Copyright 0 1993 by the Genetics Society of America Isolation and Characterization of SGEl: A Yeast Gene That Partially Suppresses the gull 1 Mutation in Multiple Copies Hiroko Amakasu, Yuriko Suzuki, MasafumiNishizawa and Toshio Fukasawa Department of Microbiology, Keio University School of Medicine, Shinjuku-ku, Tokyo 160, Japan Manuscript received August 14, 1992 Accepted for publicationMarch 6, 1993 ABSTRACT Recessive mutations of GALl 1 in Saccharomyces cerevisiae cause pleiotropic defects that include weak fermentation of galactose, a-specific sterility and slow growth on nonfermentable carbon sources. Recent experiments suggest that Gall l p functions as a “coactivator” that links transcriptional activa- tors, such as Gal4p and Grflp/Raplp/Tuflp, with the basic transcription machinery. In the present experiments we isolated a gene, SGEl, thatsuppresses galll forgrowth on ethidiumbromide/ galactose agar when the gene was present in two or more copies. The other gall 1 phenotypes were not suppressed by SGEl in the multiple-copy state. Multiple copies of SGEl increased expression of galactose-inducible genes in gall 1 yeast, presumably at the level of transcription. When SGEl was disrupted in wild-type yeast, the expression of galactose-inducible genes decreased to 50-60% of the wild-type level, presumably due to effect on transcription. Complete DNA sequence analysis revealed that SGEl encodes a predicted protein of 543 amino acids. SGEl-specific mRNA of 1.8 kilonucleotides was detected by Northern analysis along the direction of the open reading frame.The gene mapped near RAD56, at the right end of chrom&ome 16 E XPRESSION of the genes encoding a set of en- zymes required for galactose metabolism is in- ducible by exogenous galactose. Galactose induction involves an interplay of two regulatory proteins, Gal4p and GalSOp [see JOHNSTON (1 987) for review]. Each of the galactose-inducible genes has one or two sets of Gal4p-binding sites of 17bpat the respective up- stream region, which is referred to as UASG. Binding of Gal4p at UASGresults in enhancement of transcrip- tion of the gene located downstream. Gal80p antag- onizes the activating function of Gal4p, presumably by direct protein-protein interaction (LUE et al. 1987; CHASMAN and KORNBERG 1990; PARTHUN andJAEHN- ING 1990; YUN et al. 1991). In addition, normal func- tion of the GALl 1 gene (NOGI and FUKASAWA 1980), also known as SPTl3 (FASSLER and WINSTON 1989), is required for maximizing transcription of some, but not all, of the galactose-inducible genes, collectively called GAL genes in this report (SUZUKI et al. 1988). Loss-of-function mutations of the GALl 1 gene lead to reduced expression of GALl (galactokinase), GAL10 (UDPGal-4-epimerase), GAL7 (Gal-1-P uridylytrans- ferase) or GAL2 (galactose permease), but not that of MELl (a-galactosidase) or GAL80. In addition galll mutations cause a-specific sterility, presumably due to inadequate expression of a-specific genes, as well as slow growth on nonfermentable carbon sources. Ge- netic studies (NISHIZAWA et al. 1990) have suggested that Gall l p functionsasa “mediator”(KELLEHER, FLANAGAN and KORNBERG 1990) or “coactivator” (PUGH and TJIAN 1990) not only for Gal4p but also Genetics 194 675-683 (July, 1993) for Grf 1 p/Rap 1 p/Tuf 1 p, another DNA-binding tran- scriptional activator (SHOREand NASMYTH 1987; BUCHMAN, LUE and KORNBERG 1988). Thus Gall lp does notitself bind to DNA but mediates the activator function of Gal4p or Grfl p/Rapl p/Tufl p to stimu- late the basal transcription machinery, possibly by protein-protein interactions. A similar conclusion was reached through a completely different approach by HIMMELFARB et al. (1990). They isolated amutant that potentiates a weak activator, GAL4-AH, which comprises the DNA-binding domain of Gal4p of 147 amino acids and an amphipathichelical peptide of 15 amino acids. The mutation turned out to be a missense mutation within GALl 1, and was therefore designated GALllP (P stands for potentiator). They further found that Gall Ip or Gall 1 Pp, when tethered di- rectly to DNA by making a protein fusion with the DNA-binding domain of LexA, strongly activated a reporter bearing LexA-binding sites upstream of the transcriptionstartsite.Theseresultssuggestedthe existence of a direct interaction betweenGal 1 1 p and Gal4p molecules that gives rise to a complex of the two proteinsthat is a strong activator. Since then, however, no direct evidence has been obtained to prove or disprove such a model. One of the ways to get insight into thefunction of a gene is to isolate and characterize another gene(s) that suppresses the mu- tant’s phenotypes. For this reason, we have isolated and characterized a gene, SGEl, that, when present in multiple copies, suppresses the Gal- but not other phenotypes of galll null mutants. In light of these
Transcript
Copyright 0 1993 by the Genetics Society of America
Isolation and Characterization of SGEl: A Yeast Gene That Partially Suppresses the gull 1 Mutation in Multiple Copies
Hiroko Amakasu, Yuriko Suzuki, Masafumi Nishizawa and Toshio Fukasawa
Department of Microbiology, Keio University School of Medicine, Shinjuku-ku, Tokyo 160, Japan Manuscript received August 14, 1992
Accepted for publication March 6, 1993
ABSTRACT Recessive mutations of GALl 1 in Saccharomyces cerevisiae cause pleiotropic defects that include weak
