MOLECULAR AND CELLULAR BIOLOGY, Dec. 1990, p. 6103-6113 0270-7306/90/126103-11$02.00/0 Copyright © 1990, American Society for Microbiology

Vol. 10, No. 12

Role of IMEJ Expression in Regulation of Meiosis in Saccharomyces cerevisiae HAROLD E. SMITH, SOPHIA S. Y. SU, LENORE NEIGEBORN, SUZANNE E. DRISCOLL, AND AARON P. MITCHELL* Institute of Cancer Research and Department of Microbiology, College of Physicians and Surgeons, Columbia University, New York, New York 10032 Received 23 May 1990/Accepted 28 August 1990

Two signals are required for meiosis and spore formation in the yeast Saccharomyces cerevisiae: starvation and the MAT products al and a2, which determine the a/a cell type. These signals lead to increased expression of the IMEI (inducer of meiosis) gene, which is required for sporulation and sporulation-specific gene expression. We report here the sequence of the IME1 gene and the consequences of IMEI expression from the GAL] promoter. The deduced IMEI product is a 360-amino-acid protein with a tyrosine-rich C-terminal region. Expression of PGAL,-IMEJ in vegetative a/a cells led to moderate accumulation of four early sporulation-specific transcripts (IME2, SPOIl, SP013, and HOP)); the transcripts accumulated 3- to 10-fold more after starvation. Two sporulation-specific transcripts normally expressed later (SPS1 and SPS2) did not accumulate until PGALI-IMEl strains were starved, and the intact IMEI gene was not activated by PGALI-IMEl expression. In a or a cells, which lack a2 or al, expression of PGALI-IMEl led to the same pattern of IME2 and SP013 expression as in ala cells, as measured with ime2::lacZ and spol3::lacZ fusions. Thus, in wild-type strains, the increased expression of IMEI in starved a/a cells can account entirely for cell type control, but only partially for nutritional control, of early sporulation-specific gene expression. PGALI-IMEl expression did not cause growing cells to sporulate but permitted efficient sporulation of amino acid-limited cells, which otherwise sporulated poorly. We suggest that IME1 acts primarily as a positive regulator of early sporulation-specific genes and that growth arrest is an independent prerequisite for execution of the sporulation program.

Sporulation of the yeast Saccharomyces cerevisiae is a cellular differentiation pathway (reviewed in references 12 and 26). It is normally restricted to one type of cell, the a/a cell, and is induced by nitrogen starvation. These circumstances lead to arrest of the mitotic cell cycle, to expression of sporulation-specific genes, and to initiation of the sporulation program. Cells engage in meiotic DNA synthesis, recombination, and two meiotic divisions. Each of the four meiotic products is packaged into a spore, and the four spores of the cell are encased in a sac, the ascus. Sporulation thus includes meiosis and spore formation. One of the earliest unique events in starved a/a cells is elevated accumulation of IME1 RNA (22, 28, 38; reviewed in reference 27). The IME1 product is thought to play a pivotal role in activating meiosis, because multicopy IME1 plasmids permit sporulation in cells that lack the determinants of a/a cell type, the MATal and MATa2 gene products, and also permit meiotic recombination in the absence of nitrogen starvation (22, 38). Conversely, mutations that reduce IME1 expression, such as imel null mutations (22, 38) or cmkl mutations (L. Neigeborn and A. P. Mitchell, unpublished data), abolish or impede sporulation. Therefore, IME1 expression is thought to transmit both cell type and nutritional signals to stimulate meiosis (22, 38). Sporulation is accompanied by express'ion of a unique set of genes, the sporulation-specific genes. Some of these genes are essential for particular meiotic events, and others have no essential role in sporulation under laboratory conditions (1, 6, 11, 14, 17, 21, 25, 26, 33, 34, 42, 45, 47). These genes fall into early, middle, and late expression classes (1, 23, 25, 26). imel null mutations block accumulation of the IME2, *

