Copyright 0 1993 by the Genetics Society of America

Identification of Functionally Related Genes That Stimulate Early Meiotic Gene Expression in Yeast

Sophia S. Y. Su and Aaron P. Mitchell

Institute of Cancer Research and Department of Microbiology, Columbia University, New York, New York 10032 Manuscript received June 18, 1992

Accepted for publication October 3, 1992

ABSTRACT Meiosis and spore formation in the yeast Saccharomyces cerevisiae are associated with increased

expression of sporulation-specific genes. One of these genes, IME2, encodes a putative protein kinase that is a positive regulator of other sporulation-specific genes. We have isolated mutations that cause reduced expression of an ime2-lac2 fusion gene. We found mutations in IMEI , a known positive regulator of IME2, and M C K I , a known positive regulator of IMEI . We also isolated recessive mutations in 12 other genes, which we designate RIM (Regulator of g E 2 ) genes. Our analysis indicates that the defects in riml, r im8, r im9 and rim13 mutants are a consequence of diminished IMEl expression and can be suppressed by expression of IMEl from the heterologous ACT1 promoter. These rim mutations also reduced expression of an imel-HIS3 fusion, in which the HIS3 gene is expressed from the IMEl promoter, and caused reduced levels of IMEl RNA. Although the r i m l , rim??, rim9 and rim13 mutant phenotypes are similar to those of mckl mutants, we found that the defects in ime2-lac2 expression and sporulation of the mckl rim double mutants were more severe than either single mutant. In contrast, the defects of the rim rim double mutants were similar to either single mutant. The riml, r im8, r im9 and rim13 mutants also display slow growth at 17" and share a smooth colony morphology that is not evident in mckl mutants or isogenic wild-type strains. We suggest that RIMI, RIM8, RIM9 and RIM13 encode functionally related products that act in parallel to MCKI to stimulate IMEl expression.

T HE IME1 gene product plays a pivotal role in the decision to initiate meiosis and spore formation

in the yeast Saccharomyces cerevisiae. IMEl expression and sporulation respond in parallel to nutritional sig- nals; they are blocked in the presence of glucose or nitrogen. In the absence of glucose and nitrogen, IMEl expression is greater in a/a cells, which can sporulate, than in a cells or a cells, which cannot (KASSIR, GRANOT and SIMCHEN 1988). Sporulation is blocked in imel null mutants (KASSIR, GRANOT and SIMCHEN 1988) and increased expression of IMEl permits initiation of meiosis in a cells or a cells and in rich media (KASSIR, GRANOT and SIMCHEN 1988; GRANOT, MARCOLSKEE and SIMCHEN 1989; SMITH and MITCHELL 1989; SMITH et al. 1990). Therefore, the precise regulation of meiosis is achieved, in part, through regulation of IMEl expression.

One of the main roles of the IMEl product is to stimulate expression of sporulation-specific genes (SMITH and MITCHELL 1989; MITCHELL, DRISCOLL and SMITH 1990; ENCEBRECHT and ROEDER 1990; KIHARA et al. 1991). These genes are expressed at highest levels in sporulating cells and many are essen- tial for the sporulation program. One of the earliest sporulation-specific genes is IME2, which encodes a protein kinase homolog (YOSHIDA et al. 1990) that can activate many other sporulation-specific genes (Figure

Genetics 133: 67-77 (January, 1993)

l)., The IMEl product can also activate sporulation- specific genes independently of IME2. Stimulation of sporulation-specific gene expression through IME2 and through an IME2-independent mechanism sub- sequently leads to the completion of the sporulation program (MITCHELL, DRISCOLL and SMITH 1990). IMEl activity is limiting for expression of sporulation- specific genes because decreased IMEl expression leads to decreased RNA levels from IME2 and other sporulation-specific genes (SMITH and MITCHELL 1989; NEICEBORN and MITCHELL 199 1).

What are the genes that regulate IMEl expression? One set of genes affects the adenylate cyclase/cAMP- dependent protein kinase pathway (reviewed by BROACH 199 1). Reduced CAMP-dependent protein kinase activity is associated with IMEl expression in unstarved cells; increased CAMP-dependent protein kinase activity prevents IMEl expression in starved cells (SMITH and MITCHELL 1989; MATSUURA et al. 1990). Pleiotropic effects of altered CAMP-dependent protein kinase activity suggest that this pathway is involved in a starvation response. However, the signals to which this pathway responds are unclear.

A second group of genes transmits the a la cell type signal. T h e a1 and a2 products of the MAT locus are subunits of a repressor, al-a2, that determines the a/ a cell type (GOUTTE and JOHNSON 1988; DRANCINIS

a1 -a2

i\ Y +\ nitrogen+IME4

S. S. Y. Su and A. P. Mitchell

RIM13 *Specific Genes- Spore Formation

Meiosis and +

MCKl

nitrogen glucose

FIGURE 1.-Regulation of meiosis and spore formation in S. cerevisiae. IMEl is essential for meiosis. The IMEl product stimulates sporulation-specific genes. The ZME2 product stimulates sporulation-specific gene expression as well. Known positive regulators of IMEl include MCKl and ZME4. In this study, we demonstrate that RZMl, RIM8, RIM9 and RIMI? are additonal positive regulators of ZMEl that act in an MCK1-independent pathway. Known negative regulators of ZMEl include RMEl and IME2. Nitrogen prevents IMEl expression through repression of ZME4 and, possibly, effects on MCKl and RIM activities. Glucose also prevents IMEl expression, perhaps through effects on MCKl and RIM activities. al-a2, which determines the a/a cell type, permits ZMEI expression through repression of RMEl and stimulation of IME4 expression. "X" refers to a postulated repressor of IME4 (SHAH and CLANCY 1992). Epistatic relationships between IME4 and the RIM genes, or between ZME4 and MCKl, are unclear.

1990) (reviewed in HERSKOWITZ 1989). Two path- ways relay M A T information to control I M E l (Figure 1) . In one pathway, al-a2 represses an inhibitor of meiosis, the R M E l product, which blocks ZMEl expression (MITCHELL and HERSKOWITZ 1986; KAS- SIR, GRANOT and SIMCHEN 1988; COVITZ, HERSKOW- ITZ and MITCHELL 1991). In the other pathway, a l - a2 permits elevated expression of a positive regulator of meiosis, the ZME4 product, which stimulates ZMEl expression (SHAH and CLANCY 1992). It is thought that al-a2 stimulates IME4 expression indirectly by repressing a repressor, indicated by an X in Figure 1 (SHAH and CLANCY 1992). Absence of nitrogen is required for high level ZME4 expression in a/a cells. Thus, ZME4 transmits both al-a2 and nutritional sig- nals.

