The [KIL-d] Cytoplasmic Genetic Element of Yeast Results in Epigenetic Regulation of Viral M Double-Stranded RNA Gene Expression

Corresponding author: Michael J. Leibowitz, UMDNJ-Robert Wood Johnson Medical School, 675 Hoes Ln., Rm. 705, Piscataway, NJ 08854-5635., leibowit{at}umdnj.edu (E-mail).

Communicating editor: M. CARLSON

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

[KIL-d] is a cytoplasmically inherited genetic trait that causes killer virus-infected cells of Saccharomyces cerevisiae to express the normal killer phenotypes in a/ cells, but to show variegated defective killer phenotypes in a or type cells. Mating of [KIL-d] haploids results in "healing" of their phenotypic defects, while meiosis of the resulting diploids results in "resetting" of the variegated, but mitotically stable, defects. We show that [KIL-d] does not reside on the double-stranded RNA genome of killer virus. Thus, the [KIL-d] effect on viral gene expression is epigenetic in nature. Resetting requires nuclear events of meiosis, since [KIL-d] can be cytoplasmically transmitted during cytoduction without causing defects in killer virus expression. Subsequently, mating of these cytoductants followed by meiosis generates spore clones expressing variegated defective phenotypes. Cytoduction of wild-type cytoplasm into a phenotypically defective [KIL-d] haploid fails to heal, nor does simultaneous or sequential expression of both MAT alleles cause healing. Thus, healing is not triggered by the appearance of heterozygosity at the MAT locus, but rather requires the nuclear fusion events which occur during mating. Therefore, [KIL-d] appears to interact with the nucleus in order to exert its effects on gene expression by the killer virus RNA genome.

EPIGENETIC phenomena, defined as the alteration of gene expression without change (mutation) in the nucleotide sequence of the gene, occur in many genes in diverse organisms. Previously described examples (briefly reviewed by HENIKOFF and MATZKE 1997 ) of epigenetic phenomena involve alteration in expression of chromosomal genes in various organisms, generally mediated by the "heritable, but potentially reversible, changes in chromatin structure and/or DNA methylation" (HENIKOFF and MATZKE 1997 ). In this article, we describe an example of epigenetic regulation of phenotypic expression of a gene on the double-stranded (ds) RNA genome of the M satellite virus of killer virus of Saccharomyces cerevisiae. This regulation appears to involve the nucleus of the host cell and is the first example that we are aware of in which an epigenetic phenomenon alters phenotypic expression of a viral RNA genome.

Killer virus of yeast is a cytoplasmically inherited dsRNA virus of yeast (reviewed by WICKNER 1996 ). Cells harboring the virus-encapsidated M dsRNA segment secrete a protein toxin, which is lethal to yeast cells not harboring the virus, and are resistant to the toxin of the type secreted by the infected cells. Thus, wild-type killer virus-infected cells have the phenotypes of K⁺ (killing) and R⁺ (resistance), while cells lacking M dsRNA are K^-R^- in phenotype. Both toxin and resistance substances are produced by processing of the single preprotoxin polypeptide produced by translation of viral transcripts of M dsRNA. There are distinct M dsRNA species encoding different specificities of killer toxin and resistance; these include the type 1 and type 2 killer phenotypes, encoded by M₁ and M₂ dsRNA species, respectively. The different M dsRNA species show biased incompatibility (termed "exclusion") with each other, so that if both are introduced into the same cell by mating, one of the two (generally M₁ dsRNA) will out-compete the other.

All killer virus-infected cells as well as nearly all nonkillers harbor the replication-competent L-A dsRNA viral genome, which functions as a helper virus for satellites, such as M dsRNA. L-A dsRNA encodes two gene products: the capsid protein of the virions in which both M and L-A dsRNA are separately encapsidated and the virus-associated capsid-RNA polymerase fusion protein, which is produced by a -1 translational frameshift mechanism resembling that used by many retroviruses (ICHO and WICKNER 1989 ; DINMAN et al. 1991 ; DINMAN 1995 ). The capsid and capsid-RNA polymerase proteins constitute the helper function that L-A dsRNA provides for M dsRNA satellite viral genomes. A variety of naturally occurring alleles of L-A dsRNA are known that differ in their helper phenotypes (briefly reviewed by HANNIG et al. 1985 ), although the molecular basis of these differences has not been determined. These alleles include L-A-H, L-A-HN, and others. Wild-type killer cells of type 1 specificity tend to have L-A-HN dsRNA as helper for the M₁ dsRNA satellite, while in type 2 killers M₂ dsRNA tends to occur with L-A-H as helper. Despite this empirical association, both alleles of L-A are capable of providing helper function for either M dsRNA type (HANNIG et al. 1985 ). Replication of either type of M dsRNA is temperature sensitive in the presence of L-A-HN, as compared to the temperature resistance seen in the presence of L-A-H (WEINSTEIN et al. 1993 ).

