JOURNAL OF BACTERIOLOGY, Dec. 1996, p. 6865–6872 0021-9193/96/$04.0010 Copyright q 1996, American Society for Microbiology Vol. 178, No. 23 A Stationary-Phase Gene in Saccharomyces cerevisiae Is a Member of a Novel, Highly Conserved Gene Family EDWARD L. BRAUN, EDWINA K. FUGE, PAMELA A. PADILLA, AND MARGARET WERNER-WASHBURNE* Department of Biology, University of New Mexico, Albuquerque, New Mexico 87131 Received 18 December 1995/Accepted 10 September 1996 system for the study of growth control during nutrient limitation (30, 74, 77). When yeast cells are grown to stationary phase in rich, glucose-based medium, several distinct phases are observed (see references 75 and 76 for reviews). Once rapid growth begins, after lag phase, energy is derived primarily from fermentation of glucose. When glucose is exhausted, a transient growth arrest occurs (the diauxic shift), during which time cells adapt to respiratory metabolism (5, 30, 66). After this arrest, cells grow at a much lower rate for several days prior to entering stationary phase (17, 30). Stationary phase, defined as the time when there ceases to be a net increase in cell number, is a differentiated state (75, 76) during which cells enter a genetically defined offshoot of the cell cycle (41). The synthesis of most postexponential proteins in S. cerevisiae is initiated during the diauxic shift (5, 30). In dramatic contrast, synthesis of one protein, Snz1p (previously designated p35) is detected much later than other proteins (30). In this paper, we report the cloning of the S. cerevisiae SNZ1 (snooze) gene, which encodes Snz1p. Unlike many other postexponential proteins, Snz1p synthesis is not regulated by heat shock or glucose repression (30). Like Snz1p synthesis, the SNZ1 mRNA accumulates after entry into stationary phase, but not after a heat shock, suggesting that Snz1p is a component of a specific growth arrest response rather than a general stress response. On the basis of sequence analysis and lowstringency hybridization, homologs of the SNZ1 gene are present in members of all three phylogenetic domains. These analyses revealed that Snz1p is among the most evolutionarily conserved proteins yet identified (see references 34, 36, and 37). This evolutionary conservation will allow exploration of the universal nature of a growth arrest response. The proper regulation of cellular proliferation and growth in response to extracellular cues is critical for development and the maintenance of viability in all organisms (4, 45, 75, 76). The primary regulators of cellular growth and proliferation in multicellular organisms are growth factors and hormones, and the loss of this regulation may lead to neoplasia (4). Similar regulation of growth and proliferation exists in unicellular organisms, such as the budding yeast Saccharomyces cerevisiae, but nutrient availability is typically the most important extracellular cue in unicellular organisms (4, 45, 75, 76). Given the universal distribution of mechanisms for regulating cellular growth and proliferation and the obvious selective advantage of proper growth regulation, it is likely that the last common ancestor of all organisms, termed the cenancestor (22, 26), also had mechanisms to control growth and proliferation in response to nutrient limitation. Aspects of general stress responses, such as the heat shock response, are distributed universally among organisms (18, 47). During the heat shock response, synthesis of specific evolutionarily conserved proteins increases (8, 15, 18, 36, 47). The increased synthesis of the heat shock proteins occurs in response to increased temperature and other stresses in all three phylogenetic domains (defined in reference 80), suggesting that the heat shock response in extant organisms reflects the conservation of an ancestral heat shock response. Whether a similar ancestral growth arrest response also existed in the cenancestor could be confirmed by the identification of evolutionarily conserved genes involved in such a growth arrest response. We have used the budding yeast S. cerevisiae as a model * Corresponding author. Mailing address: Department of Biology, University of New Mexico, Albuquerque, NM 87131. Phone: (505) 