genetic control of l-a and l-(bc) dsrna copy number

Copyright 0 1984 by the Genetics Society of America

GENETIC CONTROL OF L-A AND L-(BC) dsRNA COPY NUMBER IN KILLER SYSTEMS OF SACCHAROMYCES

CERE VISIA E

STEVEN G. BALL,*9t CATHERINE TIRTIAUX*,t AND REED B. WICKNER*

*Section on Genetics of Sim/de Eukaryotes, Lnboratory of Biochemical Pharmacology, National Institute of Arthritis, Diabetes, and Digestive and Kidne! Diseases, National Institutes of Health, Building 4, Room

116, Bethesda, Maryland 20205; nnd tChnire de GinPtique Faculti des Sciences Agronomiques de 1’Etate 6 Gembloux,

5800 Gembloux, Belgium

Manuscript received September 27, 1983 Revised copy accepted February 27, 1984

ABSTRACT

M dsRNA in yeast encodes a toxin precursor and immunity protein, whereas L-A dsRNA encodes the 8 1,000-dalton major.protein of the intracellular particles in which both L-A and M are found. L-(BC) dsRNA(s) are found in particles with different coat proteins. We find that M dsRNA lowers the copy number of L-A, but not L-(BC). The SKI gene products lower the copy number of L-(BC), L-A, MI and MP. This is the first known interaction of L-(BC) with any element of the killer systems. The MAK3, MAKlO and PET18 gene products are necessary for L-A maintenance and replication, but mutations in these genes do not affect L-(BC) copy number. Mutations in M A K I , M A K I , MAK7, MAK17 and MAK24 do not detectably affect copy number of L-(BC) or L-A.

HE killer systems in Saccharomyces cerevisiae, determined by dsRNA virus- T like particles (VLPs), are unique among the viruses of eukaryotes in the ease with which interactions among chromosomal genes and viral elements are studied. As a result, a large number of chromosomal genes involved in viral processes have been defined (for reviews, see WICKNER 1981, 1983a; BUSSEY 1981; terminology is shown in Table 1). K1 and K2 killer strains have the ability to secrete (K+ phenotype) and to resist (R+ phenotype) a protein toxin lethal to other strains. These phenotypes rely on the presence of linear double- stranded RNAs (dsRNAs) called MI (1.8 kb; K1 and RI phenotypes) or M2 (1.5 kb; K2 and R2 phenotypes. Other dsRNAs in yeast include two different co- migrating larger species (4.5 kb) called L-A and L-(BC) (SOMMER and WICKNER 1982a,b; WICKNER and TOH-E 1982) and two lower copy number plasmids of intermediate size, T (2.7 kb) and W (2.25 kb) (W~SOLOWSKI and WICKNER 1984). L-A, L-(BC) and M are separately encapsidated in noninfectious VLPs with an RNA polymerase activity (HERRING and BEVAN 1974, 1977; WELSH, LEIBOWITZ and WICKNER 1980; WELSH and LEIBOWITZ 1980; SOMMER and WICKNER 1982b; THIELE et a l . , 1984). L-A codes for at least the major coat protein of both L-A and M particles (HOPPER et al. 1977; HARRIS 1978; BOS- Genetics 107: 199-217, June, 1984.

200 S. G. BALL, C. TIRTIAUX AND R. B. WICKNER

TABLE 1

Nomenclature

Phenotypes KI+ or K1-

K2+ or Kz- K++ K" Weak killing. RI+ or RI- R2+ or Rz-

Ability or inability to kill a lawn of sensitive K-R- or K*+Rz+ cells. Ability or inability to kill a lawn of sensitive K-R- or KI+RI+ strains. Superkiller phenotype; increased killing of sensitive lawn.

Resistance or sensitivity to the KI toxin. Resistance or sensitivity to the Kz toxin.

Nuclear genes iMAK Chromosomal gene needed for maintenance of M. MAKlO and MAK?

are also required for maintenance of [HOK], [NEX] and [EXL] (L-A dsRNA).

Chromosomal gene needed for cell growth at high temperature and for maintenance of the L-A and M dsRNAs, [HOK], [NEX], [EXL] and [RHO].

Chromosomal gene needed for maintenance of [KIL-kz] only if the cytoplasmic [NEX] gene is present.

Recessive chromosomal ski mutations produce the superkiller phenotype. All ski mutations except ski1 have a higher copy number of M dsRNA. ski1 mutants have the ability to bypass all mak defects except maklb. ski2, ski3, and ski4 bypass m k t l - l and all mak mutations except maklb, petl8, maklO and mak3. ski2, ski3, ski4, skib, ski7 and ski8 mutants are cold sensitive for growth only if M dsRNA is present.

Suppressor genes of mkt l -1 . MKS mutations can be dominant (MK.550) or recessive (mksl-I).

PET18

iMK T

SKI

MKS

Cytoplasmic geno- types

[ KIL-kl], [KIL-kz]

[KIL-n] [KIL-S]

[ KIL-sdl]

[NEXI

[KIL-01, [HOK-01, [NEX-01, [EX L-01

M I and M 2 dsRNAs

Wild-type KI or KZ killer cytoplasmic genomes carrying killing and resist-

Killer genome mutants lacking only the killing activity (neutral). Killer genome deletion mutants lacking both killing and resistance func-

tions. Such mutants are also cytoplasmic suppressive mutants of the killer genome.

ski--dependent MI dsRNA due to L-A-HN (present in wild-type killers) being replaced by L-A-E.

Wild-type KI killer genome that does not need certain MAK gene products for its maintenance.

Helper of killer. The [HOK] non-Mendelian gene enables MI to replicate in a SKI+ nuclear background.

excluder of [KIL-kz]. The [EXL] gene excludes [KIL-kp] in the absence of the [NEX] cytoplasmic trait.

Makes [KIL-kp] nonexcludable by [EXL]. But [NEX] induces the loss of [KIL-kz] in an mkt- host.

