Proc. Nati. Acad. Sci. USAVol. 81, pp. 1144-1148, February 1984Developmental Biology

Molecular cloning of hormone-responsive genes from the yeastSaccharomyces cerevisiae

(transcriptional regulation/pheromone action/poly(A)+ RNA/4-thiouridine/recombinant DNA)

GARY L. STETLER* AND JEREMY THORNERtDepartment of Microbiology and Immunology, University of California, Berkeley, CA 94720

Communicated by Marian E. Koshland, October 28, 1983

ABSTRACT A method for identifying yeast genes whosetranscription is differentially regulated was developed. Thetechnique is based on incorporation of the analog 4-thiouridineinto nascent RNAs, which allows their purification. The puri-fied RNAs are used to prepare cDNA copies for screening ofgenomic DNA libraries by hybridization. Using this proce-dure, several cloned yeast DNA segments were found whosetranscription in MATa haploids in vivo is apparently modulat-ed in dramatic fashion within 10-15 min after exposure to themating pheromone, a factor. Subsequent analysis indicatedthat these sequences fall into three major classes: (i) genes ex-pressed in vegetatively growing cells that are no longer tran-scribed after a-factor administration ("turn-off" genes); (it)genes whose expression is increased 10- to 20-fold after expo-sure of the MATa cells to a factor ("turn-up" genes); and (iii)genes that are expressed only after a-factor treatment ("turn-on" genes). The first class may encode products required forcell cycle progression; the third class may code for productsuniquely involved in the mating process.

One manifestation of peptide hormone action is the regula-tion of gene expression (1, 2). The two haploid cell types (acells and a cells) of baker's yeast (Saccharomyces cerevisi-ae) can be induced to conjugate ("mate") with each other bythe action of oligopeptide pheromones (a factor and a factor,respectively) that resemble the peptide hormones of multi-cellular eukaryotes (3). Among the responses evoked bypheromone administration are: induction of cell surface ag-glutinins, which promote adhesion between cells of the op-posite mating type; inhibition of progression past the G1phase of the cell cycle, which synchronizes the mating part-ners; activation of certain enzymes involved in cell wall bio-synthesis; and, ultimately, conversion of the normally spher-ical cells to a characteristically elongated morphology (forreview, see ref. 4). Although a growing catalogue of suchpheromone-induced changes exists, the molecular mecha-nism(s) whereby the pheromones elicit these effects is notknown nor is it known whether any of these effects reflectschanges in gene expression. Furthermore, yeast mating is agenetically complex process. The mating type locus (MAT)and at least 10 other unlinked (STE) genes appear to be in-volved in the ability of one or the other (or both) haploid celltypes to mate (5, 6). Another dozen or so genes that alsohave some effect on various aspects of the mating processhave been identified (4, 7). Aside from MAT itself, it is cur-rently unknown which, if any, of these genes have a regula-tory role and which are structural genes for products that aredirectly involved in conjugation.As one approach to understanding the biochemical basis

of pheromone action and as an independent means to identi-fy genes involved in the mating process, we devised a meth-

od to directly determine if exposure to a factor rapidly altersthe pattern of expression of specific genes in a haploids, ineither a positive or negative sense. By using this procedure,it was indeed possible to identify and isolate certain a cellgenes that are transcriptionally regulated in dramatic fashionshortly after a-factor treatment. The technique we devel-oped may be generally applicable for the identification andisolation of differentially expressed genes in other orga-nisms.