Expression of ZMEl also depends on the M C K l gene product (NEIGEBORN and MITCHELL 1991), which specifies a protein kinase homolog that cofractionates with both serinejthreonine and tyrosine kinase activi- ties (DAILEY et al. 1990). MCKI was identified on a multicopy plasmid that stimulated ZMEl expression and sporulation (NEIGEBORN and MITCHELL 199 1). In mckl mutants, IMEI expression is diminished, and sporulation is slow and inefficient. Many of the spores produced in the mutant are packaged in immature asci that persist in the population. I M E l expression from a heterologous promoter improves sporulation efficiency in the mutant but the asci remain immature. Therefore, M C K l affects IMEI expression and ascus maturation independently. M C K l is required for proper mitotic centromere function as well (SHERO and HIETER 1991). Overexpression of M C K l sup- presses certain centromere point mutations. In mckl

mutants, growth at low temperature ( 1 7 ") causes min- ichromosome instability and sensitivity to the anti- microtubule drug, Benomyl. At still lower tempera- tures ( 1 1 "), mckl mutants fail to grow. Because null mutations of M C K l lead to partial or conditional defects in I M E l expression, spore maturation, cen- tromere function and growth, alternative kinases or pathways may exist that act in parallel to M C K I . It is unclear what signal MCKI may transmit.

The pathways described above determine whether ZMEI expression is activated prior to sporulation. At later times in sporulation, ZMEl is down-regulated. Because ime2 mutants fail to down-regulate ZMEl , the IME2 gene product is formally a negative regulator of I M E l expression (SMITH and MITCHELL 1989).

Clearly, many genes involved in IME1 regulation have not been identified. For example, the substrates through which M C K l and IME2 products influence ZMEl expression as well as the genes thought to act in parallel to M C K l are unknown. We report here the identification of regulators of ZMEI expression through a screen for mutants that failed to express one of the earliest sporulation-specific genes, IME2. Our genetic screen had two special features: the uti- lization of an rmel mutation and an ime2-lac2 fusion reporter gene. The rmel mutation enabled us to use haploid strains to assay ZME2 expression. The use of haploid strains facilitated the recovery of recessive mutations and simplified dominance, complementa- tion, and segregation tests. The ime2-lac2 fusion gene enabled us to screened for mutants with diminished IME2 expression rather than for mutants with defects in more complex meiotic events. Our analysis focuses on a group of functionally related genes that act in parallel to M C K l to stimulate ZMEl expression.

69 Activation of IMEl

TABLE 1

List of yeast strains

Strain Genotype' Source

SS 1 -3B a IME2-5-lacZ::URA3 This study SS 1 -4A a IME2-5-lacZ::URA3 This study SS47-1 B a rmelA5::LEUZ IME2-5-lacZ::URAjr This study SS47-1 D a rmel A5::LEUZ IME2-5-lacZ::URA3 mcklA1::URAjr his4 This study SS47-8D a IME2-5-lacZ::URA3 This study AMP107 a This laboratoryb AMP340 a rmel A5::LEUZ IME2-5-lacZ::URA3 This study AMP343 a rmel A5::LEUZ lME2-5-lacZ::URA3 imel Al2::TRPl This study AMP374 a rmelA5::LEUZ IME2-5-lacZ::URA3 rim6-1 This study AMP375 a rmelA5::LEUZ IME2-5-lacZ::URA3 rim7-1 This study AMP524 a rmelA5::LEUZ lME2-5-lacZ::URA3 rim5-1 his4 This study AMP599 a rmelA5::LEUZ IME2-5-lacZ::URA3 riml-1 met4 This study AMP600 a rmelA5::LEUZ IME2-5-lacZ::URA3 rim21 met4 his4 This study AMP601 a rmelA5::LEUZ IME2-5-lacZ::URA3 rim3-1 met4 This study AMP637 a rmelA5::LEUZ IME2-5-lacZ::URA3 mcklA3::TRPl met4 This study AMP734 a rmelA5::LEUZ IME2-5-lacZ::URA3 arg6 This study AMP735 a rmelA5::LEUZ IME2-5-lacZ::URA3 arg6 This study AMP737 a rmel A5::LEUZ IME2-5-lacZ::URA3 met4 This study AMP744 a rmelA5::LEUZ IME2-5-lacZ::URA3 rim4-1 met4 This study AMP805 a rmelA5::LEUZ imel-13-HIS3::LEUZ arg6 his3 This laboratoryC AMP839 a rmel A5::LEUZ met4 This study SSYS a rmelA5::LEUZ IME2-5-lacZ::URA3 rim8-1 met4 This study SSY32 a rmel A5::LEUZ IME2-5-lacZ::URA3 rim9-1 met4 This study SSY37 a rmelA5::LEUZ IME2-5-lacZ::URA3 rimll-1 met4 This study SSY45 a rmelA5::LEUZ IME2-5-lacZ::URA3 riml3-1 met4 This study SSY49 a rmelA5::LEUZ IME2-5-lacZ::URA3 mckl-I4 met4 This study SSY53 a rmelA5::LEUZ IME2-5-lacZ::URA3 riml5-1 met4 This study SSY113 a rmel A5::LEU2 IME2-5-lacZ::URA3 IMEI-15::TRPl This study SSY 179 a rmelA5::LEUZ IME2-5-lacZ::URA3 imel-l3::HIS3 his3 This study SSY389 a rmelA5::LEUZ IME2-5-lacZ::URA3 IMEI-16::TRPl his3 This study AMR7 a natl-3::URA3 ade2 his3 ura3 leu2 trpl can1 R. STERNGLANZ~

a All strains except AMR7 have the additional markers ura3, leuZ::hisG, trpl::hisG, lys2 and ho::LYSP. Described in M~TCHELL, DRISCOLL and SMITH (1 990) Described in NEIGEBORN and MITCHELL (1 99 1). Described in MULLEN et al. ( 1 989).

MATERIALS AND METHODS

Strains: Yeast strains were all derived from strain SK-1 (KANE and ROTH 1974) through transformation or standard genetic manipulation (ROSE, WINSTON and HIETER 1990) and are listed in Table 1. All rim mutants analyzed in detail were derived from the rim mutant strains listed in Table 1. These strains were segregants from the second consecutive cross of each original mutant to wild-type strains. The ime lAl2 , rme lA5 , mck lA1 and mcklA3 mutations as well as the auxotrophic markers in these strains have been de- scribed previously (MITCHELL, DRISCOLL and SMITH 1990; SMITH and MITCHELL 1989; NEICEBORN and MITCHELL 199 1 ; COVITZ, HERSKOWITZ and MITCHELL 199 1).

The imel-HIS3 allele ( i m e l - l 3 ) , which places the biosyn- thetic gene HIS3 under the control of the chromosomal I M E l promoter, was described by NEICEBORN and MITCH- ELL (1 991). Because the imel-HIS3 allele is an imel deletion, it was followed in crosses by failure to complement imel mutants for sporulation.