Absence of [KIL-kl] (or [KIL-kp]), [HOK], [NEX] or [EXL].

ance functions.

1.8-kb MI and 1.5-kb M2 linear dsRNAs are the molecular determinants of [KIL-k~] and [KIL-kp].

L DSRNA COPY CONTROL 201

TABLE l-Continued

S Deletion mutants of MI, retaining only the ends of MI; S dsRNAs interfere with replication of MI (SOMERS 1973; FRIED and FINK 1978; RIDLEY and WICKNER 1983).

4.5-kb linear dsRNA. [EXL], [NEX] and [HOK] are present in various forms of L-A denoted L-A-E ([EXL] alone), L-A-HN ([HOK] and [NEX]), L-A-HE ([HOK] and [EXL]) or L-A-H ([HOK] alone).

4.5-kb linear dsRNAs unrelated to L-A. L-B and L-C show some sequence homology and the same size VLP protein. The residual L dsRNA left after curing of L-A in most strains will be called L-(BC) since its sequence relationship to either L-B or L-C is not clear.

2.7- and 2.25-kb minor dsRNAs. They do not cross hybridize with each other, other dsRNAs or cell DNA. They are cytoplasmically inherited. Heat inducibility of T and W is under control of a nowMendelian gene.

L- A

L-B and L-C

T and W

TIAN, STURGEON and TIPPER 1980; SOMMER and WICKNER 1982b), whereas M codes for a larger precursor of the killer toxin (BOSTIAN et al. 1980).

A large number of nuclear genes have been described that affect various aspects of the killer dsRNA replication. These include 28 MAK genes necessary for maintenance of M dsRNA (WICKNER and LEIBOWITZ 1976, 1979; WICKNER 1978, 1979) and 7 SKI genes, mutations of which cause increased killing, often accompanied by overproduction of M (TOH-E, GUERRY and WICKNER 1978; RIDLEY, SOMMER and WICKNER 1984). In addition to this superkiller phenotype, most ski mutations have the ability both to bypass certain mak defects (TOH-E and WICKNER 1980) and to produce cold sensitivity for cell growth by increasing M copy number (RIDLEY, SOMMER and WICKNER 1984).

The killer system has also provided a useful tool for the study of complex interactions involving extrachromosomal genetic elements, including exclusion and helper phenomena. These phenomena have revealed that the L-A molecules in different laboratory strains have different genetic properties (WICKNER 1980, 1983b; SOMMER and WICKNER 1982a,b; RIDLEY, SOMMER and WICKNER 1984) and are detailed in Table 1. [HOK] is the function of L-A that enables MI to be maintained in the absence of a ski- defect (TOH-E and WICKNER 1979; WICKNER and TOH-E 1982; RIDLEY, SOMMER and WICKNER 1984). It is likely that [HOK] is the ability of L-A to provide the major capsid protein in which MI is found (HARRIS 1978; BOSTIAN et al. 1980), but our results here suggest an alternative possibility.

In this paper we report that (1) MI and Mz dsRNAs lower L-A copy number; (2) the degree to which a particular M lowers L-A copy number is correlated with its copy number, which is determined both by chromosomal SKI genes and by cytoplasmic genes; (3) SKI gene products lower the copy number of both L-(BC) and L-A; (4) among the MAK genes tested, none affect L-(BC) copy number, and except for those whose mutants were previously shown to lose L-A, none affect L-A copy number; ( 5 ) wild-type M I dsRNAs differing in their ability to decrease L-A copy number also differ in their requirements for MAK gene products.


TABLE 2

Strains of S. cerevisiae

Nuclear Cytoplasmic Designation genotype genotype &RNAs References

18 a lysl [HOK] [NEX] [KIL-kiIis

1490 18 [HOK] [NEX] [ K I L - ~ I I A s ~ ~ ~ A

1743, K7 a arg9 [HOK] [NEX] [KIL-~IIK,

A364A a adel ade2 ura l [HOK] [NEX] [KIL-~I ]Aw~A tyrl his7 lys2 ga l l

1019, JC7 a leu1 kar l - l [HOK-o] [NEX-o] [KIL-o]

1020, J C l l a his4-15, kar l - l [HOK-o] [NEX-o] [KIL-o] 2181

1101

2176 1488

1368

2026

2028

1385

1494

SP176

SP178

SP180

2145

3544-lA 3545-15B

3609-1B

1591

1019 [HOK] [NEX] [KIL-ki]is

1020 [HOK] [NEX] [ K I L - ~ ~ I A ~ ~ ~ A

1020 [HOK] [NEXIAS~~A 1020 [HOK] [NEX] [KIL-kills

1020 [HOK] [NEX] [KIL-b]125fi

a ade5 his4 ura l can1 mktl-1

01 1eu2' cyh2 ura I his4 met13 mktl-1

[HOK] [NEX] [KIL-kp]

[HOK] [NEX] [KIL-kp]

a lysl [HOK] [NEX] [KIL-kp]

1019 [HOK] [NEX] [ K I L - ~ ~ I A s ~ ~ ~ A

1019 [HOK] [NEX] [KIL-S~]ASPOSB

1019 [HOK] [NEX] [KIL-ki]~snoss

a his1 lys2 mak4- [HOK] [NEX] [KIL-o]

a ura l mak7-1 [HOK] [NEX] [KIL-o] a adel cpa2 [HOK] [NEX] [KIL-o]

makl7-1 01 lys2 adel mktl- [HOK] [NEX] [KIL-o]

