sequence: GGTAACATAGGCAAC (SEQ ID NO: 42), Sp1 target sequences: GGGGGGGGGGGG (SEQ ID NO: 54), Egr-1 consensus target sequences: TGCGTGGGCG (SEQ ID NO: 56), p50 target sequence: GGGGATTTTCC (SEQ ID NO: 74), NF-ATc target sequence: AGGAAAA (sequence) No. 75), consensus target sequence such as p91: GAATTCCGGGAAATG (SEQ ID NO: 76), consensus target sequence such as STAT2: TTTCCCGGGAAATG (SEQ ID NO: 77) and p53 target sequence: GGACATGCCCGGGGCATGTTC (SEQ ID NO: 78) Indicated. This indicates that the protein has DNA-binding properties, and transcriptional regulation associated with diseases such as cell cycle, inflammation, apoptosis control, immune reaction, drug metabolism, cytokine production, diseases such as viral diseases, cancer, etc. It may be a factor. In addition, the protein has a high affinity for the target sequence of AREA / NIT-2: TATCTC (SEQ ID NO: 53), a consensus target sequence such as C / EBPα: AAGTGGCGAAAGAGACA (SEQ ID NO: 35), and a consensus target such as IRF-1. The sequence: Affinity was shown for GAAAAGCGAAACC (SEQ ID NO: 73).
[0153]
Proteins encoded by c-testi4004626 are YY1 target sequence: CGGCCCATCTTGCT (SEQ ID NO: 55), consensus target sequences such as Egr-1: TGCGTGGGCG (SEQ ID NO: 56), Snail target sequence: CACCTGTTTTCA (SEQ ID NO: 57), CF2 -II target sequences: GTATATATA (SEQ ID NO: 58), IRF-1, IRF-2 consensus target sequences: GAAAAGCGAAACC (SEQ ID NO: 73), NF-ATc and other consensus target sequences: AGGAAAA (SEQ ID NO: 75), and p91 The consensus target sequence such as: GAATTCCGGGAAATG (SEQ ID NO: 76) showed affinity. This indicates that the protein has DNA-binding properties and may be a transcriptional regulator associated with diseases caused by abnormalities such as immune responses and cytokine production.
[0154]
The protein encoded by c-trach 3014316 is a consensus target sequence such as GATA-1: consensus target sequence such as CCATATAAGG (SEQ ID NO: 52), Nkx-2.2, etc .: TTAAGTGGTT (SEQ ID NO: 65) and Oct-1A The sequence: ATGCAAAT (SEQ ID NO: 66) showed high affinity. This indicates that the protein has DNA-binding properties and may be a transcriptional regulator associated with diseases caused by abnormalities such as hematopoietic stem cell differentiation, hematopoietic cells, neural cells, circulatory organs, and apoptosis control. . Further, the protein includes consensus target sequences such as VBP: AGAAGCACGTGGG (SEQ ID NO: 36), USF target sequence: GTCACGTGGT (SEQ ID NO: 40), NF-1A1 and other consensus target sequences: CGTTGGGGTTTGGCACGGGGCCA (SEQ ID NO: 41), PPARα. Target sequence: CTAGGGCAAAGGTCA (SEQ ID NO: 48), PPARγ target sequence: GGTCAAAGGTCA (SEQ ID NO: 49), Snail target sequence: CACCTGTTTTCA (SEQ ID NO: 57), CDP target sequences: CCAATAATCGAT (SEQ ID NO: 63), HSF1 (short) Consensus target sequences such as: AGAAAGAAAAAGAAA (SEQ ID NO: 70), c-Ets-1_p54 and other consensus target sequences: GACAGG AAGTG (SEQ ID NO: 72), consensus target sequences such as IRF-1: GAAAAGCGGAAACC (SEQ ID NO: 73), consensus target sequences such as p91 (STAT1): GAATTCCGGGAAATG (SEQ ID NO: 76), consensus target sequences such as STAT2: TTTCCCGGGAAATG (sequence) No. 77), SRF target sequence: CCATATATGGACAT (SEQ ID NO: 80), E2 target sequence: AACCAAAAACGGTAA (SEQ ID NO: 81).
[0155]
The proteins encoded by c-best2001444 are NF-1A1 and other consensus target sequences: CTGTGGGGTTTGGCACGGGGCCA (SEQ ID NO: 41), RF-X1 target sequences: GGTAACATAGCAAC (SEQ ID NO: 42), YY1 target sequence: CGCCCCATCTTGGC (SEQ ID NO: 55) Snail target sequence: CACCTGTTTTCA (SEQ ID NO: 57), Evi-1 target sequence: AGATAGATATA (SEQ ID NO: 59), Ikaros et al. Consensus target sequence: TTGGGAGG (SEQ ID NO: 60), HNF-1A target sequence: GGTTAATGATTATAACCAC ( SEQ ID NO: 64), consensus target sequences such as Nkx-2.2: consensus such as TTAAGTGTT (SEQ ID NO: 65), Oct-1A Consensus target sequence: ATGCAAAT (SEQ ID NO: 66), consensus target sequence such as Oct-2: TATTTGCAT (SEQ ID NO: 67), consensus target sequence such as Pax-3: CGTCACGCTTTGA (SEQ ID NO: 68), Pax-1 target sequence: CCGTCCCGCTCTAGATAT (SEQ ID NO: 69), consensus target sequences such as NF-ATc: AGGAAAA (SEQ ID NO: 75), MEF-2A target sequence: CCTAAAAATA (SEQ ID NO: 79), SRF target sequences: CCATATATGGACAT (SEQ ID NO: 80) and E2 The target sequence of: AACCAAAAACGGTAA (SEQ ID NO: 81) showed affinity. Therefore, the protein has DNA binding properties, and is caused by diseases such as proliferation of hematopoietic cells and hepatocytes, generation / differentiation of immune system cells, lipid metabolism, muscle formation, such as diseases such as lifestyle-related diseases. It may be an associated transcriptional regulator.
[0156]
Example 5 Measurement of transcriptional regulatory activity (analysis of regulatory activity on disease-related transcriptional regulatory factor)
(1) Preparation of the protein of the present invention using a cell-free protein synthesis system
The regulatory activity of the protein encoded by the cDNA of the present invention was further analyzed for disease-related transcriptional regulatory factors. The protein encoded by the cDNA of each clone was prepared according to the method of Example 4 (1).
[0157]
(2) Preparation of analysis plate
The DNA used in the measurement system for the regulatory action activity of a transcriptional regulatory factor on a disease-related transcriptional regulatory factor has a base sequence to which the disease-related transcriptional regulatory factor binds, and biotin (denoted as Bio) is present on the 5 ′ side. Double-stranded DNA was used by annealing the added single-stranded DNA (denoted as oligo-A) and the single-stranded DNA having its complementary sequence (oligo-B) as described. The DNA sequence used is described below.
[0158]
Double-stranded DNA obtained by annealing biotinylated DNA containing SEQ ID NO: 87 and SEQ ID NO: 88 as a sequence recognized by the disease-related transcription regulator PPARγ
oligo-A; 5'-Bio-GGAACTAGGTCAAAGGTCATCCCCCT-3 '(SEQ ID NO: 87)
oligo-B; 3'-CCTTGATCCAGTTTCCAGTAGGGGA-5 '(SEQ ID NO: 88)
[0159]
Double-stranded DNA obtained by annealing biotinylated DNA containing SEQ ID NO: 89 and SEQ ID NO: 90 as a sequence recognized by the disease-related transcription regulatory factor p53
oligo-A; 5'-Bio-CTTGGACATGCCCGGGGCATGTCCCTC-3 '(SEQ ID NO: 89)
oligo-B; 3'-GAACCTGTACGGGGCCCGTACAGGGAG-5 '(SEQ ID NO: 90)
[0160]
Double-stranded DNA obtained by annealing biotinylated DNA containing SEQ ID NO: 91 and SEQ ID NO: 92 as a sequence recognized by the disease-related transcriptional regulatory factor NFκB
oligo-A; 5'-Bio-AGTTGAGGGGACTTTCCCAGC-3 '(SEQ ID NO: 91)
oligo-B; 3'-TCAACTCCCCCTGAAAGGGTCCG-5 '(SEQ ID NO: 92)
[0161]
Double-stranded DNA obtained by annealing biotinylated DNA containing SEQ ID NO: 93 and SEQ ID NO: 94 as a sequence recognized by the disease-related transcription regulatory factor AP-1
oligo-A; 5'-Bio-CGCTTGATGAGGTCAGCCCGGAA-3 '(SEQ ID NO: 93)
oligo-B; 3'-GCGAACTACTCAGGTCGCCCTT-5 '(SEQ ID NO: 94)
[0162]
Double-stranded DNA obtained by annealing biotinylated DNA containing SEQ ID NO: 95 and SEQ ID NO: 96 as a sequence recognized by the disease-related transcriptional regulatory factor HIF-1
oligo-A; 5'-Bio-GATCCGCCCTACGTGCTGTCTCGAGATC-3 '(SEQ ID NO: 95)
oligo-B; 3'-CTAGCGGGATGCACGACAGAGCTCAG-5 '(SEQ ID NO: 96)
[0163]
Double-stranded DNA obtained by annealing biotinylated DNA containing SEQ ID NO: 97 and SEQ ID NO: 98 as a sequence recognized by the disease-related transcriptional regulatory factor CREB
oligo-A; 5'-Bio-AGAGATTGCCCGACGTCAGAGAGCTAG-3 '(SEQ ID NO: 97)
oligo-B; 3'-TCTCTAACGGGACTGCAGTCTCTCGATC-5 '(SEQ ID NO: 98)
[0164]
A 96-well plate coated with streptavidin according to the method of BioTechniques, 32: 1168-1177 (2002) {biotin binding capacity 20 ng / well (80 pmol / well) Well), streptavidin-coated area 300 μl} or a plate attached to TransAM kit (ACTIVE MOTIF) or BD Mercury TransFactor kit was used as appropriate.
[0165]
(3) Analysis of regulatory activity against PPARγ
As an analysis sample, 1 μl of PMA-treated THP-1 cell nuclear extract solution (2.5 mg / ml ACTIVE MOTIF) containing PPARγ is diluted with 9 μl of a diluted solution {20 mM Hepes (pH 7.5), 400 mM NaCl, 20% glycerol, 0.1 mM. EDTA, 10 mM NaF, 10 μM Na₂MoO₄1 mM NaVO₃After mixing with 10 mM pNPP, 10 mM b-glycerophosphate, 1 mM DTT}, 35 μl of reaction solution {10 mM Hepes (pH 7.5), 4% glycerol, 50 mM NaCl, 0.5 mM EDTA, 1 mM MgCl₂10 μg / ml Herring sperm} and 5 μl of a cofactor solution containing the protein of the present invention prepared by the method of Example 5 (1) were used. As a positive sample, a mixture of 1 μl of PMA-treated THP-1 cell nucleus extraction solution, 9 μl diluted solution, 35 μl reaction solution and 5 μl of cofactor solution not containing the protein of the present invention was used. Further, as a negative sample, a 10 μl diluted solution, a 35 μl reaction solution and 5 μl of a cofactor solution not containing the protein of the present invention were mixed, and as a measurement blank sample, a 10 μl diluted solution and a 40 μl reaction solution were mixed. Each sample was reacted at room temperature for 30 minutes after mixing. Next, 50 μl of the above sample was added per well to the 96-well plate prepared in Example 5 (2) to which double-stranded DNA recognized by PPARγ was bound, and reacted at room temperature for 1 hour.
[0166]
Thereafter, the well is thoroughly washed with a washing buffer solution {10 mM phosphate buffer (pH 7.5), 50 mM NaCl, 0.1% Tween 20, 2.7 mM KCl}, and an antibody diluent {10 mM phosphate buffer ( 100 μl of an anti-PPAR goat antibody (0.2 μg / ml) dissolved in pH 7.5), 50 mM NaCl, 2.7 mM KCl, 10 mg / ml BSA} was added to each well, and further reacted at room temperature for 1 hour. Thereafter, the plate was thoroughly washed with a washing buffer, and 100 μl of HRP (horseradish peroxidase) -labeled anti-goat IgG antibody diluted 1000-fold with an antibody diluent was added to each well, followed by further reaction at room temperature for 1 hour. . After sufficient washing with a washing buffer, 100 μl of 1% DMSO solution containing a coloring substrate (TMB) was added to each well and reacted at room temperature for color development. Thereafter, the reaction was stopped by adding 100 μl of 0.5 M sulfuric acid solution to each well, and measurement was performed at a measurement wavelength of 450 nm and a reference wavelength of 655 nm. As the analysis result, the ratio of the difference between the measured value of the analyzed sample and the measured value of the positive sample with respect to the difference between the measured value of the positive sample and the negative sample was displayed as a percentage (%). That is, {(analysis sample value−positive sample value) / (positive sample value−negative sample value)} × 100. The results are shown in the “PPARγ evaluation” column of Table 2. In Table 2, -A is less than -50%, -B is -50% or more and less than -30%, -C is -30% or more and less than -10%, +/- D is -10% or more and 10% or more. Less than, + C is 10% or more and less than 30%, + B is 30% or more and less than 50%, and + A is 50% or more.
[0167]
[Table 2]

[0168]
The protein encoded by 4 clones of c-nt2ri3007878, c-brsn2017530, c-hchon20000473, and c-best2001444 has an activity to enhance or suppress the action of PPAR, and it may cause diabetes caused by abnormal endocrine / sugar / lipid metabolism, etc. It was speculated that it is related to lifestyle-related diseases such as obesity, circulatory system diseases due to abnormal vascular function, inflammation, cancer and the like.
