Cell-type-specific transcription in yeast

Biochimica et Biophysica A cta, 1088 (1991) 155-169 © 1991 El~vier Science Publishers B.V. 0167-4781/91/$03.50 A D O N I S 016747819100073J

BBAEX P 92201 Review

Cell-type-specific transcription in yeast

J o s e p h W . D o l a n a n d S t a n l e y F i e l d s

Department of Microbiology, ~tate University of New York at Stony Brook, Stony Brook, N Y ( U. S.A.)

(Received 5 September 1990)

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Key words: Cell-type-specific transcription; Mating type; Pheromone response; Yeast; (Saccharomyces ceret,islae )

Contents

I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 155

I!. Regulation by MAT products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 156 A. The at -a2 hypothesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 156 B. The M A T a l product . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 157 C. The MATa2 product . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 157 D. The al -a2 repressor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 159

111. The M C M I product . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 159 A. Identification of MCM1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 159 B. Role of MCMI and MCMI-al in the activation of cell-type-specific genes . . . . . . . . . . . . . . . . 160 C. Role of MCMI-a2 in the repression of a-specific genes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 161

IV. The pheromone response pathway . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 161 A. The pheromones and receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 161 B. A common intracellular pathway . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 162 C. Receptors activate a G protein . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 162 D. STES, STE7, STEI L and FUS3 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 164 E. STEI2 as a pheromone-responsive transcriptional activator . . . . . . . . . . . . . . . . . . . . . . . . . . 165

V. Yeast cell-type-specific transcription as a model system . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 167

Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 168

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . l ~g

I. Introduction

The yeast Saccharomyces cerevisiae exhibits two haploid cell types (mating types), designated a and a, each constitutively expressing a unique set of genes, the

Abbreviations: UAS, upstream activating sequence; bp, base pair; SRF, serum response factor; PRE, pheromone response element; PRTF, pheromone receptor transcription factor; ARS(s), autonomously replicating sequence(s); GRM, general regulator of mating type.

Correspondence: S. Fields. Department of Microbiology, State Uni- versity of New York at Stony Brook, Stony Brook, NY 11794, U.S.A.

a-specific genes and the a-specific genes, respectively. These genes encode products such as the mating pheromones (a-factor and a-factor) and the receptors for these pheromones. When an a cell is exposed to the pheromone secreted by a ceils, or vice versa, it responds with increased transcription of pheromone-inducible genes, increased agglutinability, arrest in the G1 stage of the cell cycle and morphological changes which produce a cell called a shmoo. These changes prepare the haploid cell for mating, which results in the formation of the third cell type, the a/a diploid. In the diploid, the a- and a-specific genes are repressed as well as the set of genes that is expressed in both haploid cell types, designated the haploid-specific genes.

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This review focuses on the two regulatory networks that control the constitutive and pheromone-induced level of cell-type-specific gene expression. One network. encoded by the mating type locus, acts as an on /o f f switch to establish which sets of cell-type-specific genes may be actively transcribed [1-41. The other network, designated the pheromone response pathway, allows cells of one type to respond to the presence of cells of the opposite type [4-6]. In addition to mediating rapid transcriptional induction, the pheromone response pathway ~ontributes significantly to the constitutive level of ceit-Upe-specific transcripts observed in cells growing in the absence of the opposite cell type. The two networks are involved in extensive cross-regulation, with many of the components of the response pathway themselves expressed in a cell-type-specific manner. The response pathway is also auto-regulated, with pheromone addition leading to increased transcription of some genes necessary for response. Finally, a protein essential in all cell types (the MCM1 protein) plays a critical role in allowing both networks to function [7-9]. This combinatorial control of transcription, mediated by cell-type-specific regulators, a signal-responsive cascade, and a ubiquitous factor, continues to provide insights into similar processes in higher eukaryotes.

i l . Regulation by MAT products

II.A. The al-u2 hypothesis

The products of the M A T locus, which is the only genetic locus that differs between a and a cells, determine which cell type will be expressed. Haploid cells that carry the MA Ta allele are a cells, haploid cells that carry the MA Ta allele are a cells and diploid cells that carry both alleles are a / a cells. MacKay and Manney [10,11] isolated mutants unable to mate and showed that some of these carry mutations at MA Tot and others carry mutations that are unlinked to the mating type locus. They proposed [11] that the mating type loci encode diffusible regulators that control the cell-type- specific expression of structural genes necessary for mating. Physiological and genetic studies of some of these mutants in M A T led to the el-a2 hypothesis [1], the main features of which are shown in Fig. 1. In an a cell, the MATal product (designated a l ) is a transcriptional activator that is essential for the expression of a-specific genes. The MA Ta2 product (designated a2) is a repressor of a-specific genes. Therefore, in the a cell, -,-specific genes are transcribed and a-specific genes are silent. In an a cell, the MA Tal product (designated' a l ) has no essential regulatory function; a cells are such because they lack the activator of a-specific genes (a l ) and the repressor of a-specific genes (a2). Therefore, in the a cell, a-specific genes are silent and a-specific genes are transcribed. In the diploid, a2 represses the a-specific

ct cell / ~ ~ a-specific genes a 2 c( 1 .],vvv~ M A T a ~ , , ~ A /-spedfic genes

2 " ~ " " ~ haploid-specifc genes

a cell

a l

MA Ta

a-specific genes

a-specific genes

haploid-specifc genes

a/~ cell

a2 L ctl ~ , , ~ ~ g e n e s

M ~ ~ ~ s p e c i f i c genes

,'x./V'w%~ haploid-specifc genes MA Ta

Fig. 1. al-a2 hypothesis showing the role of the various MAT products. The wavy lines indicate transcription; the arrows indicate that the MAT product activates transcription; the bars indicate that the

MAT product(s) represses transcription.

genes and functions with al to form a new regulatory function, designated al-a2, which represses the transcription of haploid-specific genes and of MATal . Therefore, in the a /a cell, a-, a- and haploid-specific genes are silent.

Although identified genetically by mutations that affect mating or sporulation [11-13], the M A T genes encode DNA-binding proteins that have target sites on the cell-type-specific genes. These proteins function in an absolute fashion to establish which sets of genes are transcriptionally active. They appear to have no other role than this transcriptional regulation in that deletion mutants of MA T are perfectly viable. However, both ttl and a2, as described below, carry out their functions only in cooperation with an essential protein, encoded by the MCMI gene. Thus combinatorial control occurs at one |evei by tl~e use o'f t--he same general factor foi activation and repression. At a second level, the transcriptional repressor a2 has its binding specificity changed by al to recognize a different set of cell-type- specific genes.

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ll.B. The MA Tal product

A matal mutant fails to express a-specific genes [14]. This result indicates that al is a positive activator of a-specific gene expression, as initially demonstrated for the gene encoding the a-factor receptor, STE3. Sprague et al. [14] detected STE3-specific RNA in MA Ta cells and mata2 mutants, but not in MA Ta or MA Ta/MA Ta cells or matal mutants. This result demonstrated a strict requirement for functional al protein for expression of an a-specific gene and also showed that expression of a-specific genes does not require a2 protein. To address whether provision of al is sufficient for STE3 expression, Ammeter et al. [15] assayed a and a /a cells carrying a plasmid with the MATal gene expressed from a constitutive promoter. The presence of al in a cells is sufficient to cause the expression of a-specific genes. However, production of al in an a / a diploid is not sufficient for the expression of a-specific genes, suggesting the presence of an additional level of negative regulation in the diploid [15]. A similar conclu- sion came from the analysis of a mutation in the regulatory region of MATa that causes MATal transcription in the a / a cell but does not lead to expression of an a-specific gene [16].