I mak4-1 a aro2 lys5 met13 [HOK] [NEX] [KIL-o]

ade5 cyh2 trp5 mak24-1

I

L-(BC), L-A-HN, MI

MI L-(BC), L-A-HN,

L-A-HN, MI SOMMER and WICKNER 1982b

L-(BC), L-A-HN, MI

L-(BC) CONDE and FINK 1976

L-(BC) L-(BC), L-A-HN,

MI

MI L-(BC), L-A-HN,

L-(BC), L-A-HN L-(BC), L-A-HN,

MI

MI

MP

MP

L-(BC), L-A-HN,

L-(BC), L-A-HN,

L-(BC), L-A-HN,

L-(BC), L-A-HN, Mz

MI

SI

L-(BC), L-A-HN,

L-(BC), L-A-HN,

L-(BC), L-A-HN, s3

L-(BC), L-A-HN, MI

L-(BC), L-A-HN

L-(BC), L-A-HN L-(BC), L-A-HN

L-(BC), L-A-HN

L-(BC), L-A-HN

WICKNER 1980

WICKNER 1980

WICKNER 1980

RIDLEY and WICKNER 1983




TABLE 2-Continued

Nuclear Cytoplasmic dsRNAs References Designation genotype genotype

1925

S486

S600

S596

S506

2348

s533

S648

s597

3637-8A

SP405

SP226

1101 +a SP226

2176 +a

SP226 5x47

1407

a karl-1 lysl u r a l makl-1 adel

a u r a l met13

a lys2 mksl-1 mkt l - l canl cyh2

a lys2 mktl canl cyh2 ski3-l I

a lys2 mktl canl cyh2 mksl-1

a mktl-1 met13 u r a l canl cyh2

a lys2 ski7-1 mktl canl cyh2

a ural met13 canl cyh2 mktl ski8-2

a lys2 mktl skib- I

a leu2 inktl ski4- I

a leu1 his4 lys10 karl-1 ski2-2

a adel ski2-2 mktl

SP226

SP226

a/a trpl /+

a thrl ski2-2 ura3/+ hisl/+

[HOK-01 [NEX-01 [KIL-01

[HOK-01 [NEX-01 [KIL-01

[HOK-01 [NEX-01 [KIL-01

[HOK-01 [NEX-01 [KIL-01

[HOK] [NEX]

[HOK] [NEX]

[HOK-01 [NEX-01 [KIL-01

[HOK] [NEX] [KIL-k2]

[HOK-01 [NEX-01 [KIL-01

[HOK-01 [NEX-01 [KIL-01

[HOK] [NEX] [ K I L - ~ I ~ A S ~ ~ A

[HOK-01 [NEX-O] [KIL-01

[HOK] [NEX] [ K I L - ~ I I A ~ A

[HOK] [NEX]AWAA [KIL-01

[KIL-o]

[EXL] [KIL-sd]

L-(BC)

L-(BC)

L-(BC)

L-(BC)

L-(BC), L-A-HN

L-(BC), L-A-HN

L-(BC)

L-(BC), L-A-HN, Mz

RIDLEY, SOM- MER and WICKNER 1984

MER and WICKNER 1984





RIDLEY, SOM-

RIDLEY, SOM-

RIDLEY, SOM-

RIDLEY, SOM-

RIDLEY, SOM-

L-(BC)

L-( BC)

L-(BC), L-A-HN, RIDLEY, SOM- MI MER and

WICKNER 1984

L-(BC)

L-(BC), L-A-HN, MI

L-(BC), L-A-HN

L-(BC), L-A-E, RIDLEY, SOM- MI MER and

WICKNER 1984


TABLE 2-Continued

Nuclear Designation genotype

Cytoplasmic genotype __ dsRNAs References

1408 a thrl ski2-2 [EXL] [KIL-sd] L-(BC), L-A-E, RIDLEY, SOM- MI MER and

WICKNER 1984

s 7 a [HOK] [NEX] [KIL-01 L-AHN~ S142 a thrl argl [EXL-01 [HOK-01 [NEX-01 SOMMER and

[KIL-o] WICKNER 1982a,b

A 3 B means a cytoductant produced by introducing the cytoplasm of strain A into strain B. We find that strain S7 (OLIVER et al. 1977) has only L-A-HN. It carries the [HOK] and [NEX]

cytoplasmic genes (WICKNER 1983b) and either heat curing of L-A-HN or the action of a pet18 mutation leaves no other L. L-B or L-C are not heat cured and are not dependent on pet18 for their maintenance or replication (SOMMER and WICKNER 1982a,b; WICKNER and TOH-E 1982).

MATERIALS AND METHODS

Strains and media: Many of the strains used are listed in Table 2. Growth, selection, killer and sporulation media are as previously described (WICKNER 1979, 1980).

Curing: Curing of the killer plasmid was achieved by streaking out cells either on YPAD containing 0.3 jtg/ml of cycloheximide (FINK and STYLES 1972) or by isolating colonies on YPAD at 37" or 39" (WICKNER 1974; SOMMER and WICKNER 1982a). Single colonies were picked and repurified on YPAD at 30". Clones were then tested for killing, and sensitive colonies were tested for [HOK].

Assay of killing, resistance or [HOK]: Killing was assayed by replicating cells on 4.7 MB medium which had just been seeded with a lawn of the sensitive strain 5x47. After 2 days at Z O O , killing was revealed by the presence of a zone of growth inhibition surrounding the tested strain. Resist- ance was scored as previously described (WICKNER 1980). [HOK] was assayed in sensitive haploid strains by crossing them with the ski2-2 [KIL-sdl] tester strains 1407 or 1408. The diploids were selected on SD plates (24-hr growth) and were then streaked out for single colonies. Only then were they replica-plated onto 4.7 MB medium. [HOK] strains remained perfectly stable killers, whereas [HOK-o] diploids were unstable to various degrees, ranging from complete absence of any killing to the presence of frequent K- colonies, with the remainder being very weak killers.