fermentation of galactose, a-specific sterility and slow growth on nonfermentable carbon sources. Recent experiments suggest that Gall lp functions as a “coactivator” that links transcriptional activa- tors, such as Gal4p and Grflp/Raplp/Tuflp, with the basic transcription machinery. In the present experiments we isolated a gene, SGEl, that suppresses galll for growth on ethidium bromide/ galactose agar when the gene was present in two or more copies. The other gall 1 phenotypes were not suppressed by SGEl in the multiple-copy state. Multiple copies of SGEl increased expression of galactose-inducible genes in gall 1 yeast, presumably at the level of transcription. When SGEl was disrupted in wild-type yeast, the expression of galactose-inducible genes decreased to 50-60% of the wild-type level, presumably due to effect on transcription. Complete DNA sequence analysis revealed that SGEl encodes a predicted protein of 543 amino acids. SGEl-specific mRNA of 1.8 kilonucleotides was detected by Northern analysis along the direction of the open reading frame. The gene mapped near RAD56, at the right end of chrom&ome 16
E XPRESSION of the genes encoding a set of en- zymes required for galactose metabolism is in-
ducible by exogenous galactose. Galactose induction involves an interplay of two regulatory proteins, Gal4p and GalSOp [see JOHNSTON (1 987) for review]. Each of the galactose-inducible genes has one or two sets of Gal4p-binding sites of 17 bp at the respective up- stream region, which is referred to as UASG. Binding of Gal4p at UASG results in enhancement of transcrip- tion of the gene located downstream. Gal80p antag- onizes the activating function of Gal4p, presumably by direct protein-protein interaction (LUE et al. 1987; CHASMAN and KORNBERG 1990; PARTHUN andJAEHN- ING 1990; YUN et al. 1991). In addition, normal func- tion of the GALl 1 gene (NOGI and FUKASAWA 1980), also known as SPTl3 (FASSLER and WINSTON 1989), is required for maximizing transcription of some, but not all, of the galactose-inducible genes, collectively called GAL genes in this report (SUZUKI et al. 1988). Loss-of-function mutations of the GALl 1 gene lead to reduced expression of GALl (galactokinase), GAL10 (UDPGal-4-epimerase), GAL7 (Gal-1-P uridylytrans- ferase) or GAL2 (galactose permease), but not that of MELl (a-galactosidase) or GAL80. In addition galll mutations cause a-specific sterility, presumably due to inadequate expression of a-specific genes, as well as slow growth on nonfermentable carbon sources. Ge- netic studies (NISHIZAWA et al. 1990) have suggested that Gall l p functions as a “mediator” (KELLEHER, FLANAGAN and KORNBERG 1990) or “coactivator” (PUGH and TJIAN 1990) not only for Gal4p but also
Genetics 194 675-683 (July, 1993)
for Grf 1 p/Rap 1 p/Tuf 1 p, another DNA-binding tran- scriptional activator (SHORE and NASMYTH 1987; BUCHMAN, LUE and KORNBERG 1988). Thus Gall l p does not itself bind to DNA but mediates the activator function of Gal4p or Grfl p/Rapl p/Tufl p to stimu- late the basal transcription machinery, possibly by protein-protein interactions. A similar conclusion was reached through a completely different approach by HIMMELFARB et al. (1990). They isolated a mutant that potentiates a weak activator, GAL4-AH, which comprises the DNA-binding domain of Gal4p of 147 amino acids and an amphipathic helical peptide of 15 amino acids. The mutation turned out to be a missense mutation within GALl 1, and was therefore designated GALllP (P stands for potentiator). They further found that Gall Ip or Gall 1 Pp, when tethered di- rectly to DNA by making a protein fusion with the DNA-binding domain of LexA, strongly activated a reporter bearing LexA-binding sites upstream of the transcription start site. These results suggested the existence of a direct interaction between Gal 1 1 p and Gal4p molecules that gives rise to a complex of the two proteins that is a strong activator. Since then, however, no direct evidence has been obtained to prove or disprove such a model. One of the ways to get insight into the function of a gene is to isolate and characterize another gene(s) that suppresses the mu- tant’s phenotypes. For this reason, we have isolated and characterized a gene, SGEl, that, when present in multiple copies, suppresses the Gal- but not other phenotypes of galll null mutants. In light of these
MATa adel hisl MATa ade? ura? trpl leu2 gal l I" MATa ade? ura3 MATa ade? ura3 trpl MATa ade? leu2 ura3 MATa ura3 SGEl gal l1 MATa ura3 ade? leu2/MATa ura3 ade? trpl MATa ura3 GALl 1 sgel::URA3' MATa ura3 sgel::URA3 GALl 1 MATa adel ura3 trpl hisl leu2 MATa adel ura3 trpl hisl leu2 sgel::URA3 MATa adel ura3 trpl hisl leu2 sge1::TRPl' MATa adel ura3 trpl hisl leu2 sge1::TRPl' MATa adel ura3 trpl hisl leu2 sgel::URA3 MATa adel ura3 trpl hisl leu2 gal4::GAL4-CAT-URA3 MATa adel ura3 trpl hisl leu2 ga l l 1::LEUZd MATa adel ura3 trpl hisl leu2 gall 1::LEU2 sge1::TRPl MATa adel ura3 trpl hisl leu2 gallI::LEU2 ga14::GAL4-CAT-URA? MATa sstl-2 ade2-1 can1 cyh2 his6 met1 rmel ural MATa trpl hisl rad5d
Source, derivation
Our stock This laboratory This laboratory This laboratory This laboratory This laboratory This laboratory This laboratory This laboratory This work This work This work This work This work This work This work This work This work R. CHAN H. OCAWA
a A point mutation originated from NOGI and FUKASAWA (1980). The HindIlI-Hind111 fragment of 1.8 kb containing the whole SGEl gene (see Figure 1) was replaced with URA3. The HindIII-Hind111 fragment containing the whole SGEl gene 1.8 kb long (see Figure 1) was replaced with T R P l . The SacI-EcoRV fragment of G A L l l containing 80% of its coding region (SUZUKI et a l . 1988) was replaced with LEU2. CHAN and OTTE (1982).
f S ~ ~ ~ ~ , MACHIDA and NAKAI (1980).
results, we discuss models to explain how SGEl func- tions. We sequenced S G E l , which contains an open reading frame (ORF) of 543 codons. SGEl was mapped near the right end of chromosome 16 by physical and meiotic methods.
