ALBERT

Notes: Summary In contrast to the wild-type, mutant [ANT R8] is able spontaneously to throw off stable respiratory deficient mutants. The frequency of these mutants is considerably enhanced by treatment with ethidium bromide (EB) or the azo-dye Janus green (JG). An unstable cell state with a petite-like phenotype is found in both mutant [ANT R8] and wild-type after EB-treatment. However, only in the mutant is this unstable cell state followed by the appearance of stable respiratory deficient (RD) mutants. Formation of microcolonies is observed both in [ANT R8] and wild-type. RD mutants were isolated after EB treatment. Three of them (mit-12, mit-25, and mit-30) were analyzed and mit-25 characterized in more detail. Mutant mit-25 shows mitotic segregation in the diploid state, indicating non-Mendelian mode of inheritance. The results of haploidization experiments also indicate extrachromosomal inheritance. Mit-25 shows a spontaneous rate of reversion of 10-6, which can considerably increased by EB. Mit-25 possesses enzymatically active complexes I, II, and III of the respiratory chain (the latter without cytochrome b-566), lacks complex IV and binds antimycin, showing that mitochondrial protein synthesis is functional. Several lines of evidence presented in this paper make it very likely that the lesion in the mutant mit-25 is a point mutation in mitochondrial DNA.

Notes: Summary In the antimycin-resistant mutantana r-8 of the fission yeastSchizosaccharomyces pombe (Sch.p.) spontaneous mutants were isolated showing high resistance to the aminoglycoside antibiotic paromomycin. All mutants were resistant to the structurally related antibiotic neomycin. Tetrad analysis, mitotic segregation analysis, and mitotic haploidization revealed extrachromosomal, very likely mitochondrial inheritance. In contrast to the rapid segregation of mitochondrial markers in zygotic clones ofSaccharomyces cerevisiae (S.c.) the heteroplasmic state of diploids proved to persist for at least 50 generations after zygote formation. Stationary cultures of the paromomycin-resistant mutantspar r-106 andpar r-112 contain up to 6% respiratory-deficient mutants, but no reversion to paromomycin-sensitivity was observed among 1700–1800 colonies tested. The ability of mutantana r-8 to produce spontaneously respiratory-deficient mutants could be separated from the antimycin-resistant phenotype ofana r-8.

Notes: Summary It is shown that caffeine antagonizes petiteinduction with ethidium bromide under non-growth conditions when administered during or after mutagenic treatment. Caffeine itself is shown to be a petite-inducing agent when cells are grown in liquid glucose-completemedium in the presence of the drug. A possible mode of action of caffeine in the ethidium bromide induced petite-mutagenesis is discussed.

Notes: Summary 1. Retention or loss of mitochondrial markers C 321 R , O 1 R , P 454 R , TR (gene loci RIB1, OLI1, PAR1, TSM1 respectively) has been analysed in a large number of ethidium bromide induced primary rho− clones. Retention of one or more of the four markers within a single clone was observed frequently, only 20 to 25% of clones were found to be (ToCoOoPo). Primary clones retaining two or more of the four markers were found to be mixed, i.e. the primary rho− cell contained a heterogeneous population of variously deleted mitDNA molecules which segregated into different cell lines in the corresponding primary clone. 2. A representative sample of the population of ethidium bromide induced rho− mutants has been analysed by a first subcloning performed after some 30 cell generations of vegetative multiplication in the absence of the drug. At this level the heterogeneous population of mitDNA molecules, generated by the mutagenic treatment in the primary cell, has been sorted out. The cells forming secondary clones are thus essentially homoplasmic. In contrast to primary clones, genotypes of secondary clones therefore could be determined unambigously, and the frequency of cell types can be regarded as a faithful representation of the frequency of mitDNA molecules. Retention of markers was low, in less than 2% of secondary clones one or several markers have been found. This observation has been interpreted as indicating that induction of rho− mutants by ethidium bromide is accompanied by deletion of very large sequences of mitDNA in a very large fraction of mitDNA molecules. 3. Five individual rho− clones retaining the four markers TRCRORPR have been isolated and analysed for spontaneous deletion of one or several of these markers during successive subclonings (pedigree analysis). High genetic stability (98–99.5% per cell generation) has been observed in these clones. 4. A method has been developed allowing an unambiguous determination of the order of the four markers on a circular map. It is based on the concomitant loss of two markers and retention of the other two markers (double loss/double retention analysis). The results of four out of five pedigrees of individual rho− clones analysed (spontaneous deletion) and the results of the analysis of populations of secondary rho− clones (ethidium bromide induced deletion) were in full agreement and the order of genes has beeen determined as being P-T-C-O-P. In the fifth pedigree results suggest an inversion of the T and C markers. 5. Relative distances between pairs of markers have been derived from the frequencies of separation of markers by deletion and were found to be C-T〈C-O〈T-O〈T-P〈C-P〈O-P. Linkage of the four markers could be established, and distances calculated are additive. 6. The general relevance of this approach of mapping by deletion and the methods used for the determination of order and distances of mitochondrial genes has been discussed. It is concluded that i) this attempt gives a faithful representation of the topology and of genetic distances in the wild type mitDNA molecule, and ii) that in case of relatively distant markers deletion mapping is preferable to the mapping by multifactorial rho+ x rho+ crosses.

