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Nuclear Mutations in

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 Schirosaccharomyces pombe has never been found to give rise to viable cells totally lacking mitochondrial DNA (rho"). This paper describes the isolation of rho" strains of S. pombe by very long term incubation of cells in liquid medium containing glucose, potassium acetate and

ethidium bromide. Once isolated, the rho" strains did not require potassium acetate or any other novel growth factors. These nonrespiring strains contained no mitochondrial DNA (mtDNA) detectable either by gel-blot hybridization using as probe a clone containing the entire S. pombe mtDNA, or by

1 ',6-diamidino-2-phenylindole staining of whole cells. Induction of rho" derivatives of standard laboratory strains was not reproducible from culture to culture. The cause of this irreproducibility appears to be that growth of 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, termed ptpl-I and ptp2-I. These mutations allowed reproducible ethidium bromide induction of viable rho" strains. No other phenotypes were associated with ptp mutations in rho+

strains.

I

T is well known that petite-positive yeasts, such as

Saccharomyces cereuisiae, can be efficiently and completely converted to respiratory deficient cyto- plasmic-petite mutants by treatment with ethidium

bromide (SLONIMSKI, PERRODIN and CROFT 1968).

This cytoplasmically inherited phenotype results from large deletions in the mitochondrial DNA (mtDNA) (rho-) or from the complete absence of mtDNA (rho")

(GOLDRING et al. 1970; NAGLEY and LINNANE 1970). T h e viability of rho'

S.

cereuisiae demonstrates that in this species the presence of mtDNA and the concom- itant ability to synthesize mitochondrially coded pro- teins is required only for respiratory functions.

Many other species of yeast, including Schizosac- charomyces pombe, are termed petite-negative because

rho- and rho' mutations have never been observed (AHNE et al. 1984,1988; SEITZ-MAYR and WOLF 1982; WOLF and DEL GUIDICE 1980; WOLF et al. 1976). T h e absence of such large mtDNA deletions in

S.

pombe is not due to a dependence of viability on respiration

p e r se, since many nonrespiratory mutations in both nuclear (GOFFEAU et al. 1974) and mitochondrial (AHNE et a l . 1984,1988; SEITZ-MAYR and WOLF 1982; WOLF et al. 1976) genes have been isolated. However, while previously described treatments of S. pombe with ethidium bromide 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

mannstrasse 95/III, 7400 Tiibingen. Germany. Genetics 131: 255-260 Uune, 1992)

'

Present address: Max-Planck-Institut fur Entwicklungsbiologie, Spe-

and DEL GUIDICE 1980; WOLF et 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, FROST and MORAIS

1985) and human cells (KING and ATTARDI 1989)

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 ethidium bromide. 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

Yeast strains, media and genetic methods: S. pombe

strains are listed in Table 1. PHP3 was 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,

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256 P. Haffter and Fox T. D.

TABLE 1

Strains used in this study

Strain Genotype" Source

FYCS h+, ural-161, [rho+] D. BEACH

F Y C l l h-, ade6-M210, [rho+] D. BEACH

FYC15 h+, ade6-M216, [rho+] D. BEACH

PHP3 h-, ade6-M216, leul-32, [rho+] This study PHP4 h-, ade6-M210, ptp2-1, [rho"] This study PHP14 h-, ade6-M216, leul-32, ptpl-1, [rho"] This study PHP25 h+, ade6-M216, ural-161, ptpl-1, [rho+] This study SP223 h-, ade6-M216, leul-32, ura4, [rho+] D. BEACH

genetic methods were as described (MORENO, KLAR and NURSE 199 1).

Isolation and characterization 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 in- dividual 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, WINSTON and HIETER 1988). Gel- blot 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 VOCELSTEIN 1983).

1 ',6-Diamidino-2-phenylindole (DAPI) staining was done as described (MORENO, KLAR and NURSE 199 1) except that fixation of the cells was followed by a 5-min treatment at

room temperature with 3 mg/ml Zymolyase-20T (ICN Im- munobiologicals, Inc.) in 1 M sorbitol and sequential washes

in PBS (10 mM sodium phosphate, 150 mM NaCl, 1 mM NaNS, pH 7.2) containing 1 !% Triton X-100 and PBS 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) sup- plemented with 12.5 pg/ml ethidium bromide failed. While the presence of ethidium bromide inhibited growth, no nonrespiratory mutants were induced.

