Scott D L, Clark C W
Department of Biology, Morehouse College, Atlanta, Georgia 30314, USA.
Microbios. 1995;81(326):7-16.
Understanding the mechanisms which control replication of chromosomes with multiple origins of replication have been the subject of many investigations. The plasmid, R6K, has three origins of replication, alpha, beta, and gamma. In vivo, alpha and beta are utilized with equal frequencies while the gamma origin is rarely used in the parental plasmid, or remains silent in several miniplasmids. Hence, in the present study, the multiple origin plasmid, R6K, was utilized as a model system to investigate DNA replication. A 1.6 kb PCR (polymerase chain reaction) amplified region of R6K containing the central regulatory segment (CRS) was examined to determine if membrane complexing is required for maintenance. Crude membrane extracts from exponentially growing cultures were prepared and sedimented through 30-50% (w/v) linear sucrose gradients. Fractions collected were probed using quantitative PCR which revealed two fractions containing CRS DNA. These complexes were localized in a region between the inner and outer membranes of Escherichia coli.
了解控制具有多个复制起点的染色体复制的机制一直是许多研究的主题。质粒R6K有三个复制起点，α、β和γ。在体内，α和β以相同的频率被利用，而γ起点在亲本质粒中很少被使用，或者在几个微型质粒中保持沉默。因此，在本研究中，多起点质粒R6K被用作模型系统来研究DNA复制。对包含中央调控区段（CRS）的R6K的1.6 kb聚合酶链反应（PCR）扩增区域进行了检测，以确定维持是否需要膜复合。制备了来自指数生长培养物的粗膜提取物，并通过30 - 50%（w/v）线性蔗糖梯度进行沉降。收集的组分使用定量PCR进行检测，结果显示有两个组分含有CRS DNA。这些复合物定位于大肠杆菌内膜和外膜之间的区域。