SPOIl, SPO13, SPSJ, and SPS2 transcripts (28) and block merl::lacZ expression (11). Thus, IMEJ is required for expression of early and middle sporulation-specific genes and perhaps late genes as well. Sporulation-specific gene expression and sporulation itself may be mandatory consequences of IME1 expression. Alternatively, IMEI expression may establish a permissive state which requires further direction from cell type or environmental signals for sporulation-specific transcription or for execution of the sporulation program. With this question in mind, we have examined the consequences of IMEI expression, in novel contexts, from a new promoter. MATERIALS AND METHODS Strains and genetic markers. Yeast strains were all derived from strain SK1 (a/a HOIHO) through transformation (18) or standard genetic manipulation (37) and are listed in Table 1. The imel-12, ime2-2, gal80, ho::LYS2, his4-G, and his4-N mutations have previously been described, as have the auxotrophic markers in these strains (28, 38). We note that the ho::LYS2 insertion confers a weak Lys' phenotype. The a/a diploid (strain 545) was derived from an a/a diploid (strain 537) after mild UV irradiation by screening colonies for mating-factor production through a halo assay (46). Engebrecht and Roeder observed that a/a and a/a diploids in the SK1 background were able to sporulate at a low level (strains J254 and J256 [11]). In side-by-side comparisons, we confirmed that J254 and J256 were able to sporulate and that our strain 545 was unable to sporulate. Fourteen four-spored tetrads were analyzed from an a/a/a/a tetraploid derived from crossing strains 545 and J256. Nine segregants were able to mate and able to sporulate weakly; 25 segregants were able to mate but unable to sporulate. These observations indicate that the difference in sporulation abilities

Corresponding author. 6103

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SMITH ET AL. TABLE 1. Yeast strains Genotype'

Strain 107 ....... a GAL80 108 ........a GAL80 263 ....... a imel-12::TRPI his4-G 471 ....... a imel-12::TRPI his4-G ade3 474 ........a his4-G ade3 476 ........a imel-12::TRPI his4-N arg6 477 ....... a his4-N arg6 537 ....... a/a his4-Glhis4-N arg6l+ ade3l+

MOL . CELL . BIOL . (A) pAM504 1 Kb

a Unless otherwise noted, all haploid strains have the additional markers ura3, leu2::hisG, trpl::hisG, lys2, ho::LYS2, and gaI8O::LEU2, and all diploids are homozygous for these markers.

between strains 545 and J256 derives from a genetic difference rather than from a technical difference in sporulation procedures. The ime24::1acZ fusion, a gene replacement (35) that includes LEU2 as a selectable marker, was introduced by transforming an a/a leu2IIeu2 diploid with a BglII digest of plasmid pLN1404 (Fig. 1). Southern analysis of Leu+ transformants confirmed replacement of one IME2 allele with ime24::1acZ. Meiotic analysis of one such diploid revealed single-gene segregation of the Leu+ phenotype, and all Leu+ progeny displayed the expected, recessive ime2 defect. The chromosomal PGAL,-IMEJ allele, which we designate IMEJ-14, includes TRPJ as a selectable marker. It was introduced by transforming cells with a ClaI digest of plasmid pHS153 (Fig. 1) and selecting Trp+ transformants. The structure of the integrated allele was confirmed by Southern analysis. IMEJ-14 complements imel defects in gal8O/gaI80 diploids but not in GAL8OIgaI80 or GAL801 GAL80 diploids; gal80 mutations permit expression from the GAL] promoter in the absence of galactose (43), as in sporulation media. Media and culture conditions. Synthetic growth media, sporulation plates, and liquid sporulation medium have been

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(YCPPGALI) 539 ....... a/a his4-Glhis4-N arg6l+ ade3l+ (YCpPGAL,-IMEI) 545 ....... a/a his4-Glhis4-N arg6l+ ade3l+ 571 ........a met4 (pBW2) 572 ....... a his] (pBW2) 573 ....... a/a diploid from 571 x 572 574 ....... a IMEI-14::TRPI met4 (pBW2) 575 ........a IMEI-14::TRPI met4 (pBW2) 576 ....... a/a diploid from 574 x 575 AM1318-2B ....... a imel-12:: TRPI GAL80 a imel-12::TRPI AM1318-5B ....... a imel -12:: TRPI ime24: :IacZ AM1345-2B ........ a imel-12::TRPI ime24::IacZ met4 AM1345-3C ....... a/a imel-12:: TRPllimel-12:: TRPJ ime2AM1345XT1 ....... 4::IacZlime24: :IacZ met4l+ (YCPPGALI) a/a imel-12:: TRPlIimel-12:: TRPJ ime24:: AM1345XT2 ....... 1acZ/ime24: :IacZ met4l+ (YCPPGALI_ IMEI) J254 ....... a/a crylicryl TRPJITRPJ GAL801GAL80 MERJ/merl::Tn164 J256 ....... a/a crylJcryl TRPIITRPI GAL801GAL80 MERl/merl: :Tn 164 LN764-4D ....... a ime24::lacZ met4 a IMEI-14::TRPI ime2-2::LEU2 SED16-7D ....... a IMEI -14:: TRPI SED16-11C ........ SS851-3D ....... a ime24: :IacZ (YCpP(;AL,-IMEJ) a ime24::lacZ hisl met4 (YCpPqrAL,-IMEI) SS851-7B ........ SS852-6D ....... a ime24::IacZ his] met4 (YCPP,AL,) SS852-16A ....... a ime24::lacZ hisl met4 (YCP$GALI) a imel-12::TRPI his4-G (YCp,"GAL,-IMEI) T88-2A ........

