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THE FATE OF MITOCHONDRIAL LOCI IN RHO MINUS MUTANTS INDUCED BY ULTRAVIOLET IRRADIATION OF SACCHAROMYCES CEREVZSZAE: EFFECTS OF DIFFERENT POST-IRRADIATION TREATMENTS M. HEUDE and E. MOUSTACCHI

Institut Curie, Biologie, Bdtiment 110, Centre Universitaire, 91405 Orsay Cedex, France. Manuscript received September 22, 1978 Revised copy received May 21, 1979 ABSTRACT

Three main features regarding the loss of mitochondrial genetic markers among rho- mutants induced by ultraviolet irradiation are reported: (a) the frequency of loss of six loci examined increases with UV dose; (b) preferential loss of one region of the mitochondrial genome observed in spontaneous rho- mutants is enhanced by UV; and (c) the loss of each marker results from large deletions. Marker loss in rho- mutants was also investigated under conditions that modulate rho- induction. Liquid holding of irradiated exponential or stationary phase cells, as well as a split-dose regime applied to stationary phase cells, results in rho- mutants in which the loss of markers is correlated with rho- induction: the more sensitive the cells are to rho- induction, the more frequent are the marker losses among rho- clones derived from these cells. This correlation is not found in exponential-phase cells submitted to a split-dose treatment, suggesting that a different mechanism is involved in the latter case. It is known that UV-induced pyrimidine dimers are not excised in a controlled manner in mitochondrial DNA. However, our studies indicate that an accurate repair mechanism (of the recombinational type ?) can lead to the restoration of mitochondrial genetic information in growing cells.

LTRAVIOLET irradiation (254 nm) of yeast cells induces mitochondrial Urespiratory-deficient rho- or “petite” mutants (RAUT and SIMPSON1955). This is, at least partially, due to the presence of UV-induced pyrimidine dimers in mitochondrial DNA, as evidenced by photoreactivatability of rho- induction ( SARACHEK 1958). That one or several other repair mechanisms, independent of the illumination, act on UV-induced lesions is supported by a number of reports: (1) several nuclear or mitochondrial genes, some of which are known to govern specific repair pathways, appear to control UV rho- induction (MousTACCHI 1971, 1973; MOUSTACCHI, PERLMAN and MAHLER 1976); (2) if the Wirradiated cells are incubated in the dark in a nonnutrient medium before plating Abbreviations: UV: ultraviolet of 254 nm; rho-: cytoplasmic respiratory-deficient or “petite” mutants; rho + : cytoplasmic respiratory-competent or “grande” cells; LH: dark liquid-holding of cells in nonnutrient medium at 30” with aeration; mit-: mitochondrial mutations corresponding to specific defects in mitochondrial genes. Genetics 93: 81-103 September, 1979.

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M. HEUDE A N D E. MOUSTACCHI