The ime2-lacZ fusion gene (IME2-5-ZacZ), which includes LIRA3 as a selectable marker, was introduced by transform- ing strain AMP107 with a BglII digest of plasmid pSS400 (Figure 2). Southern analysis confirmed the integration of the ime2-lacZ plasmid at the IME2 locus and the preservation of the chromosomal copy of IME2 (IME2::ime2-lacZ). We

also confirmed that IME2-5-lacZ segregated as an IME2 allele.

The PAcTI-lMEl alleles, which we designate as IMEl-15 and IME1-16, contained T R P l as a selectable marker (Figure 2). They were introduced by transforming AMP735 ( IMEl strain) and SSY 179 ( i m e l A strain) with an EcoRI digest of plasmid pSS153. Southern analysis confirmed that the gap between the EcoRI sites of pSS153 was repaired during integration (ROTHSTEIN 1991). In IMEI-I5 (IMEI::PACTI- IMEI) , IMEl was expressed from IMEl and A C T l pro- moters. In IMEl-16 ( imelA::P,+cTI-IMEl), IMEl was ex- pressed only from the A C T l promoter.

Standard procedures were used for mating, diploid iso- lation, sporulation and tetrad analysis (ROSE, WINSTON and HIETER 1990). All diploids created for tetrad analysis in this study were homozygous for rmelA5 as well as IME2-5-lacZ. Thus, rim mutations in the haploid segregants were followed by failure to express ime2-lacZ. rim mutations were also followed by failure to complement the respective ram mu- tants for ime2-lacZ expression in some crosses.

The rim rim double mutants were constructed by pairwise crosses of individual rim mutants. For example, we crossed a r iml-1 mutant to a rim8-1 mutant and analyzed the result- ing tetrads. Segregation of riml-1 and rim8-1 was followed by failure to complement r iml-1 and rim8-1 mutants, re- spectively, for ime2-ZacZ expression. Similarly, the mckl Arim

S. S. Y. SII and A. 1'. Mitcllcll 7 0

R IMEI-I5::TRPI

(IMEl::PAcrr-IMEll

( i m r l A : : P A ~ f - I M E l ) IMEI-16::TRPI

pss153

f -1 1RPI

I / M F / I I I I I A

sm sp 8 1 I\ K n, . , . , ., 11 \

I I I I 1 I sp 111x1 < \ W K n 1%

F I G U R E 'L.-Structurcs of fME2-5-larE::URA3. f M R f - f 5 : : T f ~ f ' f ; u n d fA4f<I- f6::TRf I allclcs. (A) IME2-larZ::URA3. The plasmid pSS400 was integrated into the yeast genome by homologous re- conlbination at the f M E 2 5'-promoter region. The thin and the thick solid bars represent the f M E 2 promoter and the f M E 2 coding region. respectively. The stippled bar represents lacZ sequences and the open bar represents the U R A 3 selectable marker. Bent arrows indicate transcription from the f M E 2 promoter. (R) f M E f - f 5 : : T R f f and fMEf -16: :TRf 1. The plasmid pSSl.53 was integrated into f M E f and i m e f A strains by homologous recombination at the chronnosonl;ll f M E f locus to construct fkfE!::f.,,;T/-fhfEf and imtfA::fAcr,-fMI:'f alleles, respectively. T h e thick shaded bars rep- ~'esrnt the f M E f gene. The thin shaded bars and the diagnonally striped bars represent the natural fMEl and the heterologous A C T f promoters, respectively. Rent arrows indicate transcription from he respective promoters. The horizontally striped bar respescnts fffS3 sequences present in the i m e f A strain, and the open bars wpresent the TIW f selectable marker. Restriction endonuclease sites:(B)RamHI;(S)Sall;(C)~~III;(X)XhoI;(Pv)fuull:(Sm)Smal; (Sp) Sphl; (1-1) Hindll; (K) EcoKI. The sites indicated in parentheses \\'ere lost as a consequence of cloning. The sequences are not drawn 1 0 scale.

double mutants were constructed by dissecting crosses be- tween AMP637, which contained the mckIA3::TRf l allele, and rim mutant strains. Segregation of m c k l A 3 was moni- tored through segregation of the Trp+ phenotype. Segre- gation of each rim mutation was followed by f a1 'I ure to complement respective rim mutants for ime2-lac2 expres- sion.

Media and culture conditions: Standard recipes were used for SD, SC, SC dropout media, YEPD, YEPAc, spor- ulation plates, and liquid sporulation media (ROSE, WINSTON ;und HIETER 1990; SMITH and MITCHELL 1989). Minimal sporulation plates contain 2% KAc, 2% Racto-agar and auxotrophic amino acid supplements as in SC medium. KAcXgal plates are minimal sporulation plates buffered with 0 . 2 M K P O , at pH 7 and containing 50 mg/liter of Xgal.

Sporulation ability was quantitated on plates or i n liquid cultures. For plate tests. yeast were grown on YEPD plates for one day, then replica-pl;tted to sporulation plates. For liquid culture tests, yeast were grown on YEPAc vegetative medium to mid-log phase, then transferred to 2% KAc sporulation metlitlm (SMITH et al. 1990). I n either case, we scored at least 200 cells by microscopic ex;~mination ;tfter 24 hr to quantitate sporulation ability.

All experiments were conducted at 30" except that cold sensitivity w a s scored at 17".

Plasmid construction: Diagrams of relevant plasmid in- serts are shown i n Figure 2.

The ime2-larZ fusion plasmid, pSS400, W;IS constructed by fusing the f M E 2 .i' end to the lac2 gene in plasmid YIp358R (MYERS et al. 1986). The 1.3-kbp RamHI-fvull fragment of l A I f 2 from pHSlOl, which contained nucleo- tides -983 to + I 14 of lMI:'2 (YOSHIDA et al. 1990). was eluted from an agarose gel and inserted into the BamHI and Smal sites of YIp358R. This construct fused the larZ gene in frame to I M E 2 at codon 39.

pSS153, the source of f A c r l - I M E I , was constructed i n three steps. First, primers that hybridized to the ACT1 promoter at positions -668 and -3 were made to introduce Sphl and Hindlll sites, respectively. These two primers were used i n a polymerase chain reaction wi th pBD8 1 (plasmid containing the ACT1 promoter and part of the .5' coding region) to obtain a 681-bp fragment containing the ACT1 promoter (NG and AREISON 1980). Second, the 681-bp Sphl-Hind111 promoter was mixed wi th a 2.3-kbp HindlI1- Sal1 I M E l fragment from YCp fCALI-IA4EI (SMITH et al. 1 990), and ;In 8.4-kbp Spht-Sal1 fragment of plasmid YEp24 in a single ligation mixture. Minipreps were screened for the incorporation of the ACT1 promoter and I M E I gene into YEp24 to create pRS fAc:rI- l iMEl (R. SIA, personal communication). Tllird, the 3.5-kbp Smal-Sal1 fr;tgment from pRS f A ~ : r l - I M E I , containing the entire f A ~ ; r I - l M E l gene, was ligated to integrating vector pRS304 (SIKORSKI and HIETER 1989) at the Smal and Sal1 sites to create

&Galactosidase assays: For @-galactosidase assays on plates, patches of cells grown on YEPD plates were replica- plated to KAcXgaI plates and incubated for 2 days a t 30". Patches that turned blue on these assay plates contained 10- galactosidase activity and expressed the ime2-lac2 fusion gene. We refer to the ability to express the imeZ-lac2 fusion gene as the IAC+ phenotype, and the Failure to express the f'usion as the Lac- phenotype.

