Mol Gen Genet (1995) 247:716-725

© Springer-Verlag 1995

Satoshi Harashima • Takayuki Mizuno Hiroyuki Mabuchi • Shin-ichi Yoshimitsu Ray Ramesh - Masahisa Hasebe • Atsuko Tanaka Yasuji Oshima

Mutations causing high basal level transcription that is independent of transcriptional activators but dependent on chromosomal position in

Saccharomycescerevisiae Received: 18 July 1994/Accepted 15 December 1994

Abstract Two single (bel2 and bel4) and two double (bel3 bel7 and bel5 bel6) mutations causing enhanced transcription of a gene fusion, consisting of the open reading frame of PH05 connected to the HIS5 promoter (HIS5p) integrated at the ura3 or leu2 locus, were isolated from a gcn4-disrupted mutant of Saccharomyces cerevisiae. The PH05 gene, encoding repressible acid phosphatase, in the HIS5p-PH05 construct was derepressed under amino acid starved conditions by the action of the transcriptional activator Gcn4p. The bel mutants showed temperature-sensitive cell growth and/or cell aggregation. All the mutants except bel4 also showed high levels of transcription of an intact PH05 DNA integrated at the URA3 locus in the absence of the cognate transcriptional activator, Pho4p, and in the absence of upstream activating sequences of PH05. The HIS5 and PH05 genes at their original chromosomal positions were, however, not affected by the bel2 mutation. The BEL2 gene was found to be identical with SIN4/TSF3, mutations in which cause high levels of transcription of the HO and GAL genes in the absence of their respective transcriptional activators, Swi5p and Gal4p. The effect of the bel2/sin4/tsf3 mutation on PH05 transcription was additive with the Pho4p function. Thus the effect of the bel2/sin4/tsf3 mutation is dependent on the position of PH05 in the chromosome and independent of Pho4p and Gen4p activation. Key words Basal transcription - BEL2/SIN4/TSF3 gene - GCN4 • PH04 • Yeast

Communicated by C. P. Hollenberg S. Harashima. T. Mizuno • H. Mabuchi • S. Yoshimitsu - R. Ramesh M. Hasebe • A. Tanaka. Y. Oshima ( I ~ ) Department of Biotechnology, Faculty of Engineering, Osaka University, 2-1 Yamadaoka, Suita-shi, Osaka 565, Japan

Introduction The basal level of transcription of a gene is believed to be determined by its promoter structure but to be independent of the function of gene specific transcriptional activators. It has, however, been reported that several trans-acting factors affect the basal levels of transcription of various genes in Saccharomyces cerevisiae. These are BAS1 and BAS2 (=PH02) (Arndt et al. 1987) and RAP1 (Devlin et al. 1991) which modulate the basal level of HIS4 transcription. Several negative factors have also been reported to affect the basal level of transcription. These include SIN1-SIN4 (Peterson et al. 1991; Nasmyth et al. 1987; Kruger and Herskowitz 1991; Vidal et al. 1991), SIT1-SIT4 (Arndt et al. 1987; 1989), SIR1-SIR4 (Nasmyth 1982), SPT (Roeder et al. 1985; Fassler and Winston 1988; Suzuki et al. 1988; Malone et al. 1991), SSN20 (Neigeborn et al. 1987), and MOT1 (Davis et al. 1992). Some of these were shown to suppress a non-transcriptional phenotype of several independent genes caused by deletion of the upstream activation site (UAS) and mutation of the relevant specific transcriptional activator. These mutations often confer other mutant phenotypes, such as temperature-sensitive cell growth, sporulation defects, and cell aggregation. One possible interpretation of these mutations is that they cause alterations in the structure of the nucleosome or chromatin, because nucleosome loss results in an increased basal level of transcription (Han and Grunstein 1988; Durrin et al. 1992) and the sin4 mutants were suggested to contain altered chromatin. An HMG-like protein encoded by SIN1/SPT2 (Kruger and Herskowitz 1991) was suggested to be involved in maintaining the proper structure of chromatin. To extend our understanding of the mechanism responsible for basal level transcription of genes in S. cerevisiae, we isolated mutants showing enhanced levels of repressible phosphatase (rAPase) activity

717 p r o d u c e d f r o m a H I S S p - P H 0 5 gene f u s i o n c o n s t r u c t e d b y c o n n e c t i n g t h e HIS5 p r o m o t e r to t h e P H 0 5 o p e n reading frame encoding the major fraction of rAPase ( O s h i m a 1991), w h i c h w a s i n t e g r a t e d a t t h e ura3 o r leu2 l o c u s in gcn4 m u t a n t cells. T h e HIS5 g e n e is t r a n s c r i b e d a t a l o w level w h e n cells a r e g r o w n in a m i n o a c i d - r i c h m e d i u m , a n d its t r a n s c r i p t i o n is e n h a n c e d b y s t a r v a t i o n for c e r t a i n a m i n o acids, i n c l u d i n g h i s t i d i n e , b y t h e a c t i o n o f t h e G c n 4 p p r o t e i n , b u t t h e gcn4 m u t a n t s h o w e d n o s u c h d e r e p r e s s i o n o f HIS5 ( N i s h i w a k i et al. 1987; H i n n e b u s c h 1988). H e r e we r e p o r t t h a t o n e class of m u t a n t s , bel2, w h i c h a r e alleles of t h e sin4 o r tsf3 m u t a t i o n , a n d t w o bel3 bel7 a n d bel5 be16 d o u b l e m u t a t i o n s a r e a s s o c i a t e d w i t h s i g n i f i c a n t levels of t r a n s c r i p t i o n in p h o 4 - a n d g c n 4 - cells w h e n t h e H I S 5 p - P H 0 5 c o n s t r u c t a n d t h e i n t a c t P H 0 5 g e n e w e r e i n s e r t e d a t t h e ura3 o r leu2 locus, b u t n o t a t t h e HIS5 o r P H 0 5 locus.

Materials and methods Strains and gene bank The S. cerevisiae strains used are listed in Table 1. Escherichia coli strain JA221 (Sambrook et al. 1989) was used for construction and preparation of plasmid DNAs. For cloning the BEL2 gene, the gene library CEN BANK A (Rose et al. 1987) was purchased from the American Type Culture Collection (Rockville, Md., USA).

with HindIII and BamHI, and cloned into the HindIII-BamHI gap of pSH39 to give plasmid p714. Plasmid pSH40 carrying the PH05 gene without UASs was constructed similarly by cloning the PCR product obtained with pPHO5 as template and oligonucleotides corresponding to the sequence from position - 182 to - 160 of PH05 with an additional HindIII site and CTC at the 5' end, as the forward primer, and OLI142, as the reverse primer, into the HindIIIBamHI gap of pSH39. All the primer oligonucleotides were synthesized with a DNA synthesizer (Gene Assembler Plus, Pharmacia LKB, Uppsala, Sweden). Plasmid p615 bearing a disrupted allele of PH04 (i.e. pho4: :LEU2) was constructed by insertion of a 2.1 kb SalI-XhoI fragment of the LEU2 DNA from YEpl3 into the HpaI site of the PH04 gene (Ogawa and Oshima 1990) in pUC19 after blunt-ending the SalI and XhoI ends. Plasmid pYA301 (Galtwitz and Sures 1980), carrying the ACT1 gene, was provided by D. Gallwitz.

