Zhang Zifei, Chen Weihua, Qin Zhongjun
Key laboratory of Synthetic Biology, Shanghai Institute of Plant Physiology and Ecology, Shanghai Institutes of Biological Sciences, Chinese Academy of Sciences, Shanghai 200032, China.
Wei Sheng Wu Xue Bao. 2010 Jan;50(1):41-7.
A Streptomyces strain X3 -3, growing up to 50 degrees C, was isolated from the composts prepared from swine manure and straw. This strain harbored a -7-kb plasmid pTSC2.
Cloning, sequencing and analyzing pTSC2, and identifying its replication mode as well.
The complete nucleotide sequence of pTSC2 was obtained by sub-cloning and primer-walking; the replication gene (rep), double-strand origin (dso) and single-strand origin (sso) were identified by multiple sequence alignments; and the replication intermediate was detected by Southern hybridization after neutral transfer.
The complete nucleotide sequence of pTSC2 consisted of 7516-bp in length, encoded eight proteins, four of which resembled the replication and conjugation proteins of Streptomyces plasmid pIJ101 which replicates in a rolling-circle mode. The replication elements, rep, dso and sso, were also similar with pIJ101. The rep and dso was proved as essential components for plasmid replication by transformation of both S. lividans ZX7 and thermophilic Streptomyces spp. 2C, while sso was dispensable for replication. Southern hybridization detected a large amount of single-stranded DNA accumulated during replication.
We identified that the thermophilic Streptomyces plasimd pTSC2 replicated in a rolling-circle mode. It was first reported that the plasmid from thermophilic Streptomyces was cloned and sequenced, as well as the replication of which was characterized.
从猪粪和稻草制成的堆肥中分离出一株能在50℃生长的链霉菌菌株X3 - 3。该菌株携带一个7kb的质粒pTSC2。
对pTSC2进行克隆、测序和分析，并确定其复制模式。
通过亚克隆和引物步移获得pTSC2的完整核苷酸序列；通过多序列比对鉴定复制基因（rep）、双链起点（dso）和单链起点（sso）；中性转移后通过Southern杂交检测复制中间体。
pTSC2的完整核苷酸序列长度为7516bp，编码8种蛋白质，其中4种类似于以滚环模式复制的链霉菌质粒pIJ101的复制和接合蛋白。复制元件rep、dso和sso也与pIJ101相似。通过变铅青链霉菌ZX7和嗜热链霉菌属2C的转化证明rep和dso是质粒复制的必需成分，而sso对复制是可有可无的。Southern杂交检测到复制过程中积累了大量单链DNA。
我们确定嗜热链霉菌质粒pTSC2以滚环模式复制。首次报道了嗜热链霉菌的质粒被克隆和测序，并对其复制进行了表征。
