Highlander S K, Novick R P
Department of Plasmid Biology, Public Health Research Institute of the City of New York, Inc., New York 10016.
Plasmid. 1990 Jan;23(1):1-15. doi: 10.1016/0147-619x(90)90039-f.
Plasmid pT181 is a small multicopy plasmid from Staphylococcus aureus that belongs to incompatibility group 3 and expresses two distinct types of incompatibility, Inc3A and Inc3B. Inc3A incompatibility is expressed by the primary replication control determinant, copA, which specifies two small transcripts, RNA I and RNA II, that jointly inhibit the synthesis of the rate-limiting initiator protein, RepC. Inc3B incompatibility is expressed by the leading strand replication origin and is due to competition for RepC. The copA region from each of 11 different pT181 copy number mutants was cloned onto the pT181-compatible vector, pE194, and tested for its ability to inhibit the replication of pT181 and its copy number mutants. The pT181 replication origin was also cloned and tested for its ability to inhibit the replication of the same plasmids. In general copA mutations that alter the production or sequence of RNA I and RNA II greatly reduced or completely eliminated Inc3A activity. Unlike the wild-type, all of the copy mutants were resistant to Inc3B inhibition. The separately cloned wild-type copA and ori regions each reduced the copy number of pT181 in proportion to their gene dosage, but neither blocked replication completely. It is proposed that the cloned Inc determinants cause incompatibility by interfering with the plasmid's copy correction mechanism; this interference destabilizes the plasmid even under conditions where its average copy number is not greatly reduced.
质粒pT181是一种来自金黄色葡萄球菌的小型多拷贝质粒，属于不相容群3，表达两种不同类型的不相容性，即Inc3A和Inc3B。Inc3A不相容性由主要复制控制决定簇copA表达，copA指定了两个小转录本RNA I和RNA II，它们共同抑制限速起始蛋白RepC的合成。Inc3B不相容性由前导链复制起点表达，是由于对RepC的竞争所致。将11个不同的pT181拷贝数突变体的每个copA区域克隆到与pT181相容的载体pE194上，并测试其抑制pT181及其拷贝数突变体复制的能力。还克隆了pT181复制起点并测试其抑制相同质粒复制的能力。一般来说，改变RNA I和RNA II产生或序列的copA突变会大大降低或完全消除Inc3A活性。与野生型不同，所有拷贝突变体都对Inc3B抑制有抗性。单独克隆的野生型copA和ori区域各自按其基因剂量比例降低了pT181的拷贝数，但都没有完全阻断复制。有人提出，克隆的Inc决定簇通过干扰质粒的拷贝校正机制导致不相容性；这种干扰即使在质粒平均拷贝数没有大幅降低的情况下也会使质粒不稳定。