Media, and genetic and biochemical methods The media and genetic methods used for S. cerevisiae (Rose et al. 1990; Rothstein 1983) and E. coli (Sambrook et al. 1989) were as described. Nutrient high-Pi (YPAD; 10 g of yeast extract, 20 g of bactopeptone, 0.4 g of adenine and 20 g of glucose per 1) and low-Pi (YPAD-P; YPAD depleted of inorganic phosphate) media (Bun-ya et al. 1991), and a methods to detect rAPase activity of colonies (Toh-e and Oshima 1974) and rAPase activity in cell suspensions (Toh-e et al. 1973) of S. cerevisiae were described previously. Synthetic complete medium (SC; Rose et al. 1990) and SC medium with 1/10 the concentration of tryptophan (SC-TRP) were used as repressing and derepressing conditions, respectively, for the HIS5 promoter. Manipulations of DNA and RNA were as described by Sambrook et al. (1989). U r a - clones of S. cerevisiae were selected with 5-fluoroorotic acid (5-FOA) as described (Boeke et al. 1987). Specific rAPase activities were calculated as mU per ml per U optical density of cell suspensions at 660 nm (OD66o).

Plasmids Plasmid pHRU1 (Fig. 1) was constructed by inserting a 2.1kb BamHI-HindIII fragment bearing the HIS5p-PH05 construct from plasmid pVC727A (Hwang et al. 1989) into the BamHI-HindIII gap of YIp5 (Parent et al. 1985). Plasmid pHRL1 (Fig. 1) was constructed by inserting the same 2.1 kb BamHI-HindIII fragment into the BamHI-HindIII gap of YIp32 (Parent et al. 1985). Plasmid p512 (Fig. 1) was constructed by inserting a 3 kb BamHI fragment bearing the PH05 gene from plasmid pPHO5 (Arima et al. 1983) into the BamHI site of YIp5. A modified plasmid of p512 bearing the wildtype PH05 with a 5.4 kb 5' upstream region (L-PH05 ÷) was also constructed by the following procedure: p512 was integrated at the phoS-1 locus of SH2128-CU (described later) and a 15.5 kb HindIII fragment bearing the PH05 gene (connected to a 5.4 kb 5' upstream fragment of PH05), URA3, and Amp ~ regions of p512 was isolated through E. coli transformation. A 5.5 kb HindIII-SalI fragment bearing the 5.4 kb promoter and an 82 bp region encoding the Nterminal end of PH05 was isolated by restriction of plasmid DNA prepared from the E. coli transformant and the fragment was inserted into the HindIII-SalI gap of p512 in the 5' region ofthePH05 ORF. The resultant plasmid has the same structure as p512 but the PH05 gene has a longer promoter (5.4 kb) than in p512. Plasmid pSH39 bearing PH05 as a reporter gene was described previously (Mukai et al. 1993). Plasmid p714 (Fig. 1) was constructed as follows. A 523 bp fragment of PH05 (nucleotide positions - 541 to - 19) was amplified by the polymerase chain reaction (PCR) using pPHO5 as template and an oligonucleotide corresponding to the sequence from position - 541 to - 519 of PH05 with an additional HindIII recognition sequence and CTC at the 5' end as forward primer, and an oligonucleotide designated as OLI142 (positions - 19 to - 38 of PH05 with an additional BamHI site and CTC at the 5' end) as reverse primer. The PCR product was doubly digested

Accession number of the nucleotide sequence of BEL2 The nucleotide sequence of the BEL2 gene has been deposited in the DDBJ/EMBL/GenBank nucleotide sequence data base under the accession number D12918.

Results U p - l e v e l m u t a t i o n s for b a s a l e x p r e s s i o n o f t h e HISSp-PH05 construct I n g e n e r a l , e x p r e s s i o n of t h e g e n o m i c P H 0 5 g e n e is r e p r e s s e d in h i g h - P i m e d i u m b u t d e r e p r e s s e d in l o w - P i m e d i u m a n d t h e r A P a s e a c t i v i t y o f c o l o n i e s o n p l a t e s is d e t e c t a b l e b y t h e specific s t a i n i n g m e t h o d . H e r e we e m p l o y e d this P H 0 5 O R F as a r e p o r t e r gene b u t c o n n e c t e d it to t h e HIS5 p r o m o t e r . M u t a n t s s h o w i n g inc r e a s e d r A P a s e a c t i v i t y w e r e s e l e c t e d f r o m t h e HIS5pP H 0 5 g c n 4 - s t r a i n s S H 2 1 2 8 (with t h e H I S S p - P H 0 5 c o n s t r u c t i n s e r t e d a t t h e ura3 locus), S H 2 1 5 4 (with t h e s a m e c o n s t r u c t i n s e r t e d a t t h e leu2 locus) a n d S H 2 2 5 7 (with t h e s a m e c o n s t r u c t i n s e r t e d at t h e ura3 locus) after ethyl methanesulfonate (EMS) mutagenesis. Of a p p r o x i m a t e l y 15000, 5000 a n d 1 0 0 0 0 c o l o n i e s f r o m strains SH2128, SH2154 and SH2257, respectively,

718 Table 1 The Sacchromyces cerevisiae strains used Strain

Mating type

Genotype"

Source

SH679 SH1877 SH1262 SH1273 SH2128

M A Tc~ MATc~ MATa MATa MATa

SH2154

MATa

leu2-3,112 trpl pho3-1 pho5-1 ade2 his3-532 leu2-3,112 trpl-289 ura3-1,2 pho3-1 PH05 + gcn4-103 hisl-29 leu2-3,112 trpl pho3-1 pho5-1 gcn4-103 hisl-29 trpl pho3-1 pho5-1 ura3-52 : ." [ura3-52 HIS4-1acZ] gcn4-103 hisl-29 trpl pho3-1 pho5-1 ura3-52 :." Jura3-52 HIS4-lacZ] ura3-52 : : [URA3 HIS5p-PH05] gcn4-103 hisl-29 trpl pho3-1 pho5-1 Ieu2-3,112 .": [LEU2 HIS5p-PH05]

SH2197

MATe

SH2257

MAT~

SH2301 SH2314

MATe MATc~

SH2410

MAT~

SH2634

MATe

SH2904

MATe

SH3071

MATe

SH3095

MATa

SH3182

MATa

Our stock collection Our stock collection Our stock collection Our stock collection Transformant of SH1273 with pHRU1 (Fig. 1) Transformant of SH 1262 with pHRL1 Segregant from a SH2128 x SH679 cross Segregant from a SH2128 x SH2197 cross Our stock collection Segregant from a SH2128 x SH2197 cross Segregant from a bel2-6 mutant (SH2128-M6) x SH2301 cross Transformant of SH2410 with pHRL1 (Fig. 1) Segregant from a bel5-1 bel6-1 mutant (SH2154-M4) x SH2314 cross Transformant of SH2301 with p512 (Fig. 1) Segregant from a bel2-6 mutant (transformant of SH2128-M6-CU with p512) x SH3071 cross Segregant from a bel3-1 bel7-1 mutant (transformant of SH2128-Mll-CU with p512) x SH3071 cross