MATERIALS AND METHODS
Strains and media: Saccharomyces cerevisiae strains used in this study are listed in Table 1. HSY5-3B was constructed by replacing the 2.5-kb Sad-EcoRV fragment in the GALll coding region of HSY5-3C with LEUZ. HSY5-3C-2 or HSY5-3C-5 and HSY5-3C-3 or HSY5-3C-4 were con- structed by replacing the 1.8-kb HindIII fragment encom- passing the SGEl coding region with URA3 and TRPl, respectively. HSY5-3C-CAT (chloramphenicol acetyltrans- ferase) and HSY5-3B-CAT were constructed by replacing the GAL4 locus with fragments carrying a GAL4-CAT fusion derived from pBM 1974 (GRIGGS andJoHNsToN 199 1). Rich (YPD, YPGal), synthetic complete (SD), and omission (-ur- acil or -tryptophan) media were prepared as described previously (SUZUKI et al. 1988). SGlyLac consists of 0.67% Yeast Nitrogen Base without amino acids (Difco), 0.5% casamino acids (Difco), necessary amino acids or bases at the concentrations recommended by SHERMAN (1 99 l), 2% glyc- erol and 2% lactate. Galactose was added to 2% in SGlyLac to make SGlyLacCal. EBGal agar (NOGI and FUKASAWA 1980) to test galactose fermentation contained ethidium bromide to a final concentration of 0.002% in YPGal, in which galactose is practically the only available source of carbon and energy. YPD agar for a-factor production was adjusted to pH 3.5 with acetic acid. Sporulation medium contained 1 % potassium acetate, 0.1 % yeast extract and
0.05% dextrose. Methylmethane sulfonic acid (MMS) me- dium used for RAD56 strains contained 0.01% MMS. Esch- erichia coli strains HBlOl and JM 109 were used for propa- gation of recombinant plasmids. The shuttle vectors be- tween E. coli and yeast were those described by ROSE and BROACH ( 1 99 1).
Cloning of the SGEl gene: A yeast gene library contained genomic DNA from strain S288C with an average size of 7 kb cloned into YEp24 (CARLSON and BOTSTEIN 1982). Transformation was performed by the lithium method (ITO et al. 1983) using YM4-5Aga180 carryinggal80::LEUZ as the cloning host. Ura+ transformants were first selected on uracil-lacking SD agar. Colonies were washed from the surface of SD agar and respread on EBGal agar to screen for Gal+ colonies. After proving mitotic cosegregation of Gal+ and Ura+, plasmids were recovered from the Ura+Gal+ transformants.
Plasmid constructions: Plasmids obtained in this study are as follows: pYMl101, the original isolate in this study from a YEp24-yeast gene library, contained 6.2 kb of insert DNA at the BamHI site (see Figure 1). pYMllO2 contained the 4-kb SalI fragment from pYM 1 101 in YEp24 at the SalI site. pYM 11 20 was constructed by ligating the 4-kb SalI fragment from pYMl101 into YCp50 at the SalI site. pYMll21 was constructed by self-ligation of a 1 1.8-kb EcoRI partial digestion product of pYM 1 10 1, resulting in removal of the 2.2-kb fragment containing 2-wm DNA. pHAl102T was constructed by ligating the 1.8-kb HindIII fragment of pYMllO2 into pTV3. pHA7 was constructed by removing the 1.9-kb SalI-SmaI fragment containing URA3 from YCp19-GAL7-lacZ (TAJIMA, NOGI and FUKA-
Assay of various enzymes: Yeast cell extracts for enzyme assays were prepared as described previously (TAJIMA, NOGI
SAWA 1985).
Suppressor of Yeast gall 1 Mutation 677
and FUKASAWA 1985). Yeast cells were grown exponentially for 16 hr in SGlyLacGal. When the cell density reached an absorbance at 660 nm of about 1 (2 X lo’ cells/ml), cells were collected, washed once with 10 mM Tris buffer (pH. 7.5), and resuspended in the same buffer containing 1 mM phenylmethylsulfonyl fluoride and 1 mM EDTA. Cell sus- pension was mixed with glass beads and vigorously agitated on Vortex mixer. Samples were centrifuged, and clear su- pernatant fluid was used as the enzyme source. Galactoki- nase was determined by the method described by NWI and FUKASAWA (1980). UDPGal-4-epimerase was determined by the two-step method described previously (FUKASAWA, SE- GAWA and NOGI 1982). &Galactosidase was determined as described previously in TAJIMA, NOGI and FUKASAWA (1 986). CAT was determined as described by SLEIGH (1986). Assays were performed on exponentially growing cell in tryptophan and uracil omission medium containing 5% glyc- erol, 0.1% glucose, 0.67% yeast nitrogen base and 0.5% casamino acid.
Biological assay of mating pheromones: This was car- ried out as described by JULIUS et al. (1 983). Approximately 1 O6 cells of a-factor supersensitive strain RC629 were spread on pH 3.5 YPD agar. Strains to be tested were patched on the lawn, and the plate was incubated at room temperature for 2 days.
Northern blot analysis: Total RNA was isolated from uninduced or galactose-induced yeast by the rapid method of ELDER, LOH and DAVIS ( 1 983). Five micrograms of glyox- alated RNA were fractionated by electrophoresis in 1.1 % agarose gel followed by transfer to Nylon filter membrane. The membrane was hybridized to radioactive sense or anti- sense RNAs that were synthesized usin SP6 RNA polym- erase (Takara Shuzo, Kyoto) and [a- PIUTP (3000 Ci/ mmol, Amersham) with pSP64 or pSP65 plasmids (Amer- sham kit RPN 1505) containing 0.68-kb Ssp1 fragments of SGEl DNA as templates.
DNA sequence analysis: Starting from the 2. I-kb XbaI- EcoRV fragment of pYMl10 1, a series of deletions were prepared by digestion with a combination of mung bean nuclease and exonuclease 111 from either end using Kilo- sequence deletion kit (Takara Shuzo) according to the sup- plier’s protocol. DNA sequence analysis was conducted by the dideoxy chain termination method (SANGER, NICKLEN and COULSON 1977) using DNA sequencing kit version 2 (U.S. Biochemical Corp.) according to the supplier’s proto- col.
Primer extension analysis: For primer extension analysis (MANIATIS, FRITSCH and SAMBROOK 1989), synthetic oligo- nucleotide (5’-GCACTCCTTTGTACCAAGTA-3’) com- plementary to a region from 206 through 225 from the first ATG was end-labeled with [y-’*P]dATP using polynucleo- tide kinase. Ten micrograms of total RNA were annealed to the radioactive primer at 25” for 20 hr, and an extension reaction was conducted at 42 with Rous-associated virus 2 reverse transcriptase (Takara Shuzo).