The ease of scoring for the killer phenotypes of infected cells has allowed extensive genetic studies of viral and host mutants which alter the replication, expression, or regulation of the virus (WICKNER 1996 ). Among these, perhaps the most puzzling are the diploid-dependent mutants, which harbor the [KIL-d] cytoplasmically inherited genetic element (WICKNER 1976 ). These mutants were isolated from haploid strain A364A, which is infected with wild-type (K₁⁺R₁⁺) type 1 killer virus. Phenotypically, these mutants display defective killer properties (K^-R⁺, K⁺R^-, or K^-R^- or intermediate "weak" phenotypes denoted by the w superscript), with each individual mutant phenotype being relatively stable on mitotic growth; there is an increased tendency to lose the M dsRNA segment during vegetative growth, thus generating stable K^-R^- segregants. Crossing these mutants to strains lacking M dsRNA (with or without L-A dsRNA) results in diploids that are mostly K⁺R⁺ in phenotype (or K^-R^- if the mutant parent had lost M dsRNA) and mitotically stable. The same result is obtained by crossing two [KIL-d] strains. Thus, the mating process, cell type, or ploidy of the host cell appears to affect the penetrance of the mutant phenotype. However, sporulation (meiosis) of these K⁺R⁺ diploids generates haploid spore clones which again display defects. Each clone displays a different defective phenotype, i.e., K^-R⁺, K⁺R^-, K^-R^-, K^wR⁺, K⁺R^w, etc., or even K⁺R⁺, and shows increased mitotic instability of M dsRNA. These spore clones show similar behavior on repeated backcrosses with wild-type nonkiller haploids; thus the defective phenotypes are variegated. Each defective meiotic clone is phenotypically stable, and the ratio of different phenotypes among meiotic clones tends to be quite variable and non-Mendelian in pattern of inheritance. This unusual cytoplasmically inherited diploid-dependent trait has been denoted [KIL-d]. In the original study, crosses of a [KIL-d] strain with two wild-type killer strains indicated that the mutation appeared to be recessive, since the resulting diploids and their meiotic progeny were all K⁺R⁺ in phenotype (WICKNER 1976 ). However, in other crosses described in this article we observe apparent dominance of [KIL-d]. It should be pointed out that all diploid-dependent mutants isolated to date have been obtained from mutagenized parental strain A364A (WICKNER 1976 ), which harbors the L-A-HN allele of L-A dsRNA; no other isolates of [KIL-d] have been reported. When haploid strain A364A is mutagenized, most mutants with altered killer phenotype are classical mutations in viral dsRNA or in chromosomal genes required for the replication, expression, or regulation of the cytoplasmic virions. [KIL-d] mutations represent a minority of the mutations obtained, although multiple [KIL-d] strains have been isolated (WICKNER 1976 ).

[KIL-d] confers defects on killer virus expression. These defects are not simply the result of mutations in multiple chromosomal loci, which might yield a complex pattern of segregation mimicking cytoplasmic inheritance. This conclusion is proven by the "selfing cross," in which two haploid segregants that share the same defective phenotype and are derived from a single [KIL-d] diploid are mated to each other. The selfing cross results in K⁺R⁺ dipolids that upon meiosis yield all variants of defective hapolid progeny, demonstrating that the different phenotypes of the meiotic segregants are not simply a reflection of the segregation of multiple chromosomal mutations (WICKNER 1976 ).

Although [KIL-d] was originally isolated in a search for mutants of killer virus of yeast, we show here that [KIL-d] does not map to the M or L-A dsRNA segments of killer virus. We report that [KIL-d] displays several genetic properties shared with a number of other epigenetic phenomena:

1. Regulation related to the sexual cycle, in this case with phenotypic "healing" upon mating and "resetting" of defective phenotypes upon meiosis;

2. variegation of phenotypic defects established upon meiosis, with each phenotype being mitotically stable;

3. lack of evidence of any mutation in the preprotoxin-bearing M dsRNA segment, despite the defective expression of the phenotypes encoded by this viral segment.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Media and growth conditions:
Rich (YPD), defined (SD, SC), sporulation and glycerol (YPG) media were prepared as described by KAISER et al. 1994 . For ade1 and ade2 mutant strains, adenine sulfate was added routinely at a final concentration of 0.04%. Drop-out (H) media were prepared as by WICKNER and LEIBOWITZ 1979 . For cytoduction, special drop-out medium with 3% glycerol as a carbon source was used to select for the cytoductants. For galactose-induced expression of the pGAL-HO plasmid, H-Ura drop-out medium was made with 2% galactose instead of glucose. To isolate viral dsRNA, cells were grown in rich medium containing 3% ethanol as a carbon source (WELSH et al. 1980 ; THIELE et al. 1984 ). Killer phenotype assays were carried out at 20° on 4.7 MB (rich medium buffered with sodium citrate to pH 4.7, containing 0.003% methylene blue) plates (LEIBOWITZ and WICKNER 1978 ). Canavanine was added to the H-Arg drop-out plates and supplemented minimal plates at a final concentration of 60 µg/ml for marker testing and cytoductant selection. Defined (H) medium containing 5-fluororotic acid (5-FOA) at 0.1% concentration was used to select for Ura^- colonies and to select for loss of plasmids with the URA3 marker. [KIL-0] cells were grown at 30°, while killer cells ([KIL-k]) were grown at 26° on plates for all genetic experiments, unless otherwise stated. All growth in liquid cultures was at 28°.

