Molecular genetic analysis of pheromone-independent highly transferable drug resistance plasmid of Enterococcus.

1)测定pMG1全质粒DNA序列。2)通过对序列的计算机分析，确定了假定的orf。每个OFR都设计了探针，通过Northern杂交检测其偶联过程中il的表达，结果表明转入基因产生了一些操纵子。3) ORF20插入突变体使最大操纵子的表达消失，下游操纵子的表达减少。由此可见，ORF20可能是突变基因的正调控因子。还有人建议存在负调节器。4)除了ORF2 （traA）外，所有被检测的似乎参与缀合的orf在缀合过程中都被抑制，据报道ORF2 （traA）在缀合早期被上调。5)通过与死亡受体的交配，考察受体细胞在结合中的作用。ORF42在杂交过程中表达下调，但在与死亡受体交配时表达不变。这一观察结果表明，受体必须是活的，才能启动缀合，并且需要供体和受体之间的相互作用。6)用Tn916诱变法分离出不能成为良好受体的突变体。它们在结合过程中不能下调ORF42的表达。绘制了突变图谱，确定了该基因的DNA序列。该基因与一个转录正调控因子具有显著的同源性。

1) DNA sequence of whole pMG1 plasmid was determined.2) By computer analysis of the sequence, putative ORFs were determined. Probes for each OFR was designed to examine ils expression during conjugation by Northern hybridization and it was suggested that tra genes made some operons.3) Insertion mutant of ORF20 abolished the expression of the biggest operon and reduced the expression of the operon downstream. From this observation, ORF20 was suggested to be a positive regulator of tra genes. The presence of a negative regulator was also suggested.4) All ORFs, which were examined and seemed to be involved in conjugation, were repressed during conjugation except that ORF2 (traA), had been reported to be upregulated early period of conjugation.5) The role of recipient cell in conjugation was examined by the mating with killed recipient. The expression of ORF42 was downregulated during conjugation, but was not changed in the mating with killed recipient. This observation indicated that recipient had to be alive to initiate conjugation, and interaction between donor and recipient was required.6) The mutants which could not be good recipients were isolated by Tn916 mutagenesis. They could not downregulate the ORF42 expression during conjugation. Mutation was mapped and DNA sequence of the gene was determined. This gene had significant homology with a positive regulator of transcription.