The replication of mitochondrial DNA during the cell cycle in Saccharomyces lactis.

Data accumulated in recent years that yeast mitochondria contain DNA (mDNA) with a base composition different from that of nuclear DNA (nDNA) 1 support genetic and biochemical evidence2 that these are the cytoplasmic factors determining respiratory competence. Since phenotypic expression of the respiratory function of mitochondria is under nuclear control (segregational petites),3 the synthesis of mitochondria-specific components is determined by both the nuclear and mitochondrial genomes. The above findings, as well as the extreme limitations placed by the limited size of the mitochondrial genome in yeast (ca. 107 daltons)," 4 raise the question as to whether mDNA replication is independent of nDNA replication. The recent findings in synchronous cultures of yeast that nDNA replication5 and the times of expression of several structural genes6 are periodic during the cell cycle provides a basis for examining this relationship. If the controls for mDNA replication are independent of the controls for nDNA replication, then one might expect that the population of mDNA's would either replicate continuously or randomly over the cell cycle. To investigate this relationship the replication of mDNA and nDNA in Saccharomyces lactis (Kluyveromyces lactis),7 a weakly fermentative yeast, was examined. This yeast can be readily grown with organic acids as a carbon source, conditions one might expect to derepress mitochondria formation. The present paper reports our initial findings on the properties and physiological control of mDNA in S. lactis. Evidence is also presented to show that in this yeast mDNA replication is periodic and at an interval of the cell cycle different from that of nDNA replication. Materials and Methods.-Organism and cultivation: Haploid parental (Y-14 and Y-123) or diploid (Y-14 X Y-123) stocks of Saccharomyces lactis were used. The method of isolating diploids and maintenance of stocks was described by Tingle et al.8 Cells were grown in 2% succinate synthetic medium6 (SSM) unless otherwise stated. Synchronous cultures of diploid cells in SSM were established and monitored as previously described.6 DNA isolation and centrifugation: DNA was isolated from yeast by the method described by Smith and Halvorson.9 DNA solutions were centrifuged to equilibrium (17 hr at 44,770 rpm) in either cesium chloride (1.700 gm/cc; 0.01 M tris(hydroxymethyl)aminomethane (Tris), pH 8.0) or cesium sulfate (1.554 gm/cc; 0.01 M Na borate, pH 8.5; 5 X 10-10 moles of HgCl2/4g DNA) at 250C in a Beckman model E ultracentrifuge. Escherichia coli DNA (1.710 gm/cc in CsCl) was used as a density marker. For determinations of the mDNA/nDNA ratio the DNA was centrifuged at two different concentrations (10 /g/ml for mDNA determinations; 1 ug/ml for nDNA determinations) at the same time. Microdensitometer tracings of ultraviolet photographs were made with a Joyce-Loebl chromoscan. Estimation of DNA content during synchronous growth: Fifty ml of a synchronous culture of diploid cells were withdrawn at intervals, filtered rapidly over a 25-mm diameter