[0169]
(4) Analysis of regulatory activity against p53
As an analysis sample, H containing activated p53₂O₂1 μl of treated MCF-7 cell nucleus extraction solution (2.5 mg / ml ACTIVE MOTIF) in 9 μl diluted solution {20 mM Hepes (pH 7.5), 400 mM NaCl, 20% glycerol, 0.1 mM EDTA, 10 mM NaF, 10 μM Na₂MoO₄1 mM NaVO₃After mixing with 10 mM pNPP, 10 mM b-glycerophosphate, 1 mM DTT}, 35 μl of reaction solution {20 mM Hepes (pH 7.5), 10% glycerol, 5 mM KCl, 0.5 mM EDTA, 5 mM MgCl₂A mixture of 1 mM DTT, 0.17 μg / ml poly [d (IC)]} and 5 μl of a cofactor solution containing the protein of the present invention prepared by the method of Example 5 (1) was used. H as a positive sample₂O₂A mixture of 1 μl of a treated MCF-7 cell nucleus extraction solution, 9 μl diluted solution, 35 μl reaction solution and 5 μl of a cofactor solution not containing the protein of the present invention was used. Further, as a negative sample, a 10 μl diluted solution, a 35 μl reaction solution and 5 μl of a cofactor solution not containing the protein of the present invention were mixed, and as a measurement blank sample, a 10 μl diluted solution and a 40 μl reaction solution were mixed. Each sample was reacted at room temperature for 30 minutes after mixing. Next, 50 μl of the above sample was added per well to the 96-well plate prepared in Example 5 (2) to which double-stranded DNA recognized by p53 was bound, and reacted at room temperature for 1 hour. Thereafter, the well is sufficiently washed with a washing buffer solution {10 mM phosphate buffer (pH 7.5), 50 mM NaCl, 0.1% Tween 20, 2.7 mM KCl}, and an antibody diluent {10 mM phosphate buffer 100 μl of anti-p53 rabbit antibody (0.2 μg / ml) dissolved in (pH 7.5), 50 mM NaCl, 2.7 mM KCl, 10 mg / ml BSA} was added to each well and further reacted at room temperature for 1 hour.
[0170]
Thereafter, the plate was thoroughly washed with a washing buffer solution, 100 μl of HRP (horseradish peroxidase) -labeled anti-rabbit IgG antibody diluted 1000 times with an antibody diluent was added to each well, and further reacted at room temperature for 1 hour. After thorough washing with a washing buffer, 100 μl of 1% DMSO solution containing a coloring substrate (TMB) was added to each well and reacted at room temperature for color development. Thereafter, 100 μl of 0.5 M sulfuric acid solution was added to each well to stop the reaction, and measurement was performed at a measurement wavelength of 450 nm and a reference wavelength of 655 nm. As the analysis result, the ratio of the difference between the measured value of the analyzed sample and the measured value of the positive sample with respect to the difference between the measured value of the positive sample and the negative sample was displayed as a percentage (%). That is, {(analysis sample value−positive sample value) / (positive sample value−negative sample value)} × 100. The results are shown in the “p53 evaluation” column of Table 2. In Table 2, -A is less than -50%, -B is -50% or more and less than -30%, -C is -30% or more and less than -10%, +/- D is -10% or more and 10% or more. Less than, + C is 10% or more and less than 30%, + B is 30% or more and less than 50%, and + A is 50% or more.
[0171]
It was speculated that the protein encoded by the three clones c-nt2ri3007878, c-brsn2017530, and c-testi4004626 has an activity to enhance or suppress the action of p53 and is related to cancer and the like.
[0172]
(5) Analysis of regulatory activity against NFκB
As an analysis sample, 1 μl of a TNF-α-treated HeLa cell nucleus extraction solution (2.5 mg / ml ACTIVE MOTIF) containing NFκB was added to 19 μl of a diluted solution {20 mM Hepes (pH 7.5), 350 mM NaCl, 20% glycerol, 1% Igepal. -CA630, 1 mM MgCl₂, 0.5 mM EDTA, 0.1 mM EGTA, 5 mM DTT} followed by 25 μl of reaction solution {4 mM Hepes (pH 7.5), 8% glycerol, 120 mM KCl, 1% BSA, 2 mM DTT, 10 μg / ml Herring sperm } And 5 μl of a cofactor solution containing the protein of the present invention prepared by the method of Example 5 (1) was used. As a positive sample, a mixture of 1 μl of a TNF-α-treated HeLa cell nucleus extraction solution, a 19 μl diluted solution, a 25 μl reaction solution, and 5 μl of a cofactor solution not containing the protein of the present invention was used. A negative sample was prepared by mixing a 20 μl diluted solution, a 25 μl reaction solution and 5 μl of a cofactor solution not containing the protein of the present invention, and a measurement blank sample was prepared by mixing a 20 μl diluted solution and a 30 μl reaction solution. Each sample was reacted at room temperature for 30 minutes after mixing. Next, 50 μl of the above sample was added per well to the 96-well plate prepared in Example 5 (2) to which the double-stranded DNA recognized by NFκB was bound, and reacted at room temperature for 1 hour.
[0173]
Thereafter, the wells are sufficiently washed with a washing buffer solution {10 mM phosphate buffer (pH 7.5), 50 mM NaCl, 0.1% Tween 20}, and an antibody diluent {10 mM phosphate buffer (pH 7.5), 100 μl of anti-NFκB antibody (0.2 μg / ml) dissolved in 50 mM NaCl, 0.1% Tween 20} was added to each well and allowed to react at room temperature for 1 hour. Thereafter, the plate was sufficiently washed with a washing buffer solution, 100 μl of HRP (horseradish peroxidase) -labeled anti-IgG antibody diluted with an antibody dilution solution 1000 times was added to each well, and further reacted at room temperature for 1 hour. After thorough washing with a washing buffer, 100 μl of 1% DMSO solution containing a coloring substrate (TMB) was added to each well and reacted at room temperature for color development. Thereafter, the reaction was stopped by adding 100 μl of 0.5 M sulfuric acid solution to each well, and measurement was performed at a measurement wavelength of 450 nm and a reference wavelength of 655 nm. As the analysis result, the ratio of the difference between the measured value of the analyzed sample and the measured value of the positive sample with respect to the difference between the measured value of the positive sample and the negative sample was displayed as a percentage (%). That is, {(analysis sample value−positive sample value) / (positive sample value−negative sample value)} × 100. The results are shown in the column of “NFκB evaluation” in Table 2. In Table 2, -A is less than -50%, -B is -50% or more and less than -30%, -C is -30% or more and less than -10%, +/- D is -10% or more and 10% or more. Less than, + C is 10% or more and less than 30%, + B is 30% or more and less than 50%, and + A is 50% or more.
[0174]
Proteins encoded by the three clones c-testi 4004626, c-trach 3014316, and c-best 2001444 have an activity to enhance or suppress the action of NFκB, and are immune diseases such as rheumatoid arthritis and osteoarthritis, inflammatory diseases It was speculated to be related to cancer and the like.
[0175]
(6) Analysis of regulatory activity against disease-related transcriptional regulator AP-1
As analysis samples, 1 μl of PMA containing activated AP-1 and Inmycin-treated WI-38 cell nuclear extract (2.5 mg / ml ACTIVE MOTIF) was diluted with 19 μl of a diluted solution {20 mM Hepes (pH 7.5), 400 mM NaCl, 20 % Glycerol, 0.1 mM EDTA, 10 mM NaF, 10 μM Na₂MoO₄1 mM NaVO₃After mixing with 10 mM pNPP, 10 mM b-glycophosphate, 1 mM DTT}, 25 μl reaction solution {10 mM Hepes (pH 7.5), 12% glycerol, 8 mM NaCl, 0.2 mM EDTA, 0.1% BSA, 1 mM DTT, A mixture of 0.17 μg / ml poly [d (IC)]} and 5 μl of a cofactor solution containing the protein of the present invention prepared by the method of Example 5 (1) was used. As a positive sample, PMA containing activated AP-1 and 1 μl of Inmycin-treated WI-38 cell nuclear extract solution, 19 μl diluted solution, 25 μl reaction solution and 5 μl of a cofactor solution not containing the protein of the present invention were used. As a negative sample, a 20 μl diluted solution, a 25 μl reaction solution and 5 μl of a cofactor solution not containing the protein of the present invention were mixed, and as a measurement blank sample, a 20 μl diluted solution and a 30 μl reaction solution were mixed. Each sample was reacted at room temperature for 30 minutes after mixing. Next, 50 μl of the above sample was added per well to the 96-well plate prepared in Example 5 (2) to which the double-stranded DNA recognized by AP-1 was bound, and reacted at room temperature for 1 hour. Thereafter, the well is thoroughly washed with a washing buffer solution {10 mM phosphate buffer (pH 7.5), 50 mM NaCl, 0.1% Tween 20, 2.7 mM KCl}, and an antibody diluent {10 mM phosphate buffer ( 100 μl of anti-phosphorylated-c-Jun antibody (0.4 μg / ml) dissolved in pH 7.5), 50 mM NaCl, 2.7 mM KCl, 1% BSA} was added to each well and allowed to react at room temperature for 1 hour. It was.
[0176]
Thereafter, the plate was thoroughly washed with a washing buffer solution, 100 μl of HRP (horseradish peroxidase) -labeled anti-IgG antibody diluted 1000 times with an antibody diluent was added to each well, and further reacted at room temperature for 1 hour. After thorough washing with a washing buffer, 100 μl of 1% DMSO solution containing a coloring substrate (TMB) was added to each well and reacted at room temperature for color development. Thereafter, the reaction was stopped by adding 100 μl of 0.5 M sulfuric acid solution to each well, and measurement was performed at a measurement wavelength of 450 nm and a reference wavelength of 655 nm. As the analysis result, the ratio of the difference between the measured value of the analyzed sample and the measured value of the positive sample with respect to the difference between the measured value of the positive sample and the negative sample was displayed as a percentage (%). That is, {(analysis sample value−positive sample value) / (positive sample value−negative sample value)} × 100. The result is shown in the column of “AP-1 evaluation” in Table 2. In Table 2, -A is less than -50%, -B is -50% or more and less than -30%, -C is -30% or more and less than -10%, +/- D is -10% or more and 10% or more. Less than, + C is 10% or more and less than 30%, + B is 30% or more and less than 50%, and + A is 50% or more.
[0177]
A protein encoded by 4 clones of c-nt2ri3007878, c-hchon20000473, c-testi4004626, c-trac3014316 has an activity to enhance or suppress the action of AP-1, and is related to inflammatory diseases and cancer It was speculated.
[0178]
(7) Analysis of regulatory activity against disease-related transcriptional regulatory factor HIF-1
CoCl containing HIF-1 as analysis sample₂1 μl of treated Cos-7 cell nuclear extraction solution (2.5 mg / ml ACTIVE MOTIF) 9 μl of diluted solution {20 mM Hepes (pH 7.5), 400 mM NaCl, 20% glycerol, 0.1 mM EDTA, 10 mM NaF, 10 μM Na₂MoO₄1 mM NaVO₃After mixing with 10 mM pNPP, 10 mM b-glycophosphate, 1 mM DTT}, 35 μl of reaction solution {10 mM Hepes (pH 7.5), 5% glycerol, 50 mM NaCl, 1 mM EDTA, 10 mg / ml BSA, 1 mM DTT} and Examples A mixture of 5 μl of a cofactor solution containing the protein of the present invention prepared by the method of 5 (1) was used. As a positive sample, CoCl containing HIF-1₂A mixture of 1 μl of treated Cos-7 cell nucleus extraction solution, 9 μl diluted solution, 35 μl reaction solution and 5 μl of cofactor solution not containing the protein of the present invention was used. As a negative sample, a 10 μl diluted solution, a 35 μl reaction solution and 5 μl of a cofactor solution not containing the protein of the present invention were mixed, and as a measurement blank sample, a 10 μl diluted solution and a 40 μl reaction solution were mixed. Each sample was reacted at room temperature for 30 minutes after mixing. Next, 50 μl of the above sample was added per well to the 96-well plate prepared in Example 5 (2) and bound with the double-stranded DNA recognized by HIF-1, and reacted at room temperature for 1 hour. Thereafter, the well is sufficiently washed with a washing buffer solution {10 mM phosphate buffer (pH 7.5), 50 mM NaCl, 0.1% Tween 20, 2.7 mM KCl}, and an antibody diluent {10 mM phosphate buffer 100 μl of anti-HIF-1 mouse antibody (0.25 μg / ml) dissolved in (pH 7.5), 50 mM NaCl, 2.7 mM KCl, 10 mg / ml BSA} was added to each well and allowed to react at room temperature for 1 hour. It was.
[0179]
Thereafter, the plate was thoroughly washed with a washing buffer, and 100 μl of HRP (horseradish peroxidase) -labeled anti-mouse IgG antibody diluted 1000-fold with an antibody dilution solution was added to each well and further reacted at room temperature for 1 hour. . After thorough washing with a washing buffer, 100 μl of 1% DMSO solution containing a coloring substrate (TMB) was added to each well and reacted at room temperature for color development. Thereafter, 100 μl of 0.5 M sulfuric acid solution was added to each well to stop the reaction, and measurement was performed at a measurement wavelength of 450 nm and a reference wavelength of 655 nm. As the analysis result, the ratio of the difference between the measured value of the analyzed sample and the measured value of the positive sample with respect to the difference between the measured value of the positive sample and the negative sample was displayed as a percentage (%). That is, {(analysis sample value−positive sample value) / (positive sample value−negative sample value)} × 100. The results are shown in the column of “HIF-1 evaluation” in Table 2. In Table 2, -A is less than -50%, -B is -50% or more and less than -30%, -C is -30% or more and less than -10%, +/- D is -10% or more and 10% or more. Less than, + C is 10% or more and less than 30%, + B is 30% or more and less than 50%, and + A is 50% or more.
