MOLECULAR AND CELLULAR BIOLOGY, May 1992, p. 1977-19850270-7306/92/051977-09$02.00/0

A G-Protein a Subunit from Asexual Candida albicans Functions inthe Mating Signal Transduction Pathway of Saccharomyces

cerevisiae and Is Regulated by the al-oc2 RepressorCHANCHAL SADHU,t DENISE HOEKSTRA, MICHAEL J. McEACHERN,4 STEVEN I. REED,

AND JAMES B. HICKSt*Department ofMolecular Biology, Research Institute of Scripps Clinic, La Jolla, California 92037

Received 25 September 1991/Accepted 4 February 1992

We have isolated a gene, designated CAGI, from Candida albicans by using the G-protein a-subunit cloneSCG1 of Saccharomyces cerevisiae as a probe. Amino acid sequence comparison revealed that CAG1 is morehomologous to SCG1 than to any other G protein reported so far. Homology between CAG1 and SCG1 not onlyincludes the conserved guanine nucleotide binding domains but also spans the normally variable regions whichare thought to be involved in interaction with the components of the specific signal transduction pathway.Furthermore, CAG1 contains a central domain, previously found only in SCG1. cagi null mutants of C.albicans created by gene disruption produced no readily detectable phenotype. The C. albicans CAGJ gene

complemented both the growth and mating defects of S. cerevisiae scgl null mutants when carried on either a

low- or high-copy-number plasmid. In diploid C. albicans, the CAG1 transcript was readily detectable inmycelial and yeast cells of both the white and opaque forms. However, the CAG1-specific transcript in S.cerevisiae transformants containing the C. albicans CAGi gene was observed only in haploid cells. Thistranscription pattern matches that of SCGI in S. cerevisiae and is caused by al-a2-mediated repression in

diploid cells. That is, CAGi behaves as a haploid-specific gene in S. cerevisiae, subject to control by the al-ac2mating-type regulation pathway. We infer from these results that C. albicans may have a signal transductionsystem analogous to that controlling mating type in S. cerevisiae or possibly even a sexual pathway that has so

far remained undetected.

Heterotrimeric guanine nucleotide-binding proteins (Gproteins) are important components of signal transductionsystems in eucaryotes (4). In conjunction with the serpentinetransmembrane receptors, they transduce a wide variety ofextracellular signals to the appropriate effectors within thecell. The specificity of G-protein action resides in the a

subunit, which interacts directly with the intracellular sur-face of the receptor and which contains the guanine nucleo-tide binding site. At rest, the ao subunit contains bound GDPand is complexed with the and y subunits. Upon binding ofagonist with the receptor, Ga becomes activated, exchang-ing GDP for GTP and dissociating from the 13y complex.Activation of the G-protein complex results in subsequentactivation of the target effector molecule, which carries thesignal to control sites elsewhere in the cell (12, 53).

In mammals, a large family of Ga sequences are known,while in the yeast Saccharomyces cerevisiae, two Ga genes,SCGJ (also known as GPAI) (9, 21, 35) and GPA2 (37), havebeen reported. SCGI (9), STE4, and STE18 (54) encode thea, 1B, and -y subunits, respectively of a G-protein complexthat is required for response to mating pheromones. SCGIpresumably interacts with the pheromone receptors, STE2and STE3. The inferred amino acid sequences of thesereceptors indicate that they are transmembrane proteins,each with seven membrane-spanning domains typical of thereceptors associated with G proteins in vertebrates (6, 16,

* Corresponding author.t Present address: ICOS Corporation, 22021 20th Avenue S.E.,

Bothell, WA 98021.: Present address: Department of Microbiology and Immunology,

University of California at San Francisco, San Francisco, CA 94143.

39). The role of GPA2 is not known, but it does notcomplement the loss of SCG1 for mating response (37).No sexual cycle similar to that of S. cerevisiae has been

observed in the opportunistic human pathogen Candidaalbicans. A number of C. albicans strains show variations ofcolony morphology and cell types (49, 50). Notable amongthem are the white and opaque forms of strain WO-1. Theseforms are so named because of their colony appearance.Cells of the white forms are smaller and elliptical, while theopaque cells are larger and bean shaped. Opaque forms canbe maintained as opaque at 25°C, and they can be induced togive rise to white forms by growth at 37°C. There is also an

example of a developmental switch in C. albicans betweenvegetative growth as budding (yeast) and hyphal (mycelial)forms. This switch, often referred to as germ tube formation,can be stimulated in vitro by a number of different experi-mental regimens, including changes in temperature and pH,but the most consistent effect is in response to serum factors(41). Thus, although no component has so far been charac-terized at the molecular level, it is likely that signal-sensingand transduction mechanisms are present in C. albicans. Areceptor for the adhesive glycoprotein laminin has recentlybeen detected on the surface of C. albicans hyphae (3). Inaddition, a corticosteroid-binding protein (28, 48) and pro-teins binding specifically to the mammalian C3bi factor (13)and C3d (43) have been found.No connection has yet been made between the binding

proteins and potential receptors described above and spe-cific behavior of C. albicans. We have therefore begun toexamine the sensing apparatus of C. albicans, starting at theconserved, G-protein signal transduction mechanism. In thisreport, we describe the cloning and initial characterization ofa Ga-protein gene, CAGI, from C. albicans. The primary

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TABLE 1. S. cerevisiae strains used

Strain Genotypea

IWD.. MAAITa/AM Ta [trpl leu2 his3 ura3-52 mal scgl::URA3]CSl.. AIATa, pYEp2l3-CAGI; haploid segregant of IVYDCS4.. Same as CS1CS9.. MA4Tot, pYEp2l3-CAGI; haploid segregant of IVYDCS10.. Same as CS9CS7.. MATa, pYEpl3-SCGI; haploid segregant of IVYDCS8.. Same as CS7CS11... MATa, pYEpl3-SCG1; haploid segregant of IVYDCS12.. Same as CSl1Dlll.. MAATaMAL4Tot SCGI/scgl::LEU2 [ade2-11 his3-11, 15

leu2-3,112 trpl-1 ura3-1 canl-100]CS13.. MA4Ta scgl::LEU2, pYCp5O-CAGI; haploid segregant

of DlllCS14.. Same as CS13CS15.. MATot scgl ::LEU2, pYCp5O-CAGI; haploid segregant

of DlllCS16.. Same as CS15CS26.. MA4Ta SCG1; haploid segregant of DlllCS27.. MATa SCG1; haploid segregant of Dlll

a Markers within brackets are homozygous.

sequence of this gene shows a close relationship to theSaccharomyces SCG1 gene, including a segment of theSaccharomyces gene not found in the mammalian counter-parts. The C. albicans CAG1 gene can provide nearlycomplete mating function to haploid Saccharomyces strainslacking the SCGI gene. In addition, measurement of tran-script levels has revealed not only that the C. albicans CAG1gene is transcribed efficiently from its own promoter in S.cerevisiae transformants but that it is subject to the sameregulation as is SCGJ.

