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一個人類鋅指蛋白家族新基因znf325的制作方法

文檔序號:397835閱讀:330來源:國知局
專利名稱:一個人類鋅指蛋白家族新基因znf325的制作方法
技術(shù)領(lǐng)域
本發(fā)明涉及一種人類基因,具體是鋅指蛋白家族的一個人類新基因ZNF325的cDNA全長克隆,及其它在成人各器官中的表達和在不同發(fā)育階段人類胚胎各器官中的表達。
背景技術(shù)
鋅指蛋白家族是一個巨大的超基因家族,它們廣泛的存在于各種物種中,包括酵母,果蠅,爪蟾,鼠和人。鋅指蛋白家族一般具有該家族的特征性結(jié)構(gòu)-----鋅指蛋白結(jié)構(gòu)域,是最先在果蠅中被確立的、為中樞神經(jīng)系統(tǒng)和肌肉等組織分化所必需的調(diào)控因子(Postigo et al.1997)。鋅指蛋白基因zfh-1在早期果蠅胚胎的中胚層以及晚期胚胎的多種中胚層衍生結(jié)構(gòu)中表達,參與調(diào)節(jié)表達eve基因的心臟前體細胞(EPC)的形成;還與tinman基因一同調(diào)節(jié)包括性腺中胚層在內(nèi)的外側(cè)中胚層的進一步分化(Lai Z C.,et al,1999)。zfh-1在脊椎動物中的同源基因也有相似的功能。如鋅指蛋白基因ZEB的表達產(chǎn)物ZEB-1和ZEB-2蛋白,在肌肉、中樞神經(jīng)系統(tǒng)、T淋巴細胞等組織中均有表達,而最近發(fā)現(xiàn)ZEB蛋白包含兩個獨立的區(qū)域,分別調(diào)節(jié)哺乳動物肌細胞和淋巴細胞的分化(Postigo et al.1997)。
鋅指蛋白基因家族也是人類最大的基因家族之一,很多目前已知的人類轉(zhuǎn)錄調(diào)控因子都是鋅指蛋白成員(Gou et al.,2001)。典型的鋅指蛋白為C2H2型,即Zn++離子與兩個半胱氨基酸殘基和兩個組氨酸殘基形成配位鍵。除了這四個氨基酸殘基外,還有兩個芳香族氨基酸殘基和一個亮氨酸殘基也高度保守。配位結(jié)構(gòu)加上Phe和Leu所形成的疏水核心,確定了鋅指的基本結(jié)構(gòu)和形狀。鋅指的三維結(jié)構(gòu)包含兩個短的β-折疊和一個α-螺旋。研究表明鋅指結(jié)構(gòu)對于維持DNA結(jié)構(gòu)域的結(jié)構(gòu)是必需的,在很多情況下它還與DNA直接相互作用(Coppin et al.,2002)。人們認為這種鋅指結(jié)構(gòu)在蛋白質(zhì)結(jié)合到核苷酸上時起作用,在細胞的分化和繁殖過程中,它可以激活或者抑制基因的表達,如鋅指蛋白ZEB-1,snail,hFOG等,而且,這種鋅指結(jié)構(gòu)可以識別相同的DNA序列,如ZEB-1和snail的鋅指結(jié)構(gòu)可以識別它們各自靶位點的E-BOX和E-BOX類似序列(Burke et al,.2002)。
研究表明鋅指蛋白基因在生物發(fā)育過程中充當(dāng)持家基因或調(diào)節(jié)因子的角色,也有很多鋅指蛋白在人類早期胚胎發(fā)育過程中起著重要的作用。如ZNF174是某些生長因子基因的負調(diào)節(jié)子(Williams et al.,1995);AJ18參與骨的發(fā)育(Jheon et al.,2001);Hsal1,WT1,以及TR-alpha-1等鋅指蛋白與心臟功能和疾病有關(guān),HFHZ可能對心臟肥大的病理起重要作用(Dai and Liew,1998)。鋅指蛋白GATA4和hFOG2參與心臟的形成和疾病的發(fā)生(Crispino et al.,2001)。許多鋅指蛋白參與某些腫瘤的形成,如WT1(Dennis et al.,2002)和RB基因(Lee,et al.,1987)。同時,在模型動物果蠅中,鋅指蛋白Zfh1參與調(diào)節(jié)eve基因的表達,控制心臟前體細胞(EPC)的形成,對果蠅心臟的特異分化是必需的(Su et al.,1999)。

發(fā)明內(nèi)容
本發(fā)明的目的在于提供一個人類鋅指蛋白家族新基因ZNF325的全長cDNA序列,分析它在成人各器官及人類胚胎不同發(fā)育階段中的表達,以探索該基因在人類胚胎發(fā)育過程中和成體發(fā)育中所起的作用。
本發(fā)明以本實驗室提取的腦部mRNA反轉(zhuǎn)錄所得的cDNA文庫為模板,根據(jù)C2H2型鋅指結(jié)構(gòu)及相鄰鋅指間的保守序列區(qū)域設(shè)計兼并引物A1和A2,進行PCR擴增,所得PCR擴增產(chǎn)物經(jīng)瓊脂糖凝膠點泳分離,與pUCm-T(SANGONG)載體相連接后轉(zhuǎn)化進入大腸桿菌DH5α,轉(zhuǎn)化后進一步測序分析。把所得的序列用Blastn去搜索dbEST發(fā)現(xiàn)是EST AU125576的部分序列,把ESTAU125576用blastn EST去搜索Genbank,我們發(fā)現(xiàn)了一個包括EST AU125576的EST群,它們由ESTs BG957653,BE568216,AV708178,AU125576 andAU131235組成,這個EST群屬于一個新基因。以胚胎腦文庫為模板,分別用四對引物5’-RACE anti-sense-outer/UPM,5’-RACEanti-sense-inner/UPM;3’-RACE sense-outer/NUP,3’-RACEsense-inner/NUP進行5`-RACE和3`-RACE快速擴增末端,其中5’-RACEanti-sense-outer,5’-RACE anti-sense-inner;3’-RACE sense-outer,3’-RACE sense-inner是基因特異引物,UPM和NUP由SMART RACE試劑盒提供,RACE反應(yīng)分別得到了930bp和318bp的目的片段,經(jīng)T-載體克隆、測序,它們?yōu)樵摶虻?`端和3`端,將它們拼接為全長cDNA,共2697bp,至此,我們得到了新基因的全長cDNA,經(jīng)國際人類基因命名委員會批準命名為ZNF325。
