Genetic and Molecular Characterization of Suppressors of ...Genetics 122: 29-46 (May, 1989) BUCHMAN et al. 1988). However, the sites at which these factors bind are important for SIR-mediated

Copyright 0 1989 by the Genetics Society of America

Genetic and Molecular Characterization of Suppressors of SIR4 Mutations in Saccharomyces cerevisiae

Rogene Schnell,’ Linda D’Ari, Margit FOSS, David Goodman and Jasper Rine Department of Biochemistry, University of Calgornia, Berkeley, Calgornia 94720

Manuscript received October 19, 1988 Accepted for publication February 2, 1989

ABSTRACT In order to learn more about other proteins that may be involved in repression of H M L and H M R

in Saccharomyces cerevisiae, extragenic suppressor mutations were identified that could restore repression in cells defective in SZR4, a gene required for function of the silencer elements flanking H M L and HMR. These suppressor mutations, which define at least three new genes, SANI, SANZ and SAN?, arose at the frequency expected for loss-of-function mutations following mutagenesis. All sun mutations were recessive. Suppression by sanl was allele-nonspecific, since sanl could suppress two very different alleles of SZR4, and was locus-specific since sanl was unable to suppress a SIR? mutation or a variety of mutations conferring auxotrophies. The SANl gene was cloned, sequenced, and used to construct a null allele. The null allele had the same phenotype as the EMS-induced mutations and exhibited no pleiotropies of its own. Thus, the SANl gene wag not essential. SANI-mediated suppression was neither due to compensatory mutations in interacting proteins, nor to translational missense suppression. SANl may act posttranslationally to control the stability or activity of the SIR4 protein.

T HE repression of transcription from the silent mating type loci, HML and HMR, of Saccharo-

myces cerevisiae requires the combined action of at least four proteins encoded by the SIR genes (KLAR, FOGEL and MACLEOD 1979; HABER and GEORGE 1979; RINE et al., 1979; RINE and HERSKOWITZ 1987) and regulatory sites flanking HML and HMR that are known as E and I (ABRAHAM et al . 1984; FELDMAN, HICKS and BROACH 1984). Mutations in any of the SIR genes result in loss of repression of both HML and HMR, whereas mutations in an E site or an I site result in loss of repression only of the locus adjacent to that site. The E site at HMR has been termed a “silencer” (BRAND et al . 1985) to reflect the ability of this site to block expression of a variety of genes, when they are inserted at HMR, in a manner that is independent of the orientation of the site (BRAND et al. 1985; SCHNELL and RINE 1986). The silencer is also capable of pro- moting the replication and segregation of plasmids in yeast, and the mitotic stability of these plasmids also depends upon the SIR genes, which were defined by their role in transcriptional repression (KIMMERLY and RINE 1987; BRAND, MICKLEM and NASMYTH 1987; KIMMERLY et al. 1988).

The mechanism by which the SIR proteins mediate silencer function is unknown. An analysis of proteins that bind the silencer has revealed two factors known as ABFl or SBFl and GRFl or RAPl, neither of which is encoded by any SIR gene (SHORE et al . 1987;

I Present address: Department of Genetics and Cell Biology, 250 Biolog- ical Center, University of Minnesota, St. Paul, Minnesota 55108-1095.

Genetics 122: 29-46 (May, 1989)

BUCHMAN et al . 1988). However, the sites at which these factors bind are important for SIR-mediated processes since mutant sites that no longer bind the- factors in vitro no longer mediate SIR-dependent processes in vivo (BRAND, MICKLEM and NASMYTH 1987; KIMMERLY et al. 1988). At present it is unclear whether the SIR proteins bind directly to the silencer DNA, or bind to the other proteins bound to the silencer, or act through some other mechanism.

Genetic suppressor analysis offers a general ap- proach to identifying genetic interactions between gene products (ROTH 1973). Mutations in a SIR gene result in the nonmating phenotype of an a/a diploid due to the simultaneous expression of both the a and a mating-type information present at HMRa and HMLa, respectively. The gene SUMl was identified by a recessive allele that suppresses the nonmating phenotype of mutations in the SIR2 and SIR3 genes. The absence of SUMl function bypasses the need for SIR2 and SIR3 since suml -1 suppresses null alleles of these genes (KLAR et al . 1985). suml -1 can also suppress some alleles of SIR4 but cannot suppress a null allele (A. J. S. KLAR, personal communication). The role of the SUMl gene product is enigmatic, for the recessive nature of suml-1 implies a positive control step between the SIR2 and SIR3 genes and the silencer. In order to use suppressor analysis to learn more about the relative roles of the SIR proteins in silencer function, we have identified a number of genes in which mutations were able to restore partial repression to HML and HMR in some sir4 mutants. This paper describes the characterization of these

30 R. Schnell et al.

mutations and a molecular analysis of one of t he genes identified by these mutations.

MATERIALS AND METHODS

Media and genetic methods: Yeast rich medium (YPD), minimal medium (YM) and sporulation medium were prepared as described previously (BARNES et al. 1984). Amino acids and bases were added as needed at a concentration of 30 rg/ml. Standard genetic manipulations were performed as described previously (MORTIMER and HAWTHORNE 1969). Yeast cells were transformed as described (HINNEN, HICKS and FINK 1978), except that spheroplasts were prepared with lyticase, a gift from the Schekman Laboratory. Matings between sir4 strains and SIR strains of the opposite mating type that contained complementing auxotrophic mutations were formed by mixing the two strains together on rich medium (YPD) and incubating overnight at 23". Diploid prototrophs were selected by streaking the mating mixture onto minimal medium (YM). Diploid prototrophs formed between cells of the same mating type were selected following spheroplast fusion. Approximately 5 X 10' spheroplasts prepared from each of the parent strains were mixed together in the presence of 40% polyethylene glycol 4000 (Sigma), 5% ethylene glycol, 10 mM Tris-HCI (pH 7.6), and 10 mM CaC& (0.5 ml) and incubated for 5-10 min at room temperature. The fusion mixture was added to regeneration agar and plated onto minimal medium to select prototrophic fusants.

Strains and plasmids: The strains and crosses used in this study are described in Tables 1 and 2. The plasmid pJR106, isolated from a YEp24 genomic library by complementation of a sir4-ochre mutation (sir4-351), contains a truncated SIR4 gene that encodes the carboxy-terminal75% of SIR4 (SCHNELL 1987). The plasmid pJR72, which contains the truncated SIR4 gene from pJR106 on a 3.6-kbp EcoRI-Sal1 fragment inserted into pSEY8 (EMR et al. 1986), was constructed by W. KIMMERLY. The sir4-9 allele causesa temperature sensitive nonmating phenotype (RINE and HER- SKOWITZ 1987). pJR528, a multicopy S A N l clone (see below), was constructed by inserting a 4-kbp BamHI genomic fragment containing the SANl gene into the BamHI site of YEp24.

Mutant isolation: Stationary phase cells (4 ml) were har- vested from independent cultures grown in YPD. The cells were resuspended in 1.5 ml 200 mM sodium phosphate (pH 7.0) and 0.7 ml of the cell suspension was added to 1 ml 200 mM sodium phosphate (pH 7.0) containing 50 PI ethyl- methanesulfonate (EMS). The mixtures were vortexed, then agitated gently on a rotary shaker for 1 hr at room temperature. A 0.5-ml portion of the mutagenized cell suspension was transferred to 9 ml of sterile sodium thiosulfate (5% w/ v) to inactivate the EMS. The cells were promptly washed i n 10 m l sterile water. The final suspensions, which contained approximately lo6 to lo7 viable cells per ml, were stored at 4' C for 2-4 days and plated on solid YPD medium at approximately 800 viable cells per plate. Colonies formed from the mutdgenized cells following incubation at the restrictive temperature were replica plated onto a lawn of the appropriate mating-type tester strain. Mutants that mated with the tester lawn were isolated from the original YPD plates, colony purified, and the mating ability of each mutant was retested. A summary of the mutant isolations is described in Table 3.

Nomenclature of mutants: The rsf mutants isolated in this study were so named because the suppressor mutations caused the cells to behave phenotypically as revertants of

the 2ir four mutation. Each mutant was designated by a number such as [50]rsf3.11. The number in brackets referred to the strain from which the mutant was derived (JRY50 in this case). The number to the left of the period referred to the clone from which the mutant was isolated. Thus, mutants with different clone numbers were independent. The genes defective in these mutants were designated SAN for SIR antagonist.

Dominance tests: Each mutant was tested for dominance by mating the mutant to a sir4 strain and monitoring expression of genes at HML and HMR in the resulting diploid. Mutants isolated in JRY50 (HMLa MATa HMRa s ir4-9) were mated to the strain JRY367 (HMLa matal HMRa s ir4-167) , and the ability of the resulting diploids to sporulate at 30" was tested. Both 30" and 34" are nonpermissive temperatures for sir4-9. The sir4-167 allele is uncharacterized, but has a mutant phenotype at all temperatures tested. If the suppressor mutation that restores repression of HML and HMR is dominant, the resulting diploid should be unable to sporulate since repression of HML and HMR would prevent expression of the a2-protein whose presence is required for sporulation (STRATHERN, HICKS and HERSKOWITZ 198 1). If the suppressor mutation is recessive, then HMLa and H M R a would be expressed which, together with MATa (and HMRa) expression, would lead to sporulation.

Mutants isolated from YRS23 (HMLa MATa HMRa sir4- 9 ) were mated to JRY184 (HMLa matal HMRa s ir4-351) . Both sporulation and mating phenotypes of the resulting diploids were tested following incubation at 34". If a mutation were dominant, repression of HMLa and HMRa would prevent expression of the a1 gene product, thereby preventing sporulation and enabling the diploid to exhibit the a mating type (KASSIR and SIMCHEN 1976).

Mutants isolated from YRS32 (HMLa matal HMRa sir4- 9 ) were mated to YRS41 (HMLa matal HMRa s ir4-9) and the mating ability of each of the resulting diploids was tested following incubation at 34", the restrictive temperature for the sir4-9 mutation. Complete repression of HML and HMR by a dominant suppressor mutation would inhibit a mating by preventing expression of the a1 gene, a product required to activate expression of a-specific genes (SPRAGUE, JENSEN and HERSKOWITZ 1983). By default, such a diploid would mate strongly as an a cell. Partial repression of HML and HMR by a phenotypically weak, though dominant mutation would result in a bimating phenotype (RINE et al. 1979). That is, most cells in the colony would mate with a cells and others would mate with a cells. Alternatively, a recessive mutation should result in a diploid that mates exclusively as an a cell.