MATERUILS AND METHODS

Strains, media, and plasmids. Genotypes of the S. cerevi-siae strains are listed in Table 1; the Candida strains used arelisted in Table 2. S. cerevisiae strains harboring plasmidswere cultured in appropriate selective medium. All othercultures were routinely grown in YPD medium. Yeast ge-netic manipulations were performed as described previously(46). Plasmids YEp13 and YEp213 are Escherichia coli-S.cerevisiae shuttle vectors containing the 2,um origin ofreplication and the LEU2 gene as a selective marker. YCp5Ois also an E. coli-S. cerevisiae shuttle vector and containsyeast autonomously replicating and centromeric sequencesand the URA3 gene (46). For the construction of plasmidYEp213-CAG1, Sall linkers was attached to the 1.9-kbp

TABLE 2. Candida strains used

Strain Species Strain no. orreference

3153a C. albicans ATCC 28367WO-1 C. albicans 5032032 C. albicans ATCC 3203232033 C. albicans ATCC 3203332077 C. stellatoidea ATCC 3207718814 C. claussenii ATCC 18814B4201 C. albicans 25B4365 C. stellatoidea ATCC 20408B4252 C. stellatoidea ATCC 11006CK C. krusei ATCC 34135CG C. glabrata ATCC 34138

0 500

Hindll Hpal

1000 1500

HIndlil BamHI| Clal

S S 56 &N4

2000

Hpal

16 6 I

-p --4 p -

Sau3A Rsal

FIG. 1. Partial restriction map and sequencing scheme of theCAGI clone. DNA sequencing was performed by using a combina-tion of restriction fragment subclones and synthetic oligonucleo-tides. Numbers indicate distances in base pairs.

HpaI fragment of the CAGI gene and inserted into the Sallsite of plasmid YEp213. YCp5O-CAG1 was constructed bysubcloning the 5.1-kbp EcoRI fragment in the EcoRI site ofplasmid YCp5O. Plasmid YEp13-SCG1 carries a 5.5-kbpSau3AI fragment of S. cerevisiae genomic DNA containingthe SCGI gene in the BamHI site of plasmid YEp13.

Isolation of the CAGI clone. In an effort to isolate a gene fora Ga protein from C. albicans, we screened a library ofEcoRI fragments from strain WO-1 in the A phage vectorEMBIA, using SCG1 as a probe. Hybridization was carriedout at 60°C in 0.5 M sodium phosphate buffer. Recombinantphage DNA was prepared from seven purified positiveplaques and digested with EcoRI. All seven contained a5.1-kbp fragment which yielded a strong signal when blottedand probed with SCG1. The cloned EcoRI fragment wastransferred to the Bluescript plasmid vector (StratageneInc., La Jolla, Calif.) and digested with additional restrictionenzymes, yielding the partial map shown in Fig. 1. Welocated the CAG1 sequence on the plasmid by hybridizingthe SCGI probe to blots of the gels used to map restrictionsites. The sequence of the CAGI clone was determined bythe dideoxy-chain termination method (42), using a combi-nation of subclones and synthetic oligonucleotide primers.All other recombinant DNA procedures were carried outaccording to the published protocols (30).RNA isolation. C. albicans cells were grown in YPD or in

Lee's medium (27) and harvested at different stages ofgrowth. Cells were washed in water and resuspended inRNA isolation buffer (0.1 M NaCl, 0.1 M Tris [pH 8.0], 0.05M EDTA, 1.0% sodium dodecyl sulfate [SDS]). The cellsuspension was vortexed thoroughly after the addition ofglass beads (1.5 g/ml) and an equal volume of phenol-chloroform (1:1, vol/vol). After centrifugation, the aqueousphase was collected and the organic phase was reextractedwith RNA isolation buffer. The aqueous phases were pooledand extracted repeatedly with phenol-chloroform (1:1) untilthere was no interphase. The aqueous phase was made to 2M LiCl, and RNA was precipitated overnight at 4°C (1). TheRNA precipitates were centrifuged at 10,000 rpm in a SorvallHB4 rotor, washed in 2 M LiCl, and dissolved in water.RNA was reprecipitated by the addition of 2 volumes ofethanol in the presence of 0.3 M sodium acetate (pH 5.2) at-20°C. The precipitates were washed with 70% (vol/vol)ethanol in water and dissolved in water. Concentrations ofRNA solutions were determined by measuring A260.Northern (RNA) blot analysis. Aliquots of the RNA sam-

ples were denatured by heating at 65°C in a mixture of 6%formaldehyde, 30% formamide, 1 x morpholinepropane-sulfonic acid (MOPS) buffer, and 5% glycerol containing0.04% bromophenol blue and xylene cyanol (30). The sam-

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ples were electrophoresed on 1.2% agarose gel containing6% formaldehyde in lx MOPS buffer. Electrophoresis wasconducted at room temperature in lx MOPS buffer. Afterelectrophoresis, RNA was blotted overnight on a Gene-screen (NEN Research Products) membrane by capillarytransfer using lOx SSC (lx SSC is 0.15 M NaCl plus 0.015M sodium citrate). RNA was UV cross-linked to the mem-brane (23) and prehybridized in 7% SDS-0.5 M Na2HPO4(pH 7.2) for 5 min at 60°C (8). Hybridization was in the samesolution with a 32P-labelled probe prepared by randompriming (11). After hybridization, the membrane was washedtwice in 5% SDS-50 mM Na2HPO4 at 60°C for 1 h each timeand autoradiographed.

Quantitation of mating efficiency. Quantitative determina-tion of mating efficiency was carried out as described previ-ously (38). S. cerevisiae cells (106) were mixed with thestandard tester (ilv) a or a cells (107) and collected on a GF/Cmembrane. The membrane was then incubated on a YPD-agar plate at 30°C for 2 h. Subsequently, the cells wereresuspended by dipping the membrane in 10 ml of minimalmedium (SD) and vortexing vigorously. Samples were platedon SD-agar plates to determine the number of prototrophsand on ILV dropout plates to determine the number of cellswhose efficiency was being measured. The data were nor-malized by assuming the efficiency of mating of the cellscarrying only the chromosomal SCGI gene to be 1.

