用S_1核酸酶方法测定αAC-616晶体蛋白基因转录起始点  

S1 NUCLEASE MAPPING INITIATION SITE IN THE α ACRYSTALLIN 616 GENE

作　　者：刘祥麟[1] 童坦君[2] 邵伟 Gene Lavers 陈亨[3] 

机构地区：[1]浙江医科大学肿瘤基因组,杭州310006 [2]北京医科大学生化与分子生物学系,100083 [3]纽约大学生化系,10010

出　　处：《细胞生物学杂志》1996年第3期140-143,共4页Chinese Journal of Cell Biology

摘　　要：采用BRL提供的HeLa细胞裂解物,已知该裂解物中含有RNA聚合酶Ⅱ和其他基因转录所必需的因子和成分。当αAC-616晶体蛋白基因在该体系中发生转录产生mRNA后,即与用^(32)P标记的αAC-616晶体蛋白基因探针进行分子杂交,杂交后加入S_1核酸酶消化去掉未配对的αAC-616DNA单链,保留杂交形成的RNA-DNA双链杂交体。作含脲素的聚丙酰胺凝胶电泳,用φ_x174HeaⅢDNA作分子标准参照物,结果表明转录出来的mRNA与276个脱氧核糖核苷酸形成互补杂交链,比5′-末端标记的^(32)P-晶体蛋白基因探针为短。据此在已知的晶体蛋白基因顺序图谱上判定出αAC-616晶体蛋白基因的转录起始点位于该图谱的第398位核苷酸Adenosine,距TATA盒下游第79位(A)。并对晶体蛋白基因转录的特性进行了讨论。S1 nuclease mapping technique is used to determine the possible initiation site for tran-scription in the HeLa Cell extract which containing RNA polymerase Ⅱ and other necessary transcription components. The 'Run off' experiments with S1 mapping indieate that the RNA synthesized is 276 RNA-DNA Hybrids, which is shorter than that of 5'-termini 32P-DNA probe.The initation site is at 398 position in the α A-Crystallin 616 gene in adenosine (A). This result is different from the previously proposed initiation site (Piatigrosky 1983).This study also indicates that 'TATA' box may be necessary but not sufficient for transcription. It shows that the αA-Crystallin 616 gene transcription in the HeLa cell lysate mi-ght be a ideal modei to study the crystallin gcne expression.

关 键 词：晶体蛋白基因 转录起始点 S1核酸酶 

分 类 号：Q75[生物学—分子生物学]