JP2005508600A - Nucleic acid related proteins - Google Patents

Abstract

The present invention provides human nucleic acid associated proteins (NAAP) and polynucleotides that identify and encode NAAP. The present invention also provides expression vectors, host cells, antibodies, agonists and antagonists. The present invention also provides a method for diagnosing, treating or preventing a disease associated with abnormal expression of NAAP.

Description

【０１８４】
場合によっては、最終コンセンサスポリヌクレオチド配列を確認するために、表４に示すような配列カバレッジと重複するIncyte cDNAカバレッジが得られたが、該当するIncyte cDNA識別番号は示さなかった。
【０１８５】
表５は、Incyte cDNA配列を用いて構築された完全長ポリヌクレオチド配列のための代表的なcDNAライブラリを示している。代表的なcDNAライブラリとはIncyte cDNAライブラリであり、これは、最も頻繁にはIncyte cDNA配列群によって代表されるが、これらは、上記のポリヌクレオチド配列を構築および確認するために用いられた。cDNAライブラリを作製するために用いた組織およびベクターを表５に示し、表６で説明している。
【０１８６】
本発明には、NAAPの変異体も含まれる。好適なNAAP変異配列は、NAAPの機能的或いは構造的特徴の少なくとも１つを有し、かつ該NAAPアミノ酸配列に対して少なくとも約８０％のアミノ酸配列同一性、或いは少なくとも約９０％のアミノ酸配列同一性、更には少なくとも約９５％のアミノ酸配列同一性を有する配列である。
【０１８７】
本発明はまた、NAAPをコードするポリヌクレオチドを提供する。特定の実施態様において、本発明は、NAAPをコードする、SEQ ID NO:17-32からなる一群から選択された１配列を持つポリヌクレオチド配列を提供する。SEQ ID NO:17-32のポリヌクレオチド配列には、配列表に示したように等価RNA配列をも含むが、そこでは窒素塩基チミンの出現はウラシルに置換され、糖のバックボーンはデオキシリボースではなくリボースで構成される。
【０１８８】
本発明にはまた、NAAPをコードするポリヌクレオチド配列の変異配列を含む。詳細には、このような変異体ポリヌクレオチド配列は、NAAPをコードするポリヌクレオチド配列との、少なくとも約７０％のポリヌクレオチド配列同一性、あるいは少なくとも約８５％のポリヌクレオチド配列同一性、更には少なくとも約９５％ものポリヌクレオチド配列同一性を持つこととなる。本発明の或る実施態様では、SEQ ID NO:17−32からなる群から選択されたアミノ酸配列と少なくとも約７０％、或いは少なくとも約８５％、または少なくとも約９５％もの一致率を有するようなSEQ ID NO:17−32からなる群から選択された配列を有するポリヌクレオチド配列の変異配列を含む。上記の任意のポリヌクレオチドの変異体は、NAAPの機能的若しくは構造的特徴を少なくとも１つ有するアミノ酸配列をコードし得る。
【０１８９】
更に別の例では、本発明の或るポリヌクレオチド変異配列は、NAAPをコードするポリヌクレオチド配列のスプライス変異配列である。或るスプライス変異配列はNAAPをコードするポリヌクレオチド配列との顕著な配列同一性を持つ部分複数を有し得るが、mRNAプロセッシング中のエキソン群の選択的スプライシングによって生ずる、配列の数ブロックの付加または欠失により、通常、より多数またはより少数のポリヌクレオチドを有することになる。或るスプライス変異配列には、約７０％未満、または約６０％未満、あるいは約５０％未満のポリヌクレオチド配列同一性が、NAAPをコードするポリヌクレオチド配列との間で全長に渡って見られるが、このスプライス変異配列のいくつかの部分には、NAAPをコードするポリヌクレオチド配列の各部との、少なくとも約７０％、あるいは少なくとも約８５％、または少なくとも約９５％、なおまたは１００％の、ポリヌクレオチド配列同一性を有することとなる。例えば、SEQ ID NO:29の配列を持つ或るポリヌクレオチドは、SEQ ID NO:25の配列を持つポリヌクレオチドのスプライス変異体である。上記のスプライス変異配列は何れも、NAAPの機能的或いは構造的特徴の少なくとも１つを有するアミノ酸配列をコードすることができる。
【０１９０】
遺伝暗号の縮重により、NAAPをコードする種々のポリヌクレオチド配列が作り出され、中には、既知のいかなる天然遺伝子のポリヌクレオチド配列群とも最小の類似性しか有しない配列もあることは、当業者には理解されよう。したがって本発明には、可能コドン選択に基づく組合せの選択によって産出し得るあらゆる可能なポリヌクレオチド配列のバリエーションを網羅し得る。これらの組み合わせは、天然NAAPのポリヌクレオチド配列に適用される標準的なトリプレット遺伝暗号を基に作られ、全ての変異が明確に開示されていると考慮されたい。
【０１９１】
NAAPとその変異配列とをコードするヌクレオチド配列は一般に、好適に選択されたストリンジェンシー条件下で天然NAAPのヌクレオチド配列とハイブリダイズ可能であるが、非天然コドン群を含めるなどの実質的に異なるコドン使用を有する、NAAP或いはその誘導体をコードするヌクレオチド配列を作り出すことは、有益であり得る。宿主が特定コドンを利用する頻度に基づいて、特定の真核宿主または原核宿主に発生するペプチドの発現率を高めるようにコドンを選択し得る。コードされるアミノ酸配列を改変せずに、NAAP及びその誘導体をコードするヌクレオチド配列を実質的に改変する別の理由には、天然配列から作られる転写物より例えば長い半減期など好ましい特性を備えるRNA転写物を作ることもある。
【０１９２】
本発明には、また、NAAPとその誘導体群とをコードする、DNA配列群またはその断片群を、完全に合成化学によって作り出す過程をも含む。作製後、当分野で公知の試薬類を用いて、この合成配列を任意の多くの入手可能な発現ベクター及び細胞系中に挿入し得る。更に、合成化学を用いて、NAAPまたはその任意の断片をコードする配列に突然変異を誘発し得る。
【０１９３】
更に本発明には、種々のストリンジェンシー条件下で、請求項に記載されたポリヌクレオチド配列、特にSEQ ID NO:17-32およびそれらの断片へのハイブリダイズが可能なポリヌクレオチド配列を含む（例えばChiez, R.M.およびF.Z. Regnier (1987) Methods Enzymol.152:399-407; Kimmel, A.R. (1987) Methods Enzymol. 152:507-511)。アニーリングおよび洗浄条件を含むハイブリダイゼーションの条件は、「定義」に記載した。
【０１９４】
DNAシークエンシングの方法は当分野では公知であり、本発明のいずれの実施例も、DNAシークエンシング方法を用い得る。DNAシークエンシング方法には酵素を用い得る。例えばDNAポリメラーゼIのクレノウ断片、SEQUENASE（US Biochemical, Cleveland OH）、Taqポリメラーゼ（Applied Biosystems）、熱安定性T7ポリメラーゼ（Amersham, Pharmacia Biotech, Piscataway NJ）を用い得る。あるいは、例えばELONGASE増幅システム（Life Technologies, Gaithersburg MD）において見られるように、ポリメラーゼと校正エキソヌクレアーゼとを併用し得る。好適には、MICROLAB2200液体転移システム（Hamilton, Reno, NV）、PTC200サーマルサイクラー（MJ Research, Watertown MA）およびABI CATALYST 800サーマルサイクラー（Applied Biosystems）などの装置を用いて配列の準備を自動化する。次に、ABI 373 あるいは 377 DNAシークエンシングシステム（Applied Biosystems）、MEGABACE 1000 DNAシークエンシングシステム（Molecular Dynamics, Sunnyvale CA）または当分野で公知の他のシステムを用いてシークエンシングを行う。結果として得た配列を、当分野で周知の種々のアルゴリズムを用いて分析する（例えば、Ausubel, F.M. (1997) Short Protocols in Molecular Biology, John Wiley & Sons, New York NY, 7.7ユニット、Meyers, R.A. (1995) Molecular Biology and Biotechnology, Wiley VCH, New York NY, 856-853ページを参照）。
【０１９５】
当分野で周知の、PCR法をベースにした種々の方法と、部分的ヌクレオチド配列とを利用して、NAAPをコードする核酸配列を伸長し、プロモーターや調節エレメントなど、上流にある配列を検出し得る。例えば、使用し得る方法の１つである制限部位PCR法は、ユニバーサルプライマーおよびネステッドプライマーを用いてクローニングベクター内のゲノムDNAからの未知の配列を増幅する方法である（例えば、Sarkar, G. (1993) PCR Methods Applic. 2:318-322を参照）。別の方法に逆PCR法があり、これは多岐の方向に伸長するプライマー群を用いて、環状化した鋳型から未知の配列を増幅する方法である。鋳型は、或る既知のゲノム遺伝子座およびその周辺の配列群からなる制限酵素断片群から得る(例えば Triglia, T. 他(1988) Nucleic Acids Res. 16:8186を参照)。第３の方法としてキャプチャPCR法があり、これにはヒトおよび酵母人工染色体DNAの既知の配列群に隣接するDNA断片群をPCR増幅する方法を含む(例えば Lagerstrom, M. 他(1991) PCR Methods Applic 1:111-119を参照）。この方法では、PCRを行う前に、複数の制限酵素の消化およびライゲーション反応を用い、未知の配列領域内に組換え二本鎖配列を挿入し得る。未知の配列群を検索するために用い得る他の複数の方法も当分野で公知である（例えばParker, J.D.他(1991) Nucleic Acids Res. 19:30553060を参照)。更に、PCR、ネステッドプライマー類、およびPROMOTERFINDERライブラリ類（Clontech, Palo Alto CA）を用いてゲノムDNAをウォーキングし得る。この手順は、ライブラリ類をスクリーニングする必要がなく、イントロン／エキソン接合部の発見に有用である。全てのPCRベースの方法では、市販ソフトウェア、例えばOLIGO 4.06プライマー分析ソフトウェア（National Biosciences, Plymouth MN）或いは別の好適なプログラムを用いて、長さが約２２〜３０ヌクレオチド、GC含有率が約５０％以上で、温度約６８℃〜７２℃で鋳型に対してアニーリングするように、プライマー群を設計し得る。
【０１９６】
完全長cDNA群をスクリーニングする際は、より大きなcDNA群を含むようにサイズ選択されたライブラリ群を用いるのが好ましい。更に、ランダムプライマーのライブラリ群は、しばしば遺伝子群の５'領域を有する配列を含み、オリゴd（T）ライブラリが完全長cDNAを作製できない状況に対して好適である。ゲノムライブラリ群は、５'非転写調節領域への、配列の伸長に有用であろう。
【０１９７】
市販のキャピラリー電気泳動システム群を用いて、シークエンシングまたはPCR産物のサイズを分析し、またはそのヌクレオチド配列を確認し得る。具体的には、キャピラリーシークエンシングは、電気泳動による分離のための流動性ポリマーと、４つの異なるヌクレオチドに特異的であるような、レーザで活性化される蛍光色素と、発光された波長の検出に利用するCCDカメラとを有し得る。出力／光の強度は、適切なソフトウェア（Applied Biosystems社のGENOTYPER、SEQUENCE NAVIGATORなど）を用いて電気信号に変換し得る。サンプルのロードからコンピュータ分析および電子データ表示までの全プロセスがコンピュータ制御可能である。キャピラリー電気泳動法は、特定のサンプルに少量しか存在しないようなDNA小断片のシークエンシングに特に適している。
【０１９８】
本発明の別の実施例では、NAAPをコードするポリヌクレオチド配列またはその断片を組換えDNA分子にクローニングして、適切な宿主細胞内にNAAP、その断片または機能的等価物を発現させることが可能である。遺伝暗号固有の縮重により、実質的に同じ或いは機能的に等価のアミノ酸配列をコードする別のDNA配列を産生し、これらの配列をNAAPの発現に利用し得る。
【０１９９】
種々の目的で、NAAPをコードする配列群を改変するために、当分野で一般的に既知の複数の方法を用いて、本発明のヌクレオチド配列群を組換えることができる。この組換えの多様な目的には、遺伝子産物のクローン化の、あるいはプロセッシングおよび／または発現のモディフィケーションが含まれるが、これらに限定されるものではない。遺伝子断片と合成オリゴヌクレオチドとの、ランダムなフラグメンテーションおよびPCR再アセンブリによるDNAシャッフリングを用い、ヌクレオチド配列を組み換え得る。例えば、オリゴヌクレオチドを介した部位特異的変異誘導を利用して、新規な制限部位の作製、グリコシル化パターンの改変、コドン優先の変更、スプライス変異配列の生成などを起こす突然変異を導入し得る。
【０２００】
本発明のヌクレオチドは、MOLECULARBREEDING (Maxygen Inc., Santa Clara CA; 米国特許第5,837,458号; Chang, C.-C. 他 (1999) Nat. Biotechnol. 17:793-797; Christians, F.C. 他 (1999) Nat. Biotechnol. 17:259-264; Crameri, A. 他 (1996) Nat. Biotechnol. 14:315-319)などのDNAシャッフリング技術の対象となり得る。これにより、生物活性または酵素活性、あるいは他の分子や化合物と結合する能力など、NAAPの生物学的特性を改変あるいは改良し得る。DNAシャッフリングは、PCRを介する遺伝子断片組換えを用いて遺伝子変異配列のライブラリを作製するプロセスである。ライブラリはその後、所望の特性を持つ遺伝子変異配列群を同定する、選択またはスクリーニングの手順を経る。続いて、これら好適な変異配列をプールし、更に反復してDNAシャッフリングおよび選択／スクリーニングを行い得る。かくして、「人工的な」育種および急速な分子の進化によって、多様な遺伝子が作られる。例えば、ランダムポイント突然変異を持つ単一の遺伝子の断片を組み換えて、スクリーニングし、その後所望の特性が最適化されるまでシャッフリングし得る。あるいは、所定の遺伝子の断片群を同種または異種のいずれかから得た同一遺伝子ファミリーの相同遺伝子の断片と組み換え、それによって天然に存在する複数の遺伝子の遺伝多様性を、方向付けした制御可能な方法で最大化させることができる。
【０２０１】
別の実施態様によれば、NAAPをコードする配列は、当分野で周知の化学的方法を用いて、全体あるいは一部が合成可能である（例えば、Caruthers. M.H.他(1980) Nucl. Acids Res. Symp. Ser 7:215-223; 及びHorn, T.他(1980) Nucl. Acids Res. Symp. Ser.7:225-232を参照）。別法として、化学的方法を用いてNAAP自体またはその断片を合成することが可能である。例えば、種々の液相または固相技術を用いてペプチド合成を行い得る（例えばCreighton, T.（1984）Proteins, Structures and Molecular Properties, WH Freeman, New York NY,55-60;およびRoberge, J.Y. 他(1995) Science 269:202-204等を参照)。自動合成はABI 431Aペプチドシンセサイザ（Applied Biosystems）を用いて達成し得る。更に、NAAPのアミノ酸配列または任意のその一部を、直接合成の際に改変することにより、及び／または他のタンパク質からの配列群または任意のその一部と組み合わせることにより、或る天然ポリペプチドの配列を有するポリペプチドまたは変異体ポリペプチドが作製され得る。
【０２０２】
このペプチドは、分離用高速液体クロマトグラフィーを用いて実質的に精製し得る（例えばChiez, R.M.およびF.Z. Regnier (1990) Methods Enzymol. 182:392-421を参照）。この合成ペプチドの組成は、アミノ酸分析またはシークエンシングによって確認できる（例えば前出のCreighton, 28-53ページを参照）。
【０２０３】
生物学的に活性なNAAPを発現させるために、NAAPをコードするヌクレオチド配列またはその誘導体を好適な発現ベクターに挿入し得る。好適な発現ベクターとは、好適な宿主に挿入されたコーディング配列の転写および翻訳の制御に必要なエレメント群を持つベクターである。必要なエレメントとしては、該ベクターと、NAAPをコードするポリヌクレオチド配列群とにおける調節配列群（エンハンサー、構成型及び発現誘導型のプロモーター、５'及び３'の非翻訳領域など)が含まれる。必要なエレメント群は、特長および特異性が様々である。特定の開始シグナル類を用いて、NAAPをコードする配列群の、より効果的な翻訳を達成できる。開始シグナルの例には、ATG開始コドンと、コザック配列など近傍の配列とが含まれる。NAAPをコードする配列群、その開始コドン、および上流の調節配列群が、好適な発現ベクターに挿入された場合は、更なる転写制御シグナルや翻訳制御シグナルは必要ないこともある。しかしながら、コーディング配列あるいはその断片のみが挿入された場合は、インフレームATG開始コドンなど外来性の翻訳制御シグナルが発現ベクターに含まれるようにすべきである。外来性の翻訳エレメント群および開始コドン群は、様々な天然物および合成物を起源とし得る。用いられる特定の宿主細胞系に好適なエンハンサーを含めることで発現の効率を高めることが可能である(例えば Scharf, D. 他(1994) Results Probl.Cell Differ. 20:125162を参照)。
【０２０４】
当業者に周知の方法を用いて、NAAPをコードする配列と、好適な転写および翻訳制御エレメントとを持つ発現ベクターを作製し得る。これらの方法としては、in vitro組換えDNA技術、合成技術、およびin vivo遺伝子組換え技術がある(例えば Sambrook, J. 他(1989) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, Plainview NY, 4, 8,および16-17章; Ausubel, F.M. 他(1995) Current Protocols in Molecular Biology, John Wiley & Sons, New York NY, 9, 13, および 16章を参照)。
【０２０５】
種々の発現ベクター／宿主系を利用して、NAAPをコードする配列群の保持及び発現ができる。限定するものではないがこのような発現ベクター／宿主系には、組換えバクテリオファージ、プラスミドまたはコスミドDNA発現ベクターで形質転換させた細菌や、酵母菌発現ベクターで形質転換させた酵母菌など微生物や、ウイルス発現ベクター（例えばバキュロウイルス）に感染した昆虫細胞系や、ウイルス発現ベクター（例えばカリフラワーモザイクウイルス、CaMVまたはタバコモザイクウイルス、TMV）または細菌発現ベクター（例えばTiプラスミドまたはpBR322プラスミド）で形質転換させた植物細胞系、動物細胞系がある(例えば前出 Sambrook; 前出Ausubel; Van Heeke, G.およびS.M. Schuster (1989) J. Biol. Chem. 264:55035509; Engelhard, E.K. 他(1994) Proc. Natl. Acad. Sci. USA 91:3224-3227; Sandig, V. 他(1996) Hum.Gene Ther.7:1937-1945; Takamatsu, N. (1987) EMBO J. 6:307311;『マグローヒル科学技術年鑑』(The McGraw Hill Yearbook of Science and Technology) (1992) McGraw Hill New York NY,191196; Logan, J.およびT. Shenk (1984) Proc. Natl. Acad. Sci. USA 81:36553659;およびHarrington, J.J. 他(1997) Nat. Genet. 15:345-355を参照)。レトロウイルス、アデノウイルス、ヘルペスウイルスまたはワクシニアウイルス由来の発現ベクター、または種々の細菌性プラスミド由来の発現ベクターを用いて、ヌクレオチド配列を標的器官、組織または細胞集団へ送達することができる(例えばDi Nicola, M. 他(1998) Cancer Gen. Ther. 5(6):350-356; Yu, M. 他(1993) Proc. Natl. Acad. Sci. USA 90(13):6340-6344; Buller, R.M. 他(1985) Nature 317(6040):813-815; McGregor, D.P. 他(1994) Mol. Immunol. 31(3):219-226;およびVerma, I.M.およびN. Somia (1997) Nature 389:239-242を参照)。本発明は使用される宿主細胞によって限定されるものではない。
【０２０６】
細菌系では、多数のクローニングベクターおよび発現ベクターが、NAAPをコードするポリヌクレオチド配列の使用目的に応じて選択可能である。例えば、NAAPをコードするポリヌクレオチド配列の慣例的なクローニング、サブクローニング、増殖には、PBLUESCRIPT（Stratagene, La Jolla CA）またはPSPORT１プラスミド（Life Technologies）などの多機能の大腸菌ベクターを用いることができる。ベクターのマルチクローニング部位にNAAPをコードする配列をライゲーションするとlacＺ遺伝子が破壊され、組換え分子を含む形質転換された細菌の同定のための比色スクリーニング法が可能となる。更にこれらのベクターは、クローニングされた配列におけるin vitro転写、ジデオキシのシークエンシング、ヘルパーファージによる一本鎖のレスキュー、入れ子欠失の生成にも有用であろう（例えば、Van Heeke, G.およびS.M. Schuster（1989）J. Biol. Chem. 264:55035509を参照）。例えば、抗体の産生のためなどに多量のNAAPが必要な場合は、NAAPの発現をハイレベルで誘導するベクターが使用できる。例えば、強力な誘導SP6バクテリオファージプロモーターまたは誘導T7バクテリオファージプロモーターを持つベクターが使用できる。
【０２０７】
酵母の発現系を使用してNAAPを産出することもできる。α因子、アルコールオキシダーゼ、PGHプロモーターなど、構成型あるいは誘導型のプロモーターを持つ多数のベクターが、出芽酵母菌（Saccharomyces cerevisiae）またはピキア酵母（Pichia pastoris）内で使用可能である。更に、このようなベクターは、発現したタンパク質の分泌か細胞内での保持かのどちらかを指示し、安定した増殖のために宿主ゲノムの中に外来配列を組み込むことを可能にする(例えば前出Ausubel, 1995; Bitter, G.A. 他(1987) Methods Enzymol.153:516544;およびScorer, C.A. 他(1994) Bio/Technology 12:181-184を参照)。
【０２０８】
植物系もNAAPの発現に使用可能である。NAAPをコードする配列の転写は、ウイルスプロモーター、例えば単独或いはTMV（Takamatsu, N. (1987) EMBO J 6:307-311）由来のオメガリーダー配列と組み合わせて用いられるようなCaMV由来の35S及び19Sプロモーターによって促進される。あるいは、RuBisCOの小サブユニットなどの植物プロモーター、または熱ショックプロモーターを用い得る(例えばCoruzzi, G. 他(1984) EMBO J. 3:16711680; Broglie, R. 他(1984) Science 224:838843;およびWinter, J. 他(1991) Results Probl.Cell Differ. 17:85105を参照)。これらの構成物は、直接DNA形質転換により、または病原体を媒介とする形質移入により、植物細胞内に導入し得る（例えば『マグローヒル科学技術年鑑』(The McGraw Hill Yearbook of Science and Technology) (1992) McGraw Hill New York NY,191-196ページを参照）。
【０２０９】
哺乳類細胞においては、多数のウイルスベースの発現系を利用し得る。アデノウイルスが発現ベクターとして用いられる場合、後期プロモーターと３連リーダー配列とからなるアデノウイルス転写／翻訳複合体に、NAAPをコードする配列群をライゲーションし得る。アデノウイルスゲノムの非必須E1またはE3領域へ挿入することにより、宿主細胞内でNAAPを発現する感染ウイルスを得ることができる（例えばLogan, J.およびT. Shenk（1984）Proc. Natl. Acad. Sci. USA 81:36553659を参照）。更に、ラウス肉腫ウイルス（RSV）エンハンサーなどの転写エンハンサーを用いて、哺乳動物宿主細胞における発現を増大させ得る。SV40またはEBVをベースにしたベクターを用いてタンパク質を高レベルで発現させることもできる。
【０２１０】
また、ヒト人工染色体（HAC）類を用いて、或るプラスミドに含まれ得る断片やプラスミドから発現し得る断片より大きなDNAの断片群を送達し得る。治療のために約６kb〜１０MbのHACが作製され、従来の送達方法（リポソーム、ポリカチオンアミノポリマー、またはベシクル）で送達されている(例えばHarrington, J.J. 他(1997) Nat. Genet. 15:345-355を参照)。
【０２１１】
哺乳動物系内の組換えタンパク質の長期にわたる産生のためには、細胞株におけるNAAPの安定した発現が望ましい。例えば、NAAPをコードする配列を細胞株に形質転換するために、発現ベクター類と、同じベクター上の或いは別のベクター上の選択可能マーカー遺伝子とを用い得る。用いる発現ベクターは、ウイルス起源の複製要素、および／または内因性の発現エレメント群を持ち得る。ベクターの導入後、選択培地に移す前に強化培地で約１〜２日間、細胞を増殖させ得る。選択可能マーカーの目的は選択剤への抵抗性を与えることであり、選択可能マーカーの存在により、導入した配列をうまく発現するような細胞の成長および回収が可能となる。安定的に形質転換された細胞の耐性クローンは、その細胞型に適した組織培養技術を用いて増殖可能である。
【０２１２】
任意の数の選択系を用いて、形質転換細胞株を回収できる。限定するものではないがこのような選択系には、tk⁻細胞のために用いられる単純ヘルペスウイルスのチミジンキナーゼ遺伝子と、apr⁻細胞のために用いられる単純ヘルペスウイルスのアデニンホスホリボシルトランスフェラーゼ遺伝子がある(例えばWigler, M. 他(1977) Cell 11:223-232; Lowy, I. 他(1980) Cell 22:817-823を参照)。また、選択の基礎として代謝拮抗物質、抗生物質あるいは除草剤への耐性を用いることができる。例えばdhfrはメトトレキセートに対する耐性を与え、neoはアミノグリコシッドネオマイシンおよびG-418に対する耐性を与え、alsはクロルスルフロンに対する耐性を、patはホスフィノトリシンアセチルトランスフェラーゼに対する耐性を各々与える(例えばWigler, M. 他(1980) Proc. Natl. Acad. Sci. USA 77:35673570; ColbereGarapin, F. 他(1981) J. Mol. Biol. 150:114を参照)。この他の選択可能な遺伝子、例えば、代謝のための、細胞の必要条件を改変するtrpBおよびhisDも、文献に記載がある（例えばHartman, S.C.およびR.C. Mulligan (1988) Proc. Natl. Acad. Sci. USA 85:80478051を参照）。可視マーカー類、例えばアントシアニンや、緑色蛍光タンパク質（GFP；Clontech）、βグルクロニダーゼおよびその基質であるβグルクロニド、またはルシフェラーゼおよびその基質であるルシフェリンを用い得る。これらのマーカーを用いて、トランスフォーマントを同定するだけでなく、特定のベクター系に起因する一過性あるいは安定したタンパク質発現を定量し得る（例えばRhodes, C.A. (1995) Methods Mol. Biol. 55:121131を参照）。
【０２１３】
マーカー遺伝子発現の有無によって目的の遺伝子の存在が示唆されても、その遺伝子の存在および発現の確認が必要な場合もある。例えば、NAAPをコードする配列がマーカー遺伝子配列の中に挿入された場合、NAAPをコードする配列を持つ形質転換された細胞は、マーカー遺伝子機能の欠落により同定可能である。または、単一プロモーターの制御下で、或るマーカー遺伝子を、NAAPをコードする１配列とタンデムに配置することもできる。誘導または選択に応答したマーカー遺伝子の発現は通常、タンデム遺伝子の発現も示す。
【０２１４】
一般に、NAAPをコードする核酸配列を含み且つNAAPを発現する宿主細胞は、当業者によく知られている種々の方法を用いて同定することが可能である。限定するものではないが当業者によく知られている方法には、DNA-DNA或いはDNA-RNAハイブリダイゼーションと、PCR増幅とがあり、また、核酸配列或いはタンパク質配列の検出、定量、或いはその両方を行うための、膜系、溶液ベース或いはチップベースの技術を含む、タンパク質の生物学的検定法または免疫学的検定法もある。
【０２１５】
特異的ポリクローナル抗体または特異的モノクローナル抗体を用いてNAAPの発現の検出と計測とを行うための免疫学的方法は、当分野で既知である。このような技術の例としては、酵素結合イムノソルベントアッセイ（ELISA）、ラジオイムノアッセイ（RIA）、蛍光活性化細胞選別（FACS）などが挙げられる。NAAP上の２つの非干渉エピトープに反応するモノクローナル抗体群を用いた、２部位のモノクローナルベースイムノアッセイ（two-site, monoclonal-based immunoassay）が好ましいが、競合結合試験を用いることもできる。これらのアッセイおよび他のアッセイは、当分野で周知である(例えばHampton, R. 他(1990) Serological Methods, a Laboratory Manual, APS Press, St. Paul MN, Sect.IV; Coligan, J.E. 他(1997) Current Protocols in Immunology, Greene Pub.Associates and Wiley-Interscience, New York NY;およびPound, J.D. (1998) Immunochemical Protocols, Humana Press, Totowa NJを参照)。
【０２１６】
多岐にわたる標識方法および抱合方法が当業者に知られており、様々な核酸アッセイおよびアミノ酸アッセイにこれらの方法を用い得る。NAAPをコードするポリヌクレオチドに関連する配列を検出するための、標識されたハイブリダイゼーションプローブ或いはPCRプローブを生成する方法には、オリゴ標識化、ニックトランスレーション、エンドラベリング（末端標識化)、または標識されたヌクレオチドを用いるPCR増幅が含まれる。別法として、NAAPをコードする配列、またはその任意の断片を、mRNAプローブを生成するためのベクターにクローニングすることも可能である。このようなベクターは、当分野において知られており、市販もされており、T7、T3またはSP6などの好適なRNAポリメラーゼおよび標識されたヌクレオチドを加えて、in vitroでRNAプローブの合成に用いることができる。このような方法は、例えばAmersham Pharmacia Biotech、Promega（Madison WI）、US Biochemicalなどから市販されている種々のキットを用いて実行することができる。検出を容易にするために用い得る好適なレポーター分子あるいは標識には、基質、補助因子、インヒビター、磁気粒子のほか、放射性核種、酵素、蛍光剤、化学発光剤、発色剤などがある。
【０２１７】
NAAPをコードするヌクレオチド配列で形質転換された宿主細胞は、細胞培地でのこのタンパク質の発現および回収に好適な条件下で培養される。形質転換細胞から製造されたタンパク質が分泌されるか細胞内に留まるかは、使用される配列、ベクター、あるいはその両者に依存する。NAAPをコードするポリヌクレオチド群を持つ発現ベクター類は、原核細胞膜または真核細胞膜を透過してのDMEの分泌を指示するシグナル配列群を持つように設計できることは、当業者には理解されよう。
【０２１８】
更に、宿主細胞株の選択は、挿入した配列の発現をモジュレートする能力、または発現したタンパク質を所望の形に処理する能力によって行い得る。限定するものではないがこのようなポリペプチドの修飾には、アセチル化、カルボキシル化、グリコシル化、リン酸化、脂質化およびアシル化がある。タンパク質の「プレプロ」または「プロ」形を切断する翻訳後のプロセシングを利用して、タンパク質の標的への誘導、折りたたみ、および／または活性を特定することが可能である。翻訳後の活性のための、特定の細胞装置および特徴的な機序を持つ、種々の宿主細胞（例えばCHO、HeLa、MDCK、MEK293、WI38など）は、American Type Culture Collection（ATCC, Manassas, VA）から入手可能であり、外来タンパク質の正しい修飾および処理を確実にするように選択し得る。
【０２１９】
本発明の別の実施態様では、NAAPをコードする、天然核酸配列、修飾された核酸配列、または組換え核酸配列を、或る異種配列に結合させることにより、上記した任意の宿主系内で、或る融合タンパク質の翻訳を生じ得る。例えば、市販の抗体によって認識できる異種部分を含むキメラNAAPタンパク質は、NAAP活性のインヒビターに対するペプチドライブラリのスクリーニングを促進し得る。また、異種タンパク質部分および異種ペプチド部分も、市販されている親和性マトリックスを用いて融合タンパク質の精製を促進し得る。限定されるものではないがこのような部分には、グルタチオンＳトランスフェラーゼ（GST）、マルトース結合タンパク質（MBP）、チオレドキシン（Trx）、カルモジュリン結合ペプチド（CBP）、6-His、FLAG、c-myc、赤血球凝集素（HA）がある。GSTは固定化グルタチオン上で、MBPはマルトース上で、Trxはフェニルアルシンオキシド上で、CBPはカルモジュリン上で、そして6-Hisは金属キレート樹脂上で、同族の融合タンパク質の精製を可能にする。FLAG、c-mycおよび赤血球凝集素（HA）は、これらのエピトープ標識を特異的に認識する市販されているモノクローナル抗体およびポリクローナル抗体を用いた、融合タンパク質の免疫親和性精製を可能にする。また、融合タンパク質が、NAAPをコードする配列と異種タンパク質配列との間にタンパク質分解切断部位を持つように遺伝子操作すると、精製後にNAAPが異種部分から切断され得る。融合タンパク質の発現および精製方法は、前出のAusubel（1995）10章に記載されている。市販されている種々のキットを用いて融合タンパク質の発現および精製を促進することもできる。
【０２２０】
本発明の更なる実施態様では、TNTウサギ網状赤血球可溶化液またはコムギ胚芽抽出系(Promega)を用いてin vitroで、放射能標識したNAAの合成が可能である。これらの系は、T7、T3またはSP6プロモーターと機能的に連結したタンパク質コード配列の、転写と翻訳とを結合する。翻訳は、例えば³⁵Sメチオニンのような放射能標識したアミノ酸前駆体の存在下で起こる。
【０２２１】
本発明のNAAPまたはその断片を用いて、NAAPに特異結合する化合物をスクリーニングすることができる。少なくとも１つまたは複数の試験化合物を用いて、NAAPへの特異的な結合をスクリーニングすることが可能である。試験化合物としては、抗体、オリゴヌクレオチド、タンパク質（例えば受容体）または小分子が挙げられる。
【０２２２】
或る実施態様では、このように同定された化合物は、NAAPの天然リガンドに密接に関連し、例えばリガンドやその断片であり、または天然基質や、構造的または機能的な擬態物質（mimetic）、あるいは自然結合パートナーである(例えばColigan, J.E. 他(1991) Current Protocols in Immunology 1(2):Chapter 5を参照)。同様に、この化合物は、NAAPが結合する天然受容体に、或いは例えばリガンド結合部位など少なくともこの受容体の或る断片に、密接に関連し得る。何れの場合も、既知の技術を用いてこの化合物を合理的に設計することができる。或る実施例では、これら化合物のためのスクリーニングには、分泌タンパク質として、あるいは細胞膜上でNAAPを発現する、好適な細胞群の作製が含まれる。好適な細胞には、哺乳類、酵母、ショウジョウバエ、または大腸菌からの細胞が含まれる。NAAPを発現する細胞またはNAAPを含有する細胞膜分画を試験化合物と接触させて、NAAPまたはこの化合物のどちらかの結合、刺激、または活性の阻害を分析する。
【０２２３】
或るアッセイは、単に試験化合物をポリペプチドに実験的に結合させ、蛍光色素、放射性同位体、酵素抱合体またはその他の検出可能な標識によりその結合を検出することができる。例えば、このアッセイは、少なくとも１つの試験化合物を、溶液中の、あるいは固体支持物に固定されたNAAPと混合するステップと、NAAPとこの化合物との結合を検出するステップを含み得る。別法では、標識された競合物の存在下での試験化合物の結合の検出および測定を行うことができる。更にこのアッセイは、無細胞再構成標本、化学ライブラリ、または、天然産物の混合物を用いて実施でき、試験化合物（群）は、溶液中で遊離させるか固体支持体に固定する。
【０２２４】
本発明のNAAPまたはその断片を用いて、NAAPの活性をモジュレートする化合物をスクリーニングすることが可能である。このような化合物の例には、アゴニスト、アンタゴニスト、部分的アゴニストまたは逆アゴニストなどが含まれる。或る実施例では、NAAPが少なくとも１つの試験化合物と結合する、NAAPの活性が許容される条件下でアッセイを実施し、試験化合物の存在下でのNAAPの活性と、試験化合物不在下でのNAAPの活性とを比較する。試験化合物の存在下でのNAAPの活性の変化は、NAAPの活性をモジュレートする化合物の存在を示唆する。 別法では、試験化合物を、NAAPの活性に適した条件下で、NAAPを含むin vitroまたは細胞遊離系と結合させてアッセイを実施する。これらアッセイの何れにおいても、NAAPの活性を調節する試験化合物は間接的に調節する場合があり、その際は試験化合物と直接接触する必要がない。少なくとも１つ、または複数の試験化合物をスクリーニングすることができる。
【０２２５】
別の実施態様では、胚性幹細胞（ES細胞）における相同組換えを用いて動物モデル系内で、NAAPまたはその哺乳類相同体をコードするポリヌクレオチドを「ノックアウト」する。このような技術は当技術分野において周知であり、ヒト疾患動物モデルの産生に有用である（米国特許第5,175,383号および第5,767,337号などを参照）。例えば129/SvJ細胞系などのマウスES細胞は初期のマウス胚に由来し、培地で増殖させることができる。このES細胞は、ネオマイシンホスホトランスフェラーゼ遺伝子（neo: Capecchi, M.R.（1989）Science 244:1288-1292）などのマーカー遺伝子で破壊した、目的の遺伝子を含むベクターで形質転換する。このベクターは、相同組換えにより、宿主ゲノムの対応する領域に組み込まれる。別法では、Cre-loxP系を用いて相同組換えを行い、組織特異的または発生段階特異的に目的遺伝子をノックアウトする（Marth, J.D. (1996) Clin. Invest. 97:1999-2002; Wagner, K.U. 他(1997) Nucleic Acids Res. 25:4323-4330)。形質転換したES細胞を同定し、例えばC57BL/6マウス株などから採取したマウス細胞胚盤胞に微量注入する。これらの胚盤胞を偽妊娠メスに外科的に導入し、得られるキメラ子孫の遺伝形質を特定し、これらを交配させてヘテロ接合性系またはホモ接合性系を作製する。このようにして産生した遺伝子組換え動物は、潜在的治療薬や毒性薬剤で検査することができる。
【０２２６】
NAAPをコードするポリヌクレオチドをin vitroでヒト胚盤胞由来のES細胞において操作することが可能である。ヒトES細胞は、内胚葉、中胚葉および外胚葉の細胞タイプを含む、少なくとも８つの別々の細胞系統に分化する可能性を有する。これらの細胞系統は、例えば神経細胞、造血系統および心筋細胞に分化する（Thomson, J.A. 他(1998) Science 282:1145-1147)。
【０２２７】
NAAPをコードするポリヌクレオチドを用いて、ヒト疾患をモデルとした「ノックイン」ヒト化動物（ブタ）または遺伝子組換え動物（マウスまたはラット）を作製することもできる。ノックイン技術を用いて、NAAPをコードするポリヌクレオチドの或る領域を動物ES細胞に注入し、注入した配列を動物細胞ゲノムに組み込ませる。形質転換細胞を胞胚に注入し、胞胚を上記のように移植する。遺伝子組換え子孫または近交系について試験し、潜在的医薬品を用いて処理し、ヒトの疾患の治療に関する情報を得る。別法では、例えばNAAPを乳汁内に分泌するなどLMMを過剰に発現する哺乳動物近交系は、便利なタンパク質源となり得る（Janne, J. 他(1998) Biotechnol. Annu. Rev. 4:55-74）。
【０２２８】
（治療）
NAAPの領域と核酸関連タンパク質の領域との間には、例えば配列及びモチーフの文脈における、化学的及び構造的類似性が存在する。また、NAAPを発現する組織は、前立腺腫瘍および肺腫瘍の組織と密接に関連し、また、臍帯血および臍帯血樹状細胞、および脳組織と密接に関連する。これらの組織の例は、表６を見られたい。このように、NAAPは、細胞増殖異常、神経障害、発達障害、自己免疫／炎症疾患、および感染症において、或る役割を果たすと考えられる。NAAPの発現または活性の増大に関連する疾患の治療においては、NAAPの発現または活性を低下させることが望ましい。また、NAAPの発現または活性の低下に関連する疾患の治療においては、NAAPの発現または活性を増大させることが望ましい。
【０２２９】
従って、一実施態様では、NAAPの発現または活性の低下に関連した疾患の治療または予防のために、被験者にNAAPまたはその断片や誘導体が投与され得る。限定するものではないが、このような疾患には細胞増殖異常、神経障害、発達障害、自己免疫／炎症疾患、および感染症が含まれ、細胞増殖異常には日光角化症、動脈硬化、アテローム硬化、滑液包炎、硬変、肝炎、混合性結合組織病（MCTD）、骨髄線維症、発作性夜間ヘモグロビン尿症、真性多血症、乾癬、原発性血小板血症、並びに、腺癌、白血病、リンパ腫、黒色腫、骨髄腫、肉腫、および奇形癌など癌、具体的には、副腎、膀胱、骨、骨髄、脳、乳房、頚部、胆嚢、神経節、消化管、心臓、腎臓、肝臓、肺、筋肉、卵巣、膵臓、副甲状腺、陰茎、前立腺、唾液腺、皮膚、脾臓、精巣、胸腺、甲状腺、および子宮の癌が含まれ、神経障害には、癲癇、虚血性脳血管障害、脳卒中、脳腫瘍、アルツハイマー病、ピック病、ハンチントン病、痴呆、パーキンソン病およびその他の錐体外路障害、筋萎縮性側索硬化およびその他の運動ニューロン障害、進行性神経性筋萎縮症、網膜色素変性症（色素性網膜炎）、遺伝性運動失調、多発性硬化症および他の脱髄疾患、細菌性およびウイルス性髄膜炎、脳膿瘍、硬膜下膿瘍、硬膜外膿瘍、化膿性頭蓋内血栓性静脈炎、脊髄炎および神経根炎、ウイルス性中枢神経系疾患と、プリオン病（クールー、クロイツフェルト‐ヤコブ病、およびGerstmann-Straussler-Scheinker症候群を含む）、致死性家族性不眠症、神経系性栄養病および代謝病、神経線維腫症、結節硬化症、小脳網膜血管腫症（cerebelloretinal hemangioblastomatosis）、脳３叉神経血管症候群、中枢神経系性精神遅滞および他の発達障害、脳性麻痺、神経骨格異常症、自律神経系障害、脳神経障害、脊髄疾患、筋ジストロフィー他の神経筋障害、末梢神経疾患、皮膚筋炎および多発性筋炎と、遺伝性、代謝性、内分泌性、および中毒性ミオパシーと、重症筋無力症、周期性四肢麻痺、精神障害（気分性、不安性の障害、統合失調症／分裂病）、季節性感情障害（SAD）、静座不能、健忘症、緊張病、糖尿病性ニューロパシー、遅発性ジスキネジア、ジストニー、パラノイド精神病、帯状疱疹後神経痛、トゥーレット病が含まれ、発達障害には尿細管性アシドーシス、貧血、クッシング症候群、軟骨形成不全性小人症、デュシェンヌ／ベッカー型筋ジストロフィー、癲癇、性腺形成異常、WAGR症候群（ウィルムス腫瘍、無虹彩症、尿生殖器異常、精神遅滞）、スミス‐マジェニス症候群（Smith- Magenis syndrome）、骨髄異形成症候群、遺伝性粘膜上皮異形成、遺伝性角皮症や、シャルコーマリーツース病および神経線維腫症などの遺伝性神経病、甲状腺機能低下症、水頭症や、Syndenham舞踏病（Syndenham's chorea）および脳性小児麻痺などの発作障害、二分脊椎、無脳症、頭蓋脊椎披裂、先天性緑内障、白内障、感音難聴が含まれ、自己免疫／炎症疾患には、後天性免疫不全症候群（AIDS）、アジソン病（慢性原発性副腎機能不全）、成人呼吸窮迫症候群、アレルギー、強直性脊椎炎、アミロイド症、貧血、喘息、アテローム性動脈硬化、自己免疫性溶血性貧血、自己免疫性甲状腺炎、自己免疫性多腺性内分泌カンジダ症外胚葉ジストロフィー（APECED）、気管支炎、胆嚢炎、接触皮膚炎、クローン病、アトピー性皮膚炎、皮膚筋炎、糖尿病、肺気腫、リンパ球傷害因子性偶発性リンパ球減少症、新生児溶血性疾患（胎児赤芽球症）、結節性紅斑、萎縮性胃炎、糸球体腎炎、グッドパスチャー症候群、痛風、グレーブス病、橋本甲状腺炎、過好酸球増加症、過敏性腸症候群、多発性硬化症、重症筋無力症、心筋または心膜炎症、骨関節炎、骨粗しょう症、膵炎、多発性筋炎、乾癬、ライター症候群、リウマチ様関節炎、強皮症、シェーグレン症候群、全身性アナフィラキシー、全身性エリテマトーデス、全身性強皮症、血小板減少性紫斑病、潰瘍性大腸炎、ぶどう膜炎、ウェルナー症候群、癌合併症、血液透析、体外循環、原虫感染症、蠕虫感染症、外傷が含まれ、感染症には、アデノウイルス、アレナウイルス、ブンヤウイルス（bunyavirus）、カリチウイルス（calicivirus）、コロナウイルス、フィロウイルス、ヘパドナウイルス、ヘルペスウイルス、フラビウイルス、オルソミクソウイルス、パルボウイルス、パポーバウイルス、パラミキソウイルス、ピコルナウイルス、ポックスウイルス、レオウイルス、レトロウイルス、ラブドウイルス、トガウイルスに分類されるウイルス病原体による感染や、細菌による感染（肺炎球菌感染症、ブドウ球菌、連鎖球菌、バシラス菌、コリネバクテリウム、クロストリジウム、髄膜炎菌、淋菌、リステリア、モラクセラ、キンゲラ、ヘモフィルス、レジオネラ、百日咳菌や、シゲラ、サルモネラ、カンピロバクターを含むグラム陰性腸内細菌、シュードモナス、ビブリオ、ブルセラ、野兎病菌、エルシニア、バルトネラ、norcardium、放線菌、ミコバクテリウム、spirochaetale、リケッチア、クラミジア、マイコプラズマに分類）、真菌感染(分類はアスペルギルス、ブラストミセス、皮膚糸状菌、クリプトコッカス、コクシジオイデス、malasezzia、ヒストプラスマ、または他の真菌症起因菌)、寄生虫感染(分類はプラスモディウムすなわちマラリア原虫、寄生性アメーバ、リーシュマニア、トリパノソーマ、トキソプラズマ、ニューモシスチスカリニ、腸内原虫(ジアルジアなど）、トリコモナス、組織線虫(旋毛虫など)、腸管寄生線虫(回虫など)、リンパ管フィラリア線虫、吸虫(住血吸虫など)、および条虫（サナダムシなど）)が含まれる。
【０２３０】
別の実施態様では、限定するものではないが上に列記した疾患を含む、NAAPの発現または活性の低下に関連した疾患の治療または予防のために、NAAPまたはその断片や誘導体を発現し得るベクターを被験者に投与し得る。
【０２３１】
更に別の実施態様では、限定するものではないが上に列記した疾患を含む、NAAPの発現または活性の低下に関連した疾患の治療または予防のために、実質的に精製されたNAAPを有する組成物を、好適な医薬用キャリアと共に被験者に投与し得る。
【０２３２】
更に別の実施態様では、限定するものではないが上に列記した疾患を含む、NAAPの発現または活性の低下に関連した疾患の治療または予防のために、NAAPの活性を調節するアゴニストを被験者に投与し得る。
【０２３３】
更なる実施例では、NAAPの発現または活性の増大に関連した疾患の治療または予防のために、被験者にNAAPのアンタゴニストを投与し得る。限定するものではないが、このような疾患の例には、上記した細胞増殖異常、神経障害、発達障害、自己免疫／炎症性疾患、および、感染症が含まれる。一実施態様では、NAAPと特異的に結合する抗体が直接アンタゴニストとして、或いはNAAPを発現する細胞または組織に薬剤を運ぶターゲッティング機構或いは送達機構として間接的に用いられ得る。
【０２３４】
別の実施例では、NAAPをコードするポリヌクレオチドの相補配列を発現するベクターを被験者に投与して、限定するものではないが上記した疾患を含む、NAAPの発現または活性の増大に関連した疾患の治療または予防を成し得る。
【０２３５】
別の実施態様では、本発明の任意のタンパク質、アンタゴニスト、抗体、アゴニスト、相補配列、またはベクターを、別の好適な治療薬と組み合わせて投与することもできる。併用療法で用いる好適な治療薬は、当業者が従来の医薬原理に従って選択し得る。治療薬と組み合わせることにより、上記した種々の疾患の治療または予防に相乗効果をもたらし得る。この方法により、少量の各薬剤で医薬効果をあげることが可能となり、それによって副作用の可能性を低減し得る。
【０２３６】
NAAPのアンタゴニストは、本技術分野で一般的に知られている方法を用いて製造し得る。具体的には、精製されたNAAPを用いて抗体を作るか、治療薬のライブラリをスクリーニングして、NAAPと特異結合するものを同定することが可能である。NAAPへの抗体も、本技術分野で公知の方法を用いて産生され得る。限定するものではないがこのような抗体には、ポリクローナル抗体、モノクローナル抗体、キメラ抗体、一本鎖抗体、Fab断片、およびFab発現ライブラリによって作られた断片が含まれ得る。中和抗体（すなわち二量体の形成を阻害する抗体）は通常、治療用に好適である。
【０２３７】
抗体の産生のためには、ヤギ、ウサギ、ラット、マウス、ヒトその他の種々の宿主が、NAAPまたは任意の断片の注入、または免疫原性の特性を備えるそのオリゴペプチドの注入によって免疫化され得る。宿主の種に応じて、種々のアジュバントを用いて免疫応答を高めることもできる。限定するものではないがこのようなアジュバントには、フロイントアジュバントと、水酸化アルミニウムなどのミネラルゲルアジュバントと、リゾレシチン、プルロニックポリオル、ポリアニオン、ペプチド、油性乳剤、スカシガイのヘモシアニン（KLH）、ジニトロフェノールなどの界面活性剤とがある。ヒトに用いられるアジュバントの中では、BCG（カルメット‐ゲラン杆菌）およびコリネバクテリウム‐パルバム（Corynebacterium parvum）が特に好ましい。
【０２３８】
NAAPに対する抗体を誘導するために用いるオリゴペプチド、ペプチドまたは断片は、少なくとも約５個のアミノ酸からなるアミノ酸配列を持つものが好ましく、一般的には約１０個以上のアミノ酸からなるものとなる。これらのオリゴペプチド、ペプチドまたは断片は、天然タンパク質のアミノ酸配列の一部と同一であることが望ましい。NAAPアミノ酸類の短いストレッチ群は、KLHなど他のタンパク質の配列と融合され、このキメラ分子に対する抗体群が産生され得る。
【０２３９】
NAAPに対するモノクローナル抗体は、培地内の連続した細胞株によって、抗体分子を産生する任意の技術を用いて作製することが可能である。限定するものではないがこのような技術には、ハイブリドーマ技術、ヒトＢ細胞ハイブリドーマ技術、およびEBV-ハイブリドーマ技術がある(例えばKohler, G. 他(1975) Nature 256:495497; Kozbor, D. 他(1985) J. Immunol. Methods 81:31-42; Cote, R.J. 他(1983) Proc. Natl. Acad. Sci. USA 80:20262030;およびCole, S.P. 他(1984) Mol.Cell Biol. 62:109-120を参照)。
【０２４０】
更に、「キメラ抗体」作製のために発達した、ヒト抗体遺伝子にマウス抗体遺伝子をスプライシングするなどの技術が、好適な抗原特異性および生物学的活性を備える分子を得るために用いられる(例えばMorrison, S.L. 他(1984) Proc. Natl. Acad. Sci. USA 81:68516855; Neuberger, M.S. 他(1984) Nature 312:604608;およびTakeda, S. 他(1985) Nature 314:452-454を参照)。別法では、当分野で周知の方法を用い、一本鎖抗体の産生のための記載された技術を適用して、NAAP特異的一本鎖抗体を生成する。関連した特異性を有するがイディオタイプ組成が異なるような抗体を、ランダムな組合せの免疫グロブリンライブラリ類からチェーンシャッフリングによって産生することもできる（例えばBurton D.R.（1991）Proc. Natl. Acad. Sci. USA 88:10134-10137を参照）。
【０２４１】
抗体類の産生は、リンパ球集団におけるin vivo産生の誘導によって、或いは文献に開示されているように非常に特異的な結合試薬の免疫グロブリンのライブラリまたはパネルのスクリーニングによっても行い得る(例えばOrlandi, R. 他(1989) Proc. Natl. Acad. Sci. USA 86:38333837; Winter, G. 他(1991) Nature 349:293-299を参照)。
【０２４２】
NAAPに対する特異的な結合部位を含む抗体断片も産生され得る。例えば、限定するものではないが、このような断片には、抗体分子のペプシン消化によって産生されるF（ab'）₂ 断片と、F（ab'）₂ 断片のジスルフィド架橋を還元することによって作製されるFab断片とがある。あるいは、Fab発現ライブラリを作製することによって、所望の特異性を持つモノクローナルFab断片を迅速且つ容易に同定することが可能となる（例えばHuse, W.D. 他 (1989) Science 246:1275-1281を参照）。(1989) Science 246:1275-1281等を参照)。
【０２４３】
種々の免疫学的検定（イムノアッセイ）を用いてスクリーニングし、所望の特異性を有する抗体を同定することができる。確立された特異性を有するポリクローナル抗体またはモノクローナル抗体の何れかを用いる免疫放射定量測定法または競合結合アッセイに対する数々のプロトコルは、本技術分野において公知である。このようなイムノアッセイは通常、NAAPとその特異抗体との間の複合体形成の計測を含む。２つの非干渉性NAAPエピトープに対して反応性を持つモノクローナル抗体群を用いる、２部位モノクローナルベースのイムノアッセイが一般に利用されるが、競合結合試験も利用できる(Pound、前出)。
【０２４４】
ラジオイムノアッセイ技術と共にScatchard分析などの様々な方法を用いて、NAAPに対する抗体の親和性を評価し得る。親和性を結合定数Kaで表すが、このKaは、平衡状態下の、NAAP抗体複合体のモル濃度を、遊離抗体と遊離抗原とのモル濃度で除して得られる値である。ポリクローナル抗体類は多様なNAAPエピトープに対する親和性が不均一であり、或るポリクローナル抗体試薬に関して判定したKaは、NAAP抗体群の平均の親和性または結合活性を表す。モノクローナル抗体は或る特定のNAAPエピトープに対して単一特異的であり、モノクローナル抗体の或る試薬について判定したKaは、親和性の真の測定値を表す。Ka値が１０^９〜１０^１２Ｌ／ｍｏｌの高親和性抗体試薬は、NAAP-抗体複合体が激しい操作に耐えなければならないイムノアッセイに用いるのが好ましい。Ka値が１０^６〜１０^７ｌｉｔｅｒ／ｍｏｌの低親和性抗体試薬は、NAAPが抗体から最終的に活性化状態で解離する必要がある免疫精製（immunopurification）及び類似の処理に用いるのが好ましい(Catty, D. (1988) Antibodies, Volume I: A Practical Approach. IRL Press, Washington, DC; Liddell, J. E. 及び Cryer, A. (1991) A Practical Guide to Monoclonal Antibodies, John Wiley & Sons, New York NY)。
【０２４５】
ポリクローナル抗体試薬の抗体価および結合活性を更に評価して、後に使う或る適用例に対するこのような試薬の品質および適性を決定することができる。例えば、少なくとも１〜２ｍｇ／ｍｌの特異的な抗体、好ましくは５〜１０ｍｇ／ｍｌの特異的な抗体を含むポリクローナル抗体試薬は一般に、NAAP-抗体複合体を沈殿させなければならない処理に用いられる。抗体の特異性、抗体価、結合活性、様々な適用例における抗体の品質や使用に対する指針については、一般に入手可能である（例えば前出のCatty、同Coligan 他を参照）。
【０２４６】
本発明の別の実施例では、NAAPをコードするポリヌクレオチド、またはその任意の断片や相補配列を、治療目的で使用することができる。或る実施例では、NAAPをコードする遺伝子のコーディング領域や調節領域に相補的な配列やアンチセンス分子（DNA、RNA、PNA、または修飾したオリゴヌクレオチド）を設計して遺伝子発現をモディフィケーションし得る。このような技術は当分野では周知であり、アンチセンスオリゴヌクレオチドまたはより大きな断片を、NAAPをコードする配列の制御領域、またはコード領域に沿った、さまざまな位置から設計可能である（例えばAgrawal, S.,編集（1996）Antisense Therapeutics, Humana Press Inc., Totawa NJを参照）。
【０２４７】
治療に用いる場合、アンチセンス配列を適切な標的細胞に導入するのに好適な、任意の遺伝子送達系を用いることができる。アンチセンス配列は、転写時に標的タンパク質をコードする細胞配列の少なくとも一部に相補的な配列を作製する発現プラスミドの形で、細胞内に送達し得る(例えばSlater, J.E. 他（1998）J. Allergy Clin. Immunol. 102（3）:469-475 および Scanlon, K.J. 他（1995）9（13）:1288-1296を参照）。アンチセンス配列はまた、例えばレトロウイルスやアデノ随伴ウイルスベクターなどのウイルスベクターを用いて細胞内に導入することもできる（例えばMiller, A.D.（1990）Blood 76:271、前出のAusubel、Uckert, W.およびW. Walther（1994）Pharmacol. Ther. 63（3）:323-347を参照）。その他の遺伝子送達機構には、リポソーム系、人工的なウイルスエンベロープおよび当分野で公知のその他のシステムが含まれる（例えばRossi, J.J.（1995）Br. Med. Bull. 51（1）:217-225; Boado、R.J.他(1998) J. Pharm. Sci. 87(11):1308-1315、Morris, M.C.他(1997) Nucleic Acids Res. 25(14):2730-2736を参照)。
【０２４８】
本発明の別の実施態様では、NAAPをコードするポリヌクレオチドを、体細胞若しくは生殖細胞の遺伝子治療に用いることが可能である。遺伝子治療により、（i）遺伝子欠損症を治療し（例えばＸ染色体連鎖遺伝（Cavazzana-Calvo, M.他 (2000) Science 288:669-672）により特徴付けられる重症複合型免疫不全(SCID)-X1病の場合）、先天性アデノシンデアミナーゼ（ADA）欠損症に関連する重症複合型免疫不全症候群（Blaese, R.M. 他(1995) Science 270:475-480、Bordignon, C.他 (1995) Science 270:470-475）、嚢胞性繊維症（Zabner, J.他 (1993) Cell 75:207-216: Crystal、R.G.他(1995) Hum.Gene Therapy 6:643-666、Crystal, R.G.他(1995) Hum.Gene Therapy 6:667-703）、サラセミア（thalassamia）、家族性高コレステロール血症や、第VIII因子若しくは第IX因子欠損に起因する血友病（Crystal, R.G. (1995) Science 270:404-410、Verma, I.M. および N. Somia (1997) Nature 389:239-242）、（ii）条件的致死性遺伝子産物を発現させ（例えば制御不能な細胞増殖に起因する癌の場合）、（iii）細胞内の寄生生物、例えばヒト免疫不全ウイルス(HIV)（Baltimore, D. (1988) Nature 335:395-396、Poeschla, E.他(1996) Proc. Natl. Acad. Sci. USA. 93:11395-11399）などヒトレトロウイルス、B型若しくはC型肝炎ウイルス（HBV、HCV）、Candida albicansおよびParacoccidioides brasiliensis等の寄生真菌、並びに熱帯熱マラリア原虫およびクルーズトリパノソーマ等の寄生原虫に対する防御機能を有するタンパク質を発現させることができる。NAAPの発現若しくは調節に必要な遺伝子の欠損が疾患を引き起こす場合、導入した細胞の好適な集団からNAAPを発現させて、遺伝子欠損によって起こる症状の発現を緩和することが可能である。
【０２４９】
本発明の更なる実施例では、NAAPの欠損による疾患や異常症を、NAAPをコードする哺乳類発現ベクターを作製して、これらのベクターを機械的手段によってNAAP欠損細胞に導入することによって治療する。in vivoあるいはex vitroの細胞に用いる機械的導入技術には、（i）個々の細胞内への直接的なDNA微量注射法、（ii）遺伝子銃、（iii）リポソームを介した形質移入、（iv）受容体を介した遺伝子導入、および（v）DNAトランスポゾンの使用がある（Morgan, R.A. および W.F. Anderson（1993）Annu. Rev. Biochem. 62:191-217、Ivics, Z.（1997）Cell 91:501-510; Boulay, J-L. およびH. Recipon（1998）Curr. Opin. Biotechnol. 9:445-450）。
【０２５０】
NAAPの発現に影響を及ぼし得る発現ベクターには、限定するものではないが、PCDNA 3.1、EPITAG、PRCCMV2、PREP、PVAX、PCR2-TOPOTAベクター(Invitrogen, Carlsbad CA)、PCMV-SCRIPT、PCMV-TAG、PEGSH／PERV (Stratagene, La Jolla CA)、PTET-OFF、PTET-ON、PTRE2、PTRE2-LUC、PTK-HYG (Clontech, Palo Alto CA)が含まれる。DMEを発現させるために、（i）構成的に活性なプロモーター（例えば、サイトメガロウイルス（CMV）、ラウス肉腫ウイルス（RSV）、SV40ウイルス、チミジンキナーゼ（TK）、若しくはβ−アクチンの遺伝子など）、(ii)誘導性プロモーター（例えば、市販されているT-REXプラスミド（Invitrogen）に含まれている、テトラサイクリン調節性プロモーター（Gossen, M. および H. Bujard (1992) Proc. Natl. Acad. Sci. U.S.A. 89:5547-5551; Gossen, M. 他(1995) Science 268:1766-1769; Rossi, F.M.V. および H.M. Blau (1998) Curr. Opin. Biotechnol. 9:451-456））、エクジソン誘導性プロモーター（市販されているプラスミドPVGRXRおよびPINDに含まれている：Invitrogen）、FK506／ラパマイシン誘導性プロモーター、またはRU486／ミフェプリストーン誘導性プロモーター（Rossi, F.M.V. および H.M. Blau, 前出）、または（iii）正常な個体に由来する、NAAPをコードする内因性遺伝子の天然プロモーター若しくは組織特異的プロモーターを用い得る。
【０２５１】
市販のリポソーム形質転換キット（例えばInvitrogen社のPerFect Lipid Transfection Kit）を用いれば、当業者は実験の各パラメータを最適化する努力をさほど要さず、ポリヌクレオチド群を、培養中の標的細胞群に送達し得る。別法では、リン酸カルシウム法（Graham. F.L. および A.J. Eb（1973）Virology 52:456-467）若しくは電気穿孔法（Neumann, B. 他(1982) EMBO J. 1:841845)。初代培養細胞にDNAを導入するためには、標準化された哺乳類の形質移入プロトコルの修正が必要である。
【０２５２】
本発明の別の実施例では、NAAPの発現に関連する遺伝子欠損によって起こる疾患や障害を、（i）レトロウイルス長末端反復配列（LTR）プロモーター若しくは或る独立プロモーターのコントロール下でNAAPをコードするポリヌクレオチドと、（ii）好適なRNAパッケージングシグナル群と、（iii）追加のレトロウイルス・シス作用性RNA配列と、効率的なベクター増殖に必要なコーディング配列とを伴うRev応答性エレメント（RRE）と、からなるレトロウイルスベクターを作製して治療することができる。レトロウイルスベクター（例えばPFBおよびPFBNEO）はStratagene社から市販されており、刊行データ（Riviere, I. 他(1995) Proc. Natl. Acad. Sci. USA 92:6733-6737)に基づく。該文献は特に引用を以って本明細書の一部となす。このベクターは、好適なベクター産生細胞株（VPCL）において増殖される。VPCLは、各標的細胞上の受容体への親和性を持つエンベロープ遺伝子を、またはVSVgなど汎親和性エンベロープタンパク質を発現する（Armentano, D. 他 (1987) J. Virol. 61:1647-1650、Bender, M.A. 他 (1987) J. Virol. 61:1639-1646、Adam, M.A. および A.D. Miller (1988) J. Virol. 62:3802-3806、Dull, T. 他 (1998) J. Virol. 72:8463-8471、Zufferey, R. 他 (1998) J. Virol. 72:9873-9880）。Riggに付与された米国特許第5,910,434号（「Method for obtaining retrovirus packaging cell lines producing high transducing efficiency retroviral supernatant」）において、レトロウイルスパッケージング細胞株群を得る方法が開示されており、引用を以て本明細書の一部とする。レトロウイルスベクターの増殖、細胞集団（例えばCD4⁺ Ｔ細胞）の形質導入、及び形質導入した細胞の患者への戻しは、遺伝子治療の分野では当業者に公知の方法であり、多数の文献に記載されている（Ranga, U. 他(1997) J. Virol. 71:7020-7029; Bauer, G. 他(1997) Blood 89:2259-2267; Bonyhadi, M.L. (1997) J. Virol. 71:4707-4716; Ranga, U. 他(1998) Proc. Natl. Acad. Sci. USA 95:1201-1206; Su, L. (1997) Blood 89:2283-2290)。
