Host Genes Affecting Plasmid Replication and Its Control

本文主要研究影响质粒pT181复制的两类金黄色葡萄球菌宿主突变体。迄今为止对这两类突变体的特性进行的研究表明，它们都说明了新型的宿主-质粒相互作用。第一类突变，如pcrA3，维持质粒pT181拷贝数减少，而不显著影响其遗传稳定性。pcrA突变不通过干扰质粒的控制机制而起作用，其作用不能简单地用宿主因子限制质粒复制来解释。结果表明，在pcrA宿主中存在质粒pT181 rep起始复制起始复合体的积累。这些结果表明，pcrA基因产物使质粒复制起始复合体能够有效地利用宿主酶机制进行复制。几种影响pcrA3突变反应的质粒突变已经被分离和表征。对pcrA3突变的更详细的研究将包括体内和体外复制实验以及遗传学研究。克隆pcrA基因的工作正在进行中。这将使我们能够评估其在细胞中的作用，并有助于理解其影响质粒pT181复制的方式。placC1突变是第二类突变的代表，它导致质粒pT181的拷贝数维持在更高的水平，研究发现它通过抑制作为负效应物的反转录本（ctRNA）的合成，特异性地影响了该质粒的控制机制。placC基因已被克隆，测序工作正在进行中。发现克隆的野生型等位基因与染色体上存在的placC1等位基因互补，阻止了质粒pT181的扩增。plaC基因的分离使其在宿主代谢中的作用及其表达调控方式的研究成为可能。所获得的结果将有助于确定pT181 ctRNA启动子依赖于placc基因功能的生物学意义。

The present proposal is centered on the study of two classes vof Staphylococcus aureus host mutants which affect the replication of plasmid pT181. The studies conducted so far on the characterization of these two classes of mutants have shown that both illustrate new types of host-plasmid interactions. The first class of mutations, exemplified by pcrA3, maintain plasmid pT181 at a reduced copy number, without dramatically affecting its hereditary stability. The pcrA mutations does not act by interfering with the plasmid control mechanism, and its effect can not be explained simply by a host factor limiting for plasmid replication. It was shown that in the pcrA host there is an accumulation of the plasmid pT181 Rep-origin replication initiation complex. These results suggest that the pcrA gene product enables the plasmid replication initiation complex to make efficient use of the host enzymatic machinery for replication. Several classes of plasmid mutations that affect the response to the pcrA3 mutation have been isolated and characterized. A more detailed study of the pcrA3 mutations is proposed which will include in vivo and in vitro replication experiments as well as genetical studies. Work is in progress for the cloning of the pcrA gene. This will permit us to evaluate its role in the cell and will contribute to the understanding of the way in which it affects plasmid pT181 replication. The mutation placC1, representative of the second class which leads to the maintenance of plasmid pT181 at much increased copy number, was found to specifically affect the control mechanism of this plasmid, by depressing the synthesis of the countertranscripts ( ctRNA), which act as negative effectors. The placC gene has been cloned and its sequencing is in progress. The cloned wild type allele was found to complement the placC1 allele present on the chromosome preventing the amplification of plasmid pT181. The isolation of the plaC gene makes possible the study of its role in host metabolism and of the way in which its expression is regulated. The results to be obtained will help to determine what is the biological significance of the dependence of the pT181 ctRNA promoter upon the function of the plaC gene.