Complementation tests: Mutants isolated from HMLa MATa HMRa sir4-9 (JRY50) were tested for their ability to complement each of the mutants isolated from HMLa matal HMRa sir4-9 (YRS32) by mating 8 of the mutants from- JRY50 to each of the mutants from YRS32 and determining the mating phenotypes of the resulting diploids at 34". Two alternative outcomes were predicted, depending on whether or not HML and HMR were expressed in the diploid. Non- complementing mutations would give rise to a diploid that mates as an a cell, since the homozygous suppressor mutation would repress expression of the a 1 and the a2 genes at both HML and HMR. Alternatively, suppressor mutations that complement should result in a nonmating diploid. Un- expectedly, the diploids formed by mating eight different mutants from HMLa MATa HMRa sir4-9 URY50) with each of the mutants isolated from HMLa matal HMLa sir#-9 (YRS32) were all nonmaters at 34". By this test, it appeared that all of the suppressor mutants from JRY50 complement all of the suppressor mutants from YRS32, resulting in

Suppressors of SIR4 Mutations 31

TABLE 1

Strains

Strain Genotype"

JRY4 JRY22 JRY26 JRY50 JRY79 JRY 184 JRY 187 JRY252' JRY2.5Sb JRY254b JRY294 JRY3 1 1 JRY367 JRY.527 JRY.528 JRY660 JRYl6Ol XR320-12:) XRS20-10b YRS23 YRS32 YRS4 1 YRS75 YRS77 YRS104 YRS107 YRS178 YRS277 YRS575 YRS376 YRS393 YRS401 YRS416 YRS437 YRS898 YRS932 Y RS933 YRS934 YRSl138 YRSl179 YRSl I80 YRSl183 YRSl184 YRSl185 YRSl186 [50]rsf3-11 [50]rsfl-6 [23]rsf3-1 [23]rsf4-6 [23]rsf.5.5 [23]rsf10.13

matal HMLa HMRa sir4-351 ade2 leul ura3 canl cyh2 rmel MATa ade2 his3-532 trpl-289 ura3 MATa his?A2 ura3-52 MATa sir4-9 his3-532 trpl-289am ura3-52 MATa HMLa HMRa HO ade5 his5 met4 ura4 matal HMLa HMRa sir4-351 ade2 leul ura3 MATa sir3-8 ade2-1 his4-580 leu2= lys2-oc trpl-am ura3-52 rmel Met- Tyr- h4ATa his4-260,39 leu2-I ura2-9,15,30 canl MATa thr?- I O MA Ta lys I - 1 MATa sir4-9 sanl-1 leu2-1 his3-532 his4-260,39 (ura?-52 and/or ura2,9,15,?0) MATa his4-260,39 leu2-1 ura2-9,15,30 matal HMLa HMRa sir4-167 ade2 leul ura3 canl cyh2 rmel MATa ade2-IO1 his3A200 lys2-801 ura3-52 Met- MATa ade2-I01 his3A200 lys2-801 uta?-52 tyrl

MATa sir4-9 sanl::HIS3 his3 lys2 ura3 Met- MATa sir4-351 ade2 cryl-1 canl-11 cyh2-21 his4-am leul lys2-oc rmel trpl-am tyrl-oc ura3 MATa sanl-1 his4-580 leu2-I trp5 ura3 (ura2-9,15,30 ?) MATa sir4-9 his4 leu2-I ura2-9,15,30 ura3 matal HMLa HMRa sir4-9 ade2 leul trpl-289am ura3 matal HMLa HMRa sir4-9 ade2 his3-532 ura3 MATa sir4-351 sanl-I lys2-oc trpl-am ura3 MATa sir4-351 ade2 his?-532 his4-am leu1 lys2-oc trpl-am trp5 tyrl-oc ura3 MATa sir4-351 sanl-2 ade2 lys2-oc trpl-am ura3 MATa sir4-351 sanl-2 leul trpl-am ura? (his3 and/or his4-am) MATa sir4-9 trpl-am ura3 suc2-A9 MATa sir4-9 san3-1 his4 leu2 ura3 MATa sir4-9 sanl-1 his3-532 trpl-289am trp5 ura3-52 MATa sir4-9 sanl-2 his3-532 trpl-289am ura3-52 MATa hmrA::SUP3am his4 leu2 ura3 trplam suc2A MATa hmrA::SUP3am sir4-9 his4 leu2 ura3 trplam suc2A MATa sir4-9 ade2 lys2-oc trpl-am ura3 MATa sir4-9 leu2 ura3 (his? and/or his4) Ylp5::SANl (otherwise isogenic with YRS376) MATa sanI::HIS3 ade2-I01 his3A200 lys2-801 ura?-52 Met- MATa sanl::HlS3 add-101 his3A200 lys2-801 ura3-52 tyrl MATa sanI::HIS3 ade2-101 his3A200 lys2-801 ura3-52 tyrl MATa sir4-9 san3-1 ade2 his4 leu2 ura3 MATa sir4-9 sanl-1 his3 lys2 ura3 ( trpl and/or trp5) (Met?) MATa sir4-9 sanl-1 his3 lys2 ura3 (trpl or trp5) (Met?) MATa sir4-9 sanl-2 his3 lys2 trpl-am ura3 (Met?) MATa sir4-9 sanl-2 his3 lys2 trpl-am ura3 MATa sir4-9 san2-1 his4 leu2 trpl-am ura3 MATa sir4-9 san2-1 his4 trpl-am ura3 MATa sir4-9 sanl-1 hid-532 trpl-289am trp5 ura3-52 MATa sir4-9 sanl-2 his?-532 trpl-289am ura3-52 MATa sir4-9 san3-1 his4 ham6 leu2-I ura2-9,15,30 ura3 MATa sir4-9 san2-1 his4 leu2-1 ura2-9,15,30 ura? MATa hmral sir4-9 his4 leu2-1 ura2-9,15,30 ura3 MATa hmral sir4-9 his4 leu2-1 ura2-9,15,30 ura3

MA TO^ lys 1 - 1

[23]rsf10.17 MATa hmral sir4-9 his4 leu2-1 ura2-9,15,30 ura?

a Unless otherwise noted, all strains were HMLa and HMRa and were from the laboratory strain collection. Strains were from the Herskowitz laboratory collection.

repression of HMLa, HMRa and H M R a and thus the non- sir4-9 mutant phenotype was incompletely suppressed in mating phenotype. This interpretation implied that a large these mutants. Similarly, diploids formed by mating 21 of number of genes could be mutated to produce the San- the mutants isolated from HMLa MATa HMRa sir4-9 phenotype. A more likely explanation was that this test was (YRS23) to each of the mutants isolated from HMLa matal unable to detect nonconlplementation because the muta- HMLa sir4-9 (YRS32) were weak a maters at 34" ("pap" tions do not fully repress HML and HMR. The second to"pap/+"). This result is intermediate between that ex- interpretation was consistent with the observation that the pected for full complementation (nonmating phenotype)

R. Schnell et al. 32

TABLE 2

Description of crosses

Cross Parents

JRY543 JRY527 X JRY528 XDGl [50]rsf3-11 X [23]rsf3-1 XDG2 [50]rsfl-6 X [23]rsf3-1 XDG5 JRY50 X [23]rsf3-1 XDG6 JRY50 X [23]rsf4-6 XDG7 JRY22 X [23]rsf3-1 XDG9 JRY50 X [23]rsf5.5 XDGlO JRY50 X [23]rsf10.13 XDGll JRY50 X [23]rsf10.17 XRS5 [50]rsfl-6 X [23Jrsf4-6 XRS7 [50]rsfl-6 X XR320-12a XRS15 YRS104 X YRS23 XRSPO [50]rsf3-11 X JRY252 XRS2l [50]rsfl-6 X JRY252 XRS29 [50]mfl-6 X JRY294 XRS3O JRY187 X XRSSO-lob XRS37 JRY50 X XRS20-10b XRS50 YRS277 X YRS178 XRS97 YRS933 X YRS932 XRS 102 Y RS898 X YRS4 16 XRSl12 JRY50 X JRYl6Ol XRSl13 YRS375 X JRYl6Ol XRSll4 YRS376 X JRY16Ol X R S l l 7 [50]rsf3-11 X YRS416 YRSll8 [50]rsfl-6 X YRS416 XRS124 YRS437 X YRSl l86

and for noncomplementation (strong a mating). Thus assignment of mutations to complementation groups was pre- cluded because of the intermediate phenotypes of the mutants.

Identification of hmral mutations: Three of the most efficient maters isolated from YRS23 (HMLa MATa HMRa sir4-9) behaved quite differently in the complementationtest than the 21 described above. When any of the mutants [23] rsf5.5, [23]rsf10.13, or [23]rsf10.17 were mated to any of the 25 phenotypic revertants isolated from HMLa matal HMRa sir4-9 (YRS32), the resulting diploids mated efficiently as a cells at 34". Furthermore, when each mutant was crossed to an a sir4-9 strain (JRY50), mating proficiency was inherited exclusively by MATa segregants. The simplest interpretation of these observations was that the a1 gene of HMRa had been mutated in these strains. This hypothesis was tested by determining whether the mutant strains contained a functional a1 gene. For this experiment, each strain was mated to an HMLa MATa HMRa HO strain (JRY79) and the resulting diploids were tested for their ability to sporulate. If an intact a1 gene were present in the mutant

strain, transposition of that gene from HMR to the MAT locus in the diploid would give rise to sporulation-competent a / a cells within the diploid colony. Alternatively, if no functional a1 gene were present in the mutant strain, the diploid colony would consist entirely of a/a cells. A control diploid was formed by mating an HMLa MATa HMRa sir4- 9 strain (YRS23) to the HMLa MATa HMRa HO strain URY79). After 3 days on sporulation medium, 44% of the cells fromthis diploid had sporulated. In contrast, the mutants [23]rsf5.5, [23]rsf10.13, and [23]rsf10.17, when mated to JRY79, produced diploids that were unable to sporulate. In each case, no asci were observed among 230 cells examined. This result confirmed that 3 of the 24 phenotypic suppressors that were isolated from the parent strain HMLa MATa HMRa sir4-9 (YRS23) sustained an hmral mutation that enabled them to mate. Thus, this assay proved to be a rapid method for detecting unwanted hmral mutants among suppressors of sir mutations isolated in MATa strains.