Strains, plasmids, and primers:
Strains used in this study are listed in Table 1. All [KIL-d] strains in this study are the derivatives of the original strain K30 isolated by WICKNER 1974A , WICKNER 1976 . M985 and M984 are [KIL-0] diploid strains routinely used as a sensitive lawn for testing toxin production. M21 and M1052 (1384 x 1387) are diploid strains used as tester K₁ and K₂ killers. Plasmids pJM3 and pJM9 are YCp50 plasmids carrying the MATa and MAT mating type loci, respectively (J. MARGOLSKEE, unpublished results; MITCHELL and HERSKOWITZ 1986 ). Plasmid pGAL-HO is a YCp50 plasmid harboring the HO endonuclease gene driven by the inducible GAL10 promoter (HERSKOWITZ and JENSEN 1991 ). Primers used were 8246 (5'-GAA AAA TTT TTA AAT TCA TAT AAC TCC CC-3'; ICHO and WICKNER 1989 ) and 10285 (5'-CAA CAA TGT TAT AGC CAG CGG-3').

Genetic methods:
Standard methods for tetrad dissection (KAISER et al. 1994 ) and cytoduction (CONDE and FINK 1976 ; ROSE 1996 ) were used. In the M89 M1004 cytoduction, isolation of cytoductants with the nucleus derived from M1004 was achieved by primary selection on minimal medium supplemented with leucine and canavanine and followed by selecting for growth on YPG plates. Diploids were isolated from the same cross by selection on minimal medium. Cytoduction in the opposite direction (M480 M89) was similarly performed, selecting cytoductants on H-leucine glycerol medium and confirming their Ade^- Thr^- Mat phenotype.

Elimination of M or both M and L-A dsRNAs at elevated temperature was achieved by culturing in rich liquid medium at 38°, 250 rpm, for 3–5 days (WICKNER 1974B ; WEINSTEIN et al. 1993 ). Loss of M dsRNA in strains with the R⁺ phenotype was monitored by loss of resistance to toxin and confirmed by dsRNA analysis. Loss of mitochondrial function after growth at elevated temperature was assayed by monitoring growth on YPG plates. Yeast transformation was carried out by electroporation with a Gene Pulser II (Bio-Rad, Hercules, CA) using the standard protocol (1.5 kV, 200 ohms, 25 µF, 2 mm gap cuvettes). Ethidium bromide (30 µg/ml) curing of mitochondrial DNA (GOLDRING et al. 1970 ) was performed by growth in rich liquid medium in the dark. Petite isolates were crossed with appropriate wild-type [RHO⁺] strains (M422 or M423) to determine suppressivity by [RHO^-] mitochondrial DNA of the wild-type [RHO⁺] mitochondrial genome (MICHAELIS et al. 1971 ). Only the isolates failing to show suppressivity were used as putative [RHO-0] strains for this study. Mating type tests and assays for the kar1-1 mutant phenotype (scored by papillated growth after mating on selective media) were carried out with strains M422, M423, M518, or M519 (CONDE and FINK 1976 ). Mating type conversion of pGAL-HO transformed haploid cells (HERSKOWITZ and JENSEN 1991 ) was induced for 2, 4, and 6 hr in H-Ura drop-out liquid medium containing 2% galactose as carbon source. Cells were plated at each time point onto H medium plates containing 5-FOA (0.1%) to halt induction and to select for loss of plasmid.

Killer phenotype assays:
Toxin production was assayed on a lawn of M985 or M984 sensitive diploid cells on 4.7 MB plates at 20° as described by WICKNER and LEIBOWITZ 1979 . Killer (K⁺), nonkiller (K^-), and weak killer (K^w) colonies were distinguished by the size of the halo of growth inhibition of the lawn. Killing type specificity was assayed using lawns of diploid type 1 or type 2 cells, as used for specific resistance type testing. Resistance tests were performed with both K₁ and K₂ diploid killer strains, M21 and M1052, respectively, as described by LEIBOWITZ and WICKNER 1978 . Resistance phenotypes were recorded as resistant (R⁺), nonresistant (R^-), and weakly resistant (R^w). Tests for both types specificity of killing and resistance were carried out when needed to determine the presence and/or the type of M. For killer phenotype tests of strains harboring plasmids pJM3, pJM9, or pGAL-HO, H-Ura plates buffered to pH 4.7 with sodium citrate and supplemented with methylene blue dye (0.003%) were used.

