Copyright Q 1992 by the Genetics Society of America

Nuclear Mutationsin the Petite-Negative Yeast Schixosaccharomyces pombe Allow Growth of Cells Lacking Mitochondrial DNA Pascal Haffter' and Thomas D. Fox Section of Genetics and Development, Cornell University, Zthaca, New York 14823-2703

Manuscript received September 25, 199 1 Accepted for publication February 3, 1992 ABSTRACT The fission yeast Schirosaccharomycespombe has never been found togive rise to viable cells totally lacking mitochondrial DNA (rho").This paper describes the isolation of rho" strains of S. pombe by very longtermincubation of cellsinliquidmediumcontainingglucose,potassiumacetateand rho" strains did not require potassium acetate or any other novel ethidium bromide. Once isolated, the growth factors. These nonrespiring strains contained no mitochondrial DNA (mtDNA) detectable either by gel-blot hybridization using as probea clone containing the entire S.pombe mtDNA, or by Induction of rho" derivatives of standard 1',6-diamidino-2-phenylindolestaining ofwholecells. laboratory strainswas not reproducible from culture to culture. The cause of this irreproducibility appears tobe that growthof the rho" strains of S.pombe depended on nuclear mutations that occurred in some, but not all, of the initial cultures. Two independent rho" isolates contained mutations in unlinked genes, termedptpl-I and ptp2-I. These mutations allowed reproducible ethidium bromide induction ofviable rho" strains. No other phenotypes were associated with ptp mutations in rho+ strains.

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T is well known that petite-positive yeasts, such as Saccharomycescereuisiae, can be efficiently and completelyconverted to respiratorydeficient cytoplasmic-petite mutants by treatment with ethidium bromide (SLONIMSKI,PERRODINand CROFT 1968). This cytoplasmically inherited phenotype results from large deletions in the mitochondrial DNA (mtDNA) (rho-) or from the completeabsence of mtDNA (rho") (GOLDRING et al. 1970; NAGLEY and LINNANE1970). T h e viability of rho' S. cereuisiae demonstrates that in this species the presence of mtDNA and theconcomitant ability to synthesize mitochondrially coded proteins is required only for respiratory functions. Many other species of yeast, including Schizosaccharomyces pombe, are termed petite-negative because rho- and rho' mutations have never been observed (AHNEet al. 1984,1988;SEITZ-MAYR and WOLF1982; WOLFand DELGUIDICE1980; WOLFet al. 1976). T h e absence of such large mtDNA deletionsin S. pombe is not due to a dependence of viability on respiration per se, since many nonrespiratory mutations in both nuclear (GOFFEAUet al. 1974)and mitochondrial (AHNEet al. 1984,1988;SEITZ-MAYR and WOLF1982; WOLFet al. 1976) genes have been isolated. However, while previously described treatments of S. pombe with ethidiumbromide led to reduced copy number of mtDNA and eventual cessation of growth, viable cells recovered from ethidium treated cultures contained intact and functional mitochondrial genomes (WOLF

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Present address: Max-Planck-Institut fur Entwicklungsbiologie, Spemannstrasse 95/III, 7400 Tiibingen. Germany. Genetics 131: 255-260 Uune, 1992)

and DEL GUIDICE1980; WOLFet al. 1976). These observations have led to the suggestion that S. pombe, unlike S. cereuisiae, might depend on mitochondrial gene expression for important functions not directly related to respiration. While this hypothesis is clearly plausible, it nevertheless seemed surprising that rho" strains of S. pombe could not be obtained, particularly in light of the fact that rho" derivatives of both chicken embryo fibroblasts (DFSJARDINS,FROSTand MORAIS 1985)andhuman cells (KING and ATTARDI1989) have been generated by ethidium bromide treatment. We describe here the isolation of rho' strains of S. pombe by very long term exposure of cells in culture to ethidiumbromide. Interestingly, rho' induction was not reproducible from culture to culture. We found that the cause of this irreproducibility was the fact that growth of rho' strains of S. pombe depends on nuclear mutations that occurred in some, but not all, of the initial cultures. Such nuclear mutations, which can apparently occur in at least two unlinked genes, allow reproducible induction of rho' derivatives from otherwise wild-type S. pombe. MATERIALS AND METHODS Yeaststrains,mediaandgeneticmethods: S. pombe strains are listed in Table 1. PHP3was derived from a cross between SP223 and FYC15, PHP25 from a cross between PHP14 and FYC9. PHP4 is a rho" derivative of FYCl 1; PHP14 is a rho" derivative of PHP3. Nonfermentable medium was YPEG (1 % yeast extract, 2% peptone, 3% ethanol, 3% glycerol). Other mediaand

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and P. Haffter Fox TABLE 1 Strains used in this study