Determination of mating phenotypes: A difficulty en- countered in analysis of sir4 suppressors was the variable penetrance of the suppression phenotype among the segregants. In all cases the phenotype of the suppressor was readily discernible in MATa segregants whereas the phenotype of several suppressor mutations, in particular s a d - l and san3-1, was difficult to discern in MATa segregants. Several crosses were designed so that the presence of the suppressor mutation in the MATa segregants could be in- ferred by the segregation pattern and rules of Mendelian inheritance. The segregation of suppressor mutations was determined by their effect on the mating ability of cells grown at 30 O , 34", or at both temperatures depending upon the suppressor being tested. Strains to be tested were patched onto solid medium, grown for 2 days, and replica- plated onto lawns of mating-type tester strains. Lawns were formed in the following manner. Freshly grown cells were suspended in YPD to a concentration of approximately 1.5 X 10' cells per ml. Immediately prior to replica-plating, 0.3 ml of the suspension was spread onto the surface of the solid minimal medium. Pregrowth and mating were carried out at the same temperature. After 2 days, mating proficiency was scored based upon the density of prototrophic growth within a patch of cells approximately 1.3 X 0.4 cm in size. A mating proficiency of "p" was assigned to strains that gave rise to between 1 and 10 prototrophic colonies. Mating proficiencies of "pap" (papillae), "+", "++" and "+++" represent increasing densities of prototrophic growth, with "+++" denoting confluent growth throughout the patch. The strains JRY253 and JRY254 were used as mating-type tester strains unless otherwise indicated.

Cloning SANl: Plasmids that complemented the sanl-2 mutation of YRS376 were isolated from a YCp50 plasmid library (ROSE et al. 1987). This library consists of restriction

TABLE 3

Summary of mutant isolation

Relevant genotype phenotype

Mutant

Parent HML MAT HMR strain e ) with) ("C) isolated screened quency

Survival (mates Temp Mutants Cells Mutant fre-

jRY5O (a) a (a) sir4-9 ND a cells 30 76 50,400 1.5 X lo-$ YRS23 (a) a (a) sir4-9 31 a cells 34 50 50,000 1.0 x lo-$ YRS32 (a) a1 (a) sir4-9 50 a cells 34 25 72,000 3.4 x 1 0 - ~

JRY4 ( 4 a1 (a) sir4351 40 a cells 30 0 32,000 <3 x 1RY4 (01) a1 (a) s i r4351 37 a cells 23 0 80,000 C1.5 X lo-'


pRS222 DELETIONS

FIGURE 1 .-Sequencing strategy for the SANl gene. Both strands of the entire coding sequence were sequenced. The arrows represent sequence read from a series of nested deletions of the SANl gene constructed in pEMBL18. Details are described in MATERIALS

AND METHODS.

fragments produced by a partial Sau3a digestion of genomic yeast DNA cloned into the BamHI site of YCp50, a centromere-containing yeast-Escherichia coli shuttle vector (JOHN- STON and DAVIS 1984). The library was transformed into YRS376 (a sir#-9 sanl-2 ura3) and the resulting Ura' colonies were extracted from the top agar by maceration, fol- lowed by vigorous agitation in minimal media (YM) for several hours. The suspension was diluted and spread onto supplemented YM plates (without uracil) to a density of approximately 500 cells per plate. Colonies, grown at 30°, were tested for their ability to mate by replica-plating them onto a lawn of a his3 ura3 (JRY26) on supplemented YM lacking uracil. Two days later, colonies that were unable to mate at 30 O were identified by the absence of prototrophic diploids.

Construction of a sanl disruption allele: A 1.7-kbp BamHI fragment containing the HIS3 gene (STRUHL 1985) was inserted into the SANl gene as follows (see Figure 4). The plasmid pRS2 19, which consists of the 3. l-kbp EcoRV fragment from pRS5 1 .b in the SmaI site of pSEYC58 (EMR et al. 1986), was linearized by cleavage within the insert at a unique ClaI site. The HZS3-containing fragment was li- gated into pRS219 after filling in the 5' single-stranded ends of both the plasmid and the insert. The resulting plasmid, pRS227, was digested with EcoRI to liberate a fragment that spanned the HIS3 insertion, which was used to transform JRY543 to histidine prototrophy.

DNA sequencing: For sequence analysis, recombinant plasmids were propagated in the Escherichia coli strain DIHlOl (constructed by D. ISH-HOROWICZ, provided by S. MOUNT) and single-stranded DNA was prepared and sequenced by the dideoxy-terminator method (SANGER, NICK- LEN and COUL~ON 1977). To sequence SANl a 3.1-kbp EcoRV fragment from pRS51.b was inserted into the SmaI site of pEMBL18 [provided by R. LEVIS; constructed by replacing the polylinker of pEMBL8 (DENTE, CESARENI and CORTESE 1983) with that of pUC 18 (NORRANDER, KEMPE and MESSING 19831 in both orientations, generating pRS22 1 and pRS222. pRS221 and pRS222 were digested with PstI and BamHI and nested deletions in the insert were created by exonuclease 111 and S1 nuclease treatment as described by HENIKOFF (1 984). The sequencing strategy used on the SANl gene is shown in Figure 1.

RESULTS

Isolation of mutants: Since suml-1 is able to suppress null alleles of SIR2 and SIR3, but not of SIR4, the first screen was designed to identify new mutations

capable of suppressing a sir4 nonsense mutation ( s i r 4 351). However, we were unable to identify a single suppressor mutation in a screen of 1 10,000 mutagenized colonies from JRY4. Therefore a second screen was performed to identify mutations that could suppress the temperature sensitive sir4-9 allele. Pheno- typic revertants of sir4-9 were isolated from three different strains. The conditions that were used to identify mutants from each strain are outlined in Table 3. Mutants isolated from an HMLa MATa HMRa sir4-9 strain (JRY50) and an HMLa MATa HMRa sir4-9 (YRS23) restored the ability of theses- trains to mate at a temperature nonpermissive for sir4-9 (30" or 34"). Upon retesting, mutants that mated with an efficiency of "pap/+" or greater were saved for further analysis. T h e strain HMLa matal HMRa sir4-9 (YRS32), mated as an a at 34", but at 23 " the silent mating type loci were repressed and the strain mated as an a. Each of the mutants isolated from YRS32 mated relatively weakly with an a strain ("pap" or "+") at the restrictive temperature, yet still mated strongly with an a strain ("+++"). This bimating phenotype has been observed for leaky sir alleles in strains of this genotype and indicates that the suppressor mutations do not fully suppress the sir4-9 mutation (RINE and HERSKOWITZ 1987).

Dominance tests: Mutants were tested for dominance as described in MATERIALS AND METHODS. All mutants were recessive by these tests.

sanl-1, sanl-2, san2-1 and san3-1 segregated as single nuclear mutations: Mutants from HMLa MATa HMRa sir4-9 (JRY50) that mated with an efficiency of "+" or greater at the nonpermissive temperature were chosen for further genetic analysis. T h e mutant [50]rsf3-11 was mated to an HMLa MATa HMRa sir4-9 strain (YRS416) to form the diploid X R S l l 7 . Similarly, [50]rsfl-6 was mated to YRS416 to form X R S l l 8 . X R S l l 7 and X R S l l 8 , both homozygous for sir4-9 and heterozygous for the presumptive suppressor mutation, were subjected to tetrad analysis. T h e segregation of mating ability among tetrads grown at 30" and 34" was determined. In each tetrad (1 8 tetrads from XRS 1 17 and 17 tetrads from XRS1 IS), mating ability segregated 2:2. There- fore, suppression of the sir4-9 nonmating phenotype in these mutants was due to a mutation within a single nuclear gene. Furthermore, the segregation pattern of the mutations indicted that they were able to suppress the nonmating phenotype caused by the sir4-9 mutation in a and a cells alike. T h e possibility that mating proficiency was due to intragenic reversion of sir4-9 was eliminated by crosses described later. These mutations, designated sanl-1 and sanl -2 after their behavior as Sir antagonists, were found to be alleles of the same gene (below).

Similarly, the segregation of two of the strongest a


maters isolated from HMLa MATa HMRa sir4-9 (YRS23) was analyzed by crossing each of the mutants ([23]rfs3-1 and [23]rsf4-6) to HMLa MATa HMRa sir4-9 CJRY50). The mating phenotypes of segregants from the resulting diploids XDG5 (a /a , sir4-9/sir4-9, SAN3lsun3-I) and XDG6 (a /a , sir4-9/sir4-9, SAN2/ sun2-I) were determined at 30 O . Mating ability segregated 2:2 among 15 tetrads from each diploid, indicating that a single nuclear mutation was responsible for the phenotype of each of the mutants. The analysis described below demonstrated that these mutations defined two additional genes. The mutations, designated sun2-1 (from [23]rsf4-6) and sun3-1 (from [23]rsf3-1), suppressed the nonmating phenotype of the sir4-9 mutation in both a and a cells. However, the suppressor phenotype was more pronounced among a cells than among a cells.

The difference in strength of San- phenotypes in a and a cells, and a high degree of phenotypic variability among individual sun segregants prompted a closer examination of the range of mating phenotypes caused by each sun mutation. Figure 2, A and B, illustrates the distribution of mating phenotypes among segregants from XRS117 (sunl-I /SANl, sir+ 9/sir4-9) and XRSll8 (sunI-2/SANI, sir4-9/sir4-9). The distribution of mating phenotypes among SANl segregants indicated that the phenotype of the sir4-9 mutation itself varied among different strains, most notably among a strains. The mating phenotypes of MATa sir4-9 SANl segregants ranged from nonmating (“-”) to weakly mating (“pap”), with the predom- inant class made up of weak maters. In contrast, nearly all MATa sir4-9 SANl segregants were unable to mate (“-”) at 30”.