(liquid holding or LH), lethality is reduced (PATRICK, HAYNES and URETZ 1964), but rho- induction is modified depending upon the physiological conditions of the cells at the time of irradiation. When stationary phase cells are irradiated and subjected to liquid holding, the frequency of rho- mutants increases relative to the frequency observed after immediate plating (MOUSTACCHI and ENTERIC 1970); this frequency decreases when exponential cells are treated in the same way (HEUDEand MOUSTACCHI 1973). This suggests that mitochondrial DNA metabolism is different in these two cases, and that, under appropriate conditions (e.g. exponential cells LH), an effective repair mechanism can act to restore respiratory competence of cells resulting in a rho+ phenotype; (3) fractionation of a UV-dose with an intermediate period of LH results in an and increased resistance to the lethal effects (PATRICK and HAYNES1968; PARRY PARRY 1976) and in a sensitization to rho- induction (MOUSTACCHI, HRISOHO and HEUDE, in preparation), again suggesting the intervention of mechanisms acting on damaged mitochondrial DNA. The nature of the precise mechanisms involved in the repair of mitochondrial DNA after UV irradiation is still unknown. It is known, however, that a controlled excision of pyrimidine dimers, which takes place for nuclear DNA (WATERS and MOUSTACCHI 1974a), does not act on mitochondrial DNA, but rather an extensive degradation of the mitochondrial DNA occurs during post1975). At least for UV incubation (WATERS and MOUSTACCHI 1974b; PRAKASH exponentially growing cells, this degradation is correlated with the UV dose applied (HIXON and MOUSTACCHI 1978). Extensive genetic and biochemical analysis of the mitochondrial genome of yeast over the last few years has established that the rho- mutations result from more or less extended deletions of mitochondrial DNA molecule sequences, which can be accompanied by repetitions of the retained segments (FAYE et al. 1973; BORST 1974). Genetic studies have resulted in the identification and mapping of a number of mitochondrial mutations including drug resistance and mit- markers (See DUJON,COLSONand SLONIMSKI 1977 for review). These advances now make it possible to analyze the state of mitochondrial DNA in UV-induced rho- mutants by following the fate of particular mitochondrial markers. Differences have already been reported in the relative frequency of mitochondrial marker retention in spontaneous, as well as in UV-induced rhostrains (DEUTSCH et al. 1974; MOLLOY, LINNANE and LUKINS1975). In this paper, we examine the fate of six genetic markers located in different parts of the mitochondrial genome after UV irradiation and post-UV treatments that alter rho- induction.

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MATERIALS A N D METHODS

Strains: The origin and properties of the haploid strains of Saccharomyces cereuisiae used are presented in Table 1. MH3%6D is a respiratory-sufficient haploid strain carrying four drug-resistance markers, CR321, ER221, OR144 ( 0 1 1 ) and P R 4 5 4 , which are mitochondrial genetic loci conferring resistance to chloramphenicol, erythromycin, oligomycin and paromomycin, respectively. The rho- mutants to be analyzed were derived from a UV treatment of this strain.

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F A T E O F MITOCHONDRIAL LOCI AFTER UV

The mit- mutants are the following: mit-170 ( a s p ) , mit-I40 (ozi-3) and cs-990 (located near 0, in the E-0, segment). They are used to check the loss of the three corresponding segments in the rho- clones arisen from MH32-6D. For the detection of drug-resistance markers in the rho- clones, a drug-sensitive rho+ strain, 1073, was used. Media: YEPD-glucose 2%, yeast extract 1% and bacto-peptone 2%; YPG-glycerol 3%, yeast extract 1%, bacto-peptone 2% in sodium phosphate buffer, 0.05 M, p H 6.5; selective media for drug resistance are the complete glycerol media supplemented with chloramphenicol (4 g/l), erythromycin (5 g/l) or oligomycin (3 g/l); Min-Bacto-Yeast Nitrogen Base without amino acids (Difco) (6.7 g/l) and 2% glucose. All solid media contained 3% bacto-agar (Difco). Growth conditions: Aliquots from one culture of MH32-6D were stored in a mixture (1:3 by volume) of glycerol and complete glucose medium at -70", each tube contained about 4 x 107 cells per ml. For each experiment, a cell suspension was thawed and transferred in 5 ml of liquid complete glycerol medium supplemented with 20 pg per ml histidine and 40 pg per ml adenine. This culture was allowed to grow for 24 hr at 28". A preculture was then started in 5 ml of liquid YEPD supplemented, at a concentration of 2 x 106 cells per ml and allowed to grow at 28" for five hr for exponential cultures (20 to 25% budding cells) or 24 hr for stationary cultures (less than 5% budding cells). The four tester strains (170-6D, 140-8D, cs-990-01B and 1073) were kept on YEPD slants at 4". A small inoculum was transferred in 5 ml of liquid YEPD and allowed to grow to early stationary phase before mating. Irradiation and liquid holding conditions: Irradiation and 24 h r liquid holding were performed as previously described (HEUDE, CHANETand MOUSTACCHI 1975). MH32-6D was used in the present experiments. Dose fractionation: An initial dose of 50 J per m2 was given, and the cells were platecl immediately after suitable dilution on YEPD plates. Petite analysis: After the different treatments, aliquots of the cultures were plated on complete glucose medium in order to obtain 100 to 200 colonies per plate. After five days of growth at 28", some of the plates were overlaid with tetrazolium (OGUR,SAINTJOHNand NAGAI 1975) and grandes, sectored and whole rho- colonies were scored. On the remaining TABLE 1

Genotype and origin of stocks used in this study

Strain no.