Disruption of the CAGI gene. The CAG1 gene was dis-rupted by inserting an approximately 2.5-kbp BglII-ClaIfragment of the C. albicans ADE2 gene from pMC2 (24) intothe BamHI (position 859) and ClaI (position 1038) sites of theCAG] gene. An ade2 strain of WO-1, WO-1 3/6 (J. Schmidt,University of Iowa), was then transformed, with the result-ingADE2-interrupted CAG1 gene excised as a SalI-linkeredHpaI fragment. Sixteen of the resulting transformants werethen screened by transverse alternating-field electrophoresis(TAFE) analysis for the presence of the ADE2 gene on oneof the two chromosomes carrying the CAG] gene. Fifteen ofthe sixteen clones were found to contain the ADE2 gene inone or the other chromosome carrying the CAGI gene.Southern blotting analysis of EcoRI-digested genomic DNAfrom several of these clones confirmed that one of the twocopies of CAG] was indeed interrupted.The interruption was rendered homozygous by subjecting

one of the heterozygous clones to a brief pulse ofUV light toinduce mitotic recombination. Approximately 1,000 freshlyplated cells were exposed for 5 s to a single 15-W GeneralElectric G15T8 germicidal light bulb at a distance of 30 cm,resulting in approximately 10% cell killing. Approximately1% of the resulting colonies contained one or more redsectors. These red sectors contain ade cells formed via themitotic recombinational loss of the single functional ADE2gene. Since mitotic recombination is expected to be areciprocal event, the white parts of these sectored coloniesshould contain cells that have two ADE2-disrupted CAG1genes. Ten such isolates were examined by Southern blot-ting of EcoRI-restricted DNA and by Southern blotting ofTAFE-separated chromosomes, and seven were found to behomozygous for the disruption.

Pulsed field gel electrophoresis. TAFE was carried out on aBeckman (Palo Alto, Calif.) Geneline apparatus, using theTris acetate buffer recommended by Beckman on 1.0%agarose. Electrophoresis was done in six stages as follows:100 V for 12 h with a 2-min switch time; 100 V for 36 h witha 4-min switch time; 100 V for 24 h with a 7-min switch time;80 V for 24 h with an 11-min switch time; 80 V for 24 h with

a 14-min switch time; and 80 V for 12 h with an 18-min switchtime.

Nucleotide sequence accession number. The nucleotidesequence data reported in this paper has been deposited inthe EMBL, GenBank, and DDBJ data bases under theaccession number M88113.

RESULTS

Identification of a heteromeric G-protein gene in C. albi-cans. When Southern blots containing EcoRI or HindIII-digested C. albicans genomic DNA from two differentstrains, WO-1 and 3153a, were probed with 32P-labelledSCG1, distinct bands were observed at both the reduced andnormal stringency of hybridization, indicating the presenceof a close homolog. In strain WO-1, the probe detected asingle EcoRI fragment of 5.1 kbp, while in 3153a, twofragments of 1.8 and 3.2 kbp appeared. The HindIll diges-tion patterns for the two strains were identical, showingfragments of 1.0 and 1.4 kbp (data not shown). We clonedthe 5.1-kbp EcoRI restriction fragment identified by thisprobe from strain WO-1 as described in Materials andMethods. As the nucleotide sequence was accumulated andtranslated, it was clear that the gene encoded a protein verysimilar to SCG1, and the primary amino acid sequences fellinto nearly perfect register over long stretches of the se-quence. The resulting sequence, containing a continuousreading frame of 429 amino acids, along with 514 bp of 5'flanking sequence and 80 bp at the 3' end, is presented in Fig.2.

Structural comparison of CAG1 and SCG1. Figure 3 showsan alignment of the inferred amino acid sequences for CAG],SCG1, and the gene for a human Got subunit (32). Gaxs waschosen for contrast because, of the published Ga sequences,it is the least similar to SCG1. The regions of conservationamong all three genes in the figure are thus the most likelyconserved in the GaL family. It is apparent that CAG1contains stretches of sequences along its entire length (la-beled Gl through G5) that have been postulated to beinvolved in GTP binding (Fig. 3). Overall, the primary aminoacid sequence of CAG1 is 47% homologous to that of Gas(including the conservative substitutions). Homology is par-ticularly pronounced around the regions implicated in GTPbinding (5, 17). Similar regions were also found to beconserved between CAG1 and the S. cerevisiae G proteinSCGL. However, sequence similarity between CAG1 andSCG1 extends beyond these regions. CAG1 exhibits 65%amino acid sequence identity (77% counting conservativereplacements) to SCGL.The sequence conservation between CAG1 and SCG1 is

most striking in regions that are least conserved among thenonyeast members of the family. These regions are boxed inFig. 3 and include the amino-terminal 33 residues, thecarboxy-terminal 61 residues, and part of a central segmentfound only in SCG1 and CAG1. The amino terminus of theheteromeric G-protein a subunit is thought to be necessaryfor binding to the f subunit and is also one of the morevariable regions (53). Between CAG1 and SCG1, the amino-terminal 40 residues are 60% conserved, compared with 27%for the next-best candidate. No other Ga subunit, includingthe product of the Saccharomyces GPA2 gene (37), showsmore than 23% conservation in this segment. In the pro-posed receptor binding region at the carboxy terminus (19,20, 31, 53), 46 of 53 residues are conserved (39 of 53 areidentical) between SCG1 and CAG1. None of the nonyeast

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-514 AAGCTTCATAGATTTATCAAAGAACAGGATGACGAGCATATGGAACAGAAACA

-461 TTTAGACATTTCGTTACCAACTTCATCCTCCTCAAATGCATATTCACTACCCAGCTCCATACCCCAGTACA

-390 CATTTACACAATCTTCAAGACCACAATTTGTCACCAAATTCAATAATACAAGACTAGGAAAAATTTACATA

-319 TTAAACAAGAGACGACTCTTTACATTTATAAAGAGATACAGTTTAGTCAGTTTTTGCAAAAAAGTTTGAT-248 GGTGGGTATGCTGGTGGCCAGCATTTACGTACGAGGATTGGGTTAACTTGTATTGAGAGTAGACCATATTT