Viral dsRNA isolation and analysis:
Viral dsRNA was isolated by phenol extraction of whole cells (FRIED and FINK 1978 ; THIELE et al. 1984 ). Preparations of dsRNA were assayed and individual species were isolated by agarose gel electrophoresis, which separated M dsRNA from the L dsRNA species, including the L-A allelic forms and the L-BC nonkiller virus-related dsRNA species that comigrate with L-A dsRNAs (SOMMER and WICKNER 1982 ). The M₁ and M₂ dsRNA species were distinguishable by their different electrophoretic mobilities. RNA was extracted from agarose gels using the QIAquick Gel Extraction Kit (QIAGEN, Valencia, CA).

Strand separation gel electrophoresis of agarose-purified L-A dsRNA (HANNIG et al. 1985 ) was used to determine the allelic form of L-A dsRNA. The strand separation gel was stained with ethidium bromide and L-A type determined based on mobility compared with L-A-HN and L-A-H standards.

PCR and RT-PCR methods:
Single tube RT-PCR followed by a secondary PCR amplification was performed to detect the presence of L-A dsRNA. L-A specific primers 8246 and 10285 were used in both the RT-PCR and the secondary PCR reactions. Primers were designed based on published L-A-HNB (ICHO and WICKNER 1989 ) and sequence data of different L-A isolates obtained in our laboratory (Z. TALLÓCZY, R. FELDER and M. J. LEIBOWITZ, unpublished results). The single tube RT-PCR and PCR were performed in reactions containing: 1.5 mM magnesium acetate, 86 mM potassium acetate, 25 mM Tris-chloride (pH 9.0), 8% glycerol, 1% DMSO, 0.2 mM of each dNTP, and 0.4 µM of each primer in a 50-µl reaction volume. For reverse transcription, 200 units of Superscript II reverse transcriptase (GIBCO BRL, Gaithersburg, MD) were added to the reaction after a 5 min preincubation at 95° and the reaction was run at 50° for 60 min. PCR was initiated by manual hot start using a Perkin-Elmer Cetus Thermocycler (Foster City, CA). After a 1.5-min preincubation at 94°, 2.5 units of Taq polymerase (Boehringer Mannheim, Indianapolis) and 0.015 units of Vent polymerase (New England Biolabs, Beverly, MA) were added, followed by 10 sec at 94° and 30 cycles of 10 sec at 94°, 1 min at 58° and 2.5 min at 68°. One microliter of the 50-µl RT-PCR reaction was used as template for secondary PCR. Amplification from L-A with this primer set yields a 1.4-kb PCR product.

Isolation of [KIL-d] revertants:
Reversion rates of [KIL-d] in strain M89 were measured by replating cells from a freshly grown YPAD plate onto YPAD at a density of about 200 cells/plate. After two days of incubation at 26°, colonies were replica plated onto a lawn of sensitive cells (M985) to detect K₁⁺ revertants; all such revertants were confirmed to be R₁⁺ in phenotype. Similar experiments were done with cells grown into colonies on YPAD plates containing 5 mM guanidinium hydrochloride or for three days in liquid YPAD containing ethidium bromide (30 µg/ml or 60 µg/ml). Ethidium bromide-treated colonies were also replica plated onto YPG plates to determine the number of petites. All revertants were tested by mating with M422 MATa haploid cells to prove that reversion was not due to spontaneous diploid formation.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

[KIL-d] does not map on killer virus dsRNA:
The cytoplasmic inheritance of [KIL-d] led us to test whether this trait was inherited with the dsRNA genome of killer virus. Strain M89, a meiotic segregant derived from [KIL-d] strain K30 isolated by WICKNER 1976 was used to test if [KIL-d] is inherited with the dsRNA genome of killer virus. M89 ([KIL-d] L-A-HN M₁ K₁^- R₁⁺) cells were subjected to heat curing of the viral RNAs by prolonged growth at 38°. Single colonies were isolated and their phenotypes were tested. If [KIL-d] is encoded on viral dsRNA, then it should not be inherited after this RNA is eliminated. After curing, we obtained isolates including nonkillers with two dsRNA compositions: 1 isolate with L-A-HN dsRNA only (L-A-HN M⁰) and 10 with neither L-A nor M dsRNA (L-A⁰M⁰); 1 uncured colony (containing both L-A-HN and M₁ dsRNA) was also recovered after heat treatment. The dsRNA content of the isolates was confirmed by gel electrophoresis analysis and RT-PCR. Since growth at elevated temperature can also result in mutations in mitochondrial DNA (SHERMAN 1959 ), several of these isolates were petite and were determined to be either [RHO-0] or nonsuppressive [RHO^-] mutants (see MATERIALS AND METHODS). Among these were the L-A-HN M⁰ isolate and 7 of the 10 L-A⁰ M⁰ isolates. All 12 colonies were crossed with M707, a wild-type L-A-H M₂ K₂⁺R₂⁺ strain, and the resulting diploids were tested for killer phenotypes and for their L-A dsRNA allele by strand-separation gel electrophoresis. Each diploid was then sporulated and subjected to tetrad analysis to assay for the transmission of [KIL-d].