gcn4-103 leu2-3,112 trpl pho3-1 pho5-1 ura3-52 : : [ura3-52 HIS4-lacZ] ura3-52 : : EURA3 HIS5p-PH05] gcn4-103 leu2-3,112 trpl pho3-1 pho5-1 ura3-52 : : [ura3-52 HIS4-lacZ] ura3-52 : : [URA3 HIS5p-PH05] gcn4-103 hisl-29 leu2-3,112 pho3-1 pho5-1 ura3-52 : ." [ura3-52 HIS4-lacZ] gcn4-103 leu2-3,112 trpl pho3-1 pho5-1 ura3-52 : : Jura3-52 HIS4-1acZ] ura3-52 : : [ URA3 HIS5p-PH05] bel2-6 gcn4-103 hisl-29 leu2-3,112 pho3-1 pho5-1 ura3-52 : : Jura3-52 HIS4-1acZ] bel2-1 gcn4-103 hisl-29 pho3-1 pho5-1 ura3-52 : : [ura3-52 HIS4-lacZ] leu2-3,112 .' : [LEU2 HIS5p-PH05] bel5-1 bel6-1 gcn4-103 Ieu2-3,112 hisl-29 trpl pho3-1 pho5-1 ura3-52 : : [ura3-52 HIS4-lacZ] ura3-52 : ." [ URA3 HIS5p-PH05] gcn4-103 his-29 leu2-3,112 pho3-1 pho5-1 ura3-52 : : Jura3-52 HIS4-1acZ] ura3-52 : : [URA3 PH05~ bel2-6 gcn4-103 hisl-29 trpl leu2-3,112 ura3-52 : : [URA3 PH05] ura3-52 : ." [ura3-52 HIS4-1acZ]

bel3-2 bel7-1 gcn4-103 hisl-29 1eu2-3,112 trpl pho3-1 pho5-1 ura3-52 : : Jura3-52 HIS4-1acZ] ura3-52 : : [URA3 PH05]

a The symbols ura3-52 .": [URA3 HIS5p-PH05], leu2-3,112 .": [LEU2 HIS5p-PH05] and ura3-52 : : [URA3 PH05] represent the insertion of the HIS5p-PH05 gene fusion at the ura3-52 and leu2-3,112 mutant loci, and the URA3 PH05 construct at the ura3-52 locus, respectively. The [ura3-52 HIS4-lacZ] gene fusion is that described by Lucchini et al. (1984). The other genetic symbols used are as described by Mortimer et aI. (1992)

examined on YPAD high-Pi plates, 12, 4, and 2 colonies showing the rAPase + phenotype (Bel- phenotype; basal expression level) on staining were isolated. All these parental strains have a histidine-bradytrophic mutation, hisl-29, which allowed the cells to grow without supplementation of histidine when transcription of the his1-29 allele was increased (Wolfner et al. 1975). However, these Bel- mutants showed the histidine auxotrophic phenotype, indicating that their his1-29 transcription was not enhanced. Moreover, no increase in fi-galactosidase activity from a HIS4-IacZ fusion gene inserted at the ura3 locus was observed in strains SH2128 and SH2257. Thus the Bel- phenotype might not be due to restoration of the Gcn4p function. These Bel- mutations are recessive to the wild-type counterpart. The Bel- mutants also showed temperature-sensitive cell growth at 37°C (Tsg- phenotype; Fig. 2A), cell aggregation (Flo-), rough colony morphology (Rgh-; Fig. 2B) and/or slow cell growth (Slg- ; Fig. 2C).

The original bel mutants, SH2128-M6, -M9, -Mll, and -M12, having an a mating type were crossed with either of the gcn4- BEL-- strains, SH2257 (c~) or SH2197 (c0. The diploids were sporulated and meiotic segregants having the c~ mating type and showing the Bel- phenotype were selected. These segregants and all the original bel mutants were crossed in all possible combinations and the Bel, Tsg, Slg, Flo, and Rgh phenotypes of the resulting hybrids were tested. Results showed that the original bel mutants could be classified into four groups, designated bel2 (M1 M10 mutants from SH2128, M1 M3 mutants from SH2154, and M1 and M2 mutants from SH2257), bel4 (SH2128-M12), a bel3 be17 double mutant (SH2128-Mll), and a bel5 bel6 double mutant (SH2154-M4). These bel mutants always showed the pleiotropic mutant phenotypes: SH2128-M1, -M2, -M3, -M5, -M7 also showed the Flo- phenotype; SH2128-M4 showed the Flo- Slgphenotype; SH2128-M6, -M8, -M9, SH2154-M1,-M2, -M3, and SH2257-M1 showed the Flo- Tsg- Rgh-

719 Fig. 1 Structures of the integrative plasmids used. URA3, LEU2, and PH05 are DNA fragments of Saccharornyces cerevisiae genes.HISSp and PHOSpare DNA fragmentsof the HIS5 and PH05 promoters, respectively.The PH05 DNA in p512 contains the intact PH05 gene extendingfrom -541 to ca 900 bp downstreamfrom the termination codon. Arrows represent the approximate locations and orientations of the ORFs of the relevant genes. The thin line represents the pBR322 sequence. Ampr and Ori indicate the ampicillinresistance determinant and the replication origin in Escherichia coli, respectively

HindIII

HindM

Am

~

H05

H05

~

oHRUI' HIS5p 8.3 kb ----BamHI ~"Sall

o /47

"~,~

Sail

HIS5p

pHRLiBamHI

\72L

U2AflII

SalIlXhoI PstI

HindIIIBamHI I/

s

all

HindlII BamHI Sail PstI

I

p/

PH05

SmaI /URA3 S\ailBam StuI phenotype; SH2128-M10, - M 1 2 , SH2154-M4, and SH2257-M2 showed the F l o - Slg Rgh- phenotype; and SH2128-M11 showed the Slg- phenotype. The Bel- phenotype of the bel2 and bel4 mutants was confirmed to be due to a single mutation by the fact that the Tsg-, Slg-, and/or Rgh- phenotypes co-segregated in a 2 + : 2 - fashion in 8-13 asci from each of the above diploids. In contrast, two mutants, SH2128-M11 and SH2154-M4, appeared to have double mutations, because 1 Bel- : 2 weak Bel- : 1 Bel + and 4 weak Bel- : 0 Be1 + segregations were observed in addition to the 2 Bel- :2 Bel + segregations in 7-15 asci tested when they were crossed with the BEL + strains. Also, hybrids constructed by back-crossing weak Belsegregants from the asci showing 4 weak Bel- :0 Bel + segregations with the original mutants SH2128-M11 and SH2154-M4, exhibited 2 weak Bel- :2 Bel- segregations in 10-12 asci tested. The rAPase activity of the HIS5p-PH05 construct was three- to six-fold higher in the gcn4- bel- mutants than in the gcn4- BEL + strains and the rAPase activities of the bel mutants cultivated in SC medium (repressive condition for HIS5 expression) and SC-TRP derepressible medium were not significantly different (Table 2). These results indicate that the enhanced expression of the HIS5p-PH05 construct in the bel mutants is independent of the Gcn4p function.