[0180]
The protein encoded by the three clones c-brssn2017530, c-testi4004626, c-trac3014316 has an activity to enhance or suppress the action of HIF-1, resulting from abnormal angiogenesis, abnormal intracellular oxidation, etc. It was speculated that it is related to ischemic disease, anemia, hypoxia, diabetic retinopathy, cardiovascular disease, cancer and the like.
[0181]
(8) Analysis of regulatory activity on the disease-related transcriptional regulatory factor CREB
As an analysis sample, 1 μl of Forskolin-treated WI-38 cell nuclear extract solution (2.5 mg / ml ACTIVE MOTIF) containing CREB was added to 19 μl of a diluted solution {20 mM Hepes (pH 7.5), 400 mM NaCl, 20% glycerol, 0.1 mM. EDTA, 10 mM NaF, 10 μM Na₂MoO₄1 mM NaVO₃After mixing with 10 mM pNPP, 10 mM b-glycophosphate, 1 mM DTT, 25 μl reaction solution (10 mM Hepes (pH 7.5), 4% glycerol, 50 mM NaCl, 0.5 mM EDTA, 1 mM MgCl₂1% BSA, 1 mM DTT, 10 μg / ml Herring sperm) and 5 μl of a cofactor solution containing the protein of the present invention prepared by the method of Example 5 (1) were used. As a positive sample, a mixture of 1 μl of Forskolin-treated WI-38 cell nucleus extraction solution, 19 μl diluted solution, 25 μl reaction solution and 5 μl of cofactor solution not containing the protein of the present invention was used. A negative sample was prepared by mixing a 20 μl diluted solution, a 25 μl reaction solution and 5 μl of a cofactor solution not containing the protein of the present invention, and a measurement blank sample was prepared by mixing a 20 μl diluted solution and a 30 μl reaction solution. Each sample was reacted at room temperature for 30 minutes after mixing. Next, 50 μl of the above sample was added per well to the 96-well plate prepared in Example 5 (2) to which the double-stranded DNA recognized by CREB was bound, and reacted at room temperature for 3 hours.
[0182]
Thereafter, the well is sufficiently washed with a washing buffer solution {10 mM phosphate buffer (pH 7.5), 151 mM NaCl, 0.1% Tween 20, 2.7 mM KCl}, and an antibody diluent {10 mM phosphate buffer is added. 100 μl of anti-CREB antibody (0.2 μg / ml) dissolved in (pH 7.5), 151 mM NaCl, 2.7 mM KCl, 1% BSA} was added to each well and further reacted at room temperature for 1 hour. Thereafter, the plate was thoroughly washed with a washing buffer solution, 100 μl of HRP (horseradish peroxidase) -labeled anti-IgG antibody diluted 1000 times with an antibody diluent was added to each well, and further reacted at room temperature for 1 hour. After thorough washing with a washing buffer, 100 μl of 1% DMSO solution containing a coloring substrate (TMB) was added to each well and reacted at room temperature for color development. Thereafter, 100 μl of 0.5 M sulfuric acid solution was added to each well to stop the reaction, and measurement was performed at a measurement wavelength of 450 nm and a reference wavelength of 655 nm. As the analysis result, the ratio of the difference between the measured value of the analyzed sample and the measured value of the positive sample with respect to the difference between the measured value of the positive sample and the negative sample was displayed as a percentage (%). That is, {(analysis sample value−positive sample value) / (positive sample value−negative sample value)} × 100. The result is shown in the column of “CREB evaluation” in Table 2. In Table 2, -A is less than -50%, -B is -50% or more and less than -30%, -C is -30% or more and less than -10%, +/- D is -10% or more and 10% or more. Less than, + C is 10% or more and less than 30%, + B is 30% or more and less than 50%, and + A is 50% or more.
[0183]
The protein encoded by two clones of c-testi 4004626 and c-trac 3014316 has an activity to enhance or suppress the action of CREB and is related to central diseases such as memory impairment, cardiovascular diseases, diabetes, etc. Was guessed.
[0184]
Example 6 Human tissue expression analysis
In order to examine the tissue expression variation of mRNA encoding the human protein of the present invention in normal humans and diseased patients, the PCR method was performed according to a conventional method (Higuchi R, et al., Biotechnology, 11: 1026-30 (1993)). Tissue expression analysis using was performed.
[0185]
(1) cDNA synthesis
Commercially available mRNAs extracted and purified from 9 human tissues (Biochain: normal brain (# 510005), normal hippocampus (# 510068), normal thalamus (# 510077), normal kidney (# 510030), normal liver (# 510031), normal pancreas (# 510038), normal skeletal muscle (# 510047), normal fat (# 510018), normal spleen (# 510049), and human normal peripheral blood leukocytes commercially available total RNA (manufactured by Unitech) From the purified mRNA, reverse cDNA was synthesized by reverse transcription using oligo dT as a primer, and each cDNA was synthesized from 10 human tissues (manufactured by Clontech: normal breast and breast cancer ( # HP102B), normal colon and colon cancer (# HP102C), normal lung and lung cancer (# HP104L) , Normal stomach and gastric cancer (# HP101S), Bio-Chain Co., Ltd. normal frontal lobe (# 510068), using the Alzheimer's disease frontal lobe (# 550901).
[0186]
(2) Quantification by PCR method
Expression of mRNA encoding the human protein of the present invention (c-nt2ri3007878, c-febra2001620) was performed using a light cycler quantitative PCR apparatus (Roche Diagnostics) and a LightCycler-FastStart DNA master SYBR Green I reagent. Quantified according to the protocol attached to the product. The synthetic DNA sequence used for quantitative PCR is shown below.
c-nt2ri3007878
5'-side primer: GAAGTCGAACTGCCCAAAGGG (SEQ ID NO: 99)
3 'primer: ATTCCCCGTCAAAGGAGGT (SEQ ID NO: 100)
c-febra2001620
5 'primer: GCATCCATGTTGTTTAATTAAGTG (SEQ ID NO: 101)
3'-side primer: GTGCAAAGGGTGACTACTCA (SEQ ID NO: 102)
[0187]
The quantification results were corrected using Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as an internal standard. That is, the expression level (copy number / μl) of the target gene in each tissue is divided by the expression level (copy number / μl) of GAPDH, and a constant (1 × 10⁵). The results are shown in Table 3.
[0188]
[Table 3]

[0189]
As is apparent from Table 3, c-nt2ri3007878 mRNA was strongly expressed in the pancreas, expressed in the spleen, liver, and brain, and increased in the frontal lobe of Alzheimer's disease patients (AD frontal lobe).
c-febra2001620 mRNA was expressed in pancreas, spleen, and liver, and increased in breast cancer and lung cancer.
From this result, the cDNA of the clone and the protein encoded by the cDNA can be applied to the treatment and diagnosis of cancer, Alzheimer's disease and the like. In addition, the protein encoded by the cDNA may be involved in a disease relating to a tissue in which a change in mRNA expression is observed or a tissue having a high mRNA expression level as described above.
[0190]
Example 7 Analysis results of each cDNA clone
(1) c-nt2ri3007878 (SEQ ID NOS: 1 and 11)
c-nt2ri3007878 (hereinafter referred to as “the present DNA”, and the protein encoded by the DNA is referred to as “the present protein”), as shown in SEQ ID NO: 1, consists of 6156 bases, of which from base number 227 Up to 2245 is an open reading frame (including a stop codon). The amino acid sequence predicted from the open reading frame consists of 672 amino acid residues (SEQ ID NO: 11).
[0191]
When the homology search was performed using BLAST for the amino acid sequence encoded by SEQ ID NO: 1, an NRDB protein database (a database of non-overlapping amino acid sequences created from SWISS-PROT, PIR, TREMBLE, GENPEPT, PDB) , (I) Database registration symbol X60156, zinc finger protein (ZF7) (Human) was hit. As its contents, X60156 consists of 738 amino acids, and amino acid numbers 3 to 705 in the amino acid sequence are amino acid numbers 1 to 670 of the amino acid sequence described in SEQ ID NO: 11, e-value: 0.0, and 703 amino acids. Hits were made with 58% identity across the residues. In addition, (ii) database registration symbol Q14587 and zinc finger protein 268 (human) were hit. As its contents, Q14587 consists of 947 amino acids, and amino acid numbers 81 to 774 in the amino acid sequence are amino acid numbers 6 to 670 of the amino acid sequence described in SEQ ID NO: 11, e-value: 0.0, and 698 amino acids. Hits were made with 54% identity across the residues. Furthermore, (iii) database registration symbol B32891, finger protein 2, president (Human) was hit. As its contents, B32891 consists of 651 amino acids, and amino acid numbers 2 to 618 in the amino acid sequence are amino acid numbers 85 to 670 of the amino acid sequence described in SEQ ID NO: 11, e-value: 0.0, and 617 amino acid residues. It was a hit with 58% agreement over the group. From these results, it was speculated that the protein consisting of the amino acid sequence shown in SEQ ID NO: 11 was KRAB zinc finger protein.
The protein of (i) above is described in the literature information Nucleic Acid Res. 19 (20): 5661-7, it is a zinc finger protein having a finger preceding box (FPB; synonymous with KRAB), and the protein of (ii) above is DNA Cell Biol. 14 (2): 125-36, it was clarified that monocyte differentiation / proliferation is regulated.
[0192]
Further, protein characteristics search by HMMPFAM was performed on the amino acid sequence encoded by the base sequence shown in SEQ ID NO: 1, and as a result, amino acid numbers 200 to 222, 228 to 250, 256 to 278, 284 to 306, 312 to 3 of SEQ ID NO: 11 were performed. 334, 340-362, 368-390, 396-418, 424-446, 452-474, 480-502, 508-530, 536-558, 564-586, 592-614, 620-642, 648-670 A sequence showing the characteristics of C2H2 zinc finger (amino acid sequence entered as Pfam as zf-C2H2) was found in the amino acid sequence shown.
C2H2 type zinc finger has a structural motif in which two conserved Cys and two His are coordinated to zinc ions, and is found in many DNA-binding proteins such as transcription factors.
In addition, a sequence showing the characteristics of a Kruppel-associated box in the amino acid sequence shown by amino acid numbers 6 to 46 (amino acid sequence entered as PRAm as KRAB) was also found. Kruppel-associated box (KRAB) is found in approximately 1/3 of the C2H2-type Zn finger protein, exists in the N-terminal part, acts to repress transcription, and is involved in cell differentiation and development together with the Zn finger domain. Are known.
[0193]
Furthermore, analysis by PSORT II (Trends Biochem. Mol. Biol. 58 (4): 417-424 (1996)), which is a program for predicting the intracellular localization of the protein, revealed that the protein was localized in the cell. The probability of was found to be 100.0% in the nucleus. Therefore, this protein was predicted to be localized in the nucleus.
[0194]
In order to obtain genome information, using software sim4 (Genome Res. 8, 967-974 (1998)) that maps human cDNA to the human genome sequence, mapping of this DNA to the genome was attempted. Five exons were mapped to
Noun in q24 including the position (q24.3) on chromosome 12 mapped by Disease Browser (http://www.ensembl.org/Homo_sapiens/disseaseview) in the annotation information database Ensembl of the eukaryotic genome It was found that the causal locus of spinal muscular atrophy exists in the syndrome, q24. From these, it was speculated that the present DNA may be a causative gene of these diseases and that the mutation of the present DNA can be applied to these diagnoses.
[0195]
From the above, the protein shown in SEQ ID NO: 11 was predicted to be a novel Zn finger protein having functions related to development, differentiation and formation of human organs and tissues, cell cycle and cell proliferation.
[0196]
When this protein was expressed using a wheat germ cell-free protein synthesis system, in Example 4, TBP target sequences: TATAAA (SEQ ID NO: 82), SRY, Sox-5, and Sox-9 consensus target sequences: It showed very high affinity for AAAAAACAATAGGGG (SEQ ID NO: 83), mat-Mc target sequence: TCATTGTT (SEQ ID NO: 84). This indicates that this protein has DNA binding ability and is associated with diseases caused by abnormalities such as development / differentiation of the nervous system, cartilage formation, and sex differentiation. Further, Example 5 has an activity to enhance the action of PPAR, lifestyle diseases such as diabetes and obesity due to abnormal endocrine / sugar / lipid metabolism, etc., circulatory system diseases due to abnormal vascular function, inflammation, cancer, etc. Etc. Moreover, it has the activity which suppresses the effect | action which p53 has, and it was estimated that it is related to cancer etc. Furthermore, it has the activity which suppresses the effect | action which AP-1 has, and it was estimated that it is related to an inflammatory disease, cancer, etc.
[0197]
In order to examine the expression tissue of this DNA, BLAST search is performed on dbEST (Nature Genetics 4, (4) 332-3 (1993)) using this DNA as a query, and e-value ≦ 10^-10The names of organs or tissues with 5 or more human ESTs that were hit in the above were extracted, and they were normal human eyes, uterus, placenta, intestine, immune system, fetal liver, fetal spleen, cancerous skin, and eyes . This DNA was cloned from a cDNA library derived from nervous system cells including undifferentiated NT2 cells.
DNA Cell Biol. 14 (2): 125-36 states that the BLAST hit (ii) protein (zinc finger protein protein 268) is expressed in monoblasts. Furthermore, DNA 8 (6): 377-87 (1989) shows that the protein of (iii) (finger protein 2) was cloned from a placenta-derived cDNA library.
In addition, it was shown from Example 6 that this protein is strongly expressed in the pancreas, expressed in the spleen, liver and brain, and increased in the frontal lobe of Alzheimer's disease patients.