For @-galactosidase assays on nitrocellulose filters, patches of cells were grown on YEPD plates for a day, then replica- plated to nitrocellulose filters on minimal sporulation plates. After one day at 30". the filters were placed in liquid nitrogen to permeabilize the cells. Each filter was then incubated i n 3 ml of Z buffer containing 200 pg/ml of ?<gal at 30" for at least 20 min. To terminate the reaction, the filters were removed from the assay buffer and air dried. Cells that turn blue i n this assay contained 10-galactosidase activity.

For quantitative @-galactosidase assays, cells were grown to log phase in YEPAc medium, transferred to sporulation medium. harvested 011 Millipore filters, permeabilized, and assayed w i t h ONPG as substrate (ROSE, WINSTON and HIE-

Isolation of mutants defective in imeZlacZ expression: Strains AMPJ40 ;md AMP734 were grown i n four mls of YEPD to station;lry phase, washed and suspended i n 10 mM KHPO, (pH 7), and sonicated briefly. Ethyl methylsulfonate (EMS) W;IS added to 2.5% v/v ;111d the cells were incubated at 30" to achieve 10% survival. The rnutagcnized cells were

pSSl53.

TER 1990).

Activation of IMEl

collected on a Millipore filter, washed with 10 ml of sterile water and resuspended in 4 ml of sterile water. Mutagenized cells were plated on 150 YEPAc plates to obtain 200 colonies per plate. Cells were plated on YEPAc to select against respiratory-deficient mutants, which fail to express ime2- lacZ because they fail to grow on KAcXgal plates. After 3 days, colonies were replica-plated to KAcXgal plates. Most colonies expressed ime2-lacZ and turned blue on KAcXgal plates. We screened for colonies that remained white, which included mutants defective in ime2-lacZ expression.

We anticipated that many of these white colonies would not be defective in ime2-lacZ expression but rather in the uptake of the Xgal indicator. These mutants were elimi- nated through filter &galactosidase assays in which cells were permeabilized with liquid nitrogen.

We also expected mutations within the lacZ coding region of the ime2-lac2 fusion gene. We identified 46 LacZ- mutants through crosses to AMP839, a wild-type strain lacking the ime2-lacZ fusion gene, and AMP737, a wild-type strain that provided the complementing ime2-lacZ fusion gene. Diploids formed from lacZ- mutants and AMP839 were Lac-; dip- loids formed from lacZ- mutants and AMP737 were Lac+.

Dominance and complementation tests: To test for dom- inance, each mutant was crossed to a wild-type strain of the opposite mating type (AMP73’7). In one case, the resulting diploid failed to express ime2-lacZ. This dominant defect did not segregate as a single gene trait and was not pursued. In the remaining cases, the resulting diploids expressed ime2- lacZ, indicating that these mutations were complemented by the wild-type gene. We recovered 135 recessive mutations from screening 30,000 colonies.

To test pairs of mutations for complementation, doubly heterozygous diploids were isolated by prototrophic selec- tion and tested for ime2-lac2 expression on KAcXgal plates.

Northern blots and probes: Cells were transferred during log phase growth in YEPAc to sporulation medium and samples were harvested at various times for RNA isolation (SMITH and MITCHELL 1989). Standard methods were used for preparation of Northern (RNA) blots (SMITH and MITCHELL 1989), except that nylon filters (Hybond) were used in place of nitrocellulose. Probes for ZMEl and ZME2 RNAs have been described (SMITH and MITCHELL 1989; MITCHELL, DRISCOLL and SMITH 1990). The TUB2 probe was a random-primed 1.6-kbp EcoR1-EcoR1 fragment from the TUB2 gene (NEFF et al. 1983). The ACT2 probe was random-primed plasmid pBD8 1 (provided by B. DUNN and M. A. OSLEY). We found that our preparation of the control probe, pC4 (LAW and SEGALL 1988), was a mixture of plasmids. We colony-purified one plasmid from this mixture that hybridized to a constitutive ca. 1-kb RNA (A. P. MITCH- ELL, unpublished results). We designate this plasmid pC4/ 2 .

RESULTS

Regulation of the ime2-lacZ fusion of ZME2-5- ZacZ Our primary goal was to identify genes that govern IMEI expression. Initial experiments with an imel-lacZ fusion gene gave background expression levels too high for reliable plate assays. We used an ime2-lac2 fusion gene instead to assess I M E l activity indirectly. We found that the ime2-lac2 fusion re- sponded to the same regulatory signals as the natural IME2 gene. First, ime2-lac2 expression was stimulated by starvation (Figure 3A). Increased expression was detectable by 4 hr after starvation, as expected from

A 71

0 io Hours in sporulption

B

0

Hours in sporulation

FIGURE 3.-(A) Expression of ime2-lacZ in a, (Y and a/a cells. Strains were SSI-3B (MATa IME2-5-lacZ, a), SS47-8D (MATa IME2-5-lacZ, 0). and SSI-3B X SSl-4A (MATaIMATa lME2-5-lacZ/ IME2-5-lacZ, 0). (B) Expression of ime2-lacZ in haploid strains with rmelA, imelA and mcklA mutations. Strains were wild type (SS47- 8D, O), rme lA (SS47-1B),O), rmelAimelA (AMP343), m) and rmelAmcklA (SS47-1D), 0). Strains were grown in mid-log phase in YEPAc and then shifted to sporulation medium. Samples were removed at indicated times and P-galactosidase activity was deter- mined. Points are average determinations with three to four inde- pendent cultures.

studies of ZME2 RNA levels (SMITH et al. 1990). Second, ime2-lac2 expression was restricted to ala cells; neither a cells or a cells expressed the fusion (Figure 3A). Third, mutations that affect normal IME2 expression also affected ime2-lac2 expression. T h e rmelA mutation allowed a cells and a cells to express levels of /3-galactosidase activity comparable to ala cells (Figure 3B and data not shown). The imelA and mcklA mutations caused diminished ime2- lac2 expression. Similar results have been obtained with a different ime2-lac2 fusion allele (SMITH et al. 1990; NEICEBORN and MITCHELL 199 1). Therefore, the expression of ime2-lacZ in the IME2-5-lacZ::URA3 allele accurately reflected the expression of the native IME2 gene.