MATERIALS AND METHODSLabeling and Purification of 4-Thiouridine-Substituted

RNA. Two cultures (1 liter each) of S. cerevisiae strain XS3-6b (MATa ural met2 lys2 his4-580 cryl SUP4-3) were grownat 30'C to an A600 of 1.5-1.65 (about 8 x 107 cells per ml) inSC medium (8) containing 20 ,ug of uridine per ml (in place ofuracil). Cells were collected by centrifugation at 40C for 5min at 5000 x g and washed by resuspension and recentrifu-gation in 100 ml of SD medium (8) lacking both glucose anduridine. The washed cell pellets from the two cultures wereeach resuspended in 1 liter of SC containing 75 FLM 4-thiouri-dine (Sigma) and 0.05 ,uCi (2 nM) of [6-3H]uridine (1 Ci = 3.7GBq; New England Nuclear) per ml. After incubation for 10min at 30'C with shaking, purified natural a factor (9) (gift ofH. Liao) in 0.1 M NaOAc (pH 5) was added to one culture ata final concentration of 0.3-0.4 AM (-50 units/ml); an equalvolume of 0.1 M NaOAc (pH 5) was added to the second(control) culture. After 30 min, the cells were chilled andcollected by centrifugation. Total RNA fractions of the con-trol and a-factor-treated cells were purified immediately byphenol extraction (10). Poly(A)+ RNA was selected by chro-matography on oligo(dT)-cellulose (Collaborative Research,Waltham, MA) (11) and concentrated by ethanol precipita-tion. Poly(A)+ RNA (up to 150 ,ug) was redissolved in 10 mlof 0.1% NaDodSO4/0.15 M NaCl/50 mM NaOAc, pH 5.5,heated to 65°C for 5 min, cooled rapidly in ice, and applied toa bed (1 ml) of phenylmercury agarose (12) (Affi-Gel 501,Bio-Rad) in a column (0.5 x 2 cm) at a flow rate of 5 ml/hr.The column was washed with 10 ml of the same buffer toremove nonspecifically bound RNA. The 4-thiouridine-con-taining RNA was eluted with the same buffer containing 10mM 2-mercaptoethanol, at a flow rate of 5 ml/hr. Fractions(0.5 ml) were collected and the radioactivity present in sam-ples (10 ,1u) of each was determined by liquid scintillationcounting. Fractions containing the bulk of the RNA werepooled and concentrated by ethanol precipitation.

Construction of Yeast Genomic Library. DNA purified (13)from S. cerevisiae strain 381G (MATa ade2-1 his4-580 Iys2trpl tyrl cryl SUP4-3) (6) was partially digested with eitherBamHI or Sau3A restriction endonuclease (New England

Abbreviations: kb, kilobase(s); nt, nucleotide(s).*Present address: Synergen Associates, Inc., 1885 33rd Street,Boulder, CO 80301.tTo whom reprint requests should be addressed.

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The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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BioLabs or Bethesda Research Laboratories) as recom-mended by the suppliers. Fragments in the size range 12-18kilobases (kb) were purified by sucrose gradient centrifuga-tion (14). The fragments were inserted into the bacteriophagevector X1059, using T4 DNA ligase prepared by minor modi-fications of the procedure of Tait et al. (15), and packaged invitro into phage particles, all essentially as described byKam et al. (16).Preparation of Hybridization Probes. 32P-labeled cDNA

was prepared from the 4-thiouridine-containing poly(A)+RNA by using avian myeloblastosis virus reverse transcrip-tase (gift of N. Maizels), deoxyribonucleoside [a-32P]tri-phosphates (Amersham), and random hexanucleotide prim-ers generated from calf thymus DNA (gift of S. Hughes) (17).These probes were used for differential plaque hybridiza-tions conducted by the method of Woo (18).Recombinant X phage were propagated in Escherichia coli

BNN45 (hsdR- hsdM' supE44 supF thi- met-) (19) andphage DNAs were purified as described by others (20).Whole recombinant bacteriophage X DNAs used as probeswere labeled by nick-translation (21).

Electrophoretic Analysis of RNA. Samples of poly(A)+RNA (1-2 jig), purified as above, were denatured and sub-jected to electrophoresis in gels (11 x 12.5 cm) of 1.5% agar-ose, transferred to nitrocellulose paper (Sartorius), hybrid-ized to probes, and examined by autoradiography, all byonly minor modifications of the methods of others (22, 23).