Crosses and cytoduction: Matings, diploid selection, sporulation and dissections were carried out by the usual methods (MORTIMER and HAWTHORNE 1975). Cytoduction is a technique that allows selective transfer of cytoplasm from one strain to another (CONDE and FINK 1976). In cytoduction, nuclear fusion is prevented during crosses by the presence of the karl- l mutation. This yields, after the segregation of nuclei, cells that harbor mixed cytoplasm and nuclear genes from only one or the other parent. Cytoductants are selected through the presence of suitable nuclear and cytoplasmic markers. For this purpose, recipients are usually treated with ethidium bromide to cause loss of mitochondrial DNA and, thus, loss of the ability to grow on nonfermentable carbon sources (rho').

dsRNA extrartion and analysts: dsRNA was extracted by the procedure described by FRIED and FINK (1978). Routinely, a quarter of a YPAD plate of stationary phase cells were washed in 2.5 ml of cold 50 mM EDTA, pH 7.0. The cells were incubated at 20" for 20 min in 2.5 ml of 2.5% 2-mercaptoethanol-0.05 M Tris sulfate, pH 9.3, centrifuged and resuspended in 1 ml of 0.1 % SDS- 0.1 M NaCI-10 mM Tris chloride (pH 7.5)-IO mM EDTA. An equal volume of 2/9 buffer-equilibrated phenol:% chloroform, containing 1 mg/ml of 8-hydroxyquinoline, was immediately added. Cells were then incubated at 20" for at least 0.5 hr and mixed frequently. The aqueous phase was separated by centrifugation at 4000 X g for 30 min, and nucleic acids were precipitated with 2.5 ml of ethanol. After at least 1 hr at - Z O O , the precipitate was collected by centrifugation (4000


x g, 0", 5 min). Nucleic acids were dissolved in 0.25 ml of electrophoresis buffer containing 1 mg/ml of proteinase K and stored at 4". Samples were subjected to electrophoresis at 3 V/cm for 2 hr in a 1% agarose gel containing 40 mM Tris acetate, pH 7.4, and 1 mM EDTA. These extraction and electrophoresis conditions avoided both degradation and diffusion of single-stranded rRNA. Quantification was performed by electrophoresis of various volumes of the samples (from 10 to 0.5 PI), staining the gel with 1 pg/ml of ethidium bromide and photographing it with Polaroid negative type 55 film. The negative was then scanned by a Quick Scan densitometer. Peaks were cut out and weighed. Comparisons were made only between samples run on the same gel. The amounts of M and L dsRNA were normalized using the rRNA present in the same sample as an internal standard. Calculations were then carried out only in those dilutions that showed linear decrease on dilution of both ribosomal RNA and L dsRNA. We estimate the variability of this method to be less than +50%. The validity and precision of this method for estimating relative amounts of L dsRNA were confirmed by comparing these measurements to A260nm readings performed on dsRNA extracted by complete cell lysis and CF11 chromatography, as previously described (TOH-E, GUERRY and WICKNER 1978).

Hybridization techniques: Nucleic acids from equal amounts of cells, adjusted by equalizing the amount of rRNA, from different strains were denatured and loaded on a 1% agarose slab gel. Denaturation, electrophoresis and transfer to nitrocellulose was carried out as described by THOMAS (1980). Filters were prehybridized at 50" in 50% formamide/5 X SCC/O.O2% each of bovine serum albumin, Ficoll and polyvinylpyrrolidone/200 r g of denatured sonicated calf thymus DNA/ml and hybridized in 40 ml of the same buffer, containing 1 r g of SzP-labeled denatured probe RNA. The probe was prepared using T 4 polynucleotide kinase as previously described (GOLDBACH et al. 1978). The L dsRNA was purified for labeling by two successive CFl 1 columns followed by agarose gel electrophoresis and electroelution. Occasionally, filters were rehybridized after rinsing twice at 65" in 50 mM Tris chloride (pH 8.0)/2 mM EDTA/0.5% sodium pyrophos- phate/0.02% each of bovine serum albumin, Ficoll and polyvinylpyrrolidone. Relative intensities of hybridization signals were estimated by analyzing several time exposures of autoradiographs on Kodak XAR-5 X-ray film.

RESULTS

In wild-type strains, the absence of M d s R N A results in increased L d s R N A copy number: Strains isogenic for nuclear markers, and carrying either L-(BC) + L- A + M or L-(BC) + L-A only, were constructed by curing M with cycloheximide or by growth at 37" or 39". The cured strains consistently contained more L than the uncured parent strain or colonies grown under the curing conditions but found not to have been cured (see Figure 1 and Table 3). This increase ranged from two-fold in most K2 strains to ten-fold in some K1 strains (Table 3). Furthermore, strains isogenic for nuclear genes were constructed by introducing, using cytoplasmic transfer (cytoduction), into strain 1020 either L-(BC) + L-A-HN + M1 or L-(BC) + L-A-HN from a wild-type killer strain (A364A) or a cured derivative. In all cases, for a given nuclear background and subset of plasmids, we have observed a highly reproducible increase of L when M is absent, regardless of whether the strains were constructed by curing (heat or cycloheximide) or by cytoduction.

L-A is solely responsible for the increase in L d s R N A on elimination of M dsRNA: When killer strain K7, known to carry only L-A-HN and MI (SOMMER and WICKNER 1982b), was cured of MI, a ten-fold increase in L-A-HN was observed (Figure 1). When strains carrying both L-(BC) and L-A-HN with MI were cured of M 1 , and blots of gels of their denatured RNA were hybridized with L-A only or with L-(BC) only, it was found that L-A-HN had increased about five-fold, but L-(BC) did not change on elimination of M. The example shown

206

1st K+&K- PAIR 2nd K+&K- PAIR -- I - NUCLEAR DNA - L-A ONLY

- Mi - 25s rRNA - 18s rRNA

Kt K-K+K- K t K- K-K+K- K+ K-K+

DILUTION: 1 1 1/z Vz V4 V4 1 1 '/z '/z '14 V4

FIGURE 1 .-A ten-fold increase in L-A-HN copy number in strain K7 when MI is cured. Strain K7 was streaked for single colonies on YPAD agar at 39°C. Two K I + single colonies and two K- [HOK] single colones were grown at 30°C. RNA extracted and electrophoresed on agarose gels. Equal amounts o f RNA from pairs of K I + and K- [HOK] colonies are compared and quantitated by serial dilutions. It can be seen that the four-fold diluted K- colonies have more than twice as much L as the undiluted K+ colonies. This type o f analysis, with densitometer scanning. was used to generate the data in Tables 3 and 4.

in lanes 2 and 3 of Figure 2 suggests a small decrease in L-(BC) on loss of M. but all other experiments showed no change. Thus, L-A copy number is re- pressed in the presence of M. but L-(BC) is unaffected.