The distribution of mating phenotypes of segregants from a diploid heterozygous for sun3-1 and homozygous for sir4-9 (XRS50) is shown in Figure 2C. All of the MATa, sun3-1 segregants were strong maters (greater than or equal to “++”). In contrast, MATa, sun3-1 segregants displayed a broad range of mating phenotypes, ranging from “p” to “+++,” although most exhibited a phenotype of either “pap” or “+.” From these results the following phenotypic criteria were established for assigning the sun3 genotype. MATa segregants that mated with an efficiency of“++” or greater, and MATa segregants that mated as “pap” or greater were sun3. Since MATa segregants of both mutant sun3 and wild type SAN3 genotypes were observed to mate with a “p” phenotype, the assignment of SAN3 genotypes to MATa segregants with this phenotype was ambiguous. Nevertheless, the

fraction of tetrads affected by this ambiguity was small since only 10% of the MATa segregants from XRS50 were of the “p” phenotypic class. The distribution of mating phenotypes among segregants from a diploid heterozygous for sane-1 and homozygous for sir4-9 (XRS124) is illustrated in Figure 2D. The distribution was very similar to that observed for the sun3-1 mutation. Therefore, the same criteria used for assigning the sun3-1 genotype were applied to sun2-I.

san mutations were not linked to SZR4: T o determine whether san mutations were intragenic suppressors or revertants, or whether they were extragenic suppressors of the sir4-9 mutation, the segregation of each sun mutation relative to SIR4 was examined. The mutant [50]rsf3-11 (MATa sunl-1 sir4-9) was mated to JRY252 (MATa SANI SIR4) to form the diploid XRS20. If sunl-1 were an intragenic suppressor of the sir4-9 mutation, then every segregant from the diploid would be able to mate. However, if the mutation were in some other gene, a fraction of the segregants would be nonmating recombinants of the genotype sir4-9 SANl. Among 21 tetrads, 18% (15/ 84) of the segegrants were nonmaters and hence were presnmably sir4-9 SANI. This percentage was not statistically different from the 25% expected for the segregation of unlinked genes, indicating that sunl-1 and SIR4 represent distinct genetic loci. Similarly, to determine whether sunl-2 was linked to SIR4, the diploid XRS21 (sunl-2/SANI, sir4-9/SIR4) was analyzed. Out of 26 tetrads examined, 17% (18/104) of the segregants were nonmaters. Therefore, sunl-2 was not tightly linked to SZR4.

The absence of linkage between sun2-1 and SIR4was deduced based upon the analysis of the segregation of sun2-1 relative to the tightly centromere- linked gene TRPl . Analysis of tetrads from the diploid XRS124 revealed that sun2-1 was tightly linked to a centromere (Table 4). The SIR4 gene has been mapped, and it is not centromere-linked (IVY, HICKS and KLAR 1985). Therefore, sun2-1 was not tightly linked to SIR4.

Linkage between sun3-I and SIR4 was examined by crossing the mutant [23]rfs3-1 (a sun3-1 sir4-9) to JRY22 (a SAN? SIR4). Segregation of sun3-1 in tetrads from the diploid (XDG7) was determined at 34“. From a total of 30 MATa segregants, 16 mated as “++” or greater. The remaining 14 MATa segregants were unable to mate (“-”), and were presumably of the genotype sir4-9 SAN3. Therefore, sun3-1 was not tightly linked to SIR4. (The phenotype of sun3-1 in some MATa segregants was not detectable at 30”.

FIGURE 2.-Distribution of mating phenotype in crosses involving mutations in SAN genes. Panels A , B, C and I1 represent the variable penetrance of the san mutations, and in particular the difference in the san2 and s a d phenotype betwezn a and (Y segregants. In each case, mating ability was tested at 30” . Each circle represents an individual segregant from the crosses indicated. Segregants with ;I

mating efficiency of “p” are indicated by a circle placed between the “-” and “pap” columns.

C

Suppressors of SIR4 Mutations

A XRS117 B XRS118

"- 8 sir4-9 Sm7-1 a sir4-9 SANl

"- a sir4-9 sanl-Z a sir4-9 SANl

MA Ta MATa MA Ta :MA Ta

- pap + ++ +++ - pap + ++ +++

0 SANl sir4-9

8 @ sanl-1 sir4-9

XRS50

a sir4-9 SAK3

a sir4-9 sand-1 " -

MA Ta MA Tx

- pap + ++ +++ - pap + ++ +

f E

I f f

t t

t

0 SAN3 sir4-9 t

8 san3-1 s i r 4 4 t

- pap + ++ +++ - pap + ++ +++ 8

0 SANl sir4-9

@ sanl-Z sir4-9

D

MA Ta

XRSl24

- pap + ++

8

0 SAN2

sant - 1

"- a sir4-9 SAN2 a sir4-9 san2-1

MA Tu

+++ -

slr4-9

sir4-9

35


TABLE 4

Mapping and allelism of SAN genes

Tetrad types

Diploid Mutations PD T NPD Interpretation

XRSl17 sanl-1 vs. MAT 3 12 3 Unlinked

XRSl18 sanl-2 vs. MAT 2 12 3 Unlinked

XRSSO s a d - 1 vs. MAT 1 27 6 Unlinked san3-1 vs. trpl 6 24 4 SAN3 is not centromere linked his4 vs. leu2 14 10 0

XRS124 san2-1 vs. MAT 20 47 13 Unlinked san2-1 vs. leu2 37 11 28 san2-1 vs. trpl 32 4 43 SAN2 is centromere linked leu2 vs. trpl 30 9 37

XRS20 sanl-1 vs. sir4-9 6 15 0 Not tightly linked

XRS2 1 sanl-2 vs. sir4-9 10 14 2 Not tightly linked

XRS29 sanl-1 vs. sanl-2 17 0 0 sanl-l and sanl-2 are allelic

XDG 1 sanl-1 vs. san3-1 2 8 5 Unlinked

XDG2 sanl-2 vs. s a d - 1 8 18 5 Unlinked

Therefore, MATa segregants were not included in this analysis.)

sad-Z and sanl-2 were alleles of the same gene: The allelism of sunl-1 and sunl-2 was determined by analyzing the diploid formed between an a sir4-9 sanl-1 strain (JRY294) and an a sir4-9 sunl-2 strain ([50]rsfl-6). If sunl-1 and sunl-2 were allelic, every segregant would contain a sun mutation, and therefore would be able to mate at the restrictive temperature. If the mutations were not allelic, then nonmating (sir4-9 SAN) recombinants would be recovered. Out of 17 tetrads from the diploid (XRS29), every segregant mated. The absence of nonmating, Sari+ recombinants indicated that sanl-1 and sunl-2 were probably alleles of the same gene.

Allelism of SANZ vs. SAN2: In order to determine whether sun2-1 defined a distinct gene from SANl , a diploid was formed by mating an a sir4-9 sun2-1 strain ([23]rsf4-6) to an a sir4-9 sunl-2 strain ([50]rsfl-6). The mating phenotypes of segregants from this diploid (XRS5) were determined at 34”. Since assignment of sun genotypes was not definitive for MATa segregants at 34”, this analysis was limited to MATa segregants. If sunl-1 and sun2-1 were allelic, then all MATa segregants would be maters at 34”. If sunl-1 and sun2-1 were mutations in different genes, then a fraction of the segregants (25% if sunl and sun2 are unlinked) would be nonmaters (sir4-9 SANl SAN2). Among 38 tetrads examined, 25% (19/76) of the MATa segregants were nonmaters (“-”) at 34”. Thus, SANl and SAN2 were distinct unlinked genes.

Allelism of SANl vs. SAN3: In order to determine whether SANl and SAN? were allelic, diploids were formed by mating an a sir4-9 sunl strain (both [50]rsf3-11 and [50]rsfl-6) with an a sir4-9 sun3-1

strain ([23]rsf3.1). The mating phenotypes of segregants from the diploids (XDG1 and XDG2) were tested at 23 ” , 30”, and 34 ” . Tetrads with four, three, or two “maters” were defined as PD, T, or NPD tetrads, respectively. The following criteria were used to distinguish “maters” from “nonmaters” at 30”: MATa segregants that mated with an efficiency of “+” or greater were defined as “maters” and those that mated less efficiently were defined as “nonmaters.” MATa segregants that mated with an efficiency of “pap” or greater were defined as “maters.” The tetrad ratios obtained demonstrated that SANl and SAN? were unliked genes (Table 4, XDGl and XDG2).

Allelism of SAN2 vs. SAN3: The absence of genetic linkage between SAN2 and SAN? was deduced based upon an analysis of the segregation of each gene relative to its own centromere. The centromere linkage of SAN2 was examined by analyzing the segregation of sun2-1 relative to t rp l , a tightly centromere- linked gene. These two genes displayed second division segregation (SD) only rarely whereas first division segregation (FD) was common (XRS124, SD : FD = 4 : 74), indicating that SAN2 was located only 2 cM from its centromere (PERKINS 1949; GOWANS 1965). The centromere linkage of SAN? was examined by analyzing the segregation of sun3-1 relative to trpl in tetrads from XRS50. A large fraction of the tetrads displayed second division segregation (SD : FD = 10 : 24), indicating that sun?-1 was not linked to its centromere. Since sun2-I was centromere-linked and sun?-I was not, these mutations were in separate genes.

sun mutations did not bypass the need for SZR4: In principle sun mutations could suppress the sir4-9 mutation either by bypassing the requirement for the


SIR4 gene product or by restoring SIR4 function. The inability to isolate mutations that suppressed the sir4-351 allele (a nonsense allele) suggested that sun mutations isolated as phenotypic suppressors of the sir4-9 mutation would be unable to suppress the sir4- 351 mutation. This prediction was tested directly for sun1 by fusing spheroplasts of an a sir4-9 sanl-2 strain ([50]rsfl-6) and an a sir4-351 SANl strain (XR320- 12a) and analyzing the mating phenotypes of segregants from the resulting diploid (XRS7). If sanl were not able to suppress sir4-351, then approximately 25% of the segregants would be able to mate, specifically those that had inherited both sir4-9 and sanl-2. Al- ternatively, if sun1 were able to suppress the sir4-351 allele, then 50% of the segregants would be able to mate. For this analysis, segregants that mated with an efficiency of “p” or greater were counted as “maters.” Among 35 tetrads, 37 of the segregants (25%) mated at 34” (PD:T:NPD = 8:21:6). Thus, at least in MATa cells, sanl did not suppress the sir4-351 allele. These results were extended to include cells of both mating types by crossing an a sir4-351 sanl-2 segregant obtained from XRS27 (YRS104) with an CY sir4-9 SANl strain (YRS23). The sir4-9 mutation served as a control that the sanl-2 mutation was segregating in the cross. The mating phenotypes of segregants from the diploid (XRS15) were tested at 34”. MATa segregants were defined as “maters” if they mated with an efficiency of “p” or greater and MATa segregants were defined as “maters” if they mated with an efficiency of “pap” or greater at 34”. Among 16 tetrads, 23% of the segregants were “maters,” rather than the 50% expected if sanl-2 were able to suppress both sir4 alleles. This result confirmed that sun1 was unable to suppress the sir4-351 mutation in either mating type.

