TWENTY-SIX CHROMOSOMAL GENES NEEDED T O MAINTAIN THE KILLER DOUBLE-STRANDED RNA PLASMID OF SACCHAROMYCES CEREVISIAE REED B. WICKNER

Laboratory of Biochemical Pharmacology, National institute of Arthritis, Metabolism, and Digestive Diseases, National Institutes of Health, Bethesda, Maryland 20014 Manuscript received September 9, 1977 ABSTRACT

The double-stranded RNA killer plasmid gives yeast strains carrying it both the ability to secrete R protein toxin and immunity to that toxin. This report describes a new series of mutants in chromsomal genes needed for killer plasmid maintenance (mak genes). These mutants comprise 12 complementation groups. There are a total of a t least 26 mak genes. Each mak gene product is needed for plasmid maintenance in diploids as well as in haploids. None of these mak mutations prevent the killer plasmid from entering the mak- spores in the process of meiotic sporulation. Complementation between mak mutants can be performed by mating meiotic spores from a m a k x / f plasmid-carrying diploid with a m a k y haploid. If x = y , about half the diploid clones formed lose the killer plasmid. If z # y , complementation occurs, and all of the diploid clones are killers.

HE killer plasmid (virus) of Saccharomyces cereuisiae is a 1.4 X 1O6 molecTular-weight species of double-stranded (ds) RNA encapsulated in a virus-like particle (but not known to be infectious) that confers on its host the ability to secrete a protein toxin and immunity to this toxin (WOODS and BEVAN1968; SOMERS and BEVAN1969; BUSSEY1972; BEVAN,HERRING and MITCHELL1973; KATTERMAN and FINK 1974; HERRING and BEVAN1974). Ten chromoVODKIN, somal genes have been shown to be essential for the maintenance or replication of the killer plasmid. These are pets (FINKand STYLES 1972; WICKNERand LEIBOWITZ 1976; LEIBOWITZ and WICKNER, in preparation) , maklO (SOMERS and BEVAN1969; WICKNER1977), and makl through mak8 (WICKNER 1974; WICKXER and LEIBOWITZ 1976). This paper presents a new series of mak mutants and shows, using complementation tests, that there are at least 26 mak genes. To our knowledge, this is the largest number of host genes shown to be needed for replication of any virus or plasmid in any system. MATERIALS A N D METHODS

Notation: Phenotypes: Kf or K- means ability or inability to secrete an active toxin. R+ or R- refers to resistance or sensitivity to the killer toxin. Chromosomal genes needed to MAintain the Killer plasmid are called mak genes. One exception is called pets (for petite sensitive) Genetics 8 8 : 419-425 March, 1978.

420

R. B. WICKNER

because it is needed to maintain both the killer plasmid and mitochondrial DNA (FINK and STYLE^ 1972; LEIBOWITZ and WICRNER,unpublished). Mutants of the mak type are denoted with isolation numbers, e.g., K429, until they have been mapped or placed in a complementation group. Then, they are given mak gene and allele numbers, e.g., makl8-2. The wild-type killer plasmid is denoted [KIL-k]. The absence of the killer plasmid is denoted [KIL-o]. Strains: Yeast strains used are listed in Table 1. Strain A364A was mutagenized with ethylmethane sulfonate (WICKNER1974), and K-R- clones were screened for mak mutants as described (WICKNERand LEIBOWITZ 1976). Media: Sporulation medium contained, per liter, 10 g potassium acetate, 0.5 g dextrose, 1 g TABLE 1 Strains of Saccharomyces cerevisiae

A364A

K+R+

a adel a d d gall u r d his7 lys2 tyrl

AN33

K- R-

a argl thrl [KIL-01

5x47

K- R-

aha hisl/+ trpl/+

299

K- R-

K62

a leu2 met5 mak3-I [KIL-o]

WICKNER and

490

K- R-

J1

Q

leu2 met5 mak7-I [KIL-o]