When the [KIL-d] L-A-HN M⁰ isolate was crossed with M707, the resulting diploids were K₂⁺R₂⁺ in phenotype and harbored the L-A-H and M₂ dsRNA species. Upon sporulation, the spore clones (46 viable spores of 48 dissected) were all type 2 killers with variegated defective killer phenotypes (21 of 46 defective, including 11 K⁺R^w, 1 K⁺R^-, 4 K^wR⁺, 2 K^wR^w, 2 K^wR^-, and 1 K^-R⁺) demonstrating transmission of [KIL-d] trait independent of the inheritance of L-A-HN or M₁ dsRNA.

This was confirmed when the 10 [KIL-d] L-A⁰ M⁰ isolates were crossed with M707; 8 of 10 formed L-A-H M₂ K₂⁺R₂⁺ diploids, with no apparent correlation with their [RHO] status. Upon sporulation and tetrad dissection, all spores from eight dissected type 2 killer diploids demonstrated variegated defective type 2 killer phenotypes (Table 2), again indicating that [KIL-d] does not reside on viral dsRNA. The remaining two diploids were K^-R^- and lacked M dsRNA, as shown by dsRNA analysis. Presumably, these had spontaneously lost their M₂ dsRNA.

As a control, a [KIL-d] L-A-HN M₁ isolate (M1053) that retained its L-A-HN and M₁ dsRNA during heat curing was crossed with M707 (L-A-H M₂). This mating resulted in a type 2 killer diploid, which contained L-A-H and M₂ dsRNA. Crosses of type 1 and type 2 killer cells generally yield type 1 killer diploids due to apparent biased incompatibility between M₁ and M₂ dsRNA (WICKNER 1980 ). However, in such crosses type 2 diploids can be recovered at lower frequency (WEINSTEIN et al. 1993 ). When this diploid (L-A-H M₂) was sporulated, the meiotic clones showed defective type 2 killer phenotypes (Table 3), consistent with the results of the crosses described above. Similar inheritance of variegated defects was seen when strain M89, which had not been subjected to heat curing, was crossed with strain M707 (Table 3).

Variability in the spectrum of defective phenotypes in different crosses involving [KIL-d] seems to be a property of this phenomenon. This can be seen by comparing Table 2 and Table 3. The basis of this variability is not known. In contrast, in crosses of wild-type killer (K⁺R⁺) and wild-type nonkiller (K^-R^-) strains, 4 K⁺R⁺:0 segregation of phenotypes is generally observed (WICKNER 1974A ).

Defective meiotic progeny derived from all of the above experiments were subsequently crossed with [KIL-0] testers. Healing of defective killer phenotype was observed in all such test crosses, indicating that [KIL-d] is, indeed, responsible for the defects displayed in the spore clones.

These data demonstrate that similar variegated defective gene expression is observed in the original [KIL-d] strain and in the backcrossed strains harboring either L-A-H or L-A-HN (introduced from a wild-type parent) and is independent of the M dsRNA type. The introduction of a "wild-type" M dsRNA in genetic crosses does not eliminate the epigenetic regulation by [KIL-d] in haploid cells; thus, M dsRNA does not appear to be "dominant" to [KIL-d] based on these data. Hence, the [KIL-d] phenomenon is not specific to L-A-HN and M₁ dsRNA. Furthermore, elimination of both M and L-A dsRNAs from cells does not correlate with the loss of the [KIL-d] element. Therefore, [KIL-d] cannot map on the killer virus dsRNA.

Reversion studies of [KIL-d]:
The [KIL-d] trait displays a measurable rate of spontaneous reversion to wild-type K⁺R⁺ phenotype during vegetative growth of haploids (WICKNER 1976 ). We isolated such revertants from strain M89 ([KIL-d] K^-R⁺) on YPAD medium at a frequency of approximately 10^-5, comparable to the previously published frequency of 10^-4. This relatively frequent appearance of revertants has been interpreted as consistent with a single base-pair mutation being responsible for the [KIL-d] trait (WICKNER 1976 ). However, such reversions could also represent the loss of a genetic element that either carries [KIL-d] or is required for its phenotypic expression. Ethidium bromide treatment of strain M89, at concentrations known to induce nearly complete loss of mitochondrial DNA (GOLDRING et al. 1970 ), resulted in 99.8% petite colonies, but yielded only three K⁺R⁺ revertants out of 4 x 10^-4 cells. The four orders of magnitude difference between the rate of loss of [KIL-d] and [RHO] indicates that [KIL-d] is not a mitochondrial marker, which is consistent with the persistence of [KIL-d] in petites recovered after heat curing. Strain M89 was also plated onto YPAD containing guanidinium hydrochloride, the protein denaturing agent that reversibly cures [URE3] and [PSI] yeast prions (TUITE et al. 1981 ; WICKNER 1994 ). None of the 10⁵ colonies tested reverted from K^- to K⁺ demonstrating that [KIL-d] is not a typical [URE3] or [PSI]-like yeast prion. The revertants in all experiments were confirmed to be haploids capable of mating and, therefore, not simply the result of spontaneous diploid formation.

