Mitochondrial DNA (mit-DNA) synthesis was compared in suspension cultures of Chinese hamster cells (line CHO) whose cell cycle events had been synchronized by isoleucine deprivation or mitotic selection . At hourly intervals during cell cycle progression, synchronized cells were exposed to tritiated thymidine ([3H]TdR), homogenized, and nuclei and mitochondria isolated by differential centrifugation . Mit-DNA and nuclear DNA were isolated and incorporation of radioisotope measured as counts per minute ([3H]TdR) per microgram DNA . Mit-DNA synthesis in cells synchronized by mitotic selection began after 4 h and continued for approximately 9 h . This time-course pattern resembled that of nuclear DNA synthesis . In contrast, mit-DNA synthesis in cells synchronized by isoleucine deprivation did not begin until 9-12 h after addition of isoleucine and virtually all [3H]TdR was incorporated during a 3-h interval . We have concluded from these results that mit-DNA synthesis is inhibited in CHO cells which are arrested in G i because of isoleucine deprivation and that addition of isoleucine stimulates synchronous synthesis of mit-DNA . We believe this method of synchronizing mit-DNA synthesis may be of value in studies of factors which regulate synthesis of mit-DNA .

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