WICKNER and

725 843

K- RK- R-

K403 MK35

a lysZ tyrl mak7-2 [KIL-o] a adel thrl " 2 - 1 [KIL-o]

1409-1B

K- R-

a ade2 maklO-I [KIL-o] [rho+]

1409-7D

K- R-

a ade2 maklO-l [KIL-o] [rho+]

1541-17B

K+R+

a lysl ura3 canrl makl0-1

1541-17C

K+R+

560 1532-15B 1433-13B 943 1091 701 721 714 719 722 716 552 558 531 530

KKKKKKKKKKKKKKK-

[KIL-k]

WICKNER and LEIBOWITZ 1976

ura3/+ [KIL-o]

WICKNER and LEIBOWITZ 1976 LEIBOWITZ 1976

RRRRRRRRRRR-

RRRR-

K262 K339 K425 K293 K343 K610 K431 K429 K407 K397 K379 K352 K288 K28 1 K269

[KIL-k] [rho"] a ilu3 canrl makl0-I [KIL-k] [rho"] a leu2 makll-I [KILO] a his6 u r d makl2-I [KIL-01 a lys2 his7 makl3-I [KIL-o] a leu2 makl5-I [KIL-a] a adel ural makl6-1 [KIL-o] a lysl makl7-I [KIL-o] a ural makl8-I [KIL-o] a t y r l leu2 makl8-2 [KIL-o] a his7 leu2 makl9-I [KIL-01 LY adel mak20-I [KIL-o] a a d d mak21-I [KIL-o] a adel lys2 mak22-1 [KIL-o] a leu2 mak23-I [KIL-o] a ade2 mak24-I [KIL-o] a a h 2 mak25-I [KIL-o]

LEIBOWITZ 1976 This work WICKNERand LEIBOWITZ 1976 SOMERSand BEVAN1969 SOMERS and BEVAN1969 WICKNER 1977

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KILLER

dsRNA PLASMID

421

IN SACCHAROMYCES

yeast extract, and 20 g agar. Minimal medium (SD) contained, per liter, 6.7 g Difco yeast nitrogen base without amino acids, 2 0 g dextrose, and 2 0 g agar. Rich medium (YPAD, p H approximately 6.0) contained, per liter, 10 g yeast extract, 0.4 g adenine sulfate, 20 g peptone, 20 g dextrose, and 20 g agar. 4.7 MB medium is YPAD buffered at p H 4.7 with 0.1 M sodium citrate and contains 0.003% methylene blue. Complementation iests: To perform a complementation test between two mat mutants, makx and maky, a K + R + diploid heterozygous for makx and homozygous for some auxotrophic marker is placed on sporulation medium for 3 days. The sporulated culture is scraped off the plate and digested for 20 minutes a t room temperature with a 1 : 25 dilution of Glusulase (Endo Laboratories) to free the spores from the asci. -4n aliquot of this digested culture is plated directly on minimal medium to check for prototrophs. Other aliquots are mated with three different haploid strains having auxotrophic markers complementary to that of the diploid: (1) a wild-type nonkiller to determine whether all spores carry the killer plasmid; (2) a mzkz strain to determine whether diploids require makx for maintenance of the killer plasmid; and (3) a maky strain to determine whether makx and maky are defective in the same gene. In each case, an excess of the haploid strain grown on YPAD plates is added to 0.2 ml of the digested spore suspension, vortexed, and poured onto a YPAD plate. After 6-16 hours of incubation a t 26", these mating mixtures are scraped off the YPAD plates, and appropriate dilutions are plated on SD plates at 30". Diploid clones are replicated to 4.7 MB plates previously spread with a lawn of the nonkiller toxin-sensitive strain 5x47. Killer phenotype is determined after two days of incubation of these plates at 20". The killer clones prevent growth of the lawn of 5x47 surrounding them. RESULTS