I URA3

aSIl

BEL2 is identical to SIN4/TSF3 For cloning the BEL2 DNA, one of the bel2-6 segregants, SH2410 (gcn4- ura3-), in which plasmid pHRL1 bearing the [LEU2 HIS5p-PH05] construct (Fig. 1) was integrated at the leu2 locus (i.e. SH2634), was transformed to the Ura + phenotype with DNA from the gene library CEN BANK A. Some of the Ura + transformants showing the Tsg + phenotype or decreased rAPase activity were selected. We obtained 10 Tsg + and 24 rAPase- transformants and found that all the Tsg + clones showed the rAPase- phenotype and vice versa. Plasmids were isolated from these transformants by rescue into E. coIi cells. We found that all the plasmids had a 6.0 kb insert at the BamHI site of YCp50. One of these plasmids was designated p431 (Fig. 3). We confirmed that the 6.0 kb insert of p431 contained the BEL2 gene by integrating it into a homologous site of a chromosome followed by tetrad analysis of the transformant (details not shown). The region of p431 essential for complementation of the Tsg- and rAPase + phenotypes of the bel2 mutant was narrowed down to a 4.0 kb EcoRI-BamHI/Sau3AI region by subcloning the fragment into YCp50 (Fig. 3). Sequences of the 4.0 kb BEL2 DNA (details not shown) indicated the presence of a single ORF of 2922 bp, encoding a 110 kDa protein with 974 amino acid resi-

720

bel2 x BEL

A 1 2 3 4 5 6

Strain

A

B

C

D

B BEL bel2

C

Table 2 Repressible acid phosphatase (rAPase) activity from the HIS5p-PH05 gene fusion in the bel mutants cultivated in SC and SC-TRP media

Relevant genotype

SH2128 SH2128-M1 SH2128-M6 SH2128-M7 SH2128-M11 SH2128-M12 SH2154-M4

Specific rAPase activitya in

BEL + gcn4-103 bel2-1gcn4-103 be12-6gcn4-103 bel2-7 gcn4-103 be13-2bel7-1 gcn4-103 bel4-1gcn4-103 bel5-1bel6-1 gcn4-103

SC

SC-TRP

1.8 _+ 1.1 6.8 ± 1.5 9.8 ± 2.8 7.3 ± 2.4 11.9 ± 2.6 4.8 ± 1.0 17.4 ± 2.3

1.4 _+ 0.9 6.4 ± 1.0 6.6 ± 2.1 5.0 ± 2.8 8.9 ± 2.2 4.4 ± 0.5 16.5 ± 3.2

a Cells were cultivated in SC medium at 30 ° C to growth saturation. Then, 0.2 ml of each culture was inoculated into 10 ml of SC (repressing condition) and SC-TRP (derepressing condition) media and the cultures were shaken for 12h at 30°C. The maximum rAPase activity attained during the cultivation was recorded. Specific rAPase activities (mU/ml per OD66 o of cell suspension) are means for at least three independent cultures with standard deviations

1 2 3 4 5 6 7 8 9101112 A B C D

Fig. 2A-C Pleiotropic effects of beI mutations. A Tetrad segregation of the Bel and Tsg phenotypes from a cross between the bel2-6 mutant (SH2128-M6) and the wild-type strain (SH2257). The tetrad clones were developed on a YPAD (high-P±) plate and examined for segregation of repressible acid phosphatase (rAPase) activity at 30°C by staining (left) and cell growth (Tsg phenotype) at 37°C (right). Numbers 1-6 represent ascus numbers, and A, B, C, and D represent tetrand clones of each ascus. B Rough morphology (Rgh-) of colonies of the bel2-6 mutant. Cells of the BEL ÷ strain (SH2128) and the bel2-6 mutant (SH2128-M6) were streaked on a YPAD plate and incubated at 30°C for 7 days. C Slow-growth phenotype (Slg-) of the bel4-1 mutant. A diploid constructed by mating the bel4-1 mutant (SH2128-M12) with a BEL + strain (SH2257) was sporulated and dissected. Numbers 1-12 represent ascus numbers and A, B, C, and D represent tetrad clones of each ascus. The dissection plate (YPAD agar) was incubated at 30° C for 5 days after the dissection. Spore clones developing into small-sized colonies always showed high rAPase activities, whereas those developing into normal-sized colonies showed low rAPase activities dues. C o m p a r i s o n o f t h e n u c l e o t i d e s e q u e n c e w i t h seq u e n c e s in a d a t a b a s e s h o w e d t h a t B E L 2 is t h e s a m e g e n e as SIN4 ( J i a n g a n d S t i l l m a n 1992), d e t e c t e d as a s u p p r e s s o r o f t h e swi5 m u t a t i o n ( N a s m y t h et al. 1987), a n d TSF3, w h i c h c o n f e r s h i g h levels o f e x p r e s s i o n o f t h e G A L l a n d GALIO genes i n d e p e n d e n t l y o f t h e specific t r a n s c r i p t i o n a l a c t i v a t o r G a l 4 p , a n d o f a CYC1 g e n e w i t h o u t a p r o m o t e r ( C h e n et al. 1993). W e f o u n d o n l y o n e m i n o r d e v i a t i o n b e t w e e n t h e s e D N A se

Plasmid

-2

-1

0

1

2

I

I

I

I

'

3

4

=

(kbp)

Comple mentation

+ +

p431 p436 p437 p437Sa p437Hp p456

-~URA3

p513

Fig. 3 DNA fragment complementing the bel2 mutation. The open and closed boxes at the top indicate the DNA fragment from S. cerevisiae cloned into plasmid p431. The closed box containing an open arrow labeled BEL2 indicates the region for which the nucleotide sequence was determined. The open arrow indicates the position and direction of the BEL20RF. The thin line represents the YCp50 moiety. Plasmids p436, p437, p437Sa, and p437Hp were constructed by subcloning or deletion of the indicated portion of the 6.0 kb fragment of p431. The symbols + and - indicate the ability and inability, respectively, of the plasmids to complement the beI2-6 mutation. (Abbreviations of restriction sites: B BamHI, C ClaI, Hp HpaI, RI EcoRI, RV EcoRV, Sa SalI, Sm Sinai. B/S represents a junction site of the BamHI and Sau3AI restriction ends) q u e n c e s ; n a m e l y , a n A r e s i d u e i n s e r t e d in t h e B E L 2 DNA between nucleotide positions +3466 and + 3467 ( t a k i n g t h e first n u c l e o t i d e o f t h e A T G i n i t i a t i o n c o d o n as + 1) in t h e 3' d o w n s t r e a m r e g i o n of t h e SIN40RF. W e a l s o c o n f i r m e d t h e 3.3 k b t r a n s c r i p t from the BEL2 gene described by Jiang and Stillman (1992) a n d f o u n d t h a t B E L 2 t r a n s c r i p t i o n w a s n o t affect e d b y o t h e r bel m u t a t i o n s ( d a t a n o t shown).

