Otto B, Reichard P
J Virol. 1975 Feb;15(2):259-67. doi: 10.1128/JVI.15.2.259-267.1975.
Nuclei from polyoma-infected 3T6 fibroblasts elongate in vitro the progeny strands of the replicative intermediates of polyoma DNA. When high concentrations of such nuclei were incubated, short DNA fragments were formed and subsequently added onto growing progeny strands. When nuclei were repeatedly washed with buffer containing detergent and then incubated at low concentrations. DNA synthesis was decreased. In particular, the joining process was reduced, resulting in an accumulation of short DNA fragments. All aspects of the synthetic capacity of the nuclei were restored by addition of cytoplasmic extract. Additions of purified enzymes (polynucleotide ligase from calf thymus or Escherichia coli together with E. coli DNA polymerase I) increased the joining function of the nuclei. The system can be used for the identification of the enzymatic steps concerned with polyoma DNA replication.
来自多瘤病毒感染的3T6成纤维细胞的细胞核，可在体外延长多瘤病毒DNA复制中间体的子代链。当孵育高浓度的此类细胞核时，会形成短DNA片段，随后这些片段会添加到正在生长的子代链上。当细胞核用含有去污剂的缓冲液反复洗涤，然后在低浓度下孵育时，DNA合成减少。特别是连接过程减少，导致短DNA片段积累。通过添加细胞质提取物可恢复细胞核合成能力的所有方面。添加纯化的酶（来自小牛胸腺或大肠杆菌的多核苷酸连接酶以及大肠杆菌DNA聚合酶I）可增强细胞核的连接功能。该系统可用于鉴定与多瘤病毒DNA复制相关的酶促步骤。