【０２５３】
別法では、アデノウイルス系遺伝子治療の或る送達系を用いて、NAAPの発現に関連する１つ以上の遺伝子異常を有する細胞群に、NAAPをコードするポリヌクレオチド群を送達する。アデノウイルス系ベクター類の作製およびパッケージングについては、当業者に周知である。複製欠損型アデノウイルスベクター類は、種々の免疫調節タンパク質をコードする遺伝子群を、無損傷の膵島内にインポートする目的で多様に利用し得ることが証明された（Csete, M.E. 他 (1995) Transplantation 27:263-268）。潜在的に有用なアデノウイルスベクター類はArmentanoに付与された米国特許第5,707,618号（「Adenovirus vectors for gene therapy」）に記載されており、引用することを以て本明細書の一部とする。アデノウイルスベクター類については、Antinozzi, P.A. 他 (1999) Annu. Rev. Nutr. 19:511-544 並びに、Verma, I.M. および N. Somia (1997) Nature 18:389:239-242も参照されたい。両文献は、引用を以て本明細書の一部とする。
【０２５４】
別法では、ヘルペス系遺伝子治療の送達系を用いて、NAAPの発現に関連する１つ或いは複数の遺伝子異常を有する標的細胞に、NAAPをコードするポリヌクレオチドを送達する。単純ヘルペスウイルス（HSV）系ベクター類は、HSVが親和性を持つ中枢神経細胞にNAAPを導入する際に、特に有益に思える。ヘルペス系ベクター類の作製およびパッケージングは、当業者に公知である。或る複製適格性単純ヘルペスウイルス（HSV）I型系のベクターが、或るレポーター遺伝子の、霊長類の眼への送達に用いられている（Liu, X. 他(1999) Exp.Eye Res. 169:385395)。HSV-1ウイルスベクターの作製についても、DeLucaに付与された米国特許第5,804,413号（「Herpes simplex virus strains for gene transfer」）に詳細に開示されており、該特許の引用を以て本明細書の一部とする。米国特許第5,804,413号は、ヒト遺伝子治療などの目的で、好適なプロモーターの制御下において細胞に導入される少なくとも１つの外来遺伝子を有するゲノムを持つ、組換えHSV d92の利用について記載する。上記特許はまた、ICP4、ICP27、およびICP22を欠失した組換えHSV系統の、作製および使用について開示している。HSVベクター類については、Goins, W.F. 他 (1999) J. Virol. 73:519-532 および Xu, H. 他 (1994) Dev. Biol. 163:152-161も参照されたい。両文献は、引用を以て本明細書の一部とする。クローン化したヘルペスウイルス配列群の操作、大ヘルペスウイルスゲノム（large herpesvirus genomes）の様々なセグメントを持つ複数のプラスミドを形質移入した後の組換えウイルスの産生、ヘルペスウイルスの成長および増殖、並びに細胞群へのヘルペスウイルスの感染は、当業者に公知の技術である。
【０２５５】
別法では、或るαウイルス（正の一本鎖RNAウイルス）ベクターを用いて、NAAPをコードするポリヌクレオチド群を標的細胞群に送達する。プロトタイプのαウイルスであるセムリキ森林熱ウイルス（SFV）の生物学的研究が広範に行われており、遺伝子導入ベクター類は、SFVゲノムに基づく（Garoff, H. および K.-J. Li (1998) Curr. Opin. Biotechnol. 9:464-469）。αウイルスRNAの複製中に、ウイルスのカプシドタンパク質群を通常はコードする、サブゲノムRNAが産生される。このサブゲノムRNAは、完全長ゲノムRNAよりも高レベルに複製するので、酵素活性（例えばプロテアーゼおよびポリメラーゼ）を持つウイルスタンパク質に比べてカプシドタンパク質群が過剰産生される。同様に、NAAPをコードする配列をαウイルスゲノムのカプシドをコードする領域に導入することによって、ベクター導入細胞において多数のNAAPをコードするRNAが産生され、高いレベルでNAAPが合成される。通常、αウイルスの感染は、数日以内の細胞溶解を伴う。一方、シンドビスウイルス（SIN）の或る変異体を有するハムスター正常腎臓細胞（BHK-21）群が持続的な感染を確立する能力は、αウイルス類の溶解複製を、遺伝子治療に応用し得るように好適に改変可能であることを示唆する（Dryga, S.A. 他(1997) Virology 228:74-83)。様々な宿主にαウイルスを導入できるので、様々なタイプの細胞にNAAPを導入することができる。或る集団における或るサブセットの細胞群の特異的形質導入は、形質導入前に細胞の選別を必要とし得る。αウイルスの感染性cDNAクローンの処置方法、αウイルスのcDNAおよびRNAの形質移入方法およびαウイルスの感染方法は、当業者に公知である。
【０２５６】
転写開始部位由来のオリゴヌクレオチドを用いて遺伝子発現を阻害することも可能である。転写開始部位とは、例えば開始部位から数えて約−１０と約＋１０の間である。同様に、三重らせん塩基対の形成方法を用いて阻害が可能となる。三重らせん塩基対形成は、ポリメラーゼ、転写因子または調節分子の結合のために十分に開く、二重らせんの能力を阻害するので有用である。三重らせんDNAを用いる最近の治療の進歩については文献に記載がある(例えばGee, J.E. 他(1994) in: Huber, B.E.およびB.I. Carr, Molecular and Immunologic Approaches, Futura Publishing, Mt. Kisco, NY, 163-177ページ等を参照）。また、相補配列またはアンチセンス分子を設計し、転写物がリボソームに結合するのを阻止することによってmRNAの翻訳をブロックし得る。
【０２５７】
リボザイム類は、酵素性RNA分子である。RNAの特異的切断を触媒するためにリボザイムを用い得る。リボザイム作用のメカニズムは、相補的標的RNAへのリボザイム分子の配列特異的ハイブリダイゼーションと、その後に起こる内ヌクレオチド鎖切断に関与している。例えば、人工的に作製されたハンマーヘッド型リボザイム分子が、NAAPをコードする配列の、内ヌクレオチド鎖切断を特異的且つ効果的に触媒できる可能性がある。
【０２５８】
任意の潜在的RNA標的内の特異的リボザイム切断部位を先ず同定するため、GUA、GUU、GUC配列などリボザイム切断部位に対して標的分子をスキャンする。一度同定されると、切断部位を持つ標的遺伝子の領域に対応する１５〜２０リボヌクレオチドの短いRNA配列が、そのオリゴヌクレオチドを機能不全にするような２次構造の特徴をもっていないかを評価することが可能になる。候補標的の適合性の評価も、リボヌクレアーゼ保護アッセイを用いて相補的オリゴヌクレオチド群とのハイブリダイゼーションへのアクセス可能性をテストすることによって行い得る。
【０２５９】
本発明の相補リボ核酸分子およびリボザイムは、核酸分子合成のために当分野でよく知られている任意の方法を用いて作製し得る。作製方法には、固相ホスホラミダイト化学合成など、オリゴヌクレオチドを化学的に合成する方法がある。あるいは、NAAPをコードするDNA配列のin vitroおよびin vivo転写によってRNA分子を産生し得る。このようなDNA配列は、T7やSP6などの好適なRNAポリメラーゼプロモーターを用いて多様なベクター内に取り込むことが可能である。あるいは、相補的RNAを構成的あるいは誘導的に合成するこれらのcDNA構成物を、細胞株、細胞または組織内に導入することができる。
【０２６０】
細胞内の安定性を高め、半減期を長くするためにRNA分子を修飾し得る。限定するものではないが可能な修飾としては、分子の５'末端、３'末端、あるいはその両方において隣接配列群を追加することや、分子の主鎖内においてホスホジエステラーゼ結合ではなくホスホロチオエートまたは2' O-メチルを使用することが含まれる。この概念は、本来はPNA群の産出におけるものであるが、これら全ての分子に拡大することができる。そのためには、内因性エンドヌクレアーゼによって容易には認識されない、アデニン、シチジン、グアニン、チミン、およびウリジンにアセチル−、メチル−、チオ−および同様の修飾をしたものや、非従来型塩基、例えばイノシン、クエオシン（queosine）、ワイブトシン（wybutosine）を加える。
【０２６１】
本発明の更なる実施例は、NAAPをコードするポリヌクレオチドの発現の改変に有効な化合物をスクリーニングする方法を含む。限定するものではないが特定ポリヌクレオチドの発現改変を起こすのに有効であり得る化合物には、オリゴヌクレオチド、アンチセンスオリゴヌクレオチド、三重らせん形成オリゴヌクレオチド、転写因子などのポリペプチド転写制御因子、および特定ポリヌクレオチド配列と相互作用し得る非高分子化学的実体がある。有効な化合物は、ポリヌクレオチド発現のインヒビターまたはエンハンサーのいずれかとして作用することによりポリヌクレオチド発現を改変し得る。従って、NAAPの発現または活性の増加に関連する疾患の治療においては、NAAPをコードするポリヌクレオチドの発現を特異的に阻害する化合物が治療上有用であり、NAAPの発現または活性の低下に関連する疾患の治療においては、NAAPをコードするポリヌクレオチドの発現を特異的に促進する化合物が治療上有用であり得る。
【０２６２】
或る特定ポリヌクレオチドの発現を改変する際の有効性に対して、少なくとも１個または複数個の試験化合物をスクリーニングし得る。試験化合物を得るには、当分野で公知の任意の方法を用い得る。取得方法としては、以下の場合に有効な既知化合物の化学修飾がある。ポリヌクレオチドの発現を改変する場合と、既存の、商用または専用の、天然または非天然の化合物のライブラリから選択する場合と、標的ポリヌクレオチドの化学的および／または構造的特性に基づく化合物を合理的にデザインする場合と、組合せ的にまたは無作為に生成した化合物のライブラリから選択する場合とである。NAAPをコードするポリヌクレオチドを持つサンプルを、このようにして得た試験化合物の少なくとも１つに曝露する。サンプルは例えば、無傷細胞、透過化処理した細胞、in vitro無細胞系すなわち再構成生化学系があり得る。NAAPをコードするポリヌクレオチドの発現における改変は、当分野で周知の任意の方法でアッセイする。通常は、NAAPをコードするポリヌクレオチドの配列に相補的なヌクレオチド配列を有するプローブを用いたハイブリダイゼーションにより、特定のヌクレオチドの発現を検出する。ハイブリダイゼーション量を定量することにより、１つ以上の試験化合物に曝露される、または曝露されないポリヌクレオチドの発現の比較のための基礎を形成し得る。試験化合物に曝露されるポリヌクレオチドの発現における変化の検出は、ポリヌクレオチドの発現を改変する際に試験化合物が有効であることを示す。或る特定ポリヌクレオチドの改変発現に有効な化合物のためのスクリーニングを実行でき、例えば分裂酵母（Schizosaccharomyces pombe）遺伝子発現系（Atkins, D. 他(1999) 米国特許第5,932,435号、Arndt, G.M. 他(2000) Nucleic Acids Res.28:E15) またはヒト細胞株、例えばHeLa 細胞(Clarke, M.L. 他(2000) Biochem. Biophys. Res. Commun. 268:8-13)を用いる。本発明の特定の実施例は、特異的ポリヌクレオチド配列に対するアンチセンス活性のためのオリゴヌクレオチド（デオキシリボヌクレオチド、リボヌクレオチド、ペプチド核酸、修飾オリゴヌクレオチド）の組み合わせライブラリをスクリーニングすることに関与している（Bruice, T.W. 他(1997) 米国特許第5,686,242号、Bruice, T.W. 他(2000) 米国特許第6,022,691号)。
【０２６３】
ベクターを細胞または組織に導入する多数の方法が利用可能であり、in vivo、in vitroおよびex vivoの使用に対して同程度に適している。ex vivo治療の場合、ベクターを、患者から採取した幹細胞内に導入し、クローニング増殖して同患者に自家移植で戻すことができる。トランスフェクションによる、またはリボソーム注入やポリカチオンアミノポリマーによる送達は、当分野でよく知られている方法を用いて実行することができる（例えばGoldman, C.K. 他(1997) Nat. Biotechnol. 15:462-466を参照)。
【０２６４】
上記の治療方法はいずれも、例えば、ヒト、イヌ、ネコ、ウシ、ウマ、ウサギ、サルなどの哺乳類を含めて治療が必要な全ての被験体に適用できる。
【０２６５】
本発明の或る更なる実施例は、薬剤として許容できる賦形剤と共に処方される活性成分を一般に持つ、或る組成物の投与に関する。賦形剤には例えば、糖、でんぷん、セルロース、ゴムおよびタンパク質がある。様々な処方が広く知られており、詳細はRemington's Pharmaceutical Sciences（Maack Publishing, Easton PA）の最新版に記載されている。このような組成物は、NAAP、NAAPの抗体、擬態物質、アゴニスト、アンタゴニスト、またはインヒビターなどからなる。
【０２６６】
本発明に用いる組成物は、任意の数の経路によって投与することができ、限定するものではないが経路には、経口、静脈内、筋肉内、動脈内、骨髄内、クモ膜下腔内、心室内、肺、経皮、皮下、腹腔内、鼻腔内、腸内、局所、舌下または直腸がある。
【０２６７】
肺から投与する組成物は、液状または乾燥粉末状で調製し得る。このような組成物は通常、患者が吸入する直前にエアロゾル化する。小分子（例えば伝統的な低分子量有機薬）の場合には、速効製剤のエアロゾル送達は当分野で公知である。高分子（例えばより大きなペプチドおよびタンパク質）の場合には、当該分野において肺の肺胞領域を介しての肺送達が最近向上したことにより、インスリンなどの薬剤を実質的に血液循環へ輸送することを可能にした（Patton, J.S. 他, 米国特許第5,997,848号などを参照）。肺送達は、針注射なしに投与する点で優れており、有毒な可能性のある浸透エンハンサーの必要性をなくす。
【０２６８】
本発明での使用に適した組成物には、所定の目的を達成するために必要なだけの量の活性成分を含有する組成物が含まれる。有効投与量の決定は、当業者の能力の範囲内で行う。
【０２６９】
NAAPを有する、またはその断片群を有する高分子群を直接、細胞内に送達すべく、特殊な種々の形状の組成物群が調製され得る。例えば、細胞不透過性高分子を含むリポソーム製剤は、その高分子の細胞融合と細胞内送達とを促進し得る。別法では、NAAPまたはその断片を、HIV Tat-1タンパク質の陽イオンN末端部に結合することもできる。このようにして産生された融合タンパク質類は、或るマウスモデル系の、脳を含む全ての組織の細胞群に形質導入することがわかっている（Schwarze, S.R. 他(1999) Science 285:1569-1572)。
【０２７０】
任意の化合物に対して、先ず細胞培養アッセイ、例えば新生物性細胞の細胞培養アッセイにおいて、あるいは、動物モデル、例えばマウス、ラット、ウサギ、イヌ、サルまたはブタなどにおいて、治療有効投与量を推定できる。動物モデルはまた、好適な濃度範囲および投与経路を決定するためにも用い得る。このような情報を用いて、次にヒトに対する有益な投与量および投与経路を決定することができる。
【０２７１】
治療有効投与量とは、症状や容態を回復させる、たとえばNAAPまたはその断片、NAAPの抗体、NAAPのアゴニストまたはアンタゴニスト、インヒビターなど活性処方成分の量を指す。治療有効度および毒性は、細胞培養または動物実験における標準的な薬学手法によって、例えばED₅₀（集団の５０％の治療有効量）またはLD₅₀（集団の５０％の致死量）統計を計算するなどして判定できる。毒性効果の、治療効果に対する投与量比が治療指数であり、LD₅₀/ED₅₀比として表すことができる。高い治療指数を示すような組成物が望ましい。細胞培養アッセイと動物実験とから得られたデータは、ヒトに用いる投与量の範囲での調剤に用いられる。このような組成物に含まれる投与量は、毒性を殆どあるいは全く持たず、ED₅₀を含むような血中濃度の範囲にあることが好ましい。用いられる投与形態、患者の感受性および投与の経路によって、投与量はこの範囲内で様々に変わる。
【０２７２】
正確な投与量は、治療が必要な被験者に関する要素を考慮して、現場の医者が決定することになる。充分なレベルの活性成分を与え、あるいは所望の効果を維持すべく、用法および用量を調整する。被験者に関する要素としては、疾患の重症度、患者の一般的な健康状態、患者の年齢、体重および性別、投与の時間および頻度、薬剤の配合、反応感受性および治療に対する応答などを考慮し得る。作用期間が長い組成物は、特定の製剤の半減期およびクリアランス率によって３〜４日毎に１度、１週間に１度、あるいは２週間に１度の間隔で投与し得る。
【０２７３】
通常の投与量は、投与の経路にもよるが約０.１〜１００,０００μgであり、合計で約１gまでとする。特定の投与量および送達方法に関するガイダンスは文献に記載されており、現場の医者は通常それを利用することができる。当業者は、タンパク質またはそれらのインヒビター類についての処方とは異なる、ヌクレオチドについての処方を利用することになる。同様に、ポリヌクレオチドまたはポリペプチドの送達は、特定の細胞、症状、部位などに特異的なものとなる。
【０２７４】
（診断）
別の実施例では、NAAPに特異的に結合する抗体を、NAAPの発現によって特徴付けられる障害の診断に、または、NAAPやNAAPのアゴニストまたはアンタゴニスト、インヒビターで治療を受けている患者をモニターするためのアッセイに用い得る。診断目的に有用な抗体は、上記の治療の箇所で記載した方法と同じ方法で調合される。NAAPの診断アッセイには、ヒトの体液から、あるいは細胞や組織の抽出物から、抗体および標識を用いてNAAPを検出する方法が含まれる。この抗体は修飾されたものもされていないものも可能であり、レポーター分子との共有結合または非共有結合で標識化できる。レポーター分子としては広くさまざまな種類が本分野で知られており、また使用可能であるが、そのうちのいくつかは上記で説明されている。
【０２７５】
NAAPを測定するための様々なプロトコル、例えばELISA、RIA、FACS等が当分野では周知であり、変化した、あるいは異常なレベルのNAAPの発現を診断するための基盤を提供する。正常あるいは標準的なNAAPの発現の値は、複合体の形成に適した条件下で、正常な哺乳動物、例えばヒトなどの被験体から採取した体液または細胞抽出物と、NAAPに対する抗体とを混合させることによって確定する。標準複合体形成量は、種々の方法、例えば測光法で定量できる。被験体、対照、および、生検組織からの疾患サンプルでの、NAAP発現の量を標準値と比較する。標準値と被験体との偏差が、疾患を診断するパラメータを確定する。
【０２７６】
本発明の別の実施態様によれば、NAAPをコードするポリヌクレオチドを診断のために用いることもできる。用い得るポリヌクレオチドには、オリゴヌクレオチド配列、相補的RNAおよびDNA分子、そしてPNAが含まれる。これらのポリヌクレオチドを用いて、NAAPの発現が疾患と相関し得る生検組織における遺伝子発現を検出し定量し得る。この診断アッセイを用いて、NAAPの存在の有無、更には過剰な発現を判定し、治療時のNAAPレベルの調節を監視する。
【０２７７】
或る実施形態では、NAAPまたは近縁の分子をコードする、ゲノム配列などポリヌクレオチド配列を検出可能なPCRプローブ類とのハイブリダイゼーションを、NAAPをコードする核酸配列を同定するために用いることができる。プローブが高度に特異的な領域（例えば５'調節領域）から作られている、或いはやや特異性の低い領域（例えば保存されたモチーフ）から作られているかにかかわらず、プローブの特異性と、ハイブリダイゼーション或いは増幅のストリンジェンシーとによって、そのプローブがNAAPをコードする天然配列のみを同定するかどうか、或いは対立遺伝子変異配列や関連配列を同定するかどうかが決まることとなる。
【０２７８】
プローブはまた、関連する配列の検出に利用でき、また、NAAPをコードする任意の配列との少なくとも５０％の配列同一性を有し得る。目的の本発明のハイブリダイゼーションプローブには、DNAあるいはRNAが可能であり、SEQ ID NO:17−32の配列、或いはNAAP遺伝子のプロモーター、エンハンサー、イントロンを含むゲノム配列に由来し得る。
【０２７９】
NAAPをコードするDNAに対して特異的なハイブリダイゼーションプローブの作製方法には、NAAPをコードまたはNAAP誘導体をコードするポリヌクレオチド配列をmRNAプローブの作製のためのベクターにクローニングする方法がある。mRNAプローブ作製用ベクター類は、当業者に知られており、市販されており、好適なRNAポリメラーゼと好適な標識されたヌクレオチドとを加えることによって、in vitroでRNAプローブを合成するために用い得る。ハイブリダイゼーションプローブ類は、種々のレポーターの集団によって標識され得る。レポーター集団の例としては、³²Pまたは³⁵Sなどの放射性核種が、あるいはアビジン／ビオチン結合系を介してプローブに結合されたアルカリホスファターゼなど酵素標識が挙げられる。
【０２８０】
NAAPをコードするポリヌクレオチド配列を、NAAPの発現に関係する疾患の診断に用い得る。限定するものではないが、このような疾患には細胞増殖異常、神経障害、発達障害、自己免疫／炎症疾患、および感染症が含まれ、細胞増殖異常には日光角化症、動脈硬化、アテローム硬化、滑液包炎、硬変、肝炎、混合性結合組織病（MCTD）、骨髄線維症、発作性夜間ヘモグロビン尿症、真性多血症、乾癬、原発性血小板血症、並びに、腺癌、白血病、リンパ腫、黒色腫、骨髄腫、肉腫、および奇形癌など癌、具体的には、副腎、膀胱、骨、骨髄、脳、乳房、頚部、胆嚢、神経節、消化管、心臓、腎臓、肝臓、肺、筋肉、卵巣、膵臓、副甲状腺、陰茎、前立腺、唾液腺、皮膚、脾臓、精巣、胸腺、甲状腺、および子宮の癌が含まれ、神経障害には、癲癇、虚血性脳血管障害、脳卒中、脳腫瘍、アルツハイマー病、ピック病、ハンチントン病、痴呆、パーキンソン病およびその他の錐体外路障害、筋萎縮性側索硬化およびその他の運動ニューロン障害、進行性神経性筋萎縮症、網膜色素変性症（色素性網膜炎）、遺伝性運動失調、多発性硬化症および他の脱髄疾患、細菌性およびウイルス性髄膜炎、脳膿瘍、硬膜下膿瘍、硬膜外膿瘍、化膿性頭蓋内血栓性静脈炎、脊髄炎および神経根炎、ウイルス性中枢神経系疾患と、プリオン病（クールー、クロイツフェルト‐ヤコブ病、およびGerstmann-Straussler-Scheinker症候群を含む）、致死性家族性不眠症、神経系性栄養病および代謝病、神経線維腫症、結節硬化症、小脳網膜血管腫症（cerebelloretinal hemangioblastomatosis）、脳３叉神経血管症候群、中枢神経系性精神遅滞および他の発達障害、脳性麻痺、神経骨格異常症、自律神経系障害、脳神経障害、脊髄疾患、筋ジストロフィー他の神経筋障害、末梢神経疾患、皮膚筋炎および多発性筋炎と、遺伝性、代謝性、内分泌性、および中毒性ミオパシーと、重症筋無力症、周期性四肢麻痺、精神障害（気分性、不安性の障害、統合失調症／分裂病）、季節性感情障害（SAD）、静座不能、健忘症、緊張病、糖尿病性ニューロパシー、遅発性ジスキネジア、ジストニー、パラノイド精神病、帯状疱疹後神経痛、トゥーレット病が含まれ、発達障害には尿細管性アシドーシス、貧血、クッシング症候群、軟骨形成不全性小人症、デュシェンヌ／ベッカー型筋ジストロフィー、癲癇、性腺形成異常、WAGR症候群（ウィルムス腫瘍、無虹彩症、尿生殖器異常、精神遅滞）、スミス‐マジェニス症候群（Smith- Magenis syndrome）、骨髄異形成症候群、遺伝性粘膜上皮異形成、遺伝性角皮症や、シャルコーマリーツース病および神経線維腫症などの遺伝性神経病、甲状腺機能低下症、水頭症や、Syndenham舞踏病（Syndenham's chorea）および脳性小児麻痺などの発作障害、二分脊椎、無脳症、頭蓋脊椎披裂、先天性緑内障、白内障、感音難聴が含まれ、自己免疫／炎症疾患には、後天性免疫不全症候群（AIDS）、アジソン病（慢性原発性副腎機能不全）、成人呼吸窮迫症候群、アレルギー、強直性脊椎炎、アミロイド症、貧血、喘息、アテローム性動脈硬化、自己免疫性溶血性貧血、自己免疫性甲状腺炎、自己免疫性多腺性内分泌カンジダ症外胚葉ジストロフィー（APECED）、気管支炎、胆嚢炎、接触皮膚炎、クローン病、アトピー性皮膚炎、皮膚筋炎、糖尿病、肺気腫、リンパ球傷害因子性偶発性リンパ球減少症、新生児溶血性疾患（胎児赤芽球症）、結節性紅斑、萎縮性胃炎、糸球体腎炎、グッドパスチャー症候群、痛風、グレーブス病、橋本甲状腺炎、過好酸球増加症、過敏性腸症候群、多発性硬化症、重症筋無力症、心筋または心膜炎症、骨関節炎、骨粗しょう症、膵炎、多発性筋炎、乾癬、ライター症候群、リウマチ様関節炎、強皮症、シェーグレン症候群、全身性アナフィラキシー、全身性エリテマトーデス、全身性強皮症、血小板減少性紫斑病、潰瘍性大腸炎、ぶどう膜炎、ウェルナー症候群、癌合併症、血液透析、体外循環、原虫感染症、蠕虫感染症、外傷が含まれ、感染症には、アデノウイルス、アレナウイルス、ブンヤウイルス（bunyavirus）、カリチウイルス（calicivirus）、コロナウイルス、フィロウイルス、ヘパドナウイルス、ヘルペスウイルス、フラビウイルス、オルソミクソウイルス、パルボウイルス、パポーバウイルス、パラミキソウイルス、ピコルナウイルス、ポックスウイルス、レオウイルス、レトロウイルス、ラブドウイルス、トガウイルスに分類されるウイルス病原体による感染や、細菌による感染（肺炎球菌感染症、ブドウ球菌、連鎖球菌、バシラス菌、コリネバクテリウム、クロストリジウム、髄膜炎菌、淋菌、リステリア、モラクセラ、キンゲラ、ヘモフィルス、レジオネラ、百日咳菌や、シゲラ、サルモネラ、カンピロバクターを含むグラム陰性腸内細菌、シュードモナス、ビブリオ、ブルセラ、野兎病菌、エルシニア、バルトネラ、norcardium、放線菌、ミコバクテリウム、spirochaetale、リケッチア、クラミジア、マイコプラズマに分類）、真菌感染(分類はアスペルギルス、ブラストミセス、皮膚糸状菌、クリプトコッカス、コクシジオイデス、malasezzia、ヒストプラスマ、または他の真菌症起因菌)、寄生虫感染(分類はプラスモディウムすなわちマラリア原虫、寄生性アメーバ、リーシュマニア、トリパノソーマ、トキソプラズマ、ニューモシスチスカリニ、腸内原虫(ジアルジアなど）、トリコモナス、組織線虫(旋毛虫など)、腸管寄生線虫(回虫など)、リンパ管フィラリア線虫、吸虫(住血吸虫など)、および条虫（サナダムシなど）)が含まれる。NAAPをコードするポリヌクレオチド配列は、変容したNAAP発現を検出するために患者から採取した体液或いは組織を利用する、サザーン法やノーザン法、ドットブロット法、或いはその他の膜系の技術、PCR法や、ディップスティック（dipstick）、ピン（pin）、およびマルチフォーマットのELISA式アッセイ、およびマイクロアレイに使用可能である。このような定性方法または定量方法は、当分野で公知である。
【０２８１】
或る特定の態様では、NAAPをコードするヌクレオチド配列群は、関連する障害、特に上記した障害を検出するアッセイ類において有用であり得る。NAAPをコードするヌクレオチド配列を、標準的な方法で標識化し、ハイブリダイゼーション複合体の形成に好適な条件の下、患者から採取した体液或いは組織のサンプルに加えることができる。好適なインキュベーション期間が経過したらサンプルを洗浄し、シグナルを定量して標準値と比較する。患者サンプル中のシグナルの量が、対照サンプルと較べて著しく変化している場合は、該サンプル内の、NAAPをコードするヌクレオチド配列群のレベル変化の存在が、関連する障害の存在を標示する。このようなアッセイは、動物実験、臨床試験における特定の治療効果を推定するため、あるいは個々の患者の治療をモニターするために用いることもできる。
【０２８２】
NAAPの発現に関連する疾患の診断の基準を提供するために、発現の正常すなわち標準的なプロフィールが確立される。これは、ハイブリダイゼーションあるいは増幅に好適な条件の下、動物あるいはヒトのいずれかの正常な被験体から抽出された体液あるいは細胞と、NAAPをコードする配列あるいはその断片とを混合することにより達成され得る。実質的に精製されたポリヌクレオチドを既知量で用いて行った実験から得た値を正常な被験体から得た値と比較することにより、標準ハイブリダイゼーションを定量することができる。このようにして得た標準値は、疾患の徴候を示す患者から得たサンプルから得た値と比較することができる。標準値からの偏差を用いて疾患の存在を確定する。
【０２８３】
疾患の存在が確定されて治療プロトコルが開始されると、患者の発現レベルが正常な被検者に観察されるレベルに近づき始めたかどうかを測定するため、ハイブリダイゼーションアッセイを定期的に繰り返し得る。連続アッセイから得られた結果を用いて、数日から数ヶ月の期間にわたる治療の効果を示し得る。
【０２８４】
癌に関しては、個体からの生体組織における異常な量の転写物（過少発現または過剰発現）の存在は、疾患の発生素質を示したり、実際に臨床的症状が現れる前に疾患を検出する方法を提供し得る。この種のより明確な診断により、医療の専門家が予防方法または積極的な治療法を早くから利用し、それによって癌の発生または更なる進行を防止することが可能となる。
【０２８５】
NAAPをコードする配列群から設計したオリゴヌクレオチド群の更なる診断的利用には、PCRの利用を含み得る。これらのオリゴマーは、化学的に合成するか、酵素により産生するか、あるいはin vitroで産出し得る。オリゴマーは、好ましくはNAAPをコードするポリヌクレオチドの断片、或いはNAAPをコードするポリヌクレオチドと相補的なポリヌクレオチドの断片を含み、最適化した条件下で、特定の遺伝子や条件を識別するために利用される。また、オリゴマーは、やや緩いストリンジェンシー条件下で、近縁のDNAあるいはRNA配列の検出、定量、あるいはその両方のため用いることが可能である。
【０２８６】
或る実施態様においては、NAAPをコードするポリヌクレオチド配列群に由来するオリゴヌクレオチドプライマー類を用いて、一塩基多型（SNP）を検出し得る。SNPは、多くの場合にヒトの先天性または後天性遺伝病の原因となるような置換、挿入および欠失である。限定するものではないがSNPの検出方法には、SSCP（single-stranded conformation polymorphism）および蛍光SSCP（fSSCP）がある。SSCPでは、NAAPをコードするポリヌクレオチド配列群に由来のオリゴヌクレオチドプライマー類とポリメラーゼ連鎖反応法（PCR）とを用いた、DNAの増幅を行う。このDNAは例えば、病変組織または正常組織、生検サンプル、体液などに由来し得る。DNA内のSNPは、一本鎖形のPCR生成物の２次および３次構造に差異を生じさせる。差異は非変性ゲル中でのゲル電気泳動法を用いて検出可能である。fSSCPでは、オリゴヌクレオチドプライマー類を蛍光的に標識することにより、DNAシークエンシング機などの高処理機器でアンプリマー（amplimer）類の検出が可能になる。更に、インシリコSNP（in silico SNP, isSNP）と呼ばれる配列データベース分析法は、一般的なコンセンサス配列に配列されるような個々のオーバーラップするDNA断片群の配列を比較することにより、多型性を同定し得る。これらのコンピュータベースの方法は、DNAの実験室での調製に、または統計モデルとDNA配列クロマトグラムの自動分析とを用いたシークエンシングのエラーに起因する配列変異を、フィルタリングして除去する。別の態様では、例えば高処理MASSARRAYシステム（Sequenom, Inc., San Diego CA）を用いた質量分析によりSNPを検出し、特徴付ける。
【０２８７】
NAAPの発現を定量するために用い得る方法には、ヌクレオチドの放射標識またはビオチン標識、対照核酸の共増幅（coamplification）、および標準曲線から得た結果の補間もある(例えばMelby, P.C. 他(1993) J. Immunol. Methods 159:235244; Duplaa, C. 他(1993) Anal. Biochem. 212:229236を参照)。複数サンプルの定量速度を加速するには、目的のオリゴマーまたはポリヌクレオチドを種々の希釈液中に置き、分光光度法または比色反応によって定量が迅速になるような高処理フォーマットでのアッセイを行い得る。
【０２８８】
更に別の実施例では、本明細書で記載した任意のポリヌクレオチド配列に由来するオリゴヌクレオチドまたはより長い断片を、マイクロアレイにおけるエレメントとして用いることができる。多数の遺伝子の相対発現レベルを同時にモニターする転写イメージング技術にマイクロアレイを用いることが可能である。これについては、以下に記載する。マイクロアレイはまた、遺伝子の変異体、突然変異および多型性の同定に用いることができる。この情報を用いることで、遺伝子機能を決定し、疾患の遺伝的根拠を理解し、疾患を診断し、遺伝子発現の機能としての疾病の進行／後退をモニターし、疾病治療における薬剤の活性を開発およびモニターすることができる。特に、患者にとって最もふさわしく、有効な治療法を選択するために、この情報を用いて患者の薬理ゲノムプロフィールを開発することができる。例えば、患者の薬理ゲノムプロフィールに基づき、患者に対して高度に効果的で副作用を殆ど示さない治療薬を選択することができる。
【０２８９】
別の実施例では、NAAP、NAAPの断片、NAAPに特異的な抗体を、マイクロアレイ上のエレメントとして用いることができる。マイクロアレイを用いて、上記のようなタンパク質−タンパク質相互作用、薬剤−標的相互作用および遺伝子発現プロフィールをモニターまたは測定することが可能である。
【０２９０】
或る実施態様は、或る組織または細胞タイプの転写イメージを作製する、本発明のポリヌクレオチドの使用に関連する。転写イメージは、特定の組織または細胞タイプによる、遺伝子発現の包括的パターンを表す。包括的遺伝子発現パターンは、所与の条件下で所与の時間に発現した遺伝子の数および相対存在量を定量することにより分析し得る（Seilhamer 他の米国特許第5,840,484号 「Comparative Gene Transcript Analysis」を参照。該特許は特に引用することを以って本明細書の一部となす）。したがって、特定の組織または細胞タイプの転写物または逆転写物全体に、本発明のポリヌクレオチドまたはそれらの相補体をハイブリダイズすることにより、転写イメージを作製し得る。或る実施態様では、本発明のポリヌクレオチドまたはそれらの相補体が１マイクロアレイ上に複数エレメントの１サブセットを持つような高処理フォーマットでハイブリダイゼーションを発生させる。結果として得られる転写イメージは、遺伝子活性のプロフィールを提供し得る。
【０２９１】
転写イメージは、組織、細胞株、生検またはその生体サンプルから単離した転写物を用いて作製し得る。転写イメージはしたがって、組織または生検サンプルの場合にはin vivo、細胞株の場合にはin vitroでの遺伝子発現を反映する。
【０２９２】
本発明のポリヌクレオチドの発現のプロフィールを作製する転写イメージはまた、in vitroモデル系および薬剤の前臨床評価に、あるいは工業的または天然の環境化合物の毒性試験に関連して使用し得る。全ての化合物は、作用および毒性のメカニズムを標示し、しばしば分子フィンガープリントまたは毒性シグネチャと称される、特徴的な遺伝子発現パターンを惹起する（Nuwaysir, E.F. 他（1999）Mol. Carcinog. 24:153-159、Steiner, S. および N.L. Anderson（2000）Toxicol. Lett. 112-113:467-471、該文献は特に引用を以って本明細書の一部となす）。試験化合物が、既知毒性を有する化合物のシグネチャと同一のシグネチャを有する場合には、毒性特性を共有している可能性がある。フィンガープリントまたはシグネチャは、多数の遺伝子および遺伝子ファミリーからの発現情報を含んでいる場合に、最も有用且つ正確である。理想的には、ゲノム全域にわたる発現の測定が、最高品質のシグネチャを提供する。たとえ、発現が任意の試験された化合物によって変容しない遺伝子があったとしても、それらの発現レベルを残りの発現データを正規化することに使用できるため、それらの遺伝子は重要である。正規化手順は、種々の化合物で処理した後の発現データの比較に有用である。或る毒性シグネチャのエレメント群に遺伝子機能を割り当てることは毒性機序の解釈に役立つが、毒性の予測につながる、シグネチャ群の統計的マッチングには、遺伝子機能の知識は必要でない（例えば２０００年２月２９日に米国国立環境健康科学研究所（National Institute of Environmental Health Sciences）より発行されたPress Release 00-02を参照されたい。これについてはhttp://www.niehs.nih.gov/oc/news/toxchip.htmで入手可能である）。したがって、毒性シグネチャを用いる中毒学的スクリーニングの際に、全ての発現した遺伝子配列を含めることは、重要且つ望ましい。
【０２９３】
或る実施例では、核酸を有する生体サンプルを試験化合物で処理することにより、この試験化合物の毒性を算定する。処理した生体サンプル中で発現した核酸は、本発明のポリヌクレオチドに特異的な１つ以上のプローブでハイブリダイズし、それによって本発明のポリヌクレオチドに対応する転写レベルを定量し得る。処理した生体サンプル中の転写レベルを、未処理生体サンプル中のレベルと比較する。両サンプルの転写レベルの差は、処理済サンプル中で試験化合物が引き起こす毒性反応を標示する。
【０２９４】
別の実施態様は、本発明のポリペプチド配列群を用いて或る組織または細胞タイプのプロテオームを分析することに関する。プロテオームの語は、特定の組織または細胞タイプでのタンパク質発現の包括的パターンを指す。プロテオームの各タンパク質成分は、個々に更なる分析の対象とすることができる。プロテオーム発現パターンすなわちプロフィールは、所与の条件下で所与の時間に発現したタンパク質の数および相対存在量を定量することにより分析し得る。したがって、或る細胞のプロテオームのプロフィールは、特定の組織または細胞タイプのポリペプチドを分離および分析することにより作成し得る。或る実施例では、１次元等電点電気泳動によりサンプルからタンパク質を分離し、２次元ドデシル硫酸ナトリウムスラブゲル電気泳動により分子量に応じて分離するような２次元ゲル電気泳動により、分離が達成される（前出のSteiner および Anderson）。タンパク質は、通常はクーマシーブルー、あるいは銀染色液または蛍光染色液などの物質を用いてゲルを染色することにより、分散した、独自の位置にある点としてゲル中で可視化される。各タンパク質スポットの光学密度は、通常、サンプル中のタンパク質レベルに比例する。異なるサンプル、例えば試験化合物または治療薬で処理または未処理のいずれかの生体サンプルからの、同等に位置したタンパク質スポットの光学密度を比較し、処理に関連するタンパク質スポット密度の変化を同定する。スポット内のタンパク質は、例えば化学的または酵素的切断とそれに続く質量分析を用いる標準的な方法を用いて部分的にシークエンシングする。或るスポット内のタンパク質の同一性は、その部分配列を、好適には少なくとも５個の連続するアミノ酸残基を、本発明のポリペプチド配列と比較することにより決定し得る。場合によっては、決定的なタンパク質同定のための更なる配列データが得られる。
【０２９５】
プロテオームのプロフィールは、NAAPに特異的な抗体を用いてNAAP発現レベルを定量することによっても作成可能である。或る実施態様では、マイクロアレイ上のエレメントとしてこれら抗体を用い、マイクロアレイをサンプルに曝して各アレイエレメントへのタンパク質結合レベルを検出することにより、タンパク質発現レベルを定量する（Lueking, A. 他(1999) Anal. Biochem. 270:103-111; Mendoze, L.G. 他(1999) Biotechniques 27:778-788)。検出は当分野で既知の様々な方法で行うことができ、例えば、チオール反応性またはアミノ反応性蛍光化合物とサンプル中のタンパク質を反応させ、各アレイエレメントにおける蛍光結合の量を検出し得る。
【０２９６】
プロテオームレベルでの毒性シグネチャも中毒学的スクリーニングに有用であり、転写レベルでの毒性シグネチャと並行に分析するべきである。いくつかの組織のいくつかのタンパク質については、転写物の存在量とタンパク質の存在量との相関が乏しいので（Anderson, N.L. および J. Seilhamer（1997）Electrophoresis 18:533-537）、転写イメージにはそれ程影響しないがプロテオームのプロフィールを改変するような化合物の分析において、プロテオーム毒性シグネチャは有用たり得る。更に、体液中の転写物の分析はmRNAの急速な分解のために困難なので、プロテオームのプロフィール作成はこのような場合により信頼性が高いと思われ、情報価値があり得る。
【０２９７】
別の実施例では、タンパク質を含有する生体サンプルを試験化合物で処理することにより試験化合物の毒性を算定する。処理された生体サンプル中で発現したタンパク質は、各タンパク質の量を定量し得るように分離する。各タンパク質の量を、未処理生体サンプル中の対応するタンパク質の量と比較する。両サンプルのタンパク質の量の差は、処理サンプル中の試験化合物に対する毒性反応を標示する。個々のタンパク質は、個々のタンパク質のアミノ酸残基をシークエンシングし、これら部分配列を本発明のポリペプチドと比較することにより同定する。
【０２９８】
別の実施例では、タンパク質を含有する生体サンプルを試験化合物で処理することにより試験化合物の毒性を算定する。生体サンプルから得たタンパク質は、本発明のポリペプチドに特異的な抗体を用いてインキュベートする。抗体により認識されたタンパク質の量を定量する。処理された生体サンプル中のタンパク質の量を、未処理生体サンプル中のタンパク質の量と比較する。両サンプルのタンパク質の量の差は、処理サンプル中の試験化合物に対する毒性反応を標示する。
【０２９９】
マイクロアレイは、本技術分野で既知の方法で調製し、使用し、分析する（例えばBrennan, T.M. 他（1995）米国特許第5,474,796号、Schena, M. 他（1996）Proc. Natl. Acad. Sci. USA 93:10614-10619、Baldeschweiler 他（1995）PCT出願第WO95/251116号、Shalon, D.他（1995）PCT出願第WO95/35505号、Heller, R.A. 他（1997）Proc. Natl. Acad. Sci. USA 94:2150-2155、Heller, M.J. 他（1997）米国特許第5,605,662号を参照）。様々なタイプのマイクロアレイが公知であり、詳細については、DNA Microarrays: A Practical Approach, M.Schena, 編集. (1999) Oxford University Press, Londonに記載されている。該文献は、特に引用することを以って本明細書の一部となす。
【０３００】
本発明の別の実施態様ではまた、NAAPをコードする核酸配列群を用いて、天然ゲノム配列をマッピングするのに有用なハイブリダイゼーションプローブ群を産生し得る。コード配列または非コード配列のいずれかを用いることができ、いくつかの例では、コード配列より非コード配列が好ましい。例えば、多重遺伝子族のメンバー内でのコード配列の保存により、染色体マッピング中に望ましくないクロスハイブリダイゼーションが生じる可能性がある。配列は、或る特定の染色体に、または或る染色体の或る特定領域に、または人為形成の染色体、例えば、ヒト人工染色体（HAC）、酵母人工染色体（YAC）、細菌人工染色体（BAC）、細菌P1産物、あるいは単一染色体cDNAライブラリ群に対してマッピングされる(例えばHarrington, J.J. 他(1997) Nat. Genet. 15:345-355; Price, C.M. (1993) Blood Rev. 7:127134; Trask, B.J. (1991) Trends Genet. 7:149154を参照)。一度マッピングすると、本発明の核酸配列群を用いて、例えば或る病状の遺伝を特定染色体領域の遺伝とまたは制限酵素断片長多型（RFLP）と相関させるような遺伝子連鎖地図を開発し得る（例えば、Lander, E.S.およびD. Botstein（1986）Proc. Natl. Acad. Sci. USA 83:7353-7357を参照）。
【０３０１】
蛍光原位置ハイブリッド形成法（FISH）は、他の物理的および遺伝的地図データと相関し得る(例えばHeinz-Ulrich, 他(1995) in Meyers, 前出、965-968ページを参照)。遺伝地図データの例は、種々の科学雑誌あるいはOnline Mendelian Inheritance in Man（OMIM）のウェブサイトに見られる。物理的染色体地図上の、NAAPをコードする遺伝子の位置と、特定の疾患との相関性、あるいは特定の疾患に対する素因との相関性は、この疾患と関連するDNAの領域の決定に役立ち得るため、位置を決定するクローニングの作業を促進し得る。
【０３０２】
確定した染色体マーカー類を用いた連鎖分析などの物理的マッピング技術、および染色体標本の原位置ハイブリッド形成法を用いて、遺伝地図を拡張することができる。例えばマウスなど別の哺乳類の染色体上に遺伝子を配置することにより、正確な染色体上の遺伝子座が未知でも、関連するマーカー類をしばしば明らかにし得る。この情報は、位置クローニングなどの遺伝子発見技術を用いて疾患遺伝子を探る研究者にとって価値がある。いったん疾患または症候群に関与する遺伝子（群）が、血管拡張性失調症の11q22-23領域など、特定のゲノム領域への遺伝的連鎖によって、大まかに位置決めがなされると、該領域にマップされる任意の配列は、更なる調査のための関連遺伝子あるいは調節遺伝子を提示している可能性がある(例えばGatti, R.A. 他(1988) Nature 336:577-580を参照)。転座、反転などに起因する、健常者、保有者、罹病者の三者間における染色体位置の相違を検出する場合にも、本発明のヌクレオチド配列を用い得る。
【０３０３】
本発明の別の実施態様では、NAAP、その触媒作用断片あるいは免疫原性断片またはそのオリゴペプチド群を、種々の任意の薬剤スクリーニング技術における、化合物のライブラリ群のスクリーニングに用い得る。薬剤スクリーニングに用いる断片は、溶液中に遊離しているか、固体支持物に固定されるか、細胞表面上に保持されるか、細胞内に位置することができる。NAAPと検査する薬剤との結合による複合体の形成を測定することもできる。
【０３０４】
別の薬剤スクリーニング方法は、目的のタンパク質に対して好適な結合親和性を有する化合物を高い処理能力でスクリーニングするために用いられる(例えばGeysen, 他(1984) PCT 出願番号 WO84/03564を参照)。この方法においては、多数の様々な低分子の試験用化合物を固体基板上で合成する。試験用化合物は、NAAP、或いはその断片と反応してから洗浄される。次に、結合したNAAPを本技術分野で周知の方法で検出する。精製したNAAPはまた、上記した薬剤スクリーニング技術に用いるプレート上に直接コーティングできる。別法では、非中和抗体を用いてペプチドを捕捉し、ペプチドを固体支持物に固定することもできる。
【０３０５】
別の実施例では、NAAPと特異結合可能な中和抗体がNAAPとの結合について試験化合物と競合する、競合的薬剤スクリーニングアッセイを用いることができる。この方法では、抗体を用いて、１つ以上の抗原決定基をNAAPと共有するどのペプチドの存在をも検出できる。
【０３０６】
別の実施例では、NAAPをコードするヌクレオチド配列を、将来に開発される分子生物学技術であり、現在知られているヌクレオチド配列の特性（限定はされないが、トリプレット遺伝コード、特異的な塩基対相互作用等を含む）に依存する新技術に用い得る。
【０３０７】
更に詳細説明をしなくとも、当業者であれば以上の説明を以って本発明を最大限に利用できるであろう。したがって、これ以下に記載する実施例は単なる例示目的にすぎず、いかようにも本発明を限定するものではない。
【０３０８】
本明細書において開示した全ての特許、特許出願及び刊行物、特に米国特許出願第60/257,714号、第60/260,081号、第60/262,302号、第60/266,088号、出願 [代理人整理番号（Attorney Docket No.) PF-1249 P, ファイル日は2001年10月29日]、および出願第60/263,823号は、言及することをもって本明細書の一部となす。
【０３０９】
（実施例）
１ cDNA ライブラリの作製
Incyte cDNA群の由来は、LIFESEQ GOLDデータベース (Incyte Genomics, Palo Alto CA)に記載されたcDNAライブラリ群である。幾つかの組織はホモジナイズしてグアニジニウムイソチオシアネート溶液に溶解し、他の組織はホモジナイズしてフェノールにまたは変性剤群の好適な混合液に溶解した。混合液の１例であるTRIZOL（Life Technologies）は、フェノールとグアニジンイソチオシアネートとの単相溶液である。結果として得た溶解物は、塩化セシウムのクッション液の上に重層して遠心分離するか、クロロフォルムで抽出した。イソプロパノールか、酢酸ナトリウムとエタノールか、いずれか一方、あるいは別の慣例的方法で、溶解物からRNAを沈殿させた。
【０３１０】
RNAの純度を高めるため、フェノールによるRNAの抽出および沈殿を、必要な回数繰り返した。場合によっては、DNアーゼでRNAを処理した。殆どのライブラリでは、オリゴd（T）連結常磁性粒子（Promega）、OLIGOTEXラテックス粒子（QIAGEN, Chatsworth CA）またはOLIGOTEX mRNA精製キット（QIAGEN）を用いて、ポリ（A）+RNAを単離した。別法では、別のRNA単離キット、例えばPOLY（A）PURE mRNA精製キット（Ambion, Austin TX）を用いて、組織溶解物からRNAを直接単離した。
【０３１１】
場合によってはStratagene社にRNAを提供し、対応するcDNAライブラリ群をStratagene社が作製した。そうでない場合は、UNIZAPベクターシステム（Stratagene）またはSUPERSCRIPTプラスミドシステム（Life Technologies）を用いて本技術分野で既知の推奨方法または類似の方法でcDNAを合成し、cDNAライブラリを作製した（前出のAusubel, 1997, 5.1-6.6ユニットなどを参照）。逆転写は、オリゴd（T）またはランダムプライマーを用いて開始した。合成オリゴヌクレオチドアダプターを二本鎖cDNAに連結反応させ、好適な制限酵素または酵素群でcDNAを消化した。殆どのライブラリに対して、cDNAのサイズ選択（３００〜１０００bp）は、SEPHACRYL S1000、SEPHAROSE CL2BまたはSEPHAROSE CL4Bカラムクロマトグラフィー（Amersham Pharmacia Biotech）、あるいは調製用アガロースゲル電気泳動法を用いて行った。cDNAは好適なプラスミドのポリリンカーの、適合する制限酵素部位にライゲーションされた。好適なプラスミドは、例えばPBLUESCRIPTプラスミド（Stratagene）、PSPORT1プラスミド（Life Technologies）PCDNA2.1プラスミド（Invitrogen, Carlsbad CA）、PBK-CMVプラスミド（Stratagene）、PCR2−TOPOTAプラスミド（Invitrogen）、PCMV-ICISプラスミド（Stratagene）、pIGEN（Incyte Genomics, Palo Alto CA）、pRARE (Incyte Genomics)、またはplNCY（Incyte Genomics）、またはこれらの誘導体である。組換えプラスミドは、Stratagene社のXL1-Blue、XL1-BIueMRFまたはSOLR、あるいはLife Technologies社のDH5α、DH10BまたはElectroMAX DH10Bなど適格な大腸菌細胞に形質転換した。
【０３１２】
２ ｃＤＮＡクローンの単離
実施例１のようにして得たプラスミドの、宿主細胞からの回収は、UNIZAPベクターシステム（Stratagene）を用いたin vivo切除によって、あるいは細胞溶解によって行った。プラスミドを精製する方法は、MagicまたはWIZARD Minipreps DNA精製システム（Promega）、AGTC Miniprep精製キット（Edge Biosystems, Gaithersburg MD）、QIAGEN社のQIAWELL 8 Plasmid、QIAWELL 8 Plus PlasmidおよびQIAWELL 8 Ultra Plasmid 精製システム、R.E.A.L. Prep 96プラスミド精製キットの中から少なくとも１つを用いた。プラスミドは、沈殿させた後、0.1mlの蒸留水に再懸濁して、凍結乾燥して或いは凍結乾燥しないで４℃で保管した。
【０３１３】
別法では、高処理フォーマットで直接結合PCR法を用い、宿主細胞溶解物からプラスミドDNAを増幅した（Rao, V.B.（1994）Anal. Biochem. 216:1-14）。宿主細胞の溶解および熱サイクリング過程は、単一反応混合液中で行った。サンプルを加工し、それを３８４穴プレート内で保管し、増幅したプラスミドDNAの濃度をPICOGREEN色素（Molecular Probes, Eugene OR）およびFLUOROSKANII蛍光スキャナ（Labsystems Oy, Helsinki, Finland）を用いて蛍光分析的に定量した。
【０３１４】
３ シークエンシングおよび分析
実施例２に記載したようにプラスミドから回収したIncyte cDNAを、以下に示すようにシークエンシングした。cDNAのシークエンス反応は、標準的方法あるいは高処理装置、例えばABI CATALYST 800 サーマルサイクラー（Applied Biosystems）またはPTC-200 サーマルサイクラー（MJ Research）を、HYDRAマイクロディスペンサー（Robbins Scientific）またはMICROLAB 2200（Hamilton）液体転移システムと併用して処理した。cDNAのシークエンス反応は、Amersham Pharmacia Biotech社が提供する試薬、またはABIシークエンシングキット、例えばABI PRISM BIGDYE Terminator cycle sequencing ready reaction kit（Applied Biosystems）の試薬を用いて準備した。cDNAのシークエンス反応の電気泳動的分離には、また、標識したポリヌクレオチドの検出には、MEGABACE 1000 DNAシークエンシングシステム（Molecular Dynamics）か、標準ABIプロトコルと塩基対呼び出しソフトウェアとを用いるABI PRISM 373または377シークエンシングシステム（Applied Biosystems）か、あるいはその他の本技術分野で既知の配列解析システムを用いた。cDNA配列内のリーディングフレームは、標準的方法（前出のAusubel, 1997, 7.7ユニットに概説）を用いて同定した。いくつかのcDNA配列を選択し、実施例８に開示する技術で配列を伸長させた。
【０３１５】
Incyte cDNAに由来するポリヌクレオチド配列は、ベクター、リンカーおよびポリ(A)配列を除去し、あいまいな塩基をマスクすることによって有効性を確認した。その際、BLASTと、動的プログラミングと、隣接ジヌクレオチド頻度分析とに基づく、アルゴリズムとプログラムとを用いた。次に、Incyte cDNA配列またはそれらの翻訳の問い合わせを、以下のデータベース群に対して行った。すなわち、選抜した公共のデータベース群（例えばGenBankの霊長類、げっ歯類、哺乳類、脊椎動物、真核生物のデータベースと、BLOCKS、PRINTS、DOMO、PRODOM）と、ヒト、ラット、マウス、線虫（Caenorhabditis elegans）、出芽酵母（Saccharomyces cerevisiae）、分裂酵母（Schizosaccharomyces pombe）および鵞口瘡カンジダ（Candida albicans）からの配列群を持つPROTEOMEデータベース群（Incyte Genomics, Palo Alto CA）、および、隠れマルコフモデル（HMM）ベースのタンパク質ファミリーデータベース群（PFAM等）である（HMMは、遺伝子ファミリーのコンセンサス１次構造を分析する確率的アプローチである。例えば、Eddy, S.R. (1996) Cuff. Opin. Struct. Biol. 6:361-365等を参照）。問合せは、BLAST、FASTA、BLIMPS、およびHMMERに基づくプログラムを用いて行った。Incyte cDNA配列を構築し、完全長のポリヌクレオチド配列を産出した。あるいは、GenBank cDNA群、GenBank EST群、スティッチされた配列群、ストレッチされた配列群、またはGenscan予測コード配列群（実施例４および５を参照）を用い、Incyte cDNAの集団を完全長まで伸長させた。PhredとPhrapとConsedとに基づくプログラムを用いて構築し、GenMarkとBLASTとFASTAとに基づくプログラムを用いて、cDNAの集団を、オープンリーディングフレームについてスクリーニングした。完全長ポリヌクレオチド配列を翻訳し、対応する完全長ポリペプチド配列を誘導した。あるいは、本発明のポリペプチドは、完全長翻訳ポリペプチドの任意のメチオニン残基で開始し得る。完全長ポリペプチド配列群の続いての分析としての問い合わせを、GenBankタンパク質データベース群（genpept）、SwissProt、PROTEOMEデータベース群、BLOCKS、PRINTS、DOMO、PRODOMおよびProsite等のデータベースや、PFAM等の隠れマルコフモデル（HMM）ベースのタンパク質ファミリーデータベース群に対し行った。完全長ポリヌクレオチド配列はまた、MACDNASIS PROソフトウェア（日立ソフトウェアエンジニアリング, South San Francisco CA）およびLASERGENEソフトウェア（DNASTAR）を用いて分析した。ポリヌクレオチドとポリペプチドとの配列アラインメントは、MEGALIGNマルチシークエンスアラインメントプログラム（DNASTAR）に組み込まれたCLUSTALアルゴリズムが指定する、デフォルトパラメータを用いて作製する。これは、アラインメントした配列間の一致率も計算する。
【０３１６】
表７は、Incyte cDNAと完全長配列との分析と構築とに利用したツールとプログラムとアルゴリズムとの概略と、適用可能な説明、参照文献、閾値パラメータを示す。用いたツール、プログラムおよびアルゴリズムを表７の列１に、それらの簡単な説明を列２に示す。列３は好適な参照文献であり、全ての文献は引用を以って全文を本明細書の一部となす。適用可能な場合には、列４は、２つの配列が一致する強さを評価するために用いた、スコア、確率値などのパラメータを示す（スコアが高いほど、または確率値が低いほど、２配列間の同一性が高い）。
【０３１７】
完全長ポリヌクレオチド配列およびポリペプチド配列の構築および分析に用いる上記のプログラム群は、SEQ ID NO:17−32のポリヌクレオチド配列断片の同定にも利用できる。ハイブリダイゼーション及び増幅技術に有用である約２０〜約４０００ヌクレオチドの断片群を、表４の列２に示した。
【０３１８】
４ ゲノム DNA からのコード配列の同定および編集
推定上の核酸関連タンパク質は、先ず公共のゲノム配列データベース（例えばgbpriやgbhtg）に対しGenscan遺伝子同定プログラムを実行して同定した。Genscanは汎用遺伝子同定プログラムであり、様々な生物からのゲノムDNA配列を分析する（Burge, C. および S. Karlin（1997）J. Mol. Biol. 268:78-94、Burge, C. および S. Karlin（1998）Curr. Opin. Struct. Biol. 8:346-354参照）。このプログラムは予測されたエキソン群を連結し、メチオニンから終止コドンに及ぶ、構築されたcDNA配列を形成する。Genscanの出力は、ポリヌクレオチドおよびポリペプチド配列のFASTAデータベースである。Genscanが一度に分析する配列の最大範囲は、３０kbに設定した。これらのGenscan予測cDNA配列の内、どの配列が核酸関連分子をコードするかを決定するために、コードされるポリペプチドを、PFAMモデル群に対し核酸関連分子について問合せて分析した。潜在的な核酸関連タンパク質はまた、既に核酸関連タンパク質としてアノテーションが付けられたIncyte cDNA配列に対する相同性を基に同定された。これら選択したGenscan予測配列を、次にBLAST分析により、公共データベースgenpeptおよびgbpriと比較した。必要であれば、genpeptからのトップBLASTヒットと比較することによりGenscan予測配列を編集し、余分なまたは省略されたエキソンなど、Genscanが予測した配列におけるエラーを補正した。BLAST分析はまた、Genscan予測配列の、いかなるIncyte cDNAまたは公共cDNAカバレッジ（coverage）の発見にも用いられ、したがって転写の証拠を提供した。Incyte cDNAカバレッジが利用できた場合には、この情報を用いてGenscan予測配列を補正または確認した。完全長ポリヌクレオチド配列は、実施例３に記載した構築プロセスを用いて、Incyte cDNA配列および／または公共cDNA配列でGenscan予測コード配列を構築して得た。あるいは、完全長ポリヌクレオチド配列は、編集後または非編集のGenscan予測コード配列に、完全に由来する。
【０３１９】
５ ゲノム配列データの cDNA 配列データとの統合
スティッチ配列（ Stiched Sequence ）
部分cDNA配列群を伸長させるため、実施例４に記載のGenscan遺伝子同定プログラムが予測したエキソン群を用いた。実施例３に記載したように構築した部分cDNA群を、ゲノムDNAにマッピングし、また、関連するcDNA群と、１つ以上のゲノム配列からGenscan予測されたエキソン群とを有するクラスター群に分解した。cDNAとゲノムとの情報を統合すべく、グラフ理論および動的プログラミングに基づく或るアルゴリズムを用いて各クラスターを分析し、潜在的スプライス変異配列群を産生した。配列群を続いて確認、編集または伸長し、完全長配列を創出した。区間の全長が、２つ以上の配列に在るような配列区間群をクラスター内で同定し、そのように同定された区間群は、推移性により、等しいと考えた。例えば、或る区間が、１つのcDNAと２つのゲノム配列とに在る場合、３つの区間は全て等しいと考えた。このプロセスにより、無関係だが連続したゲノム配列群を、cDNA配列で結び合わせて架橋し得る。このようにして同定した区間を、それらの親配列（parent sequences）に沿って現われる順にスティッチアルゴリズムで「縫い合わせ」、可能な限り最長の配列と変異配列群とを産生した。１種類の親配列に沿って続く区間間の連鎖（cDNA−cDNAまたはゲノム配列−ゲノム配列）は、親の種類を変える連鎖（cDNA−ゲノム配列）より優先した。結果として得たスティッチ配列群を翻訳し、BLAST分析で公共データベースgenpeptおよびgbpriと比較した。Genscanが予測した不正確なエキソン群は、genpeptからのトップBLASTヒットとの比較により補正した。必要な場合、追加cDNA配列群を用いるかゲノムDNAの検査により、配列群を更に伸長させた。
【０３２０】
ストレッチ配列（ Stretched Sequence ）
部分DNA配列群を、BLAST分析に基づく１アルゴリズムにより完全長まで伸長した。先ず、BLASTプログラムを用いて、GenBankの霊長類、げっ歯類、哺乳類、脊椎動物および真核生物のデータベースなどの公共データベースに対し、実施例３に記載されたように構築された部分cDNAを問い合わせた。次に、最も近いGenBankタンパク質相同体を、BLAST分析により、Incyte cDNA配列または実施例４に記載のGenScanエキソン予測配列のいずれかと比較した。結果として得られる高スコアリングセグメント対（HSP）を用いてキメラタンパク質を産生し、翻訳した配列をGenBankタンパク質相同体上にマッピングした。元のGenBankタンパク質相同体に対し、キメラタンパク質内では挿入または欠失が起こり得る。GenBankタンパク質相同体、キメラタンパク質またはその両方をプローブとして用い、公共のヒトゲノムデータベースから相同ゲノム配列を検索した。このようにして、部分的なDNA配列を、相同ゲノム配列の付加によりストレッチすなわち伸長した。結果として得られるストレッチ配列を検査し、完全遺伝子を含んでいるか否かを決定した。
【０３２１】
６ NAAP をコードするポリヌクレオチドの染色体マッピング
SEQ ID NO:17−32を構築するために用いた配列群を、BLAST他のSmith-Watermanアルゴリズムの実装群を用いて、Incyte LIFESEQデータベースおよび公共ドメインデータベース群の配列群と比較した。SEQ ID NO:17−32と一致するこれらのデータベースの配列を、Phrapなどの構築アルゴリズムを使用して、連続的配列及びオーバーラップした配列のクラスターに構築した（表７）。スタンフォード・ヒトゲノムセンター（SHGC）、ホワイトヘッド・ゲノム研究所（WIGR）、Genethonなどの公的な情報源から入手可能な放射線ハイブリッドおよび遺伝地図データを用いて、いずれかのクラスター化された配列が既にマッピングされているかを判定した。マッピングされた配列が或るクラスターに含まれている場合、そのクラスターの全配列が、個々の配列番号と共に、地図上の位置に割り当てられた。
【０３２２】
地図上の位置は、ヒト染色体の範囲または間隔として表される。センチモルガン間隔の地図上の位置は、染色体のpアームの末端に対して測定する（センチモルガン（cM）は、染色体マーカー間の組換え頻度に基づく計測単位である。平均して、１cMは、ヒトDNAの１メガベース（Mb）にほぼ等しい。尤も、この値は、組換えのホットスポットおよびコールドスポットに起因して広範囲に変化する）。cM距離は、各クラスタ内に配列が含まれる放射線ハイブリッドマーカー類に対して境界を提供するGenethonによってマッピングされた遺伝マーカー群に基づく。NCBI「GeneMap'99」（http://www.ncbi.nlm.nih.gov/genemap/）など一般個人が入手可能なヒトゲノム地図などの資源を用いて、既に同定された疾患遺伝子類が、上記の区間内若しくは近傍にマップされているかを判定できる。 このようにして、SEQ ID NO:20は、8番染色体の125.80から140.60センチモルガンの区間内にマップされ、またSEQ ID NO:28は、19番染色体の41.7から49.4センチモルガンの区間内にマップされた。
【０３２３】
７ ポリヌクレオチド発現の分析
ノーザン分析は、遺伝子の転写物の存在を検出するために用いられる実験技術であり、特定の細胞種または組織からのRNAが結合している膜への、標識されたヌクレオチド配列のハイブリダイゼーションに関与する（例えば前出のSambrook, 7章、同Ausubel (1995) 4章および16章を参照）。
【０３２４】
BLASTを応用した類似のコンピュータ技術を用いて、GenBankやLIFESEQ（Incyte Genomics）などのcDNAデータベースにおいて、同一または関連分子を検索した。ノーザン分析は、いくつかの膜系ハイブリダイゼーションよりも非常に速い。更に、任意の特定の一致を厳密なあるいは相同的なものとして分類するか否かを決定するため、コンピュータ検索の感度を修正することができる。検索の基準は積スコアであり、次式で定義される。
【０３２５】
【数１】