-177 TTTTTTTTGATGTGATTTTTAACATGGCTGCGGGAGTAAGCAGAAGGAAACGTTGATGTTTCAGATTTCAC

-106 CACAAAGTGTAGAGAAGAAAAAAAGGAAAGATATTTTGGGGTTTTTTCTTAATGTACATTAAAATCTGTCT

-35 TTTAGTTTACCTTTTTTTAATACCAGTATTCAATC ATG GGT TGT GGC GCT AGT GTT CCG GTTMET Gly Cys Gly Ala Ser Val Pro Val

GAT GAT GAT GAA ATT GAT CCA TTT CTT CAA GAT AAA CGT ATA AAT GAT GCT ATT

Asp Asp Asp Glu Ile Asp Pro Phe Leu Gln Asp Lys Arg Ile Asn Asp Ala Ile

GAA CAA AGT TTA CAA TTG CGT CAA CAA AAC TCG AAA AAG GGA GTC AAG TTG TTGGlu Gln Ser Leu Gln Leu Arg Gln Gln Asn Ser Lys Lys Gly Val Lys Leu Leu

TTG TTG GGT GCT GGT GAA AGT GGT AAA TCA ACA GTT TTA AAA CAA TTG AAA TTA

Leu Leu Gly Ala Gly Glu Ser Gly Lys Ser Thr Val Leu Lys Gln Leu Lys Leu

TTA CAT AAA GGT GGG TTT ACC CAA CAG GAG AGA AGA CAA TAT TCT CAT GTC ATT

Leu His Lys Gly Gly Phe Thr Gln Gln Glu Arg Arg Gln Tyr Ser His Val Ile

TGG TGT GAC GTT ATT CAA TCA ATG AAA GTT TTA ATC ATT CAA GCA AGA AAG TTGTrp Cys Asp Val Ile Gln Ser Met Lys Val Leu Ile Ile Gln Ala Arg Lys Leu

AAA ATC AAA TTA GAT TGT GAT CAG CCT AAT AAT TCA TTA ATT CCT TAT AAG CAG

Lys Ile Lys Leu Asp Cys Asp Gln Pro Asn Asn Ser Leu Ile Pro Tyr Lys Gln

ATT ATA TTA CGA AGC GAT CCT TTA AAA CAA ATA GAT GCT AGT GTT GCT GGT GGTIle Ile Leu Arg Ser Asp Pro Leu Lys Gln Ile Asp Ala Ser Val Ala Gly Gly

ACA GAT TTC CTA AAT GAT TTT GTT GTC AAG TAT AGT GAA GAA AAC AAG AAC AAGThr Asp Phe Leu Asn Asp Phe Val Val Lys Tyr Ser Glu Glu Asn Lys Asn Lys

AGA CGG TTG AAG AGT ACT GGG ACA ACA GAT ATA TGG GGT AAA GAT GAC GAT TCCArg Arg Leu Lys Ser Thr Gly Thr Thr Asp Ile Trp Gly Lys Asp Asp Asp Ser

AAT ATC AAT TCA GAT GCA ATT AAT CAA GCT TTG GAA CTG TCT TTG AAT AAA GATAsn Ile Asn Ser Asp Ala Ile Asn Gln Ala Leu Glu Leu Ser Leu Asn Lys Asp

TCT GAA CAG TTT ACT CGT CTT TCC ATA GCT GAA GCA ATC CAT AAA TTA TGG AAGSer Glu Gln Phe Thr Arg Leu Ser Ile Ala Glu Ala Ile His Lys Leu Trp Lys

TTG GAC TCG GGT ATT AAA AAG TGT TTT GAC AGG TCA AAT GAG TTC CAA TTG GAALeu Asp Ser Gly Ile Lys Lys Cys Phe Asp Arg Ser Asn Glu Phe Gln Leu Glu

GGT AGT GCT GAT TAT TAT TTC GAT AAT GTC GTC AAC TTT GCT GAT ACA AAT TAT

Gly Ser Ala Asp Tyr Tyr Phe Asp Asn Val Val Asn Phe Ala Asp Thr Asn Tyr

730 TTA TCT ACT GAT TTG GAT ATT TTA AAA GGG AGA ATT AAG ACT ACT GGT ATC ACTLeu Ser Thr Asp Leu Asp Ile Leu Lys Gly Arg Ile Lys Thr Thr Gly Ile Thr

784 GAG ACA GAT TTT TTA ATT AAA TCG TTT CAA TTT AAA GTG TTA GAT GCT GGT GGAGlu Thr Asp Phe Leu Ile Lys Ser Phe Gln Phe Lys Val Leu Asp Ala Gly Gly

838 CAA CGG TCA GTA CGT AAA AAA TGG ATC CAT TGT TTT GAA GAC ATC ACT GCT GTTGln Arg Ser Val Arg Lys Lys Trp Ile His Cys Phe Glu Asp Ile Thr Ala Val

892 TTA TTT GTT TTG GCT ATC TCT GAA TAC GAT CAA AAC CTA TTT GAA GAT GAA CGGLeu Phe Val Leu Ala Ile Ser Glu Tyr Asp Gln Asn Leu Phe Glu Asp Glu Arg

946 GTA AAT AGA ATG CAT GAG TCT ATT GTC TTG TTT GAT TCA TTG TGC AAC TCC AAAVal Asn Arg Met His Glu Ser Ile Val Leu Phe Asp Ser Leu Cys Asn Ser Lys

1000 TGG TTT GCA AAC ACC CCA TTC ATA TTA TTT TTG AAC AAA ATC GAT ATT TTC GAATrp Phe Ala Asn Thr Pro Phe Ile Leu Phe Leu Asn Lys Ile Asp Ile Phe Glu

1054 AAC ARO ATC AAA AAG AAT CCG CTA AAG AAT TAT TTC CCA GAC TAT GAT GGC AAAAsn Lys Ile Lys Lys Asn Pro Leu Lys Asn Tyr Phe Pro Asp Tyr Asp Gly Lys

1108 CCA GAC GAT ACT AAT GAA GCA ATC AAG TTT TTT GAG ACA AAT TTT TTG AAA ATAPro Asp Asp Thr Asn Glu Ala Ile Lys Phe Phe Glu Thr Asn Phe Leu Lys Ile

1162 AAT CAA ACC AAT AAA CCT ATC TAT GTT CAT CGA ACG TGT GCT ACA GAT TCA AAAAsn Gln Thr Asn Lys Pro Ile Tyr Val His Arg Thr Cys Ala Thr Asp Ser Lys

1216 TCA ATG AAA TTT GTC TTG AGT GCT GTT ACC GAC ATG ATT GTA CAA CAA AAC TTGSer Met Lys Phe Val Leu Ser Ala Val Thr Asp Met Ile Val Gln Gln Asn Leu