721

bel mutations enhance basal transcription For examination of the effect of bel mutations on transcription of the HISSp-PH05 construct in the gcn4background, total RNAs were prepared from bel2-6 (SH2128-M6), bel3-2 belT-1 (SH2128-M11), bel4-1 (SH2128-M12) and bel5-1 bel6-1 (SH2154-M4) mutants after cultivation in YPAD (high-Pi) medium, and subjected to northern analysis with 32p-labeled probes for the PH05 and HIS5 transcripts and also for that of ACT1 as an internal marker. Transcripts detected with the PH05 probe should originate from the HIS5pPH05 construct, not from the genomic pho5-1 allele, because no transcripts from this genomic allele were detected in the original BEL ÷ and bel- strains cultivated in high-Pi and low-Pi media (data not shown). We found that transcription of the HISSp-PH05 construct was enhanced in the bel mutants (Fig. 4), and that the bel5 bel6 double mutant showed significantly higher transcription than the others. The genomic HIS5 gene in the bel mutants was, however, transcribed at the same level as in BEL ÷ cells, except in the bel5 bel6 double mutant, which showed enhanced transcription. To examine whether bel mutations affect the expression of PH05 with an intact promoter, the HIS5p-PH05 construct was eliminated from the original belmutants, SH2128-M6 (bel2-6), SH2128-Mll (bel3-2 bel7-1), and SH2128-M12 (bel4-1), and from the BEL + strain, SH2128, by selecting 5-FOA resistant clones.

HIS5p-PH05 HIS5 ACT1 Fig. 4 Effects of bel mutations on transcription of the HISSp-PH05 construct integrated at the ura3-52 locus and of the genomic HIS5 gene. Total RNAs were prepared from cells of strains SH2128 (BEL +), SH2128-M6 (bel2-6), SH2128-Mll (bel3-2 bel7-1), SH2128M12 (bel4-1) and SH2154-M4 (bel5-1 bel6-1) cultivated in 10 ml of YPAD (high-Pi) medium at 30 ° C for 12 h after inoculating 0.2 mI of each saturated culture. RNA samples (each 10 p.g RNA) were electrophoresed in an agarose gel in the presence of formaldehyde, transferred to a nylon filter, blotted, and hybridized with the following probes: a 32p-labeled 0.5 kb BstEII-EcoRV fragment of PH05 D N A prepared from plasmid pPHO5 (Arima et al. 1983), a 1.0 kb BclI-HindIII fragment carrying a part of the HIS5 D N A prepared from plasmid pSH135 (Nishiwaki et al. 1987), or a 1.0 kb HindIIIXhoI fragment of ACT1 D N A prepared from plasmid pYA301 (Gallwitz and Sures 1980)

These strains were designated SH2128-CU, SH2128M6-CU, SH2128-Mll-CU, SH2128-M12-CU, and SH2128-CU. A U r a - segregant was similarly selected from a meiotic segregant, SH2904, having the bel5 bel6 ura3-52:: JURA3 HIS5p-PH05] genotype, and was designated SH2904-CU. These strains showing the rAPase- phenotype were confirmed not to carry the HIS5p-PH05 construct by Southern hybridization analysis (data not shown). Then the intact PH05 gene was integrated at the ura3-52 locus by transformation of the 5-FOA resistant strains with plasmid p512 (bearing PH05 DNA extending from nucleotide position - 5 6 2 to ca 900 bp downstream of the termination codon; Fig. 1) linearized by cutting at the StuI site in the URA3 DNA on the plasmid molecule. Another strain, SH2128-CU-pho5.': [PH05+], was constructed from SH2128-CU by replacement of the pho5-1 locus with the PH05 ÷ DNA of p512 by the gene replacement procedure (Scherer and Davis 1979). All these transformants from the bel mutants, except bel4, showed constitutive expression of PH05 inserted at the ura3 locus (Table 3). The bel2 mutant, however, could not derepress the PH05 gene at the pho5 locus in high-Pi medium (see data for SH2128-M6-CUpho5:: [PH05+]; Table 3). rAPase synthesis by the BEL ÷ transformants was, however, always repressed in high-Pi medium, irrespective of whether the PH05 + gene was inserted at the ura3 or pho5 locus. Thus, the bel2 mutant was effective in derepressing PH05 at the URA3 locus but not at the PH05 locus. When total RNAs were prepared from the above transformants cultivated in YPAD (high-Pi) and YPAD-P (low-Pi) media and subjected to northern analysis, the PH05 transcripts were clearly detected in bel2-6, bel3-2 bel71, and bel5-1 bel6-1 mutants even when the cells were cultivated in high-Pi medium (Fig. 5). Since the promoter sequence of PH05 cloned on the p512 plasmid was short (0.56 kb), PH05 expression might be affected by a cis-acting sequence in the flanking regions of the ura3 locus where the p512 molecule was inserted. To test this possibility, we examined another PH05 DNA fragment, having a 5.4 kb native 5' upstream sequence, for its expression in the bel2- background. The modified p512, bearing the 5.4 kb promoter region (i.e. L-PH05 ÷), constructed as described in Materials and methods, was integrated into the ura3-52 locus of SH2128-CU and SH2128-M6-CU. The resultant Ura ÷ transformants showed slightly lower rAPase activity, but they showed similar behaviors with respect to rAPase synthesis to that of the p512 integrants of the same host strains at the ura3 locus (Table 3). Thus we concluded that derepression of the PH05 gene inserted at the ura3 locus is caused by a trans-acting effect of the bel2 mutation. bel mutations express PH05 integrated at the ura3 locus without the function of Pho4p and UAS

722 Table 3 The bel mutants express the P H 0 5 gene inserted at the ura3 locus

Strain

Bel genotype

S H 2 1 2 8 . C U [ P H 0 5 +] b SH2128-M6-CU [ P H 0 5 + ] SH2128-M 11 [ P H 0 5 + ] SH2128-M 1 2 - C U [ P H 0 5 + ] SH2904-CU [ P H 0 5 + ] SH2128-CU [ L - P H 0 5 + ] ° SH2128-M6-CU [ L - P H 0 5 + ] SH2128-CU-pho5 : : [ P H 0 5 + ] d SH2128-M6-CU-pho5: : [ P H 0 5 + ]

BEL + bel2-6 bel3-2 bel7-1 bel4-1 bel5-1 bel6-1 BEL + bel2-6 BEL + bel2-6

Specific rAPase activity a in YPAD

YPAD-P

1.3 22.2 33.5 2.8 46.1 0.4 12.9 0.3 0.4

24.3 42.5 43.3 22.6 50.5 16.3 24.0 21.4 12.9

± 0.2 _+ 4.1 _ 2.4 _+ 0.6 ± 5.0 _ 0.4 ± 2.4 _ 0.3 ± 0.4

_+ 5.2 _+ 4.4 _ 3.5 ± 4.2 ± 6.1 ± 2.6 ± 6.9 ± 9.1 ± 3.0

Strains transformed with plasmid p512 (Fig. 1) were cultivated at 30°C in YPAD (high-P 0 and YPAD-P (low-Pi) media to growth saturation. Then, 0.2 ml of each culture was inoculated into 10 ml of fresh YPAD or YPAD-P medium. The conditions for measurement and presentation of specific rAPase activities are as described in the legend to Table 2 b Symbol [ P H 0 5 + ] represents p512 inserted into the ura3 locus after elimination of the previously inserted HISbp-PH05 construct Symbol [ L - P H 0 5 + ] indicates the P H 0 5 D N A with a 5.4 kb 5' upstream sequence inserted at the ura3 locus d Symbol pho5 : : [ P H 0 5 ÷ ] indicates the wild-type P H 0 5 D N A inserted at the pho5 locus by the gene replacement procedure (Scherer and Davis 1979)