To define the target for al activity, Jarvis et al. [17] constructed deletion mutations in the STE3 upstream regulatory region fused to the lacZ gene. This analysis identified a 43 basepair (bp) upstream activating sequence (UAS) that is essential and sufficient to render expression of the reporter gene both a-factor-inducible and al-dependent. This UAS is able to activate transcription of the reporter gene in an orientation-independent manner, although the natural orientation is more efficient than the reverse orientation. Since the STE3 UAS is sufficient to confer al-dependence to a heterologous transcript, al works at the level of transcription initiation, rather than at the level of transcript stability [17].

Contained within the 43 bp STE3 UAS is a 26 bp sequence that is highly conserved among all a-specific genes [7,17]. This 26 bp sequence consists of two elements: an imperfect palindrome of 16 bp, designated the P box, and a 10 bp element, designated the Q box (Fig. 2). A 14 bp deletion in the P box abolishes UAS activity [17]. Bender and Sprague [7], using gel-shift assays with a STE3 UAS probe, demonstrated that al binds to this 26 bp sequence, but only in the presence of an additional factor that binds at the P box. The absolute requirement for a second factor was further demonstrated by the fact that al purified from Escherichia coli is unable to bind to DNA unless supp- lemented with yeast extracts [7]. This second factor is present in extracts derived from a, a, and a / a cells as well as matal mutant cells and was designated Pheromone Receptor Transcription Factor (PRTF) [7].

PRTF was postulated to bind alone to a perfectly palindromic version of the P-box, designated P(PAL), but to require cooperative interactions with al in order to bind to the imperfect palindrome found in STE3, P(STE3). Likewise, al was postulated to be capable of binding to the Q box only through additional interactions with PRTF [7]. Consistent with these ideas, sequences homologous to the STE3 UAS are found in the upstream regulatory regions of all a-specific genes and have been shown to bind PRTF and al [7,17,18]. In all a-specific genes, the sequence of the P box diverges from the perfect palindrome at bases adjacent to the Q box. The P(PAL) sequence functions as a UAS in all three cell types, whereas P(STE3) functions only in a cells and only when adjacent to a Q box [17]. PRTF, which is the product of the MCMI gene [18-20], will be discussed in more detail in section I11.

II .C The MATa2 product

The MATa2 product plays two roles in the regulation of cell-type-specific genes. In a cells, a2 represses the transcription of a-specific genes [1,21]. In a /a diploid cells, a2 functions with the product of the MA Tal gene to repress the transcription of MATal and of haploid-specific genes [1,22-23]. These conclusions are based on genetic, physiological and biochemical studies of mata2 mutants. Mutations in mata2 are recessive and lead to the expression of a-specific functions, such as a-factor and Barrier, which degrades a-factor [26-28]. These mutants are also capable of mating as a cells, although the efficiency is significantly lower than that of wild-type a cells [1]. Northern blot analysis of STE6, a gene necessary for the secretion of a-factor, demonstrated that in cells containing a functional MA Ta2 gene (a and a / a cells), the level of STE6 RNA is at least 150-fold lower than in cells without a functional MATa2 gene (a and mata2 cells) [21]. This result confirmed that a2 negatively regulates a-specific gene expression at the level of RNA.

Johnson and Herskowitz [29] demonstrated that a2 binds directly to DNA. An a2-~8-galactosidase hybrid protein was mixed with a restriction digestion of a plasmid containing the STE6 gene, immunoprecipitated and the DNA bound by the hybrid protein analyzed by polyacrylamide gel electrophoresis. The hybrid protein bound to a single fragment of DNA [29], and DNase I footprinting revealed that it protected a 32 bp sequence. Sequences homologous to this a2 binding site are present in the upstream regulatory regions of all a-specific genes [22,29] (Fig. 2). Unlike al, a2-/i-galactosidase synthesized in E. coil is able to bind to DN s, without the aid of any other yeast proteins (but see section I11).

The a2 binding site was tested for in vivo function by placing it into the CYCI (cytochrome c) promoter fused to lcicZ and testing its ability to repress expres-

158

Consensus site Representative site Reference

A) al site CTGtCATTOAIGgACTAA'I'TAGGAAA

a I P from STE3:

m wa

CTGTCATTGTp GACACTAA'FTAGGAAA

Q I P

7,17,18

B) MCM1 site TTCCTAATTAGGAA ;'(pAL)

from S ~ 6 : • I ' r A C O T A ~ l f A G O ~ _ ~ a a ~

17,18,19,20,49

C) o2 site CATGTAATTACCNAATAAGGAAATrTACATGN

from STE6: C A T O T A A T T A C O T A A T A G G G A A A ~ r l r A O A O G O

8,21,22,29

D) al-(x2 site TC ~ TGTNN ~ NANNTACATCA

from MATal: ocTrccc~T~'ao~u~a~cA~.ATao

22,23,34

E) PRE ATa~CA

from STE12:

from STIr:

dimer site Ca- fa-

PRE adjacent to MCM1 are

l"l'r OCTA),T~GGGTAAGTA CATGATGAAACA

52,108,109,110

Fig. 2. DNA binding sites of proteins involved in tLe regulation of cell-type-specific transcription. The shapes representing the various proteins are only approximate indications of the contact sites, and in some cases (e.g., binding of a i , and of ai and a2) these contacts have not been

demonstrated directly.

sion [29]. The presence of an a2 binding site between the CYCI UAS and TATA element causes a several 100-fold ,iecrease in the activity of the promoter, as measured by a decrease in the level of fl-galactosidase activity. This decrease in promoter activity is seen in a cells and in a matal mutant but not in a cells or a mata2 mutant. The presence of the a2 binding site 5' of the UAS elements also causes a decrease in the promoter activity, but the extent of the repression is only 10-15-fold. in a cells, the a2 site is able to function as a very weak UAS when the CYCI UAS is deleted. This weak UAS activity is not seen in a cells, suggesting that the presence of a2 abolishes this activity [29].

The a2 protein contains a region with homology to the homeodomain, a sequence of 60 amino acids first identified in Drosophila homcotic and segmentation genes [30,31]. This domain has been shown subsequently to be responsible for DNA binding in a numtmr of proteins that contain it [31]. In the case of ~2, Hal! and Johnson [32] constructed deletion mutants of a2 fused to fl-galactosidase and measured the ability of these mutants to bind to DNA and to repress expression of a-specific and haploid-specific genes. This ap- proach localized the DNA binding domain of a2 to a 68 amino acid region that contains the region of homology to the homeodomain. Mutants that fail to bind

to DNA also fail to repress the expression of an a- specific gene and of a haploid-specific gene.

The a2 protein present in crude yeast extracts protects a large region of DNA (32 bp) from cleavage by DNase I, whereas a2 purified from E. coil protects only the ends of the 32 bp site, while leaving the middle of the site exposed. This observation led to a search for a second yeast protein capable of protecting the middle of the a2 binding site, and resulted in the identification of a protein designated GRM (General Regulator of Mat- ing type) [8]. GRM and a2 bind cooperatively to the DNA; a2 binds to the ends of the site and straddles GRM, which is bound to the middle of the site. GRM was subsequently found to be the same protein as PRTF, as discussed in section II1.