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FIG. 1. Plasmid insert restriction maps. (A) pAM504 insert, carried in plasmid pUC18. The thickest black line indicates the interval that was sequenced; the thinner black line indicates flanking DNA that was not sequenced. The straight arrow represents the extent of the IMEI open reading frame, and the bent arrow represents the 5' end of the IME1 RNA. (B) YCpPGAL,-IMEJ insert, carried in plasmid YCpSO. The open bar represents the GALl,10 promoter, and the bent arrow represents the 5' end of the GAL] RNA. Other symbols are as described for panel A. (C) pHS153 insert, carried in plasmid YCpSO. The shaded bar represents the TRPI gene. Other symbols are as described for panel A. (D) pLN1403 insert, carried in plasmid YEp24. The black line represents two segments derived from IME2, and the shaded bar represents a LEU2 insertion. (E) pLN1404 insert, carried in plasmid YEp24. The diagonally striped bar represents lacZ sequences, and other symbols are as for panel D. Abbreviations: B, BamHI; C,

ClaI; E, EcoRI; G, BglIl; H, Hindlll; K, KpnI; N, NcoI; Nr, NruI; P, Pstl; Pv, PvuIl; S, Sall; Sp, SphI; X, XhoI. Asterisks indicate inactivation of a site through construction; underlining refers to sites in flanking vector sequences.

described previously (28, 37, 38). YEP media contained 1% yeast extract (Difco) and 2% Bacto-Peptone (Difco) plus 2% dextrose (for YEPD) and 2% potassium acetate (for YEPAc). Solid media contained 2% agar. SAcCA media contained 0.67% yeast nitrogen base without amino acids (Difco), 2% potassium acetate, 2% Casamino Acids (Difco), 20 mg of adenine sulfate per liter, 20 mg of histidine hydrochloride per liter, and either 20 (for SAcCA+Trp) or 2 (for SAcCA-Trp) mg of tryptophan per liter. MSAc media contained 0.67% yeast nitrogen base without amino acids, 2% potassium acetate, 20 mg each of histidine hydrochloride, lysine hydrochloride, tryptophan, and leucine per liter,

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CONSTITUTIVE EXPRESSION OF A MEIOTIC ACTIVATOR

and 100 mg each of glutamate' aspartate, and arginine hydrochloride per liter. The last three amino acids were added to supplement pools of tricarboxylic acid cycle intermediates. MSAc-Lys was prepared without lysine, and MSAc-His was prepared with 2 mg of histidine per liter. All experiments were conducted at 30°C. Tryptophan limitation experiments were carried out as follows. Cells from a fresh SC-Ura (37) overnight culture were inoculated at a 1/20 dilution into SAcCA+Trp and incubated for 6 to 7 h to permit adaptation and approximately one doubling. Cells from 5 ml of culture were collected on a nitrocellulose filter and suspended in 5 ml of SAcCA-Trp. Cells from 10 ml of culture were shifted, similarly, to 5 ml of sporulation medium. A sample of the SAcCA+Trp culture was then removed for determination of His+ recombinant and sporulation frequencies. The other cultures were incubated for 24 h before determination of His+ recombinant frequencies and for 48 h before quantitation of sporulation. Histidine and lysine limitation were accomplished in essentially the same way. Cells were grown in MSAc and shifted to MSAc-His and MSAc-Lys. Sporulation was quantitated after 24 h. Wild-type a/a diploids sporulate after saturation in SAcCA and MSAc media (1 x 107 to 2 x 107 cells per ml); thus, amino acid limitation experiments were conducted at 1/10 the saturation density. Plasmids. Diagrams of relevant plasmid inserts are shown in Fig. 1. Construction of YCPPGAL,-IMEJ made use of a low-copynumber (CEN) plasmid, pBM272 (provided by M. Johnston via I. Herskowitz), which carries the URA3 gene and GALl ,10 promoter. pBM272 is a derivative of pBM150 (19) that contains both HindlIl and BamHI sites downstream of the GAL] promoter and RNA start site. YCPPGAL,-IMEJ was constructed by inserting a 2.2-kbp HindIII-SalI fragment from pAM504 into the HindIII and SalI sites of pBM272; the Sall site in pAM504 derives from vector sequences. pHS153, the source of the integrated PGAL,-IMEJ allele, was constructed in two steps. First, the 1.2-kbp EcoRI-ClaI fragment from YCpPGAL,-IMEI (containing the GAL] promoter and 5' end 6f the IME) coding region) was inserted between the EcoRI and ClaI sites of YCp5O to create pHS152-B. Second, a 1.55-kbp EcoRI fragment from plasmid pHS113-B (containing the IMEJ upstream region and the TRPI gene [38]) was inserted into the EcoRI site of pHS152-B. pHS153 contained the TRPJ gene adjacent to, and oriented divergently from, the GAL] promoter (Fig. 1). pHS154 was constructed by inserting a 2.4-kbp SphI-NruI fragment from pAM504 into the SphI and NruI sites of