277-9338. Fax: (505) 277-0304. Electronic mail address: maggieww @unm.edu. MATERIALS AND METHODS Strains and media. The following S. cerevisiae strains were used in this study: S288C (MATa gal2) (53), W303-1A (MATa ura3-1 leu2-3,112 his3-11,15 trp1-1 6865 Downloaded from http://jb.asm.org/ on February 21, 2016 by guest The regulation of cellular growth and proliferation in response to environmental cues is critical for development and the maintenance of viability in all organisms. In unicellular organisms, such as the budding yeast Saccharomyces cerevisiae, growth and proliferation are regulated by nutrient availability. We have described changes in the pattern of protein synthesis during the growth of S. cerevisiae cells to stationary phase (E. K. Fuge, E. L. Braun, and M. Werner-Washburne, J. Bacteriol. 176:5802–5813, 1994) and noted a protein, which we designated Snz1p (p35), that shows increased synthesis after entry into stationary phase. We report here the identification of the SNZ1 gene, which encodes this protein. We detected increased SNZ1 mRNA accumulation almost 2 days after glucose exhaustion, significantly later than that of mRNAs encoded by other postexponential genes. SNZ1-related sequences were detected in phylogenetically diverse organisms by sequence comparisons and low-stringency hybridization. Multiple SNZ1-related sequences were detected in some organisms, including S. cerevisiae. Snz1p was found to be among the most evolutionarily conserved proteins currently identified, indicating that we have identified a novel, highly conserved protein involved in growth arrest in S. cerevisiae. The broad phylogenetic distribution, the regulation of the SNZ1 mRNA and protein in S. cerevisiae, and identification of a Snz protein modified during sporulation in the gram-positive bacterium Bacillus subtilis support the hypothesis that Snz proteins are part of an ancient response that occurs during nutrient limitation and growth arrest. 6866 BRAUN ET AL. in 50% formamide hybridization buffer, and the blots were washed at high stringency according to the manufacturer’s recommendations. Computer analysis of data. Routine entry and analysis of DNA sequences were performed using MacDNASIS, version 3.2 (Hitachi). Database homology searches were performed using FASTA (61) and BLAST (2). SAPS (11) was used to search protein sequences for unusual sequence features. Multiple sequence alignments were conducted using CLUSTAL W (72) and corrected by visual inspection. Percent identity values correspond to the percent of identical amino acids in multiple sequence alignments, calculated with the ProtST program (1). Tentative cDNA consensus sequences were constructed from expressed sequence tags as described previously (24). Autoradiographs were digitized with 8-bit resolution using a Hewlett-Packard ScanJet IIcx/T scanner and a Power Macintosh 7100 computer. Densitometry was performed using NIH Image (developed at the National Institutes of Health and available by anonymous FTP from zippy.nimh.nih.gov), and figures were prepared using Adobe Photoshop 3.0 (Adobe Systems Incorporated, Mountain View, Calif.) running on a Macintosh IIsi or a Power Macintosh 5300cs computer. Nucleotide and protein sequences. Sequences were retrieved from GenBank, EMBL, and DDBJ using the National Center for Biotechnology Information World Wide Web site (http://www.ncbi.nlm.nih.gov). Sequences from Haemophilus influenzae (27), Methanococcus jannaschii (12), and Mycoplasma genitalium (29) were retrieved from the Institute for Genomic Research (TIGR) World Wide Web site (http://www.tigr.org). Sequenced regions of the Schizosaccharomyces pombe genome were searched using BLAST (2), available over the World Wide Web (http://www.sanger.ac.uk/;yeastpub/svw/pombe.html). The S. pombe snz gene is on cosmid c29B12 from chromosome I (sequence available by anonymous FTP from ftp.sanger.ac.uk/pub/yeast/sequences/pombe). The sequence of the SNZ1 gene was determined on both strands and shows a perfect match to cosmid 6543 (EMBL accession number, Z49807) on chromosome XIII (10). Snz protein sequences and their accession numbers are as follows: S. cerevisiae Snz1p (PIR, S55082), S. cerevisiae Snz2p (EMBL, Z71609), S. cerevisiae