ILD. The al-a2 repressor

The MATa allele codes for two detectable RNA transcripts, one corresponding to MA Tal and the other to MATa2 [24,25]. Only the MATal product has been shown to play a role in regulation of mating type while no function for the M A T a2 product has been dis- covered. Cells carrying a matal mutation still mate as a cells, indicating that the al protein is not required for mating. The al protein has a role in the a / a diploid, where al and a2 function together to form a repressor with a unique DNA binding specificity, that can repress the expression of haploid-specific genes and of MA Tal I1,22-25,331.

A DNA sequence potentially recognized by al-a2 was identified by comparing the sequences in the regulatory regions of haploid-specific genes, including HO and STE5 [22] (Fig. 2). Deletion analysis of the HO regulatory region revealed the presence of redundant elements that are similar to sequences found in the regulatory regions of MA Tal and STE5. Two different versions of this sequence found in the HO promoter were inserted into the promoter of the CYCI-IacZ gene. Expression of this gene carrying the al-a2 control element is significantly reduced in the a / a diploid relative to haploids and this reduction is not seen in a m a t a l / MA Ta diploid [221.

An analysis of MATa also indicated that this sequence confers proper diploid-specific repression. In wild-type dipioids, MA Tal transcription is totally repressed and MATa2 transcription is reduced approx. lO-fold [24,25]. Initially, a 14 bp deletion in the inter- genic region between MATa2 and MATal w ~ showii to abolish diploid-specific repression [161. Subsequent analysis focused on a 53 bp region that contains the al-a2 control element [33]. Deletion of this region results in the constitutive transcription of MATal and MA Ta2 in a / a cells. Insertion of a 28 bp fragment that contains the al-a2 control element restores proper expression of both MATa genes. The al-a2 control ele-

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ment from the MATa locus was also inserted intc, the CYCI-lacZ promoter where it confers diploid-specific repression but does not alter the normal regulation of the CYC1 promoter: significantly more ,8-galactosidase is produced from the repor,er gene when the cells are grown on a derepressing carbon source (lactate) [331. Insertion of the al-a2 element thus adds an additional level of regulation to the normal regulatory system of the C YC! gene.

The interaction of al with a2 alters the sequence recognized by a2 such that instead of binding to a- specific genes, it binds to haploid-specific genes [34 i . The 29 bp al-a2 sequence is similar to the a2 site, but with the two shorter half-sites closer together [22,34 I (see Fig. 2). This sequence arrangement led to the suggestion that a2 may contact the two half-sites in a conformation dependent on al or that a2 may bind one of the half-sites while al binds the other. Although it was not shown that al directly contacts the DNA, the presence of a homeodomain in this protein is suggestive of this function [34]. In fact, either a2 or al alone binds weakly to the al-a2 site, suggesting that each may contact a half-site (Goutte, C. and Johnson. A.D., unpublished data).

Goutte and Johnson [34] point out that the ability of a2 to acquire a new binding specificity has two ad- vantages for the a / a cell. First, the requirement for both a2 and al to produce the transcriptional pattern of the a / a cell allows the cell to monitor the presence of both an active MA Ta allele and an active MA Ta allele. Second, the a / a cell can repress two sets of genes, the a-specific and the haploid-specific, whereas the a cell requires repression of only the a-specific genes. The diploid is able to carry out these two separate functions by having a2 bind to two different targets: the a2 operator, which also functions in a cells, and the al-a2 operator, which functions only in a / a cells.

The diploid cell uses the al-a2 activity to allow sporulation in response to starvation. The rmel (regulator of meiosis) mutation permits sporulation of diploids lacking al or a2 [12], which led to the proposal that al-a2 negatively regulates RMEI, whose product inhibits meiosis [35]. Identification of the RMEI gene showed that its expression is reduced 20-fold in a / a cells [36]. Subsequent studies have shown that RME1 inhibits expression of activators of sporulation-specific genes [37-39].

IlL The M C M ! product

l lLA. Identification of MCMI

MCMI was originally identified in a search for genes required for proper functioning of autonomously replicating sequences (ARSs) [40], which are thought to be origins of DNA replication in yeast [41,42]. The mcml-I

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mutant is defective in the maintenance of plasmids that carry a specific ARS. In addition, a role for MCMI in the expression of cell-type-specific genes was revealed by mating defects associated with the mcml-I mutation; MATa mcml-I cells are sterile and MA Ta mcml-I cells mate at a reduced efficiency [9]. Sequence analysis of the cloned gene predicted an open reading frame for a protein of 286 amino acids [9,20]. The protein has homology to the helix-turn-helix DNA-binding motif, as well as a stretch of 19 aspartate and glutamate residues interrupted by a single glycine residue, and two glutamine-rich stretches. The glutamine-rich sequences are similar to polyglutamine regions in other yeast regulatory proteins, such as SSN6 [43] and HAP2 [44]. The DNA-binding domain of MCM1 has extensive sequence homology to ARG80, a small protein involved in the regulation of arginine metabolism [45], to the serum response factor (SRF) of mammalian cells [46], and to the homeotic genes agamous of Arabidopsis [47] and deficiens of the snapdragon [48] (see Ref. 47 for align- ments).

Several lines of evidence indicate that PRTF, GRM, and MCM1 represent a single protein. The DNA sequences recognized by all three proteins are very similar [18-20,49]. Jarvis et al. [19] used truncated MCM1 proteins in gel-shift assays with a DNA probe containing a P box, the binding site l'or PRTF. The DNA-pro- rein complexes containing truncated forms of MCM1 migrate faster than the DNA complex containing full- length MCMI, showing that MCM1 is part of these DNA-protein complexes. Antibodies raised against MCMI are able to alter the migration of DNA-protein complexes containing P box probes [18,19,49]. The meml-I mutation results in reduced transcription of a<pecific genes, which, based on the role of PRTF in a-specific expression, is consistent with the idea that PRTF and MCM1 are identical [9]. The GRM-dependent DNA binding activity contains a protein recognized by anti-MCM1 antiserum [50].

III.B. Role of MCMI and MCMI-al in the activation of cell-type-specific genes

In the case of a-specific genes, MCM1 plays a role in transcriptional activation, but this role may be quite limited for certain members of this set of genes. In addition, for MCM1 to activate a-specific transcription efficiently, it may require interactions with additional transcription factors bound to adjacent sites. The mcml-I mutant is reduced only about 50% for mating in a MATa background and shows a small reduction in the transcription of the a-specific gene STE2 [9], but this result must be interpreted cautiously because mcml-I is not a null mutation. The MCMI binding site as present in an a-specific gene has been tested for UAS activity by several groups. The t~2 operator from STE6,

which can bind MCM1 to its central sequence (Fig. 2), provides a very low level of activation to a CYCI-lacZ reporter gene [29]. For this gene, the actual sequences required for transcriptional activity may map greater than 100 bp upstream ef the operator [51]. A 47 bp UAS fragment from STE2 contains an MCM1 binding site adjacent to a site for a transcription factor (STE12) (Fig. 2) regulated by the pheromone response pathway [52] (see subsection IV.E). Mutation of two nucleotides in the MCM1 binding site abolishes UAS activity [20], indicating the importance of MCM1 function for activation. However, in the absence of STEI2, UAS activity is decreased by 96-fold [52]. A similan • analysis was performed with a STE2-1acZ gene deleted for its UAS [18]. Insertion of an MCM1 binding site into this reporter gene activates transcriptnon to only 8% of the level observed when the larger STE2 UAS is inserted. Thus MCM1 is necessary for STE2 transcription, but in the absence of another factor, it is insufficient to provide the wild-type level.

