Of Saccharomyces Cerevisiae Restored to Respiratory Competence

When recently arisen spontaneous petite mutants of Saccharomyces cerevzsiae are crossed, respiratory competent diploids can be recovered. Such restored strains can be divided into two groups having sectored or unsectored colony morphology, the former being due to an elevated level of spontaneous petite mutation. On the basis of petite frequency, the sectored strains can be subdivided into those with a moderate frequency (5-16%) and those with a high frequency (>60%) of petite formation. Each of the three categories of restored strains can be found on crossing two petites, suggesting either that the parental mutants contain a heterogeneous population of deleted mtDNAs at the time of mating or that different interactions can occur between the defective molecules. Restriction endonuclease analysis of mtDNA from restored strains that have a wild-type petite frequency showed that they had recovered a wild-type mtDNA fragmentation pattern. Conversely, all examined cultures from both categories of sectored strains contained aberrant mitochondrial genomes that were perpetuated without change over at least 200 generations. In addition, sectored colony siblings can have different aberrant mtDNAs. The finding that two sectored, restored strains from different crosses have identical but aberrant mtDNAs provides evidence for preferred deletion sites from the mitochondrial genome, Although it appears that mtDNAs from sectored strains invariably contain duplications, there is no apparent correlation between the size of the duplication and spontaneous petite frequency. HE yeast Saccharomyces cerevisiae spontaneously produces respiratory defiT cient mutants (petites) at the high rate of approximately 1 % per generation (EPHRUSSI 1953; NAGAI, YANACISHIMA and NAGAI 196 l ) . This mutation is irreversible, cytoplasmically inherited (EPHRUSSI 1953; WRIGHT and LEDERBERG 1957) and associated with an element termed p (SHERMAN 1963). The p I Present address: CSIRO DiLSision of Molecular Biology, P.O. Box 184. North Ryde, Sydney, N.S.W. 2113, ' Present address: Department of Genetics, LaTrobe University, Bundoora, Victoria 3083, Australia. Australia. Genetics 111: 389-402 November. 1985 390 R . J. EVANS, K . M. OAKLEY AND G. D. CLARK-WALKER factor has since been identified as mitochondrial DNA (MOUNOLOU, JAKOB and SLONIMSKI 1966; MEHROTA and MAHLER 1968; GOLDRINC et al. 1970; NACLEY and LINNANE 1970), and petite mutants have large deletions from the circular 70-80 kilobase pairs (kbp) mitochondrial genome (MOUNOLOU, JAKOB and SLONIMSKI 1966; SANDERS et al. 1973; FAYE et al. 1973; CASEY, GORDON and RABINOWITZ 1974). Investigators have subsequently been interested in the deletion mechanism. Physical studies have centered on characterizing the mtDNA retained in petite mutants and in identifying the sequences involved in the deletion process. In terms of genome organization, two broad classes of petites have been identified. In one mutant type the retained sequence is reiterated through a series of tandem duplications (FAYE et al. 1973; LOCKER, RABINOWITZ and GETZ 1974a; HEYTINC et al. 1979), deletions apparently arising by recombination at short, tandemly orientated sequences that are, in general, between 1 0-20 nucleotides long (DEZAMAROCZY, FAUGERON-FONTY and BERNARDI 1983). In the other class the remaining mtDNA is organized as inverted repeats (LOCKER, RABINOWITZ and GETZ 1974b; LAZOWSKA and SLONIMSKI 1976; HEYTINC et al. 1979) that appear to result from unequal excision between pairs of oppositely oriented, short, repeated sequences flanking the deleted segment (SOR and FUKUHARA 1983). Genetic investigations undertaken in parallel with the physical studies showed that petite mutants can retain markers from different regions of the mitochondrial genome (LINNANE et al. 1968; GINGOLD et al. 1969). In these studies, although recombination could be demonstrated between the defective mtDNA molecules on crossing established petites, restoration of respiratory competence was not observed (MICHAELIS, PETROCHILO and SLONIMSKI 1973). However, crosses performed in our laboratory at this time showed that respiratory competence could be recovered provided that spontaneous petites of recent origin were used (CLARK-WAL.KER and MIKLOS 1975). This finding demonstrated that deletions do not always involve a common sequence. During our investigations it was observed that a proportion of the respiratory competent p+ restored colonies were sectored to varying extents. Colony sectoring on selective medium has subsequently been shown to be due to a raised level of spontaneous petite mutant formation that reaches over 80% per generation in some strains (OAKLEY and CLARK-WALKER 1978). Genetic analysis revealed that the high frequency of spontaneous mutation is cytoplasmically inherited (OAKLEY and CLARK-WALKER 1978), and further experiments involving crosses between haploid petites derived from an individual high-frequency petite-forming diploid, showed that these petites, unlike those from the original parental