Ortega S, de Torrontegui G, Díaz R
Centro de Investigaciones Biológicas (C.S.I.C.), Madrid, Spain.
Mol Gen Genet. 1989 May;217(1):111-7. doi: 10.1007/BF00330949.
In vitro mutagenesis with hydroxylamine of a ParD- miniderivative of R1, pAB174, yielded mutants that were less stable in the cell than pAB174. Some of these mutants had a thermosensitive phenotype. The replication of pAB2623, one of the thermosensitive mutants, was inhibited in the cell at the restrictive temperature of 42 degrees C. The efficiency of the RepA protein of pAB2623 to promote replication of R1 in an in vitro assay was greatly reduced. Sequence analysis indicated that the repA gene of pAB2623 contains, close to its 3' end, two GC-AT transitions, separated by a single base, that change two consecutive codons of the gene. These results indicate that the phenotype of the mutant is the consequence of a mutated RepA protein and is consistent with the requirement of RepA for the in vivo replication of this plasmid.
用羟胺对R1的ParD - 微型衍生物pAB174进行体外诱变，得到的突变体在细胞中的稳定性低于pAB174。其中一些突变体具有温度敏感表型。温度敏感突变体之一pAB2623在42℃的限制温度下在细胞中的复制受到抑制。在体外试验中，pAB2623的RepA蛋白促进R1复制的效率大大降低。序列分析表明，pAB2623的repA基因在其3'端附近包含两个由单个碱基隔开的GC - AT转换，这改变了该基因的两个连续密码子。这些结果表明，突变体的表型是RepA蛋白突变的结果，并且与RepA对该质粒体内复制的需求一致。