MCM1 is able to activate transcription of a reporter gene containing the palindromic binding site P(PAL} (Fig. 2) in all three yeast cell types [17]. The transcriptional activity is very, low when P(PAL) is inserted into a promoter containing just a TATA box and an initiation site upstream of a CYCI-IacZ reporter gene. The P(PAL) sequence could result in a high level of transcription of the reporter gene when placed in the con- text of a promoter containing additional CYCI upstream sequences [17]. This high activity may indicate that MCM1 can interact with an additional factor(s) associated with the larger upstream regulatory region to produce an enhanced level of transcription.

Activation of ~t-specific genes requires the presence of both MCMI and al [7]. The MCM1 binding sites in the upstream regulatory regions of a-specific genes are degenerate and MCM1 is unable to bind efficiently to these sites, in vitro [7]. In studies using purified proteins, the binding affinity of MCM1 for the STE3 UAS was shown to be approx. 50-fold lower than the affinity of MCMI-al for the same site [53]. Similarly, MCMI binding to a fragment of the MFal gene is significantly enhanced by the presence of al [18]. Thus cooperative interactions between MCM1 and al help to recruit MCM1 to a-specific promoters. Genetic support for the role of MCM1 in a-specific transcription comes from the phenotype of the mcml-I mutant, in which transcription of two a-specific genes is reduced at least 30-fold [91.

Increased binding affinity may not be the only conse- quence of MCMI-al interactions. The MCMI-al complex interacts with a more extended region of DNA than does MCM1 alone [18,53]. For the STE3 UAS, this extension is approx. 8 bp on either side of the protection observed for MCM1 alone, and therefore is not simply on the side containing the Q-box sequence

161

[53]. These footprinting studies suggest that the presence of al alters the conformation of the MCM1-DNA complex in a manner that facilitates transcriptional activation. Either the conformation of the DNA may change, or the conformation of MCM1 may change, for example to expose an activation domain, or both. Ex- periments probing protein conformation by proteolysis suggest that it is MCM1 that changes its conformation when bound to an a-specific UAS, but not to an a- specific UAS [54]. The altered conformation may represent a transcription-competent state, and it is proposed that al binding changes the inactive MCM!-DNA complex into an active conformation [54].

The results showing limited UAS activity for an isolated MCM1 binding site suggest that the conformational change in MCM1 alone is not sufficient to provide the constitutive level of transcription. Conse- quently, a simple model of exposure of an activation domain upon binding to an a-specific gene, or to an a-specific gene in the presence of al, is inadequate. It may be that the conformational change allows MCM1 to interact with an adjacent transcription factor, for examp~= STE12. As stated earlier, transcription from the STE2 UAS is significantly reduced in a stel2 mutant, even though MCM1 is presumably able to bind and assume the altered conformation. Similarly, transcription of a-specific genes is also reduced in a stel2 mutant, even though MCMI-al are presumably able to bind. Thus, MCM1 is a crucial component of transcriptional activation of cell-type-specific genes, but MCMI alone is not sufficient for proper levels of transcription.

nal deletion mutants of ,~2 which still bind to the operator fail to repress in vivo [32], suggesting interaction between a2 and MCMI is essential for repression.

Two models have been proposed to explain repression by a2-MCM1 [8]. The masking model proposes that a2 interacts with MCM1 to mask the domain of MCM1 that interacts with the general transcription machinery, thus blocking activation. This model fails to explain how the a2 operator, when inserted into a test promoter, can cause repression of a heterologous UAS. In the locking model, a2-MCM1 interacts with the transcriptional machinery and forms a very stable complex. The transcriptional mach:.nery is thus locked in place such that a subsequent step in initiation cannot occur. The interaction of a2-MCMI with the transcriptional machinery is proposed to be stronger than the interaction of a normal transcriptional activator, allowing a2-MCM1 to out-compete the normal activator.

In addition to a2 and MCM1, the product of the SSN6 gene may also be required for repression (Keleher, C. and Johnson, A.D., unpublished data). SSN6 was identified as a negative regulator of the expression of SUC2, which encodes invertase [56]. It was observed that a ssn6 mutants can mate at low efficiency to a cells [56] and express a-specific genes [431. A direct role for SSN6 in a2 repression is suggested by the reduced ability of the a2 operator to repress transcription in ssn6 mutants, and the ability of SSN6 when bound to DNA via a heterologous DNA-binding domain to act as a transcriptional repressor (Keleher, C. and Johnson, A.D., unpublished data).

I l I .C Role of MCMI-a2 m the repression of a-specific genes

MCM1 (GRM), functioning cooperatively with a2, is required for the repression of a-specific genes in a cells. In vitro binding studies showed that purified a2 binds to two 13 bp sites at each end of the 32 bp a2 op,.rator [55], while MCM1 protects the middle 24 bp of the operator ~]. Thus the two proteins show overlapping regions of protection. Mutant operator sequences, containing deletions in the middle or at the ends of the operator, were used to show that the two proteins can bind independently [8]. However, on a wild-type operator, MCM1 and a2 bind cooperatively and form a ternary complex, with MCM1 estimated to raise the affinity of a2 for the operator by approximately 50-fold 18l.

In vivo evidence for the requirement of MCM1 in repression comes from the finding that mutant oper- ators that abolish MCM1 binding without affecting a2 binding are unable to repress transcription [8]. In addition, the N-terminal domain of a2 is required for cooperative binding of MCM1 and a2 [8], but is not required for in vitro binding of a2 [321. These N-termi-

IV. Pheromone response pathway

The products of the MAT locus are the critical determinants of cell type. The process of mating, however, is controlled by the pheromone response pathway. The binding of pheromone to its receptor activates a G protein, initiating a signal that ultimately leads to an increase in expression of genes involved in mating. In addition, this same pathway plays a major role in the constitutive level of cell-type-specific transcripts produced by cells not exposed to pheromone.

IV.A. The pheromones and receptors

The mating pheromones are small peptide hormones that are secreted into the medium by haploid cells. a-Factor, the a-specific pheromone, is encoded by the MFal and MFa2 genes [57,58] and a-factor, the a- specific pheromone, is encoded by the MFAI and MFA2 genes [59]. Both pheromones are derived from larger precursor proteins by proteolytic cleavages and, in the case of a-fa~:tor, post-translational modification, a-Fac- tor is a 13 amino acid peptide with the sequence Trp- His-Trp-Leu-Gin-Leu-Lys-Pro-Gly-Gln-Pro-Met-Tyr; it

162

is encoded by four copies in MFal and two copies in MFa2 [58]. Production of mature peptide requires the products of the STEI3 (dipeptidyl aminopeptidase A), KEXI (carboxypeptidase Y) and KEX2 (Lys-Arg endo- peptidase) genes [60-62]. a-Factor is a 12 amino acid peptide with the sequence Tyr-lle-lle-Lys-Gly-(Val or Leu)-Phe-Trp-Asp-Pro-Ala-Cys That is encoded in a single copy in both MFAI and MFA2 [59]. The MFAI gene codes for valine at position 6, while MFA2 codes for leucine. Maturation and secretion of a-factor requires the product of the STE6 gene [21], which is homologous to the multiple drug resistance gene from mammals [63,64], as well as the products of the STEI4 [65] and RAMI [66] (also called STEI6 [67]) genes. In addition to release from the precursor by proteolysis, a-factor is modified posttranslationally. The C-terminus is carboxymethylated and a farnesyl group is added [681.