Snz3p (Swiss Prot, P43545 [open reading frame YFL059w; reference 54]), Bacillus subtilis Snz (Swiss Prot, P37527), B. subtilis 32-kDa guanylylated protein (Swiss Prot, P27877), H. influenzae Snz (Swiss Prot, P45293 [TIGR, HI1647; reference 27]), Hevea brasiliensis HEVER (GenBank, M88254), M. jannaschii (TIGR, MJ0677; reference 12), Methanococcus vannielii Snz (PIR, S28731), and Stellaria longipes Snz (PIR, S33204). SNZ-related nucleotide sequences and their accession numbers are as follows: Arabidopsis thaliana T-DNA insertion site (EMBL, X71601; reference 51), and H. brasiliensis HEVER pseudogene (GenBank, M88255). cDNA sequences used to construct tentative consensus sequences for SNZ genes and their accession numbers are as follows: S. longipes cDNA (EMBL, X71601); A. thaliana cDNA 1 (GenBank, H36255, H76936, R84084, T13941, T75917, T76249; EMBL, Z37220), cDNA 2 (GenBank, N38597 and T04208), cDNA 3 (EMBL, F15381 and F15457), cDNA 4 (EMBL, Z17740), cDNA 5 (GenBank, H37367 and N65551); and Oryzae sativa cDNA 1 (DDBJ, D47302 and D47746), cDNA 2 (DDBJ, D15988). Accession numbers and gene identification numbers for additional protein sequences retrieved from the National Center for Biotechnology Information and TIGR are given in Table 3. RESULTS AND DISCUSSION Cloning of SNZ1, a gene encoding a novel postexponential protein. We previously identified Snz1p as a protein whose synthesis increases much later than that of other postexponential proteins during growth to stationary phase (30). Snz1p synthesis is not regulated by heat shock or glucose repression (30), unlike the synthesis of many other postexponential proteins (5, 30; see references 75 and 76 for reviews). To identify Snz1p, we partially purified Snz1p from S. cerevisiae cells grown to stationary phase, separated it from other proteins by two-dimensional PAGE, and sequenced three peptides (see Materials and Methods). These peptide sequences indicated that Snz1p was a previously undescribed yeast protein (Fig. 1). We obtained the SNZ1 gene by using PCR to amplify S. cerevisiae genomic DNA with degenerate oligonucleotide primers corresponding to the Snz1p peptide sequences. A fulllength SNZ1 clone was obtained from a genomic library by probing with this PCR product (see Materials and Methods). This clone contained an 891-bp open reading frame containing the peptide sequences obtained from Snz1p, indicating that this clone contains the SNZ1 gene. The SNZ1 clone was physically mapped (58) to chromosome XIII between ADH3 and ILV2. During the course of this investigation, the sequence of the yeast genome was completed, confirming this result and indicating that SNZ1 is within 1.2 kb of the CTF13 gene. Downloaded from http://jb.asm.org/ on February 21, 2016 by guest ade2-1 can1-100) (70), MW736 (MATa snz1D1 ura3-1 leu2-3,112 his3-11,15 trp1-1 ade2-1 can1-100) (this study), LP112 a/a (MATa/MATa ura3-1/ura3-1 leu2-3,112/ leu2-3,112 his3-11,15/his3-11,15 trp1-1/trp1-1 ade2-1/ade2-1 can1-100/can1-100) (62), SDD37 (MATa /MATa snz1D2/SNZ1 ura3-1/ura3-1 leu2-3,112/leu2-3,112 his3-11,15/his3-11,15 trp1-1/trp1-1 ade2-1/ade2-1 can1-100/can1-100) (this study), MW926 (MATa snz1D2 ura3-1 leu2-3,112 his3-11,15 trp1-1 ade2-1 can1-100) (this study), MW927 (MATa SNZ1 ura3-1 leu2-3,112 his3-11,15 trp1-1 ade2-1 can1100) (this study), MW930 (MATa/MATa snz1D2/snz1D2 ura3-1/ura3-1 leu23,112/leu2-3,112 his3-11,15/his3-11,15 trp1-1/trp1-1 ade2-1/ade2-1 can1-100/can1100) (this study), DS10 (MATa ura3-52 leu2-3,112 his3-11,15 Dtrp1 lys2) (78), and MW567 (MATa snz1D1 ura3-52 leu2-3,112 his3-11,15 Dtrp1 lys2) (this study). The following media were used for the cultivation of yeast: YPD (1% yeast extract, 2% peptone, 2% glucose), YP–0.05% glucose (1% yeast extract, 2% peptone, 0.05% glucose), YPAc (1% yeast extract, 2% peptone, 1% potassium acetate, pH 5.5), YPGal (1% yeast extract, 2% peptone, 2% galactose), and SC-Ura (0.67% yeast nitrogen base without amino acids, 2% glucose, supplemented with auxotrophic requirements but lacking uracil) (14, 68). Solid media contained 1.5% agar (68). YPD and YPAc for ade2 mutants were supplemented with 40 mg of adenine per ml (68). Identification of the SNZ1 gene. Snz1p was partially