The magnitude of the L-A dsRNA increase on loss of M is inherited as a cytoplasmic trait: To determine whether nuclear genes were involved in the magnitude of the L-A repression by M, L-A-HN and M I dsRNAs from strains 18 and A364A were cytoduced into several nuclear backgrounds from which L-A and M had first been eliminated. T h e cytoductants were subsequently cured of M I by heat or cycloheximide. T h e results presented in Table 3 strongly suggest that the decrease in L-A-HN copy number in the presence of M is predominantly a function of the cytoplasm and not of the nuclear background. For example, curing the MI from strain A364A's cytoplasm gives the same three- to five- fold increase in L when it is with the strain A364A nucleus, the strain 18 nucleus or the strain 1020 nucleus. But curing the M I from strain 18's cytoplasm gives an eight-fold increase in L in either the strain 18 or strain 1020 background (Table 3). At this point we do not know whether this phenotype is a function solely of M, of L-A-HN and M or of yet another cytoplasmic element. However, rho- colonies frequently generated by cycloheximide or heat behaved in the same manner as their rho' counterparts, suggesting that mitochondrial DNA at least is not involved.

Suppressive or neutrtil mutants when cured of their M or S dsRNA show the same increase as their mild-type counterparts: Several mechanisms can be imagined to explain L-A-HN copy number repression by M. M and L-A-HN could be competing for rate-limiting L-A-HN products or host gene products. Alterna-


TABLE 3

Relative dsRNA amounts in isogenic wild-type killer, suppressive and cured colonies

No. of colonies analyzed (all containing L-A-HN) dsRNA

Molar ratios of

L M o r S cured" parentb

Strain origin Genotype Method of curing S+ S- parent parent

~~

Cytoplasm M+or M-or L L

18 18 [KIL-kl] Cycloheximide curing 4 4 8 % A364A A364A [KIL-kl] Heat curing (39") 12 4 3 t o 5 $4 K7 K7 [KIL-kl] Cycloheximide and heat 12 4 10 %

1490 A364A [KIL-kl] Cycloheximide and heat 2 5 3 t o 5 $4

1101' A364A [KIL-kl] Cycloheximide and heat 4 7 3 t o 5 $4

1488' 18 [KIL-kl] Cycloheximide and heat 2 4 8 ?h

1368' 1256 [KIL-b] Heat curing (37") 2 1 10 -1

2026 1364 [KIL-kp] mktl loss of kp at 30" 1 1 2 t o 3 % 2028 1364 [KIL-k2] m k t l loss of kp at 30" 1 1 2 t o 3 % 1385 1364 [KIL-k~] Heat curing (37") 5 3 2 t o 3 %

1494 A364A [KIL-k,] Heat (37") and cyclohexi- 1 3 5 '/4

curing (39")

curing (39")

curing (39") cytoduction

curing (37")

mide curing

SP180d A8209B [KIL-kl] Heat (39") and cyclohexi- 1 3 6 '/s

SP176d A8209B [KIL-SI] Heat (39") and cyclohexi- 1 2 6 '/s

SP17Sd A8209B [KIL-s3] Heat (37" or 39") 1 3 6 %

mide curing

mide curing

" T h e molar ratio of the amount of L dsRNA in the strains cured of M or S to that in the

6 T h e molar ratio of the amount of M in the parent strain to the amount of L in the parent

' 1101, 1488 and 1368 are all isogenic nuclear strains constructed from 1020 by cytoduction. dSP180, SP176, SP178, are all isogenic nuclear strains. Note: relative amounts are not to be compared between different strains except where noted

or '. In those cases, the L copy numbers were identical within members of each group of uncured strains.

parent uncured strain.

strain.

tively, M could be actively repressing L-A-HN by an M-encoded gene product. To determine the role, if any, of M-encoded killing and resistance functions in L-A copy number, strains containing neutral or suppressive plasmids were used and compared with their isogenic nuclear wild-type K1 or cured deriva- tives (Table 3). The results show clearly that suppressive and neutral plasmids behave in the same fashion as the wild-type killer plasmids from which they were derived. S3 was previously shown to lack two-thirds of M's internal sequences (FRIED and FINK 1978). Thus, these sequences are not involved in L- A copy number control.

208 S. G. BALL. C. TIRTIAUX AND R. R. WICKNER

LANE: 1 2 3 4 5 6 7 8 9 10 11

W ' -

L-A

M I : - + - - - + + - - - - L A : + + + + + + + - - - -

L-(BC): + + + + + + + + + + + ski 2-2: - + + - - - - - + - +

FIGURE 2.-ski2-2 superkiller strains have more than 100-fold less L-A dsRNA when M I is present. Nucleic acids extracted from the same amount of stationary phase unbroken cells were loaded on Northern gels as described in text. Top, a 4-hr autoradiograph of a nitrocellulose blot hybridized with 1 pg of IO5 cpm/pg of denatured labeled L-A dsRNA from strain S7. Bottom. the same blot hybridized with L-C dsRNA from strain SI42 labeled at the same specific activity. Lanes I , 4 and 5 are extracts from strain 2176 + SP226 (ski2-2 L-(BC) L-A-HN); lane 2 is from strain I101 (SKI+ L-(BC) L-A-HN MI); lane 3 from 21 76 (SKI+ L-(BC) L-A-HN); lanes 6 and 7 are extracts from strain 1101 + SP226 (ski2-2 L-(BC) L-A-HN MI); lanes 8. 9. IO and 1 1 are extracts of a complete tetrad from cross SP209 (see Table 4).

