Luthman H, Nilsson M G, Magnusson G
J Mol Biol. 1982 Nov 15;161(4):533-50. doi: 10.1016/0022-2836(82)90406-5.
The boundaries of the origin of polyoma DNA replication have been analyzed using a set of deletion mutants. The majority of these had small deletions, 5 to 30 basepairs in size, which together removed most of the non-translated sequences of the genome. The phenotype of the mutants was characterized by analysis of infectivity, transforming ability and DNA synthesis. All mutants with reduced or abolished infectivity had corresponding defects of viral DNA synthesis. The effect of the deletion was cis-acting, since the replication of the mutants was not stimulated by the presence of wild-type DNA. Deletions causing a reduction of DNA synthesis were found at two sites. The first at the 32 base-pair inverted repeat sequence and the neighbouring A . T tract previously implicated in the initiation of DNA synthesis, and the second close to the late genes. The two sites were separated by at least 60 base-pairs of non-essential DNA. Only one mutant with a deletion at the second site was unable to express early gene functions. The mutants were constructed by linearization, shortening and recircularization of polyoma DNA inserted into the plasmid pBR322. The mutagenesis was directed at restriction endonuclease BglI or PvuII cleavage sites. The BglI-directed mutagenesis was focussed to polyoma DNA by using as a vector a derivative of pBR322 resistant to cleavage by BglI.
利用一组缺失突变体分析了多瘤病毒DNA复制起始的边界。这些突变体大多有小的缺失，大小为5至30个碱基对，它们共同去除了基因组的大部分非翻译序列。通过分析感染性、转化能力和DNA合成来表征突变体的表型。所有感染性降低或丧失的突变体都有相应的病毒DNA合成缺陷。缺失的作用是顺式作用的，因为野生型DNA的存在并不能刺激突变体的复制。在两个位点发现了导致DNA合成减少的缺失。第一个位于32个碱基对的反向重复序列和先前与DNA合成起始有关的相邻A.T序列，第二个靠近晚期基因。这两个位点被至少60个碱基对的非必需DNA隔开。只有一个在第二个位点有缺失的突变体无法表达早期基因功能。这些突变体是通过将插入质粒pBR322的多瘤病毒DNA线性化、缩短并重新环化构建的。诱变作用于限制性内切酶BglI或PvuII的切割位点。通过使用对BglI切割具有抗性的pBR322衍生物作为载体，将BglI介导的诱变作用集中于多瘤病毒DNA。