purified from yeast (S288C) cultures grown at 308C in YPD for 21 days. Snz1p was separated from other proteins by two-dimensional polyacrylamide gel electrophoresis (PAGE) as previously described (30). Tryptic cleavage and microsequencing were carried out by the microchemistry core facility at the Sloan-Kettering Memorial Cancer Center. Degenerate oligonucleotide primers corresponding to two of the tryptic peptide sequences were synthesized and used to amplify S. cerevisiae genomic DNA by the PCR. The PCR product was cloned into pGEM-5Zf(1) (Promega), generating pGPT15. The pGPT15 insert was used to probe a yeast genomic library (65), resulting in the identification of one clone (pWFY1) with an approximately 13-kb genomic insert. A 3.35-kb EcoRI fragment of pWFY1 containing the SNZ1 gene was subcloned into pBluescriptII KS2 (Stratagene), generating pWFY2. Disruption of SNZ1. The snz1D1 deletion-disruption allele (snz1D1::URA3) was constructed by inserting a 1.1-kb PstI URA3 fragment, generated from YCplac111 (31) by PCR, into the PstI-digested pWFY2 insert. This results in the removal of the DNA encoding amino acids 213 to 248 (Fig. 1). The snz1D2 deletion allele (snz1D2::URA3) was constructed by modifying the snz1D1 construct using outward-directed PCR essentially as described previously (23), except that a standard PCR rather than long PCR was performed. This modification results in the deletion of the SNZ1 start codon and the DNA encoding amino acids 1 to 248 (Fig. 1). snz1D1 and snz1D2 mutant yeast were obtained by transformation with linear snz1D1 and snz1D2 DNA and selection of transformants on SC-Ura. Yeast transformation was performed by electroporation (6). Integration of the constructs was confirmed by PCR and Southern analysis. Manipulation of nucleic acids and miscellaneous methods. Standard methods were used for the transformation of Escherichia coli, restriction enzyme mapping, and the construction of recombinant plasmids (3). Other techniques were performed according to the recommendations of the manufacturer of the reagents used. Amplification of DNA by PCR was performed with AmpliTaq DNA polymerase (Hoffman-LaRoche). The preparation of recombinant plasmids from E. coli, the isolation of restriction fragments, and the isolation of PCR products were conducted with kits from Qiagen. Isolated DNA fragments used as probes were labeled with [a-32P]dCTP (Dupont NEN) by the random primer method (Pharmacia). The sequence of SNZ1 and flanking regions was determined on both strands, using Bst polymerase (Bio-Rad). Analysis of genomic DNAs. For analysis of genomic DNA from wild-type and mutant yeast strains, genomic DNA was prepared as previously described (64), digested with the appropriate restriction enzyme, separated by agarose gel electrophoresis, and transferred to GeneScreenPlus (NEN). Hybridizations were carried out at 428C in 50% formamide hybridization buffer, and the blots were washed at high stringency. For low-stringency “zoo” blots, genomic DNAs from various organisms were digested with EcoRI and electrophoresed for 435 V z h in 0.9% agarose gels. Each gel contained S. cerevisiae genomic DNA as a control. The separated DNAs were transferred to GeneScreenPlus (NEN). These blots were probed in phosphate-sodium dodecyl sulfate (SDS) hybridization buffer (500 mM NaPi [pH 7.2], 10 mM EDTA, 7% SDS, 0.1% bovine serum albumin) at 52.58C for 48 h and washed at low stringency (two 15-min washes in 23 SSC [13 SSC is 0.15 M NaCl plus 0.015 M sodium citrate]–0.1% SDS at room temperature followed by two 15-min washes in 23 SSC–0.1% SDS at 458C). This procedure allows the detection of sequences showing at least 55 to 65% identity in nucleotide sequence (10). The SNZ1 probes used with zoo blots correspond to the DNA encoding amino acids 1 to 174 (amino-terminal probe) and amino acids 174 to 297 (carboxyl-terminal probe). The position of cloned DNAs within the yeast genome was determined by hybridization to filters containing ordered yeast DNA libraries, from the American Type Culture Collection. Hybridization and washing were conducted according to American Type Culture Collection recommendations, except that 32 P-labeled probes were used. Analysis of RNA