Pi

|

li

aa

ni

-['--'+---[----b-- -[---

I

PH05 Fig. 5 Effects of bel mutations on transcription of the P H 0 5 gene integrated at the ura3 locus. Samples of 10 gg each of total RNA, prepared from cells of S H 2 1 2 8 - C U [ P H 0 5 + ] (BEL+), SH2128-M6C U [ P H 0 5 + ] (bel2-6), S H 2 1 2 8 - M l l - C U [ P H 0 5 + ] (bel3-2 bel7-1), S H 2 1 2 8 - M 1 2 - C U [ P H 0 5 +] (bel4-1), and S H 2 9 0 4 - C U [ P H 0 5 +] (bel5-1 bel6-1) grown in YPAD high-P i ( + ) and YPAD-P low-Pi ( - ) media were fractionated and subjected to northern hybridization analysis. The 32P-labeled probes for the P H 0 5 and ACT1 transcripts were the same as those described in the legend to Fig. 4

To examine the function of Pho4p in PH05 transcription in the bel mutants, we disrupted the PH04 gene of SH3095 (bel2-6) and SH3182 (bel3-2 bel7-1), both marked with the non-transcriptional phob-1 allele in the genome and bearing the integrated intact PH05 gene at the ura3 locus, by insertion of a 6.0 kb ItindIII fragment of the pho4 : :LEU2 allele prepared from p615. Several Leu ÷ transformants were isolated and confirmed to have a disrupted pho4 gene by Southern hybridization of their genomic DNAs, after digestion with HindIII, using a 32p-labeled 3.3 kb HindIII-XhoI fragment ofPH04 DNA prepared from p615 as a probe (data not shown). The results indicated that disruption

of PH04 in the bel2-6 and bel3-2 bel7-1 mutants did not affect the level of production of rAPase in high-P± medium (Table 4). We also found that constitutive expression still occurred after removal of the promoter region of PH05. The PH05 promoter contains three UAS sites (Ogawa et al. 1994), all of which are located in a 272 bp BamHIBstEII fragment from nucleotide positions - 4 9 2 to 221. Even when an integrative plasmid pSH40, bearing the PH05 DNA lacking the promoter region (i.e. AUAS-PH05) from nucleotide positions - 5 6 2 to - 183, was introduced into SH2128-M6-CU (bel2-6) and SH2128-Mll-CU (bel3-2 bel7-1), high rAPase activity was observed in both YPAD (high-Pi) and YPAD-P (low-P0 media, whereas no appreciable derepression was observed in high-Pi medium with the same construct in a BEL + strain (SH2128-CU; Table 4). We found that bel2-6 PH04 + cells showed higher rAPase activity than did BEL + PH04 + cells in low-Pi medium. The bel2 cells with a pho4::LEU2 disrupted gene or with AUAS-PH05 showed the same levels of rAPase activity under both repressing and derepressing conditions, while bel2 PH04 + cells showed higher rAPase activity in low-Pi medium than in high-P± medium. These results indicate that the function of the bel2 mutation in derepression of PH05 is additive to, or independent of Pho4p-mediated activation. In contrast, neither disruption of PH04 nor deletion of the PH05 UASs in the bel3-2 bel7-1 mutant affected the rAPase activity, similar activity levels being observed in cells cultivated under repressing and derepressing conditions. Therefore, it appears that the bel3-2 bel7-1 double mutation is in the same pathway as the Pho4p activation of PH05 expression. The above observations also indicated that the bel3 bel7 mutation is epistatic over Pho4p activation. -

723

Table 4 Expression of the P H 0 5 O R F integrated at the ura3 locus with or without an upstream activating sequence (UAS) in the presence or absence of the transcriptional activator Pho4p in bel2-6 and bel3-2 bel7-1 mutant cells

Strain

Relevant genotype

Specific rAPase activity a in YPAD

SH3071 [ P H 0 5 SH3071 [ P H 0 5 SH3095 [ P H 0 5 SH3095 [ P H 0 5

+] + ]A p h o 4 +] +] A p h o 4 S H 3 1 8 2 [ P H 0 5 +] SH3182 [ P H 0 5 + ] A p h o 4 SH2128-CU [A U A S - P H 0 5 ] ° SH2128-M6-CU[A U A S - P H 0 5 ] SH2128-Mll-CU[A U A S - P H 0 5 ]

BEL + PH04 + BEL + pho4." : L E U 2 b bel2-6 P H 0 4 + bel2-6 p h o 4 : : LEU2 b bel3-2 bel7-1 P H 0 4 + bel3-2 bel7-1 p h o 4 : : LEU2 BEL + P H 0 4 + bel2-6 P H 0 4 + bel3-2 bel7-1 P H 0 4 +

1.8 1.2 24.4 29.3 22.2 35.2 1.1 23.5 32.3

± ± ± ± + ± _ + ±

YPAD-P 0.3 0.1 5.2 4.6 4.3 4.1 1.4 2.5 3.8

27.2 1.3 54.5 22.3 27.8 35.6 5.2 25.3 30.6

± ± ± ± ± ± + ± ±

5.2 0.3 5.5 2.8 3.5 4.6 1.2d 3.2 2.6

a Conditions for measurement and presentation of rAPase activities were as described in the legend to Table 2 b A disrupted allele o f p h o 4 (insertion of a LEU2 fragment) °A U A S - P H 0 5 represents the P H 0 5 DNA deleted for the promoter region d Possibly attributable to the P H 0 1 0 and P H O l l genes encoding a minor fraction of repressible acid phosphatase (Venter and HSrz 1989), because 5 7 U of activity was observed in untransformed host strains under derepressed conditions

Functions of bel2 mutations depend on the chromosomal positions of the target genes Transcription of the ItlS5 gene at its native genomic site was not affected by the bel2-6 or beI4-1 mutation or the bel3-2 bel7-1 double mutation, but was somewhat enhanced by bel5-1 bel6-1 double mutation (Fig. 4). Therefore, we investigated the effect of the bel2 and bel3 bel7 mutations on the expression of the PH05 gene at its normal genomic site. Strains SH2128-M6-CU ( M A T a bel2-6 pho5-1) and SH2128-Mll-CU (MATa bel3-2 bel7-1 pho5-1) were crossed with SH1877 (MA Tc~ BEL ÷ PH05 ÷) and the diploids were subjected to tetrad analysis. The rAPase activities of segregants from 10-14 asci of these diploids were tested on YPAD (high-P0 and YPAD-P (low-Pi) media. All the segregants from the bel2-6 pho5-1 xBEL ÷ PH05 + cross exhibited the rAPase- phenotype on high-P± medium but 2 rAPase ÷ :2 rAPase- segregation on low-Pi medium. Therefore, the PH05 gene at its genomic position was not expressed by the bel2 mutant under high-P~ conditions. To confirm this result, we constructed strains with the PH05 + gene inserted at the pho5 locus, with or without the bel2-6 mutation, SH2128-CU-pho5 : : [PH05 ÷] and SH2128-M6-CUphoS::[PH05+], respectively. Both the BEL ÷ and bel2 strains showed no appreciable rAPase activity in high-Pi medium but were derepressed in low-P~ medium (Table 3). A bel2 disruptant of SH1877 having the wild-type genotype for rAPase synthesis showed the same phenotype as the original SH1877 strain (data not shown). These results indicate that the bel2 mutation is effective for the PH05 gene at the URA3 locus but not at the PH05 locus. In contrast, less than two spore clones in each ascus of the bel3-2 belT-1 pho5-1 x BEL ÷ PH05 cross showed the rAPase + phenotype, even on high-P~ medium (data not shown). Therefore, the bel3 bel7