RESULTSDetection of a-Factor Regulation of MATa Cell Gene

Expression. Any method to determine whether a factor has adirect effect on the pattern of gene transcription should: (i)permit detection of both the appearance (or increase) anddisappearance (or decrease) of specific mRNA species; (ii)increase the likelihood that any observed changes representeffects on the rate of transcription of specific genes (ratherthan alterations in RNA levels due to post-transcriptionalevents); and (iii) monitor relatively immediate primary re-sponses to the pheromone (rather than long-term indirect re-sponses due to the ultimate imposition of cell cycle arrest).By isolating the newly synthesized mRNAs made in a cells

shortly after pheromone treatment and those made in un-treated a cells, cDNA probes could be prepared for screen-ing a library of yeast genomic DNA by differential hybridiza-tion. Enrichment for the nascent mRNAs made by phero-mone-treated and control cells was accomplished by theincorporation of 4-thiouridine into these molecules during abrief pulse (30 min, 0.25 of a generation). The presence of 4-thiouridine in nascent RNA permits its purification by chro-matography on phenylmercury agarose. To enhance the effi-ciency of labeling, a pyrimidine auxotroph was used. At theconcentration of 4-thiouridine employed, the rate of totalRNA synthesis was at least 90% of that observed in controlcultures in which the analog was replaced by an equivalentconcentration of uridine (results not shown).

Typically, about 1-2% of the total RNA (absorbance at260 nm) in exponentially growing cells was recovered aspoly(A)+ RNA by chromatography on oligo(dT)-cellulose.As judged by absorbance, only 3-5% of these species wereretained on phenylmercury agarose. However, 65% of theradioactivity in the total poly(A)+ RNA was present in the 4-thiouridine-containing fraction. Hence, the incorporation of4-thiouridine provided at least a 10-fold enrichment for a spe-cific class of transcripts, the newly made mRNAs. The over-all yield of 4-thiouridine-containing poly(A)+ RNA from 1-liter cultures was 3-5 ,g. cDNAs representing the nascentmRNA population from vegetatively growing a cells(cDNAv) and the nascent mRNAs species in a-factor-treat-ed a cells (cDNAp) were used as probes to screen a library of

yeast genomic DNA cloned in a bacteriophage vector,X1059. Two types of differential hybridizations were per-formed. First, duplicate filters were hybridized either withcDNAp or with cDNAv, and the hybridizations patterns werecompared. Plaques displaying clear-cut differences in theirdegree of hybridization to the two different probes werepicked for further testing. Because the average insert size inthe recombinant phages (16.5 kb) was large enough to en-code several genes, an inherent problem with the directscreen was that the presence on a phage of a gene whosetranscription is responsive to a factor might go undetected ifit is surrounded by several genes that are transcriptionallyactive under all conditions. In an attempt to minimize thisdifficulty, a second set of differential plaque hybridizationswas performed in which a 300 to 400-fold excess of unlabeledcompetitor poly(A)+ RNA was added along with the probe.To identify genes expressed only after exposure to a factor,competitor mRNA isolated from vegetatively growing cellswas added along with cDNAp. Conversely, to identify genesno longer transcribed after pheromone treatment, competi-tor mRNA isolated from pheromone-treated cells was addedalong with cDNAv. Again, autoradiograms of the pairs offilters were compared and plaques displaying obvious differ-ences in their degree of hybridization under the two differentconditions were picked for retesting.From a total of >30,000 individual plaques screened by

either of these two methods, we recovered only 6 recombi-nant phages that reproducibly behaved, upon three succes-sive plaque purifications and rescreenings, as if they carriedgenes whose transcription responded to the presence of thepheromone. Five of the phages were unique; the sixth phagecontained an insert very similar or identical to one of theother five and was not studied further. This result suggeststhat, within the limits of detection of our method, a factordoes not cause global changes in a cell gene transcription, atleast within 30 min, but does affect the transcription of ahighly selected set of genes. Nonetheless, because we chosefor further examination only those phages that showed themost pronounced signals and only obtained one duplicateisolate, we consider it likely that not all of the sequenceswhose transcription is affected by a-factor treatment wererecovered.a Factor Is Both a Positive and Negative Regulator of Gene