accumulation. Total RNA was prepared as previously described (30), electrophoresed on 1% agarose gels containing 2.5% formaldehyde, and transferred to GeneScreen (NEN). Hybridizations were carried out at 428C J. BACTERIOL. VOL. 178, 1996 A CONSERVED STATIONARY-PHASE GENE IN S. CEREVISIAE 6867 Snz1p was predicted to have a molecular mass of 32 kDa and an isoelectric point of 5.3 on the basis of sequence analysis. Snz1p migrated in two-dimensional PAGE as a protein with an apparent molecular mass of 36 kDa and an isoelectric point of 5.3, on the basis of comparison with the yeast protein map (9). Snz1p lacks long hydrophobic segments (11, 46), suggesting that it is a soluble protein. Snz1p has relatively high methionine (4.4%), valine (10.1%), and alanine (10.1%) contents relative to other yeast proteins (11). Snz1p was not predicted to contain any previously described protein motifs (10). Snz1p is a member of a highly conserved gene family. Com- parison of the predicted Snz1p protein sequence with other database sequences indicated that Snz1p is a member of a highly conserved protein family present in all three phylogenetic domains (80): eucarya, bacteria, and archaea (Fig. 1). These proteins, which we propose calling Snz proteins, share at least 45% identity and often have greater than 55% identity (Tables 1 and 2). Snz1p in S. cerevisiae actually shows more similarity to the bacterial Snz proteins than to some eukaryotic Snz proteins (Fig. 1 and Tables 1 and 2). This observation suggests that these eukaryotic Snz proteins are Snz1p paralogs, i.e., proteins encoded by genes that arose by duplication of an Downloaded from http://jb.asm.org/ on February 21, 2016 by guest FIG. 1. Sequence alignment of Snz1p and related proteins. The predicted amino acid sequence of Snz1p from S. cerevisiae was aligned with predicted Snz proteins from S. pombe, H. brasiliensis, S. longipes, M. jannaschii, M. vannielii, H. influenzae, and B. subtilis. Amino acids are boxed if they are present in the majority of sequences. The predicted Snz1 protein contains sequences corresponding to the three peptides sequenced from purified Snz1p (the peptides correspond to amino acids 83 to 102, 247 to 263, and 264 to 283). The amino-terminal sequence of the 32-kDa guanylylated protein from B. subtilis is also aligned, with amino acids identical to the B. subtilis Snz protein boxed. Conceptual translation of the M. vannielii and S. longipes SNZ sequences revealed that the similarity extends beyond the amino-terminal methionine previously reported (indicated with a star), indicating that the shorter sequences reported for M. vannielii and S. longipes represent truncated sequences (10). This suggests that the M. vannielii and S. longipes Snz proteins are actually similar in size to Snz1p. 6868 BRAUN ET AL. J. BACTERIOL. TABLE 1. Percent identity between SNZ genesa % Identity Species and gene S. cerevisiae SNZ1 S. cerevisiae SNZ1 SNZ2 SNZ3 S. pombe H. brasiliensis S. longipesb M. jannaschii M. vannieliib H. influenzae B. subtilis SNZ2 SNZ3 80.8 81.2 99.7 S. pombe H. brasiliensis S. longipesb M. jannaschii M. vannieliib H. influenzae B. subtilis 64.5 67.2 67.6 60.6 62.7 63.1 74.6 49.8 54.7 54.7 63.1 75.1 60.6 58.9 59.6 66.2 64.6 63.1 60.4 59.6 60.0 66.2 62.7 53.8 70.7 63.4 65.5 65.2 68.3 64.8 55.1 62.4 62.7 57.8 59.2 59.2 68.9 64.1 54.2 67.2 63.7 62.0 ancestral SNZ gene followed by divergence (25, 34). If the Snz1p paralogs are eliminated from the analysis, then Snz proteins all share at least 58% identity. This conservation makes the Snz proteins the most conserved of all proteins known to be present in all three phylogenetic domains (Table 3). To determine whether SNZ-related sequences were detectable in organisms for which sequence data were unavailable, SNZ1 probes were hybridized at reduced stringency with DNA isolated from a variety of organisms (Fig. 2). A SNZ1 probe corresponding to the carboxyl-terminal region of Snz1p (see Materials and Methods) hybridized with DNA isolated from organisms in each phylogenetic domain. The broad phylogenetic distribution of SNZ genes revealed by low-stringency hybridization (Fig. 2) makes it unlikely that the high degree of identity between eukaryotic and