[0198]
This protein has functions and diseases peculiar to these tissues and cells, such as melanoma, lymphoma, leukemia, glioma, neuroblastoma, uterine cancer, colon cancer, small intestine cancer, skin cancer, liver cancer, etc. Neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, chorea, inflammatory diseases such as hepatitis, dermatitis, osteochondritis, osteoarthritis, immune system diseases, diabetes, obesity, cardiovascular diseases, allergic diseases, spleen The possibility of relating to diseases, infertility, Noonan syndrome, spinal muscular atrophy, etc. can be inferred, and it can be used as a diagnostic or therapeutic target for these diseases.
[0199]
(2) c-febra2001620 (SEQ ID NO: 2, 12)
c-febra2001620 (hereinafter referred to as “the present DNA”, and the protein encoded by the DNA is referred to as “the present protein”), as shown in SEQ ID NO: 2, consists of 2146 bases, of which base number 214 Up to 1947 is an open reading frame (including a stop codon). The amino acid sequence predicted from the open reading frame consists of 777 amino acid residues (SEQ ID NO: 12).
[0200]
When the homology search was performed using BLAST for the amino acid sequence encoded by SEQ ID NO: 2, a database of non-overlapping amino acid sequences created from SWISS-PROT, PIR, TREMBLE, GENPEPT, PDB) (I) Database registration symbol AF325191, KRAB zinc finger protein HZF26 (Human) was hit. As its contents, AF325191 is composed of 470 amino acids, and amino acid numbers 2 to 468 in the amino acid sequence are amino acid numbers 17 to 483 of the amino acid sequence described in SEQ ID NO: 12, and e-value: 0.0 and 467 amino acid residues. It was a hit with 84% agreement over the group. (Ii) Database registration symbols AB075849 and KIAA1969 protein (Human) were hit. As its contents, AB075849 consists of 595 amino acids, and amino acid numbers 51 to 583 in the amino acid sequence are amino acid numbers 16 to 548 of the amino acid sequence described in SEQ ID NO: 12, e-value: 0.0 and 533 amino acid residues. It was a hit with 69% agreement over the group. Furthermore, (iii) database registration symbol, Q03923, zinc finger protein protein 85 (Zinc finger protein HPF4; Human) was hit. As its contents, Q03923 is composed of 595 amino acids, and amino acid numbers 2 to 568 in the amino acid sequence are amino acid numbers 17 to 555 of the amino acid sequence described in SEQ ID NO: 12, and e-value: 0.0 and 568 amino acid residues. It was a hit with 65% agreement over the group.
[0201]
In addition, when the protein feature search by HMMPFAM was performed for the amino acid sequence shown in SEQ ID NO: 12, amino acid numbers 188 to 210, 216 to 243, 244 to 266, 272 to 294, 300 to 322, 328 to 350 of SEQ ID NO: 12, 356 to 378, 384 to 406, 440 to 462, 468 to 490, 496 to 518, 524 to 546, an amino acid sequence represented by a zinc finger, C2H2 type region (characterized as Pfam as zf-C2H2) Amino acid sequence). This domain has a structural motif in which two conserved Cys and two His are coordinated to zinc ions, and is found in many DNA-binding proteins such as transcription factors.
Furthermore, a sequence (amino acid sequence entered as KRAB in Pfam) showing the characteristics of KRAB box region in the amino acid sequence shown in amino acid numbers 19 to 59 of SEQ ID NO: 12 was found. Kruppel-associated box (KRAB) is found in about 1/3 of the C2H2-type Zn finger protein, is present in the N-terminal part, acts upon repression of transcription when binding to DNA, and cell differentiation together with the Zn finger domain・ It is known to be involved in the outbreak.
The protein of (i) hit by BLAST is related to the regulation of monocyte differentiation / proliferation from literature information (DNA Cell. Biol. 142 (2): 125-136 (1995)) in the database, The protein of (ii) is predicted to have functions of cell signal transduction, cytoskeleton and cell motility from literature information (DNA Res. 8 (6): 319-327 (2001)) in the database. In addition, the protein of (iii) above has some of the KRAB domains constituting cofactors that repress transcription from literature information in the database (DNA Cell. Biol. 17 (11): 931-943 (1998)). It was clarified that it binds to a nuclear protein in vitro.
[0202]
Furthermore, analysis by PSORT II (Trends Biochem. Mol. Biol. 58, (4) 417-424 (1996)), which is a program for predicting the intracellular localization of the protein, revealed that the localization of the protein in the cell was detected. The probability is 100.0% in the nucleus, and the protein was predicted to be present in the nucleus. This result suggests that this protein binds to DNA in the cell nucleus and regulates the transcription of genes involved in cell differentiation / proliferation, cell cycle and the like.
[0203]
On the other hand, in order to obtain genome information, using software sim4 (Genome Res. 8, 967-974 (1998)) for mapping human cDNA to human genome sequence, we attempted to map this DNA to the genome. Six exons were mapped on the chromosome. P. (Short arm) has an exostosis and p13 has a causal locus for acute myeloid leukemia. Furthermore, the causal locus for ectodermal dysplasia, hypervaline and hyperleucine-isoleucine is No. 19. It was found on the chromosome, but its detailed position has not been identified. From these, it was speculated that the present DNA may be a causative gene of these diseases and that the mutation of the present DNA can be applied to these diagnoses.
[0204]
Based on the above, the protein shown in SEQ ID NO: 12 was predicted to be a novel Zn finger protein having functions related to the development, differentiation and formation of human organs and tissues, cell cycle and cell proliferation.
[0205]
In order to examine the expression tissue of this DNA, BLAST search is performed on dbEST (Nature Genetics 4, (4) 332-3 (1993)) using this DNA as a query, and e-value ≦ 10^-50The names of organs or tissues with 5 or more human ESTs hit in the above were extracted, and they were normal testis / eye / brain / stomach, cancerized testis / uterus / brain / lung. This DNA was cloned from a cDNA library derived from fetal brain tissue.
Further, when the literature information on the expression tissue was examined for the top 3 hits of BLAST, DNA Cell. Biol. 142 (2): 125-136 (1995), KRAB zinc finger protein HZF26 (Human) is expressed in human monoblasts by DNA Res. 8 (6): 319-327 (2001), that KIAA1969 protein (Human) is expressed in the brain, spinal cord, fetal brain, fetal liver, etc. Biol. 17 (11): 931-943 (1998) described that zinc finger protein protein 85 (Human) is highly expressed in testicular epithelioma tissue, respectively.
Moreover, it was shown from Example 6 that this protein is expressed in pancreas, spleen, and liver, and the expression increases in breast cancer and lung cancer.
[0206]
This protein has functions and diseases peculiar to these tissues and cells such as testicular cancer, melanoma, brain tumor, glioma, neuroblastoma, gastric cancer, uterine cancer, lung cancer, liver cancer, breast cancer, Alzheimer's disease, Neurodegenerative diseases such as Parkinson's disease and chorea, inflammatory diseases such as pneumonia and hepatitis, immune system diseases, allergic diseases, infertility, hypervalinemia, hyperleucine-isoleucine, ectodermal dysplasia, exostosis Possibility that it is related to symptom etc. can be presumed, and the use as a target of a diagnostic or therapeutic agent for these diseases is considered.
[0207]
(3) c-bramy 2043103 (SEQ ID NOs: 3, 13)
c-bramy2043103 (hereinafter referred to as “the present DNA”, and the protein encoded by the DNA is referred to as “the present protein”), as shown in SEQ ID NO: 3, consists of 3049 bases, of which from base number 281 Up to 2284 are open reading frames (including stop codon). The amino acid sequence predicted from the open reading frame consists of 667 amino acid residues (SEQ ID NO: 13).
[0208]
When homology search was performed using BLAST for the amino acid sequence of SEQ ID NO: 13, the database in the NRDB protein database (database of non-overlapping amino acid sequences created from SWISS-PROT, PIR, TREMBLE, GENPEPT, PDB) Registration code Q9UJW8, Zinc finger protein 180 (Human) was a hit. As its contents, Q9UJW8 consists of 692 amino acids, and 1 to 692 in the amino acid sequence are amino acid numbers 1 to 667 of the amino acid sequence described in SEQ ID NO: 13, e-value: 0.0, and 692 amino acid residues. It hit with a score of 96% match.
From the alignment of BLAST search, this protein has deleted amino acids corresponding to 25 residues of amino acid numbers 45 to 69 of Q9UJW8, and this DNA also corresponds to base numbers 398 to 472 of base sequence AF192913 corresponding to Q9UJW8. It was also found that 75 bases (between base numbers 412 and 413 of this DNA) were deleted.
[0209]
Moreover, when the protein characteristic search by HMMPFAM was performed about the amino acid sequence of sequence number 13, amino acid numbers 328-350, 356-378, 384-406, 412-434, 440-462, 468-490, 496-518, 524 -546, 552-574, 580-602, 608-630, 636-658 amino acid sequence shown in Zinc Finger, C2H2 type domain characteristic sequence (amino acid sequence entered as Pfam as zf-C2H2) It was. This domain has a structural motif in which two conserved Cys and two His are coordinated to zinc ions, and is found in many DNA-binding proteins such as transcription factors.
Furthermore, the amino acid sequence shown in amino acid numbers 47 to 87 of SEQ ID NO: 13 was found to show the characteristics of KRAB box region (amino acid sequence entered as PRAm as KRAB). Kruppel-associated box (KRAB) is found in about 1/3 of the C2H2-type Zn finger protein, is present in the N-terminal part, acts upon repression of transcription when binding to DNA, and cell differentiation together with the Zn finger domain・ It is known to be involved in the outbreak.
[0210]
In protein feature search by HMMPFAM, amino acid numbers 25 to 80 of this protein are Remorin_N region (Remorin, N-terminal region), and this region is not found in Q9UJW8. When coiled-coil is formed on the C-terminal side, this domain exists as a proline-rich region on the N-terminal side and is thought to interact with macromolecules such as DNA. In Pfam, a Zn finger domain showing DNA binding ability was detected on the C-terminal side, but this region may have a DNA binding ability, interaction, and regulation of transcriptional activity different from Q9UJW8.
[0211]
In order to clarify the exon structure of this DNA and AF192913 cDNA, software sim4 (Genome Res. 8, 967-74 (1998)) for mapping the cDNA sequence to the genomic sequence was used to align the genomic sequence. This DNA sequence was mapped to 4 exons on human chromosome 19 and the AF192913 sequence was mapped to 5 exons. The first, second, third, and fourth exons of this DNA correspond to AF192913, but this DNA lacks one third exon of AF192913. This difference is derived from the deletion portion (base numbers 398 to 472 of AF192913) of the present DNA by the BLAST search described above.
These differences may cause differences in binding and interaction with DNA, expression tissues, expression levels, and the like. From the above, the protein of SEQ ID NO: 13 is a splicing variant of Q9UJW8, Zinc finger protein 180 (Human).
[0212]
Analysis by PSORT II (Trends Biochem. Sci. 24, (1) 34-6 (1999)), which is a program for predicting intracellular localization of the protein, reveals that the probability of localization of this protein in the cell is The nucleus was 95.7% and the mitochondria was 4.3%. Since the probability of localization to the nucleus is high, this protein is considered to be a protein that binds to DNA in the cell nucleus and regulates transcription of genes involved in cell differentiation / proliferation, cell cycle, and the like.
[0213]
According to the Disease Browser (http://www.ensembl.org/Homo_sapiens/disseaseview) in the annotation information database Ensembl of the eukaryotic genome, the position (q13.31) on the 19th chromosome where this DNA is mapped is shown. Q13.2-13.3 includes progressive hereditary heart block, 3-methylglutaconic acidia causal locus, q13 autosomal dominant hearing loss, diamond-black fan anemia, palate causal locus inherited in q The causative locus for pediatric convulsions is present, and ectodermal dysplasia, hypervalineemia, and hyperleucine-isoleucine are present on chromosome 19, but their detailed location has been identified. Obviously not became. From the above, the possibility that this DNA is a causative gene of these diseases and the possibility that mutations of this DNA containing splicing variants can be applied to the diagnosis of these diseases were expected.
[0214]
When this protein was expressed using a wheat germ cell-free protein synthesis system, in Example 4, the target sequence of ER: AAGGGGAAAATGACCCC (SEQ ID NO: 46), the target sequence of RXR-α: GGTCATAGGGGT (SEQ ID NO: 47), PPAR-α: CTAGGGCAAAGGTCA (SEQ ID NO: 48), consensus target sequence such as Pax-3: CGTCAGCCTTTGA (SEQ ID NO: 68), Pax-1 target sequence: CCGTTCCCCTCTTAGATAT (SEQ ID NO: 69) and HSF1 and other consensus target sequences: AGAAAAAGAAAAAAA SEQ ID NO: 70) showed affinity. This indicates that this protein has the ability to bind to DNA, and is associated with diseases caused by abnormalities such as lipid metabolism, drug metabolism, and muscle formation, such as lifestyle-related diseases.
[0215]
In order to examine the expression tissue of this DNA, BLAST search is performed on dbEST (Nature Genetics 4, (4) 332-3 (1993)) using this DNA as a query, and e-value ≦ 10^-10In the human ESTs that were hit in, the names of organs or tissues in which 4 or more hits were found were extracted. In human normal lung, intestine, spleen, cancerous immune system / lymph system / uterus / eye / skin there were. This DNA was cloned from a cDNA library derived from the amygdala.