Expression. The number and size of the transcripts encodedby the five phages, and the magnitude and kinetics of the a-factor-induced changes in their transcription, were deter-mined. Poly(A)+ RNA was extracted from samples of a cul-ture of a cells at various intervals after their exposure to afactor (Fig. 1). The spectrum of complementary RNA mole-cules was revealed by using as probe the entire DNA of eachrecombinant phage. To ensure that the a-factor concentra-tion would remain relatively constant throughout the courseof such experiments, the a haploids used carried an sstl (alsocalled barn) mutation (24-26), which prevents a cells fromproteolytically degrading a factor (9). This analysis con-firmed that all five phages are unique and carry DNA se-quences that are transcribed differentially in vegetativelygrowing a cells and a-factor-treated a cells. Three overallclasses of responses were found (Fig. 1).The insert (10.4 kb) in one phage, XScG2, encodes two

transcripts that are no longer transcribed or are transcribedat very reduced rates, after a cells are exposed to a factor(Fig. 1A). As judged by densitometer tracings of autoradio-grams, the level of the major transcript, about 1500 nucleo-tides (nt), decays with a half-life of 30 min. We have termedthis class of response "turn-off."Two phages (XScG8 and XScG11) each encode three tran-

scripts that are present in vegetative cells; however, one ofthe transcripts on each phage shows a dramatic elevation (10to 20-fold) after pheromone treatment. The steady-state level

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of the a-factor-induced transcript coded for by XScG8 (12.2-kb insert) appeared to increase progressively in abundanceover the course of 60 min after exposure to a factor (Fig.1B). In contrast, the pheromone-induced transcript encodedby XScG11 (11.7-kb insert) reproducibly showed a so-called"burst-attentuation" response-that is, the level of tran-script found at 60 min was always less than that observed ateither 15 or 30 min (Fig. 1C). We have called these types ofresponses "turn-up."The fourth phage, XScG7 (14.0-kb insert), encodes seven

transcripts; however, three of them are present at an ex-tremely low level, virtually undetectable, in vegetativelygrowing cells. Within 15 min after exposure of the cells to afactor, these three transcripts have become nearly as abun-dant as the other transcripts encoded by the insert and theycontinue to accumulate for at least 60 min. The last phage,XScG6 (15.0-kb insert), also apparently codes for seven tran-scripts, ranging in size from 450 to 3,600 nt, the smallest ofwhich behaves very similarly to the a-factor-induced tran-scripts carried by XScG7 (results not shown). We have desig-nated this third type of response "turn-on."None of these effects could be attributed to differential

loading of RNA samples or to differential recoveries ofpoly(A)+ RNA in the preparations made from cells at thedifferent time points. For example, the exact same nitrocel-lulose filter that was used to demonstrate that XScG8 en-codes a turn-up transcript (Fig. 1B) was washed and re-probed with XScG2 and confirmed that it specifies two turn-off transcripts (Fig. 1A). Also, reprobing of all such blotswith other cloned yeast genes, specifically LEU2 (8) andLYS2 (unpublished data), indicated that the level of thesecontrol mRNAs did not vary between samples within any

given experiment by >20-30%. Finally, all but one of thephage inserts contained sequences complementary to one ormore transcripts that did not show any obvious response topheromone addition and thus served as internal standards tonormalize for the uniformity of sample application.To be certain that these responses were not due to some

minor contaminant in our preparation of natural a factor,MATa sstl cells were exposed to various concentrations ofchemically synthesized a factor and the kinetics of appear-ance of the turn-on transcripts specified by XScG7 were ex-amined by gel electrophoresis and blotting. It was clear,first, that the synthetic pheromone induced the appearanceof all three of the a-factor-regulated transcripts. Second, apronounced response was observed even at concentrationsof the pheromone as low as 0.3 nM (Fig. 2). This level ofpheromone is in good agreement with the range of concen-trations reported to be physiologically efficacious, undersimilar conditions, for evoking certain biological responses,such as agglutinin induction (28) and cell cycle arrest (24,25). Third, better resolution of the higher molecular weightspecies revealed that the induction involves only one of thetwo large (about 3600 nt) transcripts encoded by XScG7. An-other of the positively regulated pheromone-responsive tran-scripts, that encoded by XScG6, also was clearly induced at0.3 nM synthetic a factor (results not shown).