ZNF325此基因位于染色體7p22,長2697個堿基,含5個外顯子,在基因組DNA上長15kb。其開放閱讀框前5’端上游含924bp的非編碼區(qū),從925bp到2313bp為開放閱讀框,2313bp后為3’端非編碼序列。它編碼的蛋白質(zhì)全長462個氨基酸,含15個C2H2型的鋅指,它是一種典型的鋅指結(jié)構(gòu)域,由#-X-C-X(1-5)-C-X3-#-X5-#-X2-H-X(3-6)-[H/C]組成,其中X代表任何一種氨基酸,最后一個氨基酸是組氨酸或半胱氨酸,#代表對鋅指結(jié)構(gòu)起重要穩(wěn)定折疊作用的氨基酸位置。
ZNF325的基因序列已遞交到GenBank,受理號為AF505656。用JELLYFISH程序的ALIGNMENT分析其與鋅指蛋白家族的其它鋅指蛋白的同源發(fā)現(xiàn),ZNF325與RBAK同源度高達71%(見圖1)。RBAK是人們發(fā)現(xiàn)的第一個與RB相互作用的轉(zhuǎn)錄抑制因子(Skkapek et al.,2000)。而RB是一個能抑制E2F轉(zhuǎn)錄因子家族成員激活的基因表達的抑癌基因。
Northern Bolt分析表明該基因在成人的各個組織中普遍表達。進行8種正常人體組織mRNA的Northern雜交分析,結(jié)果為2.7kb的ZNF325 mRNA在成人的各個組織中普遍表達(見圖2A),此結(jié)果表明ZNF325在成體中的表達不具有細胞特異性,它可能對人體各個組織的正常功能都起重要作用。
進行16周,18周,20.5周,22周,23周和24周的人體胚胎各種組織總RNA膜的Northern雜交分析,結(jié)果表明該基因在人體不同發(fā)育時期的胚胎的表達情況與在成體的表達情況相似,ZNF325 mRNA在胚胎時期廣泛表達(見圖2B-2G),說明ZNF325可能對人類早期胚胎的正常發(fā)育起了重要作用。它的結(jié)構(gòu)和表達特征預(yù)示著它編碼的是一個具DNA結(jié)合功能的轉(zhuǎn)錄調(diào)控因子。
本發(fā)明的優(yōu)點1.提供了一個人類新基因ZNF325基因的全長cDNA序列,并描述了其編碼的蛋白產(chǎn)物與重要基因RBAK的同源性,證明了該基因是C2H2型鋅指蛋白基因家族的成員之一。
2.該基因編碼的蛋白產(chǎn)物為鋅指蛋白,是一種轉(zhuǎn)錄調(diào)控因子,并且在人類成體和胚胎組織中廣泛表達,說明該基因作為轉(zhuǎn)錄因子在胚胎發(fā)育早期起了很重要的作用,并將這種作用延續(xù)到了成體時期。
本發(fā)明通過以下附圖和實施例作進一步闡述,但并不限制本發(fā)明的范圍。


圖1.ZNF325與RBAK蛋白質(zhì)的同源比較。
圖2.ZNF325在人類組織的Northern雜交。
圖2A,2B,2C,2D,2E,2F,2G分別為成人組織,16周,18周,20.5周,22周,23周,24周胚胎組織。
圖中1胃 2腎 3骨骼肌 4肺 5腦5′大腦5″小腦 6腸 7肝 8心臟 9腎上腺 10胰腺 11脾 12肺 13膽囊 14膀胱 15舌
具體實施例方式人類鋅指蛋白家族新基因ZNF325的克隆,測序及其表達。
1.兼并引物進行PCR擴增篩選新基因根據(jù)C2H2型鋅指結(jié)構(gòu)及相鄰鋅指間的保守序列區(qū)域設(shè)計兼并引物A1和A2A1sense5’-GRNGARAARCCNUMY-3’A2antisense5’-RTGDATNCGNTGRTGVAW-3’PCR反應(yīng)以腦cDNA文庫為模板,反應(yīng)條件先94℃變性4分鐘,然后94℃變性30秒、50℃30秒、72℃30秒共30個循環(huán),最后72℃延伸7分鐘,擴增的帶,進行T-載體連接,轉(zhuǎn)化,任意挑選轉(zhuǎn)化子去測序。
測序結(jié)果去進行dbEST檢索,發(fā)現(xiàn)該片段是EST AU125576的部分片段,用此EST進行Blastn nr檢索得到一新基因,再進行dbEST檢索。2.ZNF325全長cDNA的克隆以檢索獲得的cDNA序列設(shè)計RACE引物5’-RACE anti-sense-outer 5’-CTGATGCACAGTAAGGGTTCC-3’5’-RACE anti-sense-inner 5’-AGGTCCGACATCTGGAGAAAGGC-3’3’-RACE sense-outer 5’-TGATGTATAGTGAGATAGGACC-3’3’-RACE sense-inner 5’-TCAGAGAAGCCTTCCCACATC-3’利用CLONTECH公司的SMART RACE試劑盒做5′端和3′端CLONTECH公司提供的引物MUP5′-CTAATACGACTCACTATAGGGCAAGCAGTGGTATCAACGCAGAGT-3′5′-CTAATACGACTCACTATAGGGC-3′NUP5′-AAGCAGTGGTATCAACGCAGAGT-3′具體操作按該公司protocol進行。將RACE產(chǎn)物克隆入T載體并測序獲得ZNF325的5`端和3`端序列,反應(yīng)分別得到了930bp和318bp的目的片段,將它們拼接后得到ZNF325全長cDNA,共2697bp,至此,我們得到了新基因的全長cDNA。3.Northern雜交(1)組織總RNA的提取(按王雁云等,一種快速的總RNA提取方法,生命科學(xué)研究,5(1)88-90(2001)進行)。