L-A and L-(BC) amounts are unafected by all mak or mkt mutations examined that are necessary for maintenance of the killer plasmid only: Only three nuclear genes, namely, MAK3, MAKIO and PET18 , are necessary for maintenance of

NER and TOH-E 1982). T h e mak3, maklO and pet18 mutations do not affect copy number of L-(BC) (WICKNER and TOH-E 1982). Although other ~ l a k mutations do not prevent L-A maintenance, it is not known whether they affect L-A or L-(BC) copy number in any significant manner. We have, therefore, analyzed relative dsRNA copy number from tetrads of crosses in which the diploids were heterozygous for the mak mutation under investigation. Such diploids carried L-A-HN + L-(BC), L-A-HN + L-(BC) + MI, or L-(BC) alone. Table 4 shows that consistent effects as great as two-fold were never seen in the case of L-A-HN + L-(BC) or L-(BC) alone.

When M I was present in mak-/+ diploids the mak- K- segregants showed an increase in L copy number identical with that found on curing the MAK+ segregants of MI and typical of the increase expected for the L-A-HN + MI pair used (from A364A). This increase in L was shown by blotting experiments to be due to an increase in L-A-HN (results not shown). Thus, the tnak-

L-A-HN or L-A-HE (WICKNER 1980; SOMMER and WICKNER 1982a,b; WICK-


TABLE 4

Segregation analysis of L dsRNA copy number

Cross no.

Diploid Parental strains m v x v p e

3498 3578 3577 363 1 3632 3645 3646 3648 3649

3669

3660 SP209 SP2 10 SP211 366 1 3666 3662 3667 3668 XI42 X137

1101 X 2145 3552-4B X 3544-3B 1101 X 3545-15B 1925 X 1019 1925 X 2181 3609-1B X 1019 3609-1B X 2181 1591 X 1020 1591 X 2176

mak4-I/+ mak 7- I /+ makI7-I /+ m a k l - l l + mak I - I /+ mak4-I/+ mak4- l /+ mak24-l /+ mak24-1/+

1475 X 2027 mkt 1-I/+

S486 X S600 SP226 X 1090 SP226 X 2176 SP226 X 1101 S486 X S596 2348 X S596 S486 X S599 2348 X S599

S486 X S597 S442 X S648

3637-8AA X 2348A

mks l - I /+ ski2-2/+ ski2-2/+ ski2-2/+ ski3-1 I / + ski3-I I / + ski7- l /+ ski7- l /+ ski4- l /+ ski8- l /+ ski8-2/+

Diploid dsRNAs

L-(BC) L-A-HN MI L-(BC) L-A-HN MI L-(BC) L-A-HN Mi L-(BC) L-(BC) L-A-HN L-(BC) L-(BC) L-A-HN L-(BC) L-(BC) L-A-HN

L-(BC) L-A-HN

L-(BC) L-(BC) L-(BC) L-A-HN L-(BC) L-A-HN MI L-(BC) L-(BC) L-A-HN L-(BC) L-(BC) L-A-HN L-(BC) L-(BC) L-(BC) L-A-HN

No. of tetrads

analyzed

3 3 3 4 4 5 5 5 5

L in mak- or ski- clones

L in wild-type spore clones 8

2 3 3 1 1 1 1 1 1

Ratio:

5 1

3 1 11 2 to 4 11 5 3 1 3 2 4 4 3 2 4 4 4 2 3 3 2 3

mutations tested did not directly affect L-A-HN or L-(BC) levels but only affected L-A levels by causing the loss of MI.

Comparisons of strains with different nuclear constitutions with respect to mktl-1 (Table 3) or of spores generated from crosses involving only L-A-HN, L-(BC) and the heterozygous mktl-1 mutation show that L-A-HN is also insensitive to the mktl-1 defect to our level of detection (Table 4).

In the absence of MI, both L-A-HN and L-(BC) respond to most ski mutations by increasing their copy number: Mutations in ski2, ski3, ski4, skib, ski7 and ski8 have been previously shown to increase M1 dsRNA copy number four-fold or more without having any effect on total L dsRNA (TOH-E, GUERRY and WICKNER 1978; RIDLEY, SOMMER and WICKNER 1984). In view of our results it seemed that, if M were absent from the cross, L-A dsRNA should be increased in ski- segregants compared with SKI+ segregants. Crosses were then performed in which a ski- mutation was heterozygous in the diploid that contained the three possible combinations of L and M plasmids [L-(BC) alone, L-(BC) + L-A-HN, and L-(BC) + L-A-HN + MI] (Figures 3 and 4; Table 4). To our surprise, not only did L-A respond to the presence of the ski2-2, ski3-11, ski4-1 or ski7-1 mutations by increasing its copy number five-fold, but L-(BC) behaved in a

210 S. G. BALL, C. TlRTlAUX AND R. B. WICKNER

TETRAD n"1 n"2 n"3 *+*

S K 1 2 : + - + - - + - + - + - + FIGURE 3.-A fivefold increase in L dsRNA cosegregates with the shi2-2 mutation in crosses

lacking M I dsRNA. Nucleic acids were extracted from segregants of cross SP210 (ski2-2/+ L-A- HN L-(BC); see Table 4) and electrophoresed on agarose gels. T h e relative amounts of L-A-HN + L-(BC) were compared as described in MATERlALS AND METHODS.

TETRAD n"1 n"2 A* - NUCLEAR DNA

- L dsRNA (L-(BC))

18s rRNA

SK12: - + - + - - + + FIGURE 4.-A two- to fourfold increase in L-(BC) dsRNA cosegregates with the shi2-2 mutation

in crosses lacking both M I and L-A. Equal amounts of nucleic acids, extracted from segregants of cross SP205 (shi2-2/+ L-(BC)), were electrophoresed on agarose gels. T h e relative amounts of L- (BC) were compared as in MATERIALS AND METHODS.

similar fashion. In the case of the sRi2-2 defect, sRi2-2 strains isogenic for nuclear genes and harboring various dsRNA plasmids were constructed by cytoducing M I + L-A + L-(BC) or L-A + L-(BC) into SP226. T h e results shown in Figure 5 and Table 5 indicate that, in ski2-2 mutants, total L dsRNA is 30-fold higher when MI is absent than when MI is present. T h e same conclusion was reached whether using dsRNA prepared from stationary phase cells by the method of FRIED and FINK (1 978) or from log phase cells by lysing the cells and purifying the RNA by CFl 1 chromatography (Table 5).