double mutants may express the genomic PH05 gene even in high-Pi medium. The disrupted bel2 allele enhances HIS5p-PH05 expression and causes copious cell aggregation Two disrupted alleles of the BEL2 gene were constructed. An EcoRI-SalI fragment containing the BEL2 gene prepared from p436 (Fig. 3) was cloned into the EcoRISalI gap of pBluescript II KS +. A 3.0 kb HpaI fragment containing most of the BEL2 gene (Fig. 3) was removed from the resultant plasmid and the remaining fragment was self-circularized after ligation of an 8 bp HindlII linker to the HpaI ends. The resultant plasmid was opened by digestion with HindIII and a 1.2 kb IIindlII fragment that contains the URA3 gene prepared from YEp24 (Parent et al. 1985) was inserted. This plasmid and the disrupted allele were designated p456 and bel2 .": URA3, respectively (Fig. 3). A bel2 : : TRP1 allele on plasmid p513 (Fig. 3) was constructed similarly except that an 8 bp Bg/II linker was used instead of the IIindIII linker as in the p456 construction and a 1.1 kb BamHI-BglII fragment of the TRP1 DNA prepared from YRp7 (Parent et al. 1985) was inserted. Although both of these alleles contained the intact promoter of BEL2, we could not detect any transcript at the 3.3 kb position from these disrupted alleles (data not shown). Then the 4.5 kb EcoRI-SalI fragment containing the bel2 .": URA3 allele from p456 was used to transform cells of a diploid strain, constructed by a SH2154 x SH2634 cross, having HISSp-PH05 at the leu2 locus, to the Ura ÷ phenotype by one-step gene replacement (Rothstein 1983). Disruption of the BEL2 locus in one of the two homologous chromosomes in the diploid was confirmed by Southern analysis (data not shown), and the transformant was subjected to

724

tetrad analysis. Results showed a 2 + : 2 - ratio of the Ura phenotypes in 21 asci examined, and all the Ura ÷ segregants showed five-fold higher rAPase activity than the U r a - segregants in YPAD medium (data not shown). These Ura ÷ segregants exhibited the Rghmorphology on YPAD plates and the F l o - phenotype in liquid medium as described by Chen et al. (1993) and Jiang and Stillman (1992). However, the bel2 disruptants were viable, while cell growth was retarded at 37 ° C. This is in contrast to the clear Tsg- phenotype that results from the bel2-6 mutation and inconsistent with the descriptions by Jiang and Stillman (1992) and Chen et al. (1993).

the bel2-6 mutation at least appears not to result in nucleosome loss, because bel2 mutations did not affect expression of PH05 at its genomic position, where nucleosome loss caused by repressed synthesis of histone H4 resulted in constitutive transcription of the genomic PH05 gene (Han et al. 1988). These possibilities are supported by the fact that the effect of the bel2 mutation on the PH05 gene at the URA3 or LEU2 locus was additive with Pho4p function. Thus, we speculate that the BEL genes encode proteins necessary for maintaining higher order chromosome structure or for attachment of chromosomes to the nuclear scaffold. In the bel mutants, these structures are disturbed at various positions on the chromosomes and then genes located at these positions might be expressed without a requirement for specific transcriptional activators.

Discussion The bel mutants defined six genes, BEL2, BEL3, BEL4, BEL5, BEL6, and BEL7, and the bel2 mutations (i.e. alleles of sin4 and tsf3 mutations) and the bel3 bel7 and bel5 bel6 double mutations caused a high level of basal transcription of the PH05 gene integrated at the ura3 locus under repressing conditions (Figs. 4, 5). The rAPase activities of the bel mutants were similar to the derepressed level mediated by Pho4p in wild-type cells (data with a constructed PHO wild-type strain, SH2128-CU-pho5:: [PH05+], is shown in Table 3). The bel2 mutation, however, did not affect expression of the PH05 gene at its original genomic site. In a preliminary study, the expression of similar PH05 constructs, M A Tc~ lp-PH05 and PKClp-PH05, inserted at the ura3 locus was not increased by bel2-6 and bel3-2 bel7-1 mutations (unpublished results). These observations indicate that the effect of bel mutations, at least bel2 and bel3 bel7 mutations, is not simply to stimulate transcription of the fusion gene or to stabilize the transcript. In addition, the BEL genes affect the expression of various other genes, because bel mutations result in various pleiotropic phenotypes. It has been reported that nucleosomes cover the promoter region of PH05, and that, upon derepression, they are removed by the function of Pho4p, and then the gene is derepressed (Almer et al. 1986). When nucleosomes were removed from the promoter region by genetic depletion of histone H4, several genes in S. cerevisiae were shown to be transcribed even under repressive conditions and even when UASs were deleted (Han et al. 1988; Han and Grunstein 1988; Durrin et al. 1992). The sin4 (= bel2) mutation has been shown to affect the density of superhelices in a plasmid (Jiang and Stillman 1992). It has also been found that nucleosomes located on a minichromosome are unstable toward nuclease digestion in sin4 ( = bel2) mutant cells (R. T. Simpson, NIH, Bethesda, Md., USA, personal communication). These facts suggest that the bel2 mutation (and possibly the bel3 bel7 double mutation) cause a defect in nucleosome organization. However,

Acknowledgements We thank D. Gallwitz of the Max-PlanckInstitut for plasmid pYA301. This study was partially supported by a Grant-in-Aid for Scientific Research (number 02454033) to S.H. from the Ministry of Education, Science and Culture of Japan.

References Almer A, Rudolph H, Hinnen A, H61z W (1986) Removal of positioned nucleosomes from the yeast PHO5 promoter upon PHO5 induction releases additional upstream activating DNA elements. EMBO J 5:2689 2696 Arima K, Oshima T, Kubota I, Nakamura N, Mizunaga T, Toh-e A (1983) The nucleotide sequence of the yeast PH05 gene: a putative precursor of repressible acid phosphatase contains a signal peptide. Nucleic Acids Res 11 : 1657 1672 Arndt KT, Styles C, Fink GR (1987) Multiple global regulators control HIS4 transcription in yeast. Science 237 : 874-880 Arndt KT, Styles CA, Fink GR (1989) A suppressor of a HIS4 transcriptional defect encodes a protein with homology to the catalytic subunit of protein phosphatases. Cell 56 : 527-537 Boeke JD, Trueheart J, Natsoulis G, Fink GR (1987) 5-Fluoroorotic acid as a selective agent in yeast molecular genetics. Methods Enzymol 154:164 175 Bun-ya M, Nishimura M, Harashima S, Oshima Y (1991) The PH084 gene of Saccharomyces cerevisiae encodes an inorganic phosphate transporter. Mol Cell Biol 11 : 3229-3238 Chen S, West RW Jr, Johnson SL, Gans H, Kruger B, Ma J (1993) TSF3, a global regulatory protein that silences transcription of yeast GAL genes, also mediates repression by c~2repressor and is identical to SIN4. Mol Cell Biol 13:831 840 Davis JL, Kunisawa R, Thorner J (1992) A presumptive helicase (MOT1 gene product) affects gene expression and is required for viability in the yeast Saccharomyces cerevisiae. Mol Cell Biol 12:1879-1892 Devlin C, Tice-Baldwin K, Shore D, Arndt KT (1991) RAP1 is required for BAS 1/BAS2- and GCN4-dependent transcription of the yeast HIS4 gene. Mol Cell Biol 11:3642 3651 Durrin LK, Mann RK, Grunstein, M (1992) Nucleosome loss activates CUP1 and HIS3 promoters to fully induced levels in the yeast Saccharomyces cerevisiae. Mol Cell Biol 12:1621 1629 Fassler JS, Winston F (1988) Isolation and analysis of a novel class of suppressors of Ty insertion mutations in Saccharomyces cerevisiae. Genetics 118 : 203-212 Gallwitz D, Sures I (1980) Structure of a split yeast gene: complete nucleotide sequence of the actin gene in Saccharomyces cerevisiae. Proc Natl Acad Sci USA 77:2546-2550