Isolation of mutants defective in ime2-ZacZ expres- sion: We isolated mutations that block ZME2 expres- sion in strains with the genotype MATa rmelA5 ZME2-

72 S. S. Y . Su and A. P. Mitchell

TABLE 2

Phenotypes of rim mutants

imeZ-lacZ expressiona Percent sporulation'

Strain isolates a rmelA ala rmelAfrmelA 1 day 2 days 5 days Other phenotypes' No. of

Wild type N 146 288 71 97 100 ime 1 1 1 2 1 0 0 0 mckl 1 3 86 30 46 65 Bens rim 1 4 3 28 18 47 68 Smooth colony, colds r im2 1 1 ND 51 N D ~ ND rim3 1 4 ND 0 0 ND Starv5 rim4 4 25 92 0 0 10 rim5 2 5 ND 79 ND ND

rim 7 2 12 ND 45 ND ND

rim8 2 2 58 25 49 69 Smooth colony, colds rim9 1 2 40 21 62 67 Smooth colony, colds rim 1 1 1 2 39 10 44 56 rim 12 2 2 25 58 82 ND

rim 13 1 2 45 31 50 79 Smooth colony, colds rim1 5 1 4 22 3 12 52

a &Galactosidase assays of a representative haploid and homozygous mutant diploid from each complementation group were performed a s described in MATERIALS AND METHODS after 8 hr in liquid sporulation medium. Values are the averages of at least two independent assays in Miller units. ' Sporulation ability of homozygous mutants was quantitated on sporulation plates. N o spores were observed among 1000 cells for 0% sporulation. ' Other phenotypes associated with the mutants include the following: Ben' refers to Benomyl hypersensitivity at 17" (SHERO and HIETER

1991); smooth colony refers to the smooth colony morphology; cold? refers to slow growth at 17"; and starvS refers to sensitivity to nitrogen starvation.

Not applicable. e Not determined.

5-lacZ by screening for mutants that failed to express ime2-lacZ under starvation conditions. We isolated 135 EMS-induced mutants that were white on KAcXgal plates. Among these, 55 Xgal permeability mutants and 46 1ac.Z- mutants were eliminated as described in MATERIALS AND METHODS.

We expected to isolate imel and mckl mutants. Eleven imel mutants were identified by failure to complement an i m e l A tester for ime2-lacZ expression (Table 2). Results from mckl complementation tests were inconclusive; however, subsequent analysis indi- cated that one mutation was genetically linked to the m c k l A mutation (20PD:ONPD:OT). A plasmid carry- ing the M C K l gene also complemented the ime2-lacZ expression and sporulation defects in the mutant, which verified that the mutation is a mckl allele. We refer to this mutation as mckl-14.

We crossed each of the remaining mutants to a wild- type strain (MATa r m e l A 5 I M E 2 - 5 - l a d ) and sporu- lated the resulting diploids. In each case, the ability to express ime2-lacZ segregated 2+:2-. Therefore, the defect in each mutant lies at a single genetic locus. All of the mutations are recessive since ime2-lac2 expres- sion i n heterozygous diploids was as high as in wild- type diploids (data not shown).

These 22 mutations were tested for the ability to complement each other in all pairwise combinations. Twelve complementation groups were identified (see

Table 2). These genes were designated RIM for Reg- ulator of m E 2 .

We assayed 0-galactosidase activity i n one repre- sentative mutant in each complementation group to quantitate IME2 expression under sporulating condi- tions (Table 2). In haploid a or a strains, the ime2- lacZ expression levels of rim mutants were 5-20-fold lower than the wild type. In a/a diploid strains, the ime2-lacZ expression levels of rim homozygotes were 3-IO-fold lower than the wild type. Thus, the rim mutant defects were more severe in a or a haploids than in a/a diploids. The basis for this reproducible difference is unclear. However, for the sake of uni- formity, all subsequent quantitative measurements of ime2-lacZ expression were performed on a/a diploid strains.

Sporulation efficiency of rim mutants: Because IME2 is required for sporulation, we expected rim homozygotes to have sporulation defects. The rim3 and rim4 mutants sporulated poorly (Table 2). The rim5 mutant, on the other hand, sporulated at wild- type levels. In the remaining rim mutants, sporulation efficiencies ranged from 3-58% after 1 day and in- creased to 5 2 4 2 % after 5 days. These respective sporulation abilities cosegregated with each rim mu- tation. Because rim2, rim5, rim7 and rim12 mutants sporulated almost as well as wild-type strain, we elim- inated these mutants from further studies.

Activation of I M E l 73

TABLE 3

Segregation of PAml-IMEI and rim mutations

PAcTI-IME 1 IME 1 segregants segregants

Cross Relevant

genotypen Lac+ Lac- Lac+ Lac- tetrads Total

C X ~ - 1 PA(;T~-IMEI/IMEI 26 0 26 0 13 cx3-2 PA(:TI-IMEI/IMEI 27 I b 14 14 14

MCKl/mckl - I4

RIM4/rim4-I

R l M l l / r i m l l - l

RIM15/rim15-1

R I M l / r i m l - l

RIMBlrim8-1

RIMY/rimB-l

RIM I3/rim 13- 1

cX3-3 P~cTl-IME1/IMEI 15 13 13 15 14

cx3-4 P A C ~ I - I M E I / I M E I 9 13 13 9 11

CXS-5 PA~;Tl-IMEI/IMEl 9 9 9 9 9

C X ~ - 6 PAcTj-IMEI/IMEI 20 0 11 9 10

CXS-7 PA~TI-IMEI/IMEI 24 0 14 10 12

C X ~ - 8 PAcrl-IMEI/IMEI 20 0 10 10 10

cx3-9 PA(;r,-IMEI/IMEI 14 0 4 10 7

a Diploids constructed for all the crosses were homozygous for r m e l A and ime2-lacZ.

A PAcT,-IMEI segregant that failed to express ime2-lacZ exhib- ited a slow growth phenotype which did not segregate with mckl- 14. This segregant expressed ime2-lacZ only after longer incubation o n a KAcXgal plate.

Starvation response of rim mutants: Several pre- viously identified sporulation-defective mutants fail to arrest as unbudded cells. These mutants die when starved. Such mutations affect components of the adenylate cyclase pathway (bcyl, iral and ira2), an N- terminal acetyltransferase (ard l and n a t l ) , and a pro- tein kinase required for glucose-repressible gene expression (snfl-172) (TODA et al. 1987; TANAKA, MATSUMOTO and TOH-E 1989; TANAKA et al. 1990; WHITEWAY and SZOSTAK 1985; MULLEN et al. 1989; -rHOMPSON-JAEGER et al. 199 1). We expected to iso- late mutants in this class because bey1 mutants, for example, fail to express IMEl (MATSUURA et al. 1990) and, presumably, IME2.