Transmission of [KIL-d] by cytoduction does not confer phenotypic defects:
Transmission of [KIL-d] by mating results in the phenomenon of healing. Since cytoductants have not gone through nuclear fusion to reach diploidy after mating, they can be viewed as intermediate products of the mating process. To test whether cytoplasmic mixing during cytoduction is sufficient for healing, strain M1004 (kar1-1 [KIL-0] [RHO-0]) was mated with strain M89 ([KIL-d] K^-R⁺). Using a standard cytoduction protocol, followed by selection for cells with the M1004 nucleus and mixed [RHO⁺] cytoplasm, 177 cytoductants were isolated. A total of 176 of the haploid cytoductants were K₁⁺R₁⁺ in phenotype, suggesting that healing of defective phenotypes of haploid cells can occur without nuclear fusion. All of these cytoductants were crossed with strain M423 (wild-type [KIL-0]) and all yielded K₁⁺R₁⁺ diploids. Four of these diploids were sporulated and subjected to tetrad analysis, resulting in 51 viable spores out of 120 dissected (Table 4). Thirty-six of these spores had variegated defective phenotypes, demonstrating that [KIL-d] had been transferred with the cytoplasm during the cytoduction, despite the lack of phenotypic defect in the cytoductants. Tetrad analysis of these meiotic progeny revealed no correlation between the segregation of any gene, including kar1-1, and the killer phenotypes of the spores. All defective killer spore clones became healed upon crossing with wild-type [KIL-0] strains. The one cytoductant that was K^-R^- also failed to display healing upon crossing with wild-type strain M423 (K^-R^- diploids being formed), indicating that this one cytoductant had failed to inherit M dsRNA. These results indicate that [KIL-d] can be transmitted with the cytoplasm during cytoduction, but such transmission is not sufficient for its phenotypic expression.

View this table: In this window In a new window   Table 4. K+R+ phenotype of M89M1004 cytoductants is not due to loss or suppression of [KIL-d]

Three genuine diploids recovered from the cytoduction experiment described above were K₁⁺R₁⁺ in phenotype as expected. All three were sporulated and subjected to tetrad analysis (Table 5). Out of 124 dissected spores 63 were viable, 44 of which had defective killer phenotypes. Again there was no correlation between any marker and the killer phenotypes of the spore clones. All clones with defective killer phenotype became healed upon crossing with wild-type [KIL-0] strains. Thus, [KIL-d] is transmitted cytoplasmically in crosses resulting in diploid formation or cytoduction, whether or not kar1-1 is present. However, only after meiosis is the epigenetic effect of [KIL-d] on killer phenotype seen.

Cytoduction into a [KIL-d] strain has no phenotypic effect:
To determine whether cytoduction resulted in healing of the defective phenotype of a [KIL-d] strain, cytoduction was performed in the opposite orientation to that described in the previous section, using isogenic strains. This is essentially the same as the cytoduction described in the previous section, but in the opposite direction. For this cytoduction, ethidium bromide-induced [RHO-0] derivatives of M89 were mated with M480 (kar1-1 [RHO⁺] [KIL-0]). Three [RHO-0] isolates of ethidium bromide-treated M89 were confirmed to carry L-A-HN and M₁ dsRNA and retained the K₁^-R₁⁺ phenotype. After mating M89 [RHO-0] with strain M480, cytoductants with the M89 nucleus were selected on H-Leu plates with glycerol as a carbon source, thus selecting for cytoplasmic mixing. Two of the 139 cytoductants were K^-R^- due to loss of M dsRNA, as demonstrated in a follow-up cross with M422, and were not studied further. None of the remaining 137 cytoductants showed a change in the original K₁^-R₁⁺ defective phenotype, indicating that healing had not occurred. However, when these cytoductants were crossed with M422, they all formed healed K₁⁺R₁⁺ diploids, confirming the presence of [KIL-d] in the cytoductants (Table 6). Since cytoduction did not heal the defect in the [KIL-d] recipient parent, this disproves the hypothesis that the cytoplasmic fusion step of the mating process causes healing. Given this asymmetric manner of healing during cytoduction, it is apparent that transfer of [KIL-d] along with the killer virus into a wild-type nuclear background does not allow [KIL-d] to exert its epigenetic effect on the virus. However, after mating and sporulation, the [KIL-d] element becomes active in epigenetic regulation of viral gene expression in the resulting haploid meiotic segregants. Introduction of wild-type cytoplasm by cytoduction into a haploid strain displaying epigenetic regulation of killer virus gene expression by [KIL-d] does not alter or heal this regulation.