[0216]
This protein has functions and diseases peculiar to these tissues and cells, such as cancers such as lung cancer, colon cancer, small intestine cancer, leukemia, lymphoma, uterine cancer, central diseases such as depression, anxiety, schizophrenia, pneumonia Inflammatory diseases such as immune system diseases, allergic diseases, hyperlipidemia, arteriosclerosis, splenomegaly, cleft palate, progressive hereditary heart block, 3-methylglutaconic acidia, hearing loss, anemia, pediatric convulsions, hypervalineemia, high The possibility of leucine-isoleucinemia and myogenic dysgenesis can be estimated, and it can be used as a target for diagnostics and therapeutics for these diseases.
[0217]
(4) c-brassn2013753 (SEQ ID NOs: 4, 14)
c-brsn2013753 (hereinafter referred to as “the present DNA”, and the protein encoded by the DNA is referred to as “the present protein”), as shown in SEQ ID NO: 4, consists of 2312 bases, of which base number 138 Up to 1019 are open reading frames (including stop codon). The amino acid sequence predicted from the open reading frame consists of 293 amino acid residues (SEQ ID NO: 14).
[0218]
When a homology search was performed on the amino acid sequence of SEQ ID NO: 14 using BLAST, an NRDB protein database (a database of non-overlapping amino acid sequences created from SWISS-PROT, PIR, TREMBLE, GENPEPT, PDB) ( i) Database registration symbol Q62981, Zinc finger protein OZF (Rat) was hit. As its contents, Q62981 is composed of 407 amino acids, and 116 to 407 in the amino acid sequence are amino acid numbers 1 to 293 of the amino acid sequence described in SEQ ID NO: 14, and e-value: 5 × 10 5^-176And it hit with 95% agreement over 293 amino acid residues.
(Ii) Database registration symbol Q62513, Zinc finger protein OZF (Mouse) was hit. As its contents, Q62981 is composed of 407 amino acids, and 116 to 407 in the amino acid sequence are amino acid numbers 1 to 293 of the amino acid sequence described in SEQ ID NO: 14, and e-value: 5 × 10 5^-176And it hit with 95% agreement over 293 amino acid residues.
Furthermore, (iii) Database registration symbol Q15072 and Zinc finger protein OZF (Human) were hit. As its contents, Q15072 consists of 292 amino acids, and 4 to 292 in the amino acid sequence is amino acid numbers 5 to 293 of the amino acid sequence described in SEQ ID NO: 14, and e-value: 5 × 10 5^-151And it was hit with 81% agreement over 289 amino acid residues.
[0219]
Moreover, when the protein characteristic search by HMMPFAM was performed about the amino acid sequence of sequence number 14, amino acid numbers 17-39, 45-67, 73-95, 101-123, 129-151, 157-179, 185-207, 213 A sequence showing the characteristics of Zinc Finger, C2H2 type domain (amino acid sequence entered as zf-C2H2 in Pfam) was found in the amino acid sequences shown in ˜235, 241-263, 269-291. This domain has a structural motif in which two conserved Cys and two His are coordinated to zinc ions, and is found in many DNA-binding proteins such as transcription factors.
Further, a sequence showing the characteristics of XPA protein N-terminal (amino acid sequence entered as XPA_N in Pfam) was extracted from amino acid numbers 154 to 194. XPA protein is a protein involved in the pathogenesis of pigmented scleroderma (XP), an autosomal recessive disease that induces skin cancer at a high rate by ultraviolet rays, has the ability to bind to damaged DNA, and removes and repairs DNA Responsible for the function.
The XPA protein N-terminal domain is present in this protein, but is not found in Q15072 which is a human sequence among the hit sequences. Therefore, it can be inferred that this protein has a function related to DNA repair that Q15072 does not have.
[0220]
In order to clarify the exon structure of this DNA and human hit sequence AJ011806 (DNA sequence corresponding to Q15072) cDNA, software sim4 (Genome Res. 8, 967-74) that maps the cDNA sequence to the genomic sequence. 1998)), the DNA sequence was mapped to one exon on human chromosome 19 and the AJ011806 sequence was mapped to one exon. . There is no overlap between the mapping positions of the two.
Moreover, although this DNA is a human clone, the sequence having the highest homology with this protein is the rat sequence. Therefore, the protein of SEQ ID NO: 14 is an ortholog of rat and mouse Zinc finger protein OZF, and is likely to be a novel human cDNA.
[0221]
Analysis by PSORT II (Trends Biochem. Sci. 24, (1) 34-6 (1999)), which is a program for predicting intracellular localization of the protein, reveals that the probability of localization of this protein in the cell is The nucleus was 95.7% and the mitochondria was 4.3%. Since the probability of localization to the nucleus is high, this protein is considered to be a protein that binds to DNA in the cell nucleus and regulates transcription of genes involved in cell differentiation / proliferation, cell cycle, and the like.
[0222]
According to the Disease Browser (http://www.ensembl.org/Homo_sapiens/disseaseview) in the annotation information database Ensembl of the eukaryotic genome, the position (q13.12) on the 19th chromosome where the present DNA is mapped is shown. Q13.1 includes cystinuria, cystic bone dysplasia, congenital nephrosis, q13 is autosomal dominant hearing loss, diamond-blackfan anemia, cleft palate locus, q is the gene causing hereditary childhood spasm I found out that the seat exists. Furthermore, it was revealed that ectodermal dysplasia, hypervalineemia / hyperleucine-isoleucineemia are present on chromosome 19, but their detailed positions have not been identified.
From the above, the possibility that this DNA is a causative gene of these diseases and the possibility that mutations of this DNA containing splicing variants can be applied to the diagnosis of these diseases were expected.
[0223]
When this protein was expressed using a wheat germ cell-free protein synthesis system, in Example 4, a consensus target sequence such as GATA-1: CCAGATAAGG (SEQ ID NO: 52), and a target sequence of AREA / NIT-2: It showed a very high affinity for TATCTC (SEQ ID NO: 53). This indicates that this protein has DNA binding ability and may be associated with diseases caused by abnormalities such as differentiation of hematopoietic stem cells, hematopoietic cells, neural cells, circulatory organs, and apoptosis control.
[0224]
In order to examine the expression tissue of this DNA, BLAST search is performed on dbEST (Nature Genetics 4, (4) 332-3 (1993)) using this DNA as a query, and e-value ≦ 10^-10Extracted organ names or tissue names with 4 or more hits in human ESTs hit in, and in normal human eyes / brain / intestine / lung / placenta, cancerous testis / uterus / eye / skin there were. This DNA was cloned from a substantia nigra derived cDNA library.
Eur. J. et al. Biochem. 236 (3): 991-5 (1996), human OZF is expressed in mammary epithelial cells, over-expressed in tumor cells, and Int. J. et al. Cancer 80 (3): 369-72 (1999) is highly expressed in pancreatic cancer, and is assumed to be involved in carcinogenesis and tumor development.
[0225]
Based on the above, this protein has functions and diseases peculiar to these tissues and cells, such as cancers such as melanoma, brain tumor, colon cancer, small intestine cancer, lung cancer, uterine cancer, skin cancer, testicular cancer, Parkinson's disease, chorea, etc. Neurodegenerative diseases, central diseases such as schizophrenia, inflammatory diseases such as pneumonia and dermatitis, immune system diseases, allergic diseases, infertility, cystinuria, cystic bone dysplasia, hearing loss, anemia, hematopoietic disorders, palate It can be estimated that it may be related to fissure and infantile spasm, and it can be used as a diagnostic or therapeutic target for these diseases.
[0226]
(5) c-brsn2017530 (SEQ ID NOS: 5 and 15)
c-brssn2011192 (hereinafter referred to as “the present DNA”, and the protein encoded by the DNA is referred to as “the present protein”), as shown in SEQ ID NO: 5, consists of 3839 bases, of which from base number 1582 Up to 2685 are open reading frames (including stop codon). The amino acid sequence predicted from the open reading frame consists of 367 amino acid residues (SEQ ID NO: 15).
[0227]
When homology search was performed using BLAST for the amino acid sequence of SEQ ID NO: 15, the database in the NRDB protein database (database of non-overlapping amino acid sequences created from SWISS-PROT, PIR, TREMBLE, GENPEPT, PDB) The registration symbols AF326206 and ZNF140-like transcription factor (Human) were hit. As its contents, AF326206 is composed of 399 amino acids, 33 to 399 in the amino acid sequence, amino acid numbers 1 to 367 of the amino acid sequence shown in SEQ ID NO: 15 are e-value: 0.0 and 367 amino acid residues. Hit with 99% agreement.
From the alignment of the BLAST search, it was found that the N-terminal of this protein is 32 residues shorter than AF326206, and that one residue substitution of glutamic acid to glycine exists at amino acid number 136 (corresponding to amino acid number 104 of this protein) of AF326206. This DNA differs from AF326206 by 1490 bases at the 5 ′ end, but since the translation start point is base number 1582, the difference from AF326206 is from base numbers 338 to 433 of the translation start point of AF326206, which is the N-terminal of the amino acid. This is reflected in the difference. A base corresponding to base number 743 of AF326206 (base number 1891 of this DNA) is substituted from adenine to guanine, which is the origin of amino acid substitution.
[0228]
Further, protein feature search by HMMPFAM was performed on the amino acid sequence of SEQ ID NO: 15, and the results were shown in amino acid numbers 169-191, 197-219, 225-247, 253-275, 281-303, 309-331, 337-359. A sequence showing the characteristics of Zinc Finger, C2H2 type domain (amino acid sequence entered as Pfam as zf-C2H2) was found. This domain has a structural motif in which two conserved Cys and two His are coordinated to zinc ions, and is found in many DNA-binding proteins such as transcription factors.
AF326206 has KRAB box region at amino acid numbers 4 to 44 but is not found in this protein. Since this domain is known to act as a transcriptional repressor upon binding to DNA and to participate in cell differentiation and development together with the Zn finger domain, this protein exhibits a transcriptional activity different from that of AF326206. Is expected to have some effect on
[0229]
In order to clarify the exon structure of the present DNA and AF326206 cDNA, software sim4 (Genome Res. 8, 967-74 (1998)) that maps the cDNA sequence to the genome sequence was used to align the genome sequence. This DNA sequence was mapped to 3 exons on human chromosome 19, and the AF326206 sequence was mapped to 5 exons (except poly A). The second and third exons of this DNA are almost identical to the fourth and fifth exons of AF192913, respectively, but the first exon of this DNA is longer upstream than the third exon of AF192913. In addition, the first and second exons of AF192913 do not exist in this DNA. Since the translation start position of AF192913 is base number 337, both have different translation start positions, and this causes the difference in the N-terminus on the amino acid sequence.
These differences may cause differences in binding and interaction with DNA, expression tissues, expression levels, and the like. From the above, the protein of SEQ ID NO: 15 is a splicing variant of AF326206, Zinc finger protein 140-like transcription factor (Human).
[0230]
Analysis by PSORT II (Trends Biochem. Sci. 24, (1) 34-6 (1999)), which is a program for predicting intracellular localization of the protein, reveals that the probability of localization of this protein in the cell is It was 100.0% in the nucleus. Therefore, this protein is considered to be a protein that binds to DNA in the cell nucleus and regulates transcription of genes involved in cell differentiation / proliferation, cell cycle, and the like.
[0231]
According to the Disease Browser (http://www.ensembl.org/Homo_sapiens/disseaseview) in the annotation information database Ensembl of the eukaryotic genome, the position (q13.11) on the 19th chromosome where this DNA is mapped is shown. Q13.1 includes cystinuria, congenital nephrosis, cystic bone dysgenesis locus, q13 is autosomal dominant hearing loss, diamond-blackfan anemia, cleft palate locus, and q is hereditary There is a causal locus for pediatric convulsions, and ectodermal dysplasia, hypervalineemia, and hyperleucine-isoleucine are present on chromosome 19, but their precise positions have not been identified. It became clear that there was no. From the above, the possibility that this DNA is a causative gene of these diseases and the possibility that mutations of this DNA containing splicing variants can be applied to the diagnosis of these diseases were expected.
[0232]
When this protein was expressed using a wheat germ cell-free protein synthesis system, in Example 4, the target sequence of Sp1: GGGGGGGGGG (SEQ ID NO: 54), the target sequence of Tramtrack69K: GGACCCTGC (SEQ ID NO: 61), and HOX9 High affinity was shown for target sequence: TGACAGTTTAACGA (SEQ ID NO: 62) and CDP target sequence: CCAATAATCGAT (SEQ ID NO: 63). Therefore, this protein has a DNA binding ability and is considered to be related to diseases such as diseases caused by abnormalities such as hematopoietic stem cell differentiation, apoptosis control, cytokine production, viral diseases, bacterial diseases, and cancer. Further, according to Example 5, it has an activity of strongly suppressing the action of PPAR, lifestyle diseases such as diabetes and obesity due to abnormal endocrine / sugar / lipid metabolism, circulatory system diseases due to abnormal vascular function, inflammation, etc. It was speculated that it was related to cancer. Moreover, it has the activity which suppresses the effect | action which p53 has, and it was estimated that it is related to cancer etc. Furthermore, it has an activity that enhances the action of HIF-1, and is caused by abnormal angiogenesis, abnormal intracellular oxidation, etc., ischemic disease, anemia, hypoxia, diabetic retinopathy, cardiovascular disease, cancer Etc.
[0233]
In order to examine the expression tissue of this DNA, BLAST search is performed on dbEST (Nature Genetics 4, (4) 332-3 (1993)) using this DNA as a query, and e-value ≦ 10^-50The names of organs or tissues in which 5 or more hits were found were extracted from human ESTs that were hit in, and found to be normal human brain / eye / kidney / placenta, cancerous uterus / skin / eye / testis . This DNA was cloned from a substantia nigra derived cDNA library.