The receptor for a-factor is encoded by the STE2 gene [69,70] and the receptor for a-factor is encoded by the STE3 gene [70,71]. Although these receptors do not share significant sequence homology, both appear to be structurally similar to rhodopsin and the fl-adrenergic receptor; sequence analysis predicts seven membrane- spanning domains with an extracelhilar N-terminus and a long intracellular C-terminal tail. Both receptors have sites for N-linked glycosylation in the N-terminal extracellular domain and many serine and threonine residues in the C-terminal tail, some of which serve as sites for phospho~ylat~on [72].

IV.B. A common intracollular pathway

Despite the lack of homology, both receptors appear to interact with a common intracellular response pathway. Bender and Sprague [73] expressed the genes for the receptors in a MA T-independent manner by using a galactose-inducible promoter in a matal mutant, which expresses neither a-specific genes nor a-specific genes. Cells that carry the STE2 gene express the a-factor receptor when grown on galactose and respond to a- factor treatment by induction of pheromone-responsive genes and by arrest of the cell cycle. Conversely, cells that carry the STE3 gene express a-factor receptor and respond to a-factor treatment. These experiments, and similar ones by Nakayama et al. [74], indicate that the pheromone/receptor interactions are functionally inter- changeable; cells respond to the pheromone that binds to the specific receptor that is being expressed. In wild-type cells, the M A T products determine the specificity of the pheromone response by controlling which receptor is expressed. Since matal cells are able to respond to pheromone as long as a receptor is expressed in a MAT-independent :aanner, no other a- or a-specific products are required for response.

The pheromone response pathway operates in con- juncti,~n with the M A T products to set the levels of constitutive (or uninduced) cell-type-specific transcription. Five genes, STE4, STE5, STE7, STEI I and STEI2, were originally identified by Hartwell [75] in a selecton for mutants resistant to cell cycle arrest by pheromone. These ste mutants show a defect in constitutive expression of cell-type-specific functions, e.g., a-factor production [75], which was subsequently demonstrated to be at the level of transcription [76-79]. As described below, these STE genes encode components of the pheromone response pathway. Constitutive expression of some cell-type-specific genes is critically dependent on this pathway, and almost no RNA for these genes is detected in ste mutants [78]. The expression of other genes is only decreased a few fold by these ste mutations. These differences presumably reflect the differing contribution to transcription of UAS elements not dependent on the response pathway composed of the STE products. Increased transcription due to activation of the response pathway is mediated by the pheromone response element (PRE). This sequence is sufficient for induction of a-specific genes in a cells and of haploid-specific genes in both a and a cells. The sequence that mediates induction of a-specific genes may be more complicated (see subsection IV.E). How- ever, the same sequence elements responsible for transcriptional inductioi, also mediate the response pathway's contribution to constitutive transcription.

The presence of .~ single pathway leading to the induced expression o: a-specific and a-specific genes rai:es the possibility of inappropriate expression following phe-c ~, ~ne treatment. How does the cell regulate the induction o ~ ~ aly one set of ceU-type-specific genes if it uses ,~ ;ngle pathway? Pheromone induction of transcription, dependent on the response pathway, is subject to the constraints imposed by the M A T products. In the a ceil, the presence of a2 prevents the common response pathway from inducing the expression of a-specific genes, and in the a cell, the absence of al prevents the induction of a-specific genes.

1 V.C. Receptors activate a G protein

The binding of pheromone to receptors activates a heterotrimeric G protein. The c~, fl, and "t subunits of the G protein are encoded by the GPAI (SCGI), STE4 and STEI8 genes, respectively [80-83]. The identification of these gene products as components of a G protein is based on sequence homology and the behav- ior of mutants; assembly of these proteins into a heterotrimer has not been directly demonstrated. The experiments described below have led to the following model of G protein function in the yeast pheromone response pathway (Fig. 3). In the absence of pheromone, the Ga, G~ and G v subunits associate to form an inactive bet-

Ct¢l ME "nbrane

Pheromone

G P A I ~ DP GTP GDP

G Protein Signal

Fig. 3. Model of yeast pheromone-responsive G protein function. Pheromone binds to the receptor, causing the a subunit to exchange GTP for GDP. The GTP-bound form of a dissociates from fl't, leading to the generation of an intracellular signal which activates the

response pathway.

erotrimer. By analogy to mammalian G proteins, it is assumed that in the heterotrimer the a subunit has bound GDP. The binding of pheromone to the receptor causes the exchange of GDP for GTP and the subsequent dissociation of the trimer into a free G,, subunit and a Ga~ dimer. The free Gay dimer activates an as yet unidentified effector, which generates the intracelhilar second messenger. This second messenger, which is also unidentified, activates the rest of the pheromone response pathway and the cells become mating-competent. Hydrolysis of the GTP bound to the G,, subunit is followed by the reassociation of GDP-G~ with G~v and the response pathway is turned off. Coupling of the pheromone receptors to a G protein has been indirectly demonstrated biochemically [84]. Activation of the effector by a Gay dimer differs from the mechanism of most mammalian systems, which use the G o subunit to activate the effector [85]. However, precedents for an active role for Gay do exist. Evidence suggests that in mammalian cells, phospholipase A 2 is activated by the Ga~ subunit and not by the C~ subunit [86,87].

Nakafuku et al. [80] identified GPAI based on its sequence homology to the t, subunits of mammalian G proteins. The amino acid homology between yeast GPA1 and rat Gi,, is 45~, and increases tr_, 65~ when con- servative amino acid changes are included. The sequences are most homologo, regions of the protein that are believed to be involved in GTP binding and hydrolysis. GPA1 i,. " than any of the rat Go subunits. The differet,.~ in size is mainly the conse- quence of a 110 amino acid region located near the N-terminus of the protein, which may interact with the effector molecule [88]. (;PAl is expressed only in a and a haploid cells and its expression is induced in response to pheromone treatment [81,82,89]. Dietzel and Ku0an [81] and Jahng et al. [89] independently cloned (;PAl using other genetic strategies.

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Conditional mutants were used to show that the loss of GPA1 expression leads to several phenotypes associated with response to pheromone, including arrest of cell division, an increase in the percentage of unbudded cells, changes in cellular morphology (shmooing) and increased transcription of a pheromone-inducible gene (FUSI) [82,89,90]. These phenotypes occur even in gpaI tSste2 mutants that lack the receptor for a-factor, and thus are unable to respond to exogenous pheromone [89]. Cells containing a goal null mutation undergo two or three cell divisions and then stop dividing. The cells at this stage resemble cells that have been exposed to l~heromone [81,82].