strains, fell into a small number of complementation groups (CLARK-WALKER et al. 1976). These studies suggested that abnormal mitochondrial genomes sometimes are formed on crossing recently arisen petites and that the loss of respiratory competence at high frequency is associated with a nonrandom breakdown of the restored mitochondrial genome. In the investigation described below, and in the accompanying paper (EVANS and CLARK-WALKER 1985), we provide evidence from restriction endonuclease ABERRANT MtDNA IN YEAST STRAINS 39 1 analysis of mtDNA for the existence of abnormal mitochondrial genomes in restored strains having a higher than normal frequency of spontaneous petite formation. MATERIALS AND METHODS Yeast strains: S . cereuisiae strains used in this study are different from those employed in previous wzork, due to a decrease in the mating efficiency of one of the original strains. However, as in the earlier reports, we have constructed isomitochondrial haploid parents by sporulation and ascus dissection of a diploid arising from mating D13.1A (a , p+, his3-532, t r p l ) (STRUHL et al. 1979) with 500ep(a , P O , ade l , a rg f -16 ) ; this latter strain has had its mtDNA eliminated (CLARK-WALKER 1972) and was obtained as an ascospore from a diploid produced by mating strains F and T, which have been previously described (CLARK-WALKER and MIKLOS 1975). After screening ascospores from the D13.1A X 500epdiploid, we obtained a suitable culture, T3/3 (a , p+, a d e l , arg4-16), which was used in conjunction with D13.1A as a parental strain for the production of petite mutants. Complementation tests: Isolation of recently arisen petite mutants, crossing of mutants and detection of respiratory competent restored forms have been described in detail in previous publications (CLARK-WALKER and MIKLOS 1975; OAKLEY and CLARK-WALKER 1978). Nomenclature of strains restored to respiratory competence: Restored strains are named according to their complementation experiment of origin, their D13.1A pparent, their T3/3 pparent and their colony morphology ( s = sectored, e = entire), in that order. They also may have a strain number if more than one colony of a particular morphology was picked from a single complementation-yielding matrix position. For example, the strain [ 119.6~3 was obtained from the first complementation experiment involving petite #9 derived from D 13.1 A and petite #6 derived from T3/3; it is the third picked strain of sectored colony morphology. Determination of culture petite frequencies: Strains were grown and spread on GGYP plates, as described for the selection of petites for complementation experiments; however, in this case, colonies were allowed to develop for 4-5 days at 30" to allow unequivocal identification of petite and grande colonies. Classification was principally on the basis of color, as some of the mutant strains produced respiratory competent colonies that varied in their size and shape. Mixed colonies were scored as respiratory competent, as there is a greater probability that the mutation to respiratory deficiency has occurred after, rather than before, plating (OGUR et al. 1959). Depending on the proportion of petites in the culture, as judged by preliminary spreads, between 20 and 100 GGYP plates were scored per strain. Culture petite frequencies are expressed as the number of petite colonies divided by the total number of colonies multiplied by 100%. Under conditions that do not support proliferation of petites, this value has been shown to approximate to the petite frequency per generation of the strain (OGUR et al. 1959). Other procedures: Media and culture conditions have been described previously (OAKLEY and CLARK-WALKER 1978). GlySV is a glycerol minimal medium supplemented with synthetic vitamins; GGYP contains yeast extract and bactopeptone and has glycerol as the major carbon source and glucose as a minor component. GlyYP is as GGYP, except that glycerol is the sole carbon source. For details of these media see OAKLEY and CLARK-WALKER (1978). Preparation of mtDNA was by dye-buoyant density centrifugation using bisbenzimide H 33258, as described previously (CLARK-WALKER, MCARTHUR and SRIPRAKASH 198 I). Restriction endonuclease digestion of nitDNA and electrophoretic separation of fragments in 0.8% agarose gels was as previously described (CLARK-WALKER et al. 1980), except that the enzyme-specific digestion buffers were replaced in these studies by T A buffer (O'FARRELL, KUTTER and NAKANISHI 1980). Restriction endonuclease HhaI was produced locally according to the method of GREENE et al. (1978). and Clal and PuulI were supplied by Boehringer Mannheim, West Germany. The sizes of the restriction fragments on gels were estimated according to their migration relative to fragments of known size. The marker fragments were derived from plasmid pAN124 (CHANG and COHEN 1978; MACINO and TZAGOLOFF 1979; STRUHL et al. 1979) and were the products of digestion with XhoI (14.0 kbp), EcoRl (8.0 and 6.0 kbp) and BamHI/EcoRI (4.65, 3.35, 2.35, 1.98 and 1.65 kbp). 392 R. J. EVANS, K . M . OAKLEY AND G . D. CLARK-WALKER