(2)總RNA的電泳及轉(zhuǎn)膜,均按《現(xiàn)代分子生物學(xué)實驗技術(shù)》介紹的方法進行(盧圣棟主編,第二版)。(3)Northern雜交將從CLONTECH公司買的成人組織mRNA膜和轉(zhuǎn)有20ug人體胚胎多種組織以及總胚胎組織的總RNA的尼龍膜,放入培養(yǎng)皿中,加入變性的經(jīng)42℃預(yù)熱的快速雜交液10ml(Clontech公司),在42℃條件下預(yù)雜交30分鐘,加入變性的同位素標記的ZNF325的cDNA片段,42℃雜交過夜。用2×SSC,0.05%SDS,42℃洗兩次,15分鐘/次;0.1×SSC,0.1%SDS,42℃洗兩次,15分鐘/次。(4)-80℃曝光3天,洗X光片。(5)用0.1×SSC和0.5%SDS在95℃洗10分鐘膜用β-actin cDNA重新標記,做Northern雜交進行陽性對照。
ZNF325 cDNA的核苷酸序列和編碼的氨基酸序列accaggtctg gtctacagtc gactaagtcg gtcggcccgg gcccctctgg agcanaangg 60ccttggtgag gcggacagga agggacctcg cgagcagacg cgcgcccgcg acagcaatcc 120cgccccggcc tgtcgggagc ggtggggcag aggctgcgga gccccaggag gatctgcctc 180cgctttcaca gtcctccaga tttttccaag agcagcagaa aatgaataaa tccctggggc 240cagtgtcatt caaggacgtg gctgtggact tcacccagga ggaatggcag cagctggatc 300ctgagcagaa gataacttac agggatgtga tgctggagaa ctacagcaat ctagtttctg 360tggggtatca cattatcaaa ccggatgtta tcagcaagtt ggagcaagga gaagagccat 420ggatagtaga aggagaattc ctacttcaga gctatccaga tgaagtctgg caaactgatg 480acctaataga gagaatccag gaagaggaaa ataaaccttc aaggcaaact gtgttcattg 540agaccctgat tgaagagaga ggtaatgttc ctggtaaaac ttttgatgta gaaacgaacc 600ctgttccttc aagaaaaata gcctataaaa atagcctctg tgactcatgt gaaaagtgtt 660taacgtctgt ttcagaatat attagtagtg atggaagcta tgcaagaatg aaagctgatg 720aatgtagtgg attgtgggaa atcactcctc catattaagc ttgagaaaac tcatccagga 780gatcnagctt atgatttaat caaaatgggg aaccttatac cctaaattga anaagtntta 840tcaaaaattc gtattttgga gaaacctttt gaatatattg aatgccagaa agccttccaa 900aaggacactg tttttgttaa tcacatg gaa gaa aag ccc tat aag 945Met Glu Glu Lys Pro Tyr Lys1 5tgg aat gga tct gaa ata gcc ttt ctc cag atg tcg gac ctc act 990Trp Asn Gly Ser Glu Ile Ala Phe Leu Gln Met Ser Asp Leu Thr10 15 20gta cat cag aca tct cat atg gaa atg aag ccc tat gaa tgc agt 1035Val His Gln Thr Ser His Met Glu Met Lys Pro Tyr Glu Cys Ser25 30 35gaa tgt ggg aaa tcc ttc tgt aaa aag tca aaa ttt att ata cat 1080Glu Cys Gly Lys Ser Phe Cys Lys Lys Ser Lys Phe Ile Ile His40 45 50cag agg act cac aca gga gag aaa cct tac gaa tgt aat cag tgt 1125Gln Arg Thr His Thr Gly Glu Lys Pro Tyr Glu Cys Asn Gln Cys55 60 65ggg aaa tcc ttc tgc cag aag gga acc ctt act gtg cat cag aga 1170Gly Lys Ser Phe Cys Gln Lys Gly Thr Leu Thr Val His Gln Arg70 75 80aca cac aca ggg gag aag ccc tat gaa tgt aat gaa tgt ggg aag 1215Thr His Thr Gly Glu Lys Pro Tyr Glu Cys Asn Glu Cys Gly Lys85 90 95aac ttt tac cag aag tta cac ctc att cag cat cag aga act cac 1260Asn Phe Tyr Gln Lys Leu His Leu Ile Gln His Gln Arg Thr His100 105 110tca gga gag aag ccc tat gaa tgt agt