MAT locus heterozygosity and mating type switching fail to heal in haploid cells:
M1042 (MATa ura3-52 [KIL-d] L-A-HN M₁ K₁^-R₁⁺) was transformed with centromere-containing plasmid pGAL-HO, carrying a galactose-inducible HO endonuclease gene. Cell-type switching in pGAL-HO transformants was induced by growth in 2% galactose for 2, 4, and 6 hr, after which expression was shut down and the plasmid was eliminated by growth on 5-FOA-containing H medium, and single colonies were tested for cell type. Forty-eight colonies which had undergone homothallic conversion of cell-type to MAT were phenotypically tested, and all retained the parental K^-R⁺ phenotype; thus, healing did not occur. One of these MAT derivatives was retransformed with pGAL-HO and similarly induced to undergo homothallic conversion to MATa. These convertants (48) also retained the parental defective killer phenotype. Thus, sequential expression of different alleles of MAT has no effect on the epigenetic regulation of killer virus expression by [KIL-d].

To test the role of heterozygosity at MAT in healing of the phenotypic defects in [KIL-d] diploids, M1042 and a MAT derivative generated by pGAL-HO-induced homothallic conversion were transformed by plasmids pJM3 and pJM9, which express the MATa and MAT alleles, respectively. Forty-eight transformants were isolated in each experiment. In each case, all transformants with plasmids expressing the same allele as the endogenous MAT locus mated normally with a strain of the opposite cell type, while transformants with the opposite MAT allele failed to mate. All of these transformants retained the K^-R⁺ phenotype (Table 7). Therefore, heterozygosity at MAT alone is insufficient to cause healing of the phenotypic defects in a [KIL-d] haploid strain.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

[KIL-d] is a cytoplasmically inherited genetic trait which causes killer virus-infected yeast to express normal killer phenotypes in a/ cells, but to show variegated defective killer phenotypes in a or cells. The [KIL-d] element is inherited independently of both L-A and M dsRNA disproving the model that [KIL-d] is a mutant form of viral M and/or L-A dsRNAs. [KIL-d] does not map on the mitochndrial DNA as shown by its resistance to ethidium bromide at concentrations that effectively cure the mitochondrial [RHO] genome. These results contrast with two crosses reported by WICKNER 1976 , in which [KIL-d] appeared not to be phenotypically expressed after crosses with wild-type killer strains. Strain differences between the killer strains used in different crosses or spontaneous loss of [KIL-d] complicating Wickner's experiments might account for these differences.

The previously described [PSI] and [URE3] prions of yeast (AIGLE and LACROUTE 1975 ; COX 1965 ; WICKNER 1994 ) can also be thought of as cytoplasmic elements exerting epigenetic regulation on chromosomal genes, in that [PSI] alters the phenotypic expression of SUP35 and [URE3] alters that of the URE2 gene. As has been reported for many other epigenetic elements acting on chromosomal genes, [PSI] alteration of SUP35 phenotypic expression (as measured by suppression of various auxotrophic mutations) in different isolates spontaneously developing [PSI] shows clonal variegation (DERKATCH et al. 1996 , DERKATCH et al. 1997 ); this clonal variegation resembles that seen in the different meiotic segregants containing [KIL-d]. Although [KIL-d] resembles [PSI] in its cytoplasmic inheritance pattern and clonal phenotypic variegation of the epigenetically regulated gene, the two elements are obviously quite different. [KIL-d] is resistant to detectable curing by growth in the presence of 5 mM guanidinium hydrochloride, which reversibly cures [URE3] and [PSI] (TUITE et al. 1981 ; WICKNER 1994 ). Furthermore, while the phenotypic effects of [URE3] and [PSI] are exerted on the genes that encode the proteins that constitute these prions, [KIL-d] is not encoded by the M dsRNA satellite virus genome of killer virus of yeast whose phenotypic expression is altered by its presence.

The data presented indicate that when [KIL-d] and M dsRNA are transferred into a new hapolid cell by cytoduction, both the cytoplasmic element and the viral genome are efficiently transmitted. However, the epigenetic regulation of M dsRNA phenotypic expression by [KIL-d] is not established until after the cytoductants have gone through mating and meiosis. Thus, the presence of [KIL-d] in the cytoplasm of a haploid cell does not exert an epigenetic effect on killer virus phenotypic expression until nuclear events involved in meiosis have occurred. When viewed in this manner, the results of the cytoduction of wild-type cytoplasm into a haploid cell containing [KIL-d] and M dsRNA are understandable: once epigenetic regulation of M dsRNA expression by [KIL-d] is established at meiosis, subsequent introduction of wild-type cytoplasm and any other regulatory effects of the cytoduction process have no effect on the epigenetic phenomenon, which remains mitotically stable until healed by nuclear fusion during mating. Clearly cytoduction alone is insufficient to induce the healing reaction.