prokaryotic SNZ genes is the result of horizontal gene transfer (described in references 20 and 40). Multiple bands, observed when the SNZ1 probe was hybridized with DNA isolated from several organisms, indicate that SNZ genes are frequently members of multigene families. An amino-terminal SNZ1 probe also hybridized with DNA isolated from taxa in each domain, although fewer bands were recognized in most taxa (data not shown) and a number of taxa were not recognized, including a plant (Nicotiana tabacum), a gram-negative bacterium (E. coli), and a crenarchaeote (Sulfolobus solfaraticus). These results suggest that the carboxylterminal region of Snz proteins may be more conserved than the amino-terminal region. Only weakly hybridizing bands were observed when genomic DNAs from Drosophila melanogaster and the house sparrow Passer domesticus (Fig. 2) and one ciliated protist, Tetrahymena thermophila (data not shown), were hybridized with either probe. These results suggest either that SNZ-related genes have been lost in these taxa or that they have changed enough that they are no longer readily detectable by low-stringency hybridization. SNZ genes are present in both prokaryotic domains, the bacteria and archaea (56, 57, 80). Both SNZ1 probes revealed a single band when hybridized at low stringency with DNA isolated from archaea, such as Methanococcus voltae (Fig. 2), M. jannaschii (data not shown), and S. solfaraticus (Fig. 2). Sequence analysis has revealed a SNZ gene within the archaeon M. vannielii (Fig. 1 and Table 1) (79), and the complete genomic sequence of M. jannaschii (12) has allowed the identification of a single M. jannaschii SNZ gene (Fig. 1 and Table 1), confirming the results of the low-stringency hybridization. In contrast, the carboxyl-terminal but not the amino-terminal SNZ1 probe hybridized with DNA isolated from the gramnegative bacterium E. coli (Fig. 2). Sequence analysis revealed a single SNZ gene in the gram-negative bacterium H. influenzae (Fig. 1 and Table 1) (27), which is in the same division of the proteobacteria as E. coli (57). SNZ-related sequences are absent from the phylogenetically gram-positive bacterium M. genitalium (56, 57, 63). The loss of SNZ-related sequences from M. genitalium, a bacterium with a very small genome (29, 33) that has undergone a relatively rapid evolutionary change (63, 81), may ultimately prove useful in the analysis of Snz function. SNZ genes frequently belong to multigene families. SNZ1 is a member of a multigene family in S. cerevisiae. Prior to the publication of the sequence of the yeast genome, we identified two additional SNZ genes, which we designated SNZ2 and SNZ3, on chromosomes XIV and VI, respectively (60). SNZ2 and SNZ3 are predicted to encode proteins that show a much greater similarity to Snz1p than to Snz proteins in other or- TABLE 2. Partial SNZ sequences from plantsa Species and sequence S. cerevisiae Snz1p H. brasiliensis HEVERb Identity Aligned region Identity Aligned region A. thaliana cDNA 1 cDNA 2 cDNA 3 cDNA 4 cDNA 5 T-DNA insertion site 66.3 61.2 58.5 55.4 48.8 49.2 6–87 6–58 199–297 176–260 6–41 6–134 88.6 90.5 95.7 89.2 63.4 56.4 1–104 1–75 215–309 192–276 23–58 23–151 O. sativa cDNA 1 cDNA 2 68.5 52.1 6–92 186–297 86.5 89.4 1–109 202–309 a Comparisons of partial SNZ sequences identified in A. thaliana and rice with Snz1p and the H. brasiliensis Snz protein (HEVER). Values correspond to the percent amino acid identity and the region aligned (with the amino acids numbered as shown in Fig. 1). b H. brasiliensis HEVER is the full-length SNZ gene most closely related to other plant SNZ genes, so it is included in these comparisons. Many of these partial sequences also show a high degree of identity with the SNZ gene from S. pombe (10). Downloaded from http://jb.asm.org/ on February 21, 2016 by guest a S. cerevisiae and S. pombe are fungi, H. brasiliensis and S. longipes are plants, M. jannaschii and M. vannielii are archaea, and H. influenzae and B. subtilis are bacteria. Percent amino acid identities based upon comparisons of full-length SNZ genes using ProtST (1), which excludes gaps, are presented. b These SNZ sequences do not include all of the amino terminus (Fig. 1). Comparisons