[0234]
This protein has functions and diseases peculiar to these tissues and cells, such as cancers such as brain tumors, melanoma, kidney cancer, uterine cancer, skin cancer, testicular cancer, leukemia, neurodegenerative diseases such as Parkinson's disease and chorea, schizophrenia Central diseases such as infectious diseases, inflammatory diseases such as nephritis / dermatitis, immune system diseases, allergic diseases, diabetes, obesity, ischemic diseases, cardiovascular diseases, hypoxia, diabetic retinopathy, cystinuria, nephrosis As a target for diagnostics and therapeutics for these diseases, such as hearing loss, anemia, cleft palate, pediatric convulsions, ectodermal dysplasia, hypervalineemia, and hyperleucine-isoleucineemia Can be used.
[0235]
(6) c-hcho20000473 (SEQ ID NOs: 6, 16)
c-hcho20000473 (hereinafter referred to as “the present DNA”, and the protein encoded by the DNA is referred to as “the present protein”), as shown in SEQ ID NO: 6, consists of 3588 bases, of which base number 352 Up to 2271 are open reading frames (including stop codon). The amino acid sequence predicted from the open reading frame consists of 639 amino acid residues (SEQ ID NO: 16).
[0236]
A homology search was performed on the amino acid sequence of SEQ ID NO: 16 using BLAST. In the NRDB protein database (database of non-overlapping amino acid sequences created from SWISS-PROT, PIR, TREMBLE, GENPEPT, PDB), other than genomic sequences I did not find anything showing a high score. However, in the amino acid sequence database aageneseq in GENESEQ (manufactured by Derwent), which is a commercial gene sequence patent database, the amino acid sequence described as Seq ID 893 in the registration code AAU15940 and WO01 / 55322 was hit, and Human novel secreted protein. As its contents, AAU15940 is composed of 361 amino acids, and 4 to 361 in the amino acid sequence ranges from amino acid numbers 282 to 639 of the amino acid sequence described in SEQ ID NO: 16 and e-value: 0.0 and 358 amino acid residues. It was a hit with a match of 100.0%.
The BLAST search alignment shows that this protein differs from AAU15940 by 281 residues at the N-terminus. AAU15940 differs from this protein by 3 residues. In addition, this DNA differs from AAS25927 (DNA sequence in DNA sequence database nageneseq corresponding to AAU15940) at the 5 ′ end by 1193 bases, and AAS25927 differs from this DNA by 8 bases, which causes the difference in the N-terminal amino acid sequence. It has become.
[0237]
Moreover, when the protein feature search by HMMPFAM was performed about the amino acid sequence of sequence number 16, amino acid numbers 234-256, 262-284, 290-312, 318-340, 346-368, 374-396, 402-424, 430 ˜452, 458 to 480, 486 to 508, 514 to 536, 542 to 564, 570 to 592, 598 to 620, the sequence showing the characteristics of Zinc Finger, C2H2 type domain (Pfam as zf-C2H2) The amino acid sequence to be entered was found. This domain has a structural motif in which two conserved Cys and two His are coordinated to zinc ions, and is found in many DNA-binding proteins such as transcription factors.
Furthermore, a sequence (amino acid sequence entered as PRAm as KRAB) showing the characteristics of KRAB box region was found in the amino acid sequence represented by amino acid numbers 27 to 67 of SEQ ID NO: 16. Kruppel-associated box (KRAB) is found in about 1/3 of the C2H2-type Zn finger protein, is present in the N-terminal part, acts upon repression of transcription when binding to DNA, and cell differentiation together with the Zn finger domain・ It is known to be involved in the outbreak.
KRAB box region is present in this protein but not in AAU15940. Therefore, this protein exhibits a transcriptional activity different from that of AAU15940 and may have some influence on cell differentiation.
[0238]
In order to clarify the exon structure of the present DNA and AAS25927 cDNA, software sim4 (Genome Res. 8, 967-74 (1998)) for mapping the cDNA sequence to the genome sequence was used, and alignment to the genome sequence was performed. This DNA sequence was mapped to 7 exons on the human X chromosome, and the AAS25927 sequence was mapped to 1 exon (except poly A). The seventh exon of this DNA is longer than and includes one exon of AAS25927, but the first to sixth exons of this DNA are not present in AAS25927. Among these, since the fifth and sixth exons correspond to the KRAB domain, AAU15940 is consistent with the result of HMMPFAM that lacks the KRAB domain. These differences may cause differences in binding and interaction with DNA, expression tissues, expression levels, and the like. Based on the above, the protein of SEQ ID NO: 16 is a splicing variant of the human novel secreted protein, the amino acid sequence described as Seq ID 893 in WO01 / 55322.
[0239]
Analysis by PSORT II (Trends Biochem. Sci. 24, (1) 34-6 (1999)), which is a program for predicting intracellular localization of the protein, reveals that the probability of localization of this protein in the cell is It was 95.7% in the nucleus and 4.3% in mitochondria. Therefore, this protein is considered to be a protein that binds to DNA in the cell nucleus and regulates transcription of genes involved in cell differentiation / proliferation, cell cycle, and the like.
[0240]
According to Disease Browser (http://www.ensembl.org/Homo_sapiens/disseaseview) in the annotation information database Ensembl of the eukaryotic genome, it includes the position (p11.23) on the X chromosome to which this DNA is mapped Mental retardation at p11-23, Fragile X syndrome at p11.4-11.2, Joint contractures (spinal muscular atrophy) at p11.3-11.2, Albino (eye) at p11.4-11.23 ), P11.4-11.21 with amblyopia, p11.23 with night blindness, p11.2 with papillary renal cell carcinoma, p11 with insulin-dependent diabetes, and p with congenital cataract locus It was. From the above, the possibility that this DNA is a causative gene of these diseases and the possibility that mutations of this DNA containing splicing variants can be applied to the diagnosis of these diseases were expected.
[0241]
When this protein was expressed using a wheat germ cell-free protein synthesis system, in Example 4, consensus target sequences such as HEN-1 target sequence: GGGGCGCAGCTGGCGCCC (SEQ ID NO: 38), AhR, etc .: GGGGATTGCCGTG (SEQ ID NO: 39) ), USF target sequence: GTCACGTGGT (SEQ ID NO: 40), consensus target sequence such as NF-1A1: CGTTGGGGTTTGGCACGGGGCCA (SEQ ID NO: 41), RF-X1 target sequence: GGTAACATAGGCAAC (SEQ ID NO: 42), Sp1 target sequence: GGGGG (SEQ ID NO: 54), Egr-1 target sequence: TGCGTGGGCG (SEQ ID NO: 56), p50 target sequence: GGGGATTTTCC (SEQ ID NO: 74), consensus such as NF-ATc Target sequence: AGGAAAA (SEQ ID NO: 75), consensus target sequence such as p91: GAATTCCGGGAAATG (SEQ ID NO: 76), consensus target sequence such as STAT2: TTTCCCGGGAAATG (SEQ ID NO: 77), and p53 target sequence: GGACATGCCCGGGGCATGTC (SEQ ID NO: 78) It showed very high affinity. This indicates that this protein has DNA-binding ability and may be associated with diseases such as cell cycle, inflammation, apoptosis control, immune response, drug metabolism, cytokine production, diseases such as viral diseases, and cancer. It is done. Further, Example 5 has an activity to enhance the action of PPAR, lifestyle diseases such as diabetes and obesity due to abnormal endocrine / sugar / lipid metabolism, etc., circulatory system diseases due to abnormal vascular function, inflammation, cancer, etc. Etc. Moreover, it has the activity which suppresses the effect | action which AP-1 has, and it was estimated that it is related to an inflammatory disease, cancer, etc.
[0242]
In order to examine the expression tissue of this DNA, BLAST search is performed on dbEST (Nature Genetics 4, (4) 332-3 (1993)) using this DNA as a query, and e-value ≦ 10^-10The names of organs or tissues in which 5 or more hits were found were extracted from human ESTs that were hit in, and found to be normal human brain, placenta, stomach, and mammary gland, and cancerous eyes, skin, lungs, and uterus. . This DNA was cloned from a cDNA library derived from normal cartilage primary cultured cells.
[0243]
This protein has functions and diseases peculiar to these tissues and cells, such as leukemia, lymphoma, brain tumor, stomach cancer, breast cancer, melanoma, skin cancer, lung cancer, uterine cancer, inflammatory diseases such as pneumonia and dermatitis, Presumed to be related to immune system disease, allergic disease, obesity, cardiovascular disease, infertility, mental retardation, fragile X syndrome, joint contracture, albino, amblyopia, night blindness, renal cancer, diabetes, osteoarthritis This protein can be used as a target for diagnostics and therapeutics for these diseases.
[0244]
(7) c-testi4004626 (SEQ ID NO: 7, 17)
c-testi 4004626 (hereinafter referred to as “the present DNA”, and the protein encoded by the DNA is referred to as “the present protein”) comprises 4053 bases as shown in SEQ ID NO: 7, of which base number 2097 Up to 3881 is an open reading frame (including a stop codon). The amino acid sequence predicted from the open reading frame consists of 594 amino acid residues (SEQ ID NO: 17).
[0245]
When homology search was performed using BLAST for the amino acid sequence of SEQ ID NO: 17, database registration symbol of NRDB protein database (database of non-overlapping amino acid sequences created from SWISS-PROT, PIR, TREMBLE, GENPEPT, PDB) P52736, Zinc finger protein 133 (Human) was a hit.
As its contents, P52736 consists of 654 amino acids, and 73-654 in the amino acid sequence spans amino acid numbers 13-594 of the amino acid sequence described in SEQ ID NO: 17, and e-value: 0.0 and 584 amino acid residues. Hit with 99% agreement.
The BLAST search alignment shows that this protein differs from P52736 by 12 residues at the N-terminus, and P52736 differs from this protein by 72 residues at the N-terminus. In addition, at amino acid numbers 121 and 428 (corresponding to amino acid numbers 181 and 488 of P52736) of this protein, there are substitutions of leucine to phenylalanine and histidine to arginine, respectively. In this DNA, the 5 ′ end differs from U09366 (base sequence corresponding to P52736) on the alignment by 2131 bases, but since the translation start point is base number 2097, the difference at the 5 ′ end is 35 bases. It is the cause of the difference at the N-terminus of the amino acid sequence. In addition, there are single-base substitutions from adenine to guanine and cytosine to thymine at base numbers 2282 and 2457 (corresponding to base numbers 811 and 986 of U09366), respectively, which cause amino acid substitution.
[0246]
Moreover, when the protein characteristic search by HMMPFAM was performed about the amino acid sequence of sequence number 17, amino acid numbers 154-176, 182-204, 210-232, 238-260, 266-288, 294-316, 322-344, 350 -372, 378-400, 406-428, 434-456, 462-484, 490-512, 518-540, 546-571, a sequence showing the characteristics of Zinc Finger, C2H2 type domain (in Pfam) The amino acid sequence entered as zf-C2H2) was found. This domain has a structural motif in which two conserved Cys and two His are coordinated to zinc ions, and is found in many DNA-binding proteins such as transcription factors.
P52736 has KRAB box region at amino acid numbers 1 to 41, but is not found in this protein.
Therefore, this protein shows a transcriptional activity different from that of P52736, and may have some influence on cell differentiation.
[0247]
In order to clarify the exon structure of this DNA and U09366 cDNA, software sim4 (Genome Res. 8, 967-74 (1998)) for mapping the cDNA sequence to the genomic sequence was used to align the genomic sequence. The DNA sequence was mapped to 2 exons on human chromosome 20, and the U09366 sequence was mapped to 6 exons (except poly A). The second exon of the present DNA is almost identical to the sixth exon of U09366, but the first exon of the present DNA is not present in U09366, and is located between the fifth and sixth exons of U09366. The fourth and fifth exons of U09366 correspond to the KRAB domain, and FEBS Lett. 369 (2-3): 153-7 also states that Zinc finger 133 has both A and B subdomains of KRAB box, so that both DNAs are functional as well as structural differences. It is also possible that there will be differences.
These differences may cause differences in binding and interaction with DNA, expression tissues, expression levels, and the like. From the above, the protein of SEQ ID NO: 17 is a splicing variant of P52736, Zinc finger protein 133 (Human).
[0248]
Analysis by PSORT II (Trends Biochem. Sci. 24, (1) 34-6 (1999)), which is a program for predicting intracellular localization of the protein, reveals that the probability of localization of this protein in the cell is It was 100.0% in the nucleus. Therefore, this protein is considered to be a protein that binds to DNA in the cell nucleus and regulates transcription of genes involved in cell differentiation / proliferation, cell cycle, and the like.
[0249]
According to the Disease Browser (http://www.ensembl.org/Homo_sapiens/disseaseview) in the annotation information database Ensembl of the eukaryotic genome, the position (p11.23) on the chromosome 20 where this DNA is mapped It was found that the causal locus of hereditary corneal dystrophy exists in the included p11.2. From the above, the possibility that this DNA is a causative gene of this disease and the possibility that mutations of this DNA containing splicing variants can be applied to diagnostic agents for these diseases were expected.
