LeBlanc D J, Lee L N, Abu-Al-Jaibat A
Department of Microbiology, University of Texas Health Science Center, San Antonio 78284.
Plasmid. 1992 Sep;28(2):130-45. doi: 10.1016/0147-619x(92)90044-b.
The 4.2-kb cryptic plasmid pVA380-1 has been used as a vector for the cloning of antibiotic resistance genes directly in streptococci, and in the construction of Escherichia coli/Streptococcus shuttle vectors. The results of subcloning experiments located the basic replicon of pVA380-1 within a 2.5-kb region. The nucleotide base sequence of this region was determined and contained a single complete open reading frame (ORF) encoding a 237-amino-acid peptide with a predicted size of 29 kDa. This peptide and a region of the DNA molecule 5' to the ORF encoding it shared homology with the replication protein and plus origin, respectively, of the Staphylococcus aureus plasmid pUB110. Data from Tn5 mutagenesis and complementation studies indicated that the protein product of the ORF was required for pVA380-1 replication in streptococci. Deletion of a region of the basic replicon distal to the plus origin and ORF produced an unstable derivative, and resulted in the accumulation of single-stranded replicative intermediates, consistent with the loss of a minus origin. All of these results suggest that pVA380-1 replicates by a rolling circle mode, and is most closely related to the pC194 family of single-stranded DNA plasmids.
4.2kb的隐蔽质粒pVA380 - 1已被用作直接在链球菌中克隆抗生素抗性基因以及构建大肠杆菌/链球菌穿梭载体的载体。亚克隆实验结果将pVA380 - 1的基本复制子定位在一个2.5kb的区域内。测定了该区域的核苷酸碱基序列，其包含一个单一的完整开放阅读框（ORF），编码一个237个氨基酸的肽，预测大小为29kDa。该肽以及编码它的ORF 5'端的DNA分子区域分别与金黄色葡萄球菌质粒pUB110的复制蛋白和正链起点具有同源性。来自Tn5诱变和互补研究的数据表明，ORF的蛋白质产物是pVA380 - 1在链球菌中复制所必需的。删除正链起点和ORF远端的基本复制子区域会产生一个不稳定的衍生物，并导致单链复制中间体的积累，这与负链起点的缺失一致。所有这些结果表明，pVA380 - 1通过滚环模式复制，并且与单链DNA质粒的pC194家族关系最为密切。