Nuclear genotype

Mitochondrial genotype

Origin

Use

170-6D

a ura

mit-170-

I1

Tester strain for the loss of the mit-170 segment

140-8D

a ura his, p+ mit-140-

I1

Tester strain for the loss of the mit-140 segment

cs-990-01B

a ura

P+

W-

!I

Tester strain for the loss of the cs-990 segment

1073

a leu3

p+

U+

pf

mit cs-990CS

E S OS

PS

Tester strain for the loss of the drugR markers

Note: Symbols for nuclear genotypes as in SHERMAN and LAWRENCE (1974). Mitochondrial genotype is noted following &EN et al. (1970).

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M. HEUDE AND E. MOUSTACCHI

plates, rho+ clones were distinguished from rho- clones by their color. Due to the presence of the d e 2 mutation, wild-type rho+ cells form red colonies, whereas respiratory-deficient rho- cells give rise to white colonies. The white colonies without further subcloning were randomly picked up and grown to full spots (referred as “primary” clones) on YEPD plates. The presence or absence of the different mitochondrial gentic markers was tested in every clone by the replica-cross technique (COEN et al. 1970): the matrix plates carrying the spots of primary clones were replica plated onto the lawns of the four tester strains to generate diploids; one copy of the matrix was replica plated onto glycerol medium plates in order to discard the rho+ clones that may have been selected. The replica crosses with 1073 were replica plated onto media containing the different antibiotics. The replica crosses with the mit- strains were replica plated onto glycerol. Any rho- clones that can restore the growth on glycerol after the cross with a mit- tester should contain the corresponding mitt locus. In order to avoid false negatives, it was obviously necessary to check the efficiency of mating before replica plating onto glycerol. In the case of cs-99041B, which is cryosensitive, the glycerol plates carrying the diploids derived from the cross were incubated at the nonpermissive temperature (20”). Due to the leakiness of the P R marker, we encountered some difficulties in reading the plates containing paromomycin. For this reason, the results obtained concerning the corresponding segment are not reported in the following results section. RESULTS

Only primary rho- clones, (see MATERIALS AND METHODS) at least for UVinduced rho- mutants, were analyzed in this study. We used this approach, since our interest was in the early steps of the rho+ to rho- transformation, to avoid complications due to the mitotic segregation of markers in the progeny of treated cells during the establishment of stable rho- clones. For each primary clone analyzed, we determined the loss or retention of the six markers present in the initial irradiated cell. Cells that retain two or more markers may retain them on different mitochondrial molecules. In contrast, clones exhibiting marker loss are pure with regard to this loss. Fate of the individual markers

The loss or retention of the six markers (mit-170, C, E , cs-990, O,, and mit140) was examined for each of the primary clones isolated. The results are presented according to the order of the markers on the circular genetic map of the mitochondrial D N A . In Tables 2 and 3, UV induction of rho- mutants and percent loss of markers among rho- clones on immediate and delayed plating are reported. From these data, we calculated the percent loss of markers in UVinduced rho- clones. The contribution of spontaneous rho- clones to this analysis is not negligible, particularly in the low-dose range where the induction of rho- mutants is small. In order to perform these calculations, we assumed that spontaneous rho- mutants that have retained a given marker have the same probability of losing it after a UV-treatment as a rho+ cell. It should be noted that in the low dose range (at least up to 50 J per m2), due to the sigmoidal shape of the survival curves, the difference in UV sensitivity to killing between rho+ and pre-existing rho- cells is negligible (NUNES DE LANGGUTH and GELOS

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