The gpal mutation suppresses the mating defect associated with a ste2 mutation [82,89]. However, the ste2 mutation does not suppress the growth defect of the gpal mutation, arguing that GPA1 functions at a step in the response pathway after the receptor. The phenotypes of the gpal mutants and the suppression results are consistent with mutations in GPA 1 causing a constitutive signal to be sent through the pheromone response pathway, allowing the cells to manifest a pheromone response in the absence of pheromone or receptor. Double mutants carrying gpaI and either ste4, ste5, ste7, stell or stel2 are not abte to mate, but do grow [90]. This result is consistent with these STE products working after GPA1 in the pathway and the ste mutations blocking the pheromone response signal before it can cause cell cycle arrest.

STE4, which encodes the fl subunit of the G protein, was originally identified as one of the STE genes required for r. rang in both a and a cells [11,75]. The DNA sequence of STE4 was found to be highly similar to the Ga subunits of bovine transducin and human liver G s / G i signal transducing proteins [83]. An analysis of RNA isolated from a, a, and a/a cells demonstrated that STE4 is expressed only in haploid cells [83] and is induced about two-fold by pheromone treatment [91]. A ste4 null mutant is not able to mate, produces greatly reduced levels of pheromone and fails to undergo morphological changes when exposed to the appropriate pheromone [83]. A dominant mutation, desig- i ,ted STE4 H# (Hpl stands for Haploid Lethal), has also been isolated which results in constitutive pheromone response, including cell cycle arrest [92]. Genetic analyses of the STE4 H# allele have shown that STE4 functions after the receptor but before STE5 in the response pathway [92]. Thus, the identification of the STE4 protein as the fl subunit of the G protein involved in pheromone response is based on its sequence homology to other known G F subunits, the loss of pheromone response in recessive ste4 mutants and the pbenotype of dominant STE4 Hpl mutants.

STEIS, which encodes the ), subunit of the G protein, was first identified on the basis of the response of a particular mutant to differing concentrations of

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pheromone [83[. This stel8 mutant is resistant to arrest by high levels of pheromone, but its growth is arrested by low levels of pheromone. This unusual response to pheromone concentration produced a characteristic pattern of growth when assayed on plates containing a sourc= of pheromone [83]. The STEI8 gene was cloned by screening for a transformant that no longer produced this characteristic pattern. The predicted sequence of STE18 was found to be structurally similar to the "y subt~nit of bovine transducin [83], including the sequence cysteine-aliphatic-aliphatic-X at the C- terminus. This sequence is found in several yeast proteins that are farnesylated at the cysteine residue [93]; STE18 is also membrane-bound and polyisoprenylated [94]. The expression of STE18 is haploid-specific and is not induced by pheromone [831.

The model of G protein function states that binding of pheromone to receptor causes the dissociation of G~ from Ga~, allowing free Got to transmit the response signal. If this model is correct, then overproduction of STE4 and STE18 should result in excess free Ga~ and consequently pheromone response in the absence of pheromone. Overproduction of STE4 causes _-,~!ls to arrest in an unbudded state, to exhibit abnormal cellular morphologies resembling shmoos, and to induce a FUSI-IacZ reporter gene [95-97]. Overproduction of STE18 does not produce a detectable phenotype [95-97]. However, when both STE4 and STE18 are overproduced, the level of FUSI-lacZ expression increases relative to the level seen when just STE4 is overproduced [95]. This result suggests that $1E4 is limitin~ in wild- type cells with regard to pheromone response, but that STEI8 is in excess. When STE4 is overproduced, STE18 protein becomes limiting, such that enhanced transcription of FUSI-lacZ occurs when both STE4 and STE18 are overproduced. While overproduction of STE4 re- suits in constitutive pheromone response, overproduction of GPA1 suppresses this effect, presumab!)- by titrating the free Gpv [96]. When both STE4 and GPA1 are overproduced, the growth arrest and abnormal morphology are suppressed, but the increased level of transcription of FUSI-lacZ is not suppressed [95,97]. The ability of overproduced GPA1 to suppress some, but not all, aspects of the pheromone response elicited by overproduction of STE4 demonstrates that the pheromone response is not an all-or-nothing response. This result is consistent with the observation that different concentrations of pheromone are needed for the various responses to pheromone [98]. For example, induction of agglutination in an a cell requires an a-factor concentration of only 10- i z M , but formation of shmoos requires a concentration of 10 -8 M [98].

The pheromone response pathway is controlled by the carefully balanced action of positive and negative regulators. STE4 and STE18, like the downstream STE products (STE5, STE7, STEll and STEI2), act posi-

tively in the response to pheromone, while GPA1 acts negatively to block response in the absence of pheromone. The products of the CDC36 and CDC39 genes, initially identified by Reed [99], also function in a negative manner at, or very near, the G protein. Shifting these temperature sensitive cdc mutants to the restrictive temperature results in the induction of pheromone-responsive genes (as measured by expression of a FUSI-lacZ reporter gene), arrest in G1 and changes in cellular morphology [91,100]. Double mutants carrying ste2 and either cdc36 ts or cdc39 ts are able to mate at the restrictive temperature and also exhibit other pheromone responses [91,100]. Arrest in G1 and induction of FUSI-IacZ requires a functional pheromone response pathway, as they cannot occur in strains carrying ste4, ste5, stell, stel2 or stel8 null mutations [91,100]. Assuming that yeast G proteins function in the same manner as mammalian G proteins, two possible roles for CDC36 and CDC39 have been suggested [91,100]. These proteins might activate thc intrinsic GTPase activity of GPA1 so that the cdc mutants accumulate GTP-bound (3, and free Gar. Al- ternatively, CDC36 and CDC39 might stabilize GDP- bound G~ so that, in the cdc mutants, G, would more readily exchange GDP for GTP, releasing Ga~ and activating the pathway.

The pheromone response pathway is lil~ely to be active, at a low level, even in the absence of pheromone, due to an equilibrium between the inactive heterotrimeric form of the G protein and free G a and free Gt~ ~ dimer. The free Ga~ dimer would cause the generation of a low level of second messenger and lead to a limited pheromone response. The low level of constitutive pathway activity is suggested by the effect of mutations in the STE genes that encode pathway components (STE4, STE5, STE7, STEI1 and STEI2). Mutations in any of these genes results in a decrease in the level of transcription of pheromone-inducible genes [76-79]. This decrease in transcription suggests that a functional pathway is essential for proper expression of pheromone-inducible genes, even in the absence of pheromone.

IV.D. STE5, STE7, STEI I and FUS3

STE5 was identified by mutations that blocked mating [11] or that rendered cells unresponsive to pheromone [75]. The biochemical role of the STE5 protein in pheromone response is currently unknown. Genetic evidence suggests that STE5 functions after the G protein in the response pathway [92]. The expression of STE5 is haploid-specific [101L Analysis of STE5 using its cloned gene has revealed the following (Has- son, M., Freedman, R. and l-homer, J., unpublished data). STE5 is a phosphoprotein of 917 amino acids that is predominantly localized to the nucleus. Treat- ment of cells with pheromone leads to the rapid in-

165

crease in the level of phosphorylation of the STE5 protein. Overproduction of STE5 leads to constitutive transcription of pheromone inducible genes.