[0250]
When this protein was expressed using a wheat germ cell-free protein synthesis system, in Example 4, the target sequence of YY1: CGGCCCATCTGGCT (SEQ ID NO: 55), the target sequence of Egr-1: TGCGTGGGCG (SEQ ID NO: 56), Snail target sequence: CACCTGTTTTCA (SEQ ID NO: 57), CF2-II target sequence: GTATATATA (SEQ ID NO: 58), IRF-1, IRF-2 consensus target sequences: GAAAAGCGAAAA (SEQ ID NO: 73), NF-ATc, etc. The consensus target sequence: AGGAAAA (SEQ ID NO: 75) and the consensus target sequence such as p91: GAATTCCGGGAAATG (SEQ ID NO: 76) showed affinity. From this, this protein has the ability to bind to DNA and is considered to be associated with diseases caused by abnormalities such as immune reaction and cytokine production. Moreover, it was estimated by Example 5 that it has the activity which suppresses the effect | action which p53 has, and it is related to cancer etc. Moreover, it has the activity which suppresses the effect | action which NF (kappa) B has, and it was estimated that it is related to immune diseases, inflammatory diseases, cancer, etc., such as rheumatoid arthritis and osteoarthritis. Moreover, it has the activity which suppresses the effect | action which AP-1 has, and it was estimated that it is related to an inflammatory disease, cancer, etc. In addition, it has an activity that enhances the action of HIF-1 and is caused by abnormal angiogenesis, abnormal intracellular oxidation, etc., ischemic disease, anemia, hypoxia, diabetic retinopathy, cardiovascular disease, cancer Etc. Furthermore, it has an activity that slightly enhances the action of CREB, and it was speculated that it is related to central diseases such as memory impairment, cardiovascular diseases, diabetes and the like.
[0251]
In order to examine the expression tissue of this DNA, BLAST search is performed on dbEST (Nature Genetics 4, (4) 332-3 (1993)) using this DNA as a query, and e-value ≦ 10^-50Extracted organ names or tissue names with 4 or more hits in human ESTs hit in, normal human bone, liver, pancreas, fetal heart, brain, eye, fetal liver, cancerous mammary gland It was the prostate. This DNA was cloned from a testis-derived cDNA library.
[0252]
This protein has functions and diseases peculiar to these tissues and cells, such as myeloma, liver cancer, pancreatic cancer, brain tumor, melanoma, breast cancer, prostate cancer, testicular cancer, pneumonia, pancreatitis, hepatitis, osteoarthritis Inflammatory diseases such as rheumatoid arthritis, allergic diseases, central diseases such as memory disorders, diabetes, ischemic diseases, anemia, hypoxia, diabetic retinopathy, cardiovascular diseases, infertility, Possibility that it is related to corneal dystrophy can be inferred, and its use as a target of a diagnostic or therapeutic agent for these diseases can be considered.
[0253]
(8) c-tach 3014316 (SEQ ID NOs: 8, 18)
c-trach 3014316 (hereinafter referred to as “the present DNA”, and the protein encoded by the DNA is referred to as “the present protein”), as shown in SEQ ID NO: 8, consists of 4455 bases, of which base number 1038 Up to 1937 is an open reading frame (including a stop codon). The amino acid sequence predicted from the open reading frame consists of 299 amino acid residues (SEQ ID NO: 18).
[0254]
When homology search was performed on the amino acid sequence of SEQ ID NO: 18 using BLAST, the database registration symbol of the NRDB protein database (database of non-overlapping amino acid sequences created from SWISS-PROT, PIR, TREMBLE, GENPEPT, PDB) AK0711616, FLJ00187 protein (Human) was a hit.
As its contents, AK074116 consists of 563 amino acids, and 267 to 563 in the amino acid sequence is the amino acid number 3 to 299 of the amino acid sequence described in SEQ ID NO: 18, and e-value: 5 × 10 5^-179And it hit with 100.0% agreement over 297 amino acid residues.
From the BLAST search alignment, it can be seen that this protein differs from AK074116 by 2 residues at the N-terminus, and AK074116 differs from this protein by 266 residues at the N-terminus. In this DNA, the 5 ′ end differs from AK074116 on the alignment by 1043 bases. However, since the translation start point is the base number 1038, the difference at the 5 ′ end is 6 bases. It is the cause. In AK074116, the 5 'end differs by 999 bases, but the translation start point is base number 202, which causes a difference in the N-terminal of the amino acid sequence.
[0255]
Moreover, when the protein feature search by HMMPFAM was performed about the amino acid sequence of SEQ ID NO: 18, the amino acid sequences represented by amino acid numbers 113 to 135, 141 to 163, 169 to 191, 197 to 219, 225 to 247, and 253 to 275 were obtained. A sequence showing the characteristics of Zinc Finger, C2H2 type domain (amino acid sequence entered as Pfam as zf-C2H2) was found. This domain has a structural motif in which two conserved Cys and two His are coordinated to zinc ions, and is found in many DNA-binding proteins such as transcription factors.
In addition, AK071166 extracted SCAN domain (amino acid sequence entered as PCAN in Pfam) from amino acid numbers 50 to 143, and KRAB box region (amino acid sequence entered as PRAm in KFAB) from amino acid numbers 227 to 267, respectively. These domains are not present in this protein.
SCAN domain is a conserved motif found at the N-terminus of some C2H2-type Zn finger proteins and is known to play a role in the regulation of oligomer formation and transcriptional control. Kruppel-associated box (KRAB) is found in about 1/3 of the C2H2-type Zn finger protein, and is present in the N-terminal part. When it binds to DNA, it acts as a transcriptional repressor. It is known to be involved in differentiation and development.
Therefore, this protein may exhibit a transcriptional activity different from that of AK074116 and affect cell differentiation, or may have a different action in oligomer formation.
[0256]
In order to clarify the exon structure of this DNA and AK074116 cDNA, software sim4 (Genome Res. 8, 967-74 (1998)) for mapping the cDNA sequence to the genome sequence was used to align the genome sequence. This DNA sequence was mapped to one exon on human chromosome 7, and the AK0741116 sequence was found to map to 6 exons. One exon of this DNA includes the sixth exon of AK074116 and is longer. Of the first to fifth exons of AK074116 that do not exist in this DNA, the fourth and fifth exons correspond to the KRAB domain, so that not only structural differences between them but also functional differences may occur. Conceivable.
These differences may cause differences in binding and interaction with DNA, expression tissues, expression levels, and the like. From the above, the protein of SEQ ID NO: 18 is a splicing variant of AK074116, FLJ00187 protein (Human).
[0257]
Analysis by PSORT II (Trends Biochem. Sci. 24, (1) 34-6 (1999)), which is a program for predicting intracellular localization of the protein, reveals that the probability of localization of this protein in the cell is It was 95.7% in the nucleus and 4.3% in mitochondria. Therefore, this protein is considered to be a protein that binds to DNA in the cell nucleus and regulates transcription of genes involved in cell differentiation / proliferation, cell cycle, and the like.
[0258]
According to Disease Browser (http://www.ensembl.org/Homo_sapiens/disseaseview) in the annotation information database Ensembl of the eukaryotic genome, the position (q22.1) on chromosome 7 where this DNA is mapped Q22-31.1 includes congenital diarrhea, colorectal cancer, q21.3-22 has a hemorrhagic predisposition, and the causal locus of thrombus formation tendency, q21-22 has susceptibility to malignant hyperthermia, and q has autism susceptibility These loci were found to exist. From the above, the possibility that this DNA is a causative gene of this disease and the possibility that mutations of this DNA containing splicing variants can be applied to diagnostic agents for these diseases were expected.
[0259]
When this protein was expressed using a wheat germ cell-free protein synthesis system, in Example 4, consensus target sequences such as GATA-1: consensus target sequences such as CCATAGATAGG (SEQ ID NO: 52) and Nkx-2.2 : TTAAGTG SEQ ID NO: 65) and consensus target sequences such as Oct-1A: ATGCAAAT (SEQ ID NO: 66) showed high affinity. From this, this protein has the ability to bind to DNA and is considered to be associated with diseases caused by abnormalities such as differentiation of hematopoietic stem cells, blood cells, neural cells, circulatory organs, and apoptosis control. Moreover, it was estimated by Example 5 that it has the activity which enhances the effect | action which NF (kappa) B has, and is related to immune diseases, inflammatory diseases, cancer, etc., such as rheumatoid arthritis and osteoarthritis. Moreover, it has the activity which suppresses the effect | action which AP-1 has, and it was estimated that it is related to an inflammatory disease, cancer, etc. Moreover, it has an activity that enhances the action of HIF-1 slightly strongly, and ischemic disease, anemia, hypoxia, diabetic retinopathy, cardiovascular disease caused by abnormal angiogenesis, abnormal intracellular oxidation, etc. It was speculated to be related to cancer and the like. Furthermore, it has an activity that enhances the action of CREB, and it was speculated that it is related to central diseases such as memory impairment, cardiovascular diseases, diabetes and the like.
[0260]
In order to examine the expression tissue of this DNA, BLAST search is performed on dbEST (Nature Genetics 4, (4) 332-3 (1993)) using this DNA as a query, and e-value ≦ 10^-65When we extracted the names of organs or tissues in which 5 or more hits were found in human ESTs hit in, normal human bones, skeletal muscles, eyes, pancreas, lungs, intestines, kidneys, blood, brain, placenta, The liver, fetal brain, fetal spleen, cancerous skin, eye, lung, intestine, mammary gland, uterus, brain, pancreas, and testis. This DNA was cloned from a tracheal-derived cDNA library.
[0261]
This protein has functions and diseases peculiar to these tissues and cells, such as myeloma, lung cancer, colon cancer, small intestine cancer, kidney cancer, leukemia, lymphoma, brain tumor, liver cancer, skin cancer, melanoma, breast cancer uterine cancer・ Cancers such as pancreatic cancer and testicular cancer, inflammatory diseases such as pneumonia, pancreatitis, nephritis, hepatitis, bronchitis, osteoarthritis, immune system diseases such as rheumatoid arthritis, allergic diseases such as bronchial asthma, diabetes, ischemic diseases , Anemia, hypoxia, cardiovascular diseases, central diseases such as memory impairment, skeletal muscle diseases such as muscular dystrophy / polymyositis, infertility, spleen disease, congenital diarrhea, hemorrhagic predisposition, thrombus formation tendency, The possibility of relating to malignant hyperthermia and autism can be estimated, and it can be used as a target for diagnostics and therapeutics for these diseases.
[0262]
(9) c-brhip 3009181 (SEQ ID NOs: 9 and 19)
c-brhip 3009181 (hereinafter referred to as “the present DNA”, and the protein encoded by the DNA is referred to as “the present protein”) comprises 4010 bases as shown in SEQ ID NO: 9, of which base number 1140 Up to 3506 are open reading frames (including stop codon). The amino acid sequence predicted from the open reading frame consists of 788 amino acid residues (SEQ ID NO: 19).
[0263]
When homology search was performed using BLAST for the amino acid sequence of SEQ ID NO: 19, database registration symbol of NRDB protein database (database of non-overlapping amino acid sequences created from SWISS-PROT, PIR, TREMBLE, GENPEPT, PDB) P19838, Nuclear factor NFκB p105 subunit (Human) was a hit.
As its contents, P19838 consists of 968 amino acids, and 190-968 in the amino acid sequence is amino acid numbers 10-788 of the amino acid sequence described in SEQ ID NO: 19, e-value: 0.0, and 779 amino acid residues. It hit with 100% match.
NFκB is known as one of disease-related transcriptional regulators that regulate the expression of genes involved in carcinogenesis, inflammation, and apoptosis.
From the alignment of BLAST search, this protein differs from P19838 by 9 residues at the N-terminus, and P19838 differs from this protein by 189 residues. Further, in this DNA, on alignment, M58603 (DNA sequence corresponding to P19838) differs from the 5 'end by 1165 bases, and M58603 differs from this DNA by 963 bases. This DNA has base number 1140, and M58603 has base number 398, which are the translation start points, and this causes a difference in the N-terminus of the amino acid sequence.
[0264]
When the protein feature search by HMMPFAM was performed on the amino acid sequence of SEQ ID NO: 19, the amino acid sequence shown in amino acid numbers 70 to 170 showed the sequence showing the characteristics of IPT / TIG domain (amino acid sequence entered as TIG in Pfam). I found it. This domain has an immunoglobulin-like fold and is found not only in intracellular transcription factors with DNA-binding ability, but also in cell surface receptors.
Also, Death domain (amino acid sequence entered as death in Pfam) was found at amino acid numbers 637 to 712. This domain exists at the C-terminus, and is known to interact with the cytoplasmic portion of the receptor by regulating multimer formation of cell surface receptors such as FAS and TNF, and to transmit signals that induce apoptosis. ing.
Furthermore, Ankyrin repeat (amino acid sequence entered as an ank in Pfam) was found at amino acid numbers 362 to 394, 401 to 433, 434 to 466, 470 to 502, 504 to 537, and 538 to 570. This has a function as a protein-protein interaction domain, and it is known that an ankyrin isoform has a splice variant having Death domain at the C-terminus.
Furthermore, the presence of Rel homology domain region (amino acid sequence entered as RHD in Pfam) was observed at amino acid numbers 2 to 62 of this protein. This domain consists of structurally different domains. The N-terminal domain is a domain in which a similar structure is found in p53, and the C-terminal domain has an immunoglobulin-like fold that binds to DNA.
This Rel homology domain is also present at amino acid numbers 41 to 242 of P19838, but by comparison, it can be seen that the domain of this protein is shorter and the N-terminal side of Rel homology domain is missing.
NFκB is normally inactive by forming a complex with IκB in the cytoplasm, but IκB is released by active oxygen generated by inflammation or the like, and NFκB moves to the nucleus to induce gene transcription. In addition, it is known that stimulation from extracellular TNF or IL-1 is transmitted to the NFκB / IκB complex by the protein kinase cascade, and the complex is phosphorylated and activated.
Based on the characteristics of the sequence, this protein may have a transcriptional activity and a function different from those of P19838 in the above-described pathway of Nuclear factor NFκB.
[0265]
In order to clarify the exon structure of this DNA and M58603 cDNA, software sim4 (Genome Res. 8, 967-74 (1998)) for mapping the cDNA sequence to the genomic sequence was used to align the genomic sequence. The DNA sequence was mapped to 17 exons on human chromosome 4, and the M58603 sequence was mapped to 24 exons. All exons of this DNA are almost identical to the 8th to 24th exons of M58603, but the 1st to 7th exons of M58603 are not present in this DNA.