Induction of rho' derivatives of chicken embryo fibroblasts (DESJARDINS, FROST and MORAIS 1985) and human cells (KING and ATTARDI 1989) requires the addition of uridine or uridine and pyruvate, respec- tively. [Pyrimidine biosynthesis in animal cells is de- pendent upon respiration (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 ethidium bromide-containing YES medium supplemented with 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

YES medium containing 12.5 pg/ml ethidium bro- mide and 2% potassium acetate for 17 days (MATE- RIALS AND METHODS). Cells that had grown in this culture were streaked for single colonies on YES me- dium plus

2%

potassium acetate and then replica plated to complete medium containing the nonfer-

mentable carbon sources ethanol and glycerol

(YPEG): none of the colonies grew on YPEG, sug- gesting that they might have a deficiency in mtDNA. One colony (strain PHP4) was picked for further 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 b o n a f i d e rho+ S. pombe. Like the parental strain FYC 1 1, PHP4 required adenine for growth on minimal medium and mated with the

ural strain FYCS to yield prototrophic haploid recom-

binant progeny.

To determine whether strain PHP4 contained

mtDNA, total cellular DNA was analyzed by gel-blot hybridizations (Figure 1). T h e plasmid pDG3, which contains the entire mtDNA of S. pombe in the vector pBR322 (DEL GIUDICE 1981), was used to probe for mtDNA. T h e expected restriction pattern of S. pombe

mtDNA was found for FYC 1 1, whereas no sequences hybridizing to S. pombe mtDNA could be detected in DNA isolated from PHP4 (Figure 1, A and B). Con- trol hybridizations to detect single copy nuclear DNA sequences were carried out using as probes two ran- domly chosen clones (pH1 and pH4) from a Hind111 partial genomic library of S. pombe DNA (MOLZ et 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;

MATE- RIALS AND METHODS). T h e rho' strain PHP14 (Table 1; see below) and its rho+ parent (PHP3) were com- pared. Nuclei were clearly stained in both types of cells. However, staining of mtDNA, observed as spec- kles 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" deriva- tives from several different S. pombe strains, but only some treated cultures in a given experiment yielded

rho' mutants. For example, of eight separate cultures

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FIGURE I .-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 from strain PttP4 (rho'). F.cpal

amounts of D S A were s u h j e r t e d to gel electrophoresis (MATERIAIS

A N n SIFTHOM) after digestion with restriction cn7vn~es a 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 were prolml with ["P]-laIwled D S A p r o t m

( M A T E R I A I S A N D SIFTIWM) o f rOllf$llV equal specific activitv as

follows. T h e plasmid pIX;J. carwing the entire S. pombr mitwhon-

tlrial chronrowme. was used as prohe in pmels h and R. .I'he

nurlmr probrc were 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 ~ ~ a l length of

time. whereas panel R represents a shorter e x p u r c * o f the wme

filter as in panel A .

described above. only two produced nonrespiring strains. One such strain (PHP14) obtained from treat- ment of PHP3 was characterized fullv as described above for P M P ~ and confirmed to

t

w

i

S. pomhr rho" (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 acetate. Surprisingly however. once rho" s11-1' e 111s werc

obtained their growth rate was the same on b o t h

standard glucow containing medium (YES) and YES

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 l l ~ l I k O I I ' I I I ' I I I 1 I . I l . . of S. pomhr. ' 1 ' 1 1 ~ . ~ I C X C Y ~ I I I C . I \ c l r u I i l n ~ l 111 \ I ~ I I R I A I S A Y I ) \ ~ b I I I -

OM.

plus 2% potassitm itcctatc. Thus t l w prcscncv o f

potassium ;tcct;ttc clrws not a p p c t r IO IW 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 w on 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 floc- culent pllcnotypc when grown i n liquid nlcdia.

The

S.

pornhe rho" strains carry nuclear mutations allowing their growth: ~ I I w lidlowing Iyx)thc*sis could account for the. irrcl)r'Hlrrcihility o f rho'' illdue- tion from cultrrrc t o ctrlturc*: c - t l l i t l i t r n l Iwomidc causcd a reduction i n m t D S A copv nunrlwr t o the p o i n t t h t

cell growth ceased (\VOI.F and I ~ I . C.r*rnrcx I!1XO: U'0I.F rt a/. 19i6). I ~ r t during our v c ~ y long culture periods nuclear mutations wcr<9 sclectcd t h a t ;tllo\r.c-tl

the cells to grow w i t h o t r t ttlt 1)S:I. Tlw (x-ctlrrcnce o f such nuclc;w rnt1t;ttions i n some b u t n o t a 1 1 ~ I I ~ I I I ~ C S

(cf: 1,URIA and DF.1.RRi:C.K I <)43) !r'orlkl ;lccoIIIlI for the irreprcHlucihility.