YCp5O. The ime2::lacZ fusion was constructed as follows. First, the IME2 5' end was fused to the lacZ gene in three different reading frames by inserting a 1.1-kbp BamHI-PvuII fragment from plasmid pHS101 (38) into the BamHI and SmaI sites of plasmids YEp356R, YEp357R, and YEp358R (31). Only the fusion in YEp358R was active; induction of ,-galactosidase by starvation was restricted to a/a cells and required a wild-type IMEJ allele. Subsequent nucleotide sequence analysis indicated that the YEp358R derivative, pHS122-C, contained lacZ fused in frame at codon 39 of the IME2 open reading frame (48; S. Su, unpublished results). The integrating ime2::IacZ fusion was then constructed in two steps. First, plasmid pLN1403 (Fig. 1) was constructed by partial PvuII digestion of pAM403 (38), followed by ligation with KpnI linkers to inactivate a vector PvuII site. The lacZ gene from plasmid pMC1871 (3), isolated as a 3.1-kbp SmaI-Sall

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fragment, was inserted into the unique PvuII and XhoI sites of pLN1403 to create plasmid pLN1404 (Fig. 1). This plasmid maintained IME2 fused in frame to lacZ, as in pHS122-C. Plasmid pSD103 (see Fig. 3A) was constructed by inserting an 822-bp BglII-PstI fragment from plasmid pAM504 into the BamHI and PstI sites of Bluescript plasmid SK+ (Stratagene). The YCp5O-MATx plasmid, constructed by J. Margolskee, was described previously (29). Plasmid pBW2, a multicopy plasmid that includes URA3 and a spol3::IacZ fusion, was provided by R. E. Esposito. Nucleotide sequence of IME1. Cloned segments suitable for sequencing were constructed by inserting a variety of pAM504-derived restriction fragments into M13mpl8 and M13mpl9. Dideoxy sequencing of single-stranded bacteriophage DNA was conducted with [a-35S]dATP and modified T7 DNA polymerase (Sequenase; U.S. Biochemical Corp.) as instructed by the manufacturer. Three oligonucleotides, synthesized on an Applied Biosystems 381A DNA synthesizer, were used to complete the sequence. They hybridized to positions -341 to -322 (5'-CCTCTTTT'ATGTAT'-TTAA GG-3'), -198 to -180 (5'-CATCGACTATCCTATTACG3'), and 1176 to 1157 (5'-CTTAGCAATTTGAGTGAGAC3'). The sequences of both strands of the 2.4-kbp SphI-NruI fragment were determined. DNA and protein sequences were analyzed by using the Bionet resource of Intelligenetics, Inc. Northern (RNA) blots and probes. Ceils were transferred during log-phase growth in YEPAc (107 cells per ml) to sporulation medium (38). Methods for the preparation of RNA, electrophoresis, and transfer to nitrocellulose have been described (28, 38); 10 ,ug of total RNA was loaded per lane. Probes for IMEI, IME2, SP013, SPSJ, and SPS2 RNAs have been described (28, 38). The control probe was random-primed plasmid pC4, which hybridizes to an RNA unaffected by starvation or cell type (25). The SPOIl probe was a random-primed, 1.1-kbp AccI-EcoRI restriction fragment of a SPOIl clone (1). The HOPI probe was a randomprimed BamHI-HindIll fragment from the HOPI gene (17).