Notes: Summary 1. In non-fermentable substrates growth of mutant tsm-8 cells of Saccharomyces cerevisiae is restricted to about one generation after shift from 23 to 35°C. Non-permissive conditions (35°C, glycerol) cause a gradual decrease in respiration to about 20% of the activity at permissive temperature (23°C). 2. Anaerobically grown and glucose-repressed mutant cells exhibit a decreased adaptation rate of mitochondrial functions to aerobic growth and non-fermentative growth, even at 23°C, as revealed by determination of respiratory rates and mitochondrial protein synthesis. 3. At 35°C, ρ+ cells of mutant tsm-8 are converted to ρ− cells within 6–8 generations of growth, in all fermentable substrates tested. Drugs or antibiotics as nalidixic acid, acriflavin, chloramphenicol and erythromycin, bongkrecic acid, antimycin and FCCP, as well as anaerobiosis, have little or no influence on this kinetics. A heat shock does not yield ρ− petites to a significant extent. 4. Reversion of tsm-8 cells to wild type function, which occurs spontaneously with a frequency of 10-8, is found to be due to a mitochondrial mutational event.

Notes: Summary The π-factor stability is shown to be affected by four conditional mutations, tsm-8 (mitochondrial), tsp-20, tsp-25 and tsp-30 (nuclear). Growth of mutant cells at high temperature (35°C) results in the rapid production of ρ − cells and concomittantly in the decrease of the ability to transmit mitochondrial genetic information to the ρ + progeny of crosses. Kinetics of ρ − cell formation during growth at 35°C have been compared with variations in transmission and recombination of mitochondrial markers in crosses. In all cases the transmission of mitochondrial markers of the ts-parent decreases as the number of cell generations increases. The frequencies of recombinants between mitochondrial markers either increase or decrease depending on the markers considered and the alleles of the ω-locus involved in the crosses. The results of all crosses performed have been compared with the predictions of the model for recombination and segregation of mitochondrial genes proposed by Dujon et al. (1974). This comparison indicates that the main result of high temperature treatment is a diminution of the input of mitochondrial information from the ts-parent into zygotes. Consequences of the induced variations of input follow the predictions of the model. The correlation found in ts-strains between the reduction of input in crosses and the formation of ρ − cells is discussed in terms of molecular events occurring in mitDNA molecules during high temperature induction of ρ + to ρ − mutation.

Notes: Summary An approach for the screening of mit - mutants, the isolation and preliminary classification of a series of such mutants is reported. Loss and retention of 8 mit - and 6 drug r markers in mitDNA was analyzed in populations of rho- clones derived from four yeast strains. The populations studied constitute a representative fraction of the rho- petites formed during growth at 35° C under the influence of mutation tsp-25 which is in common to the four strains. The majority of the rho- clones retained several of the markers studied. Depending on the marker regarded retention frequencies between 15% (oxi3) and 45% (oli1, cob) were observed. Loss of one and retention of the other of a pair of markers was determined in all rho- clones of the four populations. The frequencies of marker separation by rho- deletion thus obtained are assumed to reflect the distance between markers on the mitochondrial genome: the higher the frequency of separation the longer the distance between two markers. Based on these frequencies a unique order of markers on a circular map was determined. Positions of markers on a scale from 0 to 100 were found to be: cap/ery (0) — olil (16) — cob1-1354 (21) — ana101 (22) — cob2-1625 (24) — oli2 (35) — pho1 (40) — oxi3-2501 (44) — oxi3-3771 (47) — par (65) — oxi2 (79) — oxil (87) tms8 (93) —cap (100). The relevance of this map as to the faithful representation of the topology of gene loci on mitDNA is discussed. Correlation of retention frequencies of markers to their map positions reveals a pronounced polarity: mitDNA segments carrying the cob-oli1 segment prevail whereas segments retaining oxi3 are the least frequent.

Notes: Summary Uniparental inheritance of mitochondrial markers conferring resistance to 3 (3,4-dichlorphenyl)-1,1-dimethylurea (diuron or DCMU) and antimycin in the fission yeast Schizosaccharomyces pombe is promoted by delaying first division of zygotes.

Notes: Summary Crosses involving mitochondrial markers conferring resistance to antimycin (anar, AR), chloramphenicol (capr, CR), and erythromycin (eryr, ER) in cis- and trans-configuration were studied by zygote clone analysis. Mutant ana r -8 from which all other drug-resistant isolates were derived, exhibits a highly biased transmission (6.8% ana r ), in an analysis of 100 individual zygote clones. Important results of zygote clone analyses were: - Zygote clones may contain one, two, three, or four mitochondrial genotypes. - The proportion of the two parental and the two recombinant genotypes in individual zygote clones can vary almost over the entire range of percentages. - Proportions of the two corresponding recombinant types in individual clones are usually unequal. - Transmission rates of markers are higher in trans-than in cis-crosses indicating additivity of bias by two mutated alleles in coupling. - Transmission rates are different for the three markers both in cis- and trans-crosses, being lowest for C R and highest for E R . - Up to more than 80% uniform clones, expressing only one genotype, can be produced in cis- and trans-crosses. In cis-crosses always the double-sensitive parental type becomes uniform, in trans-crosses this may be the case for parental and/or recombinant genotypes. A tentative map is presented using data from cis- and trans-crosses, including a correction by omission of uniform clones. Phenomena of transmission, segregation, and formation of uniform clones are discussed with special regard to the difference brought about by fission versus budding. A comparison with relevant data from Saccharomyces cerevisiae and other organisms is presented.