Chromosomal mapping: Physical mapping: Chromosomal DNAs of yeast strains, HSY5-3C (SGEl) and HSY5-3C-2 (sgel::URA3), were prepared by the agarose plug method as described in CARLE and OLSON (1987). Cells of SGEl (HSY5-3C) or sgel null (HSY5-3C-2) strains were embedded in agarose, treated successively with Zymolyase and protein- ase K to make chromosomal DNAs naked, and subjected to pulse-field gel electrophoresis at a 60-sec pulse followed by a 90-sec pulse for 8 hr at 200 V with a contour-clamped homogeneous electric field (CHEF) apparatus. Chromo- s ~ r r ? ~ ! DNA was transferred to a Nylon filter and hybridized with a 500-bp EcoRI probe DNA fragment from pYM 1 102, which was labeled with [a-32P]dCTP using a random primer
a PC+@ $? 4 i?p r qa a a
FiAsMl -
GfY)WMONW
PW11DG - A - + pWl107
PYMl108 7 + pYM1110 -A- - PYh4lll2 -
- - la K
- pW1102,1120 c + pYM1138 luIIsl -
FIGURE 1.-Restriction map of DNA encompassing the SGEl gene. The uppermost line represents the DNA fragment contained in the original isolate that suppresses the Gal- phenotype of gall I mutant yeast judged by the growth on ethidium bromide/galactose agar. Only selected restriction sites are shown above the line. The arrow indicates an approximate position and the direction of tran- scription of SGEl mRNA. Lines under the top line indicate DNA fragments contained in subclones constructed. Each fragment was inserted into YEp24 to give a plasmid whose designation is shown to the left. Plus or minus symbols shown in the right indicate those who are able or unable to suppress the Gal- phenotype, respectively. Abbreviations of restriction sites are B, BamHI; HIII, HzndIII; Hp, HpaI; Nc, NcoI; RI, EcoRI; Sau, Sau3AI; Xb, XbaI; Xh, XhoI; S, SalI; GI, ClaI.
labeling kit (Takara Shuzo) according to the supplier’s pro- tocol. To determine the chromosomal arm in which SGEl was located, a set of Nylon filters containing A or cosmid clones of the yeast genome [see LINK and OLSON (199 1); gift of LINDA RILES of MAYNARD V. OLSON’S Jaboratory] was hybridized with the same DNA probe as above.
Meiotic mapping: Meiotic mapping was carried out accord- ing to the procedure described by SHERMAN and WAKEM (1991).
RESULTS
Cloning of the SGEl gene: During the course of cloning the GAL1 I gene in the previous study (SUZUKI et al. 1988), we isolated two identical clones, out of 13,500 Ura+ transformants, that complemented the Gal- phenotype of a gull1 point mutant (YM4- 5Aga180) for growth on EBGal agar. Analysis of var- ious subclones from one of the isolates, pYM 1 10 1, revealed that the complementation activity was lo- cated within a 1.8-kb Hind111 fragment of the insert (Figure 1). The 2-pm DNA region was removed from pYM 1 10 1, and the resulting plasmid, pYM 1 12 1, was used to transform a gall I uru3 strain (YM4-5A) after digestion with XhoI. The resulting stable Ura+Gal+ transformant was crossed with wild type strain D273- 11A. Among meiotic tetrads Gal+:Gal- segregations of 4:0, 3:l and 2:2 were found, indicating that the complementing gene was not GALII. It was desig- nated SGEl (Suppressor for Gal Eleven). When pres- ent on a YCp plasmid (pYM1120) SGEl also sup- pressed the ga l l l mutatation (data not shown). As shown in Table 2A, both YEP-SGEI and Yep-SGEI in a galll background resulted in induction of UDPGal-4-epimerase by a factor of 3.5 to give a value of about 35% the wild-type level. This level of the
678 H. Amakasu et al.
TABLE 2
Relative specific activity of galactose-inducible enzymes in various yeast strains
Crude cell extracts used as enzyme source were prepared as described in MATERIALS AND METHODS. ' YCpGAL7-lacZ (pHA7) was introduced to the respective yeast strain in addition to the indicated plasmid.
19.5 units/mg protein. Not determined. 20. I units/mg. 23.8 units/mg.
f38.2 unis/mg. 8 20. I units/mg.
24.0 units/mg. 58.3 units/mg. i
galactose-metabolizing enzymes was clearly enough to support yeast growth on EBGal agar, where galactose was practically the sole carbon source. Since the av- erage copy number of YCp plasmids is 1 to 2, and since SGEI is a unique gene as judged by Southern analysis (data not shown), we suggest that one addi- tional copy of SGEl over the chromosomal copy is necessary and sufficient to suppress the Gal- pheno- type of the gull l mutant. SGEI in high copy can also suppress a gull1 null mutation and result in enzyme induction (Table 2B and Figure 2). To test whether the effect of SGEl on the expression of galactose- metabolizing enzymes was due to enhanced transcrip- tion of GAL genes, a GAL7-lac2 fusion carried by YCp vector (pHA7) was introduced into the SGEl-carrying transformant. As shown in Table 2B, the P-galactosid- ase activity increased to a similar extent as that of UDPGal-4-epimerase or galactokinase upon induc- tion, indicating that the increased expression of galac- tose-metabolizing enzymes was due to increased tran- scription of the respective genes.
SGEZ does not suppress other phenotypes of gaZZZ: In addition to weak fermentation of galactose, the pleiotropic gull 1 mutations cause a-specific steril- ity and slow growth on nonfermentable carbon sources (FASSLER and WINSTON 1989; NISHIZAWA et ul. 1990). SGEI in high copy did not suppress the
EBGal
FIGURE 2.-Suppression of Gal- phenotype of g a l l l null yeast by multiple copies of SGE1. Cells were pregrown in uracil-omission medium (SD) for 2 days at 30°, streaked on an EBGal plate, and incubatedat 30" for 2 days. 1, HSY5-3C ( G A L l l ) carrying YEp24; 2. HSY5-3B ( g a l l l null) carrying YEp24; 3, HSY5-3B carrying pYMl102 (YEp24SGEI); 4, HSY5-3BcarryingpYM1202 (YEp24- GALII) (SUZUKI et al. 1988).
growth defect on glycerol-lactate medium (Figure 3) or the defect in pheromone production (Figure 4).