We tested the rim mutants for sensitivity to nitrogen starvation. The rim?-1 mutant was more sensitive to nitrogen limitation and accumulated a higher per- centage of budded cells (26%) than wild type (5%) after nitrogen starvation. These phenotypes cosegre- gated with rim?-I in eight tetrads. In addition, IMEI and IME2 transcript levels were very low in the rim3- 1 mutant after starvation (data not shown). Linkage or complementation tests indicated that rim?-1 is not allelic to bcyl , i ral , i ra2, a rd l or snf l . However, na t l - 3 and rim?-1 mutants did not complement each other for ime2-lacZ expression or sporulation. We conclude that rim?-1 is most likely a natl allele.

Identification of rim mutations that reduce IMEl expression: The rim tnutations may cause reduced ime2-lacZ expression by reducing IMEl expression

TABLE 4

Suppression of rim mutations by P.,cTI-IMEI

ime2-lacZ expression'

Strain Relevant

genotype" Veg Spo sporulation' Percent

cx4-1 I M E l 5 290 99 C X ~ - 2 PAcrl-IMEI 8 310 100 cx4-3 i m e l A < I < I 0 cx4-4 imelA PA~TI- IMEI 3 320 96 cx4-5 rim4-1 <1 61 0 cx4-6 rim4-1 PAcTl-IMEI < I 63 0 cx4-7 r iml l -1 < I 11 2 cx4-8 rim1 1-1 PACTI-IMEI <1 11 3 cx4-9 riml-1 < I 10 23 cx4-10 riml-1 P,ACT,-IMEl <1 140 83 cx4-1 1 rim8-I < I 1 1 23 cx4-12 rim8-1 PAcTl-IMEl <1 120 92 cx4-13 rim9-I < I 14 27 cx4-14 rim9-1 PAcTl-IMEl <1 120 76 cx4-15 riml3-1 < I 58 44 cx4-16 riml3-1 PAcTI-IMEI < I 110 72

Diploid strains were homozygous for rmelA and ime2-lacZ as well as for the mutations listed.

/3-Galactosidase assays were performed for log phase cultures in YEPAc medium (Veg) and after 8 hr in liquid sporulation medium (Spo). Values in Miller units are the averages of at least two independent determinations.

Sporulation ability was quantitated after 24 hr in sporulation medium.

levels, or may cause reduced ime2-lacZ expression despite normal IMEl expression levels. We used three tests to distinguish these possibilities. First, we exam- ined the ability of IME1, expressed from the heterol- ogous ACT1 promoter, to suppress the rim mutations. Second, we tested the ability of the rim mutants to express HIS3 from an imel-HIS3 hybrid gene. Third, we analyzed IMEl RNA accumulation in each rim mutant through Northern analysis.

For suppression studies, we analyzed meiotic tetrads from PACTI-IM~I::TRPI/IME1 RIMlrim diploids (Table 3). We found that the PAcTI-IMEI hybrid gene did not suppress the rim4-1, r im l l - I and rim15-1 mutations. Among the PACTI-IMEI (Trp") segregants in cross cx3-3, for example, the numbers of Lac+ and Lac- segregants approximated the 1: 1 ratio expected for Mendelian segregation of rim4-1. Complementa- tion tests confirmed that the ability to express ime2- lacZ (Lac+) cosegregated with RIM4 and the failure to express ime2-lacZ (Lac-) cosegregated with the rim4-1 mutation. Likewise, the ime2-lacZ expression defects of rim1 1-1 and rim15-1 were not suppressed by PACT]- IMEI (Table 3, cross cx3-4 and cx3-5). Quantitative measurements confirmed our finding for rim4-1 and rim11-I (Table 4). These results agree with other studies of rim11 mutants (MITCHELL and BOWDISH 1992). We conclude that the ime2-lac2 expression and sporulation defects in rim4-1, rim11-I and rim15-1 are not a result of defective I M E l expression.

74 S. S. Y . Su and A. P. Mitchell

TABLE 5

Effects of r i m l - I , rim8-I, rim9-I and riml3-I mutations on imel- HIS3 expression

imrl-HIS3 segregants

Cross

cx5-1 i m ~ l - H l S 3 I 1 M E l R l M l l r i m l - 1 21 19 20 cx.5-2 ime l -HlS3 l lMEl R lMXlr imX-1 10 10 10 cx.5-3 imr l -HlS3 / lMEl RIMYlr imY-1 18 24 21 cx.5-4 ime l -HlS3 / lMEl R l1M13/ r im13-1 19 27 23

Relevant Rrllotype His+ His- tetrads

Total

Segregation analysis indicated that the PA<;rl-lMEl hybrid gene did suppress mckl-14, riml-1, rim8-1, rim9-1 and rim13-1 (Table 3, cross cx3-2, cx3-6, cx3- 7, cx8-8 and CXS-9). Among the 1MEl (Trp”) segre- gants i n crosses of these mutants to PAcTl-lMEl, the numbers of Lac+ and Lac- segregants approximated the 1: l ratio expected if the rim mutations were unlinked to 1 M E l . In contrast, all of the PAcTl-lMEl (Trp+) segregants were Lac+. These results agree with previous studies of M C K l (NEIGERORN and MITCHELL 199 1). Quantitative measurements indicated that PAcTI-lMEl increased ime2-lac2 expression in a r iml- I mutant by 10-fold and increased its sporulation efficiency (Table 4). The rima-1 and rim9-1 defects were also clearly suppressed by PAeTI-IMEl (Table 4). The rimI3-1 defects are so mild that quantitative suppression by PAcTl-lMEl is inconclusive. We con- clude that the ime2-lac2 expression and sporulation defects of mckl-14, r iml-I , r im8-1 and rim9-1 mutants are suppressed by PA~TI-IMEI expression.

T o determine whether the riml-1, rim8-1, rim9-1 and rim13-1 mutations affect l M E l expression, we examined HIS3 expression from the imel-HIS3 fusion gene in these mutants. This fusion places the HIS3 coding region downstream of the I M E l promoter and untranslated leader. We crossed a strain carrying the imel-HIS3 fusion gene to these rim mutants, sporu- lated the resulting diploids and analyzed the spores for growth on plates lacking histidine (Table 5) . Among the segregants that contained the imel-HIS3 fusion gene, the numbers of His+ and His- segregants approximated the 1: 1 ratio expected for Mendelian segregation of each rim mutation. Complementation tests denionstrated that each rim mutation cosegre- gated with the His- phenotype. Therefore, r iml-1 , rim8-1, rim9-1 and r iml3-1 may reduce basal l M E l expression through the l M E l promoter or may reduce RNA stability or translation through the IMEl 5 ’ leader.

We examined l M E l RNA levels by Northern analy- sis in riml-1, rim8-1, rim9-1 and rim13-1 mutants to distinguish between defects in l M E l transcript accu- mulation or translation (Figure 4). In the wild-type strain, I M E l transcript levels were low under vegeta- t ive conditions and increased i n sporulation medium.