tat tgt gga aaa tcc ttt 1305Ser Gly Glu Lys Pro Tyr Glu Cys Ser Tyr Cys Gly Lys Ser Phe115 120 125tgc cag aag aca cac ctc aca caa cat cag aga aca cat tca gga 1350Cys Gln Lys Thr His Leu Thr Gln His Gln Arg Thr His Ser Gly130 135 140gag aga cct tat gtt tgt cat gac tgt ggg aaa acc ttc tcg cag 1395Glu Arg Pro Tyr Val Cys His Asp Cys Gly Lys Thr Phe Ser Gln145 150 155aag tca gca ctt aat gac cat cag aaa att cac aca ggt gtg aaa 1440Lys Ser Ala Leu Asn Asp His Gln Lys Ile His Thr Gly Val Lys160 165 170ctc tac aag tgt agt gaa tgt ggg aaa tgc ttc tgc cgc aag tct 1485Leu Tyr Lys Cys Ser Glu Cys Gly Lys Cys Phe Cys Arg Lys Ser175 180 185act ctc acg acc cac ctg agg acc cac aca gga gag aaa ccg tat 1530Thr Leu Thr Thr His Leu Arg Thr His Thr Gly Glu Lys Pro Tyr190 195 200gaa tgt aat gag tgt gga aaa ttc ttc tct cgg ttg tca tat ctc 1575Glu Cys Asn Glu Cys Gly Lys Phe Phe Ser Arg Leu Ser Tyr Leu205 210 215act gta cat tat aga act cat tca gga gag aaa ccc tat gaa tgt 1620Thr Val His Tyr Arg Thr His Ser Gly Glu Lys Pro Tyr Glu Cys220 225 230aat gaa tgt gga aaa acc ttc tac ctg aat tca gcc ctc atg aga 1665Asn Glu Cys Gly Lys Thr Phe Tyr Leu Asn Ser Ala Leu Met Arg235 240 245cat cag aga gtg cac aca gga gag aaa cct tac gaa tgt aat gaa 1710His Gln Arg Val His Thr Gly Glu Lys Pro Tyr Glu Cys Asn Glu250 255 260tgt gga aag tta ttc tcc cag ttg tca tac ctc act atc cat cat 1755Cys Gly Lys Leu Phe Ser Gln Leu Ser Tyr Leu Thr Ile His His265 270 275aga act cat tca gga gta aaa ccc tat gaa tgt agt gaa tgt ggg 1800Arg Thr Lis Ser Gly Val Lys Pro Tyr Glu Cys Ser Glu Cys Gly280 285 290aaa acc ttc tac cag aac tca gcc ctt tgt aga cat cgg aga ata 1845Lys Thr Phe Tyr Gln Asn Ser Ala Leu Cys Arg His Arg Arg Ile295 300 305cac aaa gga gag aag ccc tat gaa tgc tat ata tgt gga aaa ttc 1890His Lys Gly Glu Lys Pro Tyr Glu Cys Tyr Ile Cys Gly Lys Phe310 315 320ttc tct cag atg tca tac ctc act ata cat cat aga att cat tca 1935Phe Ser Gln Met Ser Tyr Leu Thr Ile His His Arg Ile His Ser325 330 335gga gag aag ccc tat gaa tgt agt gaa tgt ggg aaa acc ttc tgc 1980Gly Glu Lys Pro Tyr Glu Cys Ser Glu Cys Gly Lys Thr Phe Cys340 345 350cag aat tca gcc ctt aat cga cat cag aga aca cac aca gga gag 2025Gln Asn Ser Ala Leu Asn Arg His Gln Arg Thr His Thr Gly Glu355 360 365aaa gcc tac gaa tgt tat gaa tgt ggg aag tgc ttc tct cag atg 2070Lys Ala Tyr Glu Cys Tyr Glu Cys Gly Lys Cys Phe Ser Gln Met370 375 380tcc tat ctc act ata cat cat cga att cat tca gga gag aaa ccc 2115Ser Tyr Leu Thr Ile His His Arg Ile His Ser Gly Glu Lys Pro385 390 395ttt gaa tgt aat gag