除虫链霉菌复制起始区克隆和功能初步分析  

Isolation and Characterization of Functional Replication Origin fromStreptomyces avermitilis

作　　者：卢捷[1] 马伟[1] 茅翔[1] 覃重军[1] 姜卫红[1] 焦瑞身[1] 

机构地区：[1]中国科学院上海生命科学研究院植物生理生态研究所,上海200032

出　　处：《生物化学与生物物理学报》2002年第6期712-718,共7页

基　　金：国家自然科学基金重点资助项目 (No .10039630010 )~~

摘　　要：真细菌在复制起始区附近的基因排布顺序相当保守。根据DnaA和DnaN的氨基酸保守序列及链霉菌对密码子的偏用性 ,设计简并引物 ,以除虫链霉菌基因组DNA为模板扩增出一条约 1.4kb的片段。序列分析表明该片段含有部分的dnaA及dnaN基因 ,并在这两个基因之间有一段非编码区 ,其上有 19个典型的DnaA盒 ,推测是除虫链霉菌的复制起始区 (oriC)。与其他链霉菌已知的oriC作比较后发现除虫链霉菌的DnaA盒在位置、方向及间隔区的长度上都高度保守 ,并归纳出其保守序列为 (T/C) (T/C) (G/A/C)TCCACA(有下划线的碱基在该位置出现频率较高 )。将这段oriC插入到仅能在大肠杆菌中复制的质粒pQC15 6中 ,重组质粒可以成功转化变铅青链霉菌ZX7。对oriC分段研究发现其 3′部分对质粒的稳定性及转化效率有促进作用。Most eubacteria contain highly conservative gene clusters in the adjacent regions oforiC. According to this principle, a 1.4 kb DNA fragment containing parts ofdnaA anddnaN genes ofStreptomyces avermitiliswas cloned by degenerate PCR. Sequence analysis of this fragment indicated that it encoded two partial genes in the orderdnaA (the putative initiator protein) anddnaN (the β subunit of DNA polymerase III). The intergenic non-coding region between dnaA anddnaN was found to contain 19 putative DnaA boxes, i.e. 9 nt long DnaA protein recognition sequences. It was confirmed that the location, orientation and spacing of DnaA boxes in this intergenic region are conserved amongStreptomyces. The consensus sequence of DnaA box identified is ( T/C)( T/C)( G/A/C) TCCACA (preferred bases underlined). When this fragment was cloned intoEscherichia coli plasmid pQC156, which is otherwise non-replicative inStreptomyces, it exhibited autonomous replication activity inStreptomyces lividans, a closely relatedStreptomyces strain. Different parts of theoriC contribute unequally to the stability and transformation efficiency. The 3′ region oforiC may contain features that support stable autonomous replication. The implications of these results for the understanding of theS. avermitilis oriC replication initiation process and its future application are discussed.

关 键 词：除虫链霉菌 复制起始区 克隆 功能 ORIC DnaA盒 

分 类 号：Q785[生物学—分子生物学]