【０３２６】
積スコアは、２つの配列間の類似度と、配列が一致する長さとの両方を考慮している。積スコアは、０〜１００の正規化された値であり、次のようにして求める。BLASTスコアにヌクレオチドの配列一致率を乗じ、その積を２つの配列の短い方の長さの５倍で除する。BLASTスコアを計算するには、或る高スコアリングセグメント対（HSP）内の一致する各塩基に＋５のスコアを割り当て、各不一致塩基に−４を割り当てる。２つの配列は、２以上のHSPを共有し得る（ギャップにより隔離される）。２以上のHSPがある場合には、最高BLASTスコアのセグメント対を用いて積スコアを計算する。積スコアは、断片的オーバーラップとBLASTアラインメントの質とのバランスを表す。例えば積スコア１００は、比較した２つの配列の短い方の長さ全体にわたって１００％一致する場合のみ得られる。積スコア７０は、一端が１００％一致し、７０％オーバーラップしているか、他端が８８％一致し、１００％オーバーラップしているかのいずれかの場合に得られる。積スコア５０は、一端が１００％一致し、５０％オーバーラップしているか、７９％一致し、１００％オーバーラップしているかのいずれかの場合に得られる。
【０３２７】
或いは、NAAPをコードするポリヌクレオチド配列を、由来する組織に対して分析する。例えば幾つかの完全長配列は、少なくとも一部は、オーバーラップするIncyte cDNA配列群を用いて構築される（実施例３を参照）。各cDNA配列は、ヒト組織から作製されたcDNAライブラリに由来する。各ヒト組織は、以下の臓器／組織カテゴリーの１つに分類される。すなわち心血管系、結合組織、消化器系、胎芽構造、内分泌系、外分泌腺、女性生殖器、男性生殖器、生殖細胞、血液および免疫系、肝、筋骨格系、神経系、膵臓、呼吸器系、感覚器、皮膚、顎口腔系、非分類性／混合性または尿路である。各カテゴリーのライブラリ数を数えて、全カテゴリーの総ライブラリ数で除する。同様に、各ヒト組織は、以下の疾患／状況カテゴリーすなわち癌、細胞株、発達、炎症、神経性、外傷、心血管、プール、その他の１つに分類される。各カテゴリーのライブラリ数を数えて、全カテゴリーの総ライブラリ数で除する。得られるパーセンテージは、NAAPをコードするcDNAの、組織特異的または疾患特異的な発現を反映する。cDNA配列およびcDNAライブラリ／組織の情報は、LIFESEQ GOLD データベース（Incyte Genomics, Palo Alto CA）から得ることができる。
【０３２８】
８ ＮＡＡＰをコードするポリヌクレオチドの伸長
完全長のポリヌクレオチド配列はまた、完全長分子の適切な断片から設計したオリゴヌクレオチドプライマー群を用いて該断片を伸長させて産生した。或るプライマーは既知の断片の５'伸長を開始するべく合成し、別のプライマーは既知の断片の３'伸長を開始するべく合成した。開始プライマー群の設計にはOLIGO 4.06ソフトウェア（National Biosciences）或いは別の適切なプログラムを用い、長さが約２２〜３０ヌクレオチド、GC含有率が約５０％以上となり、約６８〜約７２℃の温度で標的配列にアニーリングするようにした。ヘアピン構造およびプライマー−プライマー二量体を生ずるようなヌクレオチド群の伸長は、全て回避した。
【０３２９】
選択したヒトcDNAライブラリ群を用い、配列を伸長した。２段階以上の伸長が必要または望ましい場合には、付加的プライマーあるいはプライマーのネステッドセットを設計した。
【０３３０】
高忠実度の増幅が、当業者によく知られている方法を利用したPCR法によって得られた。PCRは、PTC-200 サーマルサイクラー（MJ Research, Inc.）を用いて９６穴プレート内で行った。反応混合液は、鋳型DNAを有し、また、200 nmolの各プライマーを有する。また、Mg^{２ +}と（NH₄）₂SO₄と２−メルカプトエタノールとを含有する反応バッファーと、Taq DNAポリメラーゼ（Amersham Pharmacia Biotech）と、ELONGASE酵素（Life Technologies）と、Pfu DNAポリメラーゼ（Stratagene）とを含有する。プライマー対であるPCI AとPCI Bとに対し、以下のパラメータで増幅した。
ステップ1 94℃で3分間
ステップ2 94℃で15秒間
ステップ3 60℃で1分間
ステップ4 68℃で2分間
ステップ5 ステップ２、３、及び４を２０回繰り返す
ステップ6 68℃で5分間
ステップ7 4℃で保存
別法では、プライマー対であるT7とSK+に対し、以下のパラメータで増幅した。
ステップ1 94℃で3分間
ステップ2 94℃で15秒間
ステップ3 57℃で1分間
ステップ4 68℃で2分間
ステップ5 ステップ２、３、及び４を２０回繰り返す
ステップ6 68℃で5分間
ステップ7 4℃で保存
各ウェルのDNA濃度は、１× TE及び0.5μlの希釈していないPCR産物に溶解した100μlのPICOGREEN定量試薬(0.25(v/v) PICOGREEN; Molecular Probes, Eugene OR)を不透明な蛍光光度計プレート(Corning Costar, Acton MA)の各ウェルに分配してDNAが試薬と結合できるようにして測定した。サンプルの蛍光を計測してDNAの濃度を定量すべく、プレートをFluoroskan II（Labsystems Oy, Helsinki, Finland）でスキャンした。反応混合物のアリコット５〜１０μlを１％アガロースゲル上で電気泳動法によって解析し、どの反応が配列の伸長に成功したかを判定した。
【０３３１】
伸長したヌクレオチドは、脱塩および濃縮して３８４穴プレートに移し、CviJIコレラウイルスエンドヌクレアーゼ（Molecular Biology Research, Madison WI）を用いて消化し、音波処理またはせん断し、pUC 18ベクター（Amersham Pharmacia Biotech）への再連結を行った。ショットガン・シークエンシングのために、消化したヌクレオチドを低濃度（０.６〜０.８％）のアガロースゲル上で分離し、断片を切除し、寒天をAgar ACE（Promega）で消化した。伸長させたクローンをＴ４リガーゼ（New England Biolabs, Beverly MA）を用いてpUC 18ベクター（Amersham Pharmacia Biotech）に再連結し、Pfu DNAポリメラーゼ（Stratagene）で処理して制限部位のオーバーハングを満たし、適格な大腸菌細胞に形質移入した。形質移入した細胞群の選択を抗生物質を含む培地で行い、それぞれのコロニーを採取し、LB/2Xカルベニシリン培養液中の384ウェルプレート群に３７℃で一晩培養した。
【０３３２】
細胞を溶解し、Taq DNAポリメラーゼ（Amersham Pharmacia Biotech）およびPfu DNAポリメラーゼ（Stratagene）を用いて以下の手順でDNAをPCR増幅した。
ステップ1 94℃で3分間
ステップ2 94℃で15秒間
ステップ3 60℃で1分間
ステップ4 72℃で2分間
ステップ5 ステップ２、３、及び４を２９回繰り返す
ステップ6 72℃で5分間
ステップ7 4℃で保存
DNAの定量化には、上記したようにPICOGREEN試薬（Molecular Probes）を用いた。DNAの回収率が低いサンプルは、上記と同一の条件を用いて再増幅した。サンプルは20％ジメチルスルホキシド（１：２, v/v）で希釈し、DYENAMIC エネルギートランスファー シークエンシングプライマー、およびDYENAMIC DIRECT kit（Amersham Pharmacia Biotech）またはABI PRISM BIGDYE ターミネーターサイクル シークエンシングレディ反応キット（Terminator cycle sequencing ready reaction kit）（Applied Biosystems）を用いてシークエンシングした。
【０３３３】
同様に、上記手順を用いて完全長ポリヌクレオチド配列を検証した。あるいは、完全長ポリヌクレオチドを用い、上記手順で、そのような伸長のために設計したオリゴヌクレオチド類と、或る適切なゲノムライブラリとを用いて５'調節配列を得た。
【０３３４】
９ 個々のハイブリダイゼーションプローブの標識及び使用
SEQ ID NO:17−32由来のハイブリダイゼーションプローブ群を用いて、cDNA、mRNA、またはゲノムDNAをスクリーニングする。約２０塩基対からなるオリゴヌクレオチドの標識について特に記載するが、より大きなヌクレオチド断片に対しても本質的に同一の手順が用いられる。オリゴヌクレオチドは、OLIGO 4.06ソフトウェア（National Biosciences）等の最新ソフトウェアを用いて設計し、各オリゴマー５０pmolと、[γ-³²P]アデノシン３リン酸（Amersham Pharmacia Biotech）２５０μCiと、T4ポリヌクレオチドキナーゼ（DuPont NEN, Boston MA）とを化合させることにより標識する。標識したオリゴヌクレオチドは、SEPHADEX G-25超細繊分子サイズ排除デキストラン ビーズカラム（Amersham Pharmacia Biotech）を用いて実質的に精製する。Ase I、Bgl II、Eco RI、Pst I、XbaIまたはPvu II（DuPont NEN）のいずれか１つのエンドヌクレアーゼで消化されたヒトゲノムDNAの、典型的な膜ベースのハイブリダイゼーション解析において、毎分１０⁷カウントの標識されたプローブを含むアリコットを用いる。
【０３３５】
各消化物から得たDNAは、０.７％アガロースゲル上で分画してナイロン膜（Nytran Plus, Schleicher & Schuell, Durham NH）に移す。ハイブリダイゼーションは、４０℃で１６時間行う。非特異的シグナル群を除去するため、最大で例えば０.１×クエン酸ナトリウム食塩水および０.５％ドデシル硫酸ナトリウムの条件下で、ブロット群を室温で順次洗浄する。オートラジオグラフィーまたはそれに代わるイメージング手段を用いてハイブリダイゼーションパターンを視覚化し、比較する。
【０３３６】
１０ マイクロアレイ
或るマイクロアレイ上でのアレイエレメント群の結合または合成は、フォトリソグラフィ、圧電印刷（インクジェット印刷、前出のBaldeschweilerなどを参照）、機械的マイクロスポッティング技術およびこれらから派生した技術を用いて達成し得る。上記各技術において基板は、均一な、非多孔性の表面を持つ固体とすべきである（Schena（1999）前出）。推奨する基板には、シリコン、シリカ、スライドガラス、ガラスチップおよびシリコンウエハがある。あるいは、ドットブロット法またはスロットブロット法に類似した手順を利用し、熱的、紫外線的、化学的または機械的結合手順を用いて基板の表面にエレメントを配置および結合させてもよい。通常のアレイは、利用可能な、当業者に公知の方法と機械とを用いて作製でき、任意の適正数のエレメントを有し得る（例えばSchena, M. 他（1995）Science 270:467-470、Shalon. D. 他(1996) Genome Res.6:639-645、Marshall, A. および J. Hodgson（1998）Nat. Biotechnol. 16:27-31を参照）。
【０３３７】
完全長cDNA、発現配列タグ（EST）、またはそれらの断片またはオリゴマーが、マイクロアレイのエレメントと成り得る。ハイブリダイゼーションに好適な断片またはオリゴマーを、LASERGENEソフトウェア（DNASTAR）など本技術分野で公知のソフトウェアを用いて選択することが可能である。アレイエレメント群を、生体サンプル中のポリヌクレオチド群とハイブリダイズする。生体サンプル中のポリヌクレオチドは、検出を容易にするために蛍光標識などの分子タグに抱合させる。ハイブリダイゼーション後、生体サンプルからのハイブリダイズされていないヌクレオチドを除去し、蛍光スキャナを用いて各アレイエレメントでのハイブリダイゼーションを検出する。あるいは、レーザ脱離および質量スペクトロメトリを用いてもハイブリダイゼーションを検出し得る。マイクロアレイ上の或るエレメントにハイブリダイズする各ポリヌクレオチドの、相補性の度合と相対存在度とを算定し得る。 一実施態様におけるマイクロアレイの調整および使用について、以下に詳述する。
【０３３８】
組織または細胞サンプルの調製
全RNAを組織サンプル群から単離するためグアニジニウム チオシアネート法を用い、ポリ（A）⁺RNAを精製するためオリゴ（dT）セルロース法を用いる。各ポリ（A）⁺RNAサンプルを逆転写するため、MMLV逆転写酵素を用い、また、0.05pg/μlのオリゴ（dT）プライマー（21mer）、１×第一鎖バッファー、0.03unit/μlのRNアーゼ阻害因子、500μMのdATP、500μMのdGTP、500μMのdTTP、40μMのdCTP、40μMのdCTP-Cy3（BDS）またはdCTP-Cy5（Amersham Pharmacia Biotech）を用いる。逆転写反応は、GEMBRIGHTキット（Incyte）を用い、２００ｎｇのポリ（A）⁺RNAを含有する体積２５ｍｌで行う。特異的対照ポリ（A）⁺RNA群は、非コード酵母ゲノムDNAからin vitro転写により合成する。３７℃で２時間インキュベートした後、各反応サンプル（１つはＣｙ3、もう１つはＣｙ5標識）は、２.５mlの０.５M水酸化ナトリウムで処理し、８５℃で２０分間インキュベートし、反応を停止させてRNAを分解させる。サンプル群の精製には、２つの連続するCHROMA SPIN 30ゲル濾過スピンカラム（CLONTECH Laboratories, Inc. (CLONTECH), Palo Alto CA）を用いる。混合後、２つの反応サンプルのエタノール沈殿を、１mlのグリコーゲン（１mg/ml）、６０mlの酢酸ナトリウム、および３００mlの１００％エタノールで行う。サンプルは次に、SpeedVAC（Savant Instruments Inc., Holbrook NY）を用いて完全に乾燥させ、１４μlの５×SSC／０.２％SDS中で再懸濁する。
【０３３９】
マイクロアレイの調製
本発明の配列を用いて、アレイエレメントを作製する。各アレイエレメントは、クローン化したcDNAインサート群により、ベクター含有細菌細胞群から増幅する。ＰＣＲ増幅は、cDNAインサートの側面に位置するベクター配列に相補的なプライマーを用いる。３０サイクルのPCRによって、１〜２ngの初期量から５μgを超える最終量まで、アレイエレメントを増幅する。増幅したアレイエレメントは、SEPHACRYL-400（Amersham Pharmacia Biotech）を用いて精製する。
【０３４０】
精製したアレイエレメントは、ポリマーコートされたスライドグラス上に固定する。顕微鏡スライドグラス（Corning）は、０.１％のSDSおよびアセトン中で超音波洗浄し、処理中および処理後に多量の蒸留水で洗浄する。スライドグラスは、４％フッ化水素酸（VWR Scientific Products Corporation（VWR）, West Chester PA）中でエッチングし、多量の蒸留水中で洗浄し、９５％エタノール中で０.０５％アミノプロピルシラン（Sigma）を用いてコーティングする。コーティングしたスライドは、１１０℃のオーブンで硬化させる。
【０３４１】
米国特許第5,807,522号に記載されている方法を用いて、コーティングしたガラス基板にアレイエレメント群を付加する。この特許は引用を以って本明細書の一部とする。平均濃度１００ng/μlのアレイエレメントDNA１μlを、高速ロボット装置（robotic apparatus）により、開放型キャピラリープリンティングエレメント（open capillary printing element）に充填する。装置はここで、スライド毎に約５nlのアレイエレメントサンプルを置く。
【０３４２】
マイクロアレイをUV架橋するため、STRATALINKER UV架橋剤（Stratagene）を用いる。マイクロアレイの洗浄を、室温において、０.２％ＳＤＳで１回、蒸留水で３回行う。非特異結合部位をブロックするため、マイクロアレイのインキュベートをリン酸緩衝生理食塩水（PBS）（Tropix, Inc., Bedford MA）中の０.２％カゼイン中において６０℃で３０分間行った後、前に行ったように０.２％SDSおよび蒸留水で洗浄する。
【０３４３】
ハイブリダイゼーション
ハイブリダイゼーション反応に用いる９μlのサンプル混合体には、Cy3またはCy5で標識したcDNA合成産物群の各０.２μgを、５×SSC,０.２％SDSハイブリダイゼーション緩衝液中に含む。サンプル混合体は、６５℃まで５分間加熱し、マイクロアレイ表面上で等分して１.８cm²のカバーガラスで覆う。アレイを、顕微鏡用スライドよりわずかに大きい空洞を有する防水チェンバーに移す。チェンバー内を湿度100に保持するため、１４０μlの５×SSCをチェンバーの１コーナーに加える。アレイを入れたチェンバーは、６０℃で約６．５時間インキュベートする。アレイは、第１洗浄緩衝液中（１×SSC,０.１％SDS）において４５℃で１０分間洗浄し、第２洗浄緩衝液中（０.１×SSC）において４５℃で１０分間ずつ３回洗浄し、乾燥させる。
【０３４４】
検出
レポーター標識したハイブリダイゼーション複合体を検出するには、Ｃｙ3の励起のために４８８nm、Ｃｙ5の励起のために６３２nmでスペクトル線を発生し得るInnova70混合ガス10Ｗレーザ（Coherent, Inc., Santa Clara CA）を備えた顕微鏡を用いる。励起レーザ光の焦点をアレイ上に置くため、２０×顕微鏡対物レンズ（Nikon, Inc., Melville NY）を用いる。アレイを含むスライドを、顕微鏡の、コンピュータ制御のX-Yステージに置き、対物レンズを通してラスタースキャンする。本実施例で用いる１.８cm×１.８cmのアレイは、解像度２０μmでスキャンする。
【０３４５】
異なる２回のスキャンで、混合ガスマルチラインレーザは２つの蛍光色素を連続的に励起する。発光された光は、波長に基づき分離され、２つの蛍光色素に対応する２つの光電子増倍管検出器（PMT R1477, Hamamatsu Photonics Systems, Bridgewater NJ）に送られる。好適なフィルター群をアレイと光電子増倍管との間に設置して、シグナルをフィルターする。用いる蛍光色素の最大発光の波長は、Cy3では５６５nm、Cy5では６５０nmである。装置は両方の蛍光色素からのスペクトルを同時に記録し得るが、レーザ源において好適なフィルターを用いて、蛍光色素１つにつき１度スキャンし、各アレイを通常２度スキャンする。
【０３４６】
通常、各スキャンの感度を較正するため、cDNA対照種を或る既知濃度でサンプル混合体に添加し、対照種が発生するシグナル強度を用いる。アレイ上の或る特定の位置には或る相補的DNA配列が含まれ、その位置におけるシグナルの強度をハイブリダイゼーション種の重量比1：100,000で相関させる。異なる源泉（例えば代表的な試験細胞および対照細胞など）からの２つのサンプルを、各々異なる蛍光色素で標識し、異なる発現をする遺伝子群を同定するために単一のアレイにハイブリダイズする場合には、較正するcDNAのサンプルを２種の蛍光色素で標識し、ハイブリダイゼーション混合液に各々等量を加えることによって較正を行う。
【０３４７】
光電子増倍管の出力をディジタル化するため、IBMコンパチブルPCコンピュータにインストールした12ビットRTI-835Hアナログディジタル（A/D）変換ボード（Analog Devices, Inc., Norwood MA）を用いる。ディジタル化したデータは、青色（低シグナル）から赤色（高シグナル）までの擬似カラースケールへのリニア20色変換を用いて、シグナル強度がマッピングされたイメージとして表示される。データは、定量的にも分析される。２つの異なる蛍光色素を同時に励起および測定する場合には、各蛍光体の発光スペクトルを用いて、データは先ず蛍光色素間の光学的クロストーク（発光スペクトルの重なりに起因する）を補正される。
【０３４８】
グリッドが蛍光シグナルイメージ上に重ねられ、それによって各スポットからのシグナルはグリッドの各エレメントに集められる。各エレメント内の蛍光シグナルは統合され、シグナルの平均強度に応じた数値が得られる。シグナル分析に用いるソフトウェアは、GEMTOOLS遺伝子発現分析プログラム（Incyte）である。
【０３４９】
１１ 相補的ポリヌクレオチド
NAAPをコードする配列あるいはその任意の一部に対して相補的な配列は、天然NAAPの発現を検出、低減または阻害するために用いられる。約１５〜３０塩基対を含むオリゴヌクレオチドの使用について記すが、これより小さなあるいは大きな配列の断片の場合でも、本質的に同じ手順を用いる。適切なオリゴヌクレオチド群を設計するため、Oligo 4.06ソフトウェア（National Biosciences）および、NAAPのコーディング配列を用いる。転写を阻害するためには、最も独特な５'配列から相補的オリゴヌクレオチドを設計し、これを用いて、プロモーターがコーディング配列に結合するのを防止する。翻訳を阻害するには、相補的なオリゴヌクレオチドを設計して、NAAPをコードする転写物にリボソームが結合するのを防ぐ。
【０３５０】
１２ ＮＡＡＰの発現
NAAPの発現及び精製は、細菌若しくはウイルスを基にした発現系を用いて行うことができる。細菌内でATRSを発現させるには、抗生物質耐性遺伝子と、cDNA転写レベルを高める誘導性プロモーターとを有する好適なベクターにcDNAをサブクローニングする。このようなプロモーターとしては、lacオペレーター調節エレメントと併用するT5またはT7バクテリオファージプロモーター、およびtrp-lac（tac）ハイブリッドプロモーターが含まれるが、これらに限定するものではない。組換えベクターを、BL21（DE3）などの好適な細菌宿主に形質転換する。抗生物質耐性細菌にNAAPを発現させるには、イソプロピルβ−Dチオガラクトピラノシド(IPTG)で誘発する。真核細胞でのNAAPの発現は、昆虫細胞株または哺乳動物細胞株に、一般にバキュロウイルスとして知られるAutographica californica核多角体病ウイルス(AcMNPV)の組換え型を感染させて行う。バキュロウイルスの非必須ポリヘドリン遺伝子をNAAPをコードするcDNAと置換するには、相同組換えを行うか、或いは、トランスファープラスミドの媒介を伴う、細菌の媒介による遺伝子転移を行う。ウイルスの感染力は維持され、強力な多角体プロモーターによって高レベルのcDNA転写が行われる。組換えバキュロウイルスは、多くの場合は夜蛾の１種Spodoptera frugiperda（Sf9）昆虫細胞への感染に用いるが、ヒト肝細胞への感染に用いることもある。後者の感染の場合は、バキュロウイルスへの更なる遺伝的修飾が必要になる(Engelhard, E.K. 他(1994) Proc. Natl. Acad. Sci. USA 91:3224-3227; Sandig, V. 他 (1996) Hum.Gene Ther. 7:1937-1945を参照)。
【０３５１】
殆どの発現系では、NAAPが、例えばグルタチオンＳトランスフェラーゼ(GST)と、またはFLAGや6-Hisなどのペプチドエピトープ標識と合成された融合タンパク質となるため、未精製の細胞溶解物からの組換え融合タンパク質の親和性ベースの精製を、迅速に１ステップで行い得る。GSTは日本住血吸虫からの26kDaの酵素であり、タンパク質の活性および抗原性を維持した状態で、固定化したグルタチオン上での融合タンパク質の精製を可能とする（Amersham Pharmacia Biotech）。精製後、GST要素を、操作した特定の部位においてNAAPからタンパク分解的に切断することが可能である。FLAGは８アミノ酸のペプチドであり、市販されているモノクローナルおよびポリクローナル抗FLAG抗体（Eastman Kodak）を用いた免疫親和性精製を可能にする。６ヒスチジン残基が連続して伸長した6-Hisは、金属キレート樹脂上での精製を可能にする（QIAGEN）。タンパク質の発現および精製の方法は、前出のAusubel（1995）10章、16章に記載されている。これらの方法で精製したNAAPを直接用いて、以下の実施例１６、１７、１８及び１９の、適用可能なアッセイを行い得る。
【０３５２】
１３ 機能的アッセイ
NAAPの機能は、哺乳動物細胞培養系において生理的に高められたレベルでの、NAAPをコードする配列の発現によって算定する。cDNAを高いレベルで発現する強いプロモーターを持つ哺乳動物発現ベクターにcDNAをサブクローニングする。選択されるベクターとしては、PCMV SPORT（Life Technologies）およびPCR3.1（Invitrogen, Carlsbad CA）があり、どちらもサイトメガロウイルスプロモーターを持つ。リポソーム製剤あるいは電気穿孔法を用いて、５〜１０μgの組換えベクターをヒト細胞株、例えば内皮由来または造血由来の細胞株に、一過的に形質移入する。更に、標識タンパク質をコードする配列を含む１〜２μgのプラスミドを同時に形質移入する。標識タンパク質の発現により、形質移入細胞と非形質移入細胞を区別する手段が与えられる。また、標識タンパク質の発現によって、組換えベクターからのcDNA発現を正確に予想できる。標識タンパク質は、例えば緑色蛍光タンパク質（GFP；Clontech）、CD64またはCD64-GFP融合タンパク質から選択できる。自動化された、レーザー光学に基づく技術であるフローサイトメトリー（FCM）を用いて、GFPまたはCD64-GFPを発現する形質移入された細胞を同定し、それらの細胞のアポトーシス状態や他の細胞特性を評価する。FCMは、細胞死に先行するかあるいは同時に発生する現象を診断する蛍光分子の取込を検出して計量する。このような現象として挙げられるのは、ヨウ化プロピジウムによるDNA染色によって計測される核DNA含量の変化、前方散乱光と90°側方散乱光によって計測される細胞サイズと顆粒性の変化、ブロモデオキシウリジンの取込量の低下によって計測されるDNA合成の下方調節、特異抗体との反応性によって計測される細胞表面および細胞内におけるタンパク質の発現の変容、および、フルオレセイン抱合したアネキシンＶタンパク質の細胞表面への結合によって計測される原形質膜組成の変容とがある。フローサイトメトリー法については、Ormerod, M.G.（1994）Flow Cytometry, Oxford, New York NYに記述がある。
【０３５３】
遺伝子発現におけるNAAPの影響は、NAAPをコードする配列と、CD64またはCD64-GFPのどちらかとが形質移入された、高度に精製された細胞集団を用いて評価することができる。CD64またはCD64-GFPは、形質転換された細胞表面で発現し、ヒト免疫グロブリンG（IgG）の保存された複数の領域に結合する。形質転換された細胞と形質転換されない細胞とは、ヒトIgGかCD64に対する抗体のどちらかで被覆された磁気ビーズを用いて効率的に分離できる（DYNAL, Lake Success NY）。mRNAは、当業者に周知の方法で細胞から精製できる。NAAPと、目的とする他の遺伝子とをコードするmRNAの発現は、ノーザン分析やマイクロアレイ技術で分析できる。
【０３５４】
１４ ＮＡＡＰに特異的な抗体の作製
実質的に精製されたNAAPをポリアクリルアミドゲル電気泳動法（PAGE；例えばHarrington, M.G. (1990) Methods Enzymol.182:488-495を参照）または他の精製技術で作製し、これを用いて標準的なプロトコルでウサギを免疫化して抗体を作り出す。
【０３５５】
別法では、NAAPアミノ酸配列をLASERGENEソフトウェア（DNASTAR）を用いて解析して免疫原性の高い領域を決定し、対応するオリゴペプチドを合成してこれを用いて当業者に周知の方法で抗体を生産する。Ｃ末端付近の、或いは隣接する親水性領域内のエピトープなどの適切なエピトープの選択については、当分野で周知である(例えば、前出のAusubel, 1995,11章を参照)。
【０３５６】
通常は、長さ約１５残基のオリゴペプチドを、FMOCケミストリを用いるABI 431A ペプチドシンセサイザ（Applied Biosystems）を用いて合成し、N-マレイミドベンゾイル-N-ヒドロキシスクシンイミドエステル（MBS）を用いた反応によってKLH（Sigma-Aldrich, St. Louis MO）に結合させて、免疫原性を高める（例えば前出のAusubel, 1995を参照）。完全フロイントアジュバントにおいて、オリゴペプチド-KLH複合体を用いてウサギを免疫化する。得られた抗血清の抗ペプチド活性及び抗NAAP活性を試験するには、ペプチドまたはATRSを基板に結合し、１％BSAを用いてブロッキング処理し、ウサギ抗血清と反応させて洗浄し、さらに放射性ヨウ素標識されたヤギ抗ウサギIgGと反応させる。
【０３５７】
１５ 特異抗体を用いた天然ＮＡＡＰの精製
天然NAAPあるいは組換えNAAPを実質的に精製するため、NAAPに特異的な抗体群を用いるイムノアフィニティークロマトグラフィを行う。イムノアフィニティーカラムは、CNBr-活性化したSEPHAROSE（Amersham Pharmacia Biotech）のような活性化クロマトグラフィー用レジンと抗NAAP抗体とを共有結合させることにより形成する。結合後に、製造者の使用説明書に従ってこのレジンをブロックし、洗浄する。
【０３５８】
NAAPを有する培養液をイムノアフィニティーカラムに通し、NAAPを優先的に吸着できる条件で（例えば、界面活性剤の存在下で高イオン強度のバッファーで）そのカラムを洗浄する。そのカラムを、抗体とNAAPとの結合を切るような条件で（例えば、或るpH２〜３のバッファー、あるいは高濃度の、例えば尿素またはチオシアン酸イオンなどのカオトロープで）溶出させ、NAAPを回収する。
【０３５９】
１６ ＮＡＡＰと相互作用する分子の同定
NAAP、または生物学的に活性なその断片を、¹²⁵Iボルトンハンター試薬で標識する（例えば Bolton, A.E.およびW.M. Hunter（1973）Biochem. J. 133:529-539を参照）。マルチウェルプレートの各ウェルに予め配列しておいた候補の分子群を、標識したNAAPと共にインキュベートし、洗浄して、標識されたNAAP複合体を有する全てのウェルをアッセイする。様々なNAAP濃度で得られたデータを用いて、候補分子と結合したNAAPの数量、親和性、および会合についての値を計算する。
【０３６０】
別法では、NAAPと相互作用する分子を、Fields, S.および O. Song(1989, Nature 340:245-246)に記載の酵母２−ハイブリッドシステム（yeast two-hybrid system）を、または、MATCHMAKERシステム(Clontech)など２−ハイブリッドシステムに基づく市販キットを用いて分析する。
【０３６１】
NAAPはまた、ハイスループット型の酵母２ハイブリッドシステムを使用するPATHCALLINGプロセス（CuraGen Corp., New Haven CT）に用いて、遺伝子の２大ライブラリによってコードされるタンパク質間の全ての相互作用を判定できる（Nandabalan, K. 他(2000) 米国特許第6,057,101号)。
【０３６２】
１７ ＮＡＡＰ活性の実証
NAAP活性の測定を、レポーター遺伝子の転写を刺激するNAAPの能力によって行う（Liu, H. Y. 他(1997) EMBO J. 16:5289-5298)。このアッセイは、十分に特徴付けられたレポーター遺伝子作成物であるLexA_op-LacZを用いる。このLexA_op-LacZは、大腸菌LacZ酵素をコードする配列に融合されたLexA DNA転写調節エレメント（LexA_op）からなる。融合遺伝子の作成及び発現、細胞への融合遺伝子の導入、及び、LacZ酵素活性の測定方法は、当分野で周知である。NAAPをコードする配列を、LexA転写因子に由来するDNA結合ドメインとNAAPとからなる融合タンパク質、LexA-NAAPの合成を誘導するプラスミドにクローニングする。LexA-NAAP融合タンパク質をコードする得られたプラスミドを、LexA_op-LacZレポーター遺伝子を持つプラスミドと共に酵母細胞内に導入する。対照細胞と比較したLexA-NAAP形質転換細胞に関連するLacZ酵素活性の量が、NAAPによって刺激された転写の量に比例する。
【０３６３】
あるいはNAAPの活性は、亜鉛に結合する能力として測定される。5〜10 μMのサンプル溶液（2.5 mMの酢酸アンモニウム溶液中(pH 7.4)）を0.05 Mの硫酸亜鉛溶液 (Aldrich, Milwaukee WI)と、100 μMのジチオスレイトール（10%メタノールを添加）の存在下で混合する。サンプルと硫酸亜鉛溶液とのインキュベートを20分間おこなう。反応溶液をVYCACカラム（約300 Aのボアサイズ（bore size）、5 μmの粒径）(Grace Vydac, Hesperia CA)に通す。これにより、亜鉛-サンプル複合体を溶液から単離し、次に質量分析計(PE Sciex, Ontario, Canada)にかける。サンプルに結合した亜鉛の定量には、機能的原子量である63.5 Daを用いる。この原子量を観測したのはWhittal, R. M. 他((2000) Biochemistry 39:8406-8417)である。
【０３６４】
或いは、NAAPの核酸結合活性を判定する或る方法は、ポリアクリルアミドゲル泳動移動度シフトアッセイを伴う。このアッセイの調製では、NAAPは、NAAP cDNAを有する真核生物発現ベクターを用い、COS7、HeLa、若しくはCHOなど、哺乳類細胞株を形質転換することで発現される。形質転換の後、この細胞株がNAAPを発現し蓄積するのに適した条件下で、これらの細胞を４８〜７２時間インキュベーションする。可溶化した蛋白質を有する抽出物の調製は、NAAPを発現する細胞から行いうる。この方法は当分野で周知である。NAAPを有する抽出物の各部分を、[³²P]標識したRNAまたはDNAに添加する。放射性核酸の合成は、in vitro で、当分野で周知の技術でなしうる。混合物のインキュベートを25℃、RNase阻害剤とDNase阻害剤との存在下、緩衝した条件下で5-10分間おこなう。インキュベート後、サンプルの分析を、ポリアクリルアミドゲル電気泳動とその後のオートラジオグラフィとで行う。バンドがオートラジオグラム上に存在すれば、NAAPと放射性転写物との複合体の形成を示す。同様の移動度のバンドは、非形質転換細胞から調製した対照抽出物を用いて準備したサンプル中には見られないであろう。
【０３６５】
或いはNAAPのメチラーゼ活性を判定する或る方法では、供与体基質と受容体基質との間での放射標識メチル基の転移を測定する。反応混合液(50 μl の最終容量)には、15 mMのHEPES（pH 7.9）、1.5 mMのMgCl₂、10 mMジチオスレイトール、3%ポリビニルアルコール、1.5 μCi [メチル-³H]AdoMet (0.375 μMのAdoMet) (DuPont-NEN)、0.6 μgのNAAP、および受容体基質（例えば0.4 μgの [³⁵S]RNAまたは 6-メルカプトプリン(6-MP)、最大1 mMの最終濃度）を含む。反応混液を３０℃で３０分間インキュベートし、次に６５℃で５分間インキュベートする。
【０３６６】
[メチル-³H]RNAの分析法は次のとおりである。(1) 50 μlの2×ローディングバッファ(20 mMのTris-HCl（pH 7.6）、1 MのLiCl、1 mMのEDTA、1%ドデシル硫酸ナトリウム(SDS))および50 μlオリゴd(T)-セルロース(1×ローディングバッファ中に10 mg/ml)を反応混液に添加し、インキュベートを室温で振盪しながら30分間おこなう。(2)反応混液を96穴ろ過プレート(真空装置に接続)に移す。(3)各サンプルを逐次洗浄する。これには３つの2.4 mlアリコットの1×オリゴd(T)ローディングバッファ（0.5% SDSまたは 0.1% SDSを有するものと、SDSを有しないもの）を用いる。(4) RNAの溶出を、300 μlの水で96ウェル収集プレートへ行い、シンチレーションバイアル(液体シンチラントを含有)に移し、放射能を判定する。
【０３６７】
[メチル-³H]6-MPの分析法は次のとおりである。(1) 500 μlの0.5 M ホウ酸バッファ（pH 10.0）を、次に20% (v/v)でトルエンに希釈したイソアミルアルコールの2.5 mlを、反応混液に添加する。(2) サンプルの混合を、10秒間、ボルテックスで良く撹拌しておこなう。(3) 遠心分離を700g で10分間おこなった後、1.5 mlの有機相をシンチレーションバイアル( 0.5 ml 無水エタノールと液体シンチラントとを含有)に移し、放射能を判定する。(4) 結果の補正を、6-MPの、有機相への抽出について行う（約41%)。
【０３６８】
或いは、NAAPのタイプIトポイソメラーゼ活性のアッセイを、或るスーパーコイルDNA基質の弛緩(relaxation)に基づき行う。NAAPのインキュベートをその基質と共に、Mg²⁺ とATPとを欠くバッファ中で行い、反応を終了させ、生成物をアガロースゲルにロードする。変容したトポアイソマーの、スーパーコイル基質からの弁別は、電気泳動でできる。このアッセイはタイプIトポイソメラーゼ活性に特異的である。その理由は、Mg²⁺とATPとが、タイプIIトポイソメラーゼに必要な補因子だからである。
【０３６９】
NAAPのタイプIIトポイソメラーゼ活性のアッセイを、或るキネトプラストDNA（KDNA）基質の脱連環(decatenation)に基づいて行い得る。NAAPのインキュベートをKDNAと共に行い、反応を終了させ、生成物をアガロースゲルにロードする。単量体環状KDNAの、連環したKDNAとの弁別は、電気泳動でできる。タイプIトポイソメラーゼ活性とタイプIIトポイソメラーゼ活性とを測定する市販キットはTopogen (Columbus OH)で得られる。
【０３７０】
NAAPのATP依存性RNAヘリカーゼ巻き戻し活性は、ZhangおよびGrosse (1994; Biochemistry 33:3906-3912)に記載された方法で測定できる。RNA巻き戻し用の基質には ³²P-標識RNAを含み、このRNAは二本のRNAストランド(194および130ヌクレオチドの長さ)からなり、17塩基対のduplex領域を持つ。RNA基質のインキュベートを、ATP、Mg²⁺、および可変量NAAPと共に、Tris-HClバッファ中（pH 7.5）、37℃で30分間おこなう。次に一本鎖RNA産物を二本鎖RNA基質から分離するための電気泳動を10% SDSポリアクリルアミドゲルでおこない、オートラジオグラフィで定量する。回収した一本鎖RNAの量は、調製物中のNAAP量に比例する。
【０３７１】
あるいはNAAP機能のアッセイは、哺乳動物細胞培養系において生理的に高められたレベルでの、NAAPをコードする配列の発現によって算定する。cDNAを高いレベルで発現する強いプロモーターを持つ哺乳動物発現ベクターにcDNAをサブクローニングする。選択されるベクターとしては、pCMV SPORT（Life Technologies）およびpCR3.1（Invitrogen Corporation, Carlsbad CA）があり、どちらもサイトメガロウイルスプロモーターを持つ。リポソーム製剤あるいは電気穿孔法を用いて、５〜１０μgの組換えベクターをヒト細胞株、好適には内皮由来または造血由来の細胞株に、一過的に形質移入する。更に、標識タンパク質をコードする配列を含む１〜２μgのプラスミドを同時に形質移入する。
【０３７２】
標識タンパク質の発現により、形質移入細胞と非形質移入細胞を区別する手段が与えられる。また、標識タンパク質の発現によって、組換えベクターからのcDNA発現を正確に予想できる。標識タンパク質は、例えば緑色蛍光タンパク質（GFP; CLONTECH）、CD64またはCD64-GFP融合タンパク質から選択できる。自動化された、レーザー光学に基づく技術であるフローサイトメトリー（FCM）を用いて、GFPまたはCD64-GFPを発現する形質移入された細胞を同定し、それらの細胞のアポトーシス状態や他の細胞特性を評価する。
【０３７３】
FCMは、細胞死に先行するかあるいは同時に発生する現象を診断する蛍光分子の取込を検出して計量する。このような現象として挙げられるのは、ヨウ化プロピジウムによるDNA染色によって計測される核DNA含量の変化、前方散乱光と90°側方散乱光によって計測される細胞サイズと顆粒性の変化、ブロモデオキシウリジンの取込量の低下によって計測されるDNA合成の下方調節、特異抗体との反応性によって計測される細胞表面および細胞内におけるタンパク質の発現の変容、および、フルオレセイン抱合したアネキシンＶタンパク質の細胞表面への結合によって計測される原形質膜組成の変容とがある。フローサイトメトリー法については、Ormerod, M. G. (1994) Flow Cytometry, Oxford, New York NYに記述がある。
【０３７４】
遺伝子発現に与えるNAAPの影響は、NAAPをコードする配列と、CD64またはCD64-GFPのどちらかとが形質移入された、高度に精製された細胞集団を用いて評価することができる。CD64またはCD64-GFPは、形質転換された細胞表面で発現し、ヒト免疫グロブリンG（IgG）の保存された複数の領域に結合する。形質転換された細胞と形質転換されない細胞とは、ヒトIgGかCD64に対する抗体のどちらかで被覆された磁気ビーズを用いて効率的に分離できる（Dynal Biotech, Lake Success NY）。mRNAは、当業者に周知の方法で細胞から精製できる。NAAPと、目的とする他の遺伝子とをコードするmRNAの発現は、ノーザン分析やマイクロアレイ技術で分析できる。
【０３７５】
NAAPのプソイドウリジン合成酵素活性のアッセイには、トリチウム(³H) 放出アッセイを、Nurse 他((1995) RNA 1:102112)を改作して行い、RNA中の³H-放射標識Uが異性化されてプソイドウリジン(y)になるときに、ウリジル酸(U)のピリミジン成分のC₅ 位置からの³Hの放出を測定する。典型的な500 μlアッセイ混液の内容は、50 mMのHEPESバッファ(pH 7.5)、100 mM酢酸アンモニウム、5 mMジチオスレイトール、1 mMのEDTA、30 単位のRNase阻害剤、および0.1〜4.2 μMの [5³H]tRNA (約1 μCi/nmolのtRNA)である。反応を開始するには、5 μlより少量のNAAPの濃縮溶液(またはNAAP含有サンプル)を添加し、インキュベートを5分間37 ℃で行う。NAAPを加えた後、反応混液の一部を様々な経過時間(最長30分まで)に採取し、NoritSA3 (12% w/v)を含有する1 mlの0.1 MのHClに希釈して反応を停止する。停止した反応混液の遠心分離を5分間、最大速度のマイクロ遠心機で行い、上澄みのろ過を、グラスウールの詰め物を通して行う。ペレットを、1 mlの0.1 M HCl中で2回、再懸濁して洗浄した後、遠心分離する。洗浄後の上澄みの各々を、別々にグラスウールの詰め物に通し、元のろ過物と混合する。混合したろ過物の一部を、シンチレーション液(最大１０ｍｌ)と混合し、シンチレーションカウンタでカウントする。RNAから放出された³Hの量、また可溶性ろ液に存在する³Hの量が、サンプルにおけるプソイドウリジン合成酵素活性の量に比例する(Ramamurthy, V. (1999) J. Biol. Chem. 274:2222522230)。
【０３７６】
あるいはNAAPのプソイドウリジン合成酵素活性のアッセイは、30 ℃〜37 ℃で、次の混液で行う。すなわち100 mMのTrisHCl (pH 8.0)、100 mM 酢酸アンモニウム、5 mMのMgCl₂、2 mMジチオスレイトール、0.1 mMのEDTA、および1〜2 fmolの[³²P]放射標識ランオフ(runoff)転写物(産生はin vitro で、適切なRNAポリメラーゼすなわちT7またはSP6で行う)を基質として含む混液である。NAAPを加えて反応を開始させるか、反応液に加えずに対照サンプルとする。インキュベート後、RNAをフェノール-クロロホルムで抽出し、エタノール中に沈殿させる。また、完全に加水分解して3-ヌクレオチド一リン酸にするためRNase T₂を用いる。水解物の分析には2次元薄層クロマトグラフィを用い、³²P放射ラベルの量（yMPおよびUMPスポット中に存在する量）の評価を、薄層クロマトグラフィプレートをフィルムまたはPHOSPHORIMAGERスクリーン(Molecular Dynamics)に曝した後に行う。基質RNA中のウリジル酸残基の相対数を考慮してyMPとUMPとの相対量を判定し、この量を用いてtRNA分子の量に対するyの相対量を計算する(モルy /tRNAモル、またはモルy /tRNAモル/分で表す)。この量が、NAAPサンプル中のプソイドウリジン合成酵素活性の量に一致する(Lecointe, F. 他(1998) J. Biol. Chem. 273:13161323)。
【０３７７】
NAAPのN²,N²ジメチルグアノシン転移酵素((m² ₂G)メチルトランスフェラーゼ)活性の測定は、160 μl反応混液に以下を含有して行う。すなわち100 mMのTris-HCl (pH 7.5)、0.1 mMのEDTA、10 mMのMgCl₂、20 mMのNH₄Cl、1mMジチオスレイトール、6.2 μMのS-アデノシル-L-[メチル-³H]メチオニン(30〜70 Ci/mM)、8 μgのm² ₂G-欠損tRNAまたは野生型tRNA（酵母由来）および約100 μgの精製NAAPまたはNAAP含有サンプルを含む。反応液を30℃で90分間インキュベートし、氷上で冷却する。各反応液の一部を、100 μg BSAを含む1 mlの水に希釈する。1 mlの2 M HClを各サンプルに加え、酸性不溶性産物を氷上で20分間沈殿させた後、収集のためのろ過をガラス繊維フィルターで行う。収集した物質の洗浄を数回、塩酸で行い、液体シンチレーションカウンターを用いて定量する。³Hが、m² ₂Gを欠損した酸性不溶性tRNAに取り込まれる量が、NAAPサンプル中のN²,N²ジメチルグアノシン転移酵素活性の量に比例する。基質tRNAを含まない反応、または既に修飾された野生型tRNAを含む反応は対照反応として役だち、この反応では酸性不溶性³H-標識産物は得られないはずである。
【０３７８】
NAAPのポリアデニル化活性の測定には、in vitro のポリアデニル化反応を用いる。反応混合液の作製は氷上で行い、以下を含む。10 μl の 5 mM ジチオスレイトール、0.025% (v/v) Nonidet P40界面活性剤、50 mMクレアチンリン酸、6.5% (w/v)ポリビニルアルコール、0.5ユニット/μlのRNAGUARD (Amersham Pharmacia Biotech)、0.025 μg/μlクレアチン・キナーゼ、1.25 mM コルジセピン5'三リン酸、および3.75 mMのMgCl₂で、総容量25 μlとする。60 fmolのCstF、50 fmolのCPSF、240 fmolのPAP、4 μlの未精製CF II、又は部分的に精製したCF IIと、可変量CF Iを次に反応混液に加える。容量を調整して23.5 μlにするため、以下を有するバッファを加える。50 mMのTrisHCl（pH 7.9）、10% (v/v)グリセロール、および0.1 mMのNaEDTAである。最終的な硫酸アンモニウム濃度は20 mM未満とする。反応を氷上で開始するため、15 fmol の ³²P標識mRNA前駆体テンプレートと、2.5 μgの無標識tRNAを含む1.5 μlの水を加える。反応液を次にインキュベートする。これは30 ℃で75〜90分間行い、停止するには75 μl (約2倍量)のプロテイナーゼKミックス(0.2 MのTris-HCl（pH 7.9）、300 mMのNaCl、25 mMのNaEDTA、2% (w/v) SDS)、1 μlの10 mg/mlプロテイナーゼK、0.25 μlの20 mg/mlグリコーゲンおよび23.75 μlの水を加える。インキュベート後、RNAをエタノールで沈殿させ、分析を6% (w/v)ポリアクリルアミド、8.3 M尿素シーケンシングゲル上で行う。乾燥したゲルの感光を、オートラジオグラフィで、またはホスホイメージャー(phosphoimager)で行う。切断活性を判定するには、切断産物の量を、mRNA前駆体テンプレートの量と比較する。いずれかのポリペプチド成分を反応から除くことと、NAAPを代替することとは、mRNA前駆体ポリアデニル化におけるNAAPの特定の生物機能を同定する上で有用である(Ruegsegger, U. 他(1996) J. Biol. Chem. 271:6107-6113、およびその参考文献)。
【０３７９】
tRNA合成酵素活性は、[¹⁴C]−標識されたアミノ酸の存在下で基質tRNAのアミノアシル化として測定される。NAAPは緩衝液中で、[¹⁴C]−標識されたアミノ酸と好適な同族tRNA（例えば、[¹⁴C]アラニンおよび tRNA^ala）と共にインキュベートされる。¹⁴C-標識の産物は遊離[¹⁴C]アミノ酸からクロマトグラフィーによって分離され、取り込まれた[¹⁴C]はシンチレーションカウンタで測定される。¹⁴C-標識産物の量が、このアッセイのNAAPの活性に比例する。
【０３８０】
あるいはNAAP活性を測定するため、NAAP含有サンプルのインキュベートを、以下を含む溶液で行う。1 mMのATP、5 mMのHepes-KOH (pH 7.0)、2.5 mMのKCl、1.5 mM塩化マグネシウム、および0.5 mMのDTTであり、また誤ってアシル化した[¹⁴C]-GlutRNAGln (例えば1 μM)および同様の濃度の無標識Lグルタミンをも含む。反応を停止させるため3 M酢酸ナトリウム(pH 5.0)を加えた後、混液の抽出を同量の水‐飽和フェノールで行い、水相と有機相とを分離する遠心分離を15,000 × g、室温で1分間おこなう。水相の沈殿を3倍量のエタノール、-70℃で15分間おこなう。沈殿したアミノアシルtRNAを回収する遠心分離を15,000 × g、4℃で15分間おこなう。ペレットの再懸濁を25 mMのKOHで行い、脱アシル化を65℃で10分間おこない、中和を0.1 MのHClで行い(最終pH 6〜7)、真空乾燥する。乾燥ペレットの再懸濁を水中で行い、セルロースTLCプレート上にスポットする。プレートの感光を、イソプロパノール／蟻酸／水またはアンモニア／水／クロロホルム／メタノール中で行う。イメージを濃度計分析し、GluとGlnとの相対量の計算を、スポットのRf値と相対強度とに基づき行う。NAAP活性の計算を、GluがGlu-tRNA^Gln としてアシル化され転換した結果のGlnの量に基づき行う(Curnow, A.W. 他(1997) Proc. Natl. Acad. Sci. U. S. A. 94:11819-26から適用)。
【０３８１】
１８ ＮＡＡＰアゴニスト及びアンタゴニストの同定
NAAPの活性化若しくは抑制のアゴニスト若しくはアンタゴニストを、実施例１７で記述したアッセイを用いてテストし得る。アゴニストはNAAP活性の増加を引き起こし、アンタゴニストはNAAP活性の減少を引き起こす。
【０３８２】
１９ ＮＡＡＰ分泌アッセイ
或る高処理アッセイを用いて、真核細胞内に分泌されるポリペプチドを同定しうる。このようなアッセイの1例では、ポリペプチド発現ライブラリを作製するため、5'に偏向したcDNAを、或るリーダーのないβラクタマーゼ遺伝子の5'末端に融合する。βラクタマーゼは便利な遺伝子レポーターである。理由は、この酵素が高い信号／ノイズ比と、低い内因性バックグラウンド活性とを示し、他の蛋白質に融合しても活性を保持するからである。或る2重プロモーター系によって、βラクタマーゼ融合ポリペプチドの発現を、細菌又は真核細胞でなしうる。これにはlacまたはCMVプロモーターを各々用いる。
【０３８３】
ライブラリは先ず細菌(大腸菌など)に形質転換され、真核生物系で分泌されうる融合ポリペプチドをコードするライブラリメンバーが同定される。哺乳類シグナル配列は、βラクタマーゼ融合ポリペプチドが細菌のペリプラズムへ移動するよう指示する。ペリプラズムでこれはカルベニシリンへの抗生物質耐性を授ける。カルベニシリン選択された細菌の単離を固体培地で行い、個々のクローンを液体培地で成長させ、生じた培養物を用いてライブラリメンバープラスミドDNAを単離する。
【０３８４】
哺乳類細胞(293細胞など)の播種を96ウェル組織培養プレート上に、約40,000細胞／ウェルの密度で、100 μlフェノールレッドを含まないDMEに10%ウシ胎児血清(FBS) ( Life Technologies, Rockville, MD)を加えて行う。次の日に、精製プラスミドDNA(カルベニシリン耐性細菌から単離)を、15 μlのOPTI-MEM I培養液(Life Technologies)で、形質移入すべき細胞の各ウェルに25 μlの容量まで希釈する。別のプレートで、1 μlのLF2000 Reagent (Life Technologies)の希釈を25 μl/ウェルOPTI-MEM I中に行う。25 μl希釈LF2000 Reagentを次に25 μl希釈DNAと混合し、短時間混ぜ合わせ、インキュベートを20分間、室温で行う。生じたDNA-LF2000試薬複合体を次に、直接、293細胞の各ウェルに加える。細胞の形質移入はまた、適切な対照プラスミド(野生型βラクタマーゼ、リーダーのないβラクタマーゼ、又は例えばCD4融合したリーダーのないβラクタマーゼのいずれかを発現するプラスミド)で行う。形質移入の24時間後、約90 μlの細胞培地のアッセイを37^oCで、100 _MのNitrocefin (ニトロセフィン、Calbiochem, San Diego CA)及び0.5 mMのオレイン酸(Sigma, St. Louis, MO)を用い、10 mMリン酸バッファー(pH 7.0)中でおこなう。ニトロセフィンはβラクタマーゼの基質であり、加水分解すると黄色から赤へ顕著に変色する。βラクタマーゼ活性のモニターを、20分間、マイクロタイタープレートリーダーにおいて486nmで行う。486nmでの色吸収の増加が、形質移入した細胞培地でのβラクタマーゼ融合ポリペプチドの分泌に一致する。これは、融合ポリペプチドにおける真核生物シグナル配列の存在の結果である。対応するライブラリメンバープラスミドDNAのポリヌクレオチド配列分析を次に用いて、シグナル配列をコードするcDNAを同定する(記載は米国特許出願09/803,317号、ファイル日は2001年3月9日)。
【０３８５】
当業者には、本発明の要旨および精神から逸脱しない範囲での、記載した本発明の方法およびシステムの、種々の修正および変更の手段は自明であろう。本発明について説明するにあたり幾つかの実施例に関連して説明を行ったが、本発明の請求の範囲が、そのような特定の実施例に不当に制限されるべきではないことを理解されたい。分子生物学または関連分野の専門家には明らかな、本明細書に記載する本発明の実施方法の様々な修正は、明確に特許請求の範囲内にあるものとする。
【０３８６】
（表の簡単な説明）
表１は、本発明の完全長ポリヌクレオチド配列およびポリペプチド配列の命名法の概略を示す。
【０３８７】
表２は、本発明のポリペプチド群のGenBank識別番号と、最も近いGenBank相同体の注釈（annotation）と、PROTEOMEデータベース識別番号と、PROTEOMEデータベース相同体群の注釈とを示す。また、各ポリペプチドとその相同体（１つ以上）が一致する確率スコアも併せて示す。
【０３８８】
表３は、予測されるモチーフおよびドメインなど、本発明のポリヌクレオチド配列の構造的特徴を、ポリペプチドの分析に用いる方法、アルゴリズムおよび検索可能なデータベースと共に示す。
【０３８９】
表４は、本発明のポリヌクレオチド配列を構築するために用いたcDNA断片やゲノムDNA断片を、ポリヌクレオチド配列の選択した断片と共に示す。
【０３９０】
表５は、本発明のポリヌクレオチドの代表的なcDNAライブラリを示す。
【０３９１】
表６は、表５に示したcDNAライブラリの作製に用いた組織およびベクターを説明する付表である。
【０３９２】
表７は、本発明のポリヌクレオチドとポリペプチドの分析に用いたツール、プログラム、アルゴリズムを、適用可能な説明、参照文献および閾値パラメータと共に示す。
【表１】