Strain

Genotype"

Source

FYCS FYCll FYC15 PHP3 PHP4 PHP14 PHP25 SP223

h+, ural-161, [rho+] h-, ade6-M210, [rho+] h+, ade6-M216, [rho+] h-, ade6-M216, leul-32, [rho+] h-, ade6-M210, ptp2-1, [rho"] h-, ade6-M216, leul-32, ptpl-1, [rho"] h+, ade6-M216, ural-161, ptpl-1, [rho+] h-, ade6-M216, leul-32,ura4, [rho+]

D. BEACH D. BEACH D. BEACH

This study This study This study This study D. BEACH

geneticmethodswereasdescribed (MORENO, KLAR and NURSE199 1). Isolationandcharacterization of rho" strains of S. pornbe: Cultures (5 ml) of complete glucose medium (YES) supplemented with 12.5 pg/ml ethidium bromide and 2% potassium acetate were inoculated with rho+ cells from individual colonies and grown to saturation in 1 day at 30". One streaking-loop from each culture was then transferred to 5 ml of the same medium and incubated at 30" for the indicated times. Cells were restreaked for single colonies on solid YES and tested for respiration by replica plating to YPEG. Putative rho" colonies were grown up in 5 ml YES for 5 days at 30" and total DNA was isolated as described for S. cerevisiae (ROSE, WINSTONand HIETER1988). Gelblot hybridization was carried out as described (MEINKOTH and WAHL 1984). Labeled probes were prepared using [a"PIdATP by the method of random primed DNA labeling (FEINBERG and VOCELSTEIN1983). 1',6-Diamidino-2-phenylindole (DAPI) stainingwas done as described (MORENO, KLAR and NURSE199 1) except that fixation of the cells was followed by a 5-min treatment at room temperature with 3 mg/ml Zymolyase-20T (ICN Immunobiologicals, Inc.)in 1 M sorbitol and sequentialwashes in PBS (10 mM sodiumphosphate,150 mM NaCl, 1 mM NaNS,pH 7.2)containing 1% ! Triton X-100 andPBS before attachment of the cells to the coverslip. RESULTS

Ethidium bromide induction of a nonrespiratory strain of S. pombe: Initial attempts to induce rho' strains by growing the S. pombe (strain FYCl 1; Table 1) in complete medium containing glucose (YES) supplemented with 12.5 pg/ml ethidium bromide failed. While the presence of ethidiumbromideinhibited growth, no nonrespiratory mutants were induced. Induction of rho' derivatives of chicken embryo fibroblasts (DESJARDINS, FROST and MORAIS1985) and human cells (KING and ATTARDI1989) requires the addition of uridine or uridine and pyruvate, respectively. [Pyrimidine biosynthesis in animal cells is dependentuponrespiration (GR~GOIRE et al. 1984).] We, therefore, tried these supplements. However,no rho' derivatives were ever obtained from strain FYC 1 1 by treatment in ethidiumbromide-containing YES medium supplementedwith 50 pg/ml uridine and 100 &ml pyruvate. We did obtain a nonrespiring derivative of strain FYCl 1 from a culture of cells that were incubated in

T. D.

YES mediumcontaining12.5 pg/ml ethidiumbromide and 2% potassium acetate for 17 days (MATERIALS AND METHODS). Cells that had grown in this culture were streaked for single colonies on YES medium plus 2% potassium acetate andthen replica platedtocompletemediumcontaining the nonfermentable carbon sources ethanol and glycerol (YPEG): none of the colonies grew on YPEG, suggesting that they might have a deficiency in mtDNA. One colony (strain PHP4) was picked forfurther analysis. Characterization of a rho" strain of S. pombe: T o rule out the possibility that the nonrespiring strain PHP4 was a contaminant, we checked its auxotrophy and ability to mate with bonafide rho+ S. pombe. Like the parental strain FYC 11, PHP4 required adenine for growth on minimal medium and mated with the ural strain FYCS to yield prototrophic haploid recombinant progeny. To determinewhetherstrainPHP4contained mtDNA, total cellular DNA was analyzed by gel-blot hybridizations (Figure 1). The plasmid pDG3, which contains the entire mtDNA of S. pombe in the vector pBR322 (DEL GIUDICE1981), was used to probe for mtDNA. The expected restriction pattern of S. pombe mtDNA was found for FYC 11, whereas no sequences hybridizing to S. pombe mtDNA could be detected in DNA isolated from PHP4 (Figure 1, A and B). Control hybridizations to detect single copy nuclear DNA sequences were carried out using as probes two randomly chosen clones (pH1 and pH4) from a Hind111 partial genomic library of S. pombe DNA (MOLZet al. 1989). Identical patterns of hybridization to total DNA of both FYCll and PHP4 were obtained with both probes (Figure 1, C and D), indicating that our procedure was sensitive enough to detect single copy sequences and confirming that FYCl 1 and PHP4 are closely related. Thus we conclude that PHP4 is an S. pombe strain devoid of mtDNA. T o visually confirm the absence of cytoplasmically located mtDNA in rho' S. pombe we microscopically examined cells stained with DAPI (Figure 2; MATERIALS AND METHODS). The rho' strain PHP14 (Table 1; see below) and its rho+ parent (PHP3) were compared. Nuclei were clearly stained in both types of cells. However, staining of mtDNA, observed as speckles distributed throughout the cell, was evident only in rho+ cells. While the sensitivity of this method is limited, the results confirm the conclusion based on the hybridization analysis of Figure 1. Unusual features of rho" induction: We have been able to isolate ethidium bromide induced rho" derivatives from several different S. pombe strains, but only some treated cultures in a given experiment yielded rho' mutants. For example, of eight separate cultures of PHP3 treated in ethidium bromide for 15 days as