Effect of loss of SGEZ function on the expression of galactose metabolizing enzymes: The 7.4-kb AutII-CluI fragment from pYM 1 102 was self-ligated, then a 0.5-kb S G E l fragment was replaced with URA3 to generate pYMll38 (see Figure 1). The 3.6-kb fragment between the HpaI and XhoI sites was excised from pYM 1 138 and was used to transform a homo- zygous uru3 diploid strain YM428. Tetrad analysis of
Suppressor of Yeast gull 1 Mutation 679
FIGURE 3.-Effect of SGEl on the growth of gall 1 null yeast on nonfermentable carbon sources. One-loopful culture of the indi- cated strain grown in uracil-omission medium (SD) was streaked on SGlyLac plate and incubated at 30" for 2 days. 1, HSY5-3B ( g a l l l null) carrying pYM1202 (YEp24-GALII); 2, HSY5-3C ( G A L I I ) carrying YEp24; 3, HSY5-3B (gal11 null) carrying YEp24; 4, HSY5-3B (gal l1 null) carrying pYMl102 (YEp24SGEI).
FIGURE 4.-Effect of SGEl on a-factor production in gall I null yeast. Cells of the indicated strains were patched on a lawn of RC629 (MATa sstl ) cells. 1 , HSY5-3C ( G A L I I ) carrying YEp24; 2, HSY5-3B (ga l l l null) carrying pYMIl02 (YEp24SGEI); 3, HSY5-3B carrying YEp24.
Ura+ transformants revealed that Ura+:Ura- markers segregated 2:2, demonstrating that the sgel::URA3 fragment had integrated at a single site in the genome. All the tetrads exhibited 4+:0- segregation for galac- tose utilization, indicating that the disruption of SGEI did not lead to a defect in galactose utilization as judged by the growth on EBGal agar. By contrast, point mutations or disruption of G A L l l is known to result in essentially no growth on EBGal agar (NOGI and FUKASAWA 1980; SUZUKI et al. 1988). To quanti- tate the effect of loss of the SGEl function more precisely, the levels of galactokinase and UDPGal-4- epimerase activity present in the sgel null mutant were determined. Activity of &galactosidase that was di- rected by the GAL7-lac2 gene fusion was also deter- mined. As shown in Table 2C, disruption of SGEI reduced activity for all three enzymes 40-50%. Be- cause GAL7-promoted P-galactosidase activity was de- creased to the same extent as the galactose-metaboliz- ing enzymes, the effect of sgel null mutations was
TABLE 3
Effect o f SGEI on GAL4 expression
CAT activity (unitlpg
Strain Genotype Plasmid protein)"
HSY5-3C-CAT G A L l l pTV3 14.4 * 0.2 HSY5-3B-CAT gall I pTV3 14.1 * 0.2 HSY5-3B-CAT gal l1 pTV3SGEI 14.7 k 0.2
' The CAT assay was carried out as described under MATERIALS AND METHODS. One unit of cat activity is the amount of enzyme required to acetylate 1 pmol of ['4C]chloramphenicol/min at 37".
presumed to be exerted at the level of transcription. The g a l l l null mutant synthesizes the galactose-
metabolizing enzymes at 10-20% of the wild-type levels (see Table 2). To test whether the residual expression of GAL genes in g a l l l null mutants was affected by the loss of SGEI, the g a l l l sgel double- mutant yeast was constructed, and the enzyme levels were determined as above. The double null mutant expressed the galactose enzymes to a level comparable to the isogenic single gal l I mutant, indicating that the residual expression of GAL genes in the gal l I null mutant was independent of the SGEl function (Table
Multiple copies of SGEl do not affect the expres- sion of GALI: We know that the level of GAL4 expression is rate-limiting for induction of GAL gene expression (JOHNSTON and HOPPER 1982; HASHIMOTO et al. 1983), and that the expression of GAL4 itself is under the control of yet unidentified regulatory gene(s) (GRIGGS and JOHNSTON 199 1). We therefore tested whether or not GAL4 expression was influenced by multiple copies of SGEl in a g a l l l mutant strain. pTV3-SGEI (pHA1102T) or pTV3 alone was intro- duced into a yeast strain bearing a gal l I null mutation and a GAL4-CAT gene fusion (HSY5-3C-CAT, HSY5- 3B-CAT), and the activity of CAT was determined. In parallel, the CAT activity of the wild-type strain bearing the GALCCAT fusion and pTV3 was also determined. No difference was observed among these strains in the enzyme activity (Table 3). We concluded therefore that neither GALI 1 nor SGEI was involved in the expression of GAL4.
Molecular analysis of SGEl: The 1954-bp nucleo- tide sequence of the SspI-Hind111 fragment encom- passing SGEl was determined. An ORF of 543 amino acids was identified (Figure 5), which consists of 59.3% hydrophobic, 16.0% hydrophilic and 24.7% neutral amino acids. The molecular weight of Sgelp was caliculated to be 59,425. Northern blot analysis of total RNA extracted from cells grown in glucose- containing medium, detected a 1.8-kilonucleotide mRNA that was capable of encoding the SGEI ORF as judged by complementarity with the strand-specific RNA probes (Figure 6). The SGEI transcript level was not significantly changed if cells were grown on
2C).