sanl suppressed two very different leaky sir4 alleles: The allele specificity of sanl implied that, rather than obviating the need for SIR4, sanl somehow restored SIR4 function in strains that contained the sir4-9 mutation. This effect could have occurred by at least three different classes of mechanisms. First, the sanl mutations could have caused compensatory alterations in a component that interacted physically with SIR4 protein. Second, sanl could have encoded an informational suppressor, such as a missense tRNA suppressor or an altered ribosomal component. Third, sanl could have increased the level or effectiveness of a partially functional SIR4 protein. The first two models predicted that sanl would suppress only a very narrow range of sir4 mutations, whereas the third model, in which sanl increased the amount or effectiveness of the sir4-9 gene product, predicted that sanl would suppress a variety of leaky sir4 alleles. T o distinguish between these possibilities, we tested the effect of sanl on the ability of a truncated, partially functional SIR4 gene to complement the sir4-351

TABLE 5

Effect of sanl on the sir4sornplernenting activity of a truncated SIR4 gene

Mating ability of strain containing plasmid:

Relevant geno- Strain ‘YPe Trial“ pJRlO6 pJR72 YEp24

YRS77 sir4-351 SANl (1) p - - (2) pap/+ -

(2) ++ Pap -

-

YRS75 sir4-351 sanl-1 (1 ) ++ P -

YRS107 sir4-351 sanl-2 (1) ++ Pap - (2) ++ Pap -

a Strains were transformed with the indicated plasmid and the mating phenotypes of two independent transformants were determined at 34“. When transformed with a plasmid that contains the intact SIR4 gene (pRS26) every strain exhibited a wild-type (“+++”) mating ability. The plasmids pJR106 and pJR72 contained truncated versions of the SIR4 gene that consists of the carboxy-terminal 75% of the SIR4 coding region. YEp24 is a control plasmid that contains no insert.

mutation. For this experiment, the truncated SIR4 gene on plasmids pJR 106 and pJR72 was transformed into a sir4-351 sanl strains (YRS75, YRS107) and an a sir4-351 SANl strain (YRS77), and the mating phenotypes of the transformants were tested. Based upon the sequence of the SIR4 gene (MARSHALL et al. 1987), these plasmids contained a truncated SIR4gene with the capacity to encode the carboxy-terminal 75% of the wild-type SIR4 gene. The expression of these genes was driven by fortuitous promoters within the vector sequences. As summarized in Table 5, pJRlO6 and pJR72 complemented the sir4-351 mutation weakly, if at all, in the SANl strain. In the presence of the sanl mutation, however, the sir4-complementing activity of the plasmids was significantly higher. This result supported the hypothesis that sanl acted by increasing either the level or the specific activity of a partially functional SIR4 gene product.

sanl did not suppress a temperature sensitive sir3 allele: The ability of sanl to enhance repression of the silent mating-type loci could have been specific to sir4 mutants. Alternatively the mechanism of suppression could have been more general such that sanl mutations would suppress leaky mutations in any SIR gene. T o help distinguish between these possibilities, the ability of sanl to suppress the mutant phenotype of a temperature sensitive sir3 mutation was tested. For this experiment, an a sanl-1 SIR3 strain (XRSPO- lob) was mated to an a SANl sir3-8 strain (JRY187) and the mating phenotypes of the segregants from the diploid (XRS30) were tested. If sun1 were not able to suppress sir3-8, then two segregants in every tetrad would be nonmaters at 34”. Alternatively, if sun1 were able to suppress sir3-8, then more than half of the segregants would be able to mate at the high temperature. In 28 tetrads from XRS30, mating ability consistently segregated 2:2. A control diploid,


1 2 3 4

SIR4 -

-25 S

-18 S

FIGURE J . " R N A gcl-ttxnsfcr hyl~ridiz;ltion ;Inalysis of SIR4 esprcssion. Pol!(A+) R N A w a s isolated from the following strains. rlrc.~ro~~lloretic;llI~ separated on ;I 1 % agarose gel ( 1 0 pg/lane) i n tltc presence of forn~aldeh~tlc. blotted, and hvhridized to the radi- ol;lheled 4.2-khp Smal fragment isolated from pJR106: lane 1. jRY535 (.xir4::fffS3); I;me 2, YRS376 (sir4-9 sanl-2); lane 3, YRS35.5 ( s i r 4 9 s a n l - l ) ; l a w 4.JRY.50 (sir4-9). The smaller I .9-kb R N A was tr;lnscrihed from ;I gcne located near the 3' end of the SI114 gene (SCHNEIL 1985) and served ;IS ;In internal control for cquiv;llent lo;ltling i n e ; ~ h hne.

made by mating the parent of XRS3O (XRS20-lob) to an a s i r49 strain (JRY50) was analyzed to confirm the presence of the sun1 mutation. Among the tetrads analyzed from the control diploid (XRS37), 70% of the segregants were able to mate at 34". Thus, the sun1 mutation was, in fact, present in the parent strain (XRS20-lob). Based on these results, we conclude that sun1 was unable to suppress the sir3-8 mutation. T h e locus-specificity of sunl was consistent with the view that sun1 acted specifically to suppress leaky mutations in SIR4.

The effect of sanl on the abundance of SIR4 RNA: To determine whether sun1 enhanced SIR4 activity by increasing the amount of SIR4 RNA, the level of s i r49 RNA in sun1 strains (YRS375, YRS376) was compared to the level of sir4-9 RNA present in the isogenic S A N l strain (JRY50). An equivalent amount of polv(A+) RNA from each strain was separated on a denaturing agarose gel, transferred to nitrocellulose, and probed with a fragment that contains the SIR4 gene. T h e sanl mutation had no discernible effect on the level of sir4-9 RNA (Figure 3). This result was inconsistent with models in which sun1 acted by en- hancing either the synthesis or the half-life of the SIR4 messenger RNA.

Isolation of sanZ-complementing plasmids: Plas-

mids that complement the sunl-2 mutation were isolated by transforming an a sir4-9 sanl-2 uru3 strain (YRS376) with a YCp50-based yeast genomic library and testing the mating phenotypes of the resulting Ura+ transformants. A centromere-containing library was chosen to minimize gene dosage effects. To en- sure that the identification of plasmids conferring a nonmating phenotype would not be obscured by the loss of plasmids from a few cells within a colony and concomitant mating of those cells, a ura3 mating-type tester strain (JRY26) was used for these tests. Among 8000 plasmid-bearing colonies tested, 23 were unable to mate. Plasmids from 10 of the nonmating transformants were recovered, transformed into E. coli, and purified. Each plasmid was cleaved with a variety of restriction enzymes and the sizes of the fragments generated by each plasmid were compared. Eight of the plasmids contained apparently identical inserts. One representative plasmid from this group, pJR2 14, was chosen for further study. T h e inserts of the remaining two plasmids, pJR19O and pJR210, had no restriction fragments in common with each other or with pJR214. T h e purified plasmids were reintro- duced into YRS376 and the ability of the transformants to mate was tested at 30". Each of the three different plasmids (pJR190, pJR2lO and pJR214) abolished the mating ability of the a sir4-9 sunl-2 strain.

Since sun1 suppressed the Sir- phenotype in a and CY strains alike, a plasmid that contained the wild-type S A N l gene should reverse this suppression in strains of either mating type. To determine whether any of the plasmids that complement sun1 in an a strain had the same effect in an CY strain, each plasmid was transformed into CY sir4-9 sun1 strains (YRSI 180, YRSl184) and a sir4-9 sun1 strains (YRS1179, YRS 1 183). T h e mating ability of each of the resulting transformants was determined and compared to that of the same strain transformed with a control plasmid containing no insert. Only one of the plasmids, pJR190, interfered with mating in both the a and the CY strains. In contrast, the plasmids pJR210 and pJR214 conferred a nonmating phenotype in the a strains but had no effect in the CY strains. The depend- ence of pJR2 10 and pJR2 14 on mating type suggested that these plasmids inhibited mating by a mechanism that was unrelated to sunl-complementation. Of these three plasmids, only pJR 190 fully complemented sanl mutations under all conditions.

Identification of the SANZ gene: To test the hypothesis that pJR19O contained the S A N l structural gene, a portion of the insert from pJR19O was integrated into the chromosome and the site of integration relative to the SANI locus was determined. This method is based on the ability of linear DNA fragments in yeast to integrate into the genome at sites

Suppressors

SANl

- - - m" " - c. - "

c ( A ) 1

m B & ! & ! - I - x . - - I I

$68 - E E Plasmid

I II I I I pJR19O - pRS5l.b

I I pRS5O.b

w pRS52.b

y-0

SANl 5 U

c - 8 - 6 8 8 $31 83 m"

(B) I pRS5l.b

= h " - " u- = E

I I pRS219

I I pRS216 - pRS217

I pRS227 I I

, I

that are homologous to the ends of the fragments (ORR-WEAVER, SZOSTAK and ROTHSTEIN 1981). A 4- kbp BamHI fragment from the pJRl90 insert was cloned into Ylp5, a URA3-containing plasmid that is unable to replicate in yeast (STRUHL et aZ. 1979). T h e resulting plasmid (pRS2 18) was linearized by cleavage within the insert (BgZII) and used to transform an a sanl-2 sir4-9 ura3 strain (YRS376). Eight mitotically stable Ura+ transformants were tested for their ability to mate at 30". Six of the transformants lost the ability to mate upon integration of the plasmid. T h e nonmating phenotype of these transformants indicated that the sanl-2 mutation was suppressed by the integrated plasmid, and was consistent with subcloning experiments, described below, which showed that the 4-kbp BamHI fragment carried by pRS218 was suffi- cient to complement sun1 mutations. T h e site of integration of this plasmid was mapped relative to the SANl locus by mating a Ura+, nonmating transfor- mant (YRS898) to a SANl sir4-9 uru3 strain (YRS416). Among 20 tetrads from the diploid (XRSIOP), every segregant was a nonmater when grown at the high temperature. T h e absence of any mating-proficient sun1 uru3 recombinants suggested that the insert in pRS218 directed integration at or near the sanl-2 mutation. This result supported the hypothesis that pJR19O contained the SANl structural gene. Although this experiment could not rigorously eliminate the possibility that the nonmating phenotype of YRS898 was due to fortuitous reversion of the sanl-2 mutation rather than complementation, the phenotype of the SAN disruption showed that pJRl90 contained the SANl gene (see below).

of SIR4 Mutations 39

Observation: Interpretation:

Mating Phenolype Complementation?

