Polyoma virus minichromosomes: a soluble in vitro replication system
Polyoma virus minichromosomes: a soluble in vitro replication system
复制标题
多瘤病毒微型染色体：可溶性体外复制系统
作者：
B. Gourlie;M. Krauss;A. Buckler;R. Benbow;V. Pigiet
通过低渗提取分离的细胞核，从感染的3 T6细胞中分离多瘤病毒微型染色体。核提取物中体外DNA合成的动力学与完整核中观察到的相似。体外DNA合成的大部分产物与复制中间体（RI）微型染色体一起沉淀，并在1.4%琼脂糖凝胶上迁移为两条带（RI-a和RI-b）。脱氧核苷酸一磷酸掺入这些物种的动力学是一致的，在多瘤微型染色体的体外复制中存在几个限速步骤。电子显微镜分析表明，RI-α带几乎完全由RI θ结构组成，范围为46 - 87%复制，所有θ结构的一半为67 +/- 4%复制。RI-b材料更复杂，由sigma和alpha结构组成，其尾部范围为多瘤基因组长度的7 - 114%，并且较少出现连锁和多连锁二聚体结构。
Polyoma virus minichromosomes were isolated from infected 3T6 cells by hypotonic extraction of isolated nuclei. The kinetics of in vitro DNA synthesis in the nuclear extract was similar to that observed with intact nuclei. The majority of the products of in vitro DNA synthesis sedimented with replicative intermediate (RI) minichromosomes and migrated as two bands (RI-a and RI-b) on 1.4% agarose gels. The kinetics of deoxynucleotide monophosphate incorporation into these species was consistent with the existence of several rate-limiting steps in in vitro replication by polyoma minichromosomes. Electron microscope analysis showed that the RI-a band consisted almost entirely of RI theta structures ranging from 46 to 87% replicated, with one-half of all theta structures 67 +/- 4% replicated. The RI-b material was more complex, consisting of sigma and alpha structures with tails ranging from 7 to 114% of polyoma genome length and, less frequently, of linked and multiple linked dimeric structures.