【表２】

【表３】

【表４】

【表５】

【表６】

【表７】

【表８】

【表９】

【表１０】

【表１１】

【表１２】

【表１３】

【表１４】

【表１５】

【表１６】

【表１７】

【表１８】

【表１９】

【表２０】

【表２１】

【表２２】

【表２３】

【表２４】

【表２５】

【表２６】

【表２７】

【表２８】

【表２９】

【表３０】

【配列表】

[0001]
(Technical field)
The present invention relates to nucleic acid sequences and amino acid sequences of nucleic acid related proteins. The present invention also relates to diagnosis / treatment / prevention of cell proliferation abnormalities, neurological disorders, developmental disorders, autoimmune / inflammatory diseases, and infectious diseases using these sequences. The invention further relates to a calculation of the effect of foreign compounds on the expression of nucleic acid and amino acid sequences of nucleic acid related proteins.
[0002]
(Background of the Invention)
Multicellular organisms are composed of a variety of cell types that differ significantly in structure and function. Cell personality is determined by characteristic gene expression patterns. Different cell types express overlapping but unique genes during development. Spatial and temporal regulation of gene expression is crucial for the control of cell proliferation, cell differentiation, apoptosis, and other processes that contribute to the development of an organism. Furthermore, gene expression is regulated in response to extracellular signals. Extracellular signals mediate intercellular communication and coordinate the actions of various cell types. Appropriate gene regulation also ensures that cells function efficiently by expressing only those genes that require that function at a given time.
Transcription factor
Transcriptional regulatory proteins are essential for the control of gene expression. Some of these proteins function as transcription factors and initiate, activate, repress or terminate gene transcription. Transcription factors generally bind to a gene's promoter, enhancer, or upstream regulatory region in a sequence-specific manner (although some factors bind to regulatory elements within or downstream of the gene coding region). Transcription factors bind to specific regions of DNA alone or as a complex with other accessory factors (reviewed Lewin, B. (1990)Genes IV, Oxford University Press, New York, NY, and Cell Press, Cambridge, MA, page 554570).
[0003]
The double helix structure and repeat sequences of DNA produce topological and chemical features that can be recognized by transcription factors. These features include hydrogen bond donor and acceptor groups, hydrophobic patches, major and minor grooves, and repeated stretches of sequences (derived to a unique group of bends in the helix). is there. Usually, a group of DNA sequence motifs specifically recognized by a transcription factor is about 20 nucleotides in length. Multiple adjacent transcription factor binding motifs may be required for gene regulation.
[0004]
Many transcription factors incorporate DNA-binding structural motifs. These motifs consist of α helices or β sheets that bind to the major groove of DNA. Four well-characterized motifs are the helix turn helix, Zn finger, leucine zipper, and helix loop helix. Proteins having these motifs act as monomers alone or interact with DNA by forming homodimers or heterodimers.
[0005]
The helix-turn-helix motif consists of two α-helices that are connected at a fixed angle by a short chain of amino acids. One helix joins the main groove. One example of a helix-turn-helix motif is a homeobox motif, which is in a homeodomain protein. Homeodomain proteins are important in determining the anterior-posterior body axis during development and are conserved throughout the animal kingdom. Drosophila melanogaster Antennapedia and Ultrabithorax proteins are prototypical homeodomain proteins (Pabo, C.O. and R.T. Sauer (1992) Ann. Rev. Biochem. 61: 1053-1095).
[0006]
Mouse HES6 is a member of the basic helix-loop-helix transcription factor of the Hairy / Enhancer-of-split (HES) family. The HES gene acts as a nuclear effector of Notch signaling and regulates the transcriptional activity of several Notch target genes. HES6 is expressed in all neurogenic placodes, its derivatives, and the brain, and is patterned at these sites along the anteroposterior and dorsoventral axes. HES6 is also expressed in embryonic tissue, where Notch signaling controls cell fate decisions. Cell fate includes, for example, trunk, dorsal root ganglion, myotomes, and thymus. In limb buds, HES6 is expressed in skeletal muscles and tendons of expected fate. HES6 is also expressed in embryonic respiratory, urinary, and digestive epithelial cells (Vasiliauskas, D. and Stern CD (2000) Mech. Dev. 98: 133137; Pissarra, L. et al. (2000) Mech Dev 95: 275278).
[0007]
The Zn finger motif binds to zinc ions. This motif generally has a tandem repeat group of about 30 amino acids consisting of cysteine and histidine residues arranged periodically. Two examples of this sequence pattern, C2H2 and C3HC4 ("RING" fingers) have already been described (Lewin, supra). Each of the Zn finger proteins has one antiparallel β sheet and one α helix, and their proximity and conformation are maintained by zinc ions. Contact with DNA is made by the arginine residue in front of the α-helix and by the second, third, and sixth residues of the α-helix. Examples of variants of Zn finger motifs include the less well-understood cysteine-rich motifs, which bind to zinc or other metal ions. These mutant motifs may have no histidine residues and are generally non-repetitive. The Zn finger motif can be repeated in tandem within the protein so that the alpha helix of each Zn finger of the protein can contact the main groove of the DNA double helix. This repeated contact between protein and DNA results in a strong and specific DNA-protein interaction. The strength and specificity of this interaction can be regulated by the number of Zn finger motifs in the protein. Zn fingers were first identified in the DNA-binding protein group as regions that interact directly with DNA, but they also appear in various proteins that do not bind to DNA (Lodish, H. et al. (1995)Molecular Cell Biology, Scientific American Books, New York NY, 447-451). For example, Galcheva-Gargova, Z. et al. ((1996) Science 272: 1797-1802) have identified a group of Zn finger proteins that interact with various cytokine receptors.
[0008]
The C2H2 type Zn finger signature motif has a sequence of 28 amino acids, which contains two conserved Cys residues and two conserved His residues, one C2C12H3H type motif Contained within. C2H2-type motifs generally appear within multiple tandem repeats. One cysteine-rich domain with the motif Asp-His-His-Cys (DHHC-CRD) has been identified as an independent subgroup of Zn finger proteins. The DHHC-CRD region is thought to be involved in growth and development. Certain DHHC-CRD mutants show a loss of Ras function. Ras is a small membrane-bound GTP-binding protein that regulates cell growth and differentiation. On the other hand, other DHHC-CRD proteins probably function in pathways unrelated to Ras (Bartels, D.J. et al. (1999) Mol. Cell Biol. 19: 6775-6787).
[0009]
Zn finger transcription factors are often accompanied by modular sequence motifs. Examples of this motif are the Kruppel associated box (KRAB) and the SCAN domain. For example, the hypoalphalipoproteinemia susceptibility gene ZNF202 encodes a SCAN box and a KRAB domain (followed by 8 C2H2 Zn finger motifs) (Honer, C. et al. (2001) Biochim Biophys. Acta 1517: 441448). The SCAN domain is a highly conserved leucine-rich motif, consisting of about 60 amino acids and found at the amino terminus of the Zn finger transcription factor. SCAN domains are most often linked to C2H2 Zn finger motifs, and this linkage is made through their carboxyl terminus. Biochemical binding studies have identified the SCAN domain as a selective hetero- and homotypic oligomerization domain. Protein complexes mediated by SCAN domains appear to function to modulate the biological function of transcription factors (Schumacher, C. et al. (2000) J. Biol. Chem. 275: 17173-17179).
[0010]
The KRAB (Kruppel-associated box) domain is a conserved amino acid sequence spanning about 75 amino acids. The KRAB domain is found in almost one-third of the 300-700 genes that encode C2H2 Zn fingers. The KRAB domain is found N-terminal to the finger repeat. A KRAB domain is generally encoded by two exons. The KRAB-A region or box is encoded by one exon and the KRAB-B region or box is encoded by a second exon. The function of the KRAB domain is to repress transcription. To achieve transcriptional repression, either KRAB-associated protein-1 (a type of transcriptional corepressor) or KRAB-A interacting protein is recruited. Proteins with a KRAB domain appear to play a regulatory role during development (Williams, A.J. et al. (1999) Mol. Cell Biol. 19: 8526-8535). Although highly related human KRAB Zn finger proteins can be detected in all human tissues, certain subgroups are highly expressed on human T lymphocytes (Bellefroid, EJ et al. (1993) EMBO J. 12: 13631374). The ZNF85 KRAB Zn finger gene is a member of the human ZNF91 family and is highly expressed in normal adult testis, seminoma, and NT2 / D1 teratocarcinoma cell lines (Poncelet, DA et al. (1998) DNA Cell Biol. 17: 931943).
[0011]
Kruppel protein regulates Drosophila segmentation. There are about 300 genes encoding such proteins throughout the human genome. In fact, over 100 separate mRNAs (most of which are novel) encoding Kruppel multifinger proteins have been found in the human placenta. The sequence group of 106 finger repeat group in 9 kinds of these proteins is highly homologous. There are several positions within the α-helical structure that exhibit variability. Studies show that this variability affects the DNA binding specificity of these proteins (Bellefroid, E.J. et al. (1989) DNA 8: 377387).
[0012]
ZNF143 is a GLI type of human Zn finger Kruppel family protein. ZNF143 is 84% identical to Xenopus selenocysteine tRNA gene transcription activator (Staf). Staf appears to be involved in enhancing transcription of small nuclear RNA (snRNA) and snRNA type genes by RNA polymerase II (Pol II) and III (Pol III). Staf also has the ability to stimulate expression from the Pol II mRNA promoter. ZNF143 is located in chromosomal regions 7q11.2, 7q21.3-q22.1, and 11p15.3-p15.4, along with related ZNF138 and ZNF139. These regions are involved in deletions and / or translocations associated with Williams syndrome, split hand and foot disease (SHFD1), and BeckwithWiedemann syndrome, respectively, because the ZNF143 gene is a developmental and malignant disease To be involved in ZNF143 mRNA is expressed in many normal adult tissues. Examples of expression tissues include leukocytes, colon, small intestine, ovary, testis, prostate, thymus, and spleen tissues. In addition, the Ccta gene encoding the mouse chaperone is regulated by ZNF143 and another Staf family Zn finger transcription factor, ZNF76, which is active during the cell growth phase when these RNA and chaperone genes are co-regulated. (Tommerup, N. and Vissing, H. (1995) Genomics 27: 259264; Myslinski, E. et al. (1998) J. Biol. Chem. 273: 219982006; Kubota, H. et al. (2000) J. Biol. Chem. 275: 2864128648).
[0013]
The C4 motif is found in hormone-regulated proteins. C4 motifs generally have only two repeats. Many eukaryotic and viral proteins have certain conserved cysteine-rich domains, which consist of 40-60 residues and are called C3HC4 Zn fingers or RING fingers. This domain binds to two zinc atoms. It is also probably involved in mediating protein-protein interactions. The three-dimensional “crossbrace” structure of the zinc ligation system is unique to the RING domain. The spacing between cysteine groups within such domains is Cx (2) Cx (9-39) Cx (1-3) Hx (2-3) Cx (2) Cx (4-48) Cx (2) C is there. PHD fingers are some C4HC3 Zn finger-like motifs found in nuclear proteins that appear to be involved in chromatin-mediated transcriptional regulation.
[0014]
A GATA-type transcription factor has one or two Zn finger domains that specifically bind to a region of DNA having a contiguous nucleotide sequence, GATA. NMR studies show that this Zn finger has two irregular antiparallel β sheets and one α helix, followed by a long loop to the C-terminus of the finger. (Ominchinski, J.G. (1993) Science 261: 438446). The helix and the loop connecting the two β sheets contact the main groove of DNA, while the C-terminal part, which is the site that determines the binding characteristics, is wound into the minor groove and enters.
[0015]
The LIM motif has about 60 amino acid residues and contains 7 conserved cysteine residues and 1 histidine within a consensus sequence (Schmeichel, KL and Beckerle, MC (1994) Cell 79: 211 -219). The LIM family includes transcription factors and cytoskeletal proteins that can be involved in development, differentiation, and cell growth. An example actin-binding LIM protein may play a role in the regulation of cytoskeleton and cell morphogenesis (Roof, D.J. et al. (1997) J. Cell Biol. 138: 575-588). In the N-terminal domain of the actin-binding LIM protein, there are four double Zn finger motif groups and have a LIM consensus sequence. The C-terminal domain of the actin-binding LIM protein shows sequence similarity to known actin-binding proteins (eg, dematin and villin). Actin-binding LIM protein binds to F-actin. This binding takes place through the dematin-like C-terminal domain. The LIM domain appears to mediate protein-protein interactions with other LIM binding proteins.
[0016]
Myeloid cell development is controlled by a group of tissue-specific transcription factors. Myeloid Zn finger proteins (MZF) include MZF-1 and MZF-2. MZF-1 functions in the regulation of neutrophilic granulocyte development. Certain murine homologues MZF2 are expressed on myeloid cell populations, particularly cells committed to the neutrophil lineage. MZF-2 is downregulated by GCSF. It also appears to have a unique function in neutrophil development (Murai, K. et al. (1997) Genes Cells 2: 581591).
[0017]
The leucine zipper motif consists of a stretch of amino acids rich in leucine, which can form an amphipathic α-helix. This structure provides the basis for dimerization of two leucine zipper proteins. The region adjacent to the leucine zipper is usually basic, and protein dimerization results in an optimal arrangement for binding to the main groove. Proteins having such motifs are collectively referred to as bZIP transcription factors. Leucine zipper motifs are found in the proto-oncogenes Fos and Jun. Fos and Jun have AP1, a heterodimeric transcription factor. AP1 is involved in cell growth and cell lineage determination (Papavassiliou, A.G. (1995) N. Engl. J. Med. 332: 45-47).
[0018]
The mouse kreisler (kr) mutation results in segmentation abnormalities in the caudal hindbrain and defects in inner ear development. The kr cDNA encodes a basic domain-leucine zipper (bZIP) transcription factor. In this transcription factor, serine is substituted for one asparagine residue conserved within the DNA binding domain of all known bZIP family members. The identity, expression, and mutational phenotype of kr indicate the initial role in axial patterning, as well as the molecular and developmental mechanisms that govern hindbrain and ear development. (Cordes, SP and Barsh, GS (1994) Cell 79: 10251034).
[0019]
A helix-loop-helix motif (HLH) consists of one short alpha helix and a long alpha helix connected by a loop. Since this loop is flexible, the two helices can bend relative to each other and bind to DNA. Myc, a transcription factor, has a prototypical HLH motif.
[0020]
The NFκB / Rel signature defines a family of eukaryotic transcription factors. This transcription factor is involved in carcinogenesis, embryonic development, differentiation, and immune responses. Most transcription factors with a Rel homology domain (RHD) bind as a dimer to κB, a consensus DNA sequence motif. Rel family members share a highly conserved 300 amino acid domain, the Rel homology domain. The characteristic Rel C-terminal domain is involved in gene activation and cytoplasmic anchoring functions. Examples of proteins known to have RHD domains include vertebrate nuclear factor NFκB (a heterodimer of a DNA binding subunit and transcription factor p65), mammalian transcription factor RelB, and vertebrate proto-oncogene crel Crel is a protein involved in differentiation and lymphogenesis (Kabrun, N., and Enrietto, PJ (1994) Semin. Cancer Biol. 5: 103112).
[0021]
One DNA binding motif, ARID (AT-rich interaction domain), characterizes an evolutionarily conserved family of proteins. The approximately 100 residue ARID sequence is present in a series of proteins that are strongly implicated in the regulation of cell growth, differentiation, and tissue-specific gene expression. Examples of ARID proteins include Bright (a regulator of B cell-specific gene expression), dead ringer (involved in development), and MRF2 (suppresses expression from cytomegalovirus enhancers). (Dallas, P.B. et al. (2000) Mol. Cell Biol. 20: 31373146).
[0022]
The ELM2 (Egl27 and MTA1 homology 2) domain is found in the tumor metastasis-related protein MTA1 and the protein ER1. Nematode(Caenorhabditis elegans) Gene egl 27 is required for embryonic pattern formation. The human MTA1 gene is upregulated in metastatic cancers, a component of a protein complex with histone deacetylase activity and nucleosome remodeling activity (Solari, F. et al. (1999) Development 126: 24832494). The ELM2 domain is usually found at the N-terminus of the myb-like DNA binding domain. ELM2 is also seen to be associated with certain ARID DNA.
[0023]
Most transcription factors have characteristic DNA-binding motifs, and various variants of these motifs and novel motifs have been characterized so far (Faisst, S. and S Meyer (1992) Nucl. Acids Res. 20: 3-26).
Chromatin binding protein
Within the nucleus, DNA is packed into chromatin. Chromatin is a compact tissue that restricts the accessibility of DNA to transcription factors and plays a major role in gene regulation (Lewin, supra, pages 409-410). The compact structure of chromatin is determined and influenced by chromatin binding proteins. Such proteins include histones, high mobility group (HMG) proteins, and chromodomain proteins. There are five classes of histones, H1, H2A, H2B, H3 and H4, all of which are highly basic low molecular weight proteins. Nucleosomes, which are the basic units of chromatin, have 200 base pairs of DNA and two copies of H2A, H2B, H3, and H4. H1 links adjacent nucleosome groups. HMG protein is a low molecular weight non-histone protein that appears to play a role in unwinding DNA and stabilizing single-stranded DNA. Chromodomain proteins play an important role in the formation of highly compacted heterochromatin, which is transcriptionally silent (inactive).
Diseases and disorders related to gene regulation
Many tumor diseases in humans can be caused by inappropriate gene expression. Malignant cell growth can be caused by overexpression of tumor promoting genes or insufficient expression of tumor suppressor genes (Cleary, M. L. (1992) Cancer Surv. 15: 89-104). The zinc finger transcriptional regulator WT1 is a tumor suppressor protein that is inactivated in children with Wilms tumor. Oncogene bcl-6, which plays an important role in large cell lymphoma, is also a Zn finger protein (Papavassiliou, A.G. (1995) N. Engl. J. Med. 332: 45-47). Chromosomal translocations can also generate a chimeric loci that fuses the coding sequence of one gene with the regulatory region of another unrelated gene. Such an organization probably causes inappropriate gene transcription and can also lead to malignant tumors. For example, in Burkitt lymphoma, the transcription factor Myc is translocated to the immunoglobulin heavy chain locus, greatly enhancing Myc expression, resulting in rapid cell growth resulting in leukemia (Latchman, DS (1996) N. Engl. J. Med. 334: 28-33).
[0024]
In addition, the immune system responds to infection and trauma by activating a cascade of events that regulate the gradual selection, amplification, and recruitment of cellular defense mechanisms. A complex and balanced program of gene activation and repression is involved in this process. However, excessive activation of the immune system caused by inappropriate or insufficient regulation of gene expression can cause considerable damage to tissues and organs. This damage is well documented in the literature on immune responses associated with arthritis, allergens, heart attacks, strokes, and infections (Isselbacher et al.Harrison's Principles of Internal Medicine, 13 / e, McGraw Hill, Inc. and Teton Data Systems Software, 1996). The causative gene for autoimmune multiglandular endocrine insufficiency candidiasis ectodermal dystrophy (APECED) has recently been isolated and found to encode a protein with two PHD-type Zn finger motifs (Bjorses , P. et al. (1998) Hum. Mol. Genet. 7: 1547-1553).
[0025]
Furthermore, the production of multicellular organisms is based on the induction and regulation of cell differentiation at the appropriate stage of development. Central to this process is differential gene expression that confers uniqueness to cells and tissues throughout the body. Failure to regulate gene expression during development can lead to developmental disabilities. Human developmental disorders resulting from mutations in Zn finger transcriptional regulators include: Urogenital abnormalities (related to WT1); Greig craniopolysyndactyly, Pallister-Hall syndrome, and postaxial polydactyly type A (related to GLI3); and anus Townes-Brocks syndrome (SALL1-related), characterized by abnormalities in the kidneys, limbs and ears (Engelkamp, D. and van Heyningen, V. (1996) Curr. Opin. Genet. Dev. 6: 334-342; Kohlhase, J. et al. (1999) Am. J. Hum. Genet. 64: 435-445).
[0026]
Nucleic acid synthesis
Polymerase
DNA and RNA replication is a vital process for cell replication and function. The enzymes that mediate the replication of DNA and RNA are DNA polymerase and RNA polymerase, respectively, and this mediation is performed by a “templating” process. In this process, the nucleotide sequence of a DNA or RNA strand is copied to a complementary nucleic acid sequence of either DNA or RNA by complementary base pairing. However, there are fundamental differences between the two processes.
[0027]
The reaction catalyzed by DNA polymerase is the sequential addition of deoxyribonucleotides to the 3′-OH end of a polynucleotide strand (primer strand), which is paired with a second (template) strand. New DNA strands therefore grow in the 5 'to 3' direction (Alberts, B. et al. (1994)The Molecular Biology of the Cell, Garland Publishing Inc., New York, NY, pages 251-254). The substrate for this polymerization reaction is the corresponding deoxynucleotide triphosphate, which needs to base pair with the correct nucleotide on the template strand in order to be recognized by the polymerase. Since DNA exists as a double helix, each of the duplexes can serve as a template for the formation of new complementary strands. Thus, each of the two daughter cells of a dividing cell inherits a new DNA double helix, one old and one new strand. Thus, DNA is said to be “semi-conservatively” replicated by DNA polymerase. In addition to the synthesis of new DNA, DNA polymerase is also involved in repairing damaged DNA. This is the following ``Ligase"
[0028]
In contrast to DNA polymerase, RNA polymerase "transcribes" DNA into RNA using a single DNA template strand, which uses ribonucleotide triphosphate as a substrate. Like DNA polymerization, RNA polymerization proceeds in the 5 'to 3' direction by the addition of ribonucleoside monophosphate to the 3'-OH end of the growing RNA strand. DNA transcription produces messenger RNA (mRNA), which carries information for protein synthesis. DNA transcription also produces transfer RNA, ribosomal RNA, and other RNAs with structural or catalytic functions. In eukaryotic cells, three separate RNA polymerases synthesize three types of RNA (Alberts et al., Supra, pages 367-368). RNA polymerase I makes large ribosomal RNA, RNA polymerase II makes mRNA that is translated into protein, and RNA polymerase III makes various small stable RNAs (such as 5S ribosomal RNA and transfer RNA (tRNA)). In all cases, RNA synthesis is initiated by the binding of RNA polymerase to a promoter region on the DNA, which begins at the start site within the promoter. Completion of synthesis is accomplished by a stop (termination) signal in the DNA where the polymerase and the completed RNA strand are released.
Ligase
Through the process of DNA repair, accidental base changes (such as oxidative damage, hydrolytic attack, and random methylation of DNA) are corrected before DNA replication or transcription occurs. Due to the efficiency of the DNA repair process, only less than a thousandth of an accidental base change will mutate (Alberts et al., Supra, pages 245-249). Three steps common to most types of DNA repair are: (1) excision of damaged or altered bases or nucleotides by DNA nucleases. (2) Inserting the correct nucleotide into the gap left by the excised nucleotide. This is done by DNA polymerase using the complementary strand as a template. (3) Sealing with DNA ligase of the break remaining between the inserted nucleotide (s) and the existing DNA strand. In the last reaction, DNA ligase uses energy from ATP hydrolysis to activate the 5 'end of the nicked phosphodiester bond and then form a new bond with the 3'-OH of the DNA strand. Bloom syndrome is an inherited human disease, but this patient has an increased incidence of cancer due to a partial deficiency of this DNA ligation (Alberts et al., P. 247).
Nuclease
Nuclease enzymes include DNases that hydrolyze DNA and RNases that hydrolyze RNA. The two nucleases serve different purposes in nucleic acid metabolism. Nucleases hydrolyze phosphodiester bonds between adjacent nucleotides. Endonucleases hydrolyze at internal positions, and exonucleases hydrolyze at the 3 ′ or 5 ′ terminal nucleotide positions. For example, certain DNA exonuclease activities in DNA polymerases work to remove improperly paired nucleotides attached to the 3'-OH ends of DNA strands grown by polymerases I will provide a. As mentioned above, DNA endonuclease activity is involved in the excision step of the DNA repair process.
[0029]
RNases also provide various functions. For example, RNase P is a ribonucleoprotein enzyme that cleaves the 5 ′ end of tRNA precursors as part of their maturation process. RNase H digests RNA strands of certain RNA / DNA hybrids. Such hybrids occur in cells invaded by retroviruses, and RNase H is an important enzyme in the retroviral replication cycle. Pancreatic RNase is secreted into the intestine by the pancreas and hydrolyzes RNA in the ingested food. Increased RNase activity in serum and cell extracts is seen in various cancers and infectious diseases (Schein, C.H. (1997) Nat. Biotechnol. 15: 529-536). Modulation of RNase activity has been studied as a means of controlling tumor angiogenesis, allergic reactions, viral infection and replication, and fungal infection.
[0030]
Nucleic acid modification
Methylase
Methylation of specific nucleotides occurs in both DNA and RNA and provides separate functions in the two macromolecules. Methylation of cytosine residues in DNA forms 5-methylcytosine, which occurs specifically within CG sequences that are base-paired to each other within the DNA duplex. This pattern of methylation is passed between generations during DNA replication. Enzymes that do this are called "maintenance methylases" and act preferentially on CG sequences that are base paired with already methylated CG sequences. Such methylation distinguishes between active and inactive genes by preventing the binding of regulatory proteins that “turn on” the gene while allowing the binding of proteins that inactivate the gene. (Alberts et al., Pp. 448-451). In RNA metabolism, “tRNA methylase” causes one of several nucleotide modifications in tRNA. This modification affects the conformation and base pairing of this molecule and promotes recognition of the appropriate mRNA codons by specific tRNAs. The main methylation pattern is dimethylation of guanine residues, which form N, N-dimethylguanine.
Helicases and single-stranded binding proteins
The helicase enzyme destabilizes and unwinds the double helix structure of both DNA and RNA. Since DNA replication occurs on two strands almost simultaneously, the two strands must first be separated to produce a replica “fork” so that the DNA polymerase can act. Two replication proteins contribute to this process. DNA helicase and single-stranded binding protein. DNA helicase hydrolyzes ATP and uses its energy to separate DNA strands. Single-stranded binding proteins (SSB) then bind to the exposed DNA strands, but do not cover the bases and temporarily stabilize them for template removal by DNA polymerase (Alberts et al., Supra). 255-256 pages).
[0031]
RNA helicase transforms and regulates RNA conformation and secondary structure. Like DNA helicases, RNA helicases utilize energy from ATP hydrolysis to destabilize and unwind RNA duplexes. The best characterized and ubiquitous family of RNA helicases is the DEAD box family, whose name is derived from the conserved B-type ATP binding motif. This motif is characteristic for this family of proteins. Over 40 DEAD box helicases have been identified in diverse organisms ranging from bacteria, insects, yeasts, amphibians, mammals, and plants. DEAD box helicases function in a variety of processes. The processes include, for example, translation initiation, splicing, ribosome assembly, and RNA editing, transport, and stability. Examples of these RNA helicases include yeast Drs1 protein (involved in ribosomal RNA processing), yeast TIF1 and TIF2 and mammalian eIF-4A (which are essential for initiation of RNA translation), and human p68 antigen (cell growth) (Ripmaster, TL et al. (1992) Proc. Natl. Acad. Sci. USA 89: 11131-11135; Chang, T.-H. et al. (1990) Proc. Natl. Acad. Sci. USA 87: 1571-1575). These RNA helicases exhibit strong sequence homology over a stretch of about 420 amino acids. These conserved sequences include the consensus sequence of the A motif of a certain ATP-binding protein, the “DEAD box” sequence (involved in ATPase activity), the sequence SAT (involved in the actual helicase unwinding region) And there is an octapeptide consensus sequence required for RNA binding and ATP hydrolysis (Pause, A. et al. (1993) Mol. Cell Biol. 13: 6789-6798). Differences outside these conserved regions appear to reflect differences in the functional role of individual proteins (Chang, T.H. et al. (1990) Proc. Natl. Acad. Sci. USA 87: 1571-1575).
[0032]
Several DEAD box helicases play a tissue-specific and stage-specific role in spermatogenesis and embryogenesis. Overexpression of DEAD box 1 protein (DDX1) appears to play a role in the progression of neuroblastoma (Nb) and retinoblastoma (Rb) (Godbout, R. et al. (1998) J. Biol. Chem. 273: 21161-21168). These observations suggest that DDX1 promotes or enhances tumor progression by altering the normal secondary structure and expression level of RNA in cancer cells. Another group of DEAD box helicases appears to be directly or indirectly involved in tumorigenesis (discussed above in Godbout). For example, murine p68 is mutated in UV-induced tumors, and human DDX6 is located at a certain chromosomal breakpoint associated with B-cell lymphoma. Similarly, a chimeric protein consisting of DDX10 and NUP98 (one nuclear pore protein: nucleoporin) appears to be involved in the pathogenesis of several myeloid malignancies.
Topoisomerase
In addition to separating the DNA strands prior to replication, the two strands need to be “unwinded” from each other prior to separation by the DNA helicase. Proteins that perform this function are known as DNA topoisomerases. DNA topoisomerase effectively acts as a reversible nuclease to hydrolyze phosphodiesterase bonds in the DNA strand, allowing the duplex to freely rotate with respect to each other, and then unwinding the helix, Recombine the original phosphodiester bond in between. Topoisomerase is an essential enzyme responsible for the topological rearrangement of DNA produced by transcription, replication, chromatin formation, recombination, and chromosome segregation. Introduction of higher-order helical coils into the DNA can be done by passage of a processive enzyme (eg RNA polymerase) or by separation before replication of the DNA strand by helicase. During the process of DNA synthesis, storage and repair, knotting and concatenation can occur. All topoisomerase functions by breaking the phosphodiester bond in the ribose-phosphate backbone of DNA. Certain catalytic tyrosine residues in this enzyme produce reaction intermediates by nucleophilic attack on the fragile phosphodiester bond. In this intermediate, a covalent bond is formed between the enzyme and one end of the broken strand. Certain tyrosine DNA phosphodiesterases function in DNA repair by hydrolyzing this bond in the incidental dead endopoisomerase I-DNA intermediate (Pouliot, J. J. et al. (1999) Science 286: 552-555).
[0033]
There are two types of DNA topoisomerases, type I and type II. Type I topoisomerase acts as a monomer and cuts into single strands of DNA. On the other hand, type II topoisomerase acts as a homodimer and cleaves both strands. DNA topoisomerase I allows a double strand of helix to rotate about the remaining phosphodiester bond in the opposite strand by creating a single strand break in the DNA helix. DNA topoisomerase II makes temporary breaks in both strands of the DNA helix, where two double helices cross each other. This topoisomerase II can effectively separate two linked circular DNAs (Alberts et al., Supra, pages 260-262). Type II topoisomerase is almost restricted to proliferating cells (such as cancer cells) in eukaryotes. For this reason, topoisomerase II is a target for anticancer agents. Topoisomerase II is believed to be involved in multidrug resistance (MDR). The reason is that this enzyme may help repair DNA damage by DNA binding agents (such as doxorubicin or vincristine).
[0034]
The topoisomerase I family includes topoisomerase I (TopoI) and topoisomerase III (TopoIII). As the crystal structure of human topoisomerase I suggests, this enzyme partially controls the rotation about the intact DNA strand. In this “controlled rotation” model, protein-DNA interactions limit this rotation. This is driven by a torsional strain in the DNA (Stewart, L. et al. (1998) Science 379: 1534-1541). Structurally, TopoI can be recognized by a catalytic tyrosine residue in its active site region and a number of other conserved residues. TopoI appears to function during transcription. Two types of TopoIII are known in humans, which are homologous to prokaryotic topoisomerase I and have a conserved tyrosine and an active site signature specific to this family. TopoIII has been suggested to play a role in meiotic recombination. Certain TopoIII in mice are highly expressed in testis tissue, and the increase in expression is accompanied by an increase in the number of cells in the pachytene (Seki, T. et al. (1998) J. Biol. Chem. 273 : 28553-28556).
[0035]
The topoisomerase II family includes two isoenzymes (IIα and IIβ), which are encoded by separate genes. TopoII preferentially cleaves double-stranded DNA in a reproducible, non-random manner, preferentially in certain AT-rich regions, but the principle of cleavage site selectivity is unknown. Structurally, TopoII has four domains. The first two are structurally similar and probably have a distant homology with the similar domain of eukaryotic TopoI. The second domain has catalytic tyrosine and a highly conserved pentapeptide. The IIα isoform appears to be involved in unlinking DNA during chromosome segregation. Cell lines that express IIα but not IIβ suggest that IIβ is non-essential for cellular processes, but IIβ knockout mice died in the perinatal period due to a failure in neurogenesis. The fact that the main abnormality occurred mainly at late developmental events (neurogenesis) suggests that IIβ is required for DNA repair rather than mitosis (Yang, X. et al. (2000) Science 287: 131- 134).
[0036]
Topoisomerase is believed to be involved in a number of pathologies, and topoisomerase poisons have been shown to be effective antitumor agents for several human malignant cancers. TopoI is localized in the wrong place in Fanconi's anemia. It is also believed to be involved in chromosome breaks seen in this disease (Wunder, E. (1984) Hum. Genet. 68: 276281). Abbreviated TopoIII overexpression in telangiectasia ataxia (A-T) cells partially suppresses the A-T phenotype, probably due to a dominant negative mechanism. This suggests that TopoIII is deregulated in A-T (Fritz, E. et al. (1997) Proc. Natl. Acad. Sci. USA 94: 4538-4542). TopoIII also interacts with the Bloom's Syndrome gene product. It has also been suggested to have a role as a tumor suppressor (Wu, L. et al. (2000) J. Biol. Chem. 275: 9636-9644). Abnormal TopoII activity is often associated with cancer and increased cancer risk. Significantly reduced TopoII activity has been seen in some but not all A-T cell lines (Mohamed, R. et al. (1987) Biochem. Biophys. Res. Commun. 149: 233238). On the other hand, TopoII can perform DNA fragmentation in the region of the A-T gene (ATM). ATM controls all DNA damage responsive cell cycle checkpoints (Kaufmann, W.K. (1998) Proc. Soc. Exp. Biol. Med. 217: 327-334). The ability of topoisomerase to disrupt DNA has been used as a basis for antitumor drugs. Topoisomerase poisons act by increasing the intracellular number of dead-end covalent DNA-enzyme complexes, ultimately triggering the cell death pathway (Fortune, JM and N. Osheroff (2000) Prog. Nucleic Acid Res. Mol. Biol. 64: 221-253; Guichard, SM and MK Danks (1999) Curr. Opin. Oncol. 11: 482-489). Antibodies against TopoI are found in the sera of patients with systemic sclerosis, and the levels of this antibody are likely to be used as lung markers involved in this disease (Diot, E. et al. (1999) Chest 116: 715-720). Finally, the human TopoI DNA binding domain has been used as a DNA delivery vehicle for gene therapy (Chen, T.Y. et al. (2000) Appl. Microbiol. Biotechnol. 53: 558-567).
Recombinase
Genetic recombination is the process of rearrangement of DNA sequences within the genome of an organism, providing genetic variation of that organism in response to environmental changes. DNA recombination allows mutations in specific combinations of genes in an individual's genome, and can vary in the timing and level of expression of these genes (see Alberts et al., Pages 263-273, supra). See). Two broad classes of genetic recombination (universal recombination and site-specific recombination) are widely recognized. Universal recombination involves gene exchange between any pair of homologous DNA sequences. The position of these sequences is usually on two copies of the same chromosome. Recombinase, an enzyme that aids in this process, can be "nicked" somewhat randomly in one strand of a DNA duplex and exchanged for a complementary strand in the other duplex To. This process usually does not change the organization of genes in the chromosome. In site-specific recombination, the recombinase recognizes a specific nucleotide sequence in one or both of the recombining molecules. Since base pairing is not involved in this form of recombination, DNA homology between recombining molecules is not necessary. Unlike universal recombination, site-specific recombination can alter the relative position of nucleotide sequences within a chromosome.
RNA metabolism
Ribonucleic acid (RNA) is a linear single-stranded polymer (consisting of four nucleotides, ATP, CTP, UTP, and GTP). In most organisms, transcription of RNA is done as a copy of deoxyribonucleic acid (DNA), and DNA is the organism's genetic material. In retroviruses, RNA works as genetic material instead of DNA. RNA copies of genetic material either encode proteins or play various structural, catalytic, or regulatory roles within an organism. The classification of RNA is based on its intracellular localization and function. Messenger RNA (mRNA) encodes a polypeptide. Ribosomal RNA (rRNA) is combined with ribosomal proteins to form ribosomes. Ribosomes are cytoplasmic particles that translate mRNA into polypeptides. Transfer RNA (tRNA) is a cytosolic adapter molecule, and its function in mRNA translation is recognition of both mRNA codons and amino acids that match the codons. Heteronuclear RNA (hnRNA) includes mRNA precursors and other various sizes of nuclear RNA. Nuclear small RNA (snRNA) is part of the nuclear spliceosome complex. This complex removes non-coding sequences (introns) intervening in the mRNA precursor and recombines exons.
[0037]
Proteins associate with RNA during transcription of RNA from DNA, RNA processing, and translation of mRNA into protein. Proteins also associate with RNA when it is utilized for structural, catalytic, or regulatory purposes.
RNA processing
Ribosomal RNA (rRNA) is combined with ribosomal proteins to form ribosomes. Ribosomes are cytoplasmic particles that translate messenger RNA (mRNA) into a polypeptide. Eukaryotic ribosomes are composed of 60S (large) and 40S (small) subunits, which together form an 80S ribosome. In addition to 18S, 28S, 5S, and 5.8S rRNA, ribosomes contain more than 50 to 80 separate ribosomal proteins, depending on the type of organism. The classification of ribosomal proteins depends on the subunit to which they belong (ie L if associated with a large 60S subunit, S if a small 40S subunit). The Escherichia coli ribosome has been studied the most in detail and has 50 proteins, many of which are conserved in all living organisms. Elucidation of the structure of nine ribosomal proteins has been made with a resolution of less than 3.0D (ie S5, S6, S17, L1, L6, L9, L12, L14, L30), and common motif groups (for example ba-b proteins) Fold group) and acidic and basic RNA-binding motif groups located between b-strands. Most ribosomal proteins appear to be in direct contact with rRNA (reviewed in Liljas, A. and Garber, M. (1995) Curr. Opin. Struct. Biol. 5: 721727; also Woodson, SA and Leontis, NB ( 1998) Curr. Opin. Struct. Biol. 8: 294-300; see Ramakrishnan, V. and White, SW (1998) Trends Biochem. Sci. 23: 208-212).
[0038]
A variety of proteins are required for the processing of RNA transcribed in the nucleus. Pre-stages of mRNA processing include capping with methylguanosine to the 5 ′ end, polyadenylation of the 3 ′ end, and splicing to remove introns. The spliceosome complex consists of five small nuclear ribonucleoprotein particles (snRNP), U1, U2, U4, U5, and U6. Each snRNP contains one species of snRNA and about 10 proteins. Several snRNP RNA components recognize and base pair intron consensus sequences. The protein component mediates spliceosome assembly and splicing reactions. Antibodies to the snRNP protein are found in the blood of patients with systemic lupus erythematosus (Stryer, L. (1995)Biochemistry W.H. Freeman and Company, New York NY, p. 863).
[0039]
The role of heterogeneous nuclear ribonucleoprotein (hnRNP) has been identified in splicing, transport of mature RNA into the cytoplasm, and mRNA translation (Biamonti, G. et al. (1998) Clin. Exp. Rheumatol. 16: 317-326). Examples of hnRNP are the yeast proteins Hrp1p (involved in RNA cleavage and polyadenylation at the 3 ′ end), Cbp80p (involved in capping the 5 ′ end of RNA), and Npl3p (mammalian hnRNP A1 Homologue and involved in nuclear export of mRNA) (Shen, EC et al. (1998) Genes Dev. 12: 679-691). hnRNP has been shown to be an important target of autoimmune responses in rheumatic diseases (supra Biamonti).
[0040]
Many snRNP and hnRNP proteins are characterized by certain RNA recognition motifs (RRM) (reviewed by Birney, E. et al. (1993) Nucleic Acids Res. 21: 5803-5816). The RRM is approximately 80 amino acids long and forms four β strands and two α helices organized in an α / β sandwich. The RRM contains a core RNP-1 octapeptide motif and surrounding conserved sequences. In addition to the snRNP protein, examples of RNA-binding proteins having the above motif group include heteronuclear ribonucleoprotein that stabilizes nascent RNA and factors that regulate alternative splicing. Alternative splicing factors include developmentally regulated proteins, specific examples of which are lower eukaryotes (eg, Drosophila melanogaster and nematodes).Caenorhabditis elegans). Each of these proteins plays an important role in the developmental process (eg patterning and sex determination) (see, eg, Hodgkin, J. et al. (1994) Development 120: 3681-3689).
RNA stability and degradation
RNA helicase performs modification and regulation of RNA conformation and secondary structure using energy derived from ATP hydrolysis to destabilize and unwind the RNA duplex. The best characterized and ubiquitous family of RNA helicases is the DEAD box family, whose name is derived from the conserved B-type ATP binding motif. This motif is characteristic for this family of proteins. Over 40 DEAD box helicases have been identified in diverse organisms ranging from bacteria, insects, yeasts, amphibians, mammals, and plants. DEAD box helicases function in a variety of processes. The processes include, for example, translation initiation, splicing, ribosome assembly, and RNA editing, transport, and stability. Several DEAD box helicases play a tissue-specific and stage-specific role in spermatogenesis and embryogenesis. All DEAD box helicases contain several conserved sequence motifs that span approximately 420 amino acids. These motifs include A type ATP binding motif, DEAD box / B type ATP binding motif, serine / arginine / threonine tripeptide (function unknown), and C-terminal glycine rich motif (possible role is substrate Binding and rewinding). The alignment of the diverse DEAD box helicase sequences also indicates that 37 amino acid residues are identical in all of these sequences, suggesting that conservation of these residues is important for helicase function (reviews). Linder, P. et al. (1989) Nature 337: 121-122).
[0041]
Overexpression of DEAD box 1 protein (DDX1) appears to play a role in the progression of neuroblastoma (Nb) and retinoblastoma (Rb). These observations suggest that DDX1 promotes or enhances tumor progression by altering the normal secondary structure and expression level of RNA in cancer cells. Another DEAD box helicase group appears to be directly or indirectly involved in UV-induced tumors, B-cell lymphoma, and myeloid malignancies (reviewed by Godbout, R. et al. (1998) J. Biol. Chem. 273: 21161-21168).
[0042]
Ribonucleases (RNases) catalyze the hydrolysis of phosphodiester bonds within the RNA strand and thus the cleavage of the RNA. For example, RNase P is a ribonucleoprotein enzyme that cleaves the 5 ′ end of tRNA precursors as part of their maturation process. RNase H digests RNA strands of certain RNA / DNA hybrids. Such hybrids occur in cells invaded by retroviruses, and RNase H is an important enzyme in the retroviral replication cycle. The RNase H domain is often seen as a domain associated with reverse transcription. Increased RNase activity in serum and cell extracts is seen in various cancers and infectious diseases (Schein, C.H. (1997) Nat. Biotechnol. 15: 529-536). Modulation of RNase activity has been studied as a means of controlling tumor angiogenesis, allergic reactions, viral infection and replication, and fungal infection.
[0043]
Ribosomal proteins can undergo post-translational modifications and interact with other ribosome-related proteins to regulate translation. For example, the highly homologous 40S ribosomal protein S6 kinases S6K1 and S6K2 play a major role in regulating cell growth, which controls the biosynthesis of the translation components that make up protein synthesizers (eg ribosomal proteins) To do. In the case of S6K1, at least 8 phosphorylation sites appear to mediate kinase activation in a hierarchical manner (Dufner and Thomas (1999) Exp. Cell. Res. 253: 100109). Several ribosomal proteins (including L1) also function as translational repressors, which bind to the polycistronic mRNA that encodes the ribosomal protein (reviewed above, Liljas, A. and Garber, M .).
[0044]
Recent evidence suggests that many ribosomal proteins have secondary functions that are independent of their involvement in protein biosynthesis. These proteins function as regulators of cell growth and in some cases as inducers of cell death. For example, expression of the human ribosomal protein L13a has been shown to induce apoptosis. This is done by stopping cell growth in the G2 / M phase of the cell cycle. Suppression of L13a expression induces apoptosis in target cells, suggesting that this protein is required for cell survival at the proper dose. Similar results have been obtained with yeast. Here, inactivation of rp22 and rp23, which are yeast homologues of L13a, caused severe growth retardation and death. A very closely related ribosomal protein, L7, arrests cells in the G1 phase and also induces apoptosis. Thus, a subset of ribosomal proteins appear to function as cell cycle checkpoints and appear to constitute a new family of cell growth regulators.
[0045]
Individual ribosomal proteins were mapped to the surface of intact ribosomes. For this, 3D immunocryoelectronmicroscopy was used to visualize the antibodies produced against specific ribosomal proteins. Although progress has been made towards mapping L1, L7, and L12, elucidation of the structure of intact ribosomes is only 20-25D resolution, and there is a discrepancy between the different crude structures. (Frank, J. (1997) Curr. Opin. Struct. Biol. 7: 266272).
[0046]
Three separate sites have been identified on the ribosome. The aminoacyl tRNA acceptor site (A site) receives charged tRNA (the exception is the starting tRNA). The peptidyl tRNA site (P site) binds to the nascent polypeptide and amino acids from the A site are added to this extending chain. The deacylated tRNA group is released from the ribosome after binding in the escape site (E site). A review of the structure of the ribosome is given by Stryer, L. (1995)Biochemistry W.H.Freeman and Company, New York NY 888-908l pages; Lodish, H. et al. (1995)Molecular Cell Biology Scientific American Books, New York NY 119-138; and Lewin, B (1997)Genes VI See Oxford University Press, Inc. New York, NY.
[0047]
A variety of proteins are required for the processing of RNA transcribed in the nucleus. Pre-stages of mRNA processing include capping with methylguanosine to the 5 ′ end, polyadenylation of the 3 ′ end, and splicing to remove introns. The primary RNA transcript from DNA is a faithful copy of a gene with both exon and intron sequences, and the intron sequence must be excised from the RNA transcript to produce the mRNA that encodes the protein. . "Splicing" of mRNA is done in the nucleus with the aid of a large multicomponent ribonucleoprotein complex (known as the spliceosome). The spliceosome complex consists of five small nuclear ribonucleoprotein particles (snRNP), U1, U2, U4, U5, and U6. Each snRNP contains one species of snRNA and about 10 proteins. Several snRNP RNA components recognize and base pair intron consensus sequences. The protein component mediates spliceosome assembly and splicing reactions. Antibodies to the snRNP protein are found in the blood of patients with systemic lupus erythematosus (Stryer, L. (1995)Biochemistry W.H. Freeman and Company, New York NY, p. 863).
[0048]
The role of heterogeneous nuclear ribonucleoprotein (hnRNP) has been identified in splicing, export of mature RNA to the cytoplasm, and mRNA translation (Biamonti, G. et al. (1998) Clin. Exp. Rheumatol. 16: 317-326). Examples of hnRNP are the yeast proteins Hrp1p (involved in RNA cleavage and polyadenylation at the 3 ′ end), Cbp80p (involved in capping the 5 ′ end of RNA), and Npl3p (mRNA from the nucleus) Mammalian homologue of hnRNP A1 (Shen, EC et al. (1998) Genes Dev. 12: 679-691). hnRNP has been shown to be an important target of autoimmune responses in rheumatic diseases (supra Biamonti).
[0049]
Many snRNP and hnRNP proteins are characterized by certain RNA recognition motifs (RRM) (reviewed in Birney, E. et al. (1993) Nucleic Acids Res. 21: 5803-5816). The RRM is approximately 80 amino acids long and forms four β strands and two α helices organized in an α / β sandwich. Within the RRM is the core RNP-1 octapeptide motif and surrounding conserved sequences. In addition to the snRNP protein, examples of RNA-binding proteins having the above motif group include heteronuclear ribonucleoprotein that stabilizes nascent RNA and factors that regulate alternative splicing. Alternative splicing factors include developmentally regulated proteins, specific examples of which are lower eukaryotes (eg, Drosophila melanogaster and nematodes).Caenorhabditis elegans). Each of these proteins plays an important role in the developmental process (eg patterning and sex determination) (see, eg, Hodgkin, J. et al. (1994) Development 120: 3681-3689).
[0050]
The 3 ′ end of most eukaryotic mRNAs also undergoes post-transcriptional modification by polyadenylation. Polyadenylation proceeds through two enzymatically distinct steps. (i) Nucleotide strand breaks in nascent mRNA are made with cis-acting polyadenylation signals within the 3 ′ untranslated (non-coding) region, and (ii) poly (A) tract is 5 ′ added to the mRNA fragment. The presence of cis-acting RNA sequences is necessary for both steps. These sequences include 5'-AAUAAA-3 '(10-30 nucleotides upstream of the cleavage site) and less conserved GU-rich or U-rich sequence elements (10-30 nucleotides downstream of the cleavage site). Position). Cleavage factor (CstF), cleavage factor I (CF I), and cleavage factor II (CF II) are involved in the cleavage reaction, while cleavage and poly (A) addition specificity determinants (CPSF) and poly (A) Polymerase (PAP) is required for both cleavage and polyadenylation. An additional enzyme, poly (A) binding protein II (PAB II), promotes poly (A) tract elongation (Ruegsegger, U. et al. (1996) J. Biol. Chem. 271: 61076113; and references thereof. ).
translation
The correct translation of the genetic code depends on each amino acid forming a linkage with the appropriate transfer RNA (tRNA). Aminoacyl-tRNA synthetase (aaRS) is an essential protein found in all living organisms. aaRS is responsible for the activation of amino acids and their correct attachment to their cognate tRNA as the first step in protein biosynthesis. Prokaryotes have at least 20 different types of aaRS and one aaRS for each amino acid, while eukaryotes usually have two aaRS in cytosolic and mitochondrial forms for each different amino acid. The 20 aaRS enzymes can be divided into two structural classes. Class I enzymes add amino acids to the 2 ′ hydroxyl at the 3 ′ end of the tRNA, and class II enzymes add amino acids to the 3 ′ hydroxyl at the 3 ′ end of the tRNA. Each class is characterized by a unique topology of the catalytic domain. Class I enzymes have catalytic domains based on nucleotide-bound “Rossman folds”. In particular, certain consensus tetrapeptide motifs are highly conserved (Prosite Document PDOC00161, Aminoacyl-transfer RNA synthetases class-I signature). Class I enzymes are specific for arginine, cysteine, glutamic acid, glutamine, isoleucine, leucine, methionine, tyrosine, tryptophan and valine. Class II enzymes have a central catalytic domain (consisting of a seven-stranded antiparallel β-sheet domain) and an N-terminal and C-terminal regulatory domain. Class II enzymes are divided into two groups based on the heterodimeric or homodimeric structure of the enzyme, the latter being further divided by the structure of the N-terminal and C-terminal regulatory domains (Hartlein, M. and Cusack, S. (1995) J. Mol. Evol. 40: 519-530). Class II enzymes are specific for alanine, asparagine, aspartic acid, glycine, histidine, lysine, phenylalanine, proline, serine and threonine.
[0051]
Some types of aaRS also have an editing function. For example, IleRS accidentally activates valine and Val-tRNA<sup>Ile</sup> This product is removed by some hydrolysis action that destroys the mischarged product. This editing activity is located in the second catalytic site found in the connective polypeptide 1 region (CP1), a long insert within the Rossman fold domain of class I enzymes (Schimmel, P. et al. (1998) FASEB J. 12: 1599-1609). AaRS also plays a role in tRNA processing. Mature tRNAs have been shown to be charged with their respective amino acids in the nucleus before being exported to the cytoplasm. In addition, the binding with this amino acid functions as a quality control mechanism, and it is possible that tRNA is functionally maintained (Martinis, S.A. et al. (1999) EMBO J. 18: 4591-4596).
[0052]
The rate of progress of polypeptide synthesis under optimal conditions is about 40 amino acid residues / second. The ratio of erroneous capture during translation is 10<sup>-Four</sup> The main cause is that aminoacyl-t-RNA was charged with the wrong amino acid. Incorrectly charged tRNAs are cytotoxic. The reason is that they incorporate incorrect amino acid residues into the growing polypeptide. The speed of translation is presumed to be determined by a compromise between the optimal extension speed and the need for translation fidelity. For mathematical calculations, 10<sup>-Four</sup> Is the maximum allowable error rate for protein synthesis in biological systems (reviewed above by Stryer, L. and Watson, J. et al. (1987) The Benjamin / Cummings Publishing Co., Inc. Menlo Park, CA). Particularly error-prone aminoacyl-tRNA charging events are tRNA<sup>Gln</sup> Binding of glutamine (Gln). There is a mechanism to correct this mischarging, which seems to have its origin in evolution. Gln was in the last group of 20 natural amino acids used in polypeptide synthesis and appearing in nature. Gram-positive eubacteria, cyanobacteria, archaea (Archeae), and eukaryotic organelles are Gln-tRNA<sup>Gln</sup> Has a noncanonical pathway for the synthesis of Glu-tRNA<sup>Gln</sup> Glu-tRNA is an enzyme based on the transformation of Glu-tRNA synthetase GluRS<sup>Gln</sup> Amide transferase (Glu-AdT) is used. The reactions involved in the transamidation pathway are as follows (Curnow, A.W. et al. (1997) Nucleic Acids Symposium 36: 2-4).
[0053]
GluRS
tRNA<sup>Gln</sup> + Glu + ATP → Glu-tRNA<sup>Gln</sup> + AMP + PP<sub>i</sub>
Glu-AdT
Glu-tRNA<sup>Gln</sup> + Gln + ATP → Gln-tRNA<sup>Gln</sup> + Glu + ADP + P
[0054]
Asp-tRNA, a similar enzyme<sup>Asn</sup> Amidotransferase is present in primordial fungi and Asp-tRNA<sup>Asn</sup> Asn-tRNA<sup>Asn</sup>Switch to Formylase enzyme is a Met-tRNA in eubacteria<sup>fMet</sup> FMet-tRNA<sup>fMet</sup> Which appears to be a closely related enzyme. Some hydrolytic activities are also mischarged Val-tRNA<sup>Ile</sup> Have been identified (Schimmel, P. et al. (1998) FASEB J. 12: 1599-1609). One possible scenario for the evolution of Glu-AdT in primitive biological forms is the absence of a specific glutaminyl-tRNA synthetase (GlnRS)<sup>Gln</sup>An alternative route for the synthesis of In fact, the deletion of the Glu-AdT operon in Gram-positive bacteria is lethal (Curnow, A.W. et al. (1997) Proc. Natl. Acad. Sci. U.S.A. 94: 11819-11826). Although the presence of GluRS activity in other organisms has been speculated by a high degree of conservation in the natural translation machinery, GluRS has not been identified in all organisms including humans. Such enzymes appear to serve to ensure translation fidelity and reduce the synthesis of defective polypeptides.
[0055]
In addition to functions in protein synthesis, certain aminoacyl-tRNA synthetase groups play a role in cell fidelity, RNA splicing, RNA transport, apoptosis, and regulation of transcription and translation. For example, human tyrosyl-tRNA synthetase can be proteolytically cleaved into two parts with distinct cytokine activities. The C-terminal domain exhibits monocyte and leukocyte chemotaxis and stimulates the production of myeloperoxidase, tumor necrosis factor-α, and tissue factor. The N-terminal domain binds to the interleukin-8 type A receptor and functions as an interleukin 8-like cytokine. Human tyrosyl-tRNA synthetase (TyrRS) is secreted from tumor cells in the apoptotic stage. It may also accelerate apoptosis (Wakasugi, K and Schimmel, P (1999) Science 284: 147-151). Mitochondrial red mold (Neurospora crassa) TyrRS and Saccharomyces cerevisiae (S. cerevisiae) LeuRS is essential for several splicing activities of group I introns, and human mitochondrial LeuRS can be substituted for yeast LeuRS in the null strain of yeast. Several bacterial aaRSs are involved in the regulation of their own transcription or translation (above Martinis). Several aaRSs can synthesize diadenosine oligophosphates, a class of signaling molecules that have a role in cell proliferation, differentiation, and apoptosis (Kisselev, LL et al. (1998) FEBS Lett. 427: 157-163; Vartanian, A. et al. (1999) FEBS Lett. 456: 175-180).
[0056]
Autoantibodies against aminoacyl-tRNA are produced by patients with autoimmune diseases such as rheumatoid arthritis, dermatomyositis, and polymyositis. It is also correlated with complex interstitial lung disease (ILD) intensity (Freist, W. et al. (1999) Biol. Chem. 380: 623-646; Freist, W. et al. (1996) Biol. Chem. Hoppe Seyler 377: 343-356). These autoantibodies appear to be produced in response to viral infection, and Coxsackie virus has been used to induce experimental viral myositis in animals.
[0057]
Comparison of the aaRS structure between the human body and pathogens has helped in the design of new antibiotics (Schimmel, supra). Using genetically manipulated aaRS complexin vivoEnables site-specific incorporation of unnatural amino acids into proteins (Liu, D.R. et al. (1997) Proc. Natl. Acad. Sci. USA 94: 10092-10097).
tRNA Charge (charging)
Protein biosynthesis depends on each amino acid forming a linkage with the appropriate tRNA. Aminoacyl-tRNA synthetases are responsible for the activation of amino acids and correct attachment with their cognate tRNA. The 20 aminoacyl-tRNA synthetases can be divided into two structural classes. Class I enzymes add amino acids to the 2 ′ hydroxyl at the 3 ′ end of the tRNA, and class II enzymes add amino acids to the 3 ′ hydroxyl at the 3 ′ end of the tRNA. Each class is characterized by a unique topology of the catalytic domain. Class I enzymes have a catalytic domain based on nucleotide-bound “Rosman folds”. Class II enzymes have a central catalytic domain (consisting of a seven-stranded antiparallel β-sheet motif) and an N-terminal and C-terminal regulatory domain. Class II enzymes are divided into two groups based on the heterodimeric or homodimeric structure of the enzyme, the latter being further divided by the structure of the N-terminal and C-terminal regulatory domains (Hartlein, M. and Cusack, S (1995) J. Mol. Evol. 40: 519-530). Autoantibodies against aminoacyl-tRNA are produced by patients with dermatomyositis and polymyositis. It is also correlated with complex interstitial lung disease (ILD) and intensity. These autoantibodies appear to be produced in response to viral infection, and Coxsackie virus has been used to induce experimental viral myositis in animals.
tRNA Qualification
A modified ribonucleoside, pseudouridine (y), is ubiquitous in the anticodon region of transfer RNA (tRNA), large and small ribosomal RNA (rRNA), and small nuclear RNA (snRNA). y is the most common modified nucleoside present in tRNA (ie other than G, A, U, and C). Only a few yeast tRNAs unrelated to protein synthesis have no y (Cortese, R. et al. (1974) J. Biol. Chem. 249: 1103-1108). Pseudouridine synthase (pseudouridylate synthase), the enzyme responsible for the conversion of uridine to y, was first introduced in Salmonella typhimurium (Salmonella typhimurium(Arena, F. et al. (1978) Nuc. Acids Res. 5: 45234536). This enzyme has since been isolated from a number of mammals (eg, steers and mice) (Green, CJ et al. (1982) J. Biol. Chem. 257: 304552 and Chen, J. and Patton, JR (1999 ) RNA 5: 409419). tRNA pseudouridine synthase is the most widely studied member of this family. Optimal activities of these enzymes require thiol donors (such as cysteine) and monovalent cations (such as ammonia and potassium). No additional cofactors or high energy molecules (ATP or GTP) are required (Green, supra). Another eukaryotic pseudouridine synthase group has been identified and appears to be specific for rRNA (reviewed by Smith, C.M. and Steitz, J.A. (1997) Cell 89: 669672). Certain bispecific enzymes have also been identified that utilize both tRNA and rRNA substrates (Wrzesinski, J. et al. (1995) RNA 1: 437448). When y is absent within the anticodon loop of tRNA, growth is reduced in both bacteria (Singer, CE et al. (1972) Nature New Biol. 238: 7274) and yeast (Lecointe, F. (1998) 273: 13161323) However, this genetic defect is not lethal.
[0058]
Another ribonucleoside modification occurs primarily in eukaryotic cells. This is N from guanosine<sup>2</sup>, N<sup>2</sup>Dimethylguanosine (m<sup>2</sup> <sub>2</sub>G), which occurs at base position 26 or 10 of the D stem of cytosolic and mitochondrial tRNAs. This post-transcriptional modification appears to stabilize the tRNA structure by preventing the formation of alternative tRNA secondary and tertiary structures. Yeast tRNA<sup>Asp</sup> Is unique in that it does not have this modification. This modification does not occur in eubacteria, probably because the tRNA structure in these cells and organelles is constrained by the sequence, and post-transcriptional modifications do not need to prevent the formation of another structure (Steinberg, S. and Cedergren, R. (1995) RNA 1: 886-891 and references). M from guanosine<sup>2</sup> <sub>2</sub>The enzyme responsible for conversion to G is a 63 kDa S-adenosylmethionine (SAM) -dependent tRNA N<sup>2</sup>, N<sup>2</sup>Dimethylguanosine methyltransferase (also referred to as TRM1 gene product, referred to herein as TRM) (Edqvist, J. (1995) Biochimie 77: 5461). This enzyme is localized in both the nucleus and mitochondria (Li, J-M. Et al. (1989) J. Cell Biol. 109: 1411-1419). Based on studies with Xenopus TRM, base pairing at the C11-G24 and G10-C25 positions just before G26 to be modified may have certain requirements, and another structure of tRNA. The characterization also appears to be necessary for proper presentation of the G26 substrate (Edqvist. J. et al. (1992) Nuc. Acids Res. 20: 657581). Studies in yeast suggest that cells with certain weak ocher tRNA suppressors (sup3i) cannot suppress translation termination without TRM activity, so TRM regulates the frequency of suppression in eukaryotic cells Suggests a role (Niederberger, C. et al. (1999) FEBS Lett. 464: 6770). TRM also seems to ensure the proper three-dimensional structure of tRNA, which is a more general function.
Translation started
The start of translation can be divided into three stages. The first stage is a certain initial transfer RNA (Met-tRNA<sub>f</sub>) And 40S ribosomal subunit form a 43S pre-initiation complex. The second stage binds the 43S pre-initiation complex to the mRNA and then moves the complex to the correct AUG start codon. The third stage directs the 60S ribosomal subunit to the 40S subunit, producing an 80S ribosome at the start codon. Translational regulation mainly involves the first and second stages in the initiation process (V.M. Pain (1996) Eur. J. Biochem. 236: 747-771).
[0059]
Several initiation factors, many of which have multiple subunits, are involved in the complex of the initiation tRNA with the 40S ribosomal subunit. eIF2 is a guanine nucleotide binding protein that mobilizes the starting tRNA to the 40S ribosomal subunit. eIF2 associates with the initiating tRNA only when bound to GTP. eIF2B is a kind of guanine nucleotide exchange protein, and is responsible for the conversion of eIF2 from the GDP binding inactive form to the GTP binding active form. Two other factors, eIF1A and eIF3, bind to and stabilize the 40S subunit, which interacts with 18S ribosomal RNA and specific ribosomal structural proteins. eIF3 is also involved in the association of 40S ribosomal subunit with mRNA. Met-tRNA<sub>f</sub>, EIF1A, eIF3 and 40S ribosomal subunit together form a 43S pre-initiation complex (Pain, supra).
[0060]
eIF2 plays a central role in maintaining the rate-limiting step in mRNA translation. In this step, eIF2 binds GTP and MettRNAi and transfers MettRNAi to the 40S ribosomal subunit. At the end of the initiation process, GTP bound to eIF2 is hydrolyzed to GDP. The eIF2.GDP complex is released from the ribosome. The exchange of GDP bound to eIF2 with GTP is a prerequisite for binding to MettRNAi, and this exchange is mediated by a second initiation factor, eIF2B (a type of guanine nucleotide exchange factor). Its phosphorylation at the α subunit of eIF2 converts eIF2 from a substrate of eIF2B to a competitive inhibitor. Thus, phosphorylation of eIF2α effectively prevents formation of the eIF2.GTP.MettRNAi complex and inhibits overall protein synthesis. Various conditions under which eIF2α phosphorylation occurs include viral infection, apoptosis, nutrient deficiency, heme deficiency, and several types of stress. The 5 ′ untranslated region of hepatitis C virus (HCV) functions as an internal ribosome entry site (IRES) where translation of the HCV protein begins. eIF2B gamma and eIF2 gamma are intracellular factors involved in translation mediated by HCV IRES (Kimball, SR (1999) Int. J. Biochem. Cell Biol. 31: 2529; Webb, BL and Proud, CG ( 1997) Int. J. Biochem. Cell Biol. 29: 11271131; Kruger M. et al. (2000) Proc. Natl. Acad. Sci. USA 97: 85668571).
[0061]
Multiple additional factors are required for the 43S pre-initiation complex to bind to the mRNA molecule, and this process is regulated at several levels. The complex called eIF4F consists of three proteins, eIF4E, eIF4A, and eIF4G. eIF4E is the 5'-end m of mRNA<sup>7</sup>It recognizes and binds to the GTP cap, eIF4A is a bidirectional RNA-dependent helicase, and eIF4G is a kind of scaffold polypeptide. eIF4G has three binding domains. The N-terminal third of eIF4G interacts with eIF4E, the central third interacts with eIF4A, and the C-terminal third interacts with eIF3 bound to the 43S pre-initiation complex. To do. Thus, eIF4G bridges the 40S ribosomal subunit and mRNA (M.W. Hentze (1997) Science 275: 500-501).
[0062]
The ability of eIF4F to initiate 43S pre-initiation complex is regulated by the structural features of the mRNA. The mRNA molecule has an untranslated region (UTR) between the 5 ′ cap and the AUG start codon. In some mRNAs, this region forms a secondary structure that prevents binding of the 43S pre-initiation complex. The helicase activity of eIF4A appears to function to remove this secondary structure and promote binding of the 43S pre-initiation complex (Pain, supra).
Translation expansion
In the elongation process, an additional group of amino acids bind to the starting methionine to form a complete polypeptide chain. Elongation factors EF1α, EF1βγ, and EF2 are involved in the elongation of the polypeptide chain after initiation. EF1α is a GTP binding protein. In the GTP-bound form of EF1α, EF1α guides aminoacyl tRNA to the A site of the ribosome. The amino acid attached to the newly arrived aminoacyl-tRNA forms a peptide bond with the initiating methionine. GTP on EF1α is hydrolyzed to GDP, and EF1α-GDP dissociates from the ribosome. By binding EF1βγ to EF1α-GDP and dissociating GDP from EF1α, EF1α can bind to GTP and a new cycle starts.
[0063]
In turn, aminoacyl tRNAs are directed to the ribosome and EF-G (another GTP binding protein) catalyzes the transfer of tRNA from the A site to the P site and finally to the E site of the ribosome. This leads to translational processivity.
End of translation
The release factor eRF terminates the translation. eRF recognizes a stop codon in the mRNA and releases the polypeptide chain from the ribosome.
[0064]
Treacher Collins syndrome (TCS) is the most common human mandibulofacial dysostosis disorder. TCS is autosomal dominant, occurs in 1 in 50,000 live births, and about 60% is due to new mutations. The symptoms of TCS show wide variability. The cause of this disease is inferred to be interference in the development of the first and second arches. TCOF1, a TCS gene, is located on chromosome 5q31-33.3. Ten mutations have been identified in TCOF1, including nonsense mutations, insertions, deletions, or splice mutations, clearly leading to premature termination of translation. Moreover, all of them are specific to each patient's family. TCOF1 encodes a low-complexity protein (1411 amino acids), and the repetitive motif that the protein has is a mirror image of the composition of its exons. These motifs are also shared by nucleolar transport proteins in other species and are highly phosphorylated by casein kinase. Also included within the full-length TCOF1 protein sequence are nuclear and nucleolar localization signals and several polymorphisms. This data suggests that the cause of TCS is a deficiency in some nucleolar transport protein, which is critically required for human craniofacial development (Wise, CA et al. (1997) Proc. Natl. Acad. Sci. USA 94: 31103115).
breast cancer
Over 180,000 newly developed breast cancers are diagnosed each year, and the death rate from breast cancer is close to 10% of all deaths in women aged 45 to 54 years (Gish, K. (1999) AWIS Magazine 28: 7-10) . However, the survival rate based on early diagnosis of localized breast cancer is very high (97%). On the other hand, when the disease stage has progressed and the tumor has spread outside the breast, it is 22%. Current clinical breast cancer screening methods lack sensitivity and specificity, and efforts are being made to develop comprehensive gene expression profiles for breast cancer. Using this profile with conventional screening methods can improve breast cancer diagnosis and prognosis (Perou, C.M. et al. (2000) Nature 406: 747-752).
[0065]
Mutations between two genes, BRCA1 and BRCA2, are known to be a major predisposition to female breast cancer, and this mutation appears to be passed from parent to child (supra Gish). However, this type of hereditary breast cancer is only about 5% to 9% of breast cancer, and the majority of breast cancers are caused by non-hereditary mutations and mutations in breast epithelial cells.
[0066]
The relationship between expression of epidermal growth factor (EGF) and its receptor, EGFR, and human breast cancer is particularly well studied (for a review of this area, see Khazaie, K. et al. (1993) Cancer and Metastasis Rev. 12: 255274 and references therein). EGFR overexpression, particularly expression associated with downregulation of estrogen receptors, is a poor prognostic marker for breast cancer patients. In addition, EGFR expression in breast cancer metastasis is often increased compared to the primary tumor, suggesting that EGFR is involved in tumor progression and metastasis. There is accumulated evidence to support this. That is evidence that EGF has an effect on cell function with regard to the potential of metastasis. This function is, for example, cell motility, chemotaxis, secretion, and differentiation. Changes in the expression of other members of the erbB receptor family (EGFR is one of them) are also thought to be involved in breast cancer. The abundance of erbB receptors (eg HER-2 / neu, HER-3 and HER-4) and their ligands in breast cancer has shown their functional importance in the pathogenesis of breast cancer, thus targeting breast cancer treatment targets (Bacus, SS et al. (1994) Am. J. Clin. Pathol. 102: S13-S24). Other known breast cancer markers include mRNA of a human secreted frizzled protein (downregulated in breast cancer), matrix G1a protein (overexpressed in human breast cancer cells), Drg1 or RTP (expression of this gene is expressed in colon Reduced in cancer, breast cancer, prostate tumor), maspin (this tumor suppressor gene is down-regulated in invasive breast cancer), and CaN19 (a member of the S100 protein family, all of which are compared to normal breast epithelial cells in breast cancer cells) (Zhou, Z. et al. (1998) Int. J. Cancer 78: 95-99; Chen, L. et al. (1990) Oncogene 5: 1391-1395; Ulrix, W. et al. (1999) FEBS Lett 455: 23-26; Sager, R. et al. (1996) Curr. Top. Microbiol. Immunol. 213: 51-64; and Lee, SW et al. (1992) Proc. Natl. Acad. Sci. USA 89: 2504-2508).
[0067]
Cell lines derived from mammary epithelial cells of various stages of human breast cancer provide a useful model for studying the process of malignant transformation and tumor progression. The reason is that these cell lines have been shown to retain many characteristics of their parent tumor over a long culture period (Wistuba, II et al. (1998) Clin. Cancer Res. 4: 2931 -2938). Such a model is particularly useful when comparing the phenotype and molecular characteristics of human mammary epithelial cells for various stages of malignant transformation.
Preadipocytes