The STE7 and STEl l genes were also identified by mutations that rendered cells unresponsive to cell division arrest by pheromone [75]. Expression of STE7 and STEII occurs in both haploids and diploids [102], suggesting that the encoded proteins might function in a process other than mating. Nonetheless, ste7 and stell mutants do not manifest any detectable phenotype other than sterility. The nucleotide sequence of STE7 and STEII indicates that both genes are likely to encode protein kinases [103,104]. The deduced amino acid sequence of STE7 contains many of the conserved motifs of protein kinases, including the sequences Gly-Ala- Gly-Asn-Ser-Gly, Ala-Lys-Lys, Asp-Phe-Gly. and Ser- Pro-Glu, at the appropriate locations relative to one another [103]. These motifs are also conserved within the STEII sequence, and the immunoprecipitated STEI 1 protein has kinase activity [104]. In additon, the conserved lysine residue within the putative catalytic center is necessary for pheromone response [104]. The presence of a multi-copy plasmid carrying STE7 is not able to suppress the mating defect of a sic11 strain and vice versa [102]. These results indicate that the two proteins may not perform identical functions in the response pathway and an epistatic relationship can not be determined. The in vivo substrates of these two protein kinases and their state of activity in the presence or absence of pheromone are not known at this time. Some candidate substrates include the pheromone receptors, the fl subunit of the G protein (STE4), the STE5 protein and the STE12 protein, all of which are phosphorylated.

A third putative protein kinase, encoded by the FUS3 gene, is required for pheromone response and mating [105]. The FUS3 protein shares significant homology to the cdc2 (of Schizosaccharomyces pombe)/CDC28 family of protein kinases. Unlike STE7 and STEII, FUS3 is not expressed in diploids and is induced several fold in response to pheromone treatment. A positive role in pheromone response is suggested by the observation that overproduction of FUS3 renders cells more sensitive to pheromone. In addition, a fus3 mutation suppresses the growth defect of a gpal mutant, and leads to a decrease in the constitutive level of expression of pheromone-respoasive genes. While fus3 null mutants fail to exhibit any response to pheromone, point mutations were isolated that allow induced transcription and morphological changes in response to pheromone. How- ever, these mutants continue to divide in the presence of pheromone, indicating a failure to arrest in G1. The failure to arrest in G1 can be suppressed by a mutation in the CLN3 gene, which appears to encode a cyclin [106,107]. Suppression of the fus3 arrest defect by a cyclin mutation suggests that FUS3 might mediate cell

cycle arrest by regulating the activity or expression of CLN3.

! V.E. STEI2 as a pheromone-responsive transcriptional aclir.otor

The STE12 gene was first identified by Hartwell [75] in a selection for mutants resistant to cell cycle arrest by pheromone and later identified in a screen for mutants defective in expression of a-factor [77]. Northern analysis of both a and a stel2 strains revealed that the mutants have reduced levels of RNA for a-specific genes (STE2, MFAI and MFA2) and a-specific genes (STE3, MFal and MFa2) [77]. The effect of a ste12 null mutation on mRNA levels of a-specific genes is not absolute, as is the effect of a matal mutation; matal mutants fail to produce any detectable RNA from a-specific genes, whereas stel2 mutants produce a reduced, but detectable, level of RNA from these genes [78]. Expression of different cell-type-specific genes is reduced to different extents by a stel2 null mutation [78]. Expression of some genes, such as STE2, STE3 and STE6, is reduced 5- to 10-fold, while expression of other genes, such as MFAI and MFa2, is reduced at least 100-fold. The decrease in expression of a given gene is the same ir a stel2 mutant as in ste7 and stell mutants, and a ste7 stell stel2 triple mutant shows the same decrease as any of the single mutants [78]. The common phenotype argues that the products of these STE genes function in the same aspect of transcription and not at independent steps,

The STEI2 product is a DNA-binding protein that recognizes the pheromone response element (PRE) [52,108], the sequence that mediates increased transcription of responsive genes following treatment of cells with pheromone. The PRE from a-specific genes is (A/T)TGAAACA [109,110] and this sequence has been shown to be essential to render an a-specific gene (BAR1) pheromone-inducible [109 !. This sequence is found in the upstream regulatory regions of all a-specific genes, frequently in multiple copies [51,59,76,109]. PREs are also found upstream of several inducible haploid- specific genes. Multiple copies of the PRE are located upstream of FUSI [79,111], CSH1 [112,113] and STEI2 [52,108]. Induction of transcription by both a-factor and a-factor has been demonstrated for F'USI and CSHI [79,111,1131.

Gel-shift assays using DNA fragments from the regulatory regions of the a-specific genes MFAI and MFA2 demonstrated that a specific protein-DNA complex is produced by extracts from STEI2 cells, but not by extracts from stel2 cells [108]. The binding site was identified as the PRE by showing that a synthetic oligonucleotide consisting of two copies of the PRE in a head-to-tail arrangement was able to form a STEI2-deo pendent DNA-protein complex. Direct DNA binding

166

by STE12 was shown v, tth a variation of tht standard gel-shift assay: 35S-labeled STE12 forms z. specific DNA-protein complex with unlabeled DNA cantaining a PRE sequence [108]. In addition, STE12 was shown to be part of protein-DNA complexes using fragments of Tyl DNA and the STE2 gene [52] (see below).

The actual role for STEI2 in the a-factor induction of a-specific genes is less clear. A 43 bp fragment from the regulatory region of S T E 3 has been showa to be sufficient for both constitutive and a-factor induced expression. This fragment contains a P and Q box sequence that can bind MCMI and al, but lacks a clear homology to the PRE consensus [17]. In addition to PQ elements, some genes, such as the gene for a-agglutinin ( A G a l ) [114], contain an 8/8 bp match to the consensus PRE sequence, which is presumably capable of mediating a-factor induction through the binding of STE12. Other a-specific genes contain only degenerate copies of PRE sequences, within the PQ region, and elsewhere within their upstream regulatory regions, that have at best a 5 or 6 out of 8 bp match to the consensus [17,57,58]. It is not known whether STE12 can recognize

and bind to such degenerate sequences, even in the presence of MCM1 and al. Induction mediated by the PQ element may require STEI2 because STE12 is necessary for the transcription of some component essential for response, with the transcriptional effect occurring by changes in MZM1 or al. Alternatively, STEI2 may mediate induction directly by binding to the PQ element through cooperative interactions with MCM1 and/or al.

An MCM1 binding site is found 7 bp away fro:, a

PRE in the upstream regulatory region of S T E 2 [52,76] (Fig. 2). As shown by a gel-shift assay with extract from an a s te l2 null strain, a probe containing these two elements binds MCM1 [52]. Extract from an a S T E ÷ strain produces the MCM1-DNA complex and also a

slower migrating complex of MCM1-STE12-DNA. A truncated form of STE12, containing the N-terminal 469 amino acids (of 688), fails to form the MCMI- STE12-DNA complex [52]. However, the truncated STE12 does form a complex with Tyl DNA, so its inability to bind to the S T E 2 UAS is not due to a loss of the DNA-binding domain [52]. The inability of the

a-Factor/Receptor STE2

(s-Factor/Receptor STE3)

