头状链轮丝菌(Streptoverticillum caespitosus)ATCC27422染色体复制起始区(oriC)的序列分析与功能研究  

Sequence and Functional Analysis of the Chromosome Replication Origin ( oriC) of Streptoverticillum caespitosus ATCC27422

作　　者：马伟[1] 茅翔[1] 卢捷[1] 姜卫红[1] 覃重军[1] 赵国屏[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》2003年第10期930-936,共7页

基　　金：国家自然科学基金重点资助项目 (No.10 0 3 963 0 0 10 )~~

摘　　要：头状链轮丝菌 (Streptoverticillumcaespitosus)ATCC2 74 2 2染色体复制起始区 (oriC)内共有 2 2个DnaA盒结构 ;其中第 2 1、2 2个DnaA盒彼此方向相反、相互重叠 8个碱基。放线菌oriC数据库搜索发现 ,这种重叠DnaA盒在抗生素链霉菌、结核分枝杆菌等几种放线菌中也同样存在。在分枝杆菌中一般由第 1、2个DnaA盒组成 ,而在链霉菌中由最后的两个DnaA盒 (第 2 1、2 2 )组成。重叠DnaA盒保守序列为CTGTGCACAA ,长度为 10个碱基 ,即由于重叠的缘故比正常DnaA盒长 1个碱基。通过测量载体对变铅青链霉菌的转化效率研究了oriC不同部位在染色体复制起始中的功能和地位。头状链轮丝菌oriC序列的 5′端 1～ 188位的片段虽然不包含有DnaA盒结构 ,但该片段的缺失 ,造成oriC复制起始功能的完全丧失。3′端 793～ 939位片段同样没有DnaA盒结构 ,该片段的缺失 ,仅发生转化效率的降低约 4 0 % ,说明oriC的 793～ 939位序列对DNA复制起始效率以及复制子稳定性起重要作用。当oriC被克隆入载体时两端各带有一段dnaA、dnaN基因的部分序列 ,所构建的载体虽然转化效率较低 ,但转化子的菌落、菌丝形态与宿主菌原有的表型相接近 ,由此推断oriC两端的序列除了编码各自产物外 ,可能通过影响染色体DNA复制的起始效率、复制子稳定性等?Twenty-two DnaA boxes were identified in the chromosome replication origin ( oriC ) of Streptoverticillum caespitosus ATCC27422 based upon the characteristics of consensus sequences. The 21st and 22nd DnaA boxes overlapped 8 base pairs each other reversely. Compared with the oriC database of actinomycetes, similar overlapping DnaA boxes were recognized in several species of Streptomyces and Mycobacterium. These overlapping DnaA boxes were composed of the last two DnaA box (21st and 22nd) in the Streptomyces species, but the of 1st and 2nd ones in the Mycobacterium species. The consensus sequence of the overlapping DnaA box is CTGTGCACAA, one base longer than the normal DnaA box sequence presumably due to the overlapping structure. Although the DnaA boxes exist in the 189-792 bp region only, the 1-188 bp and 793-939 bp regions are also important to the DNA replication. Deletion of the 1-188 bp region may cause absolute loss of DNA replication initiation activity measured by the transformation efficiency of plasmids with truncated oriC. When the 793-939 bp region was truncated, the transformation efficiency reduced about 40%. If the oriC was cloned into a vector with partial flanking region sequences (partial dnaA and dnaN gene sequences), the transformation rate was about 4.3-fold lower than that of the construct containing the oriC region only. However, the transformants were much more similar to the host with respect to the morphology of colony and mycelium. The cis-regulatory functions of the flanking sequences, which may influence the initiation efficiency of the chromosome replication and/or the stability of replicon, are thus suggested.

关 键 词：头状链轮丝菌 染色体复制起始区 重叠DnaA盒 放线菌 

分 类 号：Q933[生物学—微生物学]