A I wild t y p e , , r i d - l , , rim#-l , rimy-1 , h r s i n s p o O 2 4 6 0 2 4 6 0 2 4 6 0 2 4 6

/ME2 - /ME1 - 1

HOPI - SPS2 - SPSl - E

t 1

1 1

control - .S”WW&sW”*.

B , wild type I I riml3-1 ~

hrsinspo 0 2 4 6 0 2 4 6

IMF2 - lMEl - 1 1

HOPI - SPS? - SPSl -

control - [ i 1 FIGURE ~ . - I : \ I ) N M ~ I H I 0 1 \I)~)l.Lll.ltlon-specific genes in r i m l - 1 ,

rimR-I. rimy-1 ; u ~ l r i m l 3 - I nlutallts. RNA w w prepared from ala diploids during grow11 i n YEl’Ac (0 hr) and at 2 , 4 and 6 hr after transfer to sporulation medium. as indicated above each lane. Strains were homozygous for the mutations indicated. (A) Northern filter A contained RNA samples from wild type. r i m l - 1 , rimX-1 and rimy-1 mutants. (D) Northern filter €3 contained RNA samples from the same wild-type strain in filter A and RNA samples from the r i m l 3 - 1 mutant. Each filter was hybridized with probes for l M E l and l M E 2 . and then stripped and hybridized with control probe pC4/2. The blots were then stripped and hybridized to a probe for H O P 1 , and then stripped again and hybridized to probes for SPS1 and SPSZ. Two H O P I RNAs were detected, but onlv the predomi- nant RNA is designated HOPI (HOLLINGSWORTH. GOETSCH and BYERS 1990).

In each of the rim mutants, 1MEl transcript levels remained low in sporulation medium. These rim mu- tations also caused reduced RNA levels from the sporulation-specific genes lME2, HOPI , SPSl and SPS2 (Figure 4). The defect in lME1 expression was not due to general defects i n RNA accumulation because TUB2 and A C T l RNA levels were normal in these mutants (Figure 5) . We conclude that these rim mutations cause reduced l M E l RNA accumulation.

These studies suggest that the primary defect in riml-1, rim8-1, rimy-1 and riml3-1 mutants is the inability to accumulate l M E l RNA. As a result of the defect in 1MBl RNA accumulation, the expression of other sporulation-specific genes is defective in these mutants. Expression of l M E l from the A C T l pro- moter suppresses the rim mutations by restoring spor- ulation-specific gene expression. Because these muta- tions are recessive, we infer that they result i n loss of gene function. Formally, R l M l , R l M 8 , R I M 9 and

Activation of I M E l 75

lanes I 2 3 4 5

TUB2 -[r ]

control -

lanes genotype TUB2 expression ACTI expression (% wild type) (P wlld lype)

I rimy-llrim9-I 72 x9

2 rima-IIrimX-I 62 72

3 riml-l/riml-l 76 X3

4 wild rype 1 0 0 I 0 0

5 riml3-llriml3-I I I7 9R

FIGURE 5,"Expression of T U B 2 and A C T I in r i m l - I , r i m R - I . r imy-1 and r i m l 3 - I mutants. R N A w a s prepared from homozygous mutants during growth in YEPAc. Northern filter contained R N A samples from strains a s indicated in the table, and hybridized with ;I probe for TUBS. The filter was stripped and hvbridized with conrrol probe pC4/2. and then stripped and hvbridized with a probe for A C T I . TUB2 and A C T 1 expression w a s quantitated from ;Icltoladiograms using densitometry. normalized relative to RNA loading control, and expressect a s a percentage of wild-type expres- sion.

R I M 1 3 are positive regulators of I M E l . These gene ~~roducts may act through the I M E l promoter or 5' leader to stimulate transcript accumulation.

The riml-1, r im8-1, r imy-1 and rim13-1 mutants shared two additional phenotypes. First, their colony morphology is not as rough as the parent strain and other SK- 1 derivatives (Table 2). Smooth colony mor- phology cosegregated with each mutation in every cross. Second, all of these mutations cause slow growth at 17". Because imel mutations do not cause smooth colony morphology or cold sensitive growth, these KIM gene products must do more than simply activate IME I expression.

RIMI, RIM8, RIM9 and RIM13 act in parallel to MCKl to activate IMEI expression: Mutations in M C K I and in R I M 1 , R I M 8 , R l M 9 and R I M 1 3 all cause diminished I M E l expression. These gene products m a y act in the same pathway or through different pathways to activate IME1 expression. I f two genes act i n the same pathway, then the phenotype of the double mutant should be the same as either single mutant. On the other hand, if two genes act through different pathways, then the phenotype of the double m u t a n t should be more severe than either single mu- tant. To distinguish between these two possibilities, we analyzed the ime2-lacZ expression levels and spor- ulation efficiencies of mcklArim double mutants and rim rim double mutants (Table 6). ime2-lacZ expres- sion i n the wild-type strain was high after 8 hr in sporulation medium. ime2-lacZ expression in m c k l A and r i m l - 1 mutants was reduced 3-fold and 20-fold, respectively. ime2-lacZ expression in the mcklArim1-1

TABLE 6

Phenotypes of mcklArim double mutants

expression' imcPlacZ

Relevant Percent

Strain genotype' Veg Spo (total)c sporulation

cx6-I Wild type 3.0 292 95 cx6-2 m c k l A 0.5 86 72 cx6-3 r i m l - I 0.1 8 24 cx6-4 rim#-I 0.2 20 55 cx6-5 rimy-I 0.1 25 49 cx6-6 rim 13- I 0.3 22 42 cx6-7 m c k l A r i m l - I 0.2 0.4 8 cx6-8 m c k l A r imR- I 0.1 0.2 5 cx6-9 m c k l A rim9-1 0.1 0.3 10 cx6-10 m c k l A r i m l 3 - 1 0.1 0.3 9

a Diploid strains were homozygous for r m e l A and ime2-lacZ as well a s for the mutations indicated. ' j3-Galactosidase assays were performed for log phase cultures i n YEPAc medium (Veg) and after 8 hr in sporulation medium (Spo). Values i n Miller units were the average of at least two independent determinations.

Sporulation ability was quantitated after 24 hr in sporulation medium. The total values include mature and immature asci.

mckl AIMCKI mckl NMCKI mckl Nmckl A riml-IIRIMI r i m l - l l r i m ~ - ~ r i d - l l r i m l - I "-

hrsinspo 0 2 4 6 0 2 4 6 0 2 4 6 I - . ~. I

control - " I .