The most important function of adipose tissue is the ability to store fat on eating and release it on an empty stomach. White adipose tissue is the main energy storage site when using excess energy, and its primary purpose is mobilization during energy deficiency. Understanding how various molecules regulate adiposity and energy balance in physiological and pathophysiological situations can develop new therapies for human obesity. Adipose tissue is also one of the important target tissues of insulin. Adipogenesis and insulin resistance in type II diabetes are linked and show an interesting relationship. Most patients with type II diabetes are obese, which then develops insulin resistance.
[0068]
Most of the previous work in adipocyte biology has utilized transformed mouse preadipocyte cell lines. The culture conditions that stimulate the differentiation of mouse preadipocytes are different from the conditions that induce the differentiation of human primary preadipocytes. Also, because primary cells are diploid,in vivoThe condition seems to reflect more than the aneuploid cell line. Understanding the gene expression profile of human adipogenesis can help understand the underlying mechanism of adiposity regulation. Furthermore, comparing the gene expression profile of adipogenesis between normal weight and obese donors allows the identification of a very important set of genes, ie potential drug targets between obesity and type II diabetes.
Peroxisome proliferator-activated receptor gamma agonist
Thiazolidinediones (TZD) act as agonists of peroxisome proliferator-activated receptor γ (PPARγ). PPARγ is a member of the nuclear hormone receptor superfamily. TZD reduces hyperglycemia, hyperinsulinemia, and hypertension, in part by promoting glucose metabolism and inhibiting gluconeogenesis. The role of PPARγ and its agonists has been demonstrated in a wide range of pathologies. This condition includes, for example, diabetes, obesity, hypertension, atherosclerosis, polycystic ovary syndrome, breast cancer, prostate cancer, liposarcoma, and colon cancer.
[0069]
The mechanism by which TZD and other PPARγ agonists enhance insulin sensitivity is not well understood, but may be related to the ability of PPARγ to promote adipogenesis. PPARγ expressed ectopically in cultured preadipocytes is a potent inducer of adipocyte differentiation. TZD can also be used in combination with other factors such as insulin to enhance human preadipocyte differentiation in the medium (Adams et al. (1997) J. Clin. Invest. 100: 3149-3153). Various TZDs produce adipogenesisin vitro The relative potency promoted by thosein vivoInsulin sensitization effect and PPARγin vitroIs proportional to both their ability to bind and activate. Interestingly, adipocytes derived from omental adipose depots are refractory to the effects of TZD. This suggests that the insulin sensitizing effects of TZDs are a result of their ability to promote adipogenesis in subcutaneous fat stores (Adams et al., Ibid). Furthermore, identification of dominant negative mutations in the PPARγ gene has been made in two non-obese cases. They had severe insulin resistance, hypertension, and overt non-insulin dependent diabetes mellitus (NIDDM) (Barroso et al. (1998) Nature 402: 880-883).
[0070]
NIDDM is the most common type of diabetes, which is a chronic metabolic disease with 143 million patients worldwide. NIDDM is characterized by abnormal glucose and lipid metabolism, which is caused by a combination of peripheral insulin resistance and defective insulin secretion. NIDDM has a complex progressive etiology and a high heritability. A number of complications are associated with diabetes (including, for example, heart disease, stroke, renal failure, retinopathy, and peripheral neuropathy) and contribute to high morbidity and mortality.
[0071]
At the molecular level, PPARγ functions as a ligand-activated transcription factor. In the presence of ligand, PPARγ forms a heterodimer with the retinoid X receptor (RXR). This dimer activates transcription of the target gene. This target gene has one or more copies of a PPARγ response element (PPRE). Many genes important for lipid storage and metabolism have PPRE and have been identified as PPARγ targets. This includes, for example, PEPCK, aP2, LPL, ACS, and FAT-P (Auwerx, J. (1999) Diabetologia 42: 1033-1049). A number of PPARγ ligands have been identified. The ligands include, for example, various fatty acid metabolites, TZD classes of synthetic drugs (eg, pioglitazone and rosiglitazone (BRL49653)), and some non-glitazone tyrosine analogues (eg GI262570 and GW1929) including. The prostaglandin derivative 15dPGJ2 is a potent endogenous ligand of PPARγ.
[0072]
PPARγ expression is very high in fat, but is hardly detected in skeletal muscle. Skeletal muscle is the main site of insulin-stimulated glucose processing in the body. Moderate expression of PPARγ is also found in the large intestine, kidney, liver, vascular smooth muscle, hematopoietic cells, and macrophages. The high expression of PPARγ in fat suggests that the sensitizing effect of TZDs is a result of transformation in the expression of one or more PPARγ regulated genes in adipose tissue. Identification of PPARγ target genes will contribute to better drug design and development of new therapeutic strategies for diabetes, obesity and other conditions.
[0073]
Systematic attempts to identify PPARγ target genes have been made in several rodent models of obesity and diabetes (Suzuki et al. (2000) Jpn. J. Pharmacol. 84: 113123; Way et al. (2001) Endocrinology 142: 1269-1277). However, there are serious shortcomings in rodent gene expression studies. It is a significant difference between adipogenesis, diabetes, and obesity in human and rodent models (Taylor (1999) Cell 97: 9-12; Gregoire et al. (1998) Physiol. Reviews 78: 783-809). ). Therefore, an unbiased technique for identifying TZD-regulated genes in primary cultures of human tissues is necessary to fully elucidate the molecular principles of diseases associated with PPARγ activity.
[0074]
Array technology can provide a simple way to explore the expression profile of a single polymorphic gene or the expression profile of many related or unrelated genes. When testing the expression of a single gene, the array is used to detect the expression of a particular gene or variant thereof. When testing an expression profile, the array provides a platform for testing: That is, which genes are tissue-specific, perform housekeeping functions, are part of a signal transduction cascade, or genes that are specifically associated with a particular genetic predisposition, condition, disease, or disorder It is a test of whether there is. Potential applications of gene expression profile generation are particularly relevant for disease diagnosis, prognosis, and treatment improvement. For example, both levels and sequences expressed in tissues from diabetic patients can be compared to levels and sequences expressed in normal tissues.
[0075]
The discovery of novel nucleic acid-related proteins and the polynucleotides that encode them can meet the needs of the art by providing new compositions. These novel compositions are useful in the diagnosis, treatment and prevention of cell proliferation abnormalities, neurological disorders, developmental disorders, autoimmune / inflammatory diseases, and infectious diseases, and the nucleic acid sequences and amino acid sequences of nucleic acid related proteins It is also useful for calculating the effects of foreign compounds on expression.
[0076]
(Summary of Invention)
The present invention is generally referred to as “NAAP”, and individually “NAAP-1,” “NAAP-2,” “NAAP-3,” “NAAP-4,” “NAAP-5,” “NAAP-6,” respectively. , "NAAP-7", "NAAP-8", "NAAP-9", "NAAP-10", "NAAP-11", "NAAP-12", "NAAP-13", "NAAP-14", " Purified polypeptides that are nucleic acid-related proteins, referred to as “NAAP-15” and “NAAP-16” are provided. In certain embodiments, the present invention provides: (a) a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-16; (b) selected from the group consisting of SEQ ID NO: 1-16 A polypeptide having a natural amino acid sequence with at least 90% identity to an amino acid sequence, (c) the biological of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-16 An isolated polypeptide selected from the group consisting of an active fragment and (d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-16 is provided . In one example, the invention provides an isolated polypeptide having the amino acid sequence of SEQ ID NO: 1-16.
[0077]
The present invention also relates to (a) a polypeptide having a certain amino acid sequence selected from the group consisting of SEQ ID NO: 1-16, and (b) a certain amino acid sequence selected from the group consisting of SEQ ID NO: 1-16 (C) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-16 Or (d) an immunogenic fragment of a polynucleotide having a certain amino acid sequence selected from the group consisting of SEQ ID NO: 1-16, isolated encoding a polypeptide selected from the group consisting of A polynucleotide is provided. In one embodiment, the polynucleotide encodes a polypeptide selected from the group consisting of SEQ ID NO: 1-16. In another embodiment, the polynucleotide is selected from the group consisting of SEQ ID NO: 17-32.
[0078]
The invention further comprises (a) a polypeptide having a certain amino acid sequence selected from the group consisting of SEQ ID NO: 1-16, and (b) a certain amino acid sequence selected from the group consisting of SEQ ID NO: 1-16 (C) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-16 And (d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-16, and a polynucleotide encoding a polypeptide selected from the group consisting of: A recombinant polynucleotide having a promoter sequence linked to is provided. In one embodiment, the present invention provides a cell transformed with this recombinant polynucleotide. In another embodiment, the present invention provides a genetic transformant having this recombinant polynucleotide.
[0079]
The present invention further relates to (a) a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-16, and (b) an amino acid selected from the group consisting of SEQ ID NO: 1-16 Biology of a polypeptide having a certain natural amino acid sequence having at least 90% identity with the sequence and a polypeptide having a certain amino acid sequence selected from the group consisting of (c) SEQ ID NO: 1-16 And a method for producing a polypeptide selected from the group consisting of: (d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-16 . The production method comprises the steps of (a) culturing a cell under conditions suitable for expression of the polypeptide, transforming the cell with a recombinant polynucleotide, and (b) The process consists of recovering the peptide. This recombinant polynucleotide has a promoter sequence operably linked to the polynucleotide encoding the polypeptide.
[0080]
The invention further comprises (a) a polypeptide having a certain amino acid sequence selected from the group consisting of SEQ ID NO: 1-16, and (b) a certain amino acid sequence selected from the group consisting of SEQ ID NO: 1-16 A polypeptide having a certain natural amino acid sequence with at least 90% identity to, (c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-16, and (D) an isolated antibody that specifically binds to a polypeptide selected from the group consisting of an immunogenic fragment of a polypeptide having a certain amino acid sequence selected from the group consisting of SEQ ID NO: 1-16 I will provide a.
[0081]
The invention further comprises (a) a polynucleotide having a polynucleotide sequence selected from the group consisting of SEQ ID NO: 17-32, (b) a polynucleotide sequence selected from the group consisting of SEQ ID NO: 17-32 A polynucleotide having a natural polynucleotide sequence with at least 90% identity to (c) a polynucleotide complementary to (a), a polynucleotide complementary to (d) (b), and (e ) An isolated polynucleotide selected from the group consisting of the RNA equivalents of (a)-(d). In one embodiment, the polynucleotide consists of at least 60 contiguous nucleotide groups.
[0082]
The present invention further provides a method of detecting a target polynucleotide in a sample. Here, the target polynucleotide was selected from (a) a polynucleotide having a certain polynucleotide sequence selected from the group consisting of SEQ ID NO: 17-32, (b) selected from the group consisting of SEQ ID NO: 17-32 A polynucleotide having a natural polynucleotide sequence having at least 90% identity to a polynucleotide sequence, (c) a polynucleotide complementary to the polynucleotide of (a), (d) a polynucleotide of (b) It has the sequence of a polynucleotide selected from the group consisting of a complementary polynucleotide and an RNA equivalent of (e) (a)-(d). The detection method comprises: (a) hybridizing the sample with a probe consisting of at least 20 consecutive nucleotide groups consisting of a sequence complementary to the target polynucleotide in the sample; and (b) The process comprises detecting the presence or absence of the hybridization complex, and optionally detecting the amount of the complex if present. The probe specifically hybridizes to the target polynucleotide under conditions such that a hybridization complex is formed between the probe and the target polynucleotide or fragment thereof. In one embodiment, the probe consists of at least 60 contiguous nucleotide groups.
[0083]
The present invention also provides a method for detecting a target polynucleotide in a sample. Here, the target polynucleotide was selected from (a) a polynucleotide having a certain polynucleotide sequence selected from the group consisting of SEQ ID NO: 17-32, (b) selected from the group consisting of SEQ ID NO: 17-32 A polynucleotide having a natural polynucleotide sequence having at least 90% identity to a polynucleotide sequence, (c) a polynucleotide complementary to the polynucleotide of (a), (d) a polynucleotide of (b) A polynucleotide having a sequence selected from the group consisting of a complementary polynucleotide and an RNA equivalent of (e) (a)-(d). The detection method includes: (a) a process of amplifying a target polynucleotide or fragment thereof using polymerase chain reaction amplification; and (b) detecting the presence or absence of the amplified target polynucleotide or fragment thereof, and the target polynucleotide or fragment thereof. If is present, it is an optional process to detect the amount.
[0084]
The invention further provides a composition comprising an effective amount of the polypeptide and a pharmaceutically acceptable excipient. An effective amount of the polypeptide is (a) a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-16, (b) a certain selected from the group consisting of SEQ ID NO: 1-16 A polypeptide comprising a natural amino acid sequence having at least 90% identity to the amino acid sequence, (c) a biological activity of a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1-16 Selected from the group consisting of a fragment and (d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-16. In one embodiment, the composition has an amino acid sequence selected from the group consisting of SEQ ID NO: 1-16. Furthermore, the present invention provides a method comprising administering the composition to a patient in need of treatment of a disease or condition associated with reduced expression of functional NAAP.
[0085]
The invention also provides (a) a polypeptide having a certain amino acid sequence selected from the group consisting of SEQ ID NO: 1-16, (b) a certain amino acid sequence selected from the group consisting of SEQ ID NO: 1-16 (C) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-16, and (d) In order to confirm the effectiveness as an agonist of a polypeptide selected from the group consisting of: an immunogenic fragment of a polypeptide having a certain amino acid sequence selected from the group consisting of SEQ ID NO: 1-16 Methods for screening compounds are provided. The screening method comprises (a) a process of exposing a sample having the polypeptide to a certain compound, and (b) a process of detecting agonist activity in the sample. Alternatively, the present invention provides a composition having an agonist compound identified by this method and an acceptable pharmaceutical excipient. In yet another alternative, the present invention provides a method of administering this composition to a patient in need of treatment of a disease or condition that involves a decrease in the expression of functional NAAP.
[0086]
The invention further comprises (a) a polypeptide having a certain amino acid sequence selected from the group consisting of SEQ ID NO: 1-16, and (b) a certain amino acid sequence selected from the group consisting of SEQ ID NO: 1-16 (C) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-16, and (d) A method for screening certain compounds for the effectiveness as an antagonist of a polypeptide selected from the group consisting of: an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-16 provide. The screening method consists of (a) exposing a sample containing the polypeptide to a certain compound, and (b) detecting antagonistic activity in the sample. In another embodiment, the present invention provides a composition having an antagonist compound identified by this method and a pharmaceutically acceptable excipient. In a further alternative, the present invention provides a method comprising administration of this composition to a patient in need of treatment of a disease or condition associated with overexpression of functional NAAP.
[0087]
The invention further comprises (a) a polypeptide having a certain amino acid sequence selected from the group consisting of SEQ ID NO: 1-16, and (b) a certain amino acid sequence selected from the group consisting of SEQ ID NO: 1-16 A polypeptide having a certain natural amino acid sequence having at least 90% identity to, (c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-16, or (D) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-16, a method of screening for a certain compound that specifically binds to a certain polypeptide selected from the group consisting of provide. The screening method comprises: (a) mixing the polypeptide with at least one test compound under appropriate conditions; (b) detecting the binding of the test compound to the polypeptide, thereby The process consists of identifying a compound that specifically binds.
[0088]
The invention further comprises (a) a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-16, and (b) an amino acid sequence selected from the group consisting of SEQ ID NO: 1-16 A polypeptide having a natural amino acid sequence with at least 90% identity, (c) a biologically active fragment of a polypeptide having a certain amino acid sequence selected from the group consisting of SEQ ID NO: 1-16, and (D) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-16, a compound that modulates the activity of a polypeptide selected from the group consisting of Provide a method of screening. The screening method comprises: (a) mixing the polypeptide with at least one test compound under conditions acceptable for the activity of the polypeptide; and (b) converting the activity of the polypeptide in the presence of the test compound. And (c) comparing the activity of the polypeptide in the presence of the test compound with the activity of the polypeptide in the absence of the test compound. A change in the activity of the polypeptide at is indicative of a compound that modulates the activity of the polypeptide.
[0089]
The present invention further provides a method of screening certain compounds for the effect of altering the expression of a target polynucleotide having a certain polynucleotide sequence selected from the group consisting of SEQ ID NO: 17-32. The method comprises (a) exposing a sample having the target polynucleotide to a compound, (b) detecting alterations in expression of the target polynucleotide, and (c) a variable amount of the compound. The process consists of comparing the expression of the target polynucleotide in the presence to the expression in the absence of the compound.
[0090]
The present invention further provides a method for calculating the toxicity of a test compound. This method includes the following processes. (A) A process of treating a biological sample having a nucleic acid group with a test compound. (B) A process of hybridizing the nucleic acid group of the treated biological sample. The following probe is used in this process. (I) a polynucleotide having a polynucleotide sequence selected from the group consisting of SEQ ID NO: 17-32; (ii) at least a polynucleotide sequence selected from the group consisting of SEQ ID NO: 17-32 A polynucleotide having a natural polynucleotide sequence with 90% identity, (iii) a polynucleotide having a sequence complementary to (i), (iv) a polynucleotide complementary to the polynucleotide of (ii), (v ) A probe consisting of at least 20 contiguous nucleotide groups of a polynucleotide selected from the group consisting of the RNA equivalents of (i) to (iv). Hybridization occurs under conditions such that a specific hybridization complex is formed between the probe and a target polynucleotide in the biological sample. The target polynucleotide is (i) a polynucleotide having a polynucleotide sequence selected from the group consisting of SEQ ID NO: 17-32, (ii) a certain selected from the group consisting of SEQ ID NO: 17-32 A polynucleotide having a natural polynucleotide sequence having at least 90% identity to the polynucleotide sequence, (iii) a polynucleotide complementary to the polynucleotide of (i), (iv) complementary to the polynucleotide of (ii) Selected from the group consisting of (v) (i) to (iv) RNA equivalents. Alternatively, the target polynucleotide has a fragment of a polynucleotide sequence selected from the group consisting of (i) to (v) above. The toxicity calculation method further includes the following processes. (C) quantifying the amount of hybridization complex, and (d) comparing the amount of hybridization complex in the treated biological sample with the amount of hybridization complex in the untreated biological sample. is there. Differences in the amount of hybridization complexes in the treated biological sample indicate the toxicity of the test compound.
[0091]
(Description of the present invention)
Although the proteins, nucleotide sequences and methods of the present invention will be described, it should be understood before that that the present invention is not limited to the specific apparatus, materials and methods described and may be modified. Also, the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the invention which is limited only by the claims. Please also understand.
[0092]
As used in the claims and in the specification, the singular forms “a” and “its” may refer to the plural unless the context clearly dictates otherwise. Please note that. Thus, for example, a reference to “a certain host cell” may include a plurality of such host cells, a “a certain antibody” may refer to one or more antibodies, and It also refers to antibody equivalents known to those skilled in the art.
[0093]
All technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art unless otherwise defined. Although any devices, materials, and methods similar or equivalent to those described herein can be used to practice or test the present invention, suitable devices, materials, and methods are now described. All publications referred to in this invention are cited for purposes of describing and disclosing cells, protocols, reagents and vectors that are reported in the publication and that may be relevant to the present invention. . No disclosure in this specification is an admission that the invention is not entitled to antedate such disclosure by virtue of prior art.
[0094]
(Definition)
The term “NAAP” refers to substantially purified NAAP obtained from all species (especially mammals including cattle, sheep, pigs, mice, horses and humans), such as natural, synthetic, semi-synthetic or recombinant. The amino acid sequence of
[0095]
The term “agonist” refers to a molecule that enhances or mimics the biological activity of NAAP. Examples of agonists can include proteins, nucleic acids, carbohydrates, small molecules and any other compound or composition, which can interact directly with NAAP or in a biological pathway involving NAAP. Modulates NAAP activity by acting on components.
[0096]
The term “allelic variant” refers to another form of the gene encoding NAAP. Allelic variant sequences can result from at least one mutation in the nucleic acid sequence, as well as altered mRNAs. It can also give rise to a group of polypeptides, but the structure or function of those polypeptides may or may not change. A gene may not have any of its native allelic variant sequences, or may have more than one. The usual mutational changes that give rise to allelic variants are generally due to natural deletions, additions or substitutions of nucleotides. These various changes can occur one or more times within a sequence, either alone or together with other changes.
[0097]
The sequence included in the “transformed / modified” nucleic acid sequence encoding NAAP is a sequence that has undergone various nucleotide deletions, insertions, or substitutions, and includes the same polypeptide as NAAP, or NAAP There are sequences that give rise to polypeptides with at least one of the functional characteristics of: This definition includes improper or unexpected hybridization to a group of allelic variants at a position that is not a normal chromosomal locus for a polynucleotide sequence encoding NAAP, and also includes a polynucleotide encoding NAAP. Of polymorphisms that are readily detectable or difficult to detect using certain oligonucleotide probes. The encoded protein may also be “transformed / modified” and may have deletions, insertions or substitutions of amino acid residues that result in a silent change resulting in a functionally equivalent NAAP. . Intentional amino acid substitutions are in terms of the polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphipathicity of the residue group, as long as NAAP activity is preserved biologically or immunologically. Can be made based on similarity. For example, negatively charged amino acids include aspartic acid and glutamic acid, and positively charged amino acids include lysine and arginine. Amino acids with uncharged polar side chains with similar hydrophilicity values include asparagine and glutamine, serine and threonine. Amino acids having uncharged side chains with similar hydrophilicity values include leucine, isoleucine and valine, glycine and alanine, and phenylalanine and tyrosine.
[0098]
The terms “amino acid” and “amino acid sequence” refer to oligopeptides, peptides, polypeptides, protein sequences, or fragments of any thereof, and refer to natural or synthetic molecules. When an “amino acid sequence” refers to the sequence of a natural protein molecule, the “amino acid sequence” and similar terms are not limited to the complete and intact amino acid sequence associated with that protein molecule described.
[0099]
The term “amplification” relates to the act of making additional replicas of a nucleic acid sequence. Amplification usually uses polymerase chain reaction (PCR) techniques known to those skilled in the art.
[0100]
The term “antagonist” refers to a molecule that inhibits or attenuates the biological activity of NAAP. Antagonists include proteins such as antibodies, nucleic acids, carbohydrates, small molecules, any molecule that interacts directly with NAAP or acts on components of biological pathways involving NAAP to regulate NAAP activity. Other compounds and compositions can be included.
[0101]
The term `` antibody '' refers to an intact immunoglobulin molecule or fragment thereof capable of binding to an epitope determinant, such as Fab, F (ab ')₂ , And refers to the Fv fragment. For the production of antibodies that bind to the NAAP polypeptide group, an intact polypeptide group can be used as an immunizing antigen, or a fragment group having the small peptide group can be used. The origin of the polypeptide or oligopeptide used to immunize animals such as mice, rats, rabbits may be in the case of RNA translation or chemical synthesis. They can also be conjugated to a carrier protein if desired. Examples of commonly used carriers that chemically bind to peptides include bovine serum albumin, thyroglobulin, and mussel hemocyanin (KLH). This bound peptide is used to immunize the animal.
[0102]
The term “antigenic determinant” refers to a region of a molecule (ie, an epitope) that makes contact with a particular antibody. When immunizing a host animal with a protein or protein fragment, multiple regions of the protein can induce the production of antibodies that specifically bind to an antigenic determinant (a specific region or three-dimensional structure of the protein). Certain antigenic determinants can compete with an intact antigen (ie, an immunogen used to elicit an immune response) for binding to certain antibodies.
[0103]
The term “aptamer” refers to a nucleic acid or oligonucleotide molecule that binds to a specific molecular target. Aptamersin vitro(E.g. SELEX (Systematic Evolution of Ligands by EXponential Enrichment), described in US Pat. No. 5,270,163). This is a process of selecting target-specific aptamer sequences from a large group of combinatorial libraries. Aptamer composition is double-stranded or single-stranded and may include deoxyribonucleotides, ribonucleotides, nucleotide derivatives, or other nucleotide-like molecules. Each nucleotide component of an aptamer may have a modified sugar group (for example, the 2'-OH group of ribonucleotides is 2'-F or 2'-NH₂This may improve desired properties such as resistance to nucleases or a longer lifetime in blood. By conjugating the aptamer with other molecules (eg, high molecular weight carriers), clearance of the aptamer from the circulatory system can be delayed. Aptamers can be specifically cross-linked with their homologous ligands, for example by photoactivation of certain cross-linking agents (see, for example, Brody, EN and L. Gold (2000) J. Biotechnol. 74: 5-13). reference).
[0104]
The term "intramer"in vivoRefers to an aptamer expressed in For example, certain RNA aptamers are expressed at high levels in the cytoplasm of leukocytes using an RNA expression system based on vaccinia virus (Blind, M. et al. (1999) Proc. Natl Acad. Sci. USA 96: 3606-3610).
[0105]
The term “spiegelmer” refers to aptamers such as left-handed nucleotide derivatives such as L-DNA and L-RNA, or left-handed nucleotide-like molecules. Aptamers with levorotatory nucleotides are resistant to degradation by natural enzymes that normally act on substrates with dextrorotatory nucleotides.
[0106]
The term “antisense” refers to any composition capable of forming base pairs with the “sense” (coding) strand of a particular nucleic acid sequence. Antisense compositions include DNA, RNA, peptide nucleic acids (PNA), modified backbone linkages such as phosphorothioic acid, methylphosphonic acid or benzylphosphonic acid, modified sugar groups such as 2 ' There are oligonucleotides with 2-methoxyethyl sugar or 2'-methoxyethoxy sugar, or oligonucleotides with modified bases such as 5-methylcytosine, 2-deoxyuracil or 7-deaza-2'-deoxyguanosine obtain. Antisense molecules can be produced by any method, such as chemical synthesis or transcription. When introduced into a cell, a complementary antisense molecule forms base pairs with the natural nucleic acid sequence produced by the cell and forms a duplex that prevents transcription or translation. The expression “negative” or “minus” may refer to the antisense strand of a reference DNA molecule, and the expression “positive” or “plus” may refer to the sense strand of a reference DNA molecule.
[0107]
The term “biologically active” refers to a protein having the structural, regulatory, or biochemical functions of a natural molecule. Similarly, the term “immunologically active” or “immunogenic” means that natural or recombinant NAAP, synthetic NAAP, or any oligopeptide thereof is suitable for a specific immune response in an appropriate animal or cell. Refers to the ability to bind to a specific antibody.
[0108]
The term “complementary” refers to the relationship between two single-stranded nucleic acid sequences that anneal by base pairing. For example, “5′-AGT-3 ′” forms a pair with its complementary sequence “3′-TCA-5 ′”.
[0109]
“A composition comprising (having) a polynucleotide sequence of” or “a composition comprising (having) an amino acid sequence of” in a broad sense refers to any composition having a specified polynucleotide sequence or amino acid sequence. Point to. The composition can include a dry formulation or an aqueous solution. A composition having a polynucleotide sequence encoding NAAP or a fragment of NAAP can be used as a hybridization probe. This probe can be stored in a lyophilized state and can be bound to a stabilizer such as a carbohydrate. In hybridization, the probe is dispersed in an aqueous solution containing a salt (eg, NaCl), a surfactant (eg, sodium dodecyl sulfate; SDS), and other components (eg, Denhart's solution, milk powder, salmon sperm DNA, etc.) Can do.
[0110]
The “consensus sequence” is subjected to repeated DNA sequence analysis to remove unnecessary bases, extended in the 5 ′ and / or 3 ′ direction using the XL-PCR kit (Applied Biosystems, Foster City CA), and sequenced again. One or more overlapping cDNAs or ESTs using a sliced nucleic acid sequence or a fragment construction computer program such as GELVIEW fragment construction system (GCG, Madison, WI) or Phrap (University of Washington, Seattle WA) Or a nucleic acid sequence constructed from genomic DNA fragments. Some sequences perform both extension and construction to produce a consensus sequence.
[0111]
The term “conservative amino acid substitution” refers to a substitution that is predicted to change little in the properties of the original protein. That is, the structure of the protein, particularly its function, is preserved and does not change significantly by substitution. The table below shows the amino acids that can be substituted for the original amino acids in the protein and are recognized as conservative amino acid substitutions.
Original residue Conservative substitution
Ala Gly, Ser
Arg His, Lys
Asn Asp, Gln, His
Asp Asn, Glu
Cys Ala, Ser
Gln Asn, Glu, His
Glu Asp, Gln, His
Gly Ala
His Asn, Arg, Gln, Glu
Ile Leu, Val
Leu Ile, Val
Lys Arg, Gln, Glu
Met Leu, Ile
Phe His, Met, Leu, Trp, Tyr
Ser Cys, Thr
Thr Ser, Val
Trp Phe, Tyr
Tyr His, Phe, Trp
Val Ile. Leu, Thr
Conservative amino acid substitutions usually include (a) the backbone structure of the polypeptide in the substitution region, eg, a β-sheet or α-helical structure, (b) the molecular charge or hydrophobicity at the substitution site, and / or (c) the side chain. Hold the most.
[0112]
The term “deletion” refers to a change in an amino acid sequence that lacks one or more amino acid residues, or a change in a nucleotide sequence that lacks one or more nucleotides.
[0113]
The term “derivative” refers to a chemically modified polynucleotide or polypeptide. For example, replacement of hydrogen with an alkyl group, acyl group, hydroxyl group or amino group can be included in chemical modification of the polynucleotide. A polynucleotide derivative encodes a polypeptide that retains at least one biological or immunological function of the natural molecule. A polypeptide derivative is a polypeptide that has been modified by the following process. That is, glycosylation, polyethylene glycolation, or any similar process that retains at least one biological or immunological function of the originating polypeptide.
[0114]
“Detectable label” refers to a reporter molecule or enzyme that is covalently or non-covalently bound to a polynucleotide or polypeptide capable of generating a measurable signal.
[0115]
“Differential expression” refers to expression of a gene or protein that is increased (upregulated), decreased (downregulated), or deleted, as determined by comparing at least two samples. Such a comparison can be made, for example, between a treated sample and an untreated sample, or a pathological sample and a healthy sample.
[0116]
“Exon shuffling” means recombination of different coding region (exon) groups. Some exons can present one structural or functional domain of the encoded protein, allowing new protein groups to be constructed with new combinations of stable substructure groups, and new protein functions Can promote the evolution of
[0117]
The term “fragment” refers to a unique portion of a NAAP or NAAP-encoding polynucleotide that is identical in sequence to its parent sequence but shorter in length than the parent sequence. Fragments can have a length that is shorter than the total length of the defined sequence minus 1 nucleotide / amino acid residue. For example, a fragment may have 5 to 1000 consecutive nucleotide or amino acid residues. Some fragments used as probes, primers, antigens, therapeutic molecules or for other purposes are at least 5, 10, 15, 20, 25, 30, 40, 50, 60, 75, 100, It can be 150, 250 or 500 consecutive nucleotide or amino acid residues. Fragments can be preferentially selected from specific regions of a molecule. For example, a polypeptide fragment is a length selected from the first 250 or 500 amino acids (or the first 25% or 50%) of a polypeptide as found within a defined sequence. Can have a continuous amino acid. These lengths are clearly given by way of example, and in embodiments of the invention can be any length supported by this specification including the sequence listing, tables and drawings.
[0118]
Certain fragments of SEQ ID NO: 17-32 have certain unique polynucleotide sequence regions. This region specifically identifies SEQ ID NO: 17-32 and differs from any other sequence in the genome from which this fragment was obtained, for example. Certain fragments of SEQ ID NO: 17-32 are useful, for example, for hybridization or amplification techniques, or for similar methods of distinguishing SEQ ID NO: 17-32 from related polynucleotide sequences. The exact length of a fragment of SEQ ID NO: 17-32 and the region of SEQ ID NO: 17-32 corresponding to that fragment is routinely determined by those skilled in the art based on the intended purpose of the fragment Can be determined.
[0119]
Certain fragments of SEQ ID NO: 1-16 are encoded by certain fragments of SEQ ID NO: 17-32. Certain fragments of SEQ ID NO: 1-16 contain a region of unique amino acid sequence that specifically identifies SEQ ID NO: 1-16. For example, certain fragments of SEQ ID NO: 1-16 are useful as immunogenic peptides in the development of antibodies that specifically recognize SEQ ID NO: 1-16. The exact length of a fragment of SEQ ID NO: 1-16 and the region of SEQ ID NO: 1-16 corresponding to this fragment is routinely determined by those skilled in the art based on the purpose of the fragment. Can do.
[0120]
A “full length” polynucleotide sequence is a sequence having at least one translation start codon (eg, methionine) followed by an open reading frame and a translation stop codon. A “full length” polynucleotide sequence encodes a “full length” polypeptide sequence.
[0121]
The term “homology” means sequence similarity, compatibility, or sequence identity of two or more polynucleotide sequences or two or more polypeptide sequences.
[0122]
The term “percent match” or “% match” as applied to a polynucleotide sequence means the percentage of residues that match between two or more polynucleotide sequences, aligned using a standardized algorithm. Since the standardization algorithm optimizes the alignment between the two sequences, a gap group can be inserted into the two sequences to be compared in a standardized and reproducible manner, so that the two sequences can be compared more significantly.
[0123]
To determine the percent identity between polynucleotide sequences, the default parameters of the CLUSTAL V algorithm built into the MEGALIGN version 3.12e sequence alignment program and the like can be used. This program is part of the LASERGENE software package (a set of molecular biological analysis programs) (DNASTAR, Madison WI). This CLUSTAL V is described in Higgins, D.G. and P.M. Sharp (1989) CABIOS 5: 151-153, Higgins, D.G. et al. (1992) CABIOS 8: 189-191. The default parameters for pairwise alignment of polynucleotide sequences are set as Ktuple = 2, gap penalty = 5, window = 4, “diagonals saved” = 4. A “weighted” residue weight table is selected by default. The percent match reported by CLUSTAL V is made as a “percent similarity” between the aligned polynucleotide sequences.
[0124]
Alternatively, the National Local Center for Biotechnology Information (NCBI) Basic Local Alignment Search Tool (BLAST) provides a set of commonly used and freely available sequence comparison algorithms (Altschul, SF et al. ( 1990) J. Mol. Biol. 215: 403-410). The BLAST algorithm is available from several sources and is also available from NCBI and the Internet (http://www.ncbi.nlm.nih.gov/BLAST/) in Bethesda, Maryland. The BLAST software suite includes a variety of sequence analysis programs, including “blastn” used to align a known polynucleotide sequence with another polynucleotide sequence from various databases. A tool called “BLAST 2 Sequences” is also available, which is used to directly compare two nucleotide sequences. “BLAST 2 Sequences” can be used interactively by accessing http://www.ncbi.nlm.nih.gov/gorf/bl2.html. The “BLAST 2 Sequences” tool can be used for both blastn and blastp (described below). The BLAST program is generally used with parameters such as gaps set to default settings. For example, to compare two nucleotide sequences, blastn can be performed using the “BLAST 2 Sequences” tool Version 2.0.12 (April 21, 2000) set to default parameters. An example of setting default parameters is shown below.
[0125]
Matrix: BLOSUM62
Reward for match: 1
Penalty for mismatch: -2
Open Gap: 5 and Extension Gap: 2 penalties
Gap x drop-off: 50
Expect: 10
Word Size: 11
Filter: on
The percent identity can be measured by the full length of a defined sequence (eg, a sequence defined by a particular SEQ ID number). Alternatively, the percentage match of shorter lengths, eg, lengths of fragments from a defined larger sequence (eg, fragments of at least 20, 30, 40, 50, 70, 100 or at least 200 consecutive nucleotides) Can also be measured. The lengths listed here are merely exemplary, and the percent identity can be measured using any fragment length supported by the sequences described herein, including tables, figures, and sequence listings. It should be understood that a certain length can be described.
[0126]
Nucleic acid sequences that do not show a high degree of identity may nevertheless encode similar amino acid sequences due to the degeneracy of the genetic code. It should be understood that this degeneracy can be used to cause changes in a nucleic acid sequence to produce a large number of nucleic acid sequences in which all nucleic acid sequences encode substantially the same protein.
[0127]
The term “percent match” or “% match” as used for a polypeptide sequence refers to the percentage of matching residues between two or more polypeptide sequences that are aligned using a standardized algorithm. Methods for polypeptide sequence alignment are known. Some alignment methods take into account conservative amino acid substitutions. Such conservative substitutions as detailed above usually conserve the structure of the polypeptide (and therefore also the function) since it preserves the charge and hydrophobicity of the substitution site.
[0128]
The percent identity between polypeptide sequences can be determined using the default parameters of the CLUSTAL V algorithm as incorporated into the MEGALIGN version 3.12e sequence alignment program (above with reference). The default parameters for pairwise alignment of polypeptide sequences using CLUSTAL V are set as Ktuple = 1, gap penalty = 3, window = 5, “diagonals saved” = 5. The PAM250 matrix is selected as the default residue weight table. As with polynucleotide alignment, the percent identity of aligned polypeptide sequence pairs is reported by CLUSTAL V as “percent similarity”.
[0129]
Alternatively, the NCBI BLAST software suite can be used. For example, when comparing two polypeptide sequences pairwise, blastp of the “BLAST 2 Sequences” tool Version 2.0.12 (April 21, 2000) can be used with default parameters set. An example of setting default parameters is shown below.
[0130]
Matrix: BLOSUM62
Open Gap: 11 and Extension Gap: 1 penalties
Gap x drop-off: 50
Expect: 10
Word Size: 3
Filter: on
The percent identity may be measured by the full length of a defined polypeptide sequence (eg, a sequence defined by a specific SEQ ID number). Alternatively, a shorter length, eg, the length of a fragment derived from a defined, larger polypeptide sequence (eg, a fragment of at least 15, 20, 30, 40, 50, 70, or at least 150 consecutive residues). The coincidence rate can also be measured. The lengths listed here are merely exemplary, and the percent identity can be measured using any fragment length supported by the sequences described herein, including tables, figures, and sequence listings. It should be understood that a certain length can be described.
[0131]
A “human artificial chromosome (HAC)” is a linear microchromosome and can have a DNA sequence of about 6 kb to 10 Mb in size. It has all the elements necessary for chromosome replication, segregation and maintenance.
[0132]
The term “humanized antibody” refers to an antibody molecule in which the amino acid sequence of the non-antigen binding region has been modified to resemble a more human antibody while retaining its original binding ability.
[0133]
“Hybridization” is the process of annealing in which a single-stranded polynucleotide forms base pairs with a complementary single strand under predetermined hybridization conditions. Specific hybridization is an indication that two nucleic acid sequences share high complementarity. Specific hybridization complexes are formed under acceptable annealing conditions and remain hybridized after one or more “washing” steps. The washing step is particularly important in determining the stringency of the hybridization process, and under more stringent conditions, nonspecific binding (ie, binding between nucleic acid strand pairs that are not perfectly matched) is reduced. Acceptable conditions for annealing nucleic acid sequences can routinely be determined by those skilled in the art. While the annealing conditions can be constant for any hybridization experiment, the wash conditions can be changed from experiment to experiment to obtain the desired stringency and thus hybridization specificity. Acceptable annealing conditions are, for example, at a temperature of 68 ° C., in the presence of about 6 × SSC, about 1% (w / v) SDS, and about 100 μg / ml shear-denatured salmon sperm DNA. is there.
[0134]
In general, the stringency of hybridization can be expressed to some extent relative to the temperature at which the wash step is performed. Such a washing temperature is usually selected to be about 5 to 20 ° C. lower than the melting point (Tm) of the specific sequence at a predetermined ionic strength and pH. This Tm is the temperature at which 50% of the target sequence hybridizes to a perfectly matched probe under conditions of a predetermined ionic strength and pH. The formula for calculating Tm and hybridization conditions for nucleic acids are well known, Sambrook, J. et al. (1989).Molecular Cloning: A Laboratory Manual, 2nd edition, 1-3, Cold Spring Harbor Press, Plainview NY, see especially chapter 9 in volume 2.
[0135]
Hybridization at high stringency conditions between polynucleotides of the invention includes wash conditions at 68 ° C. for 1 hour in the presence of about 0.2 × SSC and about 0.1% SDS. Alternatively, the temperature may be about 65 ° C, 60 ° C, 55 ° C, or 42 ° C. The SSC concentration can be varied in the range of about 0.1-2 × SSC in the presence of about 0.1% SDS. Usually, a blocking agent is used to prevent nonspecific hybridization. An example of such a blocking agent is sheared denatured salmon sperm DNA at about 100-200 μg / ml. For example, in the hybridization of RNA and DNA under specific conditions, an organic solvent such as formamide at a concentration of about 35-50% v / v can also be used. Useful variations of the wash conditions will be apparent to those skilled in the art. Hybridization can indicate evolutionary similarity between nucleotides, particularly under high stringency conditions. Evolutionary similarity strongly suggests a similar role for nucleotide groups and nucleotide-encoded polypeptides.
[0136]
The term “hybridization complex” refers to a complex of two nucleic acid sequences formed by the formation of hydrogen bonds between complementary bases. Hybridization complexes can be formed in solution (C₀t or R₀t analysis etc.). Alternatively, one nucleic acid sequence is present in a dissolved state and the other nucleic acid sequence is a solid support (eg, paper, membrane, filter, chip, pin or glass slide, or other suitable substrate that is a cell or nucleic acid thereof. Can be formed between two nucleic acid sequences that are immobilized on a substrate to which is immobilized.
[0137]
The term “insertion” or “addition” refers to a change in an amino acid sequence or nucleic acid sequence to which one or more amino acid residues or nucleotides are added, respectively.
[0138]
"Immune response" can refer to symptoms associated with inflammation, trauma, immune disorders, infectious diseases or genetic diseases and the like. These symptoms can be characterized by the expression of various factors that can affect cellular and systemic defense systems, such as cytokines, chemokines, and other signaling molecules.
[0139]
The term “immunogenic fragment” refers to a polypeptide or oligopeptide fragment of NAAP that can elicit an immune response when introduced into an organism (eg, a mammal). The term “immunogenic fragment” also refers to any polypeptide or oligopeptide fragment of NAAP useful in any antibody production method disclosed herein or well known in the art.
[0140]
The term “microarray” refers to the organization of a plurality of polynucleotides, polypeptides or other compounds on a substrate.
[0141]
The term “element” or “array element” refers to a polynucleotide, polypeptide or other compound having a specific designated position on a microarray.
[0142]
The term “modulate” or “modulate activity” refers to altering the activity of NAAP. For example, modulation can cause changes in NAAP protein activity, or binding properties, or other biological, functional, or immunological properties.
[0143]
The terms “nucleic acid” and “nucleic acid sequence” refer to nucleotides, oligonucleotides, polynucleotides or fragments thereof. The terms “nucleic acid” and “nucleic acid sequence” also refer to DNA or RNA of genomic or synthetic origin that is single or double stranded or capable of presenting a sense or antisense strand. It may refer to RNA, peptide nucleic acid (PNA), or any DNA-like or RNA-like substance.
[0144]
“Functionally linked” refers to a state in which a first nucleic acid sequence and a second nucleic acid sequence are in a functional relationship. For example, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Functionally linked DNA sequences can be very close or contiguous, and can be in the same reading frame if necessary to join two protein coding regions.
[0145]
“Peptide nucleic acid (PNA)” refers to an antisense molecule or anti-gene agent having an oligonucleotide of at least about 5 nucleotides in length attached to the peptide backbone of amino acid residues ending in lysine. Terminal lysine provides solubility to this composition. PNA binds preferentially to complementary single-stranded DNA or RNA and stops transcriptional elongation. Polyethylene glycolation can extend the lifetime of PNA in cells.
[0146]
“Post-translational modifications” of NAAP may include lipidation, glycosylation, phosphorylation, acetylation, racemization, proteolytic cleavage, and other modifications known in the art. These processes can occur synthetically or biochemically. Biochemical modifications depend on the NAAP enzyme environment and will vary with cell type.
[0147]
A “probe” refers to a nucleic acid sequence that encodes NAAP, a complementary sequence thereof, or a fragment thereof, and is used to detect the same sequence, an allelic nucleic acid sequence, or a related nucleic acid sequence. A probe is an isolated oligonucleotide or polynucleotide that is a sequence attached to a detectable label or reporter molecule. Typical labels include radioactive isotopes, ligands, chemiluminescent reagents and enzymes. A “primer” is a short nucleic acid, usually a DNA oligonucleotide, that can form complementary base pairs and anneal to a target polynucleotide. The primer can then be extended along the target DNA strand by a DNA polymerase enzyme. Primer pairs can be used for amplification (and identification) of nucleic acid sequences, for example, by polymerase chain reaction (PCR).
[0148]
The probes and primers used in the present invention usually consist of at least 15 consecutive nucleotide groups of known sequence. To increase specificity, longer probes and primers, such as probes consisting of at least 20, 25, 30, 40, 50, 60, 70, 80, 90, 100 or at least 150 consecutive nucleotides of the disclosed nucleic acid sequence And primers may also be used. Some probes and primers are much longer than this. It should be understood that nucleotides of any length supported by this specification, including tables, drawings and sequence listings may be used.
[0149]
See Sambrook, J. et al. (1989) for the preparation and use of probes and primers.Molecular Cloning: A Laboratory Manual, 2nd edition, 1-3, Cold Spring Harbor Press, Plainview NY, Ausubel, F.M., etc. (1987)Current Protocols in Molecular Biology, Greene Publ. Assoc. & Wiley-Intersciences, New York NY, Innis, M. et al. (1990)PCR Protocols, A Guide to Methods and Applications, Academic Press, San Diego CA, etc. In order to obtain a PCR primer pair from a known sequence, for example, a computer program therefor can be used, such as Primer (Version 0.5, 1991, Whitehead Institute for Biomedical Research, Cambridge MA).
[0150]
The selection of oligonucleotides to be used as primers is performed using software well known in the art for such purposes. For example, OLIGO 4.06 software is useful for selecting PCR primer pairs up to 100 nucleotides each, derived from oligonucleotides and up to 5,000 larger polynucleotides up to 32 kilobases of input polynucleotide sequences. It is also useful for analyzing sequences. Similar primer selection programs incorporate additional functionality for extended capabilities. For example, the PrimOU primer selection program (available to the general public from the Genome Center of the University of Texas Southwestern Medical Center in Dallas, Texas) can select specific primers from the megabase sequence, and thus the entire genome range. This is useful for designing primers. Using the Primer3 primer selection program (available from the Whitehead Institute / MIT Center for Genome Research, Cambridge, Mass.), You can enter a “mispriming library” that allows you to specify the sequences you want to avoid as primer binding sites. Primer3 is particularly useful for the selection of oligonucleotides for microarrays (the source code for the latter two primer selection programs can be obtained from their respective sources and modified to meet user-specific needs). The PrimerGen program (publicly available from the UK Human Genome Mapping Project in Cambridge, UK-Resource Center) designs primers based on multiple sequence alignments, thereby maximally conserving or minimally conserving aligned nucleic acid sequences Allows selection of primers that hybridize to any of the regions. Therefore, this program is useful for identifying oligonucleotides and polynucleotide fragments, whether original or conserved. Oligonucleotides and polynucleotide fragments identified by any of the above selection methods identify complete or partially complementary polynucleotides in hybridization techniques, eg, as PCR or sequencing primers, as microarray elements, or in nucleic acid samples Useful as a specific probe. The method for selecting the oligonucleotide is not limited to the above method.
[0151]
A “recombinant nucleic acid” is not a natural sequence, but a sequence that artificially combines two or more separated segments of a sequence. This artificial combination is often achieved by chemical synthesis, but more commonly by artificial manipulation of isolated segments of nucleic acids, for example by genetic engineering techniques such as those described in Sambrook (supra). To do. The term recombinant nucleic acid also includes nucleic acids in which a portion of the nucleic acid has been modified by addition, substitution or deletion. Often recombinant nucleic acids include nucleic acid sequences operably linked to promoter sequences. Such recombinant nucleic acids can be part of a vector that is used, for example, to transform a cell.
[0152]
Alternatively, such a recombinant nucleic acid can be part of a viral vector, which can be based on, for example, vaccinia virus. Such vectors can be used to inoculate a mammal and the recombinant nucleic acid is expressed to induce a protective immune response in the mammal.
[0153]
A “regulatory element” is a nucleic acid sequence normally derived from the untranslated region of a gene, including enhancers, promoters, introns, and 5 ′ and 3 ′ untranslated regions (UTRs). Regulatory elements interact with host or viral proteins that control transcription, translation or RNA stability.
[0154]
A “reporter molecule” is a chemical or biochemical component used to label a nucleic acid, amino acid or antibody. Reporter molecules include radionuclides, enzymes, fluorescent agents, chemiluminescent agents, color formers, substrates, cofactors, inhibitors, magnetic particles and other components known in the art.
[0155]
An "RNA equivalent" for a DNA sequence consists of the same linear nucleotide sequence as the reference DNA sequence, but all the nitrogenous base thymines are replaced with uracil, and the backbone of the sugar chain is deoxyribose. Without ribose.
[0156]
The term “sample” is used in its broadest sense. Samples that are presumed to contain NAAP, NAAP-encoding nucleic acid groups, or fragments thereof include body fluids, cells and cell-isolated chromosomes, organelles, and membrane extracts. There can be cells, genomic DNA, RNA, cDNA, tissue, tissue prints, etc. present in solution or fixed to a substrate.
[0157]
The terms “specific binding” and “specifically bind” refer to the interaction of a protein or peptide with an agonist, antibody, antagonist, small molecule, or any natural or synthetic binding composition. This interaction depends on the presence or absence of a specific structure of the protein (eg, an antigenic determinant or epitope) that is recognized by the binding molecule. For example, if an antibody is specific for epitope “A”, a polypeptide with epitope A may bind or bind in a reaction involving unbound labeled “A” and the antibody. If unlabeled “A” is present, the amount of labeled A bound to the antibody is reduced.
[0158]
The term “substantially purified” refers to an isolated or separated nucleic acid or amino acid sequence that has been removed from its natural environment and has removed at least about 60% of the naturally bound composition. Preferably, about 75% or more is removed, and most preferably 90% or more is removed.
[0159]
“Substitution” is the replacement of one or more amino acid residues or nucleotides with another amino acid residue or nucleotide, respectively.
[0160]
The term “substrate” refers to any suitable solid or semi-solid support, including membranes and filters, chips, slides, wafers, fibers, magnetic or non-magnetic beads, gels, tubes, plates, polymers, microparticles, capillaries. Is included. The substrate can have various surface forms such as wells, grooves, pins, channels, holes, and the like, and polynucleotides and polypeptides are bound to the substrate surface.
[0161]
A “transcription image” or “expression profile” refers to the collective pattern of gene expression by a particular cell type or tissue at a given time under given conditions.
[0162]
“Transformation” refers to the process by which exogenous DNA is introduced into a recipient cell. Transformation can occur under natural or artificial conditions according to various methods known in the art, and is any known method that inserts an exogenous nucleic acid sequence into a prokaryotic or eukaryotic host cell. Based on this method. The method of transformation is selected according to the type of host cell to be transformed. Non-limiting transformation methods include viral infection, electroporation (electroporation), heat shock, lipofection and methods using a particle gun. A “transformed cell” includes a stably transformed cell in which the introduced DNA is replicable as an autonomously replicating plasmid or as part of a host chromosome. Furthermore, cells that transiently express introduced DNA or RNA for a limited period are also included.
[0163]
As used herein, a “genetic transformant” is any organism, including but not limited to animals and plants, in which one or more cells of the organism are known by human intervention, for example, as known in the art. It has heterologous nucleic acid introduced by transformation technique. Nucleic acids are introduced into cells either directly or indirectly by introduction into cell precursors. This is done by planned genetic manipulation, for example by microinjection or by introduction of recombinant viruses. The term genetic engineering means classical crossbreeding orin vitroIt does not refer to fertilization but refers to the introduction of recombinant DNA molecules. Genetic transformants contemplated according to the present invention include bacteria, cyanobacteria, fungi and animals and plants. The isolated DNA of the present invention can be introduced into a host by methods known in the art, such as infection, transfection, transformation or transconjugation. Techniques for transferring the DNA of the present invention into such organisms are well known and are described in references such as Sambrook et al. (1989), supra.
[0164]
A “variant / mutant sequence” of a specific nucleic acid sequence is a sequence determined as a nucleic acid sequence having at least 40% sequence identity to the specific nucleic acid sequence over a certain length of one nucleic acid sequence. It is. For determination, blastn is executed by using “BLAST 2 Sequences” tool Version 2.0.9 (May 7, 1999) set as default parameters. Such nucleic acid pairs are, for example, at least 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95% for a given length, It can show 96%, 97%, 98%, 99% or more sequence identity. Variant sequences can be described, for example, as “allelic” variant sequences (described above) or “splice” variant sequences, “species” variant sequences, “polymorphic” variant sequences. Splice variant sequences may have significant identity with the reference molecule, but will usually have more or fewer polynucleotides due to alternative splicing of exons during mRNA processing. Corresponding polypeptides may have additional functional domain groups or may lack domain groups present in the reference molecule. Species variant sequences are polynucleotide sequences that vary from species to species. The resulting polypeptides usually have significant amino acid identity with each other. A polymorphic variant sequence is a variation within a polynucleotide sequence of a particular gene between certain individuals. Polymorphic variant sequences can also include “single nucleotide polymorphisms” (SNPs) that differ in one nucleotide base of the polynucleotide sequence. The presence of SNPs can indicate, for example, a particular population, condition or disease propensity.
[0165]
A “mutant sequence” of a specific polypeptide sequence was determined as a polypeptide sequence having at least 40% sequence identity to the specific polypeptide sequence over a certain length of one polypeptide sequence. Is an array. For the determination, blastp is executed by using “BLAST 2 Sequences” tool Version 2.0.9 (May 7, 1999) set as default parameters. Such polypeptide pairs are, for example, at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94% for one predetermined length of the polypeptide. 95%, 96%, 97%, 98%, 99% or more sequence identity.
[0166]
(invention)
The present invention is based on the discovery of a novel group of human nucleic acid-related proteins (NAAP) and polynucleotides encoding NAAP. It is also based on the development of diagnosis, treatment and prevention of cell proliferation abnormalities, neurological disorders, developmental disorders, autoimmune / inflammatory disorders and infectious diseases utilizing these compositions.
[0167]
Table 1 summarizes the nomenclature for the full-length polynucleotide and polypeptide sequences of the present invention. Each polynucleotide and its corresponding polypeptide correlates to one Incyte project identification number (Incyte project ID). Each polypeptide sequence was represented by a polypeptide sequence identification number (polypeptide SEQ ID NO :) and an Incyte polypeptide sequence number (Incyte polypeptide ID). Each polynucleotide sequence was represented by a polynucleotide sequence identification number (polynucleotide SEQ ID NO :) and an Incyte polynucleotide consensus sequence number (Incyte polynucleotide ID).
[0168]
Table 2 shows sequences homologous to the polypeptides of the present invention identified by BLAST analysis against the GenBank protein (genpept) database and the PROTEOME database. Columns 1 and 2 show the polypeptide sequence identification number (polypeptide SEQ ID NO :) and the corresponding Incyte polypeptide sequence number (Incyte polypeptide ID) for each polypeptide of the present invention. Column 3 shows the GenBank ID number (Genbank ID NO) of the closest GenBank homologue. Column 4 shows the probability score for a match between each polypeptide and one or more of its homologues. Column 5 indicates appropriate citations where applicable and one or more annotations of the GenBank homologue, all of which are included herein by reference.
[0169]
Table 3 shows various structural features of the polypeptides of the invention. Columns 1 and 2 show the polypeptide sequence identification number (SEQ ID NO :) and the corresponding Incyte polypeptide sequence number (Incyte polypeptide ID) for each polypeptide of the invention. Column 3 shows the number of amino acid residues of each polypeptide. Columns 4 and 5 show potential phosphorylation and glycosylation sites, respectively, as determined by the GCG sequence analysis software package MOTIFS program (Genetics Computer Group, Madison WI). Column 6 shows the amino acid residues that include the signature sequence, domain, and motif. Column 7 shows the analysis method for the analysis of the structure / function of the protein, and the applicable part further shows a searchable database used for the analysis method.
[0170]
Tables 2 and 3 together summarize the properties of each polypeptide of the present invention, which establishes that the claimed polypeptide is a nucleic acid-related protein.
[0171]
For example, SEQ ID NO: 2 has amino acid residues 210 to 768,Haemophilus influenzae) (Rd) Transcription accessory protein (tex) (GenBank ID g1573555) was determined to have 38% identity by the Basic Local Alignment Search Tool (BLAST) (see Table 2). The BLAST probability score is 7.9e-105, indicating the probability that an observed polypeptide sequence alignment will be obtained by chance. SEQ ID NO: 2 also has one S1 RNA binding domain, which searches for statistically significant matches in the conserved protein family domain PFAM database based on the Hidden Markov Model (HMM) (See Table 3). Data from BLIMPS analysis and additional BLAST analysis provide evidence to further confirm that SEQ ID NO: 2 is a transcriptional regulator.
[0172]
In another example, SEQ ID NO: 3 was determined by the Basic Local Alignment Search Tool (BLAST) to be 92% identical to zebrafish emx2 hemeoprotein (GenBank ID g1089816) (see Table 2). The BLAST probability score is 3.2e-126, which indicates the probability that a conserved polypeptide sequence alignment will be obtained by chance. SEQ ID NO: 3 also has one homeobox domain, which searches for statistically significant matches in the PFAM database of conserved protein family domains based on the Hidden Markov Model (HMM) (See Table 3). Data from BLIMPS, MOTIFS, and PROFILESCAN analyzes provide further empirical evidence that SEQ ID NO: 3 is a homeobox protein.
[0173]
In another example, SEQ ID NO: 4 was determined by BLAST analysis to be 38% identical to the Arabidopsis bromodomain-containing transcription factor (GenBank ID g6850321) with a probability score of 4.8e-52 It is. SEQ ID NO: 4 was also determined to have a bromodomain by searching for statistically significant matches in the PFAM database of conserved protein family domains based on the Hidden Markov Model (HMM) . Data from BLIMPS and PROFILESCAN analyzes provide further empirical evidence that SEQ ID NO: 4 is a bromodomain protein.
[0174]
In another example, SEQ ID NO: 5 is determined by BLAST analysis to be 58% identical to human RET finger protein-like 3 (GenBank ID g3417319) with a probability score of 2.1e-83. SEQ ID NO: 5 also has a single RING finger domain (which is characteristic of a transcription factor) in the PFAM database of conserved protein family domains based on the hidden Markov model (HMM). Was determined by searching for a significant match.
[0175]
In another example, SEQ ID NO: 6 is determined by BLAST analysis to be 34% identical to a human acidic finger protein (similar to a C3HC4 type Zn finger protein) (GenBank ID g563127) and the probability score is 1.3e-23. SEQ ID NO: 6 also has a Zn finger domain, determined by searching for statistically significant matches in the conserved PFAM database of conserved protein family domains based on the Hidden Markov Model (HMM) It was. Data from PROFILESCAN analysis and MOTIFS analysis provide further empirical evidence that SEQ ID NO: 6 is a Zn finger protein.
[0176]
In another example, SEQ ID NO: 7 is determined by BLAST analysis to be 52% identical to the human CAGH44 protein (GenBank ID g2565057) and has a probability score of 2.7e-46. SEQ ID NO: 7 also has one fork-head domain, which is statistically significant in the conserved PFAM database of conserved protein family domains based on the Hidden Markov Model (HMM). Determined by searching for a match. Data obtained from BLIMPS, MOTIFS and PROFILESCAN analyzes provide evidence to further support that SEQ ID NO: 7 has a forkhead domain and a Zn finger domain, which is characteristic of DNA binding proteins.
[0177]
In another example, SEQ ID NO: 8 was determined by the Basic Local Alignment Search Tool (BLAST) to be 91% identical to the murine OASIS CREB / ATF family transcription factor (GenBank ID g4519621) (Table 2). reference). The BLAST probability score is 9.8e-251, indicating the probability that the observed polypeptide sequence alignment will be obtained by chance. SEQ ID NO: 8 also has a bZIP transcription factor domain, which searches for statistically significant matches in the conserved protein family domain PFAM database based on the Hidden Markov Model (HMM). (See Table 3). Data from BLIMPS and MOTIFS analysis provide evidence to further confirm that SEQ ID NO: 8 is a transcription factor.
[0178]
In another example, SEQ ID NO: 11 was determined by the Basic Local Alignment Search Tool (BLAST) to be 58% identical to a human Kruppel type Zn finger protein (GenBank ID g4519270) (Table 2). reference). The BLAST probability score is 2.0e-213, indicating the probability that the observed polypeptide sequence alignment will be obtained by chance. SEQ ID NO: 11 also has one KRAB box and one C2H2-type Zn finger domain, which is statistically significant in the conserved protein family domain PFAM database based on the Hidden Markov Model (HMM). Judgment was made by searching for a significant match (see Table 3). Data from BLIMPS and MOTIFS analyzes provide further empirical evidence that SEQ ID NO: 11 is a Zn finger protein.
[0179]
As another example, SEQ ID NO: 14 was determined by the Basic Local Alignment Search Tool (BLAST) to be 64% identical to Drosophila protease (GenBank ID g2791289) (see Table 2). The BLAST probability score is 0.0, indicating the probability that an observed polypeptide sequence alignment will be obtained by chance. SEQ ID NO: 14 also has one reverse transcriptase (RNA-dependent DNA polymerase) domain, one “phage” integrase family signature sequence, and one integrase core domain, which is hidden It was determined by searching for statistically significant matches in the PFAM database of conserved protein family domains based on the Markov model (HMM) (see Table 3). Data from BLIMPS analysis and additional BLAST analysis provide evidence to further confirm that SEQ ID NO: 14 is a retrovirus-related polyprotein.
[0180]
SEQ ID NO: 1, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 15 and SEQ ID NO: 16 were analyzed in the same way. Annotated. The algorithm and parameters for the analysis of SEQ ID NO: 1-16 are described in Table 7.
[0181]
As shown in Table 4, the full-length polynucleotide sequence of the present invention was constructed using a cDNA sequence or a coding (exon) sequence derived from genomic DNA, or any combination of these two types of sequences. Column 1 shows the polynucleotide sequence identification number (polynucleotide SEQ ID NO) and the corresponding Incyte polynucleotide consensus sequence number (Incyte ID) for each polynucleotide of the invention, and the length of each polynucleotide sequence in base pairs. Yes. Column 2 identifies the cDNA and / or genomic sequences used to construct the full-length polynucleotide sequences of the present invention, eg, to identify SEQ ID NO: 17-32, or SEQ ID NO: 17- The starting nucleotide (5 ′) and ending nucleotide (3 ′) positions of a fragment of a polynucleotide sequence useful in hybridization or amplification techniques to distinguish 32 from related polynucleotide sequences are indicated.
[0182]
The polynucleotide fragment described in column 2 of Table 4 may refer to, for example, an Incyte cDNA group derived from, for example, a tissue-specific cDNA library group or a pooled cDNA library group. Alternatively, the polynucleotide fragment described in column 2 may refer to GenBank cDNA or EST that contributed to the construction (assembly) of the full-length polynucleotide sequence. In addition, the polynucleotide fragments listed in column 2 may identify sequences from the ENSEMBL (The Sanger Centre, Cambridge, UK) database (ie, sequences that include the designation “ENST”). Alternatively, the polynucleotide fragments listed in column 2 may be derived from the NCBI RefSeq Nucleotide Sequence Records database (ie, sequences containing the designation “NM” or “NT”) and from the NCBI RefSeq Protein Sequence Records. (Ie, sequences that include the designation “NP”). Alternatively, the polynucleotide fragment listed in column 2 may refer to a set consisting of both cDNA and Genscan predicted exons grouped together by an “exon-stitching” algorithm. For example, FL_XXXXXX_N in the polynucleotide sequence₁_N₂_YYYYY_N_Three_N_Four The sequence identified as is a “stitched” sequence, where XXXXXX is the identification number of the cluster of sequences to which the algorithm is applied, YYYYY is the number of predictions the algorithm creates, and N_{1,2,3 ...}Represents a specific group of exons that were manually edited during the analysis (Example 5reference). Alternatively, the polynucleotide fragments in column 2 may refer to a collection of exons joined together by an “exon-stretching” algorithm. For example, among the polynucleotide sequences, the sequence identified as FLXXXXXX_gAAAAA_gBBBBB_1_N is the “stretch” sequence identification number. Where XXXXXX is the Incyte project identification number, gAAAAA is the GenBank identification number of the human genome sequence to which the `` exon stretching '' algorithm is applied, gBBBBB is the GenBank identification number of the nearest GenBank protein homolog, or NCBI RefSeq identification number, N is specified Refers to exons of (Example 5See). When a RefSeq sequence is used as a protein homolog for the “exon stretching” algorithm, the RefSeq identifier represented by “NM”, “NP”, or “NT” is the GenBank identifier (ie, gBBBBB ) Can be used instead.
[0183]
Alternatively, the prefix code identifies a manually edited component sequence, a component sequence predicted from a genomic DNA sequence, or a component sequence derived from a combined sequence analysis method. The following table lists the constituent sequence prefix codes and examples of sequence analysis methods corresponding to the prefix codes (Examples 4 and 5See).