680
1
3 5
1 1 5
1 9 5
2 7 5
3 5 5
4 3 5
5 1 5
5 9 5
6 1 5
7 5 5
8 3 5
9 1 5
9 9 5
H. Amakasu et al.
ATTGCCAATGTAAATTAAGCTATCGGTCCGTTTA
TTTTTCTATCG~GCGGCTTAGTTAGAATGGTTGATAAACGGCTGGCCTTCGTAAAAAGAAATGACATCTTGGAACTA I 1
ATGAAGAGTACTTTGAGTTTAACTTTATGTGTTATATCGCTTCTATTAACCCTTTTTCTGGCGGCCTTGGATATTGTTAT M K S T L S L T L C V I S L L L T L F L A A L D I V I 2 7
AGTGGTTACTTTATATGATACAATTGGCATTAAGTTCCATGACTTCGGCAATATTGGTTGGTTAGTTACTGGATATGCTC V V T L Y D T I G I K F H D F C N I G W L V T G Y A L 5 4
TTTCTAATGCTGTTTTCATGTTATTATGGGGTCGCTTGGCCGAAATACTTGGTACAAAGGAGTGCTTAATGATTTCTGTT S N A V F M L L W C R L A E I L G T K E C L M I S V 8 0
ATTGTATTTGAAATAGGGTCTTTGATTTCTGCTCTTTCGAATTCAATGGCGACTCTGATTAGCGGMGAGTCGTTGCTGG I V F E I G S L I S A L S N S M A T L I S G R V V A G l O 7
CTTTGGAGGAACTGGAATTGAATCACTTGCTTTTGTAGTTGGAACATCCATTGTCCGAGAAAACCATAGAGGAATTATGA F G G S G I E S L A F V V G T S I V R E N H R G I M I
TAACGGCACTCGCTATATCGTATGTCATTGCAGAGGGAGTCGGGCCTTTTATTGGTGGTGCATTTAATGAACATTTGTCT T A L A I S Y V I A E G V C P F I G G A F N E H L S
TGGAGATGGTGCTTTTATATAAATCTTCCAATCGGTGCGTTTGCGTTCATAATATTGGCATTTTGT~CACATCTGGAGA W R W C F Y I N L P I G A F A F I I L A F C N T S G E
ACCACATCAAAAAATGTGGCTACCATCAAAAATC~TTATGAACTATGACTATGGCGAATTATTGA~GC~GTT P H Q K M W L P S K I K K I M N Y D Y C E L L K A S F 2 1 4
TTTGGAAGAATACATTTGAAGTACTTGTATTTAAACTAGACATGGTTGGGATTATTTTATCTTCAGCAGGCTTTACACTA W K N T F E V L V F K L D M V G I I L S S A G F T L 2 1 0
CTGATGTTAGGTCTTTCATTTGGTGGAkACA4CTTCCCATGGAATTCGGGTATCATTATTTGCTTTTTTACCGTGGGCCC L M L G L S F G C N N F P W N S C I I I C F F T V G P 2 6 7
TATCTTATTGTTACTATTTTGTGCTTACGACTTTCATTTTCTGTCATTATCGGGGCTTCACTATGACAACAAGCGGATCA I L L L L F C A Y D F H F L S L S G L H Y D N K R I K 2 9 4
1 0 7 5 AACCGTTACTGACATGGAATATTGCCTCAAATTGTGGCATATTTACAAGCTCCATAACAGGATTCCTTTCTTGCTTTGCT P L L T W N I A S N C G I F T S S I T C F L S C F A 3 2 0
1 1 5 5 TATGAATTACAGTCTGCTTATTTAGTCCAGCTTTATCAACTAGTATTTA~AAAGCCTACATTAGCGAGTATACATCT Y E L Q S A Y L V Q L Y Q L V F K K K P T L A S I H L l 4 7
1 2 3 5 TTGGGAACTATCAATTCCAGCTATGATTGCAACTATGGCCATAGCATATCTAAATTCAAAATATGGCATCATCAAACCGG W E L S I P A M I A T M A I A Y L N S K Y C I I K P A 3 7 4
1 1 1 5 CAATTGTTTTTGGTGTGCTTTGTGGGATTGTTGGATCTGGTTTATTTACGCTAATCAATGGCGAACTCTCTCAGTCAATA I V F G V L C G I V G S G L F T L I N G E L S Q S I 400
1 3 9 5 GGTTATTCAATTCTCCCAGGAATAGCTTTTGGTAGTATTTTCCAAGCAACGTTATTAAGCTCCCAGGTGCAGATAACATC G Y S I L P C I A F G S I F Q A T L L S S Q V Q I T S 4 2 7
1 4 7 5 AGACGATCCAGACTTTCAAAACAAGTTTATTGAAGTCACAGCTTTCAACTCGTTCGCCAAATCCTTGGGCTTTGCGTTTG D D P D F Q N K F I E V T A F N S F A K S L G F A F C 4 5 4
1 5 5 5 G A G G G A A T A T G G G G G C A A T G A T A T T C A C T G C A T C A C T C A A C A T A C C A C A A T T T C N M C A M I F T A S L K N Q M R S S Q L N I P Q F 4 8 0
1 6 3 5 ACCTCTGTAGAAACACTTTTAGCGTATAGCACAGAACATTATGATCGCCCCCAATCTTCACTATCAAAGTTCATAAACAC T S V E T L L A Y S T E H Y D G P Q S S L S K F I N T 5 0 7
1 7 1 5 AGCTATCCATGACCTTTTTTACTGCGCCTTAGGATGCTATGCTCTTTCATTCTTCTTTGGAATATTCACTTCGAGTAACA A I H . D V F Y C A L G C Y A L S F F F G I F T S S K K 5 3 4
1 7 9 5 IV \ACAACMTATCAGCCAAAAAGCAACAATGAACAATTTTCCAACGTATAAAATTAACTTATCGTAGTTCGATAAAACTA T T I S A K K Q Q ’ 5 4 1
FIGURE 5.-Nucleotide sequence and predicted amino acid sequence of the S G E l gene. The transcriptional starts determined by the experiments in Figure 7 are indicated (I) on the sequence (GenBank accession no. L11640).
glycerol-lactate or galactose (data not shown). The 5’- 15 and 18 bp upstream of the first ATG of the ORF ends of the SGEl transcripts were determined by the (Figure 7). The 5”nontranscribed region of 194 bp primer extension method at two major sites located that was sequenced contained neither UASG nor any
Suppressor of Yeast gall 1 Mutation 68 1
1 2
- 1.8knt
1 2 3 G A T C
FIGURE 6.-Northern analysis of total RNA using strand-specific probes. Lanes 1 and 2 were probed with sense and antisense RNA synthesized in vitro by the use of the SP6 system on a template derived from the S C E l ORF. Five micrograms of total RNA from the wild-type yeast were fractionated in each lane. The molecular sizes indicated on the right were deduced from positions of yeast ribosomal RNAs (3.2 and 1.6 knt) visualized in the ethidium bro- mide-stained gel.
other elements known to be involved in transcrip- tional regulation. No canonical TATA box was found within the 5”region.
SGEl was located near the right end of chromo- some 16: The SGEl probe revealed a band that cor- responded to chromosome 13 or 16. These chromo- somes were not separated under our conditions. The SGEI probe was hybridized to a set of X prime clones and found to hybridize with a clone derived from the rightmost region of chromosome 16. Since RAD56 was known to be located near the right end of chro- mosome 16 (MORTIMER et al. 1989), a strain carrying trpl sge1::TRPI (HSY5-3C-3) was crossed with a strain carrying trpl rad56 (XS2456-9B). The resulting dip- loid was subjected to tetrad analysis for the linkage between rad56, determined by MMS sensitivity, and SGEl as tryptophan prototrophy. All 61 complete tetrads studied were parental ditype, indicating that SGEl was closely linked to RAD56 within a distance of less than 0.8 cM.