725 Han M, Grunstein M (1988) Nucleosome loss activates yeast downstream promoters in vivo. Cell 55:1137-1145 Han M, Kim U-J, Kayne P, Grunstein M (1988) Depletion of histone H4 and nucleosomes activates the PH05 gene in Saccharomyces cerevisiae. EMBO J 7 : 2221-2228 Hinnebusch AG (1988) Mechanisms of gene regulation in the general control of amino acid biosynthesis in Saccharomyces cerevisiae. Microbiol Rev 52:248 273 Hwang Y-II, Harashima S, Oshima Y (1989) Improvement and application of a promoter-probe vector bearing the PH05 gene as the indicator marker in Saccharomyces cerevisiae. J Ferment Bioeng 67 : 1-7 Jiang YW, Stillman DJ (1992) Involvement of the SIN4 global transcriptional regulator in the chromatin structure of Saccharomyces cerevisiae. Moi Cell Biol 12:4503~4514 Kruger W, Herskowitz I (1991) A negative regulator of HO transcription, SIN1 (SPT2), is a nonspecific DNA-binding protein related to HMG1. Mol Cell Biol 11:4135-4146 Lucchini G, Hinnebusch AG, Chen C, Fink GR (1984) Positive regulatory interactions of the HIS4 gene of Saccharomyces cerevisiae. Mol Cell Biol 4:1326 1333 Malone EA, Clark CD, Chiang A, Winston F (1991) Mutations in SPT16/CDC68 suppress cis- and trans-acting mutations that affect promoter function in Saccharomyces cerevisiae. Mol Cell Biol 11:5710-5717 Mortimer RK, Contopoulou CR, King JS (1992) Genetic and physical maps of Saccharomyces cerevisiae, edn 11. Yeast 8:817 902 Mukai Y, Harashima S, Oshima Y (1993) Function of the Ste signal transduction pathway for mating pheromones sustains MATc~I transcription in Saccharomyces cerevisiae. Mol Cell Biol 13:2050-2060 Nasmyth KA (1982) The regulation of yeast mating-type chromatin structure by SIR: an action at a distance affecting both transcription and transposition. Cell 30 : 567-578 Nasmyth K, Seddon A, Ammerer G (1987) Cell cycle regulation of SWI5 is required for mother-cell-specific HO transcription in yeast. Cell 49 : 549 558 Neigeborn L, Celenza JL, Carlson M (1987) SSN20 is an essential gene with mutant alleles that suppress defects in SUC2 transcription in Saccharomyces cerevisiae. Mol Cell Biol 7 : 672-678 Nishiwaki K, Hayashi N, Irie S, Chung D-H, Harashima S, Oshima Y (1987) Structure of the yeast HIS5 gene responsive to general control of amino acid biosynthesis. Mol Gen Genet 208:159-167 Ogawa N, Oshima Y (1990) Functional domains of a positive regulatory protein, PHO4, for transcriptional control of the phosphatase regulon in Saccharomyces cerevisiae. Mol Cell Biol 10:2224 2236 Ogawa N, Hayashi N, Saito H, Noguchi K, Yamashita Y, Oshima Y (1994) Regulatory circuit for phosphatase genes in Saccharomyces cerevisiae: specific cis-acting sites in PHO promoters for

binding the positive regulator Pho4p. In: Torriani-Gorini A, Yagil E, Silver S (eds) Phosphate in microorganisms: cellular and molecular biology. American Society for Microbiology, Washington, DC, pp 56-62 Oshima Y (1991) Impact of the Douglas-Hawthorn model as a paradigm for elucidating cellular regulatory mechanisms in fungi. Genetics 128 : 195-201 Parent SA, Fenimore CM, Bostian KA (1985) Vector systems for the expression, analysis and cloning of DNA sequences in S. cerevisiae. Yeast 1 : 83-138 Peterson CL, Kruger W, Herskowitz I (1991) A functional interaction between the C-terminal domain of RNA polymerase II and the negative regulator SIN1. Cell 64:1t35 1143 Roeder GS, Beard C, Smith M, Karanen S (1985) Isolation and characterization of the SPT2 gene, a negative regulator of Tycontrolled yeast gene expression. Mol Cell Biol 5:1543-1553 Rose MD, Novick P, Thomas JH, Botstein D, Fink GR (1987) A Saccharomyces cerevisiae genomic plasmid bank based on a centromere-containing shuttle vector. Gene 60: 237-243 Rose MD, Winston F, Hieter P (1990) Methods in yeast genetics: a laboratory course manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York Rothstein RJ (1983) One-step gene disruption in yeast. Methods Enzymol 101 : 202-211 Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual, 2nd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York Scherer S, Davis RW (1979) Replacement of chromosome segments with altered DNA sequences constructed in vitro. Proc Natl Acad Sci USA 76:4951~4955 Suzuki Y, Nogi Y, Abe A, Fukasawa T (1988) GALl1 protein, an auxiliary transcription activator for genes encoding galactosemetabolizing enzymes in Saccharomyces cerevisiae. Mol Cell Biol 8:4991-4999 Toh-e A, Oshima Y (1974) Characterization of a dominant constitutive mutation, PHO0, for the repressible acid phosphatase synthesis in Saccharomyces cerevisiae. J Bacteriol 120:608-617 Toh-e A, Ueda Y, Kakimoto S, Oshima Y (1973) Isolation and characterization of acid phosphatase mutants in Saccharomyces cerevisiae. J Bacteriol 113:727-738 Venter U, H6rz W (1989) The acid phosphatase genes PHO10 and PHO11 in S. cerevisiae are located at the telomeres of chromosomes VIII and I. Nucleic Acids Res 17:1353 1369 Vidal M, Strich R, Esposito RE, Gaber RF (1991) RPD1 (SIN3/UME4) is required for maximal activation and repression of diverse yeast genes. Mol Cell Biol 11:6306-6316 Wolfner M, Yep D, Messenguy F, Fink GR (1975) Integration of amino acid biosynthesis into the cell cycle of Saccharomyces cerevisiae. J Mol Biol 96:273-290