These differences may cause differences in binding and interaction with DNA, expression tissues, expression levels, and the like. From the above, the protein of SEQ ID NO: 19 is a splicing variant of P19838, Nuclear factor NFκB p105 subunit (Human).
[0266]
Analysis by PSORT II (Trends Biochem. Sci. 24, (1) 34-6 (1999)), which is a program for predicting intracellular localization of the protein, reveals that the probability of localization of this protein in the cell is The nucleus was 39.1%, mitochondria 21.7%, cytoplasm 17.4%, peroxisome 8.7%, vacuole 4.3%, secretory vesicle 4.3%, and endoplasmic reticulum 4.3%.
Since NFκB moves from the cytoplasm to the nucleus and activates various transcriptions, this distribution suggests that this protein may be a splicing variant of NFκB.
[0267]
According to Disease Browser (http://www.ensembl.org/Homo_sapiens/disseaseview) in the annotation information database Ensembl of the eukaryotic genome, it includes the position (q24) on the 4th chromosome where this DNA is mapped. The cause loci for psoriasis and dentinal dysplasia are present in q, and the location is not specified, but the cause locus for neonatal neutropenia may also be present on chromosome 4. all right. From the above, the possibility that this DNA is a causative gene of this disease and the possibility that mutations of this DNA containing splicing variants can be applied to the diagnosis of these diseases were expected.
[0268]
In order to examine the expression tissue of this DNA, BLAST search is performed on dbEST (Nature Genetics 4, (4) 332-3 (1993)) using this DNA as a query, and e-value ≦ 10^-50Extracting tissues with 5 or more hits in human ESTs hit in, normal human placenta, lungs, intestines, bones, immune system, lymphatic system, spleen, fetal liver, fetal eye, cancerous The immune system, intestines, and lungs. This DNA was cloned from a hippocampal cDNA library. Pigment Cell Res. 14 (6): 456-65 (2001) states that p105 is more highly expressed in metastatic melanoma cells than in normal human melanocytes.
[0269]
This protein has functions and diseases peculiar to these tissues and cells, such as lung cancer, colon cancer, small intestine cancer, myeloma, leukemia, lymphoma, liver cancer, melanoma, Alzheimer's disease, Parkinson's disease, chorea May be related to neurodegenerative diseases such as memory impairment, inflammatory diseases such as pneumonia / hepatitis, immune system diseases, allergic diseases, spleen diseases, infertility, psoriasis, dentin dysplasia, neutropenia It can be guessed, and it can be used as a target for diagnostics and therapeutics for these diseases.
[0270]
(10) c-best 2001444 (SEQ ID NOs: 10 and 20)
c-best 2001444 (hereinafter referred to as “the present DNA”, and the protein encoded by the DNA is referred to as “the present protein”), as shown in SEQ ID NO: 10, consists of 2709 bases, of which base number 469 Up to 1437 is an open reading frame (including a stop codon). The amino acid sequence predicted from the open reading frame consists of 322 amino acid residues (SEQ ID NO: 20).
[0271]
When homology search was performed on the amino acid sequence of SEQ ID NO: 20 using BLAST, the database registration symbol of the NRDB protein database (database of non-overlapping amino acid sequences created from SWISS-PROT, PIR, TREMBLE, GENPEPT, PDB) AJ009936, Nuclear hornone receptor PRR1-C (Human) was a hit.
As its contents, AJ009936 consists of 379 amino acids, and 58 to 379 in the amino acid sequence are amino acid numbers 1 to 322 of the amino acid sequence shown in SEQ ID NO: 20, e-value: 0.0, and 322 amino acid residues. It hit with 100% match.
Pregnane X receptor (PXR) is known to form a heterodimer with Retinoid X receptor (RXR), and to induce expression of cytochrome P450 such as CYP2 and CYP3 involved in drug metabolism, and other transporters involved in xenobiotic metabolism. ing. Since cytochrome P450 is involved not only in the metabolism of many pharmaceuticals but also in drug interactions that cause drug efficacy and side effects, it is important to observe changes in the transcriptional activity of PXR. CYP3A is also involved in the metabolism of steroid hormones and bile acids, indicating that cytochrome P450, whose expression is regulated by PXR, is widely involved in xenobiotic metabolism.
From the alignment of the BLAST search, this protein is 57 residues shorter than the AJ009936 at the N-terminus. In addition, this DNA differs from AJ009936 in the 5 ′ end by 463 bases on alignment, and AJ009936 differs from this DNA by 2170 bases. This DNA has base number 469, and M58603 has base number 1771, which is the translation start point, and this causes a difference in the N-terminus of the amino acid sequence.
[0272]
When the protein feature search of the amino acid sequence of SEQ ID NO: 20 was performed by HMMPFAM, the amino acid sequence shown in amino acid numbers 136 to 317 showed a characteristic of Ligand-binding domain of nuclear homone (amino acid entered in Pfam as harmonie_rec) Sequence). This domain is known to form a hydrophobic pocket of α-helix structure in the nuclear receptor and bind to ligands such as steroid hormones with high affinity. The activity is activated or suppressed, and the expression level is regulated.
In addition, Zinc fingers, C4 type (two domains) (amino acid sequence entered as Pfam as zf-C4) was also extracted from AJ009936 at amino acid numbers 62 to 138. This domain has a conserved cysteine-rich DNA-binding region and is a eukaryotic steroid hormone, active vitamin D.₃It is found in the nuclear receptor family such as retinoic acid and thyroid hormone.
[0273]
PROSITE (Nucleic Acids Res. 30, (1) 235-8. (2002)), which is a database of amino acid patterns in which domain structures and families are classified based on similarity of protein functions and can search for functionally important sites According to AJ009936, the amino acid numbers 64 to 91 also have a STEROID_FINGER region. This part is the DNA binding region of the hormone receptor in the nucleus.
Since these two domains and regions do not exist in this protein, this protein differs from AJ009936 in the state of binding to hormones and binding to DNA in the nucleus, and as a result, the transcription activity also changes and CYP3 There is a possibility that the expression is suppressed.
[0274]
In order to clarify the exon structure of this DNA and AJ009936 cDNA, software sim4 (Genome Res. 8, 967-74 (1998)) that maps the cDNA sequence to the genomic sequence was used to align the genomic sequence. This DNA sequence was mapped to 6 exons on human chromosome 3, and the AJ009936 sequence was mapped to 10 exons. All exons of this DNA are almost identical to the 4th to 9th exons of AJ009936, but the 1st to 3rd and 10th exons of AJ009936 are not present in this DNA.
These differences may cause differences in binding and interaction with DNA, expression tissues, expression levels, and the like. From the above, the protein of SEQ ID NO: 20 is a splicing variant of AJ009936, Nuclear harmone receptor PRR1-C (Human).
[0275]
Analysis by PSORT II (Trends Biochem. Sci. 24, (1) 34-6 (1999)), which is a program for predicting intracellular localization of the protein, reveals that the probability of localization of this protein in the cell is The cytoplasm was 56.5%, the nucleus was 30.4%, the mitochondria was 8.7%, and the Golgi body was 4.3%. The probability of localization of AJ009936 is 82.6% in the nucleus, 8.7% in the cytoplasm, and 8.7% in the mitochondria, and this protein has a high probability of localization in the cytoplasm. Therefore, this protein may have a different localization from AJ009936.
[0276]
According to the Disease Browser (http://www.ensembl.org/Homo_sapiens/diseseview) in the annotation information database Ensembl of the eukaryotic genome, the position (q13.31) on the third chromosome where this DNA is mapped is shown. It was found that Chlocot-Marie-Tooth disease included in q13-21, familial hypoparathyroidism and familial tremor in q13, and the causal locus of myotonic dystrophy in q. From the above, the possibility that this DNA is a causative gene of this disease and the possibility that mutations of this DNA containing splicing variants can be applied to the diagnosis of these diseases were expected.
[0277]
When this protein was expressed using a wheat germ cell-free protein synthesis system, in Example 4, a consensus target sequence such as NF-1A1: CGTTGGGGTTTGGCACGGGGCCA (SEQ ID NO: 41), a target sequence of RF-X1: GGTAACATAGCAAC (sequence) No. 42), YY1 target sequence: CGGCCATCTTGGGCT (SEQ ID NO: 55), Snail target sequence: CACCGTTTTTCA (SEQ ID NO: 57), Evi-1 target sequence: AGATAGATATAA (SEQ ID NO: 59), Ikaros and other consensus target sequences: TTGGGAGG (SEQ ID NO: 60), HNF-1A target sequence: GGTTAATGATTACACCAC (SEQ ID NO: 64), Nkx-2.2 and other consensus target sequences: TTAAGTGGTTT (SEQ ID NO: 65), consensus target sequences such as Oct-1A: ATGCAAAT (SEQ ID NO: 66), consensus target sequences such as Oct-2: TATTTGCAT (SEQ ID NO: 67), consensus target sequences such as Pax-3: CGTCACGCTTTGA (SEQ ID NO: 68) ), Pax-1 target sequence: CCGTTCCCGCTCTAGATAT (SEQ ID NO: 69), consensus target sequences such as NF-ATc: AGGAAAA (SEQ ID NO: 75), MEF-2A target sequence: CCTAAAAATA (SEQ ID NO: 79), SRF target sequence : CCATATATGGACAT (SEQ ID NO: 80) and E2 target sequence: AACCAAAAACGGTAA (SEQ ID NO: 81). Therefore, this protein has DNA binding ability, and is caused by abnormalities such as proliferation of hematopoietic cells and hepatocytes, generation and differentiation of immune system cells, lipid metabolism, muscle formation, such as diseases such as lifestyle-related diseases. The relation of is considered. Further, according to Example 5, the activity of PPAR is slightly strongly suppressed, lifestyle diseases such as diabetes and obesity due to abnormal endocrine / sugar / lipid metabolism, etc., circulatory system diseases due to abnormal vascular function, inflammation, etc. It was speculated to be related to cancer and the like. Moreover, it has the activity which suppresses the effect | action which NF (kappa) B has, and it was estimated that it is related to immune diseases, inflammatory diseases, cancer, etc., such as rheumatoid arthritis and osteoarthritis.
[0278]
In order to examine the expression tissue of this DNA, BLAST search is performed on dbEST (Nature Genetics 4, (4) 332-3 (1993)) using this DNA as a query, and e-value ≦ 10^-04When the tissue in which the hit was found was extracted with the human EST that was hit in, the human normal fetal liver, fetal spleen, liver, stomach, intestine, and cancerous immune system were found. This DNA was cloned from a breast-derived cDNA library.
[0279]
This protein has functions and diseases peculiar to these tissues and cells, for example, cancers such as liver cancer, gastric cancer, colon cancer, small intestine cancer, leukemia and lymphoma, inflammatory diseases such as osteoarthritis, and immune systems such as rheumatoid arthritis Can be related to diseases, allergic diseases, diabetes, obesity, cardiovascular diseases, spleen diseases, infertility, familial hypoparathyroidism, familial tremor, myotonic dystrophy It can be used as a target for diagnostics and therapeutics.
[0280]
【The invention's effect】
Since the protein of the present invention and the DNA encoding the same have transcriptional regulatory activity and the like, the protein or a DNA encoding the same can be used to screen for a substance that modulates the activity, and the disease associated with the protein It is useful for the development of a medicine that can act on the like.
[0281]
[Sequence Listing]

Claims (15)

The following protein (a) or (b):
(A) a protein comprising the amino acid sequence of any one of SEQ ID NOS: 11 to 20,
(B) A protein comprising an amino acid sequence in which one or several amino acids are deleted, substituted and / or added in the amino acid sequence of any one of SEQ ID NOs: 11 to 20, and having transcriptional regulatory activity. DNA encoding the protein according to claim 1. A full-length cDNA encoding the protein of claim 1. DNA of any of the following (a) or (b);
(A) DNA having the base sequence according to any one of SEQ ID NOs: 1 to 10,
(B) a protein having a base sequence in which one or several bases are deleted, substituted and / or added in the base sequence described in any of SEQ ID NOs: 1 to 10 and having transcriptional regulatory activity DNA to do. A recombinant vector comprising the DNA according to any one of claims 2 to 4. A gene-transferred cell into which the DNA according to any one of claims 2 to 4 or the recombinant vector according to claim 5 has been introduced, or an individual comprising the cell. The protein of claim 1 produced by the cell of claim 6. A sense oligonucleotide having the same sequence as 5 to 100 consecutive bases in the base sequence of the DNA according to any one of claims 2 to 4, an antisense oligonucleotide having a sequence complementary to the sense oligonucleotide, and An oligonucleotide selected from the group consisting of oligonucleotide derivatives of the sense or antisense oligonucleotide. An antibody or a partial fragment thereof that specifically binds to the protein according to claim 1 or 7. The antibody according to claim 9, wherein the antibody is a monoclonal antibody. The antibody according to claim 10, wherein the monoclonal antibody has an action of neutralizing the transcriptional regulatory activity of the protein according to claim 1 or 7. A screening method for a substance that regulates the activity of the protein, comprising contacting the test substance with the protein according to claim 1 or 7, and measuring a change in activity of the protein by the test substance. A screening method for a substance that regulates the expression of DNA, comprising contacting the test substance with the gene-transferred cell according to claim 6 and detecting a change in the expression level of the DNA introduced into the cell. Information on at least one amino acid sequence selected from the amino acid sequence of the protein according to claim 1 and / or information on at least one base sequence selected from the base sequence of the DNA according to any one of claims 2 to 4. A computer-readable recording medium storing the data. A carrier to which the protein according to claim 1 and / or the DNA according to any one of claims 2 to 4 are bound.