tgt gga aaa gcc ttc tct cgg atg tca tac 2160Phe Glu Cys Asn Glu Cys Gly Lys Ala Phe Ser Arg Met Ser Tyr400 405 410ctc act gta cat tat aga act cat tca gga gag aaa ccc tat gag 2205Leu Thr Val His Tyr Arg Thr His Ser Gly Glu Lys Pro Tyr Glu415 420 425tgt act gaa tgt gga aaa aaa ttc tac cac aaa tca gca ttc aac 2250Cys Thr Glu Cys Gly Lys Lys Phe Tyr His Lys Ser Ala Phe Asn430 435 440agc cat cag aga att cat agg aga ggc aat atg aat gta ata gat 2295Ser His Gln Arg Ile His Arg Arg Gly Asn Met Asn Val Ile Asp445 450 455gtg gga agg ctt ctc tgaagtcaga cctcatttta tatcagagaa 2340Val Gly Arg Leu Leu460 462ccctttcagt atagtgaatc agaaactcct gcctgaagtc aaacaccttg tacatcagag 2400agttcacaca ggttagtgtg gacatcccct tgtgtgttgg actcataatc tgaagactca 2461cagaatggaa accatgatta taacaagacc acatggtata acaatactag actatagaca 2520agtaaaaatt tataaatatt aagaatgtat atacatgtca ccatggattg gaactgtttt 2580gcatatcagg gaaatcatag ccaaggggaa atctatcagt ataaggaatg tggaagacat 2640aatcctttgg aaactgttaa tactaaaaga tatgtttctg atacaatagc aaacttg2697ZNF325 cDNA的全長核苷酸序列accaggtctg gtctacagtc gactaagtcg gtcggcccgg gcccctctgg agcanaangg 60ccttggtgag gcggacagga agggacctcg cgagcagacg cgcgcccgcg acagcaatcc 120cgccccggcc tgtcgggagc ggtggggcag aggctgcgga gccccaggag gatctgcctc 180cgctttcaca gtcctccaga tttttccaag agcagcagaa aatgaataaa tccctggggc 240cagtgtcatt caaggacgtg gctgtggact tcacccagga ggaatggcag cagctggatc 300ctgagcagaa gataacttac agggatgtga tgctggagaa ctacagcaat ctagtttctg 360tggggtatca cattatcaaa ccggatgtta tcagcaagtt ggagcaagga gaagagccat 420ggatagtaga aggagaattc ctacttcaga gctatccaga tgaagtctgg caaactgatg 480acctaataga gagaatccag gaagaggaaa ataaaccttc aaggcaaact gtgttcattg 540agaccctgat tgaagagaga ggtaatgttc ctggtaaaac ttttgatgta gaaacgaacc 600ctgttccttc aagaaaaata gcctataaaa atagcctctg tgactcatgt gaaaagtgtt 660taacgtctgt ttcagaatat attagtagtg atggaagcta tgcaagaatg aaagctgatg 720aatgtagtgg attgtgggaa atcactcctc catattaagc ttgagaaaac tcatccagga 780gatcnagctt atgatttaat caaaatgggg aaccttatac cctaaattga anaagtntta 840tcaaaaattc gtattttgga gaaacctttt gaatatattg aatgccagaa agccttccaa 900aaggacactg tttttgttaa tcacatggaa gaaaagccct ataagtggaa tggatctgaa 960atagcctttc tccagatgtc ggacctcact gtacatcaga catctcatat ggaaatgaag 1020ccctatgaat gcagtgaatg tgggaaatcc ttctgtaaaa agtcaaaatt tattatacat 1080cagaggactc acacaggaga gaaaccttac gaatgtaatc agtgtgggaa atccttctgc 1140cagaagggaa cccttactgt gcatcagaga acacacacag gggagaagcc ctatgaatgt 1200aatgaatgtg ggaagaactt ttaccagaag ttacacctca ttcagcatca gagaactcac 1260tcaggagaga agccctatga atgtagttat tgtggaaaat