were performed using 225 amino acid sites. VOL. 178, 1996 A CONSERVED STATIONARY-PHASE GENE IN S. CEREVISIAE TABLE 3. Comparison of Snz proteins with other highly conserved proteinsa % Identity Protein Snz proteins Hsp70 (DnaK)b EF-1a (Tu)c EF-2 (G)d GAPDHe Enolasef Within Eucarya bacteria vs. (gram2 vs. archaea Gram2 Gram1 Gram2 Gram1 gram1) Eucarya vs. bacteria Archaea vs. bacteria 63 49 34 27 38 50 62 58 35 31 18 57 58 48 34 27 52 52 67 66 36 33 21 59 61 50 57 35 19 59 62 56 77 59 43 66 ganisms (Table 1). This is consistent with SNZ2 and SNZ3 resulting from a relatively recent gene duplication. Multiple SNZ genes were also observed in the distantly related ascomycetes S. pombe and Neurospora crassa (Fig. 2) and the chytrid fungus Blastocladiella emersonii (data not shown). During the course of this investigation, genome sequencing efforts have identified one SNZ gene from S. pombe (Fig. 1) and a SNZrelated cDNA from N. crassa (55). These results indicate that SNZ multigene families are widely distributed among the fungi. Multiple SNZ-related sequences are also present in plants. Hybridization of plant DNA samples with a SNZ1 probe at reduced stringency revealed that tobacco (N. tabacum, Fig. 2), radish (Raphanus sativus, data not shown), thale cress (A. thaliana, data not shown), and Tetramolopium spp. (a composite, data not shown) have multiple hybridizing bands, indicating that SNZ genes form a multigene family in these organisms. In fact, sequence data for A. thaliana indicate that there are at least four SNZ genes in this organism (Table 2). A SNZrelated cDNA from the rubber tree H. brasiliensis has also been identified as an mRNA (designated HEVER; Fig. 1) that accumulates as leaves mature and during exposure to ethylene or salicylic acid (69). The effect of ethylene exposure may reflect an increase in SNZ mRNA during growth arrest or senescence. Interestingly, the SNZ gene in S. pombe that has been sequenced was more closely related to plant SNZ genes than to the S. cerevisiae SNZ genes (Table 1), suggesting that the gene duplication that led to the plant SNZ genes occurred before the divergence of the plants and fungi, approximately 1 billion years ago (21). The carboxyl-terminal SNZ1 probe hybridized with three bands in DNA isolated from B. subtilis (Fig. 2), suggesting that some prokaryotes have multiple SNZ genes. Interestingly, only one of the three bands in B. subtilis was recognized by the amino-terminal probe (data not shown), underscoring the differences between these probes. One SNZ gene from B. subtilis has been identified, but the genome of B. subtilis has not been completely sequenced. We noted striking identity between the amino-terminal sequence of the predicted B. subtilis Snz protein and the amino-terminal sequence of a 32-kDa protein that is guanylylated in extracts from sporulating B. subtilis cells (52) (Fig. 1). It is very likely that the 32-kDa guanylylated protein corresponds to the B. subtilis Snz protein, given the excellent match of the two amino-terminal sequences and the correspondence in molecular weight. We believe that the single mismatch between the two amino-terminal sequences represents either a polymorphism between the strains of B. subtilis used or a sequencing error. These data suggest that starvation in B. subtilis causes changes in Snz protein accumulation, the extent of Snz protein guanylylation, or both Snz protein accumulation and modification. The SNZ1 message is induced in stationary-phase S. cerevisiae. Most yeast mRNAs that are abundant in stationary-phase cells begin to accumulate during the diauxic shift (16, 30, 74, 75, 76). In contrast, the SNZ1 message, which is detectable during exponential growth, increased only slightly during the postdiauxic phase and exhibited a 14-fold increase in accumulation during stationary phase (Fig. 3). SNZ1 mRNA accumulation increased much later than did mRNA from BCY1, FIG. 2. Identification of SNZ-related sequences in other organisms by lowstringency hybridization with SNZ1. Shown are Southern blots with DNA from the indicated species hybridized at reduced stringency with a probe corresponding to the carboxyl-terminal region of SNZ1. The species used are as follows: E.c., E. coli; B.s., B. subtilis; M.v., M. voltae; S.s., S. solfaraticus; T.e., Trypanosoma evansi; O.t., Oxytricha trifallax; N.t., N. tabacum; S.p., S. pombe; N.c., N. crassa; S.c., S. cerevisiae, D.m., D. melanogaster, P.d., P. domesticus. Autoradiographs were exposed for 2 weeks, except for that for S. cerevisiae, which was exposed overnight. Downloaded from http://jb.asm.org/ on February 21, 2016 by guest a Percent identity between highly conserved proteins. The species used are as follows: eucarya, S. cerevisiae; gram-negative bacteria, H. influenzae; gram-positive bacteria, B. subtilis; archaea, M. jannaschii. Gram2, gram negativel; Gram1, gram positive. b Hsp70 (DnaK) sequences are as follows: S. cerevisiae Ssa1p (Swiss Prot, P10591), H. influenzae (TIGR, HI1237), B. subtilis (Swiss Prot, P17820), Methanosarcina mazei (EMBL, X60265). M. jannaschii does not have a DnaK homolog, so M. mazei DnaK was used. Ssa1p shows .80% identity to other members of the same subfamily in S. cerevisiae (Ssa2p, Ssa3p, and Ssa4p), and comparisons using these proteins yield similar results (10). c EF-1a (EF-Tu) sequences used are as follows: S. cerevisiae (Swiss Prot, P02994), H. influenzae (TIGR, HI0578), B. subtilis (Swiss Prot, P33166), M. jannaschii (TIGR, MJ0324). d EF-2 (EF-G) sequences used are as follows: S. cerevisiae (Swiss Prot, P32324), H. influenzae (TIGR, HI0579), M. genitalium (TIGR, MG089), M. jannaschii (TIGR, MJ1048). An EF-G homolog in B. subtilis has not been sequenced, so the sequence from M. genitalium was used. e Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) sequences used are as follows: S. cerevisiae Tdh1p (Swiss Prot, P00360), E. coli GAPDH-B (Swiss Prot, P11603), B. subtilis (Swiss Prot, P09124), M. jannaschii (TIGR, MJ1146). H. influenzae GAPDH is orthologous to E. coli GAPDH-A, which is the result of a horizontal gene transfer from a eukaryote (20). Tdh1p shows .85% identity to the other two S. cerevisiae GAPDH isozymes, and comparisons using these proteins yield similar results (10). f Enolase sequences used are as follows: S. cerevisiae Eno1p (Swiss Prot, P00924), H. influenzae (TIGR, HI0932), B. subtilis (Swiss Prot, P37869), M. jannaschii (TIGR, MJ0232). S. cerevisiae Eno1p shows 96% identity to Eno2p, and comparisons using Eno2p yield similar results (10). 6869 6870 BRAUN ET AL. encoding the regulatory subunit of cyclic AMP (cAMP)-dependent protein kinase (see references in reference 77), and from SUC2, encoding invertase (14), which accumulated during the diauxic shift, immediately after glucose exhaustion. Likewise, SNZ1 mRNA accumulation increased after the initial increase in mRNA encoded by the HSP70-related SSA3 gene, which occurs during the diauxic shift (30, 74), concurrently with STRE (stress response/C4T-element)-regulated genes (73). This pattern of gene regulation has not been observed previously in S. cerevisiae and supports the idea that entry into stationary phase is a multistage developmental process. Additionally, the accumulation of SNZ1 mRNA in stationary phase provides the first evidence that the cell is capable of responding at the molecular level to novel signals at this late and extremely important time in the yeast life cycle. SNZ1 mRNA accumulation was maximal in stationary phase (Fig. 3, lane H), when most yeast mRNAs show substantially (.20-fold) reduced accumulation (16, 74). Although SSA3 mRNA accumulation reached its maximal level at the same time, the SSA3 mRNA began accumulating earlier. SNZ1 mRNA accumulation decreased when cells remained in stationary phase (Fig. 3, lanes J and K). This decreased accumulation probably reflects the general repression of mRNA accumulation, involving at least two different mechanisms (16), that affects many postexponential genes during stationary phase, including SSA3 (Fig. 3, lanes J and K). Despite the decrease in SNZ1 mRNA abundance, synthesis of Snz1p continues to increase in stationary phase, when measured relative to total protein synthesis (30). This may reflect increased abundance of SNZ1 mRNA relative to total mRNA during stationary phase. However, the translatability of some HSP70-related mRNAs has been shown to change as cells enter stationary phase (30