Yeast mitochondrial DNA characterization after ultraviolet irradiation.

Yeast mitochondrial (mtDNA) 3H-labelled was isolated from exponential phase cells after ultraviolet light irradiation. Both the size and amount of mtDNA were found to be reduced during a 40-h liquid-holding (LH) period in non-growth medium following irradiation as compared to the mtDNA recovered from nonirradiated cells under similar conditions. After the LH period, previously irradiated cells were resuspended in growth medium containing [14C]adenine. Double labelled mtDNA (3H and 14C) was isolated from cell samples removed during new growth. A recovery in the amount and size of mtDNA was observed in irradiated cells during new growth. These biochemical studies agree with the observed loss and recovery of mtDNA genetic markers in UV-irradiated exponential phase yeast after a period of LH and new growth resp.

Mutations and cell transformation with 2-azido-9-fluorenone oxime.

When photolyzed in situ for as little as 15 s 2-azido-9-fluorenone oxime causes Type II and Type III transformation in C3H 10T 1/2 CL8 cells. In the yeast strain Saccharomyces cerevisiae D-7, simple reversions and gene conversions occurred and also mitotic crossing over, to a lesser extent, but no mitochondril "petite" mutants occurred. No mutations or transformations were induced in the dark or by the light itself.

Resistance to adriamycin cytotoxicity among respiratory-deficient mutants in yeast.

Saccharomyces cell uptake of Adriamycin and the ensuing cytotoxic response were found to be dependent upon the ionic strength of the medium used for drug treatment. A given concentration of Adriamycin which inhibited growth in complete medium ws found to be significantly cytotoxic when administered in water. Many survivors after Adriamycin treatment in water were found to be respiratory-deficient petite mutants containing mitochondrial deoxyribonucleic acid mutations. Petite mutants arising after Adriamycin treatment were not induced but selected from the preexisting population of spontaneously derived petite mutants (normal frequency, 2%) due to an increased resistance of these mutants to killing by Adriamycin as compared with normal respiratory-sufficient cells. The responses to Adriamycin in mitochondrial deoxyribonucleic acid respiratory-deficient mutants (rho-, rho degrees, mit-) with different impaired mitochondrial functions was studied. All were similarly more resistant to killing by Adriamycin than wild-type cells. The common deficiency shared by these mutants, i.e., nonfunctioning electron transport, may play a role in protecting these mutants from Adriamycin cytotoxicity. In addition, normal cells grown on glycerol, requiring aerobic respiration for carbon source utilization were more susceptible to killing by Adriamycin than cells grown on glucose. These studies suggest that a mitochndrial function in yeast may interact with Adriamycin to potentiate a cell cytotoxic mechanism of the drug.

Direct in vitro photoaffinity labeling of DNA with daunorubicin, adriamycin, and rubidazone.

Irradiation by daylight fluorescent lamps, of a physical complex of duplex DNA with either daunorubicin, adriamycin, or rubidazone produced covalent adducts to DNA. The photoincorporated drug could not be extracted by phenol extraction which removed 99% of the non-photolyzed drug from its physical complex with DNA. The photoadduct was also stable to dialysis, Mg2+ addition, column chromatography, and thermal denaturation and alkali treatment of the DNA. The photoinduced adduct was proportional to the amount of incident irradiation, and the amount of DNA present, and as much as 30--45% of the drug which was physically associated could be photoincorporated. The drugs were not incorporated extensively into single-stranded DNA which lacks the ability to bind these antitumor agents by intercalation. Although the photochemical mechanisms of photoaffinity labeling DNA with these antineoplastic agents are unknown, this approach may prove to be useful for trageting their cellular sites of actions.

Reversal of protection by light of the ethidium bromide induced petite mutation in yeast.

An intermediate in the ethidium bromide (EB) induced petite mutation pathway may be destabilized by daylight light to cause a reversion to the normal grande phenotype. Starved cells preincubated in the dark for up to 6 h with 100 microgram/ml EB could be reverted to grandes after one hour of light exposure, whereas similarly treated cells maintained in the dark expressed the petite mutation in more than 80 percent of the population. In addition, the production of petite mutants by EB in buffer could be prevented if cell suspensions were exposed to light immediately upon the addition of EB. Photoreversal of the EB-derived petite mutation in growing cells was less efficient presumably because the availability of an energy source caused a continuation of mutation events beyond the light revertible step to a non-reversible fixation of the mutation. Cells treated with EB in growth media at 4 degrees C were more responsive to light protection and reversal of the mutation. This may be due to the cold inhibition of an enzyme which comes into play beyond the light sensitive step in the mutation pathway.

A protective effect of caffeine on the ethidium induced petite mutation in yeast.

A prerequisite for petite induction by ethidium bromide (EB) is an initial covalent attachment of the drug to cytoplasmic DNA. This DNA modification is thought to initiate repair processes. The repair inhibitor, caffeine, provided a protective effect against the ethidium induced petite mutation at caffeine concentrations known to inhibit the repair of UV damage in cytoplasmic DNA (Fig. 1). Mitochondrial DNA isolated from yeast exposed to EB in vivo was not as degraded in the presence of both drugs as with EB alone (Fig. 2).

Evidence for the dark repair of ultraviolet damage in Saccharomyces cerevisiae mitochondrial DNA.

Evidence for the dark repair of ultraviolet damage to yeast mitochondrial DNA has been observed. The ultraviolet dose necessary to inflict significant damage to both nuclear and mitochondrial DNA was determined. Cell survival at large doses of ultraviolet light was observed after immediate and delayed plating of yeast onto 1% pyruvate and 1% glucose media. In the highly lethal dose ranges of irradiation an increase in the number of normal colonies appeared after a period of liquid holding and delayed plating. This increase, demonstrated separately on 1% glucose and 1% pyruvate media suggested that the repair of both mitochondrial and nuclear DNA had occurred. After low doses of ultraviolet light an actual decrease in the number of petite survivors was seen after delayed plating, even though the total number of survivors increased. When a known repair inhibitor, caffeine, was added to the liquid holding buffer prior to the delayed plating of yeast, a marked decrease in the number of petites did not occur after delayed plating. Therefore, the decrease in the number of petite survivors after delayed plating following low doses of ultraviolet light is attributed to the repair of yeast mitochondrial DNA.