ccttttgcca gaagacacac 1320ctcacacaac atcagagaac acattcagga gagagacctt atgtttgtca tgactgtggg 1380aaaaccttct cgcagaagtc agcacttaat gaccatcaga aaattcacac aggtgtgaaa 1440ctctacaagt gtagtgaatg tgggaaatgc ttctgccgca agtctactct cacgacccac 1500ctgaggaccc acacaggaga gaaaccgtat gaatgtaatg agtgtggaaa attcttctct 1560cggttgtcat atctcactgt acattataga actcattcag gagagaaacc ctatgaatgt 1620aatgaatgtg gaaaaacctt ctacctgaat tcagccctca tgagacatca gagagtgcac 1680acaggagaga aaccttacga atgtaatgaa tgtggaaagt tattctccca gttgtcatac 1740ctcactatcc atcatagaac tcattcagga gtaaaaccct atgaatgtag tgaatgtggg 1800aaaaccttct accagaactc agccctttgt agacatcgga gaatacacaa aggagagaag 1860ccctatgaat gctatatatg tggaaaattc ttctctcaga tgtcatacct cactatacat 1920catagaattc attcaggaga gaagccctat gaatgtagtg aatgtgggaa aaccttctgc 1980cagaattcag cccttaatcg acatcagaga acacacacag gagagaaagc ctacgaatgt 2040tatgaatgtg ggaagtgctt ctctcagatg tcctatctca ctatacatca tcgaattcat 2100tcaggagaga aaccctttga atgtaatgag tgtggaaaag ccttctctcg gatgtcatac 2160ctcactgtac attatagaac tcattcagga gagaaaccct atgagtgtac tgaatgtgga 2220aaaaaattct accacaaatc agcattcaac agccatcaga gaattcatag gagaggcaat 2280atgaatgtaa tagatgtggg aaggcttctc tgaagtcaga cctcatttta tatcagagaa 2340ccctttcagt atagtgaatc agaaactcct gcctgaagtc aaacaccttg tacatcagag 2400agttcacaca ggttagtgtg gacatcccct tgtgtgttgg actcataatc tgaagactca 2460cagaatggaa accatgatta taacaagacc acatggtata acaatactag actatagaca 2520agtaaaaatt tataaatatt aagaatgtat atacatgtca ccatggattg gaactgtttt 2580gcatatcagg gaaatcatag ccaaggggaa atctatcagt ataaggaatg tggaagacat 2640aatcctttgg aaactgttaa tactaaaaga tatgtttctg atacaatagc aaacttg269權(quán)利要求
1.一個人類鋅指蛋白家族新基因ZNF325,其特征在于該基因的cDNA核苷酸序列和編碼氨基酸序列如下accaggtctg gtctacagtc gactaagtcg gtcggcccgg gcccctctgg agcanaangg 60ccttggtgag gcggacagga agggacctcg cgagcagacg cgcgcccgcg acagcaatcc 120cgccccggcc tgtcgggagc ggtggggcag aggctgcgga gccccaggag gatctgcctc 180cgctttcaca gtcctccaga tttttccaag agcagcagaa aatgaataaa tccctggggc 240cagtgtcatt caaggacgtg gctgtggact tcacccagga ggaatggcag cagctggatc 300ctgagcagaa gataacttac agggatgtga tgctggagaa ctacagcaat ctagtttctg 360tggggtatca cattatcaaa ccggatgtta tcagcaagtt ggagcaagga gaagagccat 420ggatagtaga aggagaattc ctacttcaga gctatccaga tgaagtctgg caaactgatg 480acctaataga gagaatccag gaagaggaaa ataaaccttc aaggcaaact gtgttcattg 540agaccctgat tgaagagaga ggtaatgttc ctggtaaaac ttttgatgta gaaacgaacc 600ctgttccttc aagaaaaata gcctataaaa atagcctctg tgactcatgt gaaaagtgtt 660taacgtctgt ttcagaatat attagtagtg atggaagcta tgcaagaatg aaagctgatg 720aatgtagtgg