FIGURE 7.-Mapping of the 5”ends of SGEl mRNA by primer extension. An end-labeled oligonucleotide complementary to the region of nucleotide positions from 400 through 4 19 (see Figure 5) was used to prime reverse transcription using 10 pg of total RNA from yeast strains indicated below. Lanes 1 and 2 contained RNAs from HSY5-3C-2 (5gel::LIRAjr) and HSY5-3C ( S G E l ) , respectively. Lane 3 contained denatured DNA of 298 and 220 bp. Lanes C, T , A and G represent sequence ladders corresponding to the respective bases.
DISCUSSION
We have demonstrated that two copies of SGEl are able to suppress the Gal- phenotype caused by a gall 1 mutation by increasing the expression of galactose- inducible enzymes. The suppression is not complete, and the increased level of galactose enzymes in gull I null yeast carrying Y Ep-SGEI does not reach the wild- type level. We have also shown that SGEl itself is required for full expression of GAL genes; the disrup- tion of this gene in wild-type yeast decreases galactose enzyme activity to half the wild-type level. The ob- served effect of two copies of SGEl in gall 1 null yeast as well as that of SGEl disruption in the wild type are presumed to reflect changes at the transcriptional level since expression of a GAL7-lac2 fusion is similarly affected. Since YEP-SGE1 does not suppress other phenotypes of gall 1 mutation, such as poor produc- tion of cy pheromone and slow growth on nonfer- mentable carbon source, Sgelp function is involved specifically in transcription of GALI-dependent genes. Recently, a gull I null mutation has been reported to
682 H. Amakasu et al .
decrease SUC2 expression (VALLIER and CARLSON 1991). We have found that two copies of SGEl did not affect the low expression of SUC2 in gal I I mutants (data not shown). We have excluded the possibility that multiple copies of SGEl cause enhanced tran- scription of GAL4, which in turn would suppress the Gal- phenotype of gall I yeast. One might argue that SGEI in multicopy state can suppress a gall I mutation by increasing the intracellular concentration of Gal4p for other reasons, for instance, stabilization of Gal4p. Such a possibility is also unlikely since overproduction of Gal4p by using an overexpression vector described by CHASMAN and KORNBERC (1 990) in gal I I null yeast did not suppress the mutant phenotypes (our unpub- lished observation). We have further demonstrated that disruptions of both G A L l 1 and SGEI in a cell does not result in complete shut-off of GAL gene expression. This result suggests either that Gal4p alone can stimulate transcription of GAL genes to a limited extent, or that another gene can substitute for the functions of GAL11 and/or SGEI. It has been postulated that Gall l p mediates the activation func- tion of Gal4p to the basal transcription machinery, possibly by protein-protein interaction (NISHIZAWA et al. 1990; HIMMELFARB et al. 1990). Based on these postulates, we suggest that Sgelp can link Gal4p with the basal transcription machinery if Gall Ip is missing. If this is the case, the affinity of interaction between Gall l p and Gal4p or the basal transcription machin- ery should be smaller than that of Gal1 lp , since only multiple copies of SGEl can substitute, even though partially, for Gall l p function. The fact that the spec- trum of SGEl is limited to GAL genes may suggest that Gall l p has a distinct domain specific for Gal4p- mediated transactivation. Gal4p is now known to func- tion “universally”; when introduced into any of a wide variety of eukaryotic cells, it activates transcription of a chimeric gene bearing the Gal4p-binding sequence (UASG) in its upstream region [see PTASHNE and GANN (1 990) for review]. We hope, therefore, that elucida- tion of functions of G A L l I together with SGEI may furnish an important clue to our understandings of transcription regulation not only of yeast GAL genes but also of eukaryotic genes in general. Needless to say, biochemical evidence for interaction, physical or functional, of these proteins is needed to test these ideas.
We have not been able to find genes similar to SGEl in GenBank and EMBL. Since Sgelp contains an unusually large (59.3%) amount of hydrophobic amino acids, we suspect that the protein might be an integral membrane protein. Intracellular localization of Sgelp remains to be studied.
We are indebted to DAVID BOTSTEIN for the gene library, LINDA RILES for the blots of aligned clones of yeast genome together with unpublished results, to MARK JOHNSTON for pBM1974 and HIDE-
YUKI OGAWA for XS2456-9B. We also thank members of Molecular Genetics Group of Keio University School of Medicine for discus- sions and comments to the present work. This work is supported in part by grants in aid from Human Frontier Science Project, and from Ministry of Education and Science to T.F.
LITERATURE CITED
BUCHMAN, A. R., N. F. LUE and R. D. KORNBERG, 1988 Connections between transcriptional activators, silencers, and telomeres as revealed by functional analysis of a yeast DNA- binding protein. Mol. Cell. Biol. 8: 5086-5099.
CARLE, G. F., and M. V. OLSON, 1987 Orthogonal-field-alterna- tion gel electrophoresis. Methods Enzymol. 155: 468-482
CARLSON, M., and D. BOTSTEIN, 1982 Two differentially regu- lated mRNAs with different 5’ ends encode secreted and intracellular forms of yeast invertase. Cell 28: 145-154.
CHAN, R. K., and C. A. OTTE, 1982 Isolation and genetic analysis of Saccharomyces cerevisiae mutants supersensitive to G1 arrest by a factor and LY factor pheromones. Mol. Cell. Biol. 2: 11- 20.
CHASMAN, D. I . , and R. D. KORNBERG, 1990 GAL4 protein: purification, association with GAL80 protein, and conserved domain structure. Mol. Cell. Biol. 10: 2916-2923.
ELDER, R. T . , E. Y. LOH and R. W. DAVIS, 1983 RNA from the yeast transposable element Tyl has both ends in the direct repeats, a structure similar to retrovirus RNA. Proc. Natl. Acad. Sci. USA 8 0 2432-2436.
FASSLER, J. S. , and F. WINSTON, 1989 The Saccharomyces cereuisiae SPT13IGALl I gene has both positive and negative regulatory roles in transcription. Mol. Cell. Biol. 9: 5602-5609.
FUKASAWA, T., T. SEGAWA and Y. NOGI, 1982 Uridine diphos- phate glucose-4-epimerase and galactose-1-phosphate uridyly- transferase from Saccharomyces cereuisiae. Methods Enzymol. 8 9 584-589.