12871270 AAA AAG AGT GGT ATT ATG TAG TTGCAAGAAATAGGCGATATCTTTTTTACTTTACTATTAATGT

Lys Lys Ser Gly Ile Met ---

1334 TCAGTTTAARATTTTTTGAGTTTATATCTATTT 1366

FIG. 2. DNA and deduced amino acid sequences of the CAGIclone. The putative promoter element (-169 to -152) and thetranslational stop codon (1287 to 1289) are underlined with solid andbroken lines, respectively.

gene products show greater than 50% conservation in thisregion.Both CAGI and SCGI encode unusually large Ga subunits

of 429 and 472 amino acids, respectively, compared with a

range of 325 to 375 amino acids in nonyeast genes. It hasbeen shown that for SCG1, nearly all of the size differenceresides in a 109-amino-acid segment between residues 126and 235 that is not found in any of the nonyeast homologs (9,35). Likewise, CAG1 exhibits a 73-amino-acid insert in theidentical location that shows significant amino acid sequencesimilarity (60%) to the SCG1 insert over half of its length(residues 125 to 173). The other Ga protein from S. cerevi-siae, GPA2, also contains an unusual sequence of 83 amino

CAGI 1 HMGC GA SVPVDDDEIDPFLQDKRINDAIEQSLQLRQQNSKKsOG1 1 MGCTVSTQTIGDESDPFLQNKRANDVIBQSLQL EKQRDKNGms 1 NGCLGNSKT- EDQRNEEKAQREANKKIEKQLQKDKQVYRA

G 1CAM 41 GV 1 L L L L GAGES G K ST V L K Q L K L L H KG G F TQ Q - ERR Q YS HSOG1 1 EIKLLLLGAGESGKSTVLKQLKLLHQGGFSHQ-ERLQYAQG=s 40 THRLLLLGAGESGXSTIVKQMRILHVNGFNGDSEKATKVQ

XOOOOGXGXXGKS

CAM1 83 VIWCDVIQSMKVLIIQARKLKIKLDCDQPNNSLIP- -YKQSO31 83 VIRADAIQSMKILIIQARXLGIQLDCDDPINNKDLFACKRGns 83 DIKNNLKEAIETIVAAMSNLVPPVELANPENQFRVDY ILS

CAG1 118 IILRSDPLKQIDASVAGGTDFLNDFVVKYSEENKNKRRLKSOG1 120 |ILLKAKALDYINASVAGGSDFLNDYVLKYSERYETRRRVQGas 120 VMNVPDF - - - - - - - - - - -

C4G1 158 STGTTDIWGKDDDSSNIINSDAINQAIELSLNKDSEQFTRLSSCG1 160 STGRAKAAF- DEDGNI SNVKSDTDRDAETVTQNEDVDRNNGQS ----------------------------------------

CAG1 -___---___--___--___--

SOG1 199 SSRINLQDICKDLNQEGDDQMFVRKTSKEIQGQNRRNLIHGaS 1Z7 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - D F P

C4C1 198 -- IAEAIHKLWKLDSGIKKCFDRSNEFQLBGSADYYFDNVS9G1 239 EDIAKAIRQLWNNDKGIKQCFARSNEFQLEGSAAYYFDNIGas 130 PEFYEHAKALWE- DEGVRACYERSNEYQLIDCAQYFLDKI

G2 G3C4G1 236 VNF AD TNY L ST DL D I L KG RI K T T G I T B T D F L I K S F QF K V L50G1 279 EKFASPNYVCTDEDILKGRIKTTGITETEFNIGSSKFKVLGOs 169 DVI K Q A DY VP SD Q DL L R CR V L T S G IF ET K F Q V D K V N F H M F

D -(X )n- T OJOOG 3

C0G1 276 DAGGQ RSVRIKWIHCFEDITAVLPVLAISEYDQNLFEDERSO1 319 DAGGQRSERKXWIHCFBGITAVLFVLAMSEYDQMLPEDERGas 209 DVGGQRDERRKWIQCFNDVTAI IFVVASSSYNMVIREDNQ

DXAGJXG 4

CA1 316 VNRMHESIVLFDSLCHSWWFANTPFILFLNKIDIPENKI -SOG1 359 VN RH H ES IM L F D T L L N SKW F K D T P F IL FL N KID L FE EK V -Gs 249 TNRLQEALNLFKSIWNNRWLRT ISVILFLNKQDLLAEKVL

OOOOHKXD

C4C1 355 -KKNPLKN|YFPDY DGKPDDTNEA - - - - - - - - - - - - - - -IS0G1 398 -KSMPIRKYFPDYQGRVGDAEAG - - - - - - - - - - - - - - - - LGOs 289 AGKSKIEDYFPEFARYTTPEDATPEPGEDPRVTRAKYFIR

G 5C081 378 KFFETNFLKINGTNKPIYVHRTCATDSKSMKFVLSAVTDM0111 421 KYFEK IFLSLNKTNKPIYVKRTCATDTOTMKFVLSAVTDLGas 329 DEFLRISTASGDGRHYCYPHFTCAVDTENIRRVFNDCRDI

TCATDTQ V

C0G1 418 IVQHNLKKSGIN 429SCG1 461 IIQQNLKKSGII 472as 369 IQRMHLRQYELL 380

FIG. 3. Comparison of amino acid sequences of G-protein asubunits. The single-letter amino acid code is used. Sequenceidentity or conservative replacements between CAG1 and SCG1 arein boldface; identities or conservative replacements shared by Goasare shown in boldface in that sequence. Conservative amino acidsare grouped as follows: A,G,P,S,T; D,E,N,Q; F,W,Y; H,K,R; andI,L,M,V. Stretches of amino acids (Gl to G5) presumably involvedin GTP binding and hydrolysis are overlined, and the correspondingconsensus sequences are given below, with X indicating any aminoacid and 0 and J representing hydrophobic and hydrophilic aminoacids, respectively (5). Sequences which are otherwise variable butconserved between CAG1 and SCG1 are boxed.

acids but is located in a different region of the protein, verynear the amino terminus (37). The overall similarity betweenthe deduced amino acid sequences of the C. albicans CAGIand S. cerevisiae GPA2 genes is 57%, including the conserv-ative replacements (37). A similar degree of homology wasobserved between CAG1 and the inferred amino acid se-quence of the Schizosaccharomyces pombe Ga gene (40).Chromosomal location and copy number. A Southern blot

of restriction enzyme-digested DNA from two C. albicansstrains probed with the 2.2-kbp HpaI fragment containingCAGl is shown in Fig. 4A. The cloned fragment correspondsto a single EcoRI fragment of the expected size in WO-1 andthe two fragments previously seen in 3153a. HindlIl, BglII,and XbaI digestion patterns are identical in the two strains,with the later two yielding a single band. This result strength-ens our conclusion that CAGI is present in only one chro-mosomal location and thus would be represented twice in a