Sanl

YeS I;ICIJRE 4.-Rcstriction ntap and

+ + Fb 1'l;lsmitls \+we introduced into a11 a s a n I - / s i r l - ~ s t r ~ ~ i ~ l ( Y K S J 7 5 ) ; 1 n d t h e + + Fb mating phrnotypes of the resulting

* + Fb tr;msformants were tested a t SO" using either the mating-tvlx tester strain J R Y 2 6 (a ura3 pmwl A) or JRY253 (a UR.43 ptwl B). I.';lch plasmid contains ; I n insert (rcprcseented i n the figure by solid lines) cloned i n

pRS227, pSEYC58. Both vectors contain i~ centromere. The length of

(panel A) or 0.5 kbp (panel B).

YeS sub~loning;I11;1I~sis ofthe .SA,V/ gene.

PBP YeS YCp50 or. i n the case of pRS2 I9 and

+ 4 MI the scalc bar represents either 1 kbp

PBP YeS

+ I Fb

+ + Fb

H Scale

Characterization of the SANl gene: T h e position of the SANl gene within the 9.5-kbp insert of pJR 190 was determined by subcloning analysis (Figure 4). These experiments localized the sanl-complementing function of pJR19O to a 4-kbp BamHI fragment (pRS51 .b). A smaller, 3.1-kbp EcoRV fragment (pRS2 19) from within the BamHI fragment could also complement sun I mutations. However, this activity was lost upon division of the BamHI fragment by cleavage at the CZaI site.

A gel transfer hybridization analysis of poly(A+) RNA, using the 3.1-kbp EcoRV fragment as a probe, revealed that the sun I-complementing fragment encoded a single RNA species of approximately 2.1-kb (data not shown). Hybridization analysis of genomic DNA cleaved with a variety of restriction enzymes showed that the cloned SANI gene was present in the yeast genome in only one copy and that the genomic restriction map was colinear with that of the cloned fragment (Figure 5A).

Analysis of the SANl sequence: T h e sequence of the SANl gene revealed an open reading frame of 610 codons which included the ClaI site shown to be within the SANI-complementing sequence (Figure 6). Only one open reading frame of significant size was found. T h e sequence predicted a hydrophilic protein of 65,607 daltons with a PI of approximately 5. No hydrophobic regions greater than 9 amino acids in length were observed. Therefore SANl was likely to encode a soluble protein. Analysis of the NBRF (version 32, November 1987) NBRF PIR (version 16, March 1988) and GenBank (version 55, March 1988) databases revealed no significant homologies to

R. Schnell et al. 40

A

1 2 3 4 5 6

B

1 2 3 4 5 6

-21.2

-5.15 ' 5 . 0 -4.3

-3.5

2.0 - 1.90

- 1.58

- 1.33

- 0.98 -0.83

C

1 2 3 4 5 6

21.2

5.1 5 5.0

4.3 3.5

2.0 1.90

1.58 1.33 0.98 0.83

0.56

FIGURE .5.--CeI-transfe~--hyhridi~ation analysis of genomic DNA using probes from three different san I-complementing plasmids. Total yeast DNA HX digested with one of six restriction enzymes. separated on agarose gels. and transferred to nitrocellulose filters. The filters werr hybridized using the following gel purified "2P-labelecl fragments a s probes: (A) the 4-kbp RamHl fragment from pRS5l.h (derived from p.lRI90); (B) the 3..5-khp Clal frxgment from pRS43.b (derived from pJR210); (C) the -9-kbp Sal1 fragment from pJR214. Size srand;lrrls are X DNA digested with hoth Hind111 and EcoRI. Lane numbers correspond to yeast DNA digested with Puull (lane 1); EcoRl (lane 2); !fintllII (lane 3); BamHl (lane 4); Bglll (lane 5); CIal (lane 6).

known protein sequences. Furthermore, no similari- histidine prototrophy. A mitotically stable His+ trans- ties to known sequence motifs were revealed. formant was sporulated and the phenotypes of the

Disruption of the SANZ gene: To determine segregants were analyzed. In all nine tetrads exam- whether the product of the SANl gene was important ined, His+ segregated 2:2 among four viable spores. for growth or for cell type-specific functions, a disrup- Therefore SANI was not an essential gene. To con- tion of the SANl gene was constructed in vitro and firm that the sanI::HIS3 allele integrated at the SANl integrated into the chromosome by the one-step gene locus, a His+ segregant from the transformed diploid replacement method (ROTHSTEIN 1983). A fragment was crossed to a sir4-9 his3 strain URY50) to obtain that contained the HIS3 gene was inserted into the an a sir4-9 sanI::HIS? his3 strain URY 1601), which ClaI site of pRS219 to produce pRS227. The CZaI was then mated to an a sir4-9 sanl-2 his3 strain site was at codon 2 17 in the coding sequence. Plasmid (YRS376). The mating phenotypes of segregants from complementation tests confirmed that the insertion of the resulting diploid (XRSll4) were determined at HIS? into pRS2 19 inactivated the sanl-complement- 25" and 30". Every a segregant and almost every a ing function encoded by that plasmid (Figure 4). To segregant exhibited an unambiguous San-, mating- replace the chromosomal SANI gene with the dis- proficient phenotype at 30". A small fraction of the a rupted sanI::HIS3 allele, pRS227 was cleaved with segregants (4%) were qualitatively nonmaters at 30", EcoR1, liberating a fragment that spanned the inser- yet at 25" these segregants mated more efficiently tion. The digested plasmid was used to transform an than the a sir4-9 SANl segregants obtained from a a / a &?/his3 SIR4/SIR4 diploid strain URY543) to control diploid. Thus, among 26 tetrads, no SANI


ttcattacagctatttttcatcacatttgataaatccctccctccatataagcatttact 60

agactttaaaagttgcaatgtatatctggaggtataacattgtacattacttgaaagaaa 120

agcgtcttttaacgtaaaataaataatactgttcttttttattgttactggcttcttaat 180

caacaattatatacttcgacgcaaaaagccgggtaaaacattctatctgccattgtcaaa 240

aaatatgaaagaattttttacatataactacattgcattagtatcaccaaatc~ataatg 300

aaataatagctaaaaactatatattgaaggaaatatagtttatcatatatatcctctagt..360

gaagctactatagatagaaattatttagcatttcaggatagttcgtttctcccttttttc..420

ccctttgttttctctcatagtcttgtaacctcagcttttgttcatt ATG AGT GAA AGT MET SER GLU SER

GGT CAA GAA CAA AAC AGA GGC ACA AAT ACA TCA CCA AAT AAT GCT GAA GLY GLN GLU GLN ASN ARG GLY THR ASN THR SER PRO ASN ASN ALA GLU

AAT AAT AAT AAT TCA AAT GCA GCT TCC GGT CCA CTC AAT GGT GGT GCT ASN ASN ASN ASN SER ASN ALA ALA SER GLY PRO LEU ASN GLY GLY ALA

GAG CAA ACA AGA AAC ATA ACC GTT TCC ATT CAG TAT TCC TAT TTC ACT GLU GLN THR ARG ASN ILE THR VAL SER ILE GLN TYR SER TYR PHE THR

PRO GLU ARG LEU ALA HIS LEU SER ASN ILE SER ASN ASN ASP ASN ASN CCG GAG AGA TTA GCA CAT TTG AGC AAT ATA TCT AAT AAT GAT AAT AAC

GAA AAC AAC AGC GCA GCA TCC GGT AGC ACG ATT GCC AAC GGT ACT GGG GLU ASN ASN SER ALA ALA SER GLY SER THR ILE ALA ASN GLY THR GLY

CCC AGC TTT GGC ATC GGA AAT GGG GGC CAT CAA CCC GAC GGT GCT CTT PRO SER PHE GLY ILE GLY ASN GLY GLY HIS GLN PRO ASP GLY ALA LEU

GTT TTA TCT TTT CGT GAT GTT CCT GCG TCT ACT CCA CAA GAC CGT TTG VAL LEU SER PHE ARG ASP VAL PRO ALA SER THR PRO GLN ASP ARG LEU

AAC AGC TTC ATA TCT GTT GCT GCT CAG CTT GCA ATG GAG AGA TTC AAC ASN SER PHE ILE SER VAL ALA ALA GLN LEU ALA MET GLU ARG PHE ASN

AGA CTA TTA AAT AGA CCA AAA GGA ATA TCA AAG GAT GAG TTT GAT AAG ARG LEU LEU ASN ARG PRO LYS GLY ILE SER LYS ASP GLU PHE ASP LYS

CTC CCC GTT CTG CAA GTT TCT GAT CTA CCC AAG GCC GAA GGG CCC TTA LEU PRO VAL LEU GLN VAL SER ASP LEU PRO LYS ALA GLU GLY PRO LEU

TGT AGT ATA TGT TAT GAC GAA TAT GAA GAT GAA GTT GAT TCA ACT AAA CYS SER ILE CYS TYR ASP GLU TYR GLU ASP GLU VAL ASP SER THR LYS

ALA LYS ARG LYS ARG ASP SER GLU ASN GLU GLU GLU SER GLU GLY THR GCA AAA AGA AAA AGG GAT TCT GAA AAT GAG GAG GAA TCT GAA GGA ACA

AAA AAA AGG AAG GAT AAT GAA GGT GCG CCC CTA CGC ACA ACC GCC GAT LYS LYS ARG LYS ASP ASN GLU GLY ALA PRO LEU ARG THR THR ALA ASP

AAT GAC AGT AAC CCA TCG ATT ACA AAT GCT ACG GTT GTT GAA CCG CCT ASN ASP SER ASN PRO SER ILE THR ASN ALA THR VAL VAL GLU PRO PRO

TCT ATT CCT CTC ACT GAA CAA CAA CGC ACT CTC AAT GAT GAA GAA ACA SER ILE PRO LEU THR GLU GLN GLN ARG THR LEU ASN ASP GLU GLU THR

AAT CCA AGC TAC AAA CAC TCA CCA ATC AAG TTA CCT TGT GGC CAC ATT ASN PRO SER TYR LYS HIS SER PRO ILE LYS LEU PRO CYS GLY HIS ILE

TTT GGG AGG GAA TGT ATC TAC AAA TGG TCA AGA TTA GAA AAT TCT TGT PHE GLY ARG GLU CYS ILE TYR LYS TRP SER ARG LEU GLU ASN SER CYS

CCC CTT TGT AGA CAA AAG ATC AGC GAA TCT GTA GGT GTT CAA CGT GCA PRO LEU CYS ARG GLN LYS ILE SER GLU SER VAL GLY VAL GLN ARG ALA

GCC CAA CAA GAT ACG GAT GAA GTA GCA GCT AAC GAA GCT GCT TTT GAA ALA GLN GLN ASP THR ASP GLU VAL ALA ALA ASN GLU ALA ALA PHE GLU

FIGURE 6.--Sequence of the SANZ gene.

recombinant was found. A second diploid was formed by mating the sanI::HIS3 sir4-9 strain (JRY 1601) to the sun 1-1 sir4-9 strain (YRS375). Among 45 tetrads from this diploid (XRSll3), every segregant exhibited a San- mating phenotype. Therefore, the insertion allele, sunl::HIS3, integrated at the SANl locus and sanl-1 and sanl-2 were allelic with sanl::HIS3. Fur- thermore, the phenotype of a sanl null allele paral- leled that of the original sanl-1 and sanl-2 mutations.