L DSRNA COPY CONTROL 21 1

- NUCLEAR DNA - L-A + L-( BC) - Mi - 25s rRNA - 18s rRNA

1 i L K + K- K+ K- K+ K- K+ K- K+ K-

DILUTION: 1 1 ' /z 1/2 '/5 1/5 %o %o 1/20 1/20

FIGURE MI dsRNA reduces L copy number 30-fold in ski2-2 strains. T h e isogenic strains 2340 (ski2-2 L-(BC) L-A-HN MI) SBI (ski2-2 L-(BC) L-A-HN) contain the same nuclear and mitochondrial genomes and the same L-(BC) and L-A-HN (from strain A364A). Nucleic acid extracts were serially diluted and equal volumes electrophoresed on agarose gels. T h e 1:20 dilution of the K- strain (Sa l ) has more L than the undiluted K+ strain (2340).

TABLE 5

Relative and total dsRNA amounts in nuclear isogrnic ski2-2 strains

Relative amounts of Tom1 &RNA dsRNA estimate by

cell lysis and Extraction of cell lysistnd CFI I chrom- whole cellh CFI 1 atography (X of

Cytcduction total nucleic Strain construction Cytoplasm L M L M acids)

SP226 L-(BC) 1 1 0.15 2340 1 101 + SP226 L-(BC) + L-A-HN 1 1 1 1 0.25

+ MI SB 1 2 176 + SP226 L-(BC) + L-A-HN 30 20 3

dsRNA relative amounts can be compared between the three strains. 'The method of FRIED and FINK (1978) was used for these determinations. 'The cell lysis-CFI 1 chromatography method was used for these determinations (TOH-E,

GUERRY and WICKNER 1978).

Some superkiller mutants shout more than a 100-fold decrease of L-A copy number in the presence of M I : When several ski- spores from crosses SP209 and SP211 were compared, it was clear that L dsRNA amounts in ski- K++ segregants and ski- K- spores harboring only L-(BC) were not significantly different (data not shown). Since MI does not affect L-(BC) copy number, this result suggested that most of the L in the ski- K++ strains was L-(BC). To ascertain exactly how much L-A dsRNA was left in the K++ segregants, equal cell amounts of RNA from various strains were denatured, loaded on gels, transferred to nitrocellulose filters and hybridized with an in vitro labeled L-A probe or with an L- (BC) probe (Figure 2). We found more than a 100-fold repression of L-A copy number in ski2-2 strains by M I dsRNA but, again, no effect of M I on L-(BC)


TABLE 6

Bypass of mak mutations 50 generations after cytoduction

Cytoduction donor

Strain: 1101 SP405 1488 1368 Cytoduction SKI: + ski2-2 + +

recipient Cytoplasm: [KIL-kl]assra [KIL-k~]assra [KIL-klIls [KIL-klIb

inn k 1-1 mak2-I inak3-I mak4-1 inak7-I mnk8-2 inak IO- 1 m a k l l - 1 m a k l 2 - l inak13-I ina k 14- I inak 16-1 makl7-l ma k 18- I inak22-I inak27-1

~~

+, normal killing (no K- single colonies); w, weak killing (no K- single

Strains 1101, 1488 and 1368 are isogenic nuclear strains carrying the colonies; -, presence of K- single colonies.

cytoplasm from strains A364A, 18 and 1256, respectively.

copy number. Such K++ strains have five-fold less L-A and two- to four-fold more L-(BC) than wild-type killers, leading to an L band consisting of more than 90% L-(BC) dsRNA.

One could not have predicted a priori whether the repression of L-A by increased M1 or the derepression of L-A due to the ski- mutation would predominate (be epistatic) in ski- MI L-A-HN L-(BC) strains. In fact, repression by M I is the major (epistatic) effect.

Wild-type K1 killers differing in their ability to decrease L-A copy number also differ in their requirements for MAK gene products: Strain 1368, which shows a ten-fold repression of L by MI, carries the [KIL-b] (bypass) plasmid defined by its not requiring some of the MAK genes required by the [KIL-kl] of strain A364A (ToH-E and WICKNER 1980). In isogenic nuclear strains, [KIL-b] has twice as much M I dsRNA as strain 18-derived killers and four times as much as A364A- derived killers, with which most of the genetic experiments have been carried out (Table 3). Furthermore, strains with [KIL-b] are superkillers; those with the strain 18 cytoplasm show intermediate killing, whereas A364A is a normal killer. As MI copy number, degree of killing ability and degree of L-A repression seem to correlate with bypass of mak genes in the case of [KIL-b], we have examined the bypass pattern of [KIL-b], [KIL-k1]18 and [ K I L - ~ I I A ~ ~ ~ A by scoring killing of mak- cytoductants 50 generations after transfer of the different killer cytoplasms. The results, listed in Table 6 , clearly demonstrate that [KIL-kllla has an intermediate bypass pattern between [KIL-k1]~364~ and [KIL-


b]. This observation correlates with the intermediate copy number of the MI of strain 18. All of the mak- mutations reported to be bypassed by [KIL-b] in meiosis were also bypassed in cytoduction. In addition, we have observed bypass of mak-1-1, mak8-2, makl3-1 and mak22-I. In contrast with the results obtained in meiosis, maklb-1 and makl2-1 seem to be also bypassed by [KIL- b] when it is introduced by cytoduction (see also GUERRY-KOPECKO and WICK- NER 1980).