P-Galactosidase assays. ,B-Galactosidase assays were carried out on chloroform-sodium dodecyl sulfate-permeabilized cells as described by Stern et al. (39). Culture samples were routinely frozen before assays were conducted. RNase protection assays. RNase protection assays used 10 ,ug of total RNA per sample and were done according to a standard protocol (2) except that RNase T1 alone was used for digestion. (We found that RNase A cleaved probes at internal sites.) Products were fractionated on an 8% polyacrylamide sequencing gel, and sizes were estimated by comparison with end-labeled, denatured XX174 DNA fragments. Because single-stranded DNA has greater mobility than does single-stranded RNA of the same length in these gels, we calculated the actual size of the protected probe (444 bases) as 0.925 x the extrapolated size of the protected probe from the gel (480 bases [2]). Nucleotide sequence accession number. The sequence reported is available from GenBank (accession number M37188). RESULTS Nucleotide sequence of IMEI. Subcloning of IMEJ identified a 2.4-kbp SphI-NruI DNA fragment that complemented the sporulation defect of an a/a imel-12::TRPJ/imel-12:: TRPI diploid when carried in the low-copy-number plasmid

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SMITH ET AL.

pHS154. The nucleotide sequence of this segment is shown in Fig. 2. Analysis of the sequence revealed a single long open reading frame (Fig. 2) that is oriented in the same direction as the IMEl transcript (22, 38). Evidence presented below indicates that this open reading frame encodes the IME1 product. The open reading frame has potential initiation codons at nucleotides +1 and +13. RNase protection assays indicate that the 5' end of the IMEI RNA lies approximately 240 bases upstream of the +1 AUG codon (Fig. 3): the corresponding protected band is induced by starvation and abolished by an imel deletion. Because translatiQn of most mRNAs initiates at the AUG closest to the 5' end (reviewed in reference 5), we provisionally designate the AUG at +1 the initiation codon. We note that the four nucleotides preceding +1 (CAAA) are more similar to the consensus nucleotides that precede yeast initiation codons (A/YAAJUA [5]) than are those preceding +13 (GGAT). The deduced IME1 protein product contains 360 amino acids and has a molecular weight of 41,832. The protein has no long hydrophobic segment, and the 49 acidic and 32 basic residues display little clustering. The 23 tyrosine and 26 phenylalanine residues are relatively numerous, particularly in the carboxy-terminal half of the protein: of 180 residues, 20 are tyrosine and 13 are phenylalanine. There are no consensus protein kinase A target sites (Arg-Arg-X-Ser/Thr [10]), nor are there segments similar to known nuclear localization signals (4). The IME1 product has no striking similarity to any protein in the PIR and EMBL data bases. It also has no extended homology to IME2, a protein kinase homolog (48) that can substitute for IME1 in promoting sporulation and activating sporulation-specific genes (28, 38). It also lacks sequences characteristic of transcriptional regulators, such as a zinc finger (13), helix-turn-helix (8), homeo domain (36), helixloop-helix (7), leucine zipper (24), or MCM1/SRF homology

(32).

Conditional expression of IMEI. Functional activity of the putative IMEI open reading frame was tested by placing it under control of the GAL] promoter. We used a low-copynumber YCp5O-based plasmid that contains the GALI promoter and RNA start site (19), plasmid pBM272, which we refer to as YCPPGALI. The plasmid has a HindIII site 67 nucleotides downstream of the GAL] RNA start site. The IMEI open reading frame was inserted by using a naturally occurring HindIII site, at position -31 (Fig. 2). We designate the GAL] promoter-IMEJ coding region fusion PGAL,-IMEJ and the entire plasmid YCPPGAL,-IMEl (Fig. 1). YCPPGALI-IMEJ function was assessed through its ability to complement an imel deletion. Two isogenic ala imel-12::TRPlIimel-12::TRPl diploids were constructed which differed at the GAL80 locus; GAL80 is a negative regulator of the GAL] promoter that confers galactose dependence (43). One diploid was homozygous for a gal80 deletion (strain AM1318-5B x T88-2A) and thus was able to express PGAL,-IMEJ at high levels in typical, galactose-free sporulation medium; the other diploid had a wild-type GAL80 allele (strain AM1318-2B x T88-2A) and thus was unable to express PGAL,-IMEJ at high levels in sporulation medium. The gal80 deletion homozygote was able to sporulate (68%) when it carried YCpPGAL,-IMEI but was unable to sporulate (