To determine whether SANl was required for sporulation, an a/a sanl::HZS3/sanI::HIS3 diploid (XRS97) was transformed separately with a plasmid containing no insert (YCp50), and with a plasmid containing the SANl gene (pRS5 1 .b). The sporulation efficiency of cells containing the SANI plasmid was 30 f 5.7%. The sporulation efficancy of cells

CGT ATT AGA CGA GTT TTA TAC GAC CCA ACT GCA GTG AAT AGC ACT AAC ARG ILE ARG ARG VAL LEU TYR ASP PRO THR ALA VAL ASN SER THR ASN

GAA AAT AGT TCC GCC CCA TCC GAA AAC ACG TCC AAT ACA ACG GTT CCC GLU ASN SER SER ALA PRO SER GLU ASN THR SER ASN THR THR VAL PRO

ACT ATC GGA AAT GCA AGT TCT GGC GAA CAG ATG TTA TCA AGA ACA GGC THR ILE GLY ASN ALA SER SER GLY GLU GLN MET LEU SER ARG THR GLY

TTT TTT TTA GTG CCT CAA AAC GGC CAA CCT TTA CAC AAT CCA GTC CGT PHE PHE LEU VAL PRO GLN ASN GLY GLN PRO LEU HIS ASN PRO VAL ARG

TTA CCG CCT AAT GAT AGC GAT AGA AAC GGT GTC AAC GGG CCG AGC TCA LEU PRO PRO ASN ASP SER ASP ARG ASN GLY VAL ASN GLY PRO SER SER

ACT ACT CAA AAT CCA CCC TCT AAT TCT GGT GGT TCG AAT AAC AAT CAA THR THR GLN ASN PRO PRO SER ASN SER GLY GLY SER ASN ASN ASN GLN

AGT SER

CCG PRO

CCA AAT PRO ASN

CGC ARG

CCG PRO

TGG TRP

AAT ASN

GTT VAL

CCT PRO

ccc PRO

TCC SER

ATC ILE

CCT TTG PRO LEU

GCT TCT ALA SER

GAT ASP

ACT THR

TCT SER

TTG TTC CAA LEU PHE GLN

TCG SER

GCA AGC ALA SER

TTC PHE

CCA PRO

CAC HIS

TCA SER

AGT SER

GCA ALA

GCG AAC GGT CCA AAC TCA AAT AAC ACT AGT AGT GAC GCT ACA GAC CCT ALA ASN GLY PRO ASN SER ASN ASN THR SER SER ASP ALA THR ASP PRO

CAC CAC AAC AGA CTA AGA GCC GTT TTG GAT CAC ATA TTC AAC GTT GCT HIS HIS ASN ARG LEU ARG ALA VAL LEU ASP HIS ILE PHE ASN VAL ALA

CAG AGG GGA ACT TCT GAT ACC TCT GCA ACA ACA GCA CCC GGA GCA CAA GLN ARG GLY THR SER ASP THR SER ALA THR THR ALA PRO GLY ALA GLN

ACT GTT CAC AAC CAA GGA CGT AAT GAC TCA TCG TCC TCT GAT ACA ACG THR VAL HIS ASN GLN GLY ARG ASN ASP SER SER SER SER ASP THR THR

CAA GGA AGT TCC TTT TTG GAA AAT ATT TCA CGA TTA ACA GGC CAT TTT GLN GLY SER SER PHE LEU GLU ASN ILE SER ARG LEU THR GLY HIS PHE

ACG AAT GGC TCA AGA GAC AAC AAT AAC GAC AAT AAC CAT AGC AAT GAT THR ASN GLY SER ARG ASP ASN ASN ASN ASP ASN ASN HIS SER ASN ASP

CAA CAA CGA GGT GGA AGT ACT GGT GAG AAC AAC AGA AAT AAC TTG TTT GLN GLN ARG GLY GLY SER THR GLY GLU ASN ASN ARG ASN ASN LEU PHE

TCC TCC GGT GTT GCC AGT TAT AGA AAT CAA AAT GGT GAT GTT ACT ACC SER SER GLY VAL ALA SER TYR ARG ASN GLN ASN GLY ASP VAL THR THR

GTC GAA CTA CGC AAC AAC AAT TCT GCT GCC TTT CCT CCT ACA GAC GAA VAL GLU LEU ARG ASN ASN ASN SER ALA ALA PHE PRO PRO THR ASP GLU

AAT ASN

CCC TCT PRO SER

CAA GLN

GGC GLY

CAA GLN

GGT GLY

TCA SER

AGC SER

AGT TCG SER SER

GAC ASP

ACC THR

ACC ATT CAT THR ILE HIS

AAC GAC GTC CCT AAT GAT AAC AAT GAG CAA CGA TCA TCA CAA TAA ..2299 ASN ASP VAL PRO ASN ASP ASN ASN GLU GLN ARG SER SER GLN

acaataccgcttctgaagtatgttaatatgaaaatatgtcctattggcagtcatccattt..2359

tcagacattattttctactccttttaccttttttttatctgcatctttatatatatatat..2419

catatacttatttttctttacttctacttaaagttctataatcattgttaatcagaaatt..2479

tgcatgtctccagcaagtaaatatgaaggccgagaaattaaaccaccgacataacctcct..2539

ctgaaaagtcgtttcgcaaaacagtaaaaggtgaaaacacttttaagaactgtggaacac..2599

aaactgtgttttcttaccataaaatcttttaccggaggtcgaaatcgttttctttctgat..2659

ttggaattcgacgctcgaaaagtgcagagatctcatattgttaacggactatcatctaac..2719

tttttgcataatttatacaacatgctcagat . . . 2750

containingYCp50 was 25 k 0.8%. Therefore, the product of the SANl gene was not required for sporulation.

SANl overproduction decreased repression of HMR: Since the previous results concerned the effects of SANl on mutant SIR4 product, it was of interest to determine whether SANl protein could exert an effect in a SIR cell. Specifically, since decreased SANl function seemed to restore partial SIR function, increased SANl function might decrease SIR function. To determine whether increased dosage of SANl would decrease SIR-mediated repression of HMR, a multicopy plasmid containing the SANl gene (pJR528) was introduced into a SIR4 and sir4-9 strain (YRS393 and YRS401, respectively). These strains contained both the SlJP3am gene inserted at HMR

42 R. Schnell et a l .

Number of Cells

SIR4 YEp24

SIR4 pJR528 -Tryptophan

s l r 4 - 9 Y E P ~ ~

+ Tryptophan

FIGURE 7.--l’he SIH4 trplam hmrA::SL’l’3am strain is YRSS93 and the sir#-9 frplam hmrA::SCP3am strain is YRS401. Derepres- sion of the hmrA::SUP3am locus was tested by spotting a dilution series of cells of‘e;dl genotype onto medium lacking tryptophan. The increased growth ability of dilute suspensions of the SIR4 strain transfornled with a nlulticopy S A N l plasmid (pJR528) indicated that SANl overproduction caused partial derepression of H M R . The lack of a growth difference on nledium containing tryptophan indicated that effect of S A N l overproduction w a s due specifically to H M R derepression.

and a trpl amber allele. Thus, derepression could be monitored as increased growth on medium lacking- tryptophan (SCHNELL and RINE 1986). As shown in Figure 7, the multicopy SANI clone resulted in a small yet noticeable increase in growth of the SIR4 strain in the absence of tryptophan, indicating partial loss of SIR-mediated repression. I t should be noted that this degree of derepression was small, much less than that caused by the sir4-9 mutation, and could be detected only by this assay. As expected, overproduction of SANl had no effect in the sir4-9 strain. Since overproduction of SAN 1 resulted in decreased repression in a wild-type strain, it was possible that loss of SANl function would increase repression. However, disruption of the SANI gene did not lead to increased repression of HMR by this assay (data not shown).

Plasmids that complement sun1 in MATa strains but not in MATol strains: Three different plasmids were isolated by their ability to confer a nonmating phenotype on an a sir4-9 sunl strain. The above experiments showed that only one of these plasmids,

pJR190, encoded the SANI gene. The other two plasmids, pJR2 10 and pJR2 14, complemented sun 1 in a strains but, unlike the SANI-encoding plasmid, had no effect on mating in a sir4-9 sunl strains. The mechanism by which these plasmids acted was unknown. In theory, the plasmids might interfere with the mating ability of a cells by antagonizing a-specific function(s) or by causing an increase in the level of the a 2 gene product which, in combination with a l , would repress the expression of a-specific genes. How- ever, the plasmids had no apparent effect on the mating ability of either an HMLa mutal HMRa s i r 4 351 strain (JRY 184) or an HMLa MATa HMRa strain (JRY527). Therefore, pJR2lO and pJR214 did not block a mating ability per se.

The restriction maps of pJR2lO and pJR214 showed no overlap between the inserts carried by each of these plasmids (Figure 8). Subcloning analysis of the pJR2 10 insert limited the sunl-complementing activity of that plasmid to a 3.7-kbp fragment (pRS43.b). This fragment was used as a hybridization probe of genomic DNA cleaved with various restriction enzymes (Figure 5B). Multiple large fragments in each lane hybridized to the probe. This result, combined with the restriction map of the cloned fragment, indicated that the probe shared homology with two or three separate regions within the genome. Simi- larly, a portion of the insert from pJR2 14 was used to probe genomic DNA (Figure 5C). This probe also hybridized to multiple large fragments. A comparison of the sizes of the fragments that hybridized to each probe revealed a striking similarity. For example, digestion with the enzyme Hind111 produced three fragments (4.3, 5.1 and 6.6 kbp) that hybridized to the probe from pJR2lO and five fragments that hybridized to the probe from pJR214. Three of the fragments that hybridized to pJR2 14 coincided in size with the fragments that hybridized with pJR210. These results suggested that the inserts from pJR2 10 and pJR214 shared DNA sequence homology with one another. Direct hybridization between the inserts in the two plasmids was not tested.