! CDC36 ~ G Protein: '--I CDC39 GPA1, S~E4, STE18 /

[Sigillsl] J

STES, STET0 STEll, FUS3

! $TE12 FCLN Genesq ~. L= Prodo°"J

Gene Induction

! I l l Mating Competence: Recovery: Morphological and

MFA1 / MFA2 SST2 Nuclear Modifications: (MFal / MFa2) BARI CSHt |

STE2 (STE3) CLN3 FUS1

I GPA1 STE4 STE12

IOther Geneq Products J

I Cell Cycle Arrest: FAR1

D

SE" ! Mating (

Fig. 4. Model of the pheromone response pathway. The pathway is shown for a cells treated with a-factor; the differences in a cells are shown in parentheses. Treatment with pheromone leads to the transcriptional induction of a number of genes necessary for mating, some of which are shown. in a cells, MFAI and MFA2 (Ref. 110; Michaelis, S. and Herskowitz, l., unpublished data), STE2 [70,76[ and BARI [109] are induced by a-factor; in a cells. MFal [125], MFa2 [17] and STE3 [126] are induced by a-factor; and in both celt types, genes such as GPAI [89], STE4 [911, STEI2 (Dolan. J.W. and Fields, S., unpublished data), SST2 [127], CLN3 [106], CHSI [113] and FUSI [79,111] are presumed to be induced by the appropriate pheromone, although in some cases only a-factor induction has been demonstrated. Cell cycle arrest appears to be mediated in part by the transcriptional induction of FAR1, whose gene product inhibits the synthesis or function of CLN2 [128], in part by the action of FUS3, which may antagonize the activity of CLN3 [105], and in addition by the action of another component on CLN1. Other gene products may also be

directly modified by the pathway in a manner ;hat is essential for mating to occur.

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truncated protein to bind to the STE2 UAS suggests that a region of STE12, located in the C-terminal 219 amino acids, is required for cooperative interaction with MCM1 necessary for binding to the STE2 UAS [52].

STE12 acts as a pheromone-responsive transcriptional activator (Song, O., Dolan, J.W., Yuan, Y.O. and Fields, S., unpublished data). A fusion protein consisting of the DNA-binding domain of GAL4 and the complete STE12 coding sequence activates expression of a GALI-lacZ r~porter gene only if the cells are treated with pheromone. This ability to activate via a heterologous DNA-binding domain indicates that pheromone ;.nd,:c'_io~ of transcription can occur through STE12 alone, and does not absolutely require any other protein bound at or adjacent to the PRE. In addition, STE12 is a phosphoprotein and its level of phosphorylation increases following treatment of cells with pheromone. A model of STE12 function is that the phosphorylated form of the protein is a ,~ore potent transcriptional activator than the unphosphorylated form (see Ref. 115).

The overproduction of some components of the pheromone response pathway (e.g., STE4) leads to pheromone response in the absence of pheromone. Overproduction of STE12 using a multi-copy plasmid or a galactose-inducible promoter is able to suppress the mating defect of ste2, ste5, ste7 and stell strains and to cause constitutive transcriptional induction of pheromone-inducible genes [115]. In addition, STEI2 expressed from the galactose-inducible promoter causes some cells to undergo a change in morphology, becom- ing large and misshapen and arresting as unbudded cells. These phenotypes are similar to that seen in cells responding to pheromone, indicating that overproduction of STEI2 is able to mimic certain aspects of the normal response. However, pheromone treatment clearly causes additional responses not due to transcriptional induction. A pathway of pheromone response (Fig. 4) must therefore combine the effects of the induced proteins with those of constitutively present proteins that may be themselves directly affected by pheromone.

V. Yeast cell-type-specific transcription as a model system

Control of cell-type-specific gene expression in S. cerevisiae requires the coordinated interaction of two separate regulatory networks. One network, composed of the products of the M A T locus, uses an activator (al) and two repressors (a2 and al-a2) to establish the basic cell type. Both activation by al and repression by a2 require a ubiquitous and essential factor, MCM1, which also plays a role in the function of the second regulatory network, the pheromone response pathway. In addition to the common use of MCM1, the two regulatory networks are integrated at several other levels.

While a single pathway is responsible for pheromone response in both types of haploid cells, the response pathway works in association with the MAT products to ensure the transcriptional induction of only the appropriate set of ceil-type-specific genes. Mating occurs only in haploids and, consistent with this specialization, the expression of many components of the pheromone response pathway is repressed in the diploid. This repression is mediated, at least in some cases, by the direct action of the al-a2 repressor encoded by the M A T alleles. Mutations in either the MAT locus or the STE genes result in altered expression of ceil-type- specific genes. Therefore, the constitutive and induced levels of ceil-type-specific transcripts result from the combinatorial action of two interconnected networks, each supplying transcription factors with target sites in the cell-type-specific genes.

The regulation of yeast cell-type-specific transcription has numerous parallels with transcriptional control in multicellular organisms. The al and a2 proteins are part of the large group of proteins containing a homeodomain, while MCM1 shares similarity with proteins from widely diverse species. On a functional level, these yeast proteins appear to use the same structural motifs for DNA-binding as do their metazoan counterparts. Additionally, given the considerable conservation in transcriptional mechanisms between yeast and man (see for example Refs. 116 and 117), it is reasonable to expect that insights into the activating or repressing activities of the yeast regulators will prove useful in the study of other systems. For example, the pairwise interactions of the yeast proteins al-a2 and MCMI-a2 suggest that a common property of homeodomain proteins may be interactions among themselves and with non- homeodomain prateins [31]. The role of STE12 has parallels to the regulation of the mammalian proteins jun and los. STE12 binds to its own promoter and is presumably responsible for the increased STEI2 transcription observed in response to pheromone. In this way, a transient external signal may increase both the activity and concentration of the responsive transcription factor, leading to a more prolonged change in cell-type-specific transcription. Similarly, transcription of the jun proto-oncogene is directly stimulated by its own gene product, and TPA treatment leads to a post- translational event that increases the activity of preex- isting Jun/AP-1 molecules [118]. The FUS3 protein kinase has a high degree of similarity to a mammalian protein kinase designated extracellular signal-regulated kinase 1 (ERK1) [1i9]. The ERK1 kinase may be an early intermediate in an insulin-stimulated phosphorylation cascade, suggesting another conservation between the yeast signal-responsive cascade and that of multicellular organisms.

Parallels also exist between yeast pheromone response and mammalian serum response. While SRF

168

binds tt:e serum response elemcrtt (SRE), the protein

appears 1o be constitutively presenI and no SRF moditi-

cations have been correlated with ,.erum induction [120].

Thus, SRF may interact with another protein wh6se

activity is changed by serum treatment; two candidates

are p62. thai binds directly to Ihe 5' side of the SRE [121], and p62 ~tv, that does not bind directly to the

SRE, but binds the S R F-SR E complex [122]. A c,,re

domain of S R F is capable of DNA-bi : :d ing, dimeriza-

tion, and interaction with p62 vcr [46,1231; this core

conta ins the region of homology to M C M I . If M C M I

and S R F conserve not only their D N A - b i n d i n g funct ion

but also their protein-protein contacts , it may be the

case that the proteins bound adjacent to S R F have

similarities to a l , a2 or STEI2. S R F addit ionally re-

sembles M C M I in that it is involved in t ranscr ipt ional

repression as well as activation [124]. Thus, yeast cell-

type-specific t ranscr ipt ion is likely to cont inue to serve as a useful parad igm for transcript ional regulat ion in

larger eukaryotes.

Acknowledgements

Work from our labora tory was suppor ted by gran ts

f rom the Nat ional Science Founda t ion ( D M B 8916410)

and Nat ional Inst i tutes of Health (5-T32-CA09176). We

thank George Sprague and Michael G i l m an for com-

ments on the manuscr ip t .

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Cell-type-specific transcription in yeast