T o test this lryx)tlwsis we crossed the rho" sttxin PHPI.1 to a rho' strain (FYC9) and s~mrr~l;ttcd t h e

rcsulting diploid. I'ctr;tds ~ w r c tlissc*ctccl ; I d ;I1l;ll\.Pctl for segr(*gation of four ntlclcar n1;trkcrs (nr;tting type.

ural. adrh. lru /), a l l of which segrcg;ttcd 2:2. [\e t l l c ~ n

picked a 1 1 four spores from sis o f ' IIIC. t ( ~ r ; t d ~ and treated tlletn in dtrplicatc liquid cultures w i t h cthid-

i u m bromide ( " A m R I A I s ANI) UKI'HODS). A s prctlictcd

b y

the hypothesis, t w o o f the spores from c;~ch tetrad readily vicldcd rho" cc*IIs i n 1x)th <ltlpliciItc CII~III~CS

after f c w r t h e n 8 d;tys. Sone of the otlrcr two sp)rcs

in e;tch tetrad yielded rho"cclls rqwm111cibly although, i\s exp~ctcd. three gave rise to rho" cells i n ow of t I w duplicate cultures after incuhations exceeding X d a y s . Thtts, the ithility to form rho" cells rcwlily scgrcgatcvl

2:2 in this cross. \\'e interpret this to me;\n t h ; t t thc

originally derived rho" strain. PI-IPII. c;trricd a 1111-

clear mutation ternwd p t p l - 1 (for petite-1msitivc) t h a t

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258 P. Haffter and T. D. Fox

was selected during the initial ethidium bromide treat- ment of its parent, PHPS.

T o determine whether the rho" strain PHP4, which was isolated independently of PHP14 (see above), carried a mutation in p t p l or another nuclear gene, we asked whether the presumed nuclear mutation in PHP4 was linked to p t p 2 . A ptp2-2, [rho+] strain (PHP25) was crossed to PHP4 and the resulting te- trads were dissected. The nuclear markers checked (mating type, u r d , the distinguishable alleles ade6-

M 2 2 0 and a d e 6 4 4 2 2 6 ) segregated 2:2. All four spores

from

20

tetrads were then subjected to ethidium bromide treatment for 8 days. If both strains carried

p t p l mutations we would have expected to see all four

spores in each tetrad readily give rise to rho" cells. However, if the independently isolated rho" strain carried a different nuclear mutation in an unlinked gene independent segregation would produce paren- tal ditypes (all four spores yield rho" cells), nonparental ditypes (two spores yield rho" cells and two spores do not) and tetratypes (three spores yield rho' cells and one spore does not) in a 1: 1:4 ratio. Among the

20

tetrads examined, one was a parental ditype, two were nonparental ditypes and 17 were tetratypes, fitting reasonably well to the expectation for two unlinked genes. Thus we conclude that the rho' strain PHP4 probably contains a nuclear mutation, ptp2-1, un- linked to p t p l - 2 . However, we have not verified that

ptp2-2 segregates

2:2

in a cross to wild type.

DISCUSSION

This paper describes the first isolation of S. pombe

strains totally lacking mtDNA. By extended culture of cells in complete glucose medium supplemented with ethidium bromide and potassium acetate we ob- tained viable nonrespiring strains. These rho' strains lacked detectable mtDNA as judged both by sensitive hybridization analysis and DAPI staining of whole cells.

Previous studies had shown that treatment of

S.

pombe with ethidium bromide caused a reduction in

the copy number of mtDNA and cessation of growth (WOLF and DEL GUIDICE 1980; WOLF et al. 1976). We also observed cessation of growth in cultures contain- ing ethidium bromide. However, upon extended in- cubation (approximately 15 days) some, but not all, of our cultures yielded actively growing rho" strains. Our genetic analysis indicated that these rho' strains arose as a consequence of both the depletion of mtDNA by ethidium bromide and nuclear mutations that allowed the growth of

S.

pombe cells lacking mtDNA. The existence of one such nuclear mutation, termed p t p l -

2 , was demonstrated by crossing a rho" strain to a standard laboratory rho+ strain. This cross yielded tetrads which each contained two spores that could be readily converted to rho" by ethidium bromide treat-

ment and two spores that could not. A cross between

a p t p l - 2 carrying strain and an independently isolated

rho" indicated that at least two unlinked genes could mutate to allow growth of cells lacking mtDNA.