The fact that “bypass,” in some cases, means only a more gradual loss of M I , and the correlation between MI dsRNA copy number and ability to bypass mak defects, raised the possibility that bypass is, in fact, delayed expression in time of the mak defects due to a high starting MI dsRNA copy number. Cytoduction of the [KIL-kl]A364A into the same mak- recipients from a ski2-2 donor clearly rules out this possibility (Table 6). Had we been dealing with delayed expression of the defects through high starting M1 copy number, then SP405, having the A364A killer plasmid but in five-fold higher copy number, should have shown the same bypass pattern as [KIL-b]. If, by contrast, there is genetic interaction between SKI and MAK genes, the killer plasmid from strain SP405 should show the same bypass pattern as the A364A killer plasmid from which it was derived, as is the case (Table 6).

DISCUSSION

We find that all M I and M2 killer dsRNAs analyzed reproducibly reduce L- A-HN copy number. Previous genetic evidence for antagonism between L-A- HN and M2 is the exclusion of M2 by L-A-HN in mktl or mkt2 strains (WICKNER 1980, 198313). The results obtained by curing suppressive and neutral strains have ruled out any effect of the killer or resistance functions on L-A-HN copy number. The defective killer genome S3 only has one-third of the original MI sequences left (FRIED and FINK 1978) and codes in in vitro translation experiments for only a small 8.0-kilodalton (kd) polypeptide-a remnant of the 32- kd toxin precursor (BOSTIAN et al. 1980; BOSTIAN, JAYACHANDRAN and TIPPER 1983). However, this product was not detectable in vivo, and no other sub- stantial open reading frames are found in S3 (D. J. THIELE, E. M. HANNIC and M. J. LEIBOWITZ, personal communication), making the possibility of an M product that would specifically lower L-A copy number remote.

Competition for limiting replication factors is a likely explanation for the effect of M on L-A.. The MAKIO, PET18 and M A K 3 gene products are necessary for both L-A and M maintenance; therefore, competition for one or more of these may account for the increase of L-A on loss of M. L-A copy number is insensitive to mutations in the other MAK and M K T genes we have tested. It is also possible that the competition between M and L-A is for L-A’s own gene product(s); the L-A-coded coat protein (HOPPER et al. 1977; SOMMER and WICKNER 1982b) is known to encapsulate both L-A and MI (HARRIS 1978; BOSTIAN, STURGEON and TIPPER 1980). Clearly, the increase of L-A on elimination of M is well more than what can be accounted for on a molecular weight basis (1 kb of L for 1 kb of M) or a molar basis (one molecule of L


M L-A L-( BC)

N EG N EG - SKI PRODUCTS 4

REPLICATED dsRNAs FIGURE 6.-Diagrammatic summary of interactions among SKI products and L and M dsRNAs.

The SKI products lower the copy numbers of M, L-A and L-(BC). The degree of M inhibition of L-A copy number depends on M copy number and is greater in a ski- strain than in a S K P strain.

for one molecule of M). One would thus have to assume very different needs and affinities of M and L-A for the common replication factors.

In wild-type strains, M1 requires for its replication one of several forms of L-A. All wild-type K1 killers have L-A-HN with MI. The usual explanation for this requirement is that L-A encodes the major coat protein in which M1 is encapsidated. Our work raises a second possibility. The SKI products act to lower the copy number of L-A, L-(BC) and MI. Perhaps L-A, the species present in largest amount, soaks up most of the SKI products, thereby pre- venting their acting to lower M I copy number (to zero). Another observation that supports this idea is that, in the absence of the SKI products, M1 can replicate in the absence of L-A or in the presence of an L-A variant which is inadequate to maintain M in wild-type strains (L-A-E) (RIDLEY, SOMMER and WICKNER 1984).

VODKIN, KATTERMAN and FINK (1974) noted that killers cured of M1 by cycloheximide had elevated L dsRNA, whereas spontaneous K- colonies had reduced L dsRNA. In most strains, cycloheximide cures M1 but not L-A (our unpublished observations). Presumably, VODKIN, KATTERMAN and FINK’S spontaneous K- strains lacked both M I and L-A. The fact that the suppressive plasmids behave with respect to L-A repression exactly in the same manner as their wild-type counterparts suggests that L-A copy number repression and suppression by S dsRNAs of M1 dsRNA are unrelated phenomena.

Our demonstration that L-(BC) copy number is under the control of some SKI genes is the first link between this molecule and any components of the killer system. The fact that L-(BC), L-A and M1 dsRNAs all respond to ski mutations by increasing their copy number suggests that these defects are not merely the opposite of inak mutations. Had they been such, one would expect to get a decrease both in L-A and in L-(BC) copy number in at least some mak mutants. The SKI genes could thus control negatively a very general mecha- nism in dsRNA replication or expression (Figure 6).


Most ski- mutants are cold sensitive for growth only in the presence of M (RIDLEY, SOMMER and WICKNER 1984). The fact, shown here, that ski2-2 L-A + L-(BC) cells have at least ten-fold more total dsRNA than the ski2-2 L-A + L-(BC) + MI strains, yet the former are cold resistant while the latter are cold sensitive, indicates that higher total dsRNA levels in ski- strains cannot account for the cold sensitivity. The effect is specific for high levels of M dsRNA.

Different bypass patterns of mak- mutations seem to occur frequently among different K1 killers. This variability makes it necessary to use the A364A cytoplasm for mapping purposes. Among the few killer strains examined, there seems to be a relation between superkilling, M1 copy number, ability to repress L-A copy number and bypass pattern of mak- mutations. We suspect that these properties are all causally connected, but it is not yet clear whether they are properties of L-A, M I or other cytoplasmic molecules.

We thank S. PORTER RIDLEY and STEVE S. SOMMER for many strains and for helpful discussions and STEVE S. SOMMER for the data on skid in Table 4.

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Corresponding editor: E. JONES

genetic control of l-a and l-(bc) dsrna copy number

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