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VOL. 12, 1992

A WO-1 3153aIB C E H Xl lB C E H Xl

I~~

B000o-~~~~~~'f-- ->

_ 1< < J( .*

FIG. 4. (A) Restriction enzyme digestion patterns of CAGI in C. albicans WO-1 and 3153a. DNA from the two strain was digested withBglII (lane B), ClaI (lane C), EcoRI (lane E), Hindlll (lane H), andXbal (lane X) and blotted on Zeta-probe membrane (Bio-Rad) after agarosegel electrophoresis. The blot was probed with 32P-labelled CAGI DNA and autoradiographed. (B) TAFE separation of S. cerevisiae, C.albicans (nine strains), C. krusei, and C. glabrata chromosomes. For details of the conditions of electrophoresis, see Materials and Methods.(C) Chromosomal location of CAG1. The gel shown in panel B was blotted and hybridized to 32P-labelled CAGI. In seven of the tested Calbicans strains, the hybridization occurs only to chromosome III. (In WO-1 and B4252, CAGI hybridization is to two bands of dissimilarsizes that are possibly rearranged homologs of chromosome III.)

typical diploid Candida genome. That WO-1 exhibits anexceptional restriction pattern is consistent with its unusualkaryotype (Fig. 4B). Hybridization to chromosomes sepa-rated by TAFE shows that CAG1 is located in chromosomepair III in most strains, but in WO-1 it is found in one smallerand one larger band (Fig. 4C). Other independently isolatedfragments from chromosome III also exhibit this pattern ofhybridization to WO-1 (data not shown). The smallest chro-mosome in WO-1 has been designated a supernumerarychromosome by Magee et al. (29); however, we believe itmore likely that chromosome III has undergone a rearrange-ment in this strain, as loss of this chromosome produces acell type switching defect (33). The TAFE and Southernblots together indicate that there is a single CAGI generepresented twice in the diploid Candida genome.

Expression and function of CAGI in C. albicans. As de-scribed earlier, the two best-characterized switching sys-tems known in C. albicans are the yeast-to-hyphal-formswitching (41) and the white-opaque switching of WO-1 (49,50). Northern blot analysis revealed no significant differencein the level of CAGI mRNA between the yeast and mycelialforms induced by human serum or by the synthetic mediumdeveloped by Lee et al. (27). In addition to studying theyeast and hyphal forms, we have studied the expression ofCAGI in the white and opaque forms of C. albicans WO-1(50). We found that the level of CAGI mRNA in opaque cellsis about 1.5- to 2.0-fold higher than in the white cells (datanot shown).As a test of function of the C. albicans gene, both copies

of CAGI were disrupted in an ade2 derivative of the white-opaque strain WO-1 as described in Materials and Methods.These cagl-disrupted strains were viable. Therefore, wetested them for growth rate, bud-hypha switching frequency,

and white-opaque switching. Neither the heterozygous nor

the homozygous disruptants displayed growth rates signifi-cantly different from those of the original ade strain (WO-13/6). Similarly, the disruptants showed no change in theirability to produce germ tubes in the presence of 10% humanserum. The ability to switch between the white and opaquecell types as measured by colony sectoring was also quali-tatively unaffected.

Function of CAGI in S. cerevisiae. To test the function ofthe presumptive Ga subunit gene, a 2.2-kbp HpaI fragmentcontaining the complete coding region plus 200 bp of 5'flanking sequence was transferred into yeast shuttle vectorsand introduced into S. cerevisiae strains lacking the nativeGa-subunit gene SCG1. The low-copy-number plasmidYCp5O-CAG1 was introduced into the diploid strain Dill(9), which is heterozygous for a LEU2 insertion in SCG1.The high-copy-number plasmids YEp213-CAG1 and YEp213-SCGI were introduced into diploid IVYD (21), which ishomozygous for a URA3 disruption of SCG1. Upon sporu-lation, all strains yielded tetrads containing viable spores,indicating that the CAGI gene product provides sufficientactivity to prevent the constitutive cell cycle arrest pheno-type characteristic ofsegl mutants. The Ga subunit is highlyconserved across species, and it has been shown that the ratgene encoding the Gas subunit, when engineered for expres-sion in yeast cells, can rescue segl mutants, indicating thatit can also interact successfully with the yeast cellularcomponents (e.g., the c and -y subunits) required to alleviatecell cycle arrest (9). Cells transformed with the rat gene donot, however, respond to pheromone binding, presumablybecause interaction with the receptor requires specific struc-tures not conserved between the two proteins. Therefore,

C. ALBICANS G PROTEIN 1981

~-, ocr-'Jr'J, -O,,_X, X ° . rl Q :,.~z3 LI <<mtr

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TABLE 3. Quantitative determination of mating efficiency

Efficiency ofPartial genotype Plasmid mating' as:

a a

SCGI None 1.00 1.00scgl::URA3 Ieu2 YEp213-CAG1 0.87 0.24

YEp213-CAG1 0.50 0.31scgl::URA3 leu2 YEp13-SCG1 0.43 0.21

YEp13-SCG1 0.75 0.36scgl::LEU2 ura3 YCp5O-CAG1 0.23 0.035

YCp5O-CAG1 0.31 0.011a Relative to strains carrying wild-type SCGI. Data are from two indepen-

dent experiments.

mating is a more stringent test for function of the CAGI genein a Saccharomyces host.

Standard complementation tests by replica plating showedthat on YEp213-based plasmids, the CAG1 gene can supportmating to the same qualitative degree as can the clonedSCG1 gene (data not shown). Quantitative tests for matingefficiency were performed on several segregants from eachdiploid (Table 3). We first tested the ability of CAGI tocomplement the mating defect in segl cells when expressedfrom a high-copy-number plasmid (i.e., YEp213). As acomparison, ability to complement the mating defect bySCGI expressed from a similar plasmid (i.e., YEp13) wasalso determined. As can be seen (Table 3), CAGI comple-mented the mating defect of scgl almost as efficiently as didSCGI in both the a and ao cells. We further tested theefficiency of CAGl to complement the mating defect of scglwhen expressed from a low-copy-number plasmid (i.e.,YCp5O). When present on a low-copy-number plasmid,CAG1 also restored the mating defect of scgl cells. How-

A°- _ 3 U)6 7)

l123 45i6 78 9

SCGI-

0e

.as

ever, the efficiency of mating was significantly lower in acells than in a cells.