Intergenic cross-complementation by sunlcom- plementing plasmids: The cloned SANl gene (pJR 190) was used to test the feasibility of using plasmid complementation analysis to assign sun mutations to complementation groups. For this test, pJR19O was introduced into a sir4-9 and a sir4-9 strains that contained a mutation in either sunl, sun2, or sun3, and the mating phenotypes of the resulting transformants were determined. Unexpectedly, the ability of pJR19O to complement sun2 and sun3 mutations depended upon the mating type of the strain. In the CY strains, pJR19O complemented the sun1 mutations but not sun2-1 or sun3-I. However, in the a strains, pJR190 complemented all of the sun muta-


Observation: interpretation:

Plasmid Phenotype Mating

cornDlementation? san 1

H

pJR214

pJRP10

pRS42

pRS43.b

pRS4l.a + - pRSM + 4

pRS45 + 4

Yes

Yes

Yes FIGURE 8,"Restriction maps of

No other SANf-complementing plasmids.

N3 Plasmids were introduced into an a sir+ 9 sanl-1 strain (YRS375) and the mating

ants were tested at 30" using the mating- type tester strain JRY26. The box in the line for pRS42 represents nucleotides that were removed.

r b phenotypes o f the resulting transform-

- 1 kbp

tions. Since the SANl-containing plasmid exhibited cross-complementation in a strains but not in a strains, complementation tests using the cloned SANl gene were reliable only if the mutations were in a strains.

To determine whether the ability of pJR2 10 and pJR214 to complement sanl extended to sun2 and sun? as well, the plasmids were transformed into both a sir4-9 and a sir4-9 strains that contained a mutation in either SAN2 or SAN3, and the mating phenotypes of the resulting transformants were determined. In a strains, pJR2 10 and pJR2 14 complemented both sun2 and sun3 mutations, although the plasmids had no effect in a strains. Therefore, pJR2 10 and pJR2 14 were unlikely to encode either SAN2 or SAN3. Rather, these plasmids might have encoded SAN genes that had not yet been defined mutationally. The pattern that emerged from these data was that within a strains, a sun-complementing plasmid complemented any sun mutation, but that within LY strains, the plasmid complemented only alleles of the gene encoded by that plasmid.

DISCUSSION

Mutations were isolated that restored the Sir+ phenotype to strains containing the temperature-sensitive sir4-9 mutation. A total of 150 such mutants, all recessive, were isolated from three sir4-9 strains, The assignment of sun mutations to complementation groups using a standard complementation test was not possible. This failure was probably due to the partial suppression of the Sir- phenotype by sun mutations, since the test would only have detected noncomplementation if HML and HMR were fully repressed in a homozygous sun diploid.

Genetic analysis of four independent sun mutations

showed that each mutation segregated as a single nuclear gene and suppressed the sir4-9 phenotype, at least partially in both MATa and MATa strains. No pleiotropies were observed for any of the mutations. Two of the sun mutations were alleles of a single gene, designated SANl. The other two mutaticns defined two additional loci, SAN2 and SAN3. The SAN genes appeared unlinked to each other and to SIR4. A survey of the mating efficiencies of many different segregants with the sir4-9 sanl genotype revealed considerable phenotypic variability among the strains. Overall, sun1 suppressed the Sir- phenotype equally well in strains of either mating type. In contrast, both sun2 and sun3 suppressed the Sir- phenotype much more effectively in MATa strains than in MATa strains.

The dramatic difference between the effect of sun2 and sun3 mutations on the mating efficiency of MATa sir4-9 strains relative to MATa sir4-9 strains suggested that sun2 and sun3 mutants repressed HMRa more than HMLa. The ability of a gene to affect the repression of one silent mating-type locus more than the other has recently been demonstrated for the ARDl gene (WHITEWAY et ul. 198'7). Mutations in this gene severely disrupt the repression of HML yet have only a slight effect on the expression of genes at HMR. By analogy, sun2 and sun3 might discriminate between the HML and HMR loci. A study of the E silencers of HMR and HML indicates that different combinations of factors may act on the two silencers. Specifically HMR E is bound by both AilFl and GRFl , whereas HML E is bound by GRFl but not ABFl (BUCHMAN et ul. 1988). Furthermore, both the ABFl and GRFl binding sites contribute to silencer function of HMR E (KIMMERLY et ul. 1988). Perhaps the suppression of sir4 mutations by sun2 and san3 involved ABF 1, either directly or indirectly.


In order to determine whether sun1 was allele- specific for sir4-9, sun1 's ability to suppress other sir mutations was tested. These experiments showed that sanl was unable to suppress sir4-351, an ochre mutation. Thus sun1 did not obviate the need for SIR4 in the regulation of H M L and H M R . However, sun1 was not completely allele-specific since sanl enhanced the sir4-complementing activity of plasmids that encoded a partially functional SIR4 gene product. The sir4-9 allele and the cloned SIR4 gene used in this study both exhibited partial or leaky SIR4 activity. Aside from their leaky phenotypes, these alleles appeared to have little in common. The sir#-9 mutation, presumably a missense mutation, resided within the 3' one- half of the SIR4 gene (our unpublished observations), whereas the cloned SIR4 gene used in this study consisted of a truncated version of the wild-type gene and encoded only the carboxy-terminal 75% of the SIR4 protein. Since the sunI::HIS3 presumptive null allele had the same phenotype as sunl-1 and sunl-2, suppression of sir4 mutations by sun1 mutations was not due to compensatory mutations in interacting proteins.

Although the sequence of the SANl gene did not, by homology with known proteins, provide insight into the function of SANl protein, the sequence did provide additional strong evidence against the notion that sun2 mutations were informational suppressors. Missense informational suppressors are most com- monly altered tRNA genes, but can also arise from changes in ribosomal components, and elongation factors, all of which would be essential for viability (re- viewed in MURGOLA 1985; SHERMAN 1982). In principle, missense mutations could also be suppressed by altered aminoacyl-tRNA synthetases that misacylate a tRNA species. However, SANl clearly encoded a protein larger than all known ribosomal proteins (OTAKA and KOBATA 1978) and that had no homology to elongation factors or aminoacyl tRNA synthetases, and was not an essential gene. Furthermore, increased dosage of SANl resulted in decreased repression of H M R in Sir' strains as measured by the hmrA::SUP3um allele. This result showed that SANl protein could have an effect in the absence of any sir mutation. Thus both homology and genetic data indicated that SANl played a somewhat specific role in mating-type regulation rather than a more general role in translational fidelity.

sun1 may have compensated for leaky sir4 mutations by increasing the specific activity or level of a partially functional SIR4 gene product. By this model, the wild-type SANl gene would encode a negative regulator of SIR4. For example, SANl may have fa- cilitated the degradation of SIR4, much as the E. coli hflA protein degrades the cII protein of bacterio- phage X (HOYT et ul. 1982). Trans-acting regulators

that alter the stability of a protein have also been found in eukaryotic organisms and may represent a widely used strategy for the fine-tuning of gene expression (e.g., BEWLEY and LAURIE-ALBERG 1984; KING and MCDONALD 1987).

Increasing the dosage of the SIR3 gene is also capable of suppressing some alleles of SIR4 (IVY, KLAR and HICKS 1986). Hence, it was formally possible that sanl mutations suppressed the sir#-9 defect by increasing the level of SIR3 function. This regulation would have to occur posttranscriptionally, since sanl did not increase the level of SIR? RNA (data not shown). However, since sanl could not suppress the temperature sensitive sir3-8 mutation even at semi-restrictive temperatures, sun1 was unlikely to increase the functional dose of SIR3 product. This interpretation required several assumptions regarding the sir3-8 gene product. For example, if the sir3-8 mutation affected the synthesis of SIR3 rather than its function, cells grown at the high temperature would contain no SIR3 protein. Under these conditions elimination of a post- translational regulator of SIR3 would not suppress the mutation.

At face value, the ability of the SANl gene on a centromere plasmid to complement mutations in sun2- 1 and sun3-1 was surprising. Two factors may have contributed to this intergenic complementation by the SANl gene on the centromere plasmid. First, centromere-containing plasmids can be present in one to three copies per cell (CLARKE and CARBON 1980). Second, genes located on centromere-containing plasmids are often transcribed at a higher level than their chromosomal counterparts (MARCZYNSKI and JAEHN- ING 1986). Thus, the complementation by the SANI- bearing CEN plasmid is probably another example of elevated-dosage-dependent complementation. Never- theless, the suppression of sun2 and sun3 by modest increases in SAN 1 suggest that the three genes partic- ipate in a related process. In addition, although pJRZ10 and pJR2 14 did not contain the structural genes for SANI, SAN2 or SAN3, they did contain partially homologous sequences that may encode func- tionally related products. These factors may bear upon why a presumptive null allele of S A N l had a somewhat weak phenotype.

In contrast to the sun mutations described here, the sum]-I mutation, isolated as an extragenic suppressor of a sir2 mutation, is neither allele-specific nor locus- specific (KLAR et ul. 1985). suml-2 suppresses sir2 null and sir3 null mutations, and weakly suppresses sir4- 351. Thus, SUM1 appears to be a positive regulator of the silent mating-type loci. Because the suml-1 mutation arose by an extremely rare event, and because it appears to bypass much of the requirement for SIR function, this mutation is particularly enigmatic. In view of the fact that suml-1 was the only


Sir+ revertant observed among 75,000 heavily mutagenized cells (3.5% survival; KLAR et al. 1985), it is not surprising that an analogous suppressor of sir4- 351 was not found in this study, in which cells were mutagenized much less severely (30-50% survival). Since extragenic suppressors of the sir4-35Z mutation were not obtained from cells that gave rise to many mutations in test genes, it is possible that sumZ-Z may not be a simple loss-of-function mutation. It is apparent that mutational saturation of SAN-like and SUM- like genes has not yet been approached.

We thank GEORJANA BARNES, MICHAEL BASSON, WARREN GISH, WILLIAM KIMMERLY and MARY THORSNESS for many helpful dis- cussions during the course of this work. In addition, we thank AMY AXELROD, PATRICIA LAURENSON, FRANK MCNALLY and LORRAINE PILLUS for critically reviewing the manuscript.

This research was supported by U.S. Public Health Service grants from the National Institutes of Health to J.R. and by U.S. Public Health Service Training Grant GM07232.

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Communicating editor: M. CARLSON

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Genetic and Molecular Characterization of Suppressors of ...Genetics 122: 29-46 (May, 1989) BUCHMAN et al. 1988). However, the sites at which these factors bind are important for SIR-mediated