The rho' strains studied here grew slowly on glucose containing medium and not at all on medium contain- ing ethanol and glycerol as carbon sources, as ex- pected. In addition they exhibited a flocculent (clumpy) phenotype in liquid media. Matings between rho' and rho+ strains were less efficient than standard crosses, probably due to the slow growth and clumpi- ness of the rho' cells. However, the rho" S. pombe strains had no other detectable phenotypes. In this regard they resemble

S.

cerevisiae rho' strains and differ from

rho' animal cell lines, which exhibit growth require-

ments for uridine (DESJARDINS, FROST and MORAIS

1985) and, also, in the case of human cells, pyruvate

(KING and ATTARDI 1989). Although potassium ace- tate appeared to promote the induction of viable rho"

S.

pombe cells from standard laboratory strains under

the conditions used here, its role in the process is unclear. Once established, the rho' strains grew equally well in the presence or absence of potassium acetate.

rho+ strains of S. pombe carrying either the ptp2-1

or ptP2-2 mutations had no detectable phenotypes

other than their ability to undergo loss of mtDNA reproducibly. Unfortunately, we were only able to score this phenotype by long term incubation in liquid medium containing ethidium bromide, making ge- netic analysis cumbersome. One important question that remains unanswered is whether the p t p mutations are dominant (likely due to gain of function) or reces- sive (likely due to loss of function). Owing to the instability of

S.

pombe diploids, we were unable to maintain haploid-free liquid cultures long enough to test dominance.

An important conclusion from this study is that the petite-negative property of S. pombe is not due to the presence of a unique mitochondrial gene whose expression is centrally important to the viability of the cell. Single nuclear mutations (either spontaneous or induced by ethidium bromide) can overcome the ina- bility of wild-type S. pombe to grow without mtDNA, without causing any other detectable phenotype. It is interesting to compare this situation with an approxi- mate converse in

S.

cerewisiae. In this well studied petite-positive yeast, a single nuclear mutation in a gene termed op2 (or p e t s ) prevents the growth of rho-

and rho' cells, in effect converting

s.

cerewisiae into a

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teins (KOLAROV, KOLAROVA and NELSON 1990). In view of the fact that incubation of both wild-type S.

pombe and opI mutant S. cerevisiue in medium contain-

ing ethidium bromide leads to cessation of growth after a few generations, it is tempting to speculate that

the p t p mutations described here might affect the

activity of ATP/ADP translocators.

A strain of the petite-negative budding yeast, Kluy-

veromyces lactis, that tolerates rho- and rho" mutations

has been derived by fusion of K . lactis and S. cerevisiae

protoplasts (GALEOTTI and CLARK-WALKER 1983;

HARDY, GALEOTTI and CLARK-WALKER 1989; MA- LESZKA and CLARK-WALKER 1989). Karyotypic analy- sis of this fusant by gel electrophoresis indicated that it had a substantially intact

K.

lactis genome, although a large deletion of rDNA genes was detected (MA-

LESZKA and CLARK-WALKER 1989). It remains unclear why this K . lactis strain tolerates rho' mutations: it may have acquired

S.

cerevisiae genes during protoplast fusion that allow it to do so, or it might have a mutation in a K . lactis gene analogous to the p t p

mutations described here.

S. cerevisiae strains lacking mtDNA have proven to

be very useful for genetic analysis of mitochondrial functions (for reviews see COSTANZO and

Fox

1990; DUJON 198 1). In particular, rho" strains of S. cerevisiae

have provided excellent recipients for mitochondrial transformation

(Fox

et al. 199 1 ;

FOX,

SANFORD and MCMULLIN 1988) allowing the introduction of new or altered genetic material into the mitochondrial ge- nome (FOLLEY and

Fox

199 l ; THORSNESS and

Fox

1990). Although we do not yet understand the nature of the nuclear mutations described here that allow growth of S . pombe cells lacking mtDNA, the availa- bility of such rho" strains may prove useful for the analysis of mitochondrial functions in that species as well.

We thank D. BEACH for gifts of strains and the genomic library of S. pombe, and L. DEL GIUDICE for the gift of plasmid pDG3. We also thank R. BUTOW for suggesting acetate, although we still do not understand its role. P.H. was a recipient of a fellowship from the Swiss National Science Foundation. This work was supported by a grant (GM29362) from the U.S. National Institutes of Health.

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Figure

TABLE 1 YES medium  containing  12.5 mide and 2% potassium acetate  for 17 days
FIGURE I pbmbr. tlrial chronrowme. amounts (lanes (MATERIAIS undiptcci (lanes nurlmr  probrc  were lanm ANn time

References

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