Transcription of CAGI in S. cerevisiae. To estimate thelevel at which CAGJ is expressed in S. cerevisiae, weperformed Northern blot analysis on several haploid segre-gants and diploids made by mating those segregants withstandard haploid strains. As noted above, the gene CAGI issmaller than SCG1, and the transcript is correspondinglyshorter. It was therefore possible to probe both transcriptson the same blot. Figure 5 shows the transcription patternsof a set of strains probed first with SCGI (Fig. 5A) and then,without washing the blot, probed again with CAGl (Fig. 5B).Lane 1 contains RNA from an untransformed haploid strainof S. cerevisiae, and lane 9 contains RNA from an untrans-formed diploid strain of C. albicans. Lanes 7 and 8 representscgl haploid Saccharomyces segregants containing the plas-mid-borne CAGI and SCGJ genes, respectively. Since thelengths and the specific activities of the probes used are insimilar ranges, comparison of the lanes indicates that thetransformed strains produce approximately equal amountsof CAGI and SCGJ RNAs and about fourfold more than isfound in untransformed strains. The transcript produced bythe plasmid-borne CAGI gene appears to be slightly shorterthan that from the native CAGI gene in C. albicans, possiblybecause of altered initiation or termination.An unanticipated result was obtained when the diploid

Saccharomyces strains were compared (Fig. 5, lanes 2 to 6).SCGI is a haploid-specific gene and is therefore turned off inthe MATaMIATo diploid (lane 2) by the action of theproducts of the MATal andAM Ta2 genes. MATa and AMTaare alleles of the same locus and encode regulatory proteinsthat define the mating types of haploid cells. The al and a2proteins are normally found together only in A 4Ta/A 4Ta,diploid cells, in which they act together to turn off the set ofhaploid specific genes, including the G-protein genes SCG1,

CD cD

{X C-D --)

l 2 3 4 5 6 7 8 9B.

SCGI-- *_

*. V

-CAGIYEP21 3:CAGI

Probe: SCGI SCGI +CAGIFIG. 5. Transcription of CAGI and SCGI in S. cerevisiae. Haploid S. cerevisiae strains carrying either YEp213-CAG1 or YEp13-SCG1

were mated to standard haploid strains, and diploids were selected. Total RNA was isolated from the haploid and diploid strains and analyzedby Northern blot analysis. (A) SCGI probe; (B) SCGI plus CAGI probe. Lanes: 1, haploid S. cerevisiae; 2, diploid S. cerevisiae; 3 and 4,diploid S. cerevisiae; 7, haploid S. cerevisiae (all carrying YEp-CAGI); 5 and 6, diploid S. cerevisiae; 8, haploid S. cerevisiae (all carryingYEp-SCGI); 9, C. albicans WO-1.

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TABLE 4. Comparison of al-a2 binding sitesSite Sequencea

CAGI..... GATGTCATTTTTAACATGMA Ta...... AATGTAGAAAAGTACATCConsensus...... C7AbTGT

C2GA1

a Boldface letters indicate the symmetrical highly conserved domains. Theconsensus sequence is a summary of 10 half-sites shown to be active in vivo(15).

STE4, and STE18 (51). The transcript level of the plasmid-borne SCGJ gene is only slightly lower in the diploid strains(lanes 5 and 6) than in the haploid strains, indicating that thenormal control site may not be present on the clonedfragment. In contrast, the transcript from the plasmid-borneCAGI gene is not detectable in the diploids (Fig. SB, lanes 3and 4) and is thus apparently under tight regulation by theproducts of the Saccharomyces mating-type genes, specifi-cally the al and a2 proteins.CAGI contains an al-a2 binding site. Examination of the 5'

flanking sequence of CAGI revealed a potential al-a2 bind-ing site between positions -152 and -169. The al-a2 bindingsites consist of two highly conserved symmetrical half-sitesof five base pairs separated by eight less conserved basepairs and were first noted by Miller et al. (34). Goutte andJohnson (14) have subsequently confirmed by footprintingand by gel retardation assay that this sequence is a bindingsite for the combined al and a2 proteins and that it issufficient to confer haploid-specific gene regulation. Thepotential al-a2 binding site upstream of CAG1, as well as aconsensus sequence (15) derived from several confirmedal-a2 binding sites, is shown in Table 4.We tested the upstream region of the CAGI gene for al-a2

binding by gel retardation assays, using purified componentssupplied by C. Goutte in the laboratory of A. Johnson(University of California, San Francisco). Two neighboringrestriction fragments from the upstream region of CAGIwere used as probes. Fragment I (310 bp) is the HindIII-HpaI fragment between positions -514 and -204. FragmentII contains the putative al-a2 binding site (underlined in Fig.2) and is located between positions -204 and +44. FragmentD contains the bona fide al-a2 binding site from the MA4Talgene (14) cloned into a polylinker from which it can beisolated as part of a 125-bp fragment. Results of the gelretardation assay are shown in Fig. 6. Migration of fragmentI is unaffected in the presence of either al alone or combinedal-a2 (lanes 1 and 2). The migration pattern of fragment IIwas also unchanged in the presence of al alone (lanes 3 and11). However, an al-a2 extract showed a significant retar-dation of fragment II (lane 4), indicating an interactionbetween this fragment and the combined al-a2 extract. Totest the specificity of this interaction, increasing levels offragment D were added as competitor (lanes 5 to 7). Theamount of bound fragment II is reduced nearly to zero by theaddition of this sequence but is unaffected by the addition ofa sequence from the upstream region of the S. cerevisiaeGALl gene (lanes 8 to 10). The CAGI upstream region thuscontains an al-a2 binding site, and it is our working hypoth-esis that the CAGI gene is under an as yet undeterminedform of cell type regulation, perhaps involving regulatorsthat are homologs of the Saccharomyces A4T gene prod-ucts.

MATil GALal1 al1 --- r- l+ + (nM) (nM) no

al a2 a1 a2 4 8 20 4 8 20 extract

FragmentFragment 11-i_- __ --

1 2 3 4 5 6 7 8 9 10 1IFIG. 6. Binding of S. cerevisiae al and a2 proteins to CAG1

DNA. Binding of the al and a2 proteins to two neighboringfragments of a CAGI clone was tested in a gel retardation assay.Fragment I spans from positions -504 to -204; fragment II spansfrom positions -204 to +44. Lanes: 1 and 2, no binding of fragmentI in the presence of al alone and of a2 alone, respectively; 3 and 4,binding of fragment II to Al and al-a2, respectively; 5 to 7 and 8 to10, binding of al-a2 to fragment II in the presence of increasingconcentrations of the S. cerevisiae AM Tal and GAL] genes, respec-tively; 11, migration of fragment II alone.