GRIGGS, D. W., and M. JOHNSTON, 1991 Regulated expression of the GAL4 activator gene in yeast provides a sensitive genetic switch for glucose repression. Proc. Natl. Acad. Sci. USA 88:
HASHIMOTO, H., Y. KIKUCHI, Y. NOGI and T. FUKASAWA, 1983 Regulation of expression of the galactose gene cluster in Saccharomyces cereuisiae. Isolation and characterization of the regulatory gene GAL4. Mol. Gen. Genet. 191: 31-38.
HIMMELFARB, H. J., J. PEARLBERG, D. H. LAST and M. PTASHNE, 1990 GALl 1P: a yeast mutation that potentiates the effect of weak GAL4-derived activators. Cell 63: 1299-1 309.
ITO, H., Y. FUKUDA, K. MURATA and A. KIMURA, 1983 Transformation of intact yeast cells treated with alkali cations. J. Bacteriol. 153: 163-168.
JOHNSTON, M., 1987 A model fungal gene regulatory mechanism: the GAL genes of Saccharomyces cereuisiae. Microbiol. Rev. 51: 458-476.
JOHNSTON, S. A., and J. E. HOPPER, 1982 Isolation of the yeast regulatory gene GAL4 and analysis of its dosage effects on the galactose/melibiose regulon. Proc. Natl. Acad. Sci. USA 7 9 6971-6975.
JULIUS, D., L. BLAIR, A. BRAKE, G. SPRAGUE and J. THORNER, 1983 Yeast a factor is processed from a larger precursor po1ypeptide:the essential role of a membrane-bound dipeptidyl aminopeptidase. Cell 32: 839-852.
KELLEHER, R. J., 111, P. M. FLANAGAN and R. D. KORNBERG, 1990 A novel mediator between activator proteins and the RNA polymerase 11 transcription apparatus. Cell 61: 120% 1215.
LINK, A. J., and M. V. OLSON, 1991 Physical map of the Saccha- romyces cereuisiae genome at 1 10-kilobase resolution. Genetics 127: 681-698.
LUE, N . F., D. I. CHASMAN, A. R. BUCHMAN and R. D. KORNBERG,
8597-860 1.
Suppressor of Yeast gall 1 Mutation 683
1987 Interaction of GAL4 and GAL80 gene regulatory pro- teins in vitro. Mol. Cell. Biol. 7: 3446-3451.
MANIATIS, T., E. F. FRITSCH and J. SAMBROOK, 1989 Analysis of RNA by primer extension, pp. 7.79-7.87 in Molecular Cloning: A Laboratory Manual, Ed. 2. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.
MORTIMER, R. K., D. SCHILD, C. R. CONTOPOULOU and J. A. KANS, 1989 Genetic map of Saccharomyces cerevisiae, Edition 10. Yeast 5: 321-403.
NISHIZAWA, M., Y. SUZUKI, Y. NOGI, K. MATSUMOTO and T. FUKASAWA, 1990 Yeast Gall 1 protein mediates the transcrip- tional activation signal of two different transacting factors, Gal4 and general regulatory factor l/repressor/activator site binding protein I/translation upstream factor. Proc. Natl. Acad. Sci.
NOGI, Y., and T. FUKASAWA, 1980 A novel mutation that affects utilization of galactose in Saccharomyces cerevisiae. Curr. Genet.
PARTHUN, M. R., and J. A. JAEHNING, 1990 Purification and characterization of the yeast transcriptional activator GAL4. J. Biol. Chem. 265: 209-213.
PTASHNE, M., and A. A. F. GANN, 1990 Activators and targets.
PUGH, B. F., and R. TJIAN, 1990 Mechanism of transcriptional activation by Spl: evidence for coactivators. Cell 61: 1 187- 1197.
ROSE, M. D., and J. R. BROACH, 1991 Cloning genes by comple- mentation in yeast. Method Enzymol. 194 195-230.
SAEKI, T., 1. MACHIDA and S. NAKAI, 1980 Genetic control of diploid recovery after y-irradiation in the yeast Saccharomyces cerevisiae. Mutat. Res. 73: 251-265.
SANGER, F., S. NICKLEN and A. R. COULSON, 1977 DNA sequenc-
USA 87: 5373-5377.
2: 1 15-1 20.
Nature 346: 329-33 1.
ing with chain terminating inhibitors. Proc. Natl. Acad. Sci.
SHERMAN, F., 1991 Getting started with yeast. Method Enzymol.
SHERMAN, F., and P. WAKEM, 1991 Mapping yeast genes. Methods Enzymol. 194 38-57.
SHORE, D., and K. NASMYTH, 1987 Purification and cloning of a DNA binding protein from yeast that binds to both silencer and activator elements. Cell 51: 721-732.
SLEIGH, M. J., 1986 A nonchromatographic assay for expression of the chloramphenicol acetyltransferase gene in eucaryotic cells. Anal. Biochem. 156 251-256.
SUZUKI, Y., Y. NOGI, A. ABE and T. FUKASAWA, 1988 G A L l l protein, an auxiliary transcription activator for genes encoding galactose-metabolizing enzymes in Saccharomyces cereuisiae. Mol. Cell. Biol. 8 4991-4999.
TAJIMA, M., Y. NOGI and T. FUKASAWA, 1985 Primary structure of the Saccharomyces cerevisiae GAL7 gene. Yeast 1: 67-77.
TAJIMA, M., Y. NocrandT. FUKASAWA, 1986 Duplicate upstream activating sequences in the promoter region of the Saccharo- myces cerevisiae GAL7 gene. Mol. Cell. Biol. 6 246-256.
VALLIER, L. G., and M. CARLSON, 1991 New SNF genes, G A L l l and GRRl affect SUC2 expression in Saccharomyces cerevisiae. Genetics 129 675-684.
WINTER, E., F. YAMAMOTO, C. ALMOGUERA and M. PERUCHO, 1985 A method to detect and characterize point mutations in transcribed genes: amplification and overexpression of the mutant c-Ki-ras allele in human tumor cells. Proc. Natl. Acad. Sci. USA 82: 7575-7579.
YUN, S. J., Y. HIRAOKA, M. NISHIZAWA, K. TAKIO, K. TITANI, Y. NOGI and T . FUKASAWA, 1991 Purification and characteriza- tion of the yeast negative regulatory protein GAL80. J. Biol. Chem. 266: 693-697.