The original naming of the diploid-dependent genetic element (WICKNER 1976 ) suggested that either ploidy or MAT locus status was critical in the epigenetic regulation by [KIL-d] of killer virus phenotypic expression. Since neither haploids expressing both alleles of MAT simultaneously (MAT transformation experiments) or sequentially (homothallic conversion experiments) show alterations in the phenotypic defect of killer virus in the presence of [KIL-d], it is clear that healing of the defects seen upon mating is not due to regulation by MAT. On the other hand, ploidy itself is not sufficient to explain this regulation either, since when [KIL-d] and M dsRNA are simultaneously introduced into the cytoplasm of a wild-type cell by cytoduction, the resulting haploid cytoductants express the wild-type killer phenotype (as would a diploid healed after mating), even though they are not diploid.

If neither ploidy nor MAT status explains regulation of the [KIL-d] epigenetic phenomenon, how can this be understood? A cytoplasmically inherited altered protein (with conformational or post-translational modifications) whose phenotypic expression is "reset" by meiosis and "healed" by nuclear fusion would formally explain these observations. However, the asymmetric results of cytoduction and variegation upon meiosis are not obviously predictable from this model without additional assumptions. We propose that the cytoplasmically inherited [KIL-d] element, like most epigenetic elements previously described, acts on a chromatin target in the cell nucleus, whose expression is altered by this action, resulting in alteration of expression of the killer virus M dsRNA genome segment. Since many chromosomal genes are known to be required for the replication, expression, and regulation of this viral genome segment, involvement of nuclear gene(s) in this epigenetic process in not unprecedented. This heuristic model includes the following components:

1. The cytoplasmically inherited [KIL-d] element can only exert its effect on its nuclear target by gaining accessibility to that target by nuclear alterations that occur during meiosis. This explains the lack of phenotypic effect of [KIL-d] in cells that have received it by cytoduction.

2. Upon meiosis, when [KIL-d] gains access to the nucleus, it might act either on different genetic loci or on the same locus in different ways, thus establishing variegation. This interaction must result in some change which is potentially reversible (consistent with the relatively high frequency of reversion) but which is generally mitotically stable (consistent with the clonal stability of the variegated phenotypes after meiosis). Since in many other epigenetic phenomena, the underlying mechanism is an alteration in chromatin structure and/or DNA methylation state that is mitotically stable but potentially reversible (HENIKOFF and MATZKE 1997 ), such an alteration seems like a strong possibility for the event that triggers resetting of the defective phenotype by meiosis, and its reversal might be the basis of the healing that occurs upon mating.

3. The [KIL-d] element is cytoplasmically inherited but has not been localized to any known plasmid or virus of yeast. This, together with its unusual genetic properties, is consistent with [KIL-d] possibly being a cytoplasmically inherited form of a protein, formally bearing some resemblance to the prions previously described in yeast. However, its properties are clearly different from typical [PSI] and [URE3] prions, in that alteration of phenotype is not seen upon treatment with guanidinium hydrochloride. Since all reported [KIL-d] elements isolated to date have arisen from the parental strain A364A (WICKNER 1976 ; HANNIG 1985 ), it is possible that this strain contains an allele of the putative [KIL-d] generating gene whose product is more likely to spontaneously assume the [KIL-d] conformation. Since all reported [KIL-d] isolates have been obtained after mutagenesis, it seems likely that the speculated gene encoding this protein requires additional mutations in order to spontaneously adopt the [KIL-d] conformation at detectable frequencies. However, once [KIL-d] is established, it can readily be transmitted to other strains with a wild-type nucleus. This model resembles the prions of mammals, in which mutant forms of the PrP protein are more likely to spontaneously form disease-causing prions, and yet such prions are readily transmissible to organisms with the wild-type allele of the PrP gene (PRUSINER 1997 ).

This model, based entirely on genetic evidence, allows us to make molecular predictions which are testable using the power of yeast genetics.

	ACKNOWLEDGMENTS

We thank R. B. WICKNER (National Institutes of Health) for helpful discussions and D. E. GEORGOPOULOS for excellent technical assistance with some aspects of this work. We thank J. D. DINMAN, T. G. KINZY and S. W. PELTZ for critical reading of this manuscript. This work was partially supported by a grant from the Army Research Office (DAAL03-92-G-0212; M.J.L.), and fellowships from the Foundation of the Hungarian Academy of Engineering-Rubik International Foundation and the Soros Foundation-Hungary (Z.T.).

Manuscript received February 24, 1998; Accepted for publication May 21, 1998.

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