DISCUSSIONIn this communication, we report the cloning and initial

characterization of a putative heterotrimeric G-protein gene,CAG1, from the pathogenic yeast C. albicans. Of the manyGa genes reported so far, the deduced amino acid sequenceof CAGI exhibits maximum homology to the product of theS. cerevisiae Gat gene SCG1. This may be an indication ofthe close evolutionary relationship between Saccharomycesand Candida specits. C. albicans has been placed in thegroup Fungi Imperfecti, the group of fungi that are notknown to have arty mating phase. However, sequenceanalyses of small ribosomal subunit RNAs and 5S rRNAreveal that C. albicans belongs to the order Endomycetales,to which the family Saccharomycetaceae also belongs (7,18). Significant degrees of DNA sequence homology be-tween a number of C. albicans genes and the correspondingS. cerevisiae genes have been reported (2, 36, 44). Compar-ison ofSCGI and CAGI coding-region DNA sequences (datanot shown) showed that the two genes are 62.5% homolo-gous, which attests to the close phylogenetic relationshipbetween the two yeast species. On the basis of the primarysequence relationships among the different G-protein a.subunits, Strathmann and Simon (52) divided them into threeclasses, Gs, Gq, and Gi. Our sequence comparison indicatesthat the G proteins encoded by the C. albicans CAGI geneand the S. cerevisiae SCGJ gene probably constitute adifferent class.

In addition to the structural similarities characteristic ofthe heteromeric G-protein family as a whole, the two yeastgenes show a strong similarity in regions dedicated tospecific cellular interactions which is also reflected at thefunctional level. The C. albicans gene CAGI complementedthe two major phenotypes, namely, growth and matingdefects of scgl S. cerevisiae cells.

In S. cerevisiae, SCGJ mediates mating pheromone signaltransduction by interacting with the transmembrane recep-

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tors and the cognate ,B and -y subunits. The Ga subunitencoded by SCG1, in response to the pheromone-boundreceptor, is believed to release the 0-y complex. It is the 13ycomplex which carries out the subsequent step in a positivemanner. In segl S. cerevisiae cells, the a and -y subunits arefree to interact with the downstream components of themating pathway even in the absence of the pheromones,thereby resulting in a constitutive activation of the pathway(9). Expression of mammalian Ga subunits alleviates thegrowth defect of scgl mutation, suggesting that the heterol-ogous Ga proteins can interact with the S. cerevisiae 3-ycomplex to prevent activation of the pathway (9, 22). Com-plementation of the growth defect of segl S. cerevisiae cellsby the C. albicans CAGJ gene similarly points to thesuccessful interaction between CAG1 and the S. cerevisiae13y complex.Though the region(s) of Ga subunits involved in the

interaction with the Py complex and the effector molecule isnot yet defined, several lines of evidence suggest that in bothmammalian systems and S. cerevisiae, the C terminus of Goasubunits is sufficient for the interaction with the cognatereceptors (19, 20, 31, 53). This region of the a subunits of theG proteins is quite variable, reflecting the different specific-ities of interaction with the cognate receptor. All of themammalian Ga subunits tested thus far have failed tointeract with the mating pheromone receptors of S. cerevi-siae (22). Our data show that CAG1 can complement themating defect in S. cerevisiae. Hence, it is conceivable thatCAGI performs a similar role in a mating pathway in C.albicans which has thus far escaped detection. However,close examination of the molecular mechanism of the matingpheromone signal transduction in S. cerevisiae points tosome degree of flexibility built into the system. There is noobvious sequence similarity between the a- and a-factortransmembrane receptors STE2 and STE3 (6, 16, 39), andonly one G protein, i.e., SCG1, is sufficient in transducingthe signal generated by both of them. Therefore, it seemsjust as likely that in its native environment, CAGI maysupport a signal from some other type of receptor, the ligandof which has nothing to do with mating. As mentionedearlier, certain cell surface molecules of C. albicans havebeen reported to bind specifically to human extracellularproteins such as laminin and C3d. Though the nature ofphysiological changes of C. albicans in response to the hostligands is not yet known, it is possible that CAGI plays a rolein the signal transduction pathway employed by any suchreceptor.

In S. cerevisiae, three key regulatory proteins, al, a2, andal, provide cell-type-specific expression of particular sets ofgenes. al acts as a positive regulator of a-specific genes. a2protein represses a-specific genes in haploid a mating-typecells. In a/a diploid cells, a2 in combination with al proteininhibits expression of haploid-specific genes (51). The al-a2specific repression is mediated by a specific 18-bp-longsequence (Table 4) which has been found in the upstreamregion of several haploid-specific genes of S. cerevisiae (34,47). DNA sequence analysis shows the presence of such aputative cis-acting element in the 5' region of the C. albicansCAG1 gene. The observed mating-type control of the CAG1gene at the transcriptional level, and the in vitro binding ofthe S. cerevisiae al and a2 proteins to its upstream region,suggests that this element could be functional.

It is conceivable that in C. albicans, the putative al-a2control site is not active but rather is a fossil, left over froma time when the two species shared a common ancestor.Comparison of the available DNA sequences (data not

shown) that are upstream of CAGI and SCGI does notreveal any obvious sequence conservation, making thislatter idea seem unlikely and further strengthening the notionthat the putative al-a2 binding site has been conservedupstream of CAG1 in order to fulfill a specific biological rolein the cell. It has been observed that the homeodomain genesthat control development of more complex organisms suchas Drosophila and Xenopus species contain regions of ho-mology with the al and a2 proteins (26, 45). It is possiblethat the use of regulatory network similar to that of al-a2 ismore widespread in nature than has been documented todate. The presence of an apparently functional al-a2 bindingsite upstream of the CAGl gene may point to the existence ofa similar regulatory circuit in C. albicans.

ACKNOWLEDGMENTS

We thank J. Kurjan, R. Kelly, M. B. Kurtz, J. Schmidt, and D.Soll for providing plasmids and yeast strains. We thank C. Goutteand A. D. Johnson for generous help with the gel shift experiments.We also thank A. Steinhardt for excellent technical assistance andK. Sadhu for help with the figures.

This work was supported by a grant from National Institutes ofHealth to J.B.H. M.J.M. was supported by a fellowship from theDamon Runyon-Walter Winchell Cancer Fund.

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