Badaracco G, Ginelli E, Sacchi N, Sora S, Plevani P
Eur J Biochem. 1981 Aug;118(2):247-54. doi: 10.1111/j.1432-1033.1981.tb06393.x.
A procedure to prepare crude extracts from single colonies of Saccharomyces cerevisiae is described. Partially purified extracts catalyzed DNA synthesis directed by single-stranded fd DNA. Maximum activity requires the presence of ribonucleoside triphosphates although extensive DNA synthesis is observed in the presence of only deoxynucleoside triphosphates. Alkaline sucrose gradient analysis demonstrated that initiation of new DNA chains occurs in vitro and the DNA products synthesized are heterogeneous in size, Isopycnic analysis of the products of ribonucleotide-initiated fd DNA replication showed covalent linkage between the initiator RNA and the newly synthesized DNA. The fd-replicating capacity of extracts prepared from cell-division-cycle mutants, defective in events controlling DNA initiation of elongation, showed an increased thermosensitivity in the DNA replication reaction in vitro.
本文描述了一种从酿酒酵母单菌落制备粗提物的方法。部分纯化的提取物催化由单链fd DNA指导的DNA合成。尽管仅在存在脱氧核苷三磷酸的情况下观察到大量的DNA合成，但最大活性需要核糖核苷三磷酸的存在。碱性蔗糖梯度分析表明，新DNA链的起始发生在体外，合成的DNA产物大小不均一。对核糖核苷酸起始的fd DNA复制产物的等密度分析表明，起始RNA与新合成的DNA之间存在共价连接。从细胞分裂周期突变体制备的提取物的fd复制能力在控制DNA起始延伸的事件中存在缺陷，在体外DNA复制反应中表现出增加的热敏感性。