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pomhrrho" Schirosaccharomycrs

1 . M . I R I ~ , - I ~ \ l ' l ~ I , l l l l l l l ~ , l~l / l ~ ~ ' l l ' l l l ' ~ l , l Il kl O~ l I I ' I I I ' I of S. pomhr. '1'11~. ~ I C X C Y ~ I I I \ C c. l r u I i l n ~ l111 \ I ~ I I R I A I S A Y I ) OM.

plus 2% potassitm

FIGUREI .-Cel-blot hybridization analvsk of' l o t ; t l I)%..\ from S. pbmbr. l a n e s 1-4 contained total DSh from strain FY(:l I (rho'). lanm 5-8 rontnined total BSA fromstrain PttP4 (rho'). F.cpal amounts of D S A were s u h j e r t e d to gel electrophoresis (MATERIAIS ANn SIFTHOM) after digestion with restriction cn7vn~esa s follows: u n d i p t c c i (lanes I , 5). Shnl (lanes 2.6). Ilindlll (lanes 3. i ) .E m K l (lanes 4. 8 ) . Filters wereprolml with ["P]-laIwled D S A protm ( M A T E R I A I S A N D SIFTIWM) o f rOllf$llV equal specificactivitv as follows. T h e plasmid pIX;J. carwing the entire S. pombr mitwhontlrial chronrowme. was used as prohe in pmels h and R. .I'he nurlmrprobrcwere pH1 in pmel C and ptt4 in pmel I). l'hc filtem for panel A. C and D were expowd h r an e q ~ ~ length al of time. whereas panel R represents a shorter e x p u r c * o fthe wme filter as in panel A .

described above.only two produced nonrespiring strains. One such strain (PHP14) obtained from treatment of PHP3 was characterized fullv as described and confirmed to tw iS. pomhr rho" above for P M P ~ (not shown). T h e inclusion of potassium acetate in the ethidiunr bromide-containing medium appears to be necessary for the induction of rho" mutants from standard rho' laboratory strains since we werc never able to obtain rho" strains by treatment in media lacking potassium e 111swerc acetate. Surprisingly however.once rho" s11-1' obtained their growth rate was the same on b o t h standard glucow containing medium (YES) and YES

II1I.Il..

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t l w prcscncv o f potassium ;tcct;ttc clrws not appctr IOIW comlwnsating for a metalmlic dclicicwcy o f ' 1 1 w rho" strains and its role in their induction is unclc;tr. Not surprisingly. a l l tllc rho" strains studicd grcw substantially s l o ~ r won conrplctc medium contititling glucose ( w i t h or w i t h o u t potassium ;tcct;ttc) t h a n t l w corresponding rho' strains. They also cshihitctl ;I flocculent pllcnotypc when grown i n liquid nlcdia. The S.pornhe rho" strains carry nuclear mutations allowingtheir growth: ~ I I wlidlowing Iyx)thc*sis could account for the. irrcl)r'Hlrrcihility o f rho'' illduetion from cultrrrct o ctrlturc*:c - t l l i t l i t r n l Iwomidc causcd a reduction i n mtDSA copv nunrlwr t o the p o i n t t h t cell growthceased (\VOI.F and I ~ I C.r*rnrcx . I!1XO: U'0I.F rt a/. 19i6).I ~ r during t our v c ~ ylong culture periods nuclear mutations wcr

Nuclear mutations in the petite-negative yeast Schizosaccharomyces pombe allow growth of cells lacking mitochondrial DNA.

The fission yeast Schizosaccharomyces pombe has never been found to give rise to viable cells totally lacking mitochondrial DNA (rho(o)). This paper d...
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