[0184]
In some cases, Incyte cDNA coverage overlapping with the sequence coverage as shown in Table 4 was obtained to confirm the final consensus polynucleotide sequence, but no corresponding Incyte cDNA identification number was given.
[0185]
Table 5 shows a representative cDNA library for full-length polynucleotide sequences constructed using Incyte cDNA sequences. A representative cDNA library is an Incyte cDNA library, which is most often represented by a group of Incyte cDNA sequences, which were used to construct and confirm the polynucleotide sequences described above. The tissues and vectors used to create the cDNA library are shown in Table 5 and described in Table 6.
[0186]
The present invention also includes NAAP variants. Preferred NAAP variant sequences have at least one functional or structural characteristic of NAAP and are at least about 80% amino acid sequence identity or at least about 90% amino acid sequence identity to the NAAP amino acid sequence. Or a sequence having at least about 95% amino acid sequence identity.
[0187]
The present invention also provides a polynucleotide encoding NAAP. In certain embodiments, the present invention provides a polynucleotide sequence having a sequence selected from the group consisting of SEQ ID NO: 17-32 encoding NAAP. The polynucleotide sequence of SEQ ID NO: 17-32 also includes an equivalent RNA sequence as shown in the sequence listing, where the occurrence of the nitrogen base thymine is replaced by uracil and the sugar backbone is not deoxyribose. Consists of ribose.
[0188]
The present invention also includes variant sequences of polynucleotide sequences encoding NAAP. In particular, such a variant polynucleotide sequence has at least about 70% polynucleotide sequence identity, or at least about 85% polynucleotide sequence identity with a polynucleotide sequence encoding NAAP, and at least There will be as much as about 95% polynucleotide sequence identity. In certain embodiments of the invention, the SEQ ID NO: 17-32 has an amino acid sequence selected from the group consisting of at least about 70%, alternatively at least about 85%, or at least about 95% identity A variant sequence of the polynucleotide sequence having a sequence selected from the group consisting of ID NO: 17-32 is included. Any of the polynucleotide variants described above may encode an amino acid sequence having at least one functional or structural characteristic of NAAP.
[0189]
In yet another example, certain polynucleotide variant sequences of the invention are splice variant sequences of polynucleotide sequences encoding NAAP. Some splice variant sequences may have multiple portions with significant sequence identity to a polynucleotide sequence encoding NAAP, but may result from the addition of a few blocks of sequences resulting from alternative splicing of exons during mRNA processing or Deletions will usually have more or fewer polynucleotides. For some splice variant sequences, less than about 70%, or less than about 60%, or less than about 50% of the polynucleotide sequence identity is found over the entire length with the polynucleotide sequence encoding NAAP. A portion of this splice variant sequence may comprise at least about 70%, or at least about 85%, or at least about 95%, or even 100% of the polynucleotide with each portion of the polynucleotide sequence encoding NAAP. It will have sequence identity. For example, a polynucleotide having the sequence of SEQ ID NO: 29 is a splice variant of a polynucleotide having the sequence of SEQ ID NO: 25. Any of the above splice variant sequences can encode an amino acid sequence having at least one functional or structural characteristic of NAAP.
[0190]
The degeneracy of the genetic code creates a variety of polynucleotide sequences that encode NAAP, some of which are known in the art to have minimal similarity to any known native gene polynucleotide sequences. Will be understood. Thus, the present invention may cover all possible polynucleotide sequence variations that can be produced by selection of combinations based on possible codon selection. These combinations should be considered based on the standard triplet genetic code applied to the native NAAP polynucleotide sequence, with all mutations clearly disclosed.
[0191]
Nucleotide sequences that encode NAAP and its variant sequences are generally hybridizable to nucleotide sequences of native NAAP under suitably selected stringency conditions, but include substantially different codons, such as including non-natural codon groups. It may be beneficial to create a nucleotide sequence encoding NAAP or a derivative thereof having use. Codons can be selected to increase the expression rate of peptides generated in a particular eukaryotic or prokaryotic host based on the frequency with which the host utilizes the particular codon. Another reason for substantially altering the nucleotide sequence encoding NAAP and its derivatives without altering the encoded amino acid sequence is to provide RNA with favorable properties such as longer half-life than transcripts made from the native sequence Sometimes a transcript is made.
[0192]
The present invention also includes a process in which DNA sequences or fragments thereof encoding NAAP and its derivatives are completely generated by synthetic chemistry. After production, this synthetic sequence can be inserted into any of a number of available expression vectors and cell lines using reagents known in the art. In addition, synthetic chemistry can be used to mutagenize sequences encoding NAAP or any fragment thereof.
[0193]
The invention further includes polynucleotide sequences that are capable of hybridizing to the claimed polynucleotide sequences, particularly SEQ ID NO: 17-32 and fragments thereof, under various stringency conditions (eg, Chiez, RM and FZ Regnier (1987) Methods Enzymol. 152: 399-407; Kimmel, AR (1987) Methods Enzymol. 152: 507-511). Hybridization conditions, including annealing and washing conditions, are described in “Definitions”.
[0194]
Methods for DNA sequencing are known in the art, and any of the embodiments of the present invention can use DNA sequencing methods. Enzymes can be used for DNA sequencing methods. For example, Klenow fragment of DNA polymerase I, SEQUENASE (US Biochemical, Cleveland OH), Taq polymerase (Applied Biosystems), thermostable T7 polymerase (Amersham, Pharmacia Biotech, Piscataway NJ) can be used. Alternatively, a polymerase and a proofreading exonuclease can be used in combination, as seen, for example, in the ELONGASE amplification system (Life Technologies, Gaithersburg MD). Preferably, sequence preparation is automated using equipment such as the MICROLAB 2200 liquid transfer system (Hamilton, Reno, NV), the PTC200 thermal cycler (MJ Research, Watertown MA) and the ABI CATALYST 800 thermal cycler (Applied Biosystems). The sequencing is then performed using the ABI 373 or 377 DNA sequencing system (Applied Biosystems), the MEGABACE 1000 DNA sequencing system (Molecular Dynamics, Sunnyvale CA) or other systems known in the art. The resulting sequence is analyzed using various algorithms well known in the art (eg, Ausubel, F.M. (1997)Short Protocols in Molecular Biology, John Wiley & Sons, New York NY, 7.7 units, Meyers, R.A. (1995)Molecular Biology and BiotechnologyWiley VCH, New York NY, pages 856-853).
[0195]
Using various PCR-based methods well-known in the art and partial nucleotide sequences, NAAP-encoding nucleic acid sequences can be extended to detect upstream sequences such as promoters and regulatory elements. obtain. For example, one of the methods that can be used is the restriction site PCR method, which uses a universal primer and a nested primer to amplify an unknown sequence from genomic DNA in a cloning vector (eg, Sarkar, G. ( 1993) PCR Methods Applic. 2: 318-322). Another method is reverse PCR, which amplifies an unknown sequence from a circularized template using a group of primers extending in various directions. The template is obtained from a group of restriction enzymes consisting of a certain known genomic locus and its surrounding sequences (see, eg, Triglia, T. et al. (1988) Nucleic Acids Res. 16: 8186). A third method is capture PCR, which includes PCR amplification of DNA fragments adjacent to known sequences of human and yeast artificial chromosomal DNA (eg Lagerstrom, M. et al. (1991) PCR Methods). (See Applic 1: 111-119). In this method, a recombinant double-stranded sequence can be inserted into an unknown sequence region using multiple restriction enzyme digestion and ligation reactions prior to PCR. Several other methods that can be used to search for unknown sequences are also known in the art (see, eg, Parker, J.D. et al. (1991) Nucleic Acids Res. 19: 30553060). In addition, genomic DNA can be walked using PCR, nested primers, and PROMOTERFINDER libraries (Clontech, Palo Alto CA). This procedure is useful for discovery of intron / exon junctions without the need to screen libraries. All PCR-based methods use commercially available software such as OLIGO 4.06 primer analysis software (National Biosciences, Plymouth MN) or another suitable program, about 22-30 nucleotides in length and about 50% GC content. Thus, the primer group can be designed to anneal to the template at a temperature of about 68 ° C. to 72 ° C.
[0196]
When screening for full-length cDNA groups, it is preferable to use library groups that are size-selected to include larger cDNA groups. In addition, random primer libraries often contain sequences having the 5 ′ region of the gene cluster and are suitable for situations in which an oligo d (T) library cannot produce a full-length cDNA. Genomic libraries may be useful for extending sequences into the 5 ′ non-transcribed regulatory region.
[0197]
Commercially available capillary electrophoresis systems can be used to analyze the size of sequencing or PCR products or confirm their nucleotide sequences. Specifically, capillary sequencing is a flowable polymer for electrophoretic separation, a laser-activated fluorescent dye that is specific for four different nucleotides, and detection of the emitted wavelength. And a CCD camera to be used. The output / light intensity can be converted to an electrical signal using appropriate software (eg, Applied Biosystems GENOTYPER, SEQUENCE NAVIGATOR, etc.). The entire process from sample loading to computer analysis and electronic data display can be computer controlled. Capillary electrophoresis is particularly suitable for sequencing small DNA fragments that are present in small amounts in a particular sample.
[0198]
In another embodiment of the invention, a polynucleotide sequence encoding NAAP or a fragment thereof can be cloned into a recombinant DNA molecule to express NAAP, a fragment or functional equivalent thereof in a suitable host cell. It is. Due to the inherent degeneracy of the genetic code, other DNA sequences encoding substantially the same or functionally equivalent amino acid sequences can be produced and used for NAAP expression.
[0199]
For various purposes, the nucleotide sequences of the present invention can be recombined using a number of methods generally known in the art to modify the sequences encoding NAAP. The various purposes of this recombination include, but are not limited to, cloning of the gene product or modification of processing and / or expression. Nucleotide sequences can be recombined using random fragmentation of gene fragments and synthetic oligonucleotides and DNA shuffling by PCR reassembly. For example, site-directed mutagenesis via oligonucleotides can be used to introduce mutations that create new restriction sites, alter glycosylation patterns, change codon preference, generate splice variant sequences, and the like.
[0200]
Nucleotides of the present invention are disclosed in MOLECULARBREEDING (Maxygen Inc., Santa Clara CA; U.S. Patent No. 5,837,458; Chang, C.-C. et al. (1999) Nat. Biotechnol. 17: 793-797; Christians, FC et al. (1999) Nat. Biotechnol. 17: 259-264; Crameri, A. et al. (1996) Nat. Biotechnol. 14: 315-319). This can modify or improve the biological properties of NAAP, such as biological or enzymatic activity, or the ability to bind other molecules and compounds. DNA shuffling is a process of creating a library of gene mutation sequences using PCR-mediated gene fragment recombination. The library is then subjected to a selection or screening procedure that identifies a group of genetic variants with the desired characteristics. These suitable mutant sequences can then be pooled and further repeated for DNA shuffling and selection / screening. Thus, “artificial” breeding and rapid molecular evolution create diverse genes. For example, single gene fragments with random point mutations can be recombined, screened, and then shuffled until the desired properties are optimized. Alternatively, a group of fragments of a given gene can be recombined with fragments of homologous genes of the same gene family obtained from either the same species or different species, thereby controlling the genetic diversity of multiple naturally occurring genes. Can be maximized in a way.
[0201]
According to another embodiment, the NAAP-encoding sequence can be synthesized in whole or in part using chemical methods well known in the art (eg, Caruthers. MH et al. (1980) Nucl. Acids Res Symp. Ser 7: 215-223; and Horn, T. et al. (1980) Nucl. Acids Res. Symp. Ser. 7: 225-232). Alternatively, NAAP itself or a fragment thereof can be synthesized using chemical methods. For example, peptide synthesis can be performed using various liquid phase or solid phase techniques (eg, Creighton, T. (1984)Proteins, Structures and Molecular Properties, WH Freeman, New York NY, 55-60; and Roberge, J.Y. et al. (1995) Science 269: 202-204). Automated synthesis can be achieved using an ABI 431A peptide synthesizer (Applied Biosystems). Further, certain natural polypeptides may be modified by directly altering the amino acid sequence of NAAP or any part thereof during direct synthesis and / or in combination with sequences from other proteins or any part thereof. Polypeptides or mutant polypeptides having the sequence can be made.
[0202]
This peptide can be substantially purified using preparative high performance liquid chromatography (see, eg, Chiez, R.M. and F.Z. Regnier (1990) Methods Enzymol. 182: 392-421). The composition of this synthetic peptide can be confirmed by amino acid analysis or sequencing (see, for example, Creighton, pages 28-53 above).
[0203]
In order to express biologically active NAAP, a nucleotide sequence encoding NAAP or a derivative thereof may be inserted into a suitable expression vector. A suitable expression vector is a vector having elements necessary for controlling transcription and translation of a coding sequence inserted into a suitable host. Necessary elements include regulatory sequences in the vector and polynucleotide sequences encoding NAAP (enhancers, constitutive and expression-inducible promoters, 5 ′ and 3 ′ untranslated regions, etc.). Necessary elements vary in feature and specificity. Specific initiation signals can be used to achieve more effective translation of NAAP-encoding sequences. Examples of initiation signals include ATG initiation codons and nearby sequences such as Kozak sequences. If the NAAP-encoding sequence, its initiation codon, and upstream regulatory sequences are inserted into a suitable expression vector, no additional transcriptional or translational control signals may be required. However, if only the coding sequence or a fragment thereof is inserted, an exogenous translational control signal such as an in-frame ATG start codon should be included in the expression vector. Exogenous translation elements and initiation codons can originate from a variety of natural and synthetic products. Inclusion of an enhancer suitable for the particular host cell system used can increase the efficiency of expression (see, eg, Scharf, D. et al. (1994) Results Probl. Cell Differ. 20: 125162).
[0204]
Methods which are well known to those skilled in the art can be used to create expression vectors with sequences encoding NAAP and suitable transcriptional and translational control elements. These methods include:in vitroRecombinant DNA technology, synthesis technology, andin vivoGenetic engineering techniques (eg Sambrook, J. et al. (1989)Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, Plainview NY, 4, 8, and 16-17; Ausubel, F.M., et al. (1995)Current Protocols in Molecular Biology, John Wiley & Sons, New York NY, 9, 13, and 16).
[0205]
A variety of expression vector / host systems can be utilized to retain and express sequences encoding NAAP. Such expression vector / host systems include, but are not limited to, microorganisms such as bacteria transformed with recombinant bacteriophage, plasmid or cosmid DNA expression vectors, yeast transformed with yeast expression vectors, Transformed with an insect cell line infected with a viral expression vector (eg baculovirus), a viral expression vector (eg cauliflower mosaic virus, CaMV or tobacco mosaic virus, TMV) or a bacterial expression vector (eg Ti plasmid or pBR322 plasmid) Plant cell lines, animal cell lines (e.g., Sambrook; supra Ausubel; Van Heeke, G. and SM Schuster (1989) J. Biol. Chem. 264: 55035509; Engelhard, EK et al. (1994) Proc. Natl. Acad. Sci. USA 91: 3224-3227; Sandig, V. et al. (1996) Hum.Gene Ther.7: 1937-1945; Takamatsu, N. (1987) EMBO J. 6: 307311; Technology Yearbook "(The McGraw Hill Yearbook of Science and Technology(1992) McGraw Hill New York NY, 191196; Logan, J. and T. Shenk (1984) Proc. Natl. Acad. Sci. USA 81: 36553659; and Harrington, JJ et al. (1997) Nat. Genet. 15: (See 345-355). Nucleotide sequences can be delivered to target organs, tissues or cell populations using expression vectors derived from retroviruses, adenoviruses, herpesviruses or vaccinia viruses, or expression vectors derived from various bacterial plasmids (eg Di Nicola , M. et al. (1998) Cancer Gen. Ther. 5 (6): 350-356; Yu, M. et al. (1993) Proc. Natl. Acad. Sci. USA 90 (13): 6340-6344; Buller, RM (1985) Nature 317 (6040): 813-815; McGregor, DP et al. (1994) Mol.Immunol. 31 (3): 219-226; and Verma, IM and N. Somia (1997) Nature 389: 239- 242). The present invention is not limited by the host cell used.
[0206]
In bacterial systems, a number of cloning and expression vectors may be selected depending upon the use intended for polynucleotide sequences encoding NAAP. For example, a multifunctional E. coli vector such as PBLUESCRIPT (Stratagene, La Jolla CA) or PSPORT1 plasmid (Life Technologies) can be used for routine cloning, subcloning and propagation of a polynucleotide sequence encoding NAAP. Ligation of the NAAP-encoding sequence into the vector's multicloning site disrupts the lacZ gene, allowing a colorimetric screening method for identification of transformed bacteria containing recombinant molecules. Furthermore, these vectors are in the cloned sequencein vitroIt may also be useful for transcription, dideoxy sequencing, single-stranded rescue with helper phage, generation of nested deletions (eg, Van Heeke, G. and SM Schuster (1989) J. Biol. Chem. 264: 55035509 See). For example, when a large amount of NAAP is required for antibody production or the like, a vector that induces NAAP expression at a high level can be used. For example, vectors with a strong inducible SP6 bacteriophage promoter or an inducible T7 bacteriophage promoter can be used.
[0207]
A yeast expression system can also be used to produce NAAP. Many vectors with constitutive or inducible promoters, such as alpha factor, alcohol oxidase, and PGH promoter,Saccharomyces cerevisiae) Or Pichia yeast (Pichia pastoris) Can be used. In addition, such vectors direct either secretion of the expressed protein or retention within the cell, allowing integration of foreign sequences into the host genome for stable growth (e.g., prior to See Ausubel, 1995; Bitter, GA et al. (1987) Methods Enzymol. 153: 516544; and Scorer, CA et al. (1994) Bio / Technology 12: 181-184).
[0208]
Plant systems can also be used for NAAP expression. Transcription of NAAP-encoding sequences can be achieved by viral promoters such as CaMV-derived 35S and 19S as used alone or in combination with omega leader sequences from TMV (Takamatsu, N. (1987) EMBO J 6: 307-311). Promoted by a promoter. Alternatively, plant promoters such as the small subunit of RuBisCO, or heat shock promoters can be used (e.g. Coruzzi, G. et al. (1984) EMBO J. 3: 16711680; Broglie, R. et al. (1984) Science 224: 838843; and Winter, J. et al. (1991) Results Probl. Cell Differ. 17: 85105). These constructs can be introduced into plant cells by direct DNA transformation or by pathogen-mediated transfection (eg, “Mugglow Hill Science and Technology Yearbook” (The McGraw Hill Yearbook of Science and Technology) (1992) McGraw Hill New York NY, see pages 191-196).
[0209]
In mammalian cells, a number of virus-based expression systems can be utilized. When an adenovirus is used as an expression vector, a group of sequences encoding NAAP can be ligated to an adenovirus transcription / translation complex consisting of a late promoter and a triple leader sequence. By inserting into the nonessential E1 or E3 region of the adenovirus genome, infectious viruses that express NAAP in host cells can be obtained (see, eg, Logan, J. and T. Shenk (1984) Proc. Natl. Acad. Sci. USA 81: 36553659). In addition, transcription enhancers such as the Rous sarcoma virus (RSV) enhancer can be used to increase expression in mammalian host cells. Proteins can also be expressed at high levels using vectors based on SV40 or EBV.
[0210]
In addition, human artificial chromosomes (HACs) can be used to deliver fragments of DNA that are larger than fragments that can be contained in a plasmid or that can be expressed from a plasmid. Approximately 6 kb to 10 Mb of HAC is made for therapy and is delivered by conventional delivery methods (liposomes, polycation amino polymers, or vesicles) (eg Harrington, JJ et al. (1997) Nat. Genet. 15: 345). See -355).
[0211]
For long-term production of recombinant proteins in mammalian systems, stable expression of NAAP in cell lines is desirable. For example, expression vectors and selectable marker genes on the same vector or on different vectors can be used to transform NAAP-encoding sequences into cell lines. The expression vector used can have replication elements of viral origin and / or endogenous expression elements. After the introduction of the vector, the cells can be grown in enhanced medium for about 1-2 days before being transferred to the selective medium. The purpose of the selectable marker is to confer resistance to the selective agent, and the presence of the selectable marker allows growth and recovery of cells that successfully express the introduced sequence. Resistant clones of stably transformed cells can be propagated using tissue culture techniques appropriate to the cell type.
[0212]
Any number of selection systems can be used to recover transformed cell lines. Such a selection system is not limited to tk⁻Herpes simplex virus thymidine kinase gene used for cells and apr⁻There are herpes simplex virus adenine phosphoribosyltransferase genes used for cells (see eg Wigler, M. et al. (1977) Cell 11: 223-232; Lowy, I. et al. (1980) Cell 22: 817-823 ). Moreover, tolerance to antimetabolites, antibiotics or herbicides can be used as a basis for selection. For example, dhfr confers resistance to methotrexate, neo confers resistance to aminoglycoside neomycin and G-418, als confers resistance to chlorsulfuron, pat confers resistance to phosphinothricin acetyltransferase (e.g. Wigler, M (1980) Proc. Natl. Acad. Sci. USA 77: 35673570; ColbereGarapin, F. et al. (1981) J. Mol. Biol. 150: 114). Other selectable genes, such as trpB and hisD that modify cellular requirements for metabolism, are also described in the literature (eg Hartman, SC and RC Mulligan (1988) Proc. Natl. Acad. Sci. USA 85: 80478051). Visible markers such as anthocyanins, green fluorescent protein (GFP; Clontech), β-glucuronidase and its substrate β-glucuronide, or luciferase and its substrate luciferin can be used. These markers can be used not only to identify transformants, but also to quantify transient or stable protein expression due to a particular vector system (eg Rhodes, CA (1995) Methods Mol. Biol. 55: 121131).
[0213]
Even if the presence or absence of marker gene expression suggests the presence of the target gene, the presence and expression of the gene may need to be confirmed. For example, if a sequence encoding NAAP is inserted into a marker gene sequence, transformed cells having a sequence encoding NAAP can be identified by the lack of marker gene function. Alternatively, a marker gene can be placed in tandem with one sequence encoding NAAP under the control of a single promoter. Expression of a marker gene in response to induction or selection usually also indicates tandem gene expression.
[0214]
In general, host cells containing a nucleic acid sequence encoding NAAP and expressing NAAP can be identified using a variety of methods well known to those skilled in the art. Non-limiting methods well known to those skilled in the art include DNA-DNA or DNA-RNA hybridization, PCR amplification, and detection, quantification, or both of nucleic acid or protein sequences. There are also protein bioassays or immunoassays, including membrane-based, solution-based or chip-based techniques to do
[0215]
Immunological methods for detecting and measuring NAAP expression using specific polyclonal or specific monoclonal antibodies are known in the art. Examples of such techniques include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), fluorescence activated cell sorting (FACS) and the like. A two-site, monoclonal-based immunoassay using a group of monoclonal antibodies that react with two non-interfering epitopes on NAAP is preferred, but a competitive binding test can also be used. These and other assays are well known in the art (e.g., Hampton, R. et al. (1990)Serological Methods, a Laboratory Manual, APS Press, St. Paul MN, Sect. IV; Coligan, J.E. et al. (1997)Current Protocols in Immunology, Greene Pub.Associates and Wiley-Interscience, New York NY; and Pound, J.D. (1998)Immunochemical Protocols, Humana Press, Totowa NJ).
[0216]
A wide variety of labeling and conjugation methods are known to those skilled in the art and can be used in various nucleic acid and amino acid assays. Methods for generating labeled hybridization probes or PCR probes to detect sequences related to NAAP-encoding polynucleotides include oligo-labeling, nick translation, end-labeling, or labeling PCR amplification using the synthesized nucleotides is included. Alternatively, the NAAP-encoding sequence, or any fragment thereof, can be cloned into a vector for generating mRNA probes. Such vectors are known in the art and are also commercially available, with the addition of a suitable RNA polymerase such as T7, T3 or SP6 and labeled nucleotides,in vitroCan be used for the synthesis of RNA probes. Such methods can be performed using various kits commercially available from, for example, Amersham Pharmacia Biotech, Promega (Madison WI), US Biochemical, and the like. Suitable reporter molecules or labels that can be used to facilitate detection include substrates, cofactors, inhibitors, magnetic particles, as well as radionuclides, enzymes, fluorescent agents, chemiluminescent agents, color formers, and the like.
[0217]
Host cells transformed with a nucleotide sequence encoding NAAP are cultured under conditions suitable for expression and recovery of this protein in cell culture media. Whether a protein produced from a transformed cell is secreted or remains intracellular depends on the sequence used, the vector, or both. Those skilled in the art will appreciate that expression vectors having NAAP-encoding polynucleotides can be designed to have a signal sequence that directs secretion of DME across a prokaryotic or eukaryotic membrane.
[0218]
Furthermore, selection of the host cell line may be made by its ability to modulate the expression of the inserted sequences or to process the expressed protein in the desired form. Such polypeptide modifications include, but are not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation and acylation. Post-translational processing that cleaves the “prepro” or “pro” form of the protein can be used to identify induction, folding, and / or activity of the protein to the target. Various host cells (eg CHO, HeLa, MDCK, MEK293, WI38, etc.) with specific cellular equipment and characteristic mechanisms for post-translational activity are available from the American Type Culture Collection (ATCC, Manassas, VA). And can be selected to ensure correct modification and processing of the foreign protein.
[0219]
In another embodiment of the invention, in any host system described above, by linking a natural nucleic acid sequence, a modified nucleic acid sequence, or a recombinant nucleic acid sequence encoding NAAP to a heterologous sequence, Translation of certain fusion proteins can occur. For example, a chimeric NAAP protein that contains a heterologous moiety that can be recognized by a commercially available antibody can facilitate the screening of peptide libraries for inhibitors of NAAP activity. Also, heterologous protein portions and heterologous peptide portions can facilitate the purification of the fusion protein using a commercially available affinity matrix. Such moieties include, but are not limited to, glutathione S transferase (GST), maltose binding protein (MBP), thioredoxin (Trx), calmodulin binding peptide (CBP), 6-His, FLAG, c-myc, There is hemagglutinin (HA). GST on immobilized glutathione, MBP on maltose, Trx on phenylarsine oxide, CBP on calmodulin, and 6-His on metal chelate resins allow for purification of cognate fusion proteins. FLAG, c-myc and hemagglutinin (HA) allow immunoaffinity purification of fusion proteins using commercially available monoclonal and polyclonal antibodies that specifically recognize these epitope tags. Also, if the fusion protein is genetically engineered to have a proteolytic cleavage site between the sequence encoding NAAP and the heterologous protein sequence, NAAP can be cleaved from the heterologous portion after purification. Methods for expression and purification of the fusion protein are described in Ausubel (1995) chapter 10 above. Various commercially available kits can also be used to facilitate the expression and purification of the fusion protein.
[0220]
In a further embodiment of the invention, a TNT rabbit reticulocyte lysate or wheat germ extract system (Promega) is used.in vitroThus, it is possible to synthesize radiolabeled NAA. These systems combine transcription and translation of protein coding sequences operably linked to a T7, T3 or SP6 promoter. Translation is for example³⁵Occurs in the presence of a radiolabeled amino acid precursor such as S-methionine.
[0221]
The NAAP or fragment thereof of the present invention can be used to screen for compounds that specifically bind to NAAP. At least one or more test compounds can be used to screen for specific binding to NAAP. Test compounds include antibodies, oligonucleotides, proteins (eg receptors) or small molecules.
[0222]
In certain embodiments, the compound thus identified is closely related to a natural ligand of NAAP, such as a ligand or a fragment thereof, or a natural substrate, a structural or functional mimetic, Or a natural binding partner (e.g. Coligan, JE et al. (1991)Current Protocols in Immunology 1 (2): See Chapter 5). Similarly, the compound may be closely related to the natural receptor to which NAAP binds, or at least some fragment of the receptor, such as the ligand binding site. In either case, the compound can be rationally designed using known techniques. In some embodiments, screening for these compounds includes the generation of suitable cell populations that express NAAP as secreted proteins or on the cell membrane. Suitable cells include cells from mammals, yeast, Drosophila, or E. coli. Cells expressing NAAP or cell membrane fractions containing NAAP are contacted with a test compound and analyzed for binding, stimulation, or inhibition of activity of either NAAP or this compound.
[0223]
Some assays simply allow the test compound to be conjugated experimentally to the polypeptide and the binding detected by a fluorescent dye, radioisotope, enzyme conjugate or other detectable label. For example, the assay can include mixing at least one test compound with NAAP in solution or immobilized on a solid support, and detecting binding of NAAP to the compound. Alternatively, detection and measurement of test compound binding in the presence of labeled competitor can be performed. In addition, this assay can be performed using cell-free reconstituted specimens, chemical libraries, or natural product mixtures, where the test compound (s) are released in solution or immobilized on a solid support.
[0224]
The NAAP or fragment thereof of the present invention can be used to screen for compounds that modulate NAAP activity. Examples of such compounds include agonists, antagonists, partial agonists or inverse agonists. In certain embodiments, the assay is performed under conditions in which NAAP binds at least one test compound and the activity of NAAP is tolerated, and the activity of NAAP in the presence of the test compound and in the absence of the test compound. Compare with NAAP activity. A change in NAAP activity in the presence of the test compound suggests the presence of a compound that modulates NAAP activity. Alternatively, the test compound comprises NAAP under conditions suitable for NAAP activity.in vitroAlternatively, the assay is performed in conjunction with a cell release system. In any of these assays, the test compound that modulates NAAP activity may be indirectly regulated, in which case no direct contact with the test compound is required. At least one or more test compounds can be screened.
[0225]
In another embodiment, homologous recombination in embryonic stem cells (ES cells) is used to “knock out” a polynucleotide encoding NAAP or a mammalian homolog thereof in an animal model system. Such techniques are well known in the art and are useful for the production of animal models of human disease (see, eg, US Pat. Nos. 5,175,383 and 5,767,337). For example, mouse ES cells such as the 129 / SvJ cell line are derived from early mouse embryos and can be grown in culture. This ES cell is transformed with a vector containing the gene of interest disrupted with a marker gene such as the neomycin phosphotransferase gene (neo: Capecchi, M.R. (1989) Science 244: 1288-1292). This vector is integrated into the corresponding region of the host genome by homologous recombination. Alternatively, homologous recombination is performed using the Cre-loxP system, and the target gene is knocked out in a tissue-specific or developmental stage-specific manner (Marth, JD (1996) Clin. Invest. 97: 1999-2002; KU et al. (1997) Nucleic Acids Res. 25: 4323-4330). Transformed ES cells are identified and microinjected into mouse cell blastocysts collected from, for example, the C57BL / 6 mouse strain. These blastocysts are surgically introduced into pseudopregnant females, the genetic traits of the resulting chimeric progeny are identified, and they are crossed to create heterozygous or homozygous systems. The transgenic animals produced in this way can be tested with potential therapeutics and toxic drugs.
[0226]
Polynucleotide encoding NAAPin vitroCan be manipulated in ES cells derived from human blastocysts. Human ES cells have the potential to differentiate into at least eight separate cell lineages, including endoderm, mesoderm and ectoderm cell types. These cell lines differentiate into, for example, neural cells, hematopoietic lineages and cardiomyocytes (Thomson, J.A. et al. (1998) Science 282: 1145-1147).
[0227]
Polynucleotides encoding NAAP can also be used to create “knock-in” humanized animals (pigs) or transgenic animals (mouse or rat) modeled on human disease. Using knock-in technology, a region of a polynucleotide encoding NAAP is injected into animal ES cells and the injected sequence is integrated into the animal cell genome. Transformed cells are injected into blastula and transplanted as described above. Test for genetically modified progeny or inbreds and treat with potential medicines to obtain information on the treatment of human disease. Alternatively, mammalian inbred lines that overexpress LMM, such as secreting NAAP into milk, can be a convenient source of protein (Janne, J. et al. (1998) Biotechnol. Annu. Rev. 4:55 -74).
[0228]
(Treatment)
There are chemical and structural similarities, for example in the context of sequences and motifs, between the region of NAAP and the region of nucleic acid-related proteins. Tissues expressing NAAP are also closely associated with prostate and lung tumor tissues, and closely associated with umbilical cord blood and cord blood dendritic cells, and brain tissue. See Table 6 for examples of these organizations. Thus, NAAP is thought to play a role in cell proliferation abnormalities, neurological disorders, developmental disorders, autoimmune / inflammatory diseases, and infectious diseases. In the treatment of diseases associated with increased NAAP expression or activity, it is desirable to reduce NAAP expression or activity. It is also desirable to increase NAAP expression or activity in the treatment of diseases associated with decreased NAAP expression or activity.
[0229]
Thus, in one embodiment, NAAP or a fragment or derivative thereof can be administered to a subject for the treatment or prevention of a disease associated with decreased NAAP expression or activity. Such diseases include, but are not limited to, cell proliferation abnormalities, neurological disorders, developmental disorders, autoimmune / inflammatory diseases, and infectious diseases, which include actinic keratosis, arteriosclerosis, atheroma Sclerosis, bursitis, cirrhosis, hepatitis, mixed connective tissue disease (MCTD), myelofibrosis, paroxysmal nocturnal hemoglobinuria, polycythemia vera, psoriasis, primary thrombocythemia, and adenocarcinoma, Cancers such as leukemia, lymphoma, melanoma, myeloma, sarcoma, and teratocarcinoma, specifically adrenal gland, bladder, bone, bone marrow, brain, breast, neck, gallbladder, ganglion, gastrointestinal tract, heart, kidney, liver Includes lung, muscle, ovary, pancreas, parathyroid gland, penis, prostate, salivary gland, skin, spleen, testis, thymus, thyroid, and uterine cancer. Neurological disorders include epilepsy, ischemic cerebrovascular disorder, stroke , Brain tumor, Alzheimer's disease, Pick's disease, Huntington's disease, Dementia, Parkinson's disease and other extrapyramidal disorders, amyotrophic lateral sclerosis and other motor neuron disorders, progressive neuromuscular atrophy, retinitis pigmentosa (retinitis pigmentosa), hereditary ataxia, frequent occurrence Sclerosis and other demyelinating diseases, bacterial and viral meningitis, brain abscess, subdural abscess, epidural abscess, purulent intracranial thrombophlebitis, myelitis and radiculitis, viral Central nervous system diseases and prion diseases (including Kuru, Creutzfeldt-Jakob disease, and Gerstmann-Straussler-Scheinker syndrome), lethal familial insomnia, neurotrophic and metabolic diseases, neurofibromatosis, nodules Sclerosis, cerebelloretinal hemangioblastomatosis, cerebral trigeminal vascular syndrome, central nervous system mental retardation and other developmental disorders, cerebral palsy, neuroskeletal disorders, autonomic nervous system disorders, cranial nerve disorders Spinal cord disease, muscular dystrophy and other neuromuscular disorders, peripheral neuropathy, dermatomyositis and polymyositis, hereditary, metabolic, endocrine and toxic myopathy, myasthenia gravis, periodic limb paralysis, mental disorders ( Mood, anxiety disorder, schizophrenia / schizophrenia), seasonal emotional disorder (SAD), restlessness, amnesia, catatonia, diabetic neuropathy, tardive dyskinesia, dystonia, paranoid psychosis, postherpetic zoster Neuralgia, Tourette's disease, developmental disorders include tubular acidosis, anemia, Cushing syndrome, chondrogenic dystrophy, Duchenne / Becker muscular dystrophy, epilepsy, gonadal malformation, WAGR syndrome (Wilms tumor, no (Iris, genitourinary abnormalities, mental retardation), Smith- Magenis syndrome, myelodysplastic syndrome, hereditary mucosal epithelium Seizure disorders such as dysplasia, hereditary keratosis, hereditary neuropathies such as Charcot-Marie-Tooth disease and neurofibromatosis, hypothyroidism, hydrocephalus, Syndenham's chorea and cerebral palsy , Spina bifida, anencephaly, craniotomy, congenital glaucoma, cataract, sensorineural hearing loss, autoimmune / inflammatory diseases include acquired immune deficiency syndrome (AIDS), Addison's disease (chronic primary adrenal insufficiency) ), Adult respiratory distress syndrome, allergy, ankylosing spondylitis, amyloidosis, anemia, asthma, atherosclerosis, autoimmune hemolytic anemia, autoimmune thyroiditis, autoimmune multiglandular endocrine candidiasis ectoderm Dystrophies (APECED), bronchitis, cholecystitis, contact dermatitis, Crohn's disease, atopic dermatitis, dermatomyositis, diabetes, emphysema, lymphocytic factor-induced incident lymphopenia Hemolytic disease (fetal erythroblastosis), erythema nodosum, atrophic gastritis, glomerulonephritis, Goodpasture syndrome, gout, Graves' disease, Hashimoto's thyroiditis, hypereosinocytosis, irritable bowel syndrome, multiple occurrences Systemic sclerosis, myasthenia gravis, myocardial or pericardial inflammation, osteoarthritis, osteoporosis, pancreatitis, polymyositis, psoriasis, Reiter syndrome, rheumatoid arthritis, scleroderma, Sjogren's syndrome, systemic anaphylaxis, systemic Lupus erythematosus, systemic scleroderma, thrombocytopenic purpura, ulcerative colitis, uveitis, Werner syndrome, cancer complications, hemodialysis, extracorporeal circulation, protozoal infection, helminth infection, trauma, infection Adenoviruses, arenaviruses, bunyaviruses, caliciviruses, coronaviruses, filoviruses, hepadnaviruses, herpes virus Infection by viral pathogens classified as Rus, Flavivirus, Orthomyxovirus, Parvovirus, Papovavirus, Paramyxovirus, Picornavirus, Poxvirus, Reovirus, Retrovirus, Rhabdovirus, Togavirus, and infection by bacteria (Grams including pneumococcal infections, staphylococci, streptococci, bacillus, corynebacterium, clostridium, meningococcus, gonorrhea, listeria, molaxella, kingella, hemophilus, legionella, pertussis and gram, salmonella, campylobacter Negative enterobacteriaceae, Pseudomonas, Vibrio, Brucella, wild gonorrhoeae, Yersinia, Bartonella, norcardium, actinomycetes, mycobacterium, spirochaetale, rickettsia, chlamydia, mycoplasma), fungal infection (classification is Spergillus, Blast myces, Dermatophytes, Cryptococcus, Coccidioides, malasezzia, histoplasma, or other fungal pathogens, parasite infection (classified as Plasmodium or Plasmodium parasite, parasitic amoeba, Leishmania, trypanosoma, toxoplasma, Pneumocystis carinii, intestinal protozoa (such as Giardia), Trichomonas, tissue nematode (such as Trichinella), intestinal parasitic nematode (such as roundworm), lymphatic filaria nematode, fluke (such as schistosomiasis), and tapeworm (such as tapeworm) )) Is included.
[0230]
In another embodiment, a vector capable of expressing NAAP or a fragment or derivative thereof for the treatment or prevention of diseases associated with decreased NAAP expression or activity, including but not limited to the diseases listed above. Can be administered to a subject.
[0231]
In yet another embodiment, a composition having substantially purified NAAP for the treatment or prevention of diseases associated with decreased NAAP expression or activity, including but not limited to the diseases listed above. The product can be administered to a subject together with a suitable pharmaceutical carrier.
[0232]
In yet another embodiment, an agonist that modulates NAAP activity is provided to a subject for the treatment or prevention of a disease associated with a decrease in NAAP expression or activity, including but not limited to the diseases listed above. Can be administered.
[0233]
In a further example, NAAP antagonists may be administered to a subject for the treatment or prevention of diseases associated with increased NAAP expression or activity. Examples of such diseases include, but are not limited to, cell proliferation abnormalities, neurological disorders, developmental disorders, autoimmune / inflammatory diseases, and infectious diseases as described above. In one embodiment, an antibody that specifically binds to NAAP can be used directly as an antagonist or indirectly as a targeting or delivery mechanism that delivers the drug to cells or tissues that express NAAP.
[0234]
In another example, a vector expressing a complementary sequence of a polynucleotide encoding NAAP is administered to a subject to treat a disease associated with increased NAAP expression or activity, including but not limited to the diseases described above. Can be treated or prevented.
[0235]
In another embodiment, any protein, antagonist, antibody, agonist, complementary sequence, or vector of the invention can be administered in combination with another suitable therapeutic agent. Suitable therapeutic agents for use in combination therapy can be selected by one skilled in the art according to conventional pharmaceutical principles. When combined with a therapeutic agent, it can provide a synergistic effect in the treatment or prevention of the various diseases described above. This method makes it possible to increase the pharmaceutical effect with a small amount of each drug, thereby reducing the possibility of side effects.
[0236]
An antagonist of NAAP can be produced using methods generally known in the art. Specifically, antibodies can be made using purified NAAP or a library of therapeutic agents can be screened to identify those that specifically bind to NAAP. Antibodies to NAAP can also be produced using methods known in the art. Such antibodies can include, but are not limited to, polyclonal antibodies, monoclonal antibodies, chimeric antibodies, single chain antibodies, Fab fragments, and fragments produced by Fab expression libraries. Neutralizing antibodies (ie, antibodies that inhibit dimer formation) are usually suitable for therapy.
[0237]
For the production of antibodies, various hosts such as goat, rabbit, rat, mouse, human and others can be immunized by injection of NAAP or any fragment or injection of its oligopeptide with immunogenic properties. . Depending on the host species, various adjuvants can be used to enhance the immune response. Such adjuvants include, but are not limited to, Freund's adjuvant, mineral gel adjuvants such as aluminum hydroxide, lysolecithin, pluronic polyol, polyanion, peptide, oily emulsion, mussel hemocyanin (KLH), dinitrophenol, etc. There is a surfactant. Among adjuvants used in humans, BCG (Bacille Calmette-Guerin) and Corynebacterium parvum (Corynebacterium parvumIs particularly preferred.
[0238]
The oligopeptide, peptide or fragment used for inducing an antibody against NAAP preferably has an amino acid sequence consisting of at least about 5 amino acids, and generally consists of about 10 or more amino acids. These oligopeptides, peptides or fragments are preferably identical to part of the amino acid sequence of the natural protein. Short stretches of NAAP amino acids can be fused with sequences of other proteins such as KLH to generate antibodies to this chimeric molecule.
[0239]
Monoclonal antibodies against NAAP can be made using any technique that produces antibody molecules by continuous cell lines in the culture medium. Such technologies include, but are not limited to, hybridoma technology, human B cell hybridoma technology, and EBV-hybridoma technology (eg, Kohler, G. et al. (1975) Nature 256: 495497; Kozbor, D. et al. ( (1985) J. Immunol. Methods 81: 31-42; Cote, RJ et al. (1983) Proc. Natl. Acad. Sci. USA 80: 20262030; and Cole, SP et al. (1984) Mol. Cell Biol. 62: 109- (See 120).
[0240]
In addition, techniques developed for the production of “chimeric antibodies”, such as splicing mouse antibody genes into human antibody genes, can be used to obtain molecules with suitable antigen specificity and biological activity (eg Morrison , SL et al. (1984) Proc. Natl. Acad. Sci. USA 81: 68516855; Neuberger, MS et al. (1984) Nature 312: 604608; and Takeda, S. et al. (1985) Nature 314: 452-454). Alternatively, NAAP-specific single chain antibodies are generated using methods described in the art and applying the described techniques for the production of single chain antibodies. Antibodies with related specificity but different idiotype composition can also be generated by chain shuffling from random combinations of immunoglobulin libraries (eg, Burton DR (1991) Proc. Natl. Acad. Sci. USA). 88: 10134-10137).
[0241]
Antibody production in lymphocyte populationsin vivoIt can also be done by induction of production or by screening a library or panel of immunoglobulins of very specific binding reagents as disclosed in the literature (e.g. Orlandi, R. et al. (1989) Proc. Natl. Acad. Sci. USA 86: 38333837; Winter, G. et al. (1991) Nature 349: 293-299).
[0242]
Antibody fragments that contain specific binding sites for NAAP can also be produced. For example, without limitation, such fragments include F (ab ′) produced by pepsin digestion of antibody molecules.₂ Fragment and F (ab ')₂ And Fab fragments made by reducing the disulfide bridges of the fragments. Alternatively, a Fab expression library can be generated to quickly and easily identify monoclonal Fab fragments with the desired specificity (see, eg, Huse, WD et al. (1989) Science 246: 1275-1281) . (1989) Science 246: 1275-1281 etc.).
[0243]
A variety of immunoassays (immunoassays) can be screened to identify antibodies with the desired specificity. Numerous protocols for immunoradiometric assays or competitive binding assays using either polyclonal or monoclonal antibodies with established specificity are known in the art. Such immunoassays usually involve the measurement of complex formation between NAAP and its specific antibody. A two-site monoclonal-based immunoassay using a group of monoclonal antibodies reactive to two non-interfering NAAP epitopes is commonly used, but competitive binding studies are also available (Pound, supra).
[0244]
Various methods such as Scatchard analysis in conjunction with radioimmunoassay techniques can be used to assess the affinity of an antibody for NAAP. The affinity is represented by a binding constant Ka, which is a value obtained by dividing the molar concentration of the NAAP antibody complex under equilibrium conditions by the molar concentration of free antibody and free antigen. Polyclonal antibodies have heterogeneous affinities for various NAAP epitopes, and Ka as determined for a given polyclonal antibody reagent represents the average affinity or binding activity of NAAP antibody groups. Monoclonal antibodies are monospecific for certain NAAP epitopes, and the Ka determined for certain reagents of the monoclonal antibody represents a true measure of affinity. Ka value is 10⁹-10¹²L / mol high affinity antibody reagents are preferably used in immunoassays where the NAAP-antibody complex must withstand harsh manipulations. Ka value is 10⁶-10⁷The liter / mol low affinity antibody reagent is preferably used for immunopurification and similar treatments where NAAP must eventually dissociate from the antibody in an activated state (Catty, D. (1988)Antibodies, Volume I: A Practical ApproachIRL Press, Washington, DC; Liddell, J. E. and Cryer, A. (1991)A Practical Guide to Monoclonal Antibodies, John Wiley & Sons, New York NY).
[0245]
The antibody titer and binding activity of the polyclonal antibody reagent can be further evaluated to determine the quality and suitability of such a reagent for certain applications used later. For example, polyclonal antibody reagents containing at least 1-2 mg / ml of specific antibody, preferably 5-10 mg / ml of specific antibody are generally used in processes where NAAP-antibody complexes must be precipitated. Antibody specificity, antibody titer, binding activity, and guidelines for antibody quality and use in various applications are generally available (see, eg, Catty, Coligan et al., Supra).
[0246]
In another embodiment of the invention, a polynucleotide encoding NAAP, or any fragment or complementary sequence thereof, can be used for therapeutic purposes. In some embodiments, gene expression can be modified by designing sequences or antisense molecules (DNA, RNA, PNA, or modified oligonucleotides) that are complementary to the coding and regulatory regions of the gene encoding NAAP. obtain. Such techniques are well known in the art, and antisense oligonucleotides or larger fragments can be designed from various positions along the control region of the sequence encoding NAAP, or along the coding region (eg, Agrawal, S., Editing (1996)Antisense Therapeutics, Humana Press Inc., Totawa NJ).
[0247]
For use in therapy, any gene delivery system suitable for introducing an antisense sequence into a suitable target cell can be used. Antisense sequences can be delivered into cells in the form of expression plasmids that produce sequences complementary to at least a portion of the cellular sequence encoding the target protein during transcription (eg, Slater, JE et al. (1998) J. Allergy). Clin. Immunol. 102 (3): 469-475 and Scanlon, KJ et al. (1995) 9 (13): 1288-1296). Antisense sequences can also be introduced into cells using viral vectors such as retroviruses and adeno-associated virus vectors (eg, Miller, AD (1990) Blood 76: 271, supra, Ausubel, Uckert, W And W. Walther (1994) Pharmacol. Ther. 63 (3): 323-347). Other gene delivery mechanisms include liposome systems, artificial viral envelopes and other systems known in the art (eg, Rossi, JJ (1995) Br. Med. Bull. 51 (1): 217-225). Boado, RJ et al. (1998) J. Pharm. Sci. 87 (11): 1308-1315; Morris, MC et al. (1997) Nucleic Acids Res. 25 (14): 2730-2736).
[0248]
In another embodiment of the invention, a polynucleotide encoding NAAP may be used for somatic or germ cell gene therapy. Gene therapy treats (i) gene deficiency (eg, severe combined immunodeficiency (SCID) − characterized by X chromosome linkage inheritance (Cavazzana-Calvo, M. et al. (2000) Science 288: 669-672) X1 disease), severe combined immunodeficiency syndrome associated with congenital adenosine deaminase (ADA) deficiency (Blaese, RM et al. (1995) Science 270: 475-480, Bordignon, C. et al. (1995) Science 270: 470-475), Cystic fibrosis (Zabner, J. et al. (1993) Cell 75: 207-216: Crystal, RG et al. (1995) Hum. Gene Therapy 6: 643-666, Crystal, RG et al. (1995) Hum Gene Therapy 6: 667-703), thalassamia, familial hypercholesterolemia, and hemophilia caused by factor VIII or factor IX deficiency (Crystal, RG (1995) Science 270: 404-410 , Verma, IM and N. Somia (1997) Nature 389: 239-242), (ii) expressing a conditional lethal gene product (eg in the case of cancer resulting from uncontrolled cell growth), (i ii) Intracellular parasites such as human immunodeficiency virus (HIV) (Baltimore, D. (1988) Nature 335: 395-396, Poeschla, E. et al. (1996) Proc. Natl. Acad. Sci. USA. 93 : 11395-11399) and other human retroviruses, hepatitis B or C virus (HBV, HCV),Candida albicansandParacoccidioides brasiliensisAnd the like, and proteins having a protective function against parasitic protozoa such as Plasmodium falciparum and Trypanosoma cruzi can be expressed. When a gene deficiency required for NAAP expression or regulation causes a disease, NAAP can be expressed from a suitable population of introduced cells to alleviate the expression of symptoms caused by the gene deficiency.
[0249]
In a further embodiment of the invention, diseases and abnormalities caused by NAAP deficiency are treated by preparing mammalian expression vectors encoding NAAP and introducing these vectors into NAAP-deficient cells by mechanical means.in vivoOrex vitroThe mechanical introduction techniques used in the cells include (i) direct microinjection of DNA into individual cells, (ii) gene guns, (iii) liposome-mediated transfection, (iv) receptors Mediated gene transfer and (v) use of DNA transposons (Morgan, RA and WF Anderson (1993) Annu. Rev. Biochem. 62: 191-217, Ivics, Z. (1997) Cell 91: 501-510 Boulay, JL. And H. Recipon (1998) Curr. Opin. Biotechnol. 9: 445-450).
[0250]
Expression vectors that can affect NAAP expression include, but are not limited to, PCDNA 3.1, EPITAG, PRCCMV2, PREP, PVAX, PCR2-TOPOTA vectors (Invitrogen, Carlsbad CA), PCMV-SCRIPT, PCMV-TAG, PEGSH / PERV (Stratagene, La Jolla CA), PTET-OFF, PTET-ON, PTRE2, PTRE2-LUC, PTK-HYG (Clontech, Palo Alto CA) are included. To express DME, (i) a constitutively active promoter (eg, cytomegalovirus (CMV), rous sarcoma virus (RSV), SV40 virus, thymidine kinase (TK), or β-actin gene) (Ii) inducible promoters (eg tetracycline regulatable promoters (Gossen, M. and H. Bujard (1992) Proc. Natl. Acad. Sci, contained in the commercially available T-REX plasmid (Invitrogen)) USA 89: 5547-5551; Gossen, M. et al. (1995) Science 268: 1766-1769; Rossi, FMV and HM Blau (1998) Curr. Opin. Biotechnol. 9: 451-456)), ecdysone-inducible promoter. (Included in commercially available plasmids PVGRXR and PIND: Invitrogen), FK506 / rapamycin-inducible promoter, or RU486 / mifepristone-inducible promoter (Rossi, FMV and HM Blau, supra), or iii) from normal individuals may use native promoter or a tissue specific promoter of an endogenous gene encoding NAAP.
[0251]
By using a commercially available liposome transformation kit (for example, Invitrogen's PerFect Lipid Transfection Kit), those skilled in the art do not need much effort to optimize the parameters of the experiment, and the polynucleotide group can be used as the target cell group in culture. Can be delivered. Alternatively, the calcium phosphate method (Graham. F.L. and A.J. Eb (1973) Virology 52: 456-467) or electroporation (Neumann, B. et al. (1982) EMBO J. 1: 841845). In order to introduce DNA into primary culture cells, modifications to standardized mammalian transfection protocols are required.
[0252]
In another embodiment of the invention, a disease or disorder caused by a gene defect associated with NAAP expression is encoded by (i) NAAP under the control of a retroviral long terminal repeat (LTR) promoter or some independent promoter. A Rev responsive element (RRE) with a polynucleotide, (ii) a suitable group of RNA packaging signals, (iii) additional retroviral cis-acting RNA sequences, and coding sequences necessary for efficient vector propagation And a retroviral vector consisting of Retroviral vectors (eg PFB and PFBNEO) are commercially available from Stratagene and are based on published data (Riviere, I. et al. (1995) Proc. Natl. Acad. Sci. USA 92: 6733-6737). This document is specifically incorporated herein by reference. This vector is propagated in a suitable vector producing cell line (VPCL). VPCL expresses envelope genes with affinity for receptors on each target cell, or pan-affinity envelope proteins such as VSVg (Armentano, D. et al. (1987) J. Virol. 61: 1647-1650, Bender, MA et al. (1987) J. Virol. 61: 1639-1646, Adam, MA and AD Miller (1988) J. Virol. 62: 3802-3806, Dull, T. et al. (1998) J. Virol. 72: 8463-8471, Zufferey, R. et al. (1998) J. Virol. 72: 9873-9880). US Pat. No. 5,910,434 to “Rigg” (“Method for obtaining retrovirus packaging cell lines producing high transducing efficiency retroviral supernatant”) discloses a method for obtaining a group of retroviral packaging cell lines, which is incorporated herein by reference. As part of Retroviral vector propagation, cell population (eg CD4⁺ Transduction of T cells) and return of the transduced cells to the patient is a method well known to those skilled in the art of gene therapy and has been described in numerous references (Ranga, U. et al. (1997). J. Virol. 71: 7020-7029; Bauer, G. et al. (1997) Blood 89: 2259-2267; Bonyhadi, ML (1997) J. Virol. 71: 4707-4716; Ranga, U. et al. (1998) Proc Natl. Acad. Sci. USA 95: 1201-1206; Su, L. (1997) Blood 89: 2283-2290).
[0253]
Alternatively, a delivery system of adenoviral gene therapy is used to deliver a group of polynucleotides encoding NAAP to a group of cells having one or more genetic abnormalities associated with NAAP expression. The production and packaging of adenoviral vectors is well known to those skilled in the art. Replication-deficient adenovirus vectors have been demonstrated to be able to be used in a variety of ways to import genes encoding various immunoregulatory proteins into intact islets (Csete, ME et al. (1995) Transplantation 27: 263-268). Potentially useful adenoviral vectors are described in US Pat. No. 5,707,618 (“Adenovirus vectors for gene therapy”) to Armentano, which is incorporated herein by reference. See also Antinozzi, P.A. et al. (1999) Annu. Rev. Nutr. 19: 511-544 and Verma, I.M. and N. Somia (1997) Nature 18: 389: 239-242 for adenoviral vectors. Both documents are hereby incorporated by reference.
[0254]
Alternatively, a herpes gene therapy delivery system is used to deliver a polynucleotide encoding NAAP to target cells having one or more genetic abnormalities associated with NAAP expression. Herpes simplex virus (HSV) -based vectors seem particularly beneficial when introducing NAAP into central neurons where HSV has an affinity. The production and packaging of herpes vectors is known to those skilled in the art. A replication competent herpes simplex virus (HSV) type I vector has been used to deliver a reporter gene to the primate eye (Liu, X. et al. (1999) Exp. Eye Res. 169: 385395). The construction of HSV-1 viral vectors is also disclosed in detail in US Pat. No. 5,804,413 (“Herpes simplex virus strains for gene transfer”) to DeLuca, which is incorporated herein by reference. And US Pat. No. 5,804,413 describes the use of recombinant HSV d92 with a genome having at least one foreign gene introduced into a cell under the control of a suitable promoter for purposes such as human gene therapy. The patent also discloses the generation and use of recombinant HSV lines lacking ICP4, ICP27, and ICP22. For HSV vectors, see also Goins, W.F. et al. (1999) J. Virol. 73: 519-532 and Xu, H. et al. (1994) Dev. Biol. 163: 152-161. Both documents are hereby incorporated by reference. Manipulation of cloned herpesvirus sequences, production of recombinant virus after transfection with multiple plasmids with various segments of large herpesvirus genomes, herpesvirus growth and proliferation, and cell populations Infection with herpesviruses is a technique known to those skilled in the art.
[0255]
Alternatively, certain alphavirus (positive single stranded RNA virus) vectors are used to deliver a group of polynucleotides encoding NAAP to a group of target cells. Biological studies of the prototype alphavirus Semliki Forest Fever Virus (SFV) have been extensive and gene transfer vectors based on the SFV genome (Garoff, H. and K.-J. Li (1998 ) Curr. Opin. Biotechnol. 9: 464-469). During the replication of alpha viral RNA, subgenomic RNA is produced that normally encodes the viral capsid proteins. Since this subgenomic RNA replicates to a higher level than the full-length genomic RNA, the capsid proteins are overproduced compared to viral proteins with enzymatic activity (eg, proteases and polymerases). Similarly, by introducing a sequence encoding NAAP into a region encoding the capsid of the α virus genome, RNA encoding a large number of NAAP is produced in the vector-introduced cell, and NAAP is synthesized at a high level. Usually, infection with alpha virus is accompanied by cell lysis within a few days. On the other hand, the ability of a group of normal hamster kidney cells (BHK-21) having a certain variant of Sindbis virus (SIN) to establish a persistent infection can apply lytic replication of α viruses to gene therapy (Dryga, SA et al. (1997) Virology 228: 74-83). Since α viruses can be introduced into various hosts, NAAP can be introduced into various types of cells. Specific transduction of a subset of cells in a population may require cell sorting prior to transduction. Methods for treating alphavirus infectious cDNA clones, alphavirus cDNA and RNA transfection methods, and alphavirus infection methods are known to those skilled in the art.
[0256]
It is also possible to inhibit gene expression using an oligonucleotide derived from the transcription start site. The transcription start site is, for example, between about −10 and about +10 from the start site. Similarly, inhibition can be achieved using triple helix base pairing methods. Triple helix base pairing is useful because it inhibits the ability of the double helix to open sufficiently for the binding of polymerases, transcription factors or regulatory molecules. Recent therapeutic advances using triple helix DNA are described in the literature (e.g. Gee, J.E. et al. (1994) in: Huber, B.E. and B.I.Carr,Molecular and Immunologic Approaches, Futura Publishing, Mt. Kisco, NY, pages 163-177). In addition, complementary sequences or antisense molecules can be designed to block the translation of mRNA by preventing transcripts from binding to ribosomes.
[0257]
Ribozymes are enzymatic RNA molecules. Ribozymes can be used to catalyze the specific cleavage of RNA. The mechanism of ribozyme action involves sequence specific hybridization of the ribozyme molecule to complementary target RNA and subsequent internal nucleotide strand breaks. For example, it is possible that an artificially produced hammerhead ribozyme molecule can specifically and effectively catalyze internal nucleotide strand breakage of a sequence encoding NAAP.
[0258]
To identify specific ribozyme cleavage sites within any potential RNA target, the target molecule is scanned for ribozyme cleavage sites such as GUA, GUU, and GUC sequences. Once identified, assess whether a short RNA sequence of 15-20 ribonucleotides corresponding to the region of the target gene with the cleavage site has secondary structural features that render the oligonucleotide dysfunctional. Is possible. Assessment of the suitability of candidate targets can also be performed by testing accessibility to hybridization with complementary oligonucleotide groups using a ribonuclease protection assay.
[0259]
The complementary ribonucleic acid molecules and ribozymes of the present invention can be made using any method well known in the art for nucleic acid molecule synthesis. As a production method, there is a method of chemically synthesizing oligonucleotides such as solid phase phosphoramidite chemical synthesis. Alternatively, the DNA sequence encoding NAAPin vitroandin vivoRNA molecules can be produced by transcription. Such DNA sequences can be incorporated into a variety of vectors using a suitable RNA polymerase promoter such as T7 or SP6. Alternatively, these cDNA constructs that synthesize complementary RNA constitutively or inducibly can be introduced into cell lines, cells or tissues.
[0260]
RNA molecules can be modified to increase intracellular stability and half-life. Possible modifications include, but are not limited to, the addition of flanking sequences at the 5 'end, 3' end, or both of the molecule, or phosphorothioate or 2 'O rather than phosphodiesterase linkages in the main chain of the molecule. -Use of methyl is included. This concept is originally in the production of PNA groups, but can be extended to all these molecules. For this purpose, acetyl-, methyl-, thio- and similar modifications of adenine, cytidine, guanine, thymine, and uridine that are not easily recognized by endogenous endonucleases, or unconventional bases such as inosine , Queosine, wybutosine.
[0261]
Further embodiments of the invention include methods for screening for compounds effective in altering the expression of a polynucleotide encoding NAAP. Compounds that may be effective in causing, but not limited to, modification of expression of specific polynucleotides include oligonucleotides, antisense oligonucleotides, triplex-forming oligonucleotides, polypeptide transcriptional regulators such as transcription factors, and specific There are non-polymeric chemical entities that can interact with polynucleotide sequences. Effective compounds can alter polynucleotide expression by acting as either inhibitors or enhancers of polynucleotide expression. Thus, in the treatment of diseases associated with increased NAAP expression or activity, compounds that specifically inhibit the expression of polynucleotides encoding NAAP are therapeutically useful and are associated with decreased NAAP expression or activity. In the treatment of disease, compounds that specifically promote expression of a polynucleotide encoding NAAP may be therapeutically useful.
[0262]
At least one or more test compounds can be screened for effectiveness in altering the expression of a particular polynucleotide. Any method known in the art can be used to obtain the test compound. As an acquisition method, there is chemical modification of a known compound effective in the following cases. Rationalize compounds based on chemical and / or structural characteristics of the target polynucleotide, when modifying the expression of the polynucleotide, selecting from a library of existing, commercial or dedicated, natural or non-natural compounds And selecting from a library of compounds generated combinatorially or randomly. A sample having a polynucleotide encoding NAAP is exposed to at least one of the test compounds thus obtained. Samples include, for example, intact cells, permeabilized cells,in vitroThere can be a cell-free or reconstituted biochemical system. Alterations in the expression of a polynucleotide encoding NAAP are assayed by any method well known in the art. Usually, the expression of a specific nucleotide is detected by hybridization using a probe having a nucleotide sequence complementary to the sequence of a polynucleotide encoding NAAP. Quantifying the amount of hybridization can form the basis for comparison of expression of polynucleotides that are exposed to or not exposed to one or more test compounds. Detection of a change in the expression of the polynucleotide exposed to the test compound indicates that the test compound is effective in altering the expression of the polynucleotide. Screening for compounds effective for altered expression of certain polynucleotides can be performed, such as fission yeast (Schizosaccharomyces pombe) Gene expression system (Atkins, D. et al. (1999) US Pat. No. 5,932,435, Arndt, GM et al. (2000) Nucleic Acids Res. 28: E15) or human cell lines such as HeLa cells (Clarke, ML et al. (2000) Biochem. Biophys. Res. Commun. 268: 8-13). Certain embodiments of the present invention involve screening a combinatorial library of oligonucleotides (deoxyribonucleotides, ribonucleotides, peptide nucleic acids, modified oligonucleotides) for antisense activity against specific polynucleotide sequences ( Bruice, TW et al. (1997) US Pat. No. 5,686,242, Bruice, TW et al. (2000) US Pat. No. 6,022,691).
[0263]
Numerous methods for introducing vectors into cells or tissues are available,in vivo,in vitroandex vivoIs equally suitable for use.ex vivoIn the case of treatment, the vector can be introduced into stem cells taken from the patient, cloned and expanded back to the patient by autologous transplantation. Delivery by transfection or by ribosome injection or polycation amino polymers can be carried out using methods well known in the art (eg Goldman, CK et al. (1997) Nat. Biotechnol. 15: 462- 466).
[0264]
Any of the above treatment methods can be applied to all subjects in need of treatment including mammals such as humans, dogs, cats, cows, horses, rabbits, monkeys, and the like.
[0265]
Certain further embodiments of the invention relate to the administration of certain compositions generally having an active ingredient formulated with a pharmaceutically acceptable excipient. Excipients include, for example, sugar, starch, cellulose, gum and protein. Various prescriptions are widely known, detailsRemington's Pharmaceutical Sciences(Maack Publishing, Easton PA). Such compositions consist of NAAP, NAAP antibodies, mimetics, agonists, antagonists, inhibitors, and the like.
[0266]
The compositions used in the present invention can be administered by any number of routes including, but not limited to, oral, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, There are intraventricular, lung, transdermal, subcutaneous, intraperitoneal, intranasal, intestinal, topical, sublingual or rectal.
[0267]
Compositions administered from the lungs can be prepared in liquid or dry powder form. Such compositions are usually aerosolized just before the patient inhales. In the case of small molecules (eg traditional low molecular weight organic drugs), aerosol delivery of fast acting formulations is known in the art. In the case of macromolecules (eg, larger peptides and proteins), recent improvements in pulmonary delivery through the alveolar region of the lung in the art can substantially transport drugs such as insulin into the blood circulation. (See Patton, JS et al., US Pat. No. 5,997,848, etc.). Pulmonary delivery is superior in administration without needle injection and eliminates the need for penetration enhancers that may be toxic.
[0268]
Compositions suitable for use in the present invention include compositions that contain as much active ingredient as necessary to achieve a given purpose. The determination of an effective dose is within the ability of those skilled in the art.
[0269]
A variety of specially shaped composition groups can be prepared to deliver macromolecules with NAAP or fragments thereof directly into cells. For example, a liposomal formulation comprising a cell-impermeable polymer can facilitate cell fusion and intracellular delivery of the polymer. Alternatively, NAAP or a fragment thereof can be attached to the cationic N-terminal part of the HIV Tat-1 protein. The fusion proteins thus produced have been shown to transduce cell groups of all tissues, including the brain, of a mouse model system (Schwarze, SR et al. (1999) Science 285: 1569- 1572).
[0270]
For any compound, a therapeutically effective dose can be estimated initially in cell culture assays, eg, cell culture assays of neoplastic cells, or in animal models such as mice, rats, rabbits, dogs, monkeys or pigs . The animal model may also be used to determine the appropriate concentration range and route of administration. Such information can then be used to determine beneficial doses and routes for administration to humans.
[0271]
A therapeutically effective dose refers to the amount of an active formulation ingredient, such as NAAP or a fragment thereof, an antibody of NAAP, an agonist or antagonist of NAAP, an inhibitor, etc., that restores symptoms and conditions. Therapeutic efficacy and toxicity can be determined by standard pharmaceutical techniques in cell culture or animal experiments, for example ED₅₀(Therapeutically effective amount of 50% of the population) or LD₅₀(50% lethal dose of population) Can be determined by calculating statistics. The dose ratio of toxic effect to therapeutic effect is the therapeutic index, and LD₅₀/ ED₅₀It can be expressed as a ratio. Compositions that exhibit high therapeutic indices are desirable. Data obtained from cell culture assays and animal experiments are used in the formulation of dosage ranges for human use. The dosage contained in such compositions has little or no toxicity, and ED₅₀It is preferable to be in the range of the blood concentration that contains Depending on the mode of administration used, patient sensitivity and route of administration, the dosage will vary within this range.
[0272]
The exact dosage will be determined by the practitioner in light of factors related to the subject that requires treatment. Dosage forms and dosages are adjusted to provide sufficient levels of the active ingredient or to maintain the desired effect. Factors related to the subject may include the severity of the disease, the patient's general health, the patient's age, weight and gender, time and frequency of administration, drug formulation, response sensitivity and response to treatment. Long-acting compositions may be administered once every 3-4 days, once a week, or once every two weeks, depending on the half-life and clearance rate of the particular formulation.
[0273]
The usual dose depends on the route of administration, but is about 0.1 to 100,000 μg, up to a total of about 1 g. Guidance on specific doses and delivery methods is described in the literature and is usually available to practitioners. One skilled in the art will utilize a formulation for nucleotides that is different from that for proteins or their inhibitors. Similarly, delivery of polynucleotides or polypeptides will be specific to particular cells, symptoms, sites, etc.
[0274]
(Diagnosis)
In another example, an antibody that specifically binds to NAAP is used to diagnose a disorder characterized by NAAP expression or to monitor patients treated with NAAP or an agonist or antagonist or inhibitor of NAAP. Can be used in the assay. Antibodies useful for diagnostic purposes are formulated in the same manner as described above for the treatment site. NAAP diagnostic assays include methods of detecting NAAP from human body fluids or from cell or tissue extracts using antibodies and labels. The antibody can be modified or unmodified and can be labeled covalently or non-covalently with a reporter molecule. A wide variety of reporter molecules are known in the art and can be used, some of which have been described above.
[0275]
Various protocols for measuring NAAP, such as ELISA, RIA, FACS, etc., are well known in the art and provide a basis for diagnosing altered or abnormal levels of NAAP expression. Normal or standard NAAP expression values are obtained by mixing body fluids or cell extracts collected from normal mammals, such as human subjects, with antibodies to NAAP under conditions suitable for complex formation. To confirm. The amount of standard complex formation can be quantified by various methods such as photometry. The amount of NAAP expression in subject, control, and disease samples from biopsy tissue is compared to a standard value. The deviation between the standard value and the subject establishes the parameter for diagnosing the disease.
[0276]
According to another embodiment of the present invention, a polynucleotide encoding NAAP may be used for diagnosis. Polynucleotides that can be used include oligonucleotide sequences, complementary RNA and DNA molecules, and PNA. These polynucleotides can be used to detect and quantify gene expression in biopsy tissues where NAAP expression can be correlated with disease. This diagnostic assay is used to determine the presence of NAAP, as well as overexpression, and to monitor the regulation of NAAP levels during treatment.
[0277]
In certain embodiments, hybridization with PCR probes capable of detecting polynucleotide sequences, such as genomic sequences, encoding NAAP or closely related molecules can be used to identify nucleic acid sequences encoding NAAP. . Whether the probe is made from a highly specific region (eg, a 5 ′ regulatory region) or a slightly less specific region (eg, a conserved motif), The stringency of hybridization or amplification will determine whether the probe will identify only the native sequence encoding NAAP, or whether it will identify allelic variants or related sequences.
[0278]
Probes can also be used to detect related sequences and can have at least 50% sequence identity with any sequence encoding NAAP. The hybridization probe of the present invention of interest can be DNA or RNA, and can be derived from the sequence of SEQ ID NO: 17-32, or a genomic sequence including the promoter, enhancer, and intron of the NAAP gene.
[0279]
As a method for preparing a hybridization probe specific for DNA encoding NAAP, there is a method of cloning a polynucleotide sequence encoding NAAP or a NAAP derivative into a vector for preparing an mRNA probe. Vectors for making mRNA probes are known to those skilled in the art and are commercially available, by adding a suitable RNA polymerase and a suitable labeled nucleotide,in vitroCan be used to synthesize RNA probes. Hybridization probes can be labeled with various reporter populations. Examples of reporter groups include³²P or³⁵Examples include enzyme labels such as alkaline phosphatase in which a radionuclide such as S is bound to a probe via an avidin / biotin binding system.
[0280]
A polynucleotide sequence encoding NAAP may be used in the diagnosis of diseases associated with NAAP expression. Such diseases include, but are not limited to, cell proliferation abnormalities, neurological disorders, developmental disorders, autoimmune / inflammatory diseases, and infectious diseases, which include actinic keratosis, arteriosclerosis, atheroma Sclerosis, bursitis, cirrhosis, hepatitis, mixed connective tissue disease (MCTD), myelofibrosis, paroxysmal nocturnal hemoglobinuria, polycythemia vera, psoriasis, primary thrombocythemia, and adenocarcinoma, Cancers such as leukemia, lymphoma, melanoma, myeloma, sarcoma, and teratocarcinoma, specifically adrenal gland, bladder, bone, bone marrow, brain, breast, neck, gallbladder, ganglion, gastrointestinal tract, heart, kidney, liver Includes lung, muscle, ovary, pancreas, parathyroid gland, penis, prostate, salivary gland, skin, spleen, testis, thymus, thyroid, and uterine cancer. Neurological disorders include epilepsy, ischemic cerebrovascular disorder, stroke , Brain tumor, Alzheimer's disease, Pick's disease, Huntington's disease, Dementia, Parkinson's disease and other extrapyramidal disorders, amyotrophic lateral sclerosis and other motor neuron disorders, progressive neuromuscular atrophy, retinitis pigmentosa (retinitis pigmentosa), hereditary ataxia, frequent occurrence Sclerosis and other demyelinating diseases, bacterial and viral meningitis, brain abscess, subdural abscess, epidural abscess, purulent intracranial thrombophlebitis, myelitis and radiculitis, viral Central nervous system diseases and prion diseases (including Kuru, Creutzfeldt-Jakob disease, and Gerstmann-Straussler-Scheinker syndrome), lethal familial insomnia, neurotrophic and metabolic diseases, neurofibromatosis, nodules Sclerosis, cerebelloretinal hemangioblastomatosis, cerebral trigeminal vascular syndrome, central nervous system mental retardation and other developmental disorders, cerebral palsy, neuroskeletal disorders, autonomic nervous system disorders, cranial nerve disorders Spinal cord disease, muscular dystrophy and other neuromuscular disorders, peripheral neuropathy, dermatomyositis and polymyositis, hereditary, metabolic, endocrine and toxic myopathy, myasthenia gravis, periodic limb paralysis, mental disorders ( Mood, anxiety disorder, schizophrenia / schizophrenia), seasonal emotional disorder (SAD), restlessness, amnesia, catatonia, diabetic neuropathy, tardive dyskinesia, dystonia, paranoid psychosis, postherpetic zoster Neuralgia, Tourette's disease, and developmental disorders include tubular acidosis, anemia, Cushing's syndrome, chondrogenic dwarfism, Duchenne / Becker muscular dystrophy, epilepsy, gonadal dysplasia, WAGR syndrome (Wilms tumor, no (Iris, genitourinary abnormalities, mental retardation), Smith- Magenis syndrome, myelodysplastic syndrome, hereditary mucosal epithelium Seizure disorders such as dysplasia, hereditary keratosis, hereditary neuropathies such as Charcot-Marie-Tooth disease and neurofibromatosis, hypothyroidism, hydrocephalus, Syndenham's chorea and cerebral palsy , Spina bifida, anencephaly, craniotomy, congenital glaucoma, cataract, sensorineural hearing loss, autoimmune / inflammatory diseases include acquired immune deficiency syndrome (AIDS), Addison's disease (chronic primary adrenal insufficiency) ), Adult respiratory distress syndrome, allergy, ankylosing spondylitis, amyloidosis, anemia, asthma, atherosclerosis, autoimmune hemolytic anemia, autoimmune thyroiditis, autoimmune multiglandular endocrine candidiasis ectoderm Dystrophies (APECED), bronchitis, cholecystitis, contact dermatitis, Crohn's disease, atopic dermatitis, dermatomyositis, diabetes, emphysema, lymphocytic factor-induced accidental lymphopenia, newborn Hemolytic disease (fetal erythroblastosis), erythema nodosum, atrophic gastritis, glomerulonephritis, Goodpasture syndrome, gout, Graves' disease, Hashimoto's thyroiditis, hypereosinocytosis, irritable bowel syndrome, multiple occurrences Systemic sclerosis, myasthenia gravis, myocardial or pericardial inflammation, osteoarthritis, osteoporosis, pancreatitis, polymyositis, psoriasis, Reiter syndrome, rheumatoid arthritis, scleroderma, Sjogren's syndrome, systemic anaphylaxis, systemic Lupus erythematosus, systemic scleroderma, thrombocytopenic purpura, ulcerative colitis, uveitis, Werner syndrome, cancer complications, hemodialysis, extracorporeal circulation, protozoal infection, helminth infection, trauma, infection Adenoviruses, arenaviruses, bunyaviruses, caliciviruses, coronaviruses, filoviruses, hepadnaviruses, herpes virus Infection by viral pathogens classified as Rus, Flavivirus, Orthomyxovirus, Parvovirus, Papovavirus, Paramyxovirus, Picornavirus, Poxvirus, Reovirus, Retrovirus, Rhabdovirus, Togavirus, and infection by bacteria (Grams including pneumococcal infections, staphylococci, streptococci, bacillus, corynebacterium, clostridium, meningococcus, gonorrhea, listeria, molaxella, kingella, hemophilus, legionella, pertussis and gram, salmonella, campylobacter Negative enterobacteriaceae, Pseudomonas, Vibrio, Brucella, wild gonorrhoeae, Yersinia, Bartonella, norcardium, actinomycetes, mycobacterium, spirochaetale, rickettsia, chlamydia, mycoplasma), fungal infection (classification is Spergillus, Blast myces, Dermatophytes, Cryptococcus, Coccidioides, malasezzia, histoplasma, or other fungal pathogens, parasitic infection (classified as Plasmodium or Plasmodium parasite, parasitic amoeba, Leishmania, trypanosoma, toxoplasma, Pneumocystis carinii, intestinal protozoa (such as Giardia), Trichomonas, tissue nematode (such as Trichinella), intestinal parasitic nematode (such as roundworm), lymphatic filaria nematode, fluke (such as schistosomiasis), and tapeworm (such as tapeworm) )) Is included. Polynucleotide sequences encoding NAAP can be obtained from Southern, Northern, dot blot, or other membrane-based technologies, PCR methods, and the like that utilize body fluids or tissues collected from patients to detect altered NAAP expression. Can be used for dipstick, dipstick, pin, and multi-format ELISA format assays, and microarrays. Such qualitative or quantitative methods are known in the art.
[0281]
In certain embodiments, nucleotide sequences encoding NAAP may be useful in assays that detect related disorders, particularly those mentioned above. Nucleotide sequences encoding NAAP can be labeled by standard methods and added to body fluids or tissue samples taken from patients under conditions suitable for the formation of hybridization complexes. After a suitable incubation period, the sample is washed and the signal is quantified and compared to a standard value. If the amount of signal in the patient sample is significantly changed compared to the control sample, the presence of a level change in the NAAP-encoding nucleotide sequence group in the sample is indicative of the presence of the associated disorder. Such assays can also be used to estimate specific therapeutic effects in animal experiments, clinical trials, or to monitor individual patient treatment.
[0282]
In order to provide a basis for diagnosis of diseases associated with NAAP expression, a normal or standard profile of expression is established. This is accomplished by mixing body fluids or cells extracted from either normal animal or human subjects with sequences encoding NAAP or fragments thereof under conditions suitable for hybridization or amplification. obtain. Standard hybridization can be quantified by comparing values obtained from experiments conducted with known amounts of substantially purified polynucleotides to values obtained from normal subjects. The standard value thus obtained can be compared with the value obtained from a sample obtained from a patient showing signs of disease. Deviation from standard values is used to determine the presence of the disease.
[0283]
Once the presence of the disease is established and the treatment protocol is initiated, the hybridization assay can be repeated periodically to determine whether the patient's expression level has begun to approach levels observed in normal subjects. The results obtained from continuous assays can be used to show the effect of treatment over a period of days to months.
[0284]
For cancer, the presence of an abnormal amount of transcripts (underexpressed or overexpressed) in living tissue from an individual indicates a predisposition to the disease or a method to detect the disease before clinical symptoms appear. Can be provided. This type of clearer diagnosis allows medical professionals to take advantage of preventive or aggressive treatment early on, thereby preventing the development or further progression of cancer.
[0285]
Further diagnostic uses of oligonucleotides designed from NAAP-encoding sequences can include the use of PCR. These oligomers can be synthesized chemically, produced enzymatically, orin vitroCan yield. Oligomers preferably contain a fragment of a polynucleotide encoding NAAP, or a fragment of a polynucleotide complementary to a polynucleotide encoding NAAP, and are used to identify specific genes and conditions under optimized conditions. Is done. Oligomers can also be used for the detection, quantification, or both of closely related DNA or RNA sequences under moderately stringent conditions.
[0286]
In some embodiments, oligonucleotide primers derived from NAAP-encoding polynucleotide sequences can be used to detect single nucleotide polymorphisms (SNPs). SNPs are substitutions, insertions and deletions that often cause human congenital or acquired genetic diseases. Although not limited, SNP detection methods include single-stranded conformation polymorphism (SSCP) and fluorescent SSCP (fSSCP). In SSCP, DNA amplification is performed using oligonucleotide primers derived from a polynucleotide sequence group encoding NAAP and polymerase chain reaction (PCR). This DNA can be derived from, for example, diseased or normal tissue, biopsy samples, body fluids, and the like. SNPs in DNA make a difference in the secondary and tertiary structure of single-stranded PCR products. Differences can be detected using gel electrophoresis in non-denaturing gels. In fSSCP, oligonucleotide primers can be fluorescently labeled to detect amplimers with high-throughput equipment such as DNA sequencing machines. Furthermore, a sequence database analysis method called in silico SNP (in silico SNP, isSNP) is used to compare polymorphisms by comparing the sequences of individual overlapping DNA fragments that are arranged in a general consensus sequence. Can be identified. These computer-based methods filter out sequence variations that result from sequencing errors in the laboratory preparation of DNA or using statistical models and automated analysis of DNA sequence chromatograms. In another embodiment, SNPs are detected and characterized, for example, by mass spectrometry using a high throughput MASSARRAY system (Sequenom, Inc., San Diego CA).
[0287]
Methods that can be used to quantify NAAP expression include nucleotide radiolabeling or biotin labeling, coamplification of control nucleic acids, and interpolation of results obtained from standard curves (eg Melby, PC et al. (1993) ) J. Immunol. Methods 159: 235244; Duplaa, C. et al. (1993) Anal. Biochem. 212: 229236). To accelerate the quantification rate of multiple samples, the oligomer or polynucleotide of interest can be placed in various dilutions and assayed in a high-throughput format that allows rapid quantitation by spectrophotometric or colorimetric reactions .
[0288]
In yet another example, oligonucleotides or longer fragments derived from any of the polynucleotide sequences described herein can be used as elements in the microarray. Microarrays can be used in transcriptional imaging techniques that simultaneously monitor the relative expression levels of multiple genes. This is described below. Microarrays can also be used to identify genetic variants, mutations and polymorphisms. Use this information to determine gene function, understand the genetic basis of the disease, diagnose the disease, monitor disease progression / regression as a function of gene expression, and develop drug activity in disease treatment And can be monitored. In particular, this information can be used to develop a pharmacogenomic profile of the patient to select the most appropriate and effective treatment for the patient. For example, based on a patient's pharmacogenomic profile, a therapeutic agent that is highly effective for the patient and has few side effects can be selected.
[0289]
In another example, NAAP, a fragment of NAAP, or an antibody specific for NAAP can be used as an element on the microarray. Microarrays can be used to monitor or measure protein-protein interactions, drug-target interactions and gene expression profiles as described above.
[0290]
Certain embodiments relate to the use of the polynucleotides of the invention to produce a transcription image of a tissue or cell type. A transcript image represents a global pattern of gene expression by a particular tissue or cell type. Comprehensive gene expression patterns can be analyzed by quantifying the number and relative abundance of genes expressed at a given time under given conditions (Seilhamer et al. US Pat. No. 5,840,484 “Comparative Gene Transcript Analysis”). Which patent is hereby incorporated by reference). Thus, a transcript image can be generated by hybridizing a polynucleotide or a complement thereof of the invention to the entire tissue or cell type transcript or reverse transcript. In certain embodiments, hybridization is generated in a high throughput format such that the polynucleotides of the invention or their complements have a subset of multiple elements on a microarray. The resulting transcript image can provide a profile of gene activity.
[0291]
Transcript images can be generated using transcripts isolated from tissues, cell lines, biopsies or biological samples thereof. Transcript images are therefore in the case of tissue or biopsy samplesin vivoIn the case of cell linesin vitroReflects gene expression in
[0292]
Transcript images that produce the expression profiles of the polynucleotides of the invention are also:in vitroIt can be used for preclinical evaluation of model systems and drugs, or in connection with toxicity tests of industrial or natural environmental compounds. All compounds display a mechanism of action and toxicity, eliciting a characteristic gene expression pattern often referred to as a molecular fingerprint or toxicity signature (Nuwaysir, EF et al. (1999) Mol. Carcinog. 24: 153 -159, Steiner, S. and NL Anderson (2000) Toxicol. Lett. 112-113: 467-471, which is specifically incorporated herein by reference). If a test compound has the same signature as that of a compound with known toxicity, it may share toxic properties. A fingerprint or signature is most useful and accurate when it contains expression information from multiple genes and gene families. Ideally, measurement of expression across the genome provides the highest quality signature. Even if there are genes whose expression is not altered by any tested compound, those genes are important because their expression levels can be used to normalize the remaining expression data. The normalization procedure is useful for comparing expression data after treatment with various compounds. Assigning gene function to elements of a toxic signature helps to interpret the toxic mechanism, but knowledge of gene function is not required for statistical matching of signature groups that leads to prediction of toxicity (eg, 2000 2 See Press Release 00-02 issued by the National Institute of Environmental Health Sciences on May 29. http://www.niehs.nih.gov/oc/ available at news / toxchip.htm). Therefore, it is important and desirable to include all expressed gene sequences in toxicological screening using toxicity signatures.
[0293]
In one embodiment, the toxicity of a test compound is calculated by treating a biological sample with nucleic acid with the test compound. Nucleic acid expressed in the treated biological sample can be hybridized with one or more probes specific for the polynucleotide of the invention, thereby quantifying the transcription level corresponding to the polynucleotide of the invention. The transcription level in the treated biological sample is compared to the level in the untreated biological sample. Differences in transcription levels between the two samples indicate the toxic response caused by the test compound in the treated sample.
[0294]
Another embodiment relates to analyzing the proteome of a tissue or cell type using the polypeptide sequences of the present invention. The term proteome refers to the global pattern of protein expression in a particular tissue or cell type. Each protein component of the proteome can be individually subject to further analysis. Proteomic expression patterns or profiles can be analyzed by quantifying the number and relative abundance of proteins expressed at a given time under a given condition. Thus, a proteomic profile of a cell can be generated by isolating and analyzing polypeptides of a particular tissue or cell type. In some embodiments, the separation is achieved by two-dimensional gel electrophoresis in which the protein is separated from the sample by one-dimensional isoelectric focusing and separated according to molecular weight by two-dimensional sodium dodecyl sulfate slab gel electrophoresis. (Steiner and Anderson, above). Proteins are visualized in the gel as dispersed, unique points by staining the gel with a substance such as Coomassie Blue, or silver stain or fluorescent stain. The optical density of each protein spot is usually proportional to the protein level in the sample. The optical density of equally located protein spots from different samples, eg, biological samples either treated or untreated with the test compound or therapeutic agent, are compared to identify changes in protein spot density associated with the treatment. Proteins within the spot are partially sequenced using standard methods using, for example, chemical or enzymatic cleavage followed by mass spectrometry. The identity of a protein within a spot can be determined by comparing its subsequence, preferably at least 5 consecutive amino acid residues, to the polypeptide sequence of the invention. In some cases, additional sequence data is obtained for definitive protein identification.
[0295]
Proteomic profiles can also be generated by quantifying NAAP expression levels using antibodies specific for NAAP. In some embodiments, these antibodies are used as elements on a microarray, and protein expression levels are quantified by exposing the microarray to a sample and detecting the level of protein binding to each array element (Lueking, A. et al. (1999). Anal. Biochem. 270: 103-111; Mendoze, LG et al. (1999) Biotechniques 27: 778-788). Detection can be performed in various ways known in the art, for example, a thiol-reactive or amino-reactive fluorescent compound can be reacted with a protein in the sample to detect the amount of fluorescent binding in each array element.
[0296]
Toxicity signatures at the proteome level are also useful for toxicological screening and should be analyzed in parallel with toxicity signatures at the transcriptional level. For some proteins in some tissues, the correlation between transcript abundance and protein abundance is poor (Anderson, NL and J. Seilhamer (1997) Electrophoresis 18: 533-537). Proteome toxicity signatures may be useful in the analysis of compounds that do not affect so much but alter the proteome profile. Furthermore, since analysis of transcripts in body fluids is difficult due to the rapid degradation of mRNA, proteome profiling may be more reliable in such cases and may be informative.
[0297]
In another example, the toxicity of a test compound is calculated by treating a biological sample containing the protein with the test compound. Proteins expressed in the treated biological sample are separated so that the amount of each protein can be quantified. The amount of each protein is compared to the amount of the corresponding protein in the untreated biological sample. The difference in the amount of protein in both samples is indicative of a toxic response to the test compound in the treated sample. Individual proteins are identified by sequencing the amino acid residues of the individual proteins and comparing these subsequences to the polypeptides of the invention.
[0298]
In another example, the toxicity of a test compound is calculated by treating a biological sample containing the protein with the test compound. The protein obtained from the biological sample is incubated with an antibody specific for the polypeptide of the present invention. The amount of protein recognized by the antibody is quantified. The amount of protein in the treated biological sample is compared to the amount of protein in the untreated biological sample. The difference in the amount of protein in both samples is indicative of a toxic response to the test compound in the treated sample.
[0299]
Microarrays are prepared, used and analyzed by methods known in the art (eg, Brennan, TM et al. (1995) US Pat. No. 5,474,796, Schena, M. et al. (1996) Proc. Natl. Acad. Sci. USA 93: 10614-10619, Baldeschweiler et al. (1995) PCT application WO95 / 251116, Shalon, D. et al. (1995) PCT application WO95 / 35505, Heller, RA et al. (1997) Proc. Natl. Acad. Sci USA 94: 2150-2155, Heller, MJ et al. (1997) US Pat. No. 5,605,662). Various types of microarrays are known, for detailsDNA Microarrays: A Practical Approach, M. Schena, edited. (1999) Oxford University Press, London. This document is incorporated herein by reference in particular.
[0300]
In another embodiment of the invention, a group of nucleic acid sequences encoding NAAP may also be used to produce a group of hybridization probes useful for mapping native genomic sequences. Either coding or non-coding sequences can be used, and in some cases, non-coding sequences are preferred over coding sequences. For example, conservation of coding sequences within members of a multigene family can result in unwanted cross-hybridization during chromosomal mapping. The sequence may be on a particular chromosome, or on a particular region of a chromosome, or an artificially formed chromosome such as a human artificial chromosome (HAC), a yeast artificial chromosome (YAC), a bacterial artificial chromosome (BAC), Bacterial P1 product, or mapped to a single chromosome cDNA library group (e.g. Harrington, JJ et al. (1997) Nat. Genet. 15: 345-355; Price, CM (1993) Blood Rev. 7: 127134; Trask , BJ (1991) Trends Genet. 7: 149154). Once mapped, gene linkage maps can be developed using the nucleic acid sequences of the present invention, eg, to correlate the inheritance of a disease state with the inheritance of a particular chromosomal region or with restriction enzyme fragment length polymorphism (RFLP) ( See, for example, Lander, ES and D. Botstein (1986) Proc. Natl. Acad. Sci. USA 83: 7353-7357).
[0301]
Fluorescence in situ hybridization (FISH) can be correlated with other physical and genetic map data (see, eg, Heinz-Ulrich, et al. (1995) in Meyers, supra, pages 965-968). Examples of genetic map data can be found in various scientific journals or the Online Mendelian Inheritance in Man (OMIM) website. The correlation between the location of the gene encoding NAAP on a physical chromosomal map and a particular disease or a predisposition to a particular disease can help determine the region of DNA associated with this disease Can facilitate the cloning task of determining the position.
[0302]
Genetic maps can be expanded using physical mapping techniques such as linkage analysis with established chromosomal markers, and in situ hybridization of chromosomal specimens. By placing a gene on the chromosome of another mammal, such as a mouse, the associated markers can often be revealed even if the exact chromosomal locus is unknown. This information is valuable for researchers searching for disease genes using gene discovery techniques such as positional cloning. Once a gene (s) involved in a disease or syndrome is roughly positioned by genetic linkage to a specific genomic region, such as the 11q22-23 region of vasodilatory ataxia, it is mapped to that region Any sequence may represent a relevant or regulatory gene for further investigation (see, eg, Gatti, RA et al. (1988) Nature 336: 577-580). The nucleotide sequence of the present invention can also be used for detecting a difference in chromosomal location among the healthy person, the owner, and the affected person due to translocation, inversion, and the like.
[0303]
In another embodiment of the invention, NAAP, a catalytic fragment thereof or an immunogenic fragment or oligopeptide group thereof may be used for screening a library of compounds in any of a variety of drug screening techniques. Fragments used for drug screening can be free in solution, fixed to a solid support, retained on the cell surface, or located within the cell. Complex formation due to binding of NAAP to the agent being tested can also be measured.
[0304]
Another drug screening method is used to screen compounds with suitable binding affinity for the protein of interest with high throughput (see, eg, Geysen, et al. (1984) PCT Application No. WO84 / 03564). In this method, a large number of different small molecule test compounds are synthesized on a solid substrate. The test compound is washed after reacting with NAAP or a fragment thereof. The bound NAAP is then detected by methods well known in the art. Purified NAAP can also be coated directly onto plates used in the drug screening techniques described above. Alternatively, non-neutralizing antibodies can be used to capture the peptide and immobilize the peptide on a solid support.
[0305]
In another example, a competitive drug screening assay can be used in which neutralizing antibodies capable of specific binding to NAAP compete with the test compound for binding to NAAP. In this method, antibodies can be used to detect the presence of any peptide that shares one or more antigenic determinants with NAAP.
[0306]
In another embodiment, the NAAP-encoding nucleotide sequence is a molecular biology technique that will be developed in the future, and features of the nucleotide sequence currently known (including but not limited to triplet genetic code, specific base pairing) It can be used for new technologies that depend on (including interactions, etc.).
[0307]
Without further details, those skilled in the art will be able to make full use of the present invention with the above description. Accordingly, the examples described below are for illustrative purposes only and do not limit the invention in any way.
[0308]
All patents, patent applications and publications disclosed herein, especially U.S. Patent Application Nos. 60 / 257,714, 60 / 260,081, 60 / 262,302, 60 / 266,088, applications [attorney docket number (Attorney Docket No.) PF-1249 P, file date October 29, 2001], and application 60 / 263,823 are hereby incorporated by reference.
[0309]
(Example)
1 cDNA Library creation
The Incyte cDNA group is derived from the cDNA library group described in the LIFESEQ GOLD database (Incyte Genomics, Palo Alto CA). Some tissues were homogenized and dissolved in guanidinium isothiocyanate solution, and other tissues were homogenized and dissolved in phenol or a suitable mixture of denaturants. TRIZOL (Life Technologies), which is an example of a mixed solution, is a single-phase solution of phenol and guanidine isothiocyanate. The resulting lysate was layered on a cesium chloride cushion solution and centrifuged or extracted with chloroform. RNA was precipitated from the lysate using either isopropanol, sodium acetate and ethanol, or another conventional method.
[0310]
To increase RNA purity, RNA extraction and precipitation with phenol was repeated as many times as necessary. In some cases, RNA was treated with DNase. In most libraries, poly (A) + RNA was isolated using oligo d (T) -linked paramagnetic particles (Promega), OLIGOTEX latex particles (QIAGEN, Chatsworth CA) or OLIGOTEX mRNA purification kit (QIAGEN). Alternatively, RNA was isolated directly from tissue lysates using another RNA isolation kit, such as POLY (A) PURE mRNA purification kit (Ambion, Austin TX).
[0311]
In some cases, RNA was provided to Stratagene, and the corresponding cDNA libraries were created by Stratagene. If not, cDNA was synthesized using the UNIZAP vector system (Stratagene) or SUPERSCRIPT plasmid system (Life Technologies) using the recommended or similar methods known in the art to create a cDNA library (Ausubel, supra). , 1997, 5.1-6.6 units, etc.). Reverse transcription was initiated using oligo d (T) or random primers. A synthetic oligonucleotide adapter was ligated to double stranded cDNA and the cDNA was digested with a suitable restriction enzyme or group of enzymes. For most libraries, cDNA size selection (300-1000 bp) was performed using SEPHACRYL S1000, SEPHAROSE CL2B or SEPHAROSE CL4B column chromatography (Amersham Pharmacia Biotech) or preparative agarose gel electrophoresis. The cDNA was ligated to suitable restriction enzyme sites on the polylinker of a suitable plasmid. Suitable plasmids include, for example, PBLUESCRIPT plasmid (Stratagene), PSPORT1 plasmid (Life Technologies) PCDNA2.1 plasmid (Invitrogen, Carlsbad CA), PBK-CMV plasmid (Stratagene), PCR2-TOPOTA plasmid (Invitrogen), PCMV-ICIS plasmid ( Stratagene), pIGEN (Incyte Genomics, Palo Alto CA), pRARE (Incyte Genomics), or plNCY (Incyte Genomics), or derivatives thereof. The recombinant plasmid was transformed into qualified E. coli cells such as Stratagene XL1-Blue, XL1-BIueMRF or SOLR, or Life Technologies DH5α, DH10B or ElectroMAX DH10B.
[0312]
2 Isolation of cDNA clones
Example 1The UNIZAP vector system (Stratagene) was used to recover the plasmid obtained as described above from the host cell.in vivoThis was done by excision or by cell lysis. Plasmid purification methods include Magic or WIZARD Minipreps DNA purification system (Promega), AGTC Miniprep purification kit (Edge Biosystems, Gaithersburg MD), QIAGEN QIAWELL 8 Plasmid, QIAWELL 8 Plus Plasmid and QIAWELL 8 Ultra Plasmid purification systems, REAL At least one of the Prep 96 plasmid purification kits was used. The plasmid was precipitated and then resuspended in 0.1 ml distilled water and stored at 4 ° C. either lyophilized or not lyophilized.
[0313]
Alternatively, plasmid DNA was amplified from host cell lysates using direct binding PCR in a high-throughput format (Rao, V.B. (1994) Anal. Biochem. 216: 1-14). Host cell lysis and thermal cycling processes were performed in a single reaction mixture. Samples are processed and stored in 384-well plates, and the concentration of amplified plasmid DNA is determined fluorimetrically using PICOGREEN dye (Molecular Probes, Eugene OR) and FLUOROSKANII fluorescence scanner (Labsystems Oy, Helsinki, Finland) Quantified.
[0314]
3 Sequencing and analysis
Example 2The Incyte cDNA recovered from the plasmid as described in 1 was sequenced as shown below. Sequencing of cDNA can be performed using standard methods or high-throughput equipment such as ABI CATALYST 800 thermal cycler (Applied Biosystems) or PTC-200 thermal cycler (MJ Research), HYDRA microdispenser (Robbins Scientific) or MICROLAB 2200 (Hamilton) liquid. Treated in conjunction with the transfer system. A cDNA sequencing reaction was prepared using a reagent provided by Amersham Pharmacia Biotech, or an ABI sequencing kit such as ABI PRISM BIGDYE Terminator cycle sequencing ready reaction kit (Applied Biosystems). For electrophoretic separation of cDNA sequencing reactions, and for detection of labeled polynucleotides, ABI PRISM 373 or MEGABACE 1000 DNA sequencing system (Molecular Dynamics) or using standard ABI protocol and base pairing software A 377 sequencing system (Applied Biosystems) or other sequence analysis system known in the art was used. Reading frames within the cDNA sequences were identified using standard methods (reviewed in Ausubel, 1997, 7.7 units supra). Select several cDNA sequences,Example 8The sequence was extended with the technique disclosed in.
[0315]
Polynucleotide sequences derived from Incyte cDNA were validated by removing vector, linker and poly (A) sequences and masking ambiguous bases. At that time, an algorithm and a program based on BLAST, dynamic programming, and adjacent dinucleotide frequency analysis were used. Next, the following database group was queried for Incyte cDNA sequences or their translations. That is, a selection of public databases (eg GenBank primates, rodents, mammals, vertebrates, eukaryotes and BLOCKS, PRINTS, DOMO, PRODOM) and humans, rats, mice, nematodes (Caenorhabditis elegans), Budding yeast (Saccharomyces cerevisiae), Fission yeast (Schizosaccharomyces pombe) And Candida Candida (Candida albicans) PROTEOME databases (Incyte Genomics, Palo Alto CA) with sequences from Hidden Markov Model (HMM) -based protein family databases (PFAM, etc.) (HMM is the consensus primary structure of gene families) (See, for example, Eddy, SR (1996) Cuff. Opin. Struct. Biol. 6: 361-365). Queries were made using programs based on BLAST, FASTA, BLIMPS, and HMMER. The Incyte cDNA sequence was constructed to yield the full length polynucleotide sequence. Alternatively, GenBank cDNA group, GenBank EST group, stitched sequence group, stretched sequence group, or Genscan predicted coding sequence group (Examples 4 and 5The Incyte cDNA population was extended to full length. It was constructed using a program based on Phred, Phrap, and Consed, and a population of cDNA was screened for open reading frames using a program based on GenMark, BLAST, and FASTA. The full length polynucleotide sequence was translated and the corresponding full length polypeptide sequence was derived. Alternatively, the polypeptides of the present invention can begin at any methionine residue of the full-length translated polypeptide. Inquiries for subsequent analysis of full-length polypeptide sequences, including GenBank protein databases (genpept), SwissProt, PROTEOME databases, BLOCKS, PRINTS, DOMO, PRODOM and Prosite, and hidden Markov models such as PFAM (HMM) Base protein family database group. Full-length polynucleotide sequences were also analyzed using MACDNASIS PRO software (Hitachi Software Engineering, South San Francisco CA) and LASERGENE software (DNASTAR). Sequence alignments between polynucleotides and polypeptides are generated using default parameters specified by the CLUSTAL algorithm incorporated into the MEGALIGN multi-sequence alignment program (DNASTAR). This also calculates the percent identity between aligned sequences.
[0316]
Table 7 gives an overview of the tools, programs and algorithms used for analysis and construction of Incyte cDNA and full-length sequences, and applicable descriptions, references and threshold parameters. The tools, programs and algorithms used are shown in column 1 of Table 7 and a brief description thereof is shown in column 2. Column 3 is a preferred reference, all of which are incorporated herein by reference in their entirety. Where applicable, column 4 shows the parameters, such as score, probability value, etc., used to evaluate the strength with which the two sequences match (the higher the score or the lower the probability value, the 2 High identity between sequences).
[0317]
The above set of programs used for the construction and analysis of full-length polynucleotide sequences and polypeptide sequences can also be used to identify polynucleotide sequence fragments of SEQ ID NO: 17-32. Groups of fragments of about 20 to about 4000 nucleotides that are useful in hybridization and amplification techniques are shown in column 2 of Table 4.
[0318]
4 Genome DNA And editing of coding sequences from
Putative nucleic acid-related proteins were first identified by running the Genscan gene identification program against public genome sequence databases (eg gbpri and gbhtg). Genscan is a universal gene identification program that analyzes genomic DNA sequences from various organisms (Burge, C. and S. Karlin (1997) J. Mol. Biol. 268: 78-94, Burge, C. and S Karlin (1998) Curr. Opin. Struct. Biol. 8: 346-354). This program links predicted exons together to form a constructed cDNA sequence that spans from methionine to a stop codon. The output of Genscan is a FASTA database of polynucleotide and polypeptide sequences. The maximum range of sequences that Genscan analyzes at one time was set to 30 kb. To determine which of these Genscan predicted cDNA sequences encode nucleic acid-related molecules, the encoded polypeptides were analyzed by querying the PFAM model group for nucleic acid-related molecules. Potential nucleic acid-related proteins were also identified based on homology to Incyte cDNA sequences already annotated as nucleic acid-related proteins. These selected Genscan predicted sequences were then compared to the public databases genpept and gbpri by BLAST analysis. If necessary, Genscan predicted sequences were edited by comparison with the top BLAST hits from genpept to correct errors in the sequence predicted by Genscan, such as extra or omitted exons. BLAST analysis was also used to find any Incyte cDNA or public cDNA coverage of the Genscan predicted sequence, thus providing evidence of transcription. When Incyte cDNA coverage was available, this information was used to correct or confirm the Genscan predicted sequence. The full-length polynucleotide sequence isExample 3The Genscan predicted coding sequence was constructed with Incyte cDNA sequences and / or public cDNA sequences using the construction process described in. Alternatively, the full-length polynucleotide sequence is completely derived from the edited or unedited Genscan predicted coding sequence.
[0319]
5 of genome sequence data cDNA Integration with sequence data
Stitch arrangement ( Stiched Sequence )
In order to extend the partial cDNA sequence group,Example 4Exon groups predicted by the Genscan gene identification program described in 1) were used.Example 3The partial cDNA groups constructed as described above were mapped to genomic DNA and resolved into cluster groups with related cDNA groups and exons predicted from Genscan from one or more genomic sequences. To integrate the cDNA and genome information, each cluster was analyzed using an algorithm based on graph theory and dynamic programming to produce a set of potential splice variants. The sequences were subsequently confirmed, edited or extended to create a full length sequence. A sequence segment group in which the total length of the segment is in two or more sequences was identified in the cluster, and the segment groups identified as such were considered to be equal due to transitivity. For example, if a section is in one cDNA and two genomic sequences, all three sections were considered equal. This process allows unrelated but contiguous genomic sequences to be cross-linked by joining them with cDNA sequences. The sections thus identified were “stitched” with the stitch algorithm in the order they appear along their parent sequences, producing the longest possible sequence and variant sequences. Linkage between sections (cDNA-cDNA or genomic sequence-genomic sequence) that continues along one type of parent sequence takes precedence over linkages that change the type of parent (cDNA-genomic sequence). The resulting stitch sequences were translated and compared with public databases genpept and gbpri by BLAST analysis. The incorrect exon group predicted by Genscan was corrected by comparison with the top BLAST hits from genpept. If necessary, the sequences were further extended using additional cDNA sequences or by examining genomic DNA.
[0320]
Stretch arrangement ( Stretched Sequence )
Partial DNA sequences were extended to full length by one algorithm based on BLAST analysis. First, using the BLAST program, GenBank's primate, rodent, mammalian, vertebrate and eukaryotic databases,Example 3We interrogated partial cDNAs constructed as described in. The closest GenBank protein homologue is then analyzed by BLAST analysis using the Incyte cDNA sequence orExample 4Compared to any of the GenScan exon predicted sequences described in. The resulting high scoring segment pair (HSP) was used to produce a chimeric protein and the translated sequence was mapped onto the GenBank protein homologue. Insertions or deletions can occur in the chimeric protein relative to the original GenBank protein homologue. Homologous genomic sequences were searched from public human genome databases using GenBank protein homologues, chimeric proteins or both as probes. In this way, the partial DNA sequence was stretched or extended by the addition of homologous genomic sequences. The resulting stretch sequence was examined to determine whether it contained the complete gene.
[0321]
6 NAAP Chromosome Mapping of Polynucleotides Encoding DNA
The sequences used to construct SEQ ID NO: 17-32 were compared with sequences from the Incyte LIFESEQ database and public domain databases using BLAST et al.'S implementation of the Smith-Waterman algorithm. The sequences in these databases that matched SEQ ID NO: 17-32 were constructed into clusters of contiguous and overlapping sequences using a construction algorithm such as Phrap (Table 7). Using the radiation hybrid and genetic map data available from public sources such as the Stanford Human Genome Center (SHGC), Whitehead Genome Research Institute (WIGR), Genethon, etc., any clustered sequences have already been Judged whether it was mapped. If the mapped sequence was contained in a cluster, the entire sequence of that cluster was assigned to a map location along with the individual SEQ ID numbers.
[0322]
The location on the map is expressed as a range or interval of human chromosomes. The map position of the centimorgan interval is measured relative to the end of the p-arm of the chromosome (centiorgan (cM) is a unit of measure based on the frequency of recombination between chromosomal markers. On average, 1 cM is Equivalent to 1 megabase (Mb) of human DNA, although this value varies widely due to recombination hot and cold spots). The cM distance is based on a set of genetic markers mapped by Genethon that provide boundaries for radiation hybrid markers whose sequences are contained within each cluster. NCBI “GeneMap'99” (http://www.ncbi.nlm.nih.gov/genemap/) and other resources such as human genome maps that can be obtained by general individuals have already identified disease genes. It can be determined whether it is mapped in or near the section. Thus, SEQ ID NO: 20 maps within the segment from 125.80 to 140.60 centimorgans on chromosome 8 and SEQ ID NO: 28 maps within the 41.7 to 49.4 centmorgan segment on chromosome 19. It was.
[0323]
7 Analysis of polynucleotide expression
Northern analysis is an experimental technique used to detect the presence of a transcript of a gene and involves the hybridization of labeled nucleotide sequences to a membrane to which RNA from a particular cell type or tissue is bound. (See, for example, Sambrook, Chapter 7 and Ausubel (1995) chapters 4 and 16 above).
[0324]
Using similar computer techniques applying BLAST, we searched for identical or related molecules in cDNA databases such as GenBank and LIFESEQ (Incyte Genomics). Northern analysis is much faster than some membrane-based hybridizations. In addition, the sensitivity of computer searches can be modified to determine whether any particular match is classified as exact or homologous. The search criterion is a product score, which is defined by the following equation.
[0325]
[Expression 1]