In contrast, the negatively regulated pheromone-respon-sive transcripts specified by XScG2 did not show a signifi-cant decrease in their abundance at 30 min unless the cellswere exposed to a concentration of synthetic a factor of 2nM or above (results not shown). Similarly, induction of thepositively regulated transcripts encoded by XScG8 andXScG11 was not apparent below 2 nM a factor. At least one

A

4362 -21171595 --1444 "-676-

C4362 -

211 7-15951307 <676 -

0 15 30 60

4362 -- B

0 15 30 60

4362 -- D

2 11 7 - XO:1444 --E1307 tt,

676

0 15 30 60

676 -

368-15 30 4560

FIG. 1. Time course for transcriptional responses of yeast genes to a-factor mating pheromone. S. cerevisiae RC634 (MATa sstl-3 ade2-1his6 metl ural canl cyh2 rmel GAL) (24) was grown to a cell density of 5 x 107 per ml in SC medium buffered at pH 5 with 20 mM sodiumsuccinate and exposed to a final concentration of a factor of 0.24 AtM; samples of the culture were removed at the times (in min) indicated.Poly(A)+ RNA was isolated, subjected to electrophoresis, and transferred to nitrocellulose. The filter-bound RNA was then hybridized to 32P-

labeled DNA isolated from each of the indicated pheromone-responsive phage clones. Molecular weight markers were a mixture of fragmentsfrom three separate digests (EcoRI, Taq I, and Rsa I) of pBR322 (19) denatured by formaldehyde treatment (22). Probes were: XScG2 (A),XScG8 (B), XScG11 (C), and, XScG7 (D).

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FIG. 2. Concentration dependence for transcriptional effects ofsynthetic a factor. S. cerevisiae RC634 (MATa sstl-3) was cultivat-ed and treated with synthetic a factor (Serva) at the concentrationsindicated, and, after 30 min, RNA was isolated and analyzed by hy-bridization to the XScG7 probe, all as described in the legend to Fig.1, except that vanadyl ribonucleoside complexes (10-20 mM) werepresent during washing and lysis of the cells as an additional precau-tion against ribonuclease action (27). Lane 1, no a factor added; lane2, 0.06 nM a factor; lane 3, 0.3 nM a factor; and lane 4, 2 nM afactor.

biological response elicited from a cells, morphological elon-gation, is known to require levels of the pheromone in thisrange (3).There exist a number of temperature-conditional muta-

tions that cause yeast cells to arrest their cell division cycleapparently at the same stage as that brought about by expo-sure to mating pheromone (29, 30). One such lesion is thecdc28 mutation (29, 31). None of the transcripts describedhere exhibited altered expression due to the imposition ofcell cycle arrest per se because the abundance of these spe-cies was unchanged in poly(A)+ RNA isolated from MATacdc28 mutants shifted to the restrictive temperature for 60min (results not shown).

DISCUSSIONWe have identified five genomic clones of S. cerevisiae DNAthat appear to encode a variety of polyadenylated RNAswhose intracellular concentrations change during the re-sponse of a cells to a factor. The screening procedure usedto isolate these clones was dependent on differences in theamounts of newly synthesized mRNA rather than on differ-ences in the steady-state level of total mRNA. Althoughpost-transcriptional mechanisms may play a role in control-ling the levels of these pheromone-regulated transcripts, weare unaware of effects of this magnitude and rapidity that areattributable solely to changes in RNA stability. For thesereasons, we consider it very likely that the rate of transcrip-tion of these genes is controlled by the pheromone.Because the tactic of labeling nascent RNA with 4-thiouri-