A new series of mak mutants was obtained and distinguished from other types of K-R- mutants as previously outlined (WICKNER and LEIBOWITZ 1976). The mak mutants discussed here yielded only K-R- diploids in crosses with [ K I L O ] strains and K+R+ diploids in crosses with +[KlL-k] strains. The latter diploids showed 2 K+R+ : 2 K-R- segregation in each case (data not shown). To prform complementation tests between these mak mutants, it was necessary to prepare diploid cells carrying both mak mutations and, at least transiently, the killer plasmid. Since mak- haploid strains ordinarily lack the killer plasmid, this cannot be done by simply mating the mak mutants with each other. This can be done, however, in the case of makl0-1, utilizing the fact (WICKNER. 1977) that, in the absence of the mitochondrial genome, the maklO gene is not needed for killer plasmid maintenance. Thus, a makl0-I [rho"] [KILk] strain was mated with various mak- [rho+] [ K I L O ] strains, and diploid clones were examined for their killer phenotype. As previously shown, a maklO-l/maklO-l [rho+] diploid cannot maintain the killer plasmid and becomes [ K I L O ] (WICKNER 1977; Table 2). The results in Table 2 indicate that all the mak mutants tested complemented makl0-1. This method works only for makZO since, of the mak mutants tested, only makl0-l is bypassed by the absence of mitochondrial DNA. A more general method for doing complementation tests was suggested by the and BEVAN(1969) that makl0-l spores from a K+R+ makZ0finding of SOMERS lJ+diploid carry the killer plasmid. Mak- spores carry the killer plasmid: Germinating spores from mak-/-t [KILk] diploids were mated en masse with a wild-type [ K I L O ] strain, and the

+

422

R. B. WICKNER TABLE 2 Complementation tests with makl0-1 mak- [rho+] [KIL-01 mutant tested

mak10-I

K339 K425 K343 K61O K431 K429 K407 K403 K397 K379 K352 K288 K281 K269

makf0-I [rho"] [KIL-I;] tester 1541-17C

1541-17B

Diploid pheno'types* all K- Rall K- Rmost K+R+ all K+R+ rare K- Rall K+R+ all K+R+ all K+R+ all K+R+ all K+R+ all K+R+ all K+R+ all K+R+ all K+R+ all K+R+ all K+R+ all K+R+ all K+R+ all K+R+ all K+R+ all K+R+ all K+R+ all K+R+ all K+R+

I

* Strains were mated on rich medium, and diploids were selected en masse on minimal medium. Diploids were then streaked for single colonies on minimal medium, and their killer and resistance phenotypes determined (20-40 diploid single colonies for each cross).

killer phenotype of the diploid clones was determined (see MATERIALS AND I n each case (Table 3, first column of data), greater than 97% of the diploids formed were killers. These results indicate that, for all mak mutants tested, almost all the spores carried the killer plasmid. Selection against 1C-Rdiploids was avoided by(1) using a vast excess of K-R- haploids over K+R+ spores in the mating mixture; (2) performing the matings at pH 6 where the toxin is inactive; and ( 3 ) growing the diploid clones at 30" where the toxin is essentially inactive. That these measures were effective is indicated by the outcome of the crosses described in the next section. Diploids require the mak genes for killer plasmid maintenance: When the same germinating spores from a m a k x l f [ K I L k ] diploid were mated with a makx [ K I L O ] haploid strain, 14% to 57% of the diploids (usually 30% to 50%) were K-. This indicates that K- diploids formed in such matings are not strongly selected against and that makx/makx diploids cannot maintain the killer plasmid. Since several doublings occur between the time of spore-to-cell matings and selection f o r diploids on minimal medium, it is possible that the occasional low frequency (14% to 18%) of K- diploids found in some of these matings may be due to slower growth of makx/makx homozygous diploids than of makx/+ diploids. This effect was observed in the case of mak20-2 and mak21-1 and was minimized by plating for diploids after only 6-8 hours of spore-to-cell mating. Complementation tests: The results above suggest that if the makx [KIL-k] METHODS).

dsRNA PLASMID

KILLER

423

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