[0326]
The product score takes into account both the similarity between two sequences and the length with which the sequences match. The product score is a normalized value from 0 to 100, and is determined as follows. Multiply the BLAST score by the nucleotide sequence identity and divide the product by 5 times the shorter length of the two sequences. To calculate the BLAST score, a score of +5 is assigned to each matching base in a high scoring segment pair (HSP) and -4 is assigned to each mismatched base. Two sequences can share more than one HSP (separated by gaps). If there are two or more HSPs, the product score is calculated using the segment pair with the highest BLAST score. The product score represents the balance between the fractional overlap and the quality of the BLAST alignment. For example, a product score of 100 is obtained only if there is a 100% match over the shorter length of the two sequences compared. The product score 70 is obtained when either one end is 100% coincident and 70% overlap, or the other end is 88% coincident and 100% overlap. A product score of 50 is obtained if either end is 100% coincident and 50% overlap, or 79% coincidence and 100% overlap.
[0327]
Alternatively, the polynucleotide sequence encoding NAAP is analyzed against the tissue from which it was derived. For example, some full-length sequences are constructed, at least in part, using overlapping Incyte cDNA sequences (Example 3See). Each cDNA sequence is derived from a cDNA library made from human tissue. Each human tissue is classified into one of the following organ / tissue categories. Cardiovascular system, connective tissue, digestive system, embryonic structure, endocrine system, exocrine gland, female genital organs, male genital organs, germ cells, blood and immune system, liver, musculoskeletal system, nervous system, pancreas, respiratory system, Sensory organs, skin, stomatognathic system, non-classified / mixed or urinary tract. Count the number of libraries in each category and divide by the total number of libraries in all categories. Similarly, each human tissue is classified into one of the following disease / situation categories: cancer, cell line, development, inflammation, neurological, trauma, cardiovascular, pool, etc. Count the number of libraries in each category and divide by the total number of libraries in all categories. The resulting percentage reflects tissue-specific or disease-specific expression of cDNA encoding NAAP. cDNA sequence and cDNA library / tissue information can be obtained from the LIFESEQ GOLD database (Incyte Genomics, Palo Alto CA).
[0328]
8 Extension of polynucleotides encoding NAAP
Full-length polynucleotide sequences were also produced by extending the fragments using oligonucleotide primers designed from the appropriate fragments of the full-length molecule. One primer was synthesized to initiate a 5 ′ extension of a known fragment and another primer was synthesized to initiate a 3 ′ extension of a known fragment. The OLIGO 4.06 software (National Biosciences) or another appropriate program was used to design the starting primer group, and the length was about 22-30 nucleotides, the GC content was about 50% or more, and the temperature was about 68-72 ° C. To anneal to the target sequence. All elongations of nucleotide groups that resulted in hairpin structures and primer-primer dimers were avoided.
[0329]
The sequence was extended using selected human cDNA libraries. Where more than one extension was necessary or desirable, additional primers or nested sets of primers were designed.
[0330]
High fidelity amplification was obtained by PCR using methods well known to those skilled in the art. PCR was performed in a 96-well plate using a PTC-200 thermal cycler (MJ Research, Inc.). The reaction mixture has template DNA and 200 nmol of each primer. Mg^{2 +}And (NH_Four)₂SO_FourAnd a reaction buffer containing 2-mercaptoethanol, Taq DNA polymerase (Amersham Pharmacia Biotech), ELONGASE enzyme (Life Technologies), and Pfu DNA polymerase (Stratagene). For the primer pair PCI A and PCI B, amplification was performed with the following parameters.
Step 1 3 minutes at 94 ° C
Step 2 15 seconds at 94 ° C
Step 3 1 minute at 60 ℃
Step 4 at 68 ° C for 2 minutes
Step 5 Repeat steps 2, 3, and 4 20 times
Step 6 at 68 ° C for 5 minutes
Step 7 Store at 4 ℃
In another method, the primer pair T7 and SK + were amplified with the following parameters.
Step 1 3 minutes at 94 ° C
Step 2 15 seconds at 94 ° C
Step 3 1 minute at 57 ℃
Step 4 at 68 ° C for 2 minutes
Step 5 Repeat steps 2, 3, and 4 20 times
Step 6 at 68 ° C for 5 minutes
Step 7 Store at 4 ℃
The DNA concentration in each well is 1 x TE and 100 µl of PICOGREEN quantification reagent (0.25 (v / v) PICOGREEN; Molecular Probes, Eugene OR) dissolved in 0.5 µl of undiluted PCR product. It was distributed to each well of (Corning Costar, Acton MA) and measured so that DNA could bind to the reagent. Plates were scanned with Fluoroskan II (Labsystems Oy, Helsinki, Finland) to measure sample fluorescence and quantify DNA concentration. An aliquot of 5-10 μl of the reaction mixture was analyzed by electrophoresis on a 1% agarose gel to determine which reaction was successful in extending the sequence.
[0331]
Extended nucleotides are desalted and concentrated, transferred to a 384-well plate, digested with CviJI cholera virus endonuclease (Molecular Biology Research, Madison WI), sonicated or sheared, and pUC 18 vector (Amersham Pharmacia Biotech) Re-linked to. For shotgun sequencing, digested nucleotides were separated on a low concentration (0.6-0.8%) agarose gel, fragments were excised, and agar was digested with Agar ACE (Promega). Elongated clones were religated to pUC 18 vector (Amersham Pharmacia Biotech) using T4 ligase (New England Biolabs, Beverly MA) and treated with Pfu DNA polymerase (Stratagene) to fill restriction site overhangs E. coli cells were transfected. Transfected cells were selected on a medium containing antibiotics, and each colony was collected and cultured overnight at 37 ° C. in a 384 well plate group in LB / 2X carbenicillin culture.
[0332]
Cells were lysed and DNA was PCR amplified using Taq DNA polymerase (Amersham Pharmacia Biotech) and Pfu DNA polymerase (Stratagene) as follows.
Step 1 3 minutes at 94 ° C
Step 2 15 seconds at 94 ° C
Step 3 1 minute at 60 ℃
Step 4 at 72 ° C for 2 minutes
Step 5 Repeat steps 2, 3, and 4 29 times
Step 6 5 minutes at 72 ° C
Step 7 Store at 4 ℃
For quantification of DNA, PICOGREEN reagent (Molecular Probes) was used as described above. Samples with low DNA recovery were reamplified using the same conditions as above. Samples are diluted with 20% dimethyl sulfoxide (1: 2, v / v), and the DYENAMIC energy transfer sequencing primer and the DYENAMIC DIRECT kit (Amersham Pharmacia Biotech) or ABI PRISM BIGDYE terminator cycle sequencing ready reaction kit (Terminator cycle sequencing) ready reaction kit) (Applied Biosystems).
[0333]
Similarly, full-length polynucleotide sequences were verified using the above procedure. Alternatively, 5 ′ regulatory sequences were obtained using full-length polynucleotides using the oligonucleotides designed for such extension and some appropriate genomic library in the above procedure.
[0334]
9 Labeling and use of individual hybridization probes
Hybridization probes from SEQ ID NO: 17-32 are used to screen cDNA, mRNA, or genomic DNA. Although specifically described for labeling oligonucleotides of about 20 base pairs, essentially the same procedure is used for larger nucleotide fragments. Oligonucleotides were designed using the latest software, such as OLIGO 4.06 software (National Biosciences).³²Label by combining P] adenosine triphosphate (Amersham Pharmacia Biotech) 250 μCi with T4 polynucleotide kinase (DuPont NEN, Boston MA). The labeled oligonucleotide is substantially purified using a SEPHADEX G-25 ultrafine molecular size exclusion dextran bead column (Amersham Pharmacia Biotech). In a typical membrane-based hybridization analysis of human genomic DNA digested with any one of Ase I, Bgl II, Eco RI, Pst I, XbaI or Pvu II (DuPont NEN), 10⁷An aliquot containing a count of labeled probes is used.
[0335]
DNA obtained from each digest is fractionated on a 0.7% agarose gel and transferred to a nylon membrane (Nytran Plus, Schleicher & Schuell, Durham NH). Hybridization is performed at 40 ° C. for 16 hours. In order to remove non-specific signal groups, the blot groups are washed sequentially at room temperature, for example under conditions of up to 0.1 × sodium citrate saline and 0.5% sodium dodecyl sulfate. Visualize and compare hybridization patterns using autoradiography or alternative imaging means.
[0336]
10 Microarray
Combining or synthesizing array elements on a microarray can be accomplished using photolithography, piezoelectric printing (see inkjet printing, Baldeschweiler, etc.), mechanical microspotting techniques, and techniques derived therefrom. . In each of the above technologies, the substrate should be a solid with a uniform, non-porous surface (Schena (1999) supra). Recommended substrates include silicon, silica, glass slides, glass chips and silicon wafers. Alternatively, elements similar to dot blot or slot blot methods may be utilized to place and bind elements to the surface of the substrate using thermal, ultraviolet, chemical or mechanical bonding procedures. Conventional arrays can be made using available methods and machinery known to those skilled in the art and can have any suitable number of elements (eg, Schena, M. et al. (1995) Science 270: 467-470 Shalon. D. et al. (1996) Genome Res. 6: 639-645, Marshall, A. and J. Hodgson (1998) Nat. Biotechnol. 16: 27-31).
[0337]
Full-length cDNAs, expressed sequence tags (ESTs), or fragments or oligomers thereof can be elements of the microarray. Fragments or oligomers suitable for hybridization can be selected using software known in the art such as LASERGENE software (DNASTAR). The array element group is hybridized with the polynucleotide group in the biological sample. The polynucleotide in the biological sample is conjugated to a molecular tag such as a fluorescent label to facilitate detection. After hybridization, unhybridized nucleotides from the biological sample are removed and hybridization at each array element is detected using a fluorescent scanner. Alternatively, hybridization can also be detected using laser desorption and mass spectrometry. The degree of complementarity and relative abundance of each polynucleotide that hybridizes to an element on the microarray can be calculated. The preparation and use of the microarray in one embodiment is detailed below.
[0338]
Tissue or cell sample preparation
The guanidinium thiocyanate method is used to isolate total RNA from tissue samples, and poly (A)⁺The oligo (dT) cellulose method is used to purify RNA. Each poly (A)⁺MMLV reverse transcriptase was used to reverse transcribe RNA samples, and 0.05 pg / μl oligo (dT) primer (21mer), 1 × first strand buffer, 0.03 unit / μl RNase inhibitor, 500 μM Use dATP, 500 μM dGTP, 500 μM dTTP, 40 μM dCTP, 40 μM dCTP-Cy3 (BDS) or dCTP-Cy5 (Amersham Pharmacia Biotech). Reverse transcription reaction was performed using GEMBRIGHT kit (Incyte) and 200 ng poly (A).⁺Perform in a volume of 25 ml containing RNA. Specific control poly (A)⁺RNA groups are derived from non-coding yeast genomic DNAin vitroSynthesize by transcription. After incubation at 37 ° C for 2 hours, each reaction sample (one Cy3 and the other Cy5 labeled) was treated with 2.5 ml of 0.5M sodium hydroxide and incubated at 85 ° C for 20 minutes to react. To break down RNA. Two consecutive CHROMA SPIN 30 gel filtration spin columns (CLONTECH Laboratories, Inc. (CLONTECH), Palo Alto CA) are used for sample group purification. After mixing, ethanol precipitation of the two reaction samples is performed with 1 ml glycogen (1 mg / ml), 60 ml sodium acetate, and 300 ml 100% ethanol. The sample is then completely dried using SpeedVAC (Savant Instruments Inc., Holbrook NY) and resuspended in 14 μl of 5 × SSC / 0.2% SDS.
[0339]
Microarray preparation
Array elements are made using the sequences of the present invention. Each array element is amplified from a vector-containing bacterial cell group by a cloned cDNA insert group. PCR amplification uses primers complementary to the vector sequence located on the side of the cDNA insert. Array elements are amplified by 30 cycles of PCR from an initial amount of 1-2 ng to a final amount in excess of 5 μg. The amplified array element is purified using SEPHACRYL-400 (Amersham Pharmacia Biotech).
[0340]
The purified array element is fixed on a polymer-coated slide glass. Microscope slides (Corning) are ultrasonically washed in 0.1% SDS and acetone, and washed with plenty of distilled water during and after treatment. The slides were etched in 4% hydrofluoric acid (VWR Scientific Products Corporation (VWR), West Chester PA), washed in copious amounts of distilled water, and 0.05% aminopropylsilane (Sigma) in 95% ethanol. ) To coat. Coated slides are cured in an oven at 110 ° C.
[0341]
Array elements are added to the coated glass substrate using the method described in US Pat. No. 5,807,522. This patent is hereby incorporated by reference. 1 μl of array element DNA with an average concentration of 100 ng / μl is filled into an open capillary printing element by means of a robotic apparatus. The instrument now places approximately 5 nl of array element samples per slide.
[0342]
STRATALINKER UV crosslinker (Stratagene) is used to UV crosslink the microarray. The microarray is washed once with 0.2% SDS and three times with distilled water at room temperature. In order to block non-specific binding sites, microarray incubation was performed in 60% at 30 ° C. in 0.2% casein in phosphate buffered saline (PBS) (Tropix, Inc., Bedford MA) before Wash with 0.2% SDS and distilled water as done.
[0343]
Hybridization
The 9 μl sample mixture used for the hybridization reaction contains 0.2 μg of each of the cDNA synthesis products labeled with Cy3 or Cy5 in 5 × SSC, 0.2% SDS hybridization buffer. The sample mixture is heated to 65 ° C. for 5 minutes and aliquoted on the microarray surface to 1.8 cm.²Cover with a cover glass. The array is transferred to a waterproof chamber with a cavity slightly larger than the microscope slide. To keep the chamber at 100 humidity, add 140 μl of 5 × SSC to one corner of the chamber. The chamber with the array is incubated at 60 ° C. for about 6.5 hours. The array is washed in the first wash buffer (1 × SSC, 0.1% SDS) for 10 minutes at 45 ° C. and in the second wash buffer (0.1 × SSC) for 10 minutes at 45 ° C. 3 Wash once and dry.
[0344]
detection
To detect reporter labeled hybridization complexes, an Innova 70 mixed gas 10 W laser (Coherent, Inc., Santa Clara CA) capable of generating spectral lines at 488 nm for Cy3 excitation and 632 nm for Cy5 excitation. A microscope equipped with is used. A 20 × microscope objective (Nikon, Inc., Melville NY) is used to focus the excitation laser light on the array. The slide containing the array is placed on the microscope's computer-controlled XY stage and raster scanned through the objective lens. The 1.8 cm × 1.8 cm array used in this example scans with a resolution of 20 μm.
[0345]
In two different scans, the mixed gas multiline laser sequentially excites the two fluorescent dyes. The emitted light is separated based on the wavelength and sent to two photomultiplier detectors (PMT R1477, Hamamatsu Photonics Systems, Bridgewater NJ) corresponding to the two fluorescent dyes. A suitable filter group is placed between the array and the photomultiplier tube to filter the signal. The maximum emission wavelength of the fluorescent dye used is 565 nm for Cy3 and 650 nm for Cy5. The instrument can record spectra from both fluorochromes simultaneously, but scans once for each fluorochrome and usually scans each array twice using a suitable filter in the laser source.
[0346]
Usually, to calibrate the sensitivity of each scan, a cDNA control species is added to the sample mixture at some known concentration and the signal intensity generated by the control species is used. A particular location on the array contains a complementary DNA sequence, and the intensity of the signal at that location is correlated at a hybridization species weight ratio of 1: 100,000. When two samples from different sources (such as a representative test cell and a control cell) are labeled with different fluorescent dyes and hybridized to a single array to identify gene groups with different expression Performs calibration by labeling a sample of cDNA to be calibrated with two fluorescent dyes and adding equal amounts of each to the hybridization mixture.
[0347]
A 12-bit RTI-835H analog-to-digital (A / D) conversion board (Analog Devices, Inc., Norwood MA) installed on an IBM-compatible PC computer is used to digitize the photomultiplier tube output. The digitized data is displayed as an image in which the signal intensity is mapped using a linear 20 color conversion from a blue (low signal) to red (high signal) pseudo color scale. Data is also analyzed quantitatively. When two different fluorescent dyes are excited and measured simultaneously, using the emission spectra of each phosphor, the data is first corrected for optical crosstalk between the fluorescent dyes (due to overlapping emission spectra).
[0348]
A grid is overlaid on the fluorescent signal image, whereby the signal from each spot is collected on each element of the grid. The fluorescence signals within each element are integrated to give a numerical value depending on the average intensity of the signal. The software used for signal analysis is the GEMTOOLS gene expression analysis program (Incyte).
[0349]
11 Complementary polynucleotides
A sequence complementary to a sequence encoding NAAP or any portion thereof is used to detect, reduce or inhibit the expression of native NAAP. Although the use of oligonucleotides containing about 15-30 base pairs is described, essentially the same procedure is used for smaller or larger sequence fragments. Oligo 4.06 software (National Biosciences) and NAAP coding sequences are used to design appropriate oligonucleotide groups. In order to inhibit transcription, a complementary oligonucleotide is designed from the most unique 5 'sequence and used to prevent the promoter from binding to the coding sequence. To inhibit translation, a complementary oligonucleotide is designed to prevent the ribosome from binding to transcripts encoding NAAP.
[0350]
12 NAAP expression
NAAP expression and purification can be performed using bacterial or viral based expression systems. To express ATRS in bacteria, the cDNA is subcloned into a suitable vector having an antibiotic resistance gene and an inducible promoter that increases the level of cDNA transcription. Such promoters include, but are not limited to, the T5 or T7 bacteriophage promoter in combination with the lac operator regulatory element and the trp-lac (tac) hybrid promoter. The recombinant vector is transformed into a suitable bacterial host such as BL21 (DE3). To express NAAP in antibiotic resistant bacteria, induce with isopropyl β-D thiogalactopyranoside (IPTG). NAAP expression in eukaryotic cells is commonly known as baculovirus in insect cell lines or mammalian cell linesAutographica californicaInfected with a recombinant form of nuclear polyhedrosis virus (AcMNPV). To replace the baculovirus nonessential polyhedrin gene with a cDNA encoding NAAP, either homologous recombination or bacterial-mediated gene transfer with transfer plasmid mediation is performed. The infectivity of the virus is maintained and a high level of cDNA transcription is performed by a strong polyhedron promoter. Recombinant baculoviruses are often a type of night owlSpodoptera frugiperda(Sf9) Used for infection of insect cells, but may also be used for infection of human hepatocytes. In the case of the latter infection, further genetic modification to baculovirus is required (Engelhard, EK et al. (1994) Proc. Natl. Acad. Sci. USA 91: 3224-3227; Sandig, V. et al. (1996) ) See Hum. Gene Ther. 7: 1937-1945).
[0351]
In most expression systems, NAAP becomes a fusion protein synthesized with, for example, glutathione S transferase (GST) or peptide epitope tags such as FLAG and 6-His, so recombinant fusion from unpurified cell lysates Protein affinity-based purification can be performed rapidly in one step. GST is a 26 kDa enzyme from Schistosoma japonicum that allows purification of the fusion protein on immobilized glutathione while maintaining protein activity and antigenicity (Amersham Pharmacia Biotech). After purification, the GST element can be proteolytically cleaved from NAAP at the specific engineered site. FLAG is an 8-amino acid peptide that allows immunoaffinity purification using commercially available monoclonal and polyclonal anti-FLAG antibodies (Eastman Kodak). 6-His, in which 6 histidine residues are continuously extended, allows purification on metal chelate resins (QIAGEN). Methods for protein expression and purification are described in Ausubel (1995) chapters 10 and 16 above. Using NAAP purified by these methods directly,Examples 16, 17, 18 and 19Applicable assays can be performed.
[0352]
13 Functional assays
The function of NAAP is assessed by the expression of sequences encoding NAAP at physiologically elevated levels in mammalian cell culture systems. Subclone the cDNA into a mammalian expression vector with a strong promoter that expresses cDNA at high levels. Vectors selected include PCMV SPORT (Life Technologies) and PCR3.1 (Invitrogen, Carlsbad CA), both of which have a cytomegalovirus promoter. Using a liposomal formulation or electroporation, 5-10 μg of the recombinant vector is transiently transfected into a human cell line, eg, an endothelial or hematopoietic cell line. In addition, 1-2 μg of plasmid containing the sequence encoding the labeled protein is simultaneously transfected. The expression of the labeled protein provides a means to distinguish between transfected and non-transfected cells. Moreover, the expression of cDNA from the recombinant vector can be accurately predicted by the expression of the labeled protein. The labeled protein can be selected from, for example, green fluorescent protein (GFP; Clontech), CD64 or CD64-GFP fusion protein. Identify transfected cells expressing GFP or CD64-GFP using flow cytometry (FCM), an automated, laser-optical technology, and determine the apoptotic status and other cellular properties of those cells evaluate. FCM detects and measures the uptake of fluorescent molecules that diagnose phenomena that precede or occur simultaneously with cell death. These phenomena include changes in nuclear DNA content as measured by DNA staining with propidium iodide, changes in cell size and granularity as measured by forward and 90 ° side scatter, bromodeoxy Down-regulation of DNA synthesis measured by decreased uridine incorporation, cell surface and altered protein expression measured by reactivity with specific antibodies, and cell surface of fluorescein-conjugated annexin V protein And changes in the plasma membrane composition measured by binding to. For flow cytometry, see Ormerod, M.G. (1994).Flow Cytometry, There is a description in Oxford, New York NY.
[0353]
The effect of NAAP on gene expression can be assessed using highly purified cell populations transfected with sequences encoding NAAP and either CD64 or CD64-GFP. CD64 or CD64-GFP is expressed on the surface of transformed cells and binds to a conserved region of human immunoglobulin G (IgG). Transformed and non-transformed cells can be efficiently separated using magnetic beads coated with either human IgG or antibodies to CD64 (DYNAL, Lake Success NY). mRNA can be purified from cells by methods well known to those skilled in the art. Expression of mRNA encoding NAAP and other genes of interest can be analyzed by Northern analysis or microarray technology.
[0354]
14 Production of antibodies specific for NAAP
Substantially purified NAAP is made by polyacrylamide gel electrophoresis (PAGE; see, for example, Harrington, MG (1990) Methods Enzymol. 182: 488-495) or other purification techniques and used to produce standard Immunize rabbits with a simple protocol to produce antibodies.
[0355]
Alternatively, the NAAP amino acid sequence is analyzed using LASERGENE software (DNASTAR) to determine the highly immunogenic region, the corresponding oligopeptide is synthesized and used to generate antibodies in a manner well known to those skilled in the art. Produce. The selection of suitable epitopes, such as those near the C-terminus or in adjacent hydrophilic regions, is well known in the art (see, eg, Ausubel, 1995, chapter 11 above).
[0356]
Typically, an approximately 15-residue oligopeptide is synthesized using an ABI 431A peptide synthesizer (Applied Biosystems) using FMOC chemistry and by a reaction using N-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS). Bind to KLH (Sigma-Aldrich, St. Louis MO) to increase immunogenicity (see, eg, Ausubel, 1995, supra). Rabbits are immunized with oligopeptide-KLH conjugate in complete Freund's adjuvant. To test the anti-peptide activity and anti-NAAP activity of the obtained antiserum, the peptide or ATRS was bound to a substrate, blocked with 1% BSA, washed with a rabbit antiserum, washed, and further radioactive. React with iodine labeled goat anti-rabbit IgG.
[0357]
15 Purification of natural NAAP using specific antibodies
In order to substantially purify natural NAAP or recombinant NAAP, immunoaffinity chromatography using a group of antibodies specific for NAAP is performed. The immunoaffinity column is formed by covalently binding an activation chromatography resin such as CNBr-activated SEPHAROSE (Amersham Pharmacia Biotech) and an anti-NAAP antibody. After binding, the resin is blocked and washed according to the manufacturer's instructions.
[0358]
The culture solution containing NAAP is passed through an immunoaffinity column, and the column is washed under conditions that allow preferential adsorption of NAAP (for example, with a high ionic strength buffer in the presence of a surfactant). The column is eluted under conditions that break the binding between the antibody and NAAP (eg, with some pH 2-3 buffer, or a high concentration of a chaotrope such as urea or thiocyanate ion) to recover NAAP. .
[0359]
16 Identification of molecules that interact with NAAP
NAAP, or a biologically active fragment thereof,¹²⁵Label with I Bolton Hunter reagent (see, eg, Bolton, A.E. and W.M. Hunter (1973) Biochem. J. 133: 529-539). Candidate molecules pre-sequenced in each well of the multiwell plate are incubated with labeled NAAP, washed and all wells with labeled NAAP complex are assayed. Data obtained at various NAAP concentrations are used to calculate values for the quantity, affinity, and association of NAAP bound to the candidate molecule.
[0360]
Alternatively, the molecule that interacts with NAAP may be selected from the yeast two-hybrid system described in Fields, S. and O. Song (1989, Nature 340: 245-246), or MATCHMAKER. Analyze using a commercial kit based on a 2-hybrid system such as the system (Clontech).
[0361]
NAAP can also be used in the PATHCALLING process (CuraGen Corp., New Haven CT) using a high-throughput yeast two-hybrid system to determine all interactions between proteins encoded by the two large libraries of genes ( Nandabalan, K. et al. (2000) US Pat. No. 6,057,101).
[0362]
17 Demonstration of NAAP activity
NAAP activity is measured by the ability of NAAP to stimulate transcription of the reporter gene (Liu, H. Y. et al. (1997) EMBO J. 16: 5289-5298). This assay is a well-characterized reporter gene construct, LexA_opUse -LacZ. This LexA_op-LacZ is a LexA DNA transcriptional regulatory element (LexA) fused to a sequence encoding the E. coli LacZ enzyme._op). Methods for creating and expressing fusion genes, introducing fusion genes into cells, and measuring LacZ enzyme activity are well known in the art. The sequence encoding NAAP is cloned into a plasmid that induces the synthesis of LexA-NAAP, a fusion protein consisting of a DNA binding domain derived from a LexA transcription factor and NAAP. The resulting plasmid encoding the LexA-NAAP fusion protein was transformed into LexA_op-Introduced into a yeast cell together with a plasmid having a LacZ reporter gene. The amount of LacZ enzyme activity associated with LexA-NAAP transformed cells compared to control cells is proportional to the amount of transcription stimulated by NAAP.
[0363]
Alternatively, NAAP activity is measured as the ability to bind to zinc. 5-10 μM sample solution (in 2.5 mM ammonium acetate solution (pH 7.4)) in the presence of 0.05 M zinc sulfate solution (Aldrich, Milwaukee WI) and 100 μM dithiothreitol (with 10% methanol) Mix under. Incubate the sample with the zinc sulfate solution for 20 minutes. The reaction solution is passed through a VYCAC column (bore size of about 300 A, particle size of 5 μm) (Grace Vydac, Hesperia CA). Thereby, the zinc-sample complex is isolated from the solution and then subjected to a mass spectrometer (PE Sciex, Ontario, Canada). A functional atomic weight of 63.5 Da is used for the determination of zinc bound to the sample. This atomic weight was observed by Whittal, R. M. et al. ((2000) Biochemistry 39: 8406-8417).
[0364]
Alternatively, one method of determining NAAP nucleic acid binding activity involves a polyacrylamide gel migration mobility shift assay. In preparing this assay, NAAP is expressed by transforming a mammalian cell line, such as COS7, HeLa, or CHO, using a eukaryotic expression vector with NAAP cDNA. Following transformation, the cells are incubated for 48-72 hours under conditions suitable for the cell line to express and accumulate NAAP. Preparation of an extract with solubilized protein can be performed from cells expressing NAAP. This method is well known in the art. For each part of the extract with NAAP, [³²P] Add to labeled RNA or DNA. The synthesis of radioactive nucleic acidsin vitro Thus, this can be achieved by techniques well known in the art. Incubate the mixture at 25 ° C. in the presence of RNase inhibitor and DNase inhibitor under buffered conditions for 5-10 minutes. After incubation, the sample is analyzed by polyacrylamide gel electrophoresis followed by autoradiography. The presence of a band on the autoradiogram indicates the formation of a complex between NAAP and the radioactive transcript. Similar mobility bands will not be seen in samples prepared with control extracts prepared from non-transformed cells.
[0365]
Alternatively, one method of determining methylase activity of NAAP measures the transfer of radiolabeled methyl groups between the donor and acceptor substrates. The reaction mixture (50 μl final volume) contains 15 mM HEPES (pH 7.9), 1.5 mM MgCl₂, 10 mM dithiothreitol, 3% polyvinyl alcohol, 1.5 μCi [methyl-^ThreeH] AdoMet (0.375 μM AdoMet) (DuPont-NEN), 0.6 μg NAAP, and a receptor substrate (eg, 0.4 μg [³⁵S] RNA or 6-mercaptopurine (6-MP), final concentration up to 1 mM). The reaction mixture is incubated at 30 ° C. for 30 minutes and then at 65 ° C. for 5 minutes.
[0366]
[Methyl-^ThreeThe analysis method of H] RNA is as follows. (1) 50 μl 2 × loading buffer (20 mM Tris-HCl (pH 7.6), 1 M LiCl, 1 mM EDTA, 1% sodium dodecyl sulfate (SDS)) and 50 μl oligo d (T)- Cellulose (10 mg / ml in 1 × loading buffer) is added to the reaction mixture and the incubation is performed for 30 minutes with shaking at room temperature. (2) Transfer the reaction mixture to a 96-well filtration plate (connected to a vacuum device). (3) Wash each sample sequentially. This uses three 2.4 ml aliquots of 1 × oligo d (T) loading buffer (with 0.5% SDS or 0.1% SDS and without SDS). (4) RNA is eluted into a 96-well collection plate with 300 μl of water and transferred to a scintillation vial (containing liquid scintillant) to determine radioactivity.
[0367]
[Methyl-^ThreeThe analysis method of H] 6-MP is as follows. (1) Add 500 μl of 0.5 M borate buffer (pH 10.0) and then 2.5 ml of isoamyl alcohol diluted in toluene at 20% (v / v) to the reaction mixture. (2) Mix the sample by vortexing for 10 seconds. (3) After centrifuging at 700 g for 10 minutes, transfer 1.5 ml of the organic phase to a scintillation vial (containing 0.5 ml absolute ethanol and liquid scintillant) and determine the radioactivity. (4) The result is corrected for extraction of 6-MP into the organic phase (approximately 41%).
[0368]
Alternatively, NAAP type I topoisomerase activity assays are based on the relaxation of certain supercoiled DNA substrates. Incubate NAAP with its substrate, Mg²⁺ The reaction is terminated and the product is loaded onto an agarose gel. Discrimination of the transformed topoisomer from the supercoil substrate can be done by electrophoresis. This assay is specific for type I topoisomerase activity. The reason is Mg²⁺And ATP are cofactors required for type II topoisomerase.
[0369]
An assay for NAAP type II topoisomerase activity can be performed based on the decatenation of certain kinetoplast DNA (KDNA) substrates. Incubation of NAAP is performed with KDNA to terminate the reaction and load the product onto an agarose gel. Discrimination of monomer circular KDNA from linked KDNA can be performed by electrophoresis. A commercial kit for measuring type I topoisomerase activity and type II topoisomerase activity is obtained from Topogen (Columbus OH).
[0370]
NAAP's ATP-dependent RNA helicase unwinding activity can be measured by the method described in Zhang and Grosse (1994; Biochemistry 33: 3906-3912). RNA unwinding substrate³²Contains P-labeled RNA, which consists of two RNA strands (194 and 130 nucleotides in length) and has a 17 base pair duplex region. Incubation of RNA substrate, ATP, Mg²⁺, And with variable amount of NAAP in Tris-HCl buffer (pH 7.5) at 37 ° C. for 30 minutes. Next, electrophoresis for separating the single-stranded RNA product from the double-stranded RNA substrate is performed on a 10% SDS polyacrylamide gel, and quantified by autoradiography. The amount of single stranded RNA recovered is proportional to the amount of NAAP in the preparation.
[0371]
Alternatively, an assay for NAAP function is assessed by expression of a sequence encoding NAAP at a physiologically elevated level in a mammalian cell culture system. Subclone the cDNA into a mammalian expression vector with a strong promoter that expresses cDNA at high levels. Vectors selected include pCMV SPORT (Life Technologies) and pCR3.1 (Invitrogen Corporation, Carlsbad CA), both of which have a cytomegalovirus promoter. Using a liposomal formulation or electroporation, 5-10 μg of the recombinant vector is transiently transfected into a human cell line, preferably an endothelial or hematopoietic cell line. In addition, 1-2 μg of plasmid containing the sequence encoding the labeled protein is simultaneously transfected.
[0372]
The expression of the labeled protein provides a means to distinguish between transfected and non-transfected cells. Moreover, expression of cDNA from the recombinant vector can be accurately predicted by expressing the labeled protein. The labeled protein can be selected from, for example, green fluorescent protein (GFP; CLONTECH), CD64 or CD64-GFP fusion protein. Identify transfected cells expressing GFP or CD64-GFP using flow cytometry (FCM), an automated, laser-optical technology, and determine the apoptotic status and other cellular properties of those cells evaluate.
[0373]
FCM detects and measures the uptake of fluorescent molecules that diagnose phenomena that precede or occur simultaneously with cell death. These phenomena include changes in nuclear DNA content as measured by DNA staining with propidium iodide, changes in cell size and granularity as measured by forward and 90 ° side scatter, bromodeoxy Down-regulation of DNA synthesis measured by decreased uridine uptake, cell surface and altered protein expression measured by reactivity with specific antibodies, and cell surface of fluorescein-conjugated annexin V protein And changes in the plasma membrane composition measured by binding to. For flow cytometry, see Ormerod, M. G. (1994).Flow Cytometry, Oxford, New York NY.
[0374]
The effect of NAAP on gene expression can be assessed using highly purified cell populations transfected with sequences encoding NAAP and either CD64 or CD64-GFP. CD64 or CD64-GFP is expressed on the surface of transformed cells and binds to a conserved region of human immunoglobulin G (IgG). Transformed and non-transformed cells can be efficiently separated using magnetic beads coated with either human IgG or antibodies to CD64 (Dynal Biotech, Lake Success NY). mRNA can be purified from cells by methods well known to those skilled in the art. Expression of mRNA encoding NAAP and other genes of interest can be analyzed by Northern analysis or microarray technology.
[0375]
NAAP pseudouridine synthase activity assay includes tritium (^ThreeH) A release assay was performed by adapting Nurse et al. ((1995) RNA 1: 102112)^ThreeWhen H-radiolabeled U is isomerized to pseudouridine (y), the pyrimidine component C of uridylic acid (U)_Five From position^ThreeMeasure H release. A typical 500 μl assay mix consists of 50 mM HEPES buffer (pH 7.5), 100 mM ammonium acetate, 5 mM dithiothreitol, 1 mM EDTA, 30 units RNase inhibitor, and 0.1-4.2 μM. [Five^ThreeH] tRNA (about 1 μCi / nmol tRNA). To initiate the reaction, add less than 5 μl of a concentrated solution of NAAP (or a sample containing NAAP) and incubate for 5 minutes at 37 ° C. After adding NAAP, a portion of the reaction mixture is collected at various elapsed times (up to 30 minutes) and diluted in 1 ml of 0.1 M HCl containing NoritSA3 (12% w / v) to react. Stop. Centrifugation of the stopped reaction mixture is carried out in a microcentrifuge at maximum speed for 5 minutes, and the supernatant is filtered through a glass wool pad. The pellet is washed by resuspension twice in 1 ml 0.1 M HCl and then centrifuged. Each of the washed supernatants is passed separately through a glass wool stuffing and mixed with the original filtrate. A part of the mixed filtrate is mixed with scintillation liquid (maximum 10 ml) and counted with a scintillation counter. Released from RNA^ThreeThe amount of H and also present in the soluble filtrate^ThreeThe amount of H is proportional to the amount of pseudouridine synthase activity in the sample (Ramamurthy, V. (1999) J. Biol. Chem. 274: 2222522230).
[0376]
Alternatively, the NAAP pseudouridine synthase activity assay is performed at 30 ° C. to 37 ° C. in the following mixture. That is, 100 mM TrisHCl (pH 8.0), 100 mM ammonium acetate, 5 mM MgCl₂2 mM dithiothreitol, 0.1 mM EDTA, and 1-2 fmol [³²P] radiolabeled runoff transcript (production isin vitro And a suitable RNA polymerase (performed with T7 or SP6) as a substrate. Start the reaction by adding NAAP, or do not add to the reaction solution and use as a control sample. After incubation, RNA is extracted with phenol-chloroform and precipitated in ethanol. Also, RNase T for complete hydrolysis to 3-nucleotide monophosphate₂Is used. Two-dimensional thin layer chromatography is used to analyze hydrolyzate,³²Assessment of the amount of P-radiation label (the amount present in yMP and UMP spots) is performed after exposing the thin layer chromatography plate to film or PHOSPHORIMAGER screen (Molecular Dynamics). Determine the relative amount of yMP and UMP considering the relative number of uridylate residues in the substrate RNA, and use this amount to calculate the relative amount of y to the amount of tRNA molecules (mol y / tRNA mol, Or expressed in moles y / tRNA moles / minute). This amount corresponds to the amount of pseudouridine synthase activity in the NAAP sample (Lecointe, F. et al. (1998) J. Biol. Chem. 273: 13161323).
[0377]
NAAP N², N²Dimethylguanosine transferase ((m² ₂Measurement of G) methyltransferase) activity is carried out in a 160 μl reaction mixture containing: That is, 100 mM Tris-HCl (pH 7.5), 0.1 mM EDTA, 10 mM MgCl₂20 mM NH_FourCl, 1 mM dithiothreitol, 6.2 μM S-adenosyl-L- [methyl-^ThreeH] methionine (30-70 Ci / mM), 8 μg m² ₂Contains G-deficient or wild-type tRNA (yeast derived) and approximately 100 μg of purified NAAP or NAAP-containing sample. Incubate the reaction at 30 ° C. for 90 minutes and cool on ice. Dilute a portion of each reaction in 1 ml of water containing 100 μg BSA. 1 ml of 2 M HCl is added to each sample and the acidic insoluble product is allowed to settle on ice for 20 minutes before filtration for collection through a glass fiber filter. The collected material is washed several times with hydrochloric acid and quantified using a liquid scintillation counter.^ThreeH is m² ₂The amount incorporated into acidic insoluble tRNA lacking G is the amount of N in the NAAP sample.², N²It is proportional to the amount of dimethylguanosine transferase activity. Reactions that do not contain a substrate tRNA or that already contain a modified wild-type tRNA serve as a control reaction, in which the reaction is acidic insoluble^ThreeNo H-labeled product should be obtained.
[0378]
To measure NAAP polyadenylation activity,in vitro The polyadenylation reaction is used. The reaction mixture is made on ice and includes: 10 μl of 5 mM dithiothreitol, 0.025% (v / v) Nonidet P40 surfactant, 50 mM creatine phosphate, 6.5% (w / v) polyvinyl alcohol, 0.5 units / μl RNAGUARD (Amersham Pharmacia Biotech), 0.025 μg / μl creatine kinase, 1.25 mM cordycepin 5 ′ triphosphate, and 3.75 mM MgCl₂The total volume is 25 μl. 60 fmol CstF, 50 fmol CPSF, 240 fmol PAP, 4 μl unpurified CF II, or partially purified CF II and a variable amount of CF I are then added to the reaction mixture. To adjust the volume to 23.5 μl, add a buffer with: 50 mM TrisHCl (pH 7.9), 10% (v / v) glycerol, and 0.1 mM NaEDTA. The final ammonium sulfate concentration should be less than 20 mM. To start the reaction on ice, 15 fmol³²Add P-labeled mRNA precursor template and 1.5 μl water containing 2.5 μg unlabeled tRNA. The reaction is then incubated. This is done at 30 ° C. for 75-90 minutes and to stop, 75 μl (approximately 2 volumes) proteinase K mix (0.2 M Tris-HCl, pH 7.9, 300 mM NaCl, 25 mM NaEDTA, 2 % (w / v SDS), 1 μl of 10 mg / ml proteinase K, 0.25 μl of 20 mg / ml glycogen and 23.75 μl of water. Following incubation, RNA is precipitated with ethanol and analysis is performed on a 6% (w / v) polyacrylamide, 8.3 M urea sequencing gel. The dried gel is sensitized by autoradiography or by a phosphoimager. To determine cleavage activity, the amount of cleavage product is compared to the amount of mRNA precursor template. Removing either polypeptide component from the reaction and substituting NAAP is useful for identifying specific biological functions of NAAP in mRNA precursor polyadenylation (Ruegsegger, U. et al. (1996) J. Biol. Chem. 271: 6107-6113, and references thereof).
[0379]
tRNA synthetase activity is [¹⁴C] —measured as aminoacylation of substrate tRNA in the presence of labeled amino acid. NAAP is in buffer, [¹⁴C] -labeled amino acid and a suitable cognate tRNA (eg, [¹⁴C] alanine and tRNA^ala).¹⁴C-labeled product is free [¹⁴[C] Chromatographically separated and incorporated from amino acids [¹⁴C] is measured with a scintillation counter.¹⁴The amount of C-labeled product is proportional to the activity of NAAP in this assay.
[0380]
Alternatively, to measure NAAP activity, the NAAP-containing sample is incubated with a solution containing: 1 mM ATP, 5 mM Hepes-KOH (pH 7.0), 2.5 mM KCl, 1.5 mM magnesium chloride, and 0.5 mM DTT, and misacylated [¹⁴C] -GlutRNAGln (eg 1 μM) and similar concentrations of unlabeled L-glutamine are also included. To stop the reaction, 3 M sodium acetate (pH 5.0) was added, and the mixture was extracted with the same amount of water-saturated phenol. Do for 1 minute. Precipitate the aqueous phase with 3 volumes of ethanol at -70 ° C for 15 minutes. Centrifuge to collect the precipitated aminoacyl-tRNA at 15,000 xg for 15 minutes at 4 ° C. The pellet is resuspended with 25 mM KOH, deacylated for 10 minutes at 65 ° C., neutralized with 0.1 M HCl (final pH 6-7) and vacuum dried. Resuspend the dried pellet in water and spot on a cellulose TLC plate. Plates are exposed in isopropanol / formic acid / water or ammonia / water / chloroform / methanol. The image is subjected to densitometer analysis, and the relative amount of Glu and Gln is calculated based on the Rf value and relative intensity of the spot. Glu calculated GAP-tRNA for NAAP activity^Gln (According to Curnow, A.W. et al. (1997) Proc. Natl. Acad. Sci. U. S. A. 94: 11819-26).
[0381]
18 Identification of NAAP agonists and antagonists
An agonist or antagonist of NAAP activation or inhibition,Example 17Can be tested using the assay described in. Agonists cause an increase in NAAP activity and antagonists cause a decrease in NAAP activity.
[0382]
19 NAAP secretion assay
Certain high throughput assays can be used to identify polypeptides that are secreted into eukaryotic cells. In one example of such an assay, a 5′-biased cDNA is fused to the 5 ′ end of an unleaded β-lactamase gene to generate a polypeptide expression library. β-lactamase is a convenient gene reporter. The reason is that this enzyme shows a high signal / noise ratio and low endogenous background activity, and retains activity even when fused to other proteins. Certain double promoter systems may allow expression of β-lactamase fusion polypeptides in bacteria or eukaryotic cells. For this, the lac or CMV promoter is used, respectively.
[0383]
The library is first transformed into a bacterium (such as E. coli) to identify library members that encode a fusion polypeptide that can be secreted in a eukaryotic system. The mammalian signal sequence directs the β-lactamase fusion polypeptide to migrate into the bacterial periplasm. In the periplasm this confers antibiotic resistance to carbenicillin. Carbenicillin-selected bacteria are isolated in solid media, individual clones are grown in liquid media, and the resulting culture is used to isolate library member plasmid DNA.
[0384]
Seeding mammalian cells (such as 293 cells) on 96-well tissue culture plates at a density of approximately 40,000 cells / well, 10% fetal bovine serum (FBS) in DME without 100 μl phenol red (Life Technologies, Rockville, MD) is added. The next day, purified plasmid DNA (isolated from carbenicillin resistant bacteria) is diluted to a volume of 25 μl in each well of cells to be transfected with 15 μl of OPTI-MEM I medium (Life Technologies). In a separate plate, dilute 1 μl LF2000 Reagent (Life Technologies) in 25 μl / well OPTI-MEM I. 25 μl diluted LF2000 Reagent is then mixed with 25 μl diluted DNA, mixed briefly and incubated for 20 minutes at room temperature. The resulting DNA-LF2000 reagent complex is then added directly to each well of 293 cells. Cell transfection is also performed with an appropriate control plasmid (a plasmid that expresses either a wild-type β-lactamase, a leader-free β-lactamase, or a leader-free β-lactamase, eg, a CD4 fused). 24 hours after transfection, approximately 90 μl of cell culture assay should be performed 37^oC. Use 100_M Nitrocefin (nitrocefin, Calbiochem, San Diego Calif.) And 0.5 mM oleic acid (Sigma, St. Louis, Mo.) in 10 mM phosphate buffer (pH 7.0). Nitrocefin is a substrate for β-lactamase, which, when hydrolyzed, changes color significantly from yellow to red. β-lactamase activity is monitored at 486 nm in a microtiter plate reader for 20 minutes. The increase in color absorption at 486 nm is consistent with the secretion of β-lactamase fusion polypeptide in transfected cell media. This is a result of the presence of a eukaryotic signal sequence in the fusion polypeptide. Polynucleotide sequence analysis of the corresponding library member plasmid DNA is then used to identify the cDNA encoding the signal sequence (as described in US patent application 09 / 803,317, file date March 9, 2001).
[0385]
It will be apparent to those skilled in the art that various modifications and variations can be made to the described method and system of the present invention without departing from the spirit or spirit of the invention. While the invention has been described with reference to several embodiments, it should be understood that the scope of the invention should not be unduly limited to such specific embodiments. . Various modifications of the practice of the invention described herein that are apparent to those skilled in molecular biology or related fields are intended to be within the scope of the following claims.
[0386]
(Short description of the table)
Table 1 outlines the nomenclature for the full-length polynucleotide and polypeptide sequences of the present invention.
[0387]
Table 2 shows the GenBank identification number of the polypeptides of the present invention, the annotation of the closest GenBank homolog, the PROTEOME database identification number, and the annotation of the PROTEOME database homolog. Also shown is the probability score for each polypeptide and its homologues (one or more) matching.
[0388]
Table 3 shows the structural features of the polynucleotide sequences of the invention, such as predicted motifs and domains, along with the methods, algorithms and searchable databases used for polypeptide analysis.
[0389]
Table 4 shows the cDNA and genomic DNA fragments used to construct the polynucleotide sequences of the present invention, along with selected fragments of the polynucleotide sequences.
[0390]
Table 5 shows a representative cDNA library of the polynucleotides of the present invention.
[0390]
Table 6 is an appendix explaining the tissues and vectors used for the preparation of the cDNA library shown in Table 5.
[0392]
Table 7 shows the tools, programs, and algorithms used to analyze the polynucleotides and polypeptides of the present invention, along with applicable descriptions, references and threshold parameters.
[Table 1]