dine could be applied under a variety of conditions imposedby an investigator, the method we have described should begenerally useful for identifying and isolating genes that aretranscribed differentially under other circumstances-for ex-ample, shifts of temperature-conditional mutants from onetemperature to another, transfer of cells from one carbonsource to another, or after heat shock of normal cells.One of the more remarkable aspects of our findings is the

variety of apparent transcriptional responses to the phero-mone. These effects appear to define pheromone-responsivegenes of three types: (i) genes not expressed, or expressed atvery low levels, in vegetatively growing cells that are rapidlyinduced after exposure to a factor; (ii) genes expressed in the

absence of the pheromone that show a greatly elevated levelof expression after pheromone treatment; and (iii) genes thatare no longer expressed after pheromone administration.The products of the genes in the first category are likely to befunctions specifically involved in the mating process-forexample, agglutinins or products of the various STE genes.The products of the genes in the second category may not

encode mating-specific functions because they are expressedto a certain extent in the absence of pheromone and are ex-pressed equivalently in both haploid cell types and in dip-loids (unpublished results). Therefore, these genes may codefor functions that have some role in normal vegetativegrowth but are required at elevated levels in conjugatingcells. An example of such functions might be cell wall bio-synthetic enzymes, like chitin synthase and glucan synthase,which have been reported to be induced after pheromonetreatment (ref. 32; S. Zinker, personal communication).Because one response ultimately elicited by pheromone

treatment is arrest of the cell division cycle, the products ofthe genes in the turn-off class might be functions that controlthe decision to enter a new cell cycle. Conversely, it is possi-ble that the observed changes in these mRNAs reflect theonset of G1 arrest. In this regard, however, based on theirlength, the transcripts encoded by XScG2 are unlikely to bethe mRNAs for histones H2A and H2B (33), HO endonucle-ase (23, 34), or CDC9 (L. Prakash and S. Reed, personalcommunication), all of which appear to be present at greatlyreduced levels in cells arrested in the G1 phase of the cellcycle.Because it can be considered well established that a factor

is a truly pleiotropic effector molecule at the cellular level (3,4), it was reassuring to see the diversity of its evoked re-sponses reflected at the level of mRNA metabolism. Themechanism by which a single kind of effector generates posi-tive, negative, and apparently transient transcriptional re-sponses in a single target cell is not yet known. It is possiblethat there are several discrete classes of receptors for a fac-tor, each of which generates a different gene-specific type ofinternal signal. This seems unlikely because mutations in asingle gene, STE2, abolish all known biological responses tothe pheromone (6) and also eliminate detectable binding ofradioactive a factor by a cells (35). An alternative explana-tion for the multiplicity of effects elicited by a factor is that itgenerates a single internal signal that interacts with intracel-lular regulatory molecules to affect the expression of specificgenes differently. Evidence from both in vitro and in vivostudies (36, 37) suggests that the signal generated upon theinteraction of a factor with its a cell receptor may be a reduc-tion in the intracellular concentration of cAMP. This pertur-bation in cAMP level could initiate a cascade of events lead-ing to the transcriptional induction of some genes and therepression of others. In fact, addition of cAMP itself hasbeen shown to elicit both positive and negative transcription-al responses in another fungal system-namely, during thedevelopmental program of Dictyostelium discoideum (38,39). Also, it has been shown recently that the transcriptionaleffects of those peptide hormones and growth factors thatmodulate cAMP levels in pituitary cells are correlated withthe degree of phosphorylation of a chromatin-associated ba-sic protein (2).

We appreciate greatly the technical assistance of Wayne Snowdonand Leo Levinson. This work was supported by an American Can-cer Society Postdoctoral Fellowship (PF-1882) to G.L.S. and by anaward from the Cancer Research Coordinating Committee of theUniversity of California and a Research Grant (GM21841) from theNational Institutes of Health to J.T.

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