FIGURE 6.-Expression of / . M E 1 and l.llE2 i n r iml-1 and m r k l A r i m l - 1 double mutant. RNA was prepared from a/n diploids during growth in YEPAc (0 hr) and 2.4, and 6 hr after transfer to sporulation medium. as indicated above each lane. Strains were m c k l A / M C K I r i m l - l / R I M l , m c k l A / M C K I r i m l - l / r i m l - l and mcklA/mchlAriml-l/rim-l. A single Northern filter was hybridized with probes for I M E l and I M E 2 , and hen stripped and hybridized with control probe pC4/2.

mutant was reduced 700-fold. Northern analysis con- firmed that the mcklAriml-1 mutant had even lower IME1 RNA levels than the r i m l - 1 mutant (Figure 6). The sporulation defect of the mckIArim1-1 mutant was also more severe than either single mutant (Table 6) . Similarly, the mcklArim8-1, mcklArim9-1, and mcklAriml3-1 mutants displayed more extreme ime2- lacZ expression and sporulation defects than the single mutants. ime2-lac2 expression levels and sporulation efficiencies of all pairwise double mutants of r i m l - 1 , rim8-1, rim9-1 and r iml3-1 were comparable to those of the single mutants (Table 7). We conclude that R I M l , R I M R , R I M 9 and R I M 1 3 act in a single pathway to stimulate I M E l expression and that the RIM118191 1 3 pathway is independent of the MCKl pathway.

DISCUSSION

We have isolated 34 recessive mutations that cause defects in ime2-lacZ expression. These mutations de-

S. S. Y . Su and A. P. Mitchell 76

TABLE 7

Phenotypes of rim rim double mutants

ime2-lacZ expression' Percent

Strain genotypea "eg Spo (t0tal)C Relevant sporulation

cx7-1 Wild type 1.6 270 90 cx7-2 riml-1 0.2 28 30 cx7-3 rim8-1 0.2 58 44 cx7-4 rimy-1 0.3 40 40 cx7-5 riml3-1 0.2 45 42 cx7-6 riml-1 rim8-1 0.3 30 24 cx7-7 riml-1 rimy-1 0.2 30 28 cx7-8 riml-1 rim13-1 0.2 30 23 cx7-9 rim8-1 rimy-1 0.2 54 41 cx7-10 rim8-1 riml3-1 0.2 24 33 cx7-11 rimy-1 riml3-1 0.3 65 40

a Strains were homozygous for rmelA and ime2-lacZ as well as for the mutations indicated. ' @-Galactosidase assays were performed for log phase cultures in YEPAc medium (Veg) and after 8 hr in sporulation medium (Spo). Values in Miller units are the averages of two independent determinations.

Sporulation ability was quantitated after 24 hr in sporulation medium.

fined 14 genes, including the previously identified genes IMEI , MCKl and NATI . The finding that most RIM genes were defined by single isolates suggests that repeated application of our screen may yield mutations in additional genes. The limited size of the screen may explain the absence of ime4 and rim16 mutations (SHAH and CLANCY 1992; MITCHELL and BOWDISH 1992), which should have been detectable.

Although IME2 is required for sporulation, several mutants with ime2-lac2 expression defects had mild sporulation defects. rim mutations that cause no spor- ulation defects may affect only ime2-lac2 expression and not expression of the natural IME2 gene. Muta- tions with severe effects on ho-lac2 expression and not on the natural HO RNA have been reported previ- ously (BREEDEN and NASMYTH 1987). On the other hand, most mutations cause defects in both ime2-lac2 expression and sporulation. The partial sporulation defects in these mutants are consistent with reduced /ME2 expression.

Why do several mutants display only partial defects in ime2-lacZ expression levels and sporulation? One explanation is that these mutations may cause a partial loss of RIM gene function. The r iml l - I mutation described in this study is an example because rim11 alleles described elsewhere cause an absolute sporu- lation defect (MITCHELL and BOWDISH 1992). An- other possible explanation is that these mutations cause a complete loss of RIM gene function, but that activity from a second pathway can partially suppress the mutant defects. In this study, we found that the residual ability of rim1/8/9/13 mutants to express ime2-ZacZ and to sporulate is largely due to the activity of MCK I.

RIMl , R IM8, R IM9 and RIM13 (which we designate the R I M l pathway genes) stimulate IME2 expression indirectly, by stimulating IMEl transcript accumula- tion. MCKl also stimulates IMEl transcript accumu- lation. Our measurements of ime2-lac2 expression and sporulation indicate that defects in R I M l pathway genes and MCKl are additive. Measurements of IMEl RNA levels in a mckIArimI-1 double mutant is con- sistent with our conclusion. Thus, R I M l pathway genes and MCKl act independently to stimulate IMEl expression.

Additional phenotypes of mckl and rim1/8/9/1? mutants support the conclusion that these genes func- tion in distinct pathways. For example, mckl mutants grow well at 17" but are Benomyl hypersensitive at that temperature (SHERO and HIETER 1991). rim1/8/ 9 / 1 3 mutants grow poorly at 17" but are not Benomyl hypersensitive (S. SU, unpublished results). Another example is that mckl mutants accumulate immature asci. rim1/8/9/13 mutants accumulate higher levels of immature asci than wild-type strains but, unlike mckl mutants, the asci mature after prolonged incubation. Finally, mckl mutants have rough colony morphology like our isogenic wild-type strains, whereas rim1/8/9/ l? mutants have a distinct smooth colony morphology. All of these observations suggest that the RIM1 path- way genes function independently from M C K l .

We suggest that the R I M l pathway gene products are positive regulators of IMEl because recessive mu- tations in these genes reduce IMEI expression. The basis of the pleiotropic phenotypes of the R I M l path- way mutants is unclear. We anticipate that suppressor analysis may reveal target genes that influence colony morphology and growth at low temperatures. With regard to IMEI expression, one question is what the relationship is between the R I M l pathway genes and IME4. They may either act in the same pathway or distinct pathways. A second question is what signal the R I M l pathway transmits. Any of the signals that gov- ern IMEl expression, which include al-a2, glucose, and nitrogen, may influence R I M l pathway activity. The R I M l pathway may also respond to the IME2- dependent signal that down-regulates IMEl expres- sion. A final question is what the relationship is be- tween the RIM1/8/9/1? gene products. For example, they may act in a linear pathway or as subunits of a protein complex. Molecular analysis of the R I M l path- way genes may provide insight into the signals that they transmit and their mechanisms of action.

We thank members of this laboratory for their assistance and advice. We are grateful to LENORE NEIGEBORN, MARIAN CARLSON and DAVID FIGURSKI for discussion and comments on this manu- script. We also thank B. DUNN, M. A. OSLEY, S. POWERS, M. SCIDMORE, S. KURTZ, M. CARLSON and R. STERNGLANZ for provid- ing plasmids and strains for this study. This work was supported by U.S. Public Health Service grant GM3951 and by funds from the Irma T. Hirschl Charitable Trust and the Searle Scholars Program/

Activation of ZMEl 77 ~1 I he Chicago Community Trust. S.S.Y.S. was supported, in part, as ;I predoctoral trainee in the Cellular, Molecular, and Biophysical Studies Training Program (GM 08224), and on a Cancer Biology training grant.

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Communicating editor: E. W. JONES