Oertel W, Goulian M
J Bacteriol. 1979 Nov;140(2):333-41. doi: 10.1128/jb.140.2.333-341.1979.
Cells of Saccharomyces cerevisiae permeabilized by treatment with ether take up and incorporate exogenous deoxynucleoside triphosphate into deoxyribonucleic acid (DNA). With rho(+) strains, more than 95% of the product was mitochondrial DNA (mtDNA). This report characterizes ether-permeabilized yeast cells and describes studies on the mechanism of mtDNA synthesis with this system. The initial rate of in vitro mtDNA synthesis with one strain (X2180-1Brho(+)) was close to the rate of mtDNA replication in vivo. The extent of synthesis after 45 min was sufficient for the duplication of about 25% of the total mtDNA in the cells. The incorporated radioactivity resulting from in vitro DNA synthesis appeared in fragments that were an average of 30% mitochondrial genome size. Density-labeling experiments showed that continuous strands of at least 7 kilobases after denaturation, and up to 25 kilobase pairs before denaturation, were synthesized by this system. Pulse-chase experiments demonstrated that a large proportion of DNA product after short labeling times appeared in 0.25-kilobase fragments (after denaturation), which served as precursors of high-molecular-weight DNA. It is not yet clear whether the short pieces participate in a mechanism of discontinuous replication similar to that of bacterial and animal cell chromosomal DNA or whether they are related to the rapidly turning over, short initiation sequence of animal cell mtDNA. In rho(0) strains, which lack mtDNA, the initial rate of nuclear DNA synthesis in vitro was 1 to 2% of the average in vivo rate. With temperature-sensitive DNA replication mutants (cdc8), the synthesis of nuclear DNA was temperature sensitive in vitro as well, and in vitro DNA synthesis was blocked in an initiation mutant (cdc7) that was shifted to the restrictive temperature before the ether treatment.
经乙醚处理而透性化的酿酒酵母细胞能够摄取外源脱氧核苷三磷酸并将其掺入脱氧核糖核酸（DNA）中。对于ρ⁺菌株，超过95%的产物是线粒体DNA（mtDNA）。本报告对经乙醚透性化的酵母细胞进行了表征，并描述了用该系统对mtDNA合成机制的研究。用一个菌株（X2180 - 1Bρ⁺）进行体外mtDNA合成的初始速率接近体内mtDNA复制的速率。45分钟后的合成程度足以使细胞中约25%的总mtDNA发生复制。体外DNA合成产生的掺入放射性出现在平均为线粒体基因组大小30%的片段中。密度标记实验表明，该系统合成的变性后至少7千碱基的连续链，以及变性前高达25千碱基对的链。脉冲追踪实验表明，短时间标记后大部分DNA产物出现在0.25千碱基的片段中（变性后），这些片段作为高分子量DNA的前体。目前尚不清楚这些短片段是否参与类似于细菌和动物细胞染色体DNA的不连续复制机制，或者它们是否与动物细胞mtDNA快速周转的短起始序列有关。在缺乏mtDNA的ρ⁰菌株中，体外核DNA合成的初始速率是体内平均速率的1%至2%。对于温度敏感的DNA复制突变体（cdc8），体外核DNA合成也对温度敏感，并且在一个起始突变体（cdc7）中，在乙醚处理前转移到限制温度时，体外DNA合成被阻断。