[Table 2]

[Table 3]

[Table 4]

[Table 5]

[Table 6]

[Table 7]

[Table 8]

[Table 9]

[Table 10]

[Table 11]

[Table 12]

[Table 13]

[Table 14]

[Table 15]

[Table 16]

[Table 17]

[Table 18]

[Table 19]

[Table 20]

[Table 21]

[Table 22]

[Table 23]

[Table 24]

[Table 25]

[Table 26]

[Table 27]

[Table 28]

[Table 29]

[Table 30]

[Sequence Listing]

Claims (87)

An isolated polypeptide selected from the group consisting of the following (a) to (e):
(A) a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-16 (SEQ ID NOs: 1 to 16) (b) from SEQ ID NO: 1-2 and SEQ ID NO: 4-16 A polypeptide having a natural amino acid sequence which is at least 90% identical to an amino acid sequence selected from the group (c) a natural such that at least 95% is identical to the amino acid sequence of SEQ ID NO: 3 A polypeptide having an amino acid sequence (d) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-16 (e) selected from the group consisting of SEQ ID NO: 1-16 Immunogenic fragments of polypeptides having amino acid sequences 2. The isolated polypeptide of claim 1 having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-16. 2. An isolated polynucleotide encoding the polypeptide of claim 1. An isolated polynucleotide encoding the polypeptide of claim 2. 5. The isolated polynucleotide of claim 4 having a polynucleotide sequence selected from the group consisting of SEQ ID NO: 17-32. A recombinant polynucleotide having a promoter sequence operably linked to the polynucleotide of claim 3. A cell transformed with the recombinant polynucleotide according to claim 6. A genetic transformant comprising the recombinant polynucleotide according to claim 6. A method for producing the polypeptide of claim 1, comprising:
(A) a cell transformed with a recombinant polynucleotide having a promoter sequence operably linked to a polynucleotide encoding the polypeptide of claim 1 under conditions suitable for expression of said polypeptide. Culturing, and
And (b) recovering the polypeptide so expressed. 10. The method of claim 9, wherein the polypeptide has an amino acid sequence selected from the group consisting of SEQ ID NO: 1-16. An isolated antibody that specifically binds to the polypeptide of claim 1. An isolated polynucleotide selected from the group consisting of the following (a) to (g).
(A) a polynucleotide having a polynucleotide sequence selected from the group consisting of SEQ ID NO: 17-32 (b) a certain poly selected from the group consisting of SEQ ID NO: 17-18, SEQ ID NO: 20-32 A polynucleotide having a native polynucleotide sequence that is at least 90% identical to the nucleotide sequence (c) a natural polynucleotide sequence such that at least 95% is identical to the polynucleotide sequence of SEQ ID NO: 19 A polynucleotide complementary to the polynucleotide (e) (b), the polynucleotide complementary to the polynucleotide (f) (c), g) RNA equivalents of (a)-(f) 13. An isolated polynucleotide having at least 60 contiguous nucleotides of the polynucleotide of claim 12. A method for detecting a target polynucleotide having the sequence of the polynucleotide of claim 12 in a sample comprising:
(A) hybridizing the sample with a probe having at least 20 consecutive nucleotide groups having a sequence complementary to the target polynucleotide in the sample, wherein the probe is a hybridization complex with the probe; Specifically hybridizing to the target polynucleotide under conditions formed with the target polynucleotide or fragments thereof;
(B) detecting the presence or absence of the hybridization complex, and optionally detecting the amount of the complex if present. 15. The method of claim 14, wherein the probe comprises at least 60 consecutive nucleotides. A method for detecting a target polynucleotide having the sequence of the polynucleotide of claim 12 in a sample comprising:
(A) amplifying the target polynucleotide or fragment thereof using polymerase chain reaction amplification;
(B) detecting the presence or absence of the amplified target polynucleotide or a fragment thereof, and optionally detecting the amount of the target polynucleotide or a fragment thereof, if present. . A composition comprising the polypeptide of claim 1 and a pharmaceutically acceptable excipient. 18. The composition of claim 17, wherein said polypeptide has an amino acid sequence selected from the group consisting of SEQ ID NO: 1-16. 18. A method of treating a disease or condition associated with reduced expression of functional NAAP, comprising administering the composition of claim 17 to a patient in need of such treatment. A method of screening a compound to confirm the effectiveness of the polypeptide of claim 1 as an agonist, comprising:
(A) exposing a sample having the polypeptide of claim 1 to a compound;
(B) a screening method comprising the step of detecting agonist activity in the sample. 21. A composition comprising an agonist compound identified by the method of claim 20 and also having a pharmaceutically acceptable excipient. 22. A method of treating a disease or condition associated with reduced expression of functional NAAP, comprising administering the composition of claim 21 to a patient in need of such treatment. A method of screening a compound to confirm the effectiveness of the polypeptide of claim 1 as an antagonist comprising:
(A) exposing a sample having the polypeptide of claim 1 to a compound;
(B) a screening method comprising the step of detecting antagonist activity in the sample. 24. A composition comprising an antagonist compound identified by the method of claim 23 and a pharmaceutically acceptable excipient. 25. A method of treating a disease or condition associated with overexpression of functional NAAP, comprising administering the composition of claim 24 to a patient in need of such treatment. A method for screening for a compound that specifically binds to the polypeptide of claim 1, comprising:
(A) mixing the polypeptide of claim 1 with at least one test compound under suitable conditions;
(B) detecting the binding of the polypeptide of claim 1 to a test compound and thereby identifying the compound that specifically binds to the polypeptide of claim 1, . A method of screening for compounds that modulate (modulate) the activity of the polypeptide of claim 1 comprising:
(A) mixing the polypeptide of claim 1 with at least one test compound under conditions that permit the activity of the polypeptide of claim 1;
(B) calculating the activity of the polypeptide of claim 1 in the presence of a test compound;
(C) comparing the activity of the polypeptide of claim 1 in the presence of the test compound with the activity of the polypeptide of claim 1 in the absence of the test compound, A method wherein the change in activity of the polypeptide of claim 1 in the presence of is indicative of a compound that modulates the activity of the polypeptide of claim 1. A method of screening for compounds effective to alter the expression of a target polynucleotide having the sequence of claim 5 comprising:
(A) exposing a sample having the target polynucleotide to a compound under conditions suitable for expression of the target polynucleotide;
(B) detecting altered expression of the target polynucleotide;
(C) comparing the expression of the target polynucleotide in the presence of a variable amount of the compound and in the absence of the compound. A method for calculating the toxicity of a test compound comprising:
(A) treating a biological sample having a nucleic acid group with the test compound;
(B) hybridizing a nucleic acid group of the treated biological sample with a probe having at least 20 consecutive nucleotide groups of the polynucleotide of claim 12, wherein the hybridization comprises the probe and the probe 13. A polynucleotide sequence of the polynucleotide of claim 12 or a sequence thereof, wherein the target polynucleotide is formed under conditions that form a specific hybridization complex with a target polynucleotide of a biological sample. The process, which is a polynucleotide having fragments;
(C) quantifying the yield of the hybridization complex;
(D) comparing the amount of the hybridization complex in the treated biological sample with the amount of the hybridization complex in an untreated biological sample, A difference in the amount of the hybridization complex in a biological sample is indicative of the toxicity of the test compound. A diagnostic test for a condition or disease associated with the expression of NAAP in a biological sample, comprising:
(A) mixing the biological sample with the antibody of claim 11 under conditions suitable for the antibody to bind to the polypeptide and to form a complex of antibody and polypeptide; ,
(B) detecting the complex, wherein the presence of the complex correlates with the presence of the polypeptide in the biological sample. The antibody of claim 11, comprising
An antibody comprising any one of (a) a chimeric antibody, (b) a single chain antibody, (c) a Fab fragment, (d) an F (ab ′) ₂ fragment, and (e) a humanized antibody. 12. A composition comprising the antibody of claim 11 and an acceptable excipient. 33. A method for diagnosing a symptom or disease associated with NAAP expression in a subject, comprising the step of administering to the subject an effective amount of the composition of claim 32. 33. The composition of claim 32, wherein the antibody is labeled. 35. A method of diagnosing a symptom or disease associated with NAAP expression in a subject, comprising the step of administering to the subject an effective amount of the composition of claim 34. A method for preparing a polyclonal antibody having the specificity of the antibody of claim 11, comprising:
(A) immunizing an animal with a polypeptide comprising an amino acid sequence selected from the group having SEQ ID NO: 1-16 or an immunogenic fragment thereof under conditions that elicit an antibody response;
(B) isolating the antibody from the animal;
(C) screening said isolated antibody with said polypeptide, thereby identifying a polyclonal antibody that specifically binds to a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-16 A process comprising the steps of: 37. A polyclonal antibody produced by the method of claim 36. 38. A composition comprising the polyclonal antibody of claim 37 and a suitable carrier. A method for producing a monoclonal antibody having the specificity of the antibody according to claim 11,
(A) immunizing an animal with a polypeptide comprising an amino acid sequence selected from the group having SEQ ID NO: 1-16 or an immunogenic fragment thereof under conditions that elicit an antibody response;
(B) isolating cells producing antibodies from said animal;
(C) fusing the immortalized cell with the antibody-producing cell to form a hybridoma cell that produces a monoclonal antibody;
(D) culturing the hybridoma cells;
(E) isolating from the culture a monoclonal antibody that specifically binds to a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-16. 40. A monoclonal antibody produced by the method of claim 39. 41. A composition comprising the monoclonal antibody of claim 40 and a suitable carrier. 12. The antibody of claim 11, wherein the antibody is produced by screening a Fab expression library. 12. Antibody according to claim 11, characterized in that it is produced by screening a recombinant immunoglobulin library. A method of detecting in a sample a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-16,
(A) incubating the antibody of claim 11 with a sample under conditions that allow specific binding of the antibody to the polypeptide;
(B) a step of detecting specific binding, wherein the specific binding indicates that a polypeptide having a certain amino acid sequence selected from the group consisting of SEQ ID NO: 1-16 is present in the sample A method characterized by: A method for purifying a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-16 from a sample comprising:
(A) incubating the antibody of claim 11 with a sample under conditions that allow specific binding of the antibody to the polypeptide;
(B) separating the antibody from the sample to obtain a purified polypeptide having an amino acid sequence selected from the group having SEQ ID NO: 1-16. 14. A microarray, wherein at least one element of the microarray is a polynucleotide according to claim 13. A method for generating an expression profile of a sample having a polynucleotide comprising:
(A) labeling a sample polynucleotide;
(B) contacting the elements of the microarray of claim 46 with the labeled polynucleotides of the sample under conditions suitable for formation of a hybridization complex;
(C) quantifying the expression of the polynucleotide group in the sample. An array comprising various nucleotide molecules attached to unique physical locations on a solid substrate, wherein at least one said nucleotide molecule is capable of specifically hybridizing with at least 30 consecutive nucleotides of a target polynucleotide 13. An array having an initial oligonucleotide or polynucleotide sequence, wherein the target polynucleotide is the polynucleotide of claim 12. 49. The array of claim 48, wherein the initial sequence of the oligonucleotide or polynucleotide is completely complementary to at least 30 contiguous nucleotides of the target polynucleotide. 49. The array of claim 48, wherein the initial sequence of the oligonucleotide or polynucleotide is completely complementary to at least 60 contiguous nucleotides of the target polynucleotide. 49. The array of claim 48, wherein the initial sequence of the oligonucleotide or polynucleotide is completely complementary to the target polynucleotide. 49. The array of claim 48, wherein the array is a microarray. 49. The array of claim 48, further comprising the target polynucleotide hybridized to a nucleotide molecule having a first sequence of the oligonucleotide or polynucleotide. 49. The array of claim 48, wherein a linker connects at least one of the nucleotide molecules and the solid substrate. 49. The array of claim 48, wherein each unique physical location on the substrate comprises a plurality of nucleotide molecules, wherein the plurality of nucleotide molecules at any single unique physical location have the same sequence. And each of the unique physical locations on the substrate comprises nucleotide molecules having a sequence that is different from the sequence of the nucleotide molecules at another unique physical location on the substrate. 2. The polypeptide of claim 1 having the amino acid sequence of SEQ ID NO: 1. The polypeptide of claim 2 having the amino acid sequence of SEQ ID NO: 1. 4. The polypeptide of claim 3 having the amino acid sequence of SEQ ID NO: 1. 5. The polypeptide of claim 4, having the amino acid sequence of SEQ ID NO: 1. 6. The polypeptide of claim 5 having the amino acid sequence of SEQ ID NO: 1. 7. The polypeptide of claim 6, having the amino acid sequence of SEQ ID NO: 1. 8. The polypeptide of claim 7, having the amino acid sequence of SEQ ID NO: 1. 9. The polypeptide of claim 8, having the amino acid sequence of SEQ ID NO: 1. 10. The polypeptide of claim 9, having the amino acid sequence of SEQ ID NO: 1. 11. The polypeptide of claim 10, having the amino acid sequence of SEQ ID NO: 1. 12. The polypeptide of claim 11 having the amino acid sequence of SEQ ID NO: 1. 13. The polypeptide of claim 12, having the amino acid sequence of SEQ ID NO: 1. 14. The polypeptide of claim 13, having the amino acid sequence of SEQ ID NO: 1. 15. The polypeptide of claim 14, having the amino acid sequence of SEQ ID NO: 1. 16. The polypeptide of claim 15, having the amino acid sequence of SEQ ID NO: 1. 17. The polypeptide of claim 16, having the amino acid sequence of SEQ ID NO: 1. The polynucleotide of Claim 12 having the polynucleotide sequence of SEQ ID NO: 17. The polynucleotide of Claim 12 having the polynucleotide sequence of SEQ ID NO: 18. The polynucleotide of Claim 12 having the polynucleotide sequence of SEQ ID NO: 19. The polynucleotide of Claim 12 having the polynucleotide sequence of SEQ ID NO: 20. The polynucleotide of Claim 12 having the polynucleotide sequence of SEQ ID NO: 21. The polynucleotide of Claim 12 having the polynucleotide sequence of SEQ ID NO: 22. The polynucleotide of Claim 12 having the polynucleotide sequence of SEQ ID NO: 23. The polynucleotide of Claim 12 having the polynucleotide sequence of SEQ ID NO: 24. The polynucleotide of Claim 12 having the polynucleotide sequence of SEQ ID NO: 25. The polynucleotide of Claim 12 having the polynucleotide sequence of SEQ ID NO: 26. The polynucleotide of Claim 12 having the polynucleotide sequence of SEQ ID NO: 27. The polynucleotide of claim 12 having the polynucleotide sequence of SEQ ID NO: 28. The polynucleotide of claim 12 having the polynucleotide sequence of SEQ ID NO: 29. The polynucleotide of claim 12 having the polynucleotide sequence of SEQ ID NO: 30. The polynucleotide of Claim 12 having the polynucleotide sequence of SEQ ID NO: 31. The polynucleotide of Claim 12 having the polynucleotide sequence of SEQ ID NO: 32.