attgtgggaa atcactcctc catattaagc ttgagaaaac tcatccagga 780gatcnagctt atgatttaat caaaatgggg aaccttatac cctaaattga anaagtntta 840tcaaaaattc gtattttgga gaaacctttt gaatatattg aatgccagaa agccttccaa 900aaggacactg tttttgttaa tcacatg gaa gaa aag ccc tat aag 945Met Glu Glu Lys Pro Tyr Lys1 5tgg aat gga tct gaa ata gcc ttt ctc cag atg tcg gac ctc act 990Trp Asn Gly Ser Glu Ile Ala Phe Leu Gln Met Ser Asp Leu Thr10 15 20gta cat cag aca tct cat atg gaa atg aag ccc tat gaa tgc agt 1035Val His Gln Thr Ser His Met Glu Met Lys Pro Tyr Glu Cys Ser25 30 35gaa tgt ggg aaa tcc ttc tgt aaa aag tca aaa ttt att ata cat 1080Glu Cys Gly Lys Ser Phe Cys Lys Lys Ser Lys Phe Ile Ile His40 45 50cag agg act cac aca gga gag aaa cct tac gaa tgt aat cag tgt 1125Gln Arg Thr His Thr Gly Glu Lys Pro Tyr Glu Cys Asn Gln Cys55 60 65ggg aaa tcc ttc tgc cag aag gga acc ctt act gtg cat cag aga 1170Gly Lys Ser Phe Cys Gln Lys Gly Thr Leu Thr Val His Gln Arg70 75 80aca cac aca ggg gag aag ccc tat gaa tgt aat gaa tgt ggg aag 1215Thr His Thr Gly Glu Lys Pro Tyr Glu Cys Asn Glu Cys Gly Lys85 90 95aac ttt tac cag aag tta cac ctc att cag cat cag aga act cac 1260Asn Phe Tyr Gln Lys Leu His Leu Ile Gln His Gln Arg Thr His100 105 110tca gga gag aag ccc tat gaa tgt agt tat tgt gga aaa tcc ttt 1305Ser Gly Glu Lys Pro Tyr Glu Cys Ser Tyr Cys Gly Lys Ser Phe115 120 125tgc cag aag aca cac ctc aca caa cat cag aga aca cat tca gga 1350Cys Gln Lys Thr His Leu Thr Gln His Gln Arg Thr His Ser Gly130 135 140gag aga cct tat gtt tgt cat gac tgt ggg aaa acc ttc tcg cag 1395Glu Arg Pro Tyr Val Cys His Asp Cys Gly Lys Thr Phe Ser Gln145 150 155aag tca gca ctt aat gac cat cag aaa att cac aca ggt gtg aaa 1440Lys Ser Ala Leu Asn Asp His Gln Lys Ile His Thr Gly Val Lys160 165 170ctc tac aag tgt agt gaa tgt ggg aaa tgc ttc tgc cgc aag tct 1485Leu Tyr Lys Cys Ser Glu Cys Gly Lys Cys Phe Cys Arg Lys Ser175 180 185act ctc acg acc cac ctg agg acc cac aca gga gag aaa ccg tat 1530Thr Leu Thr Thr His Leu Arg Thr His Thr Gly Glu Lys Pro Tyr190 195 200gaa tgt aat gag tgt gga aaa ttc ttc tct cgg ttg tca tat ctc 1575Glu Cys Asn Glu Cys Gly Lys Phe Phe Ser Arg Leu Ser Tyr Leu205 210 215act gta cat tat aga act cat tca gga gag aaa ccc tat gaa tgt 1620Thr Val His Tyr Arg Thr His Ser Gly Glu Lys Pro Tyr Glu Cys220 225 230aat gaa tgt gga aaa acc ttc tac ctg aat tca gcc ctc atg aga 1665Asn Glu Cys Gly Lys Thr Phe Tyr Leu Asn Ser Ala Leu Met Arg235 240 245cat cag aga gtg cac aca gga gag aaa cct tac gaa tgt aat gaa 1710His Gln Arg Val His Thr Gly Glu Lys Pro Tyr Glu Cys Asn Glu250 255 260tgt gga aag tta ttc tcc cag ttg tca tac ctc act atc cat cat 1755Cys Gly Lys Leu Phe Ser Gln Leu Ser Tyr Leu Thr Ile His His265 270 275aga act cat tca gga gta aaa ccc tat gaa tgt agt gaa tgt ggg 1800Arg Thr His Ser Gly Val Lys Pro Tyr Glu Cys Ser Glu Cys Gly280 285 290aaa acc ttc tac cag aac tca gcc ctt tgt aga cat cgg aga ata 1845Lys Thr Phe Tyr Gln Asn Ser Ala Leu Cys Arg His Arg Arg Ile295 300 305cac aaa gga gag aag ccc tat gaa tgc tat ata tgt gga aaa ttc 1890His Lys Gly Glu Lys Pro Tyr Glu Cys Tyr Ile Cys Gly Lys Phe310 315 320ttc tct cag atg tca tac ctc act ata cat cat aga att cat tca 1935Phe Ser Gln Met Ser Tyr Leu Thr Ile His His Arg Ile His Ser325 330 335gga gag aag ccc tat gaa tgt agt gaa tgt ggg aaa acc ttc tgc 1980Gly Glu Lys Pro Tyr Glu Cys Ser Glu Cys Gly Lys Thr Phe Cys340 345 350cag aat tca gcc ctt aat cga cat cag aga aca cac aca gga gag 2025Gln Asn Ser Ala Leu Asn Arg His Gln Arg Thr His Thr Gly Glu355 360 365aaa gcc tac gaa tgt tat gaa tgt ggg aag tgc ttc tct cag atg 2070Lys Ala Tyr Glu Cys Tyr Glu Cys Gly Lys Cys Phe Ser Gln Met370 375 380tcc tat ctc act ata cat cat cga att cat tca gga gag aaa ccc 2115Ser Tyr Leu Thr Ile His His Arg Ile His Ser Gly Glu Lys Pro385 390 395ttt gaa tgt aat gag tgt gga aaa gcc ttc tct cgg atg tca tac 2160Phe Glu Cys Asn Glu Cys Gly Lys Ala Phe Ser Arg Met Ser Tyr400 405 410ctc act gta cat tat aga act cat tca gga gag aaa ccc tat gag 2205Leu Thr Val His Tyr Arg Thr His Ser Gly Glu Lys Pro Tyr Glu415 420 425tgt act gaa tgt gga aaa aaa ttc tac cac aaa tca gca ttc aac 2250Cys Thr Glu Cys Gly Lys Lys Phe Tyr His Lys Ser Ala Phe Asn430 435 440agc cat cag aga att cat agg aga ggc aat atg aat gta ata gat 2295Ser His Gln Arg Ile His Arg Arg Gly Asn Met Asn Val Ile Asp445 450 455gtg gga agg ctt ctc tgaagtcaga cctcatttta tatcagagaa 2340Val Gly Arg Leu Leu460 462ccctttcagt atagtgaatc agaaactcct gcctgaagtc aaacaccttg tacatcagag 2400agttcacaca ggttagtgtg gacatcccct tgtgtgttgg actcataatc tgaagactca 2461cagaatggaa accatgatta taacaagacc acatggtata acaatactag actatagaca 2520agtaaaaatt tataaatatt aagaatgtat atacatgtca ccatggattg gaactgtttt 2580gcatatcagg gaaatcatag ccaaggggaa atctatcagt ataaggaatg tggaagacat 2640aatcctttgg aaactgttaa tactaaaaga tatgtttctg atacaatagc aaacttg269全文摘要
一個人類鋅指蛋白家族新基因ZNF325,涉及一種人類基因,它的cDNA全長2697bp,位于染色體7p22,編碼的蛋白質(zhì)全長462個氨基酸,含15個C
文檔編號C12N15/12GK1427009SQ0213975
公開日2003年7月2日 申請日期2002年11月12日 優(yōu)先權(quán)日2002年11月12日
發(fā)明者吳秀山, 李永青, 周亮, 朱傳炳, 袁婺洲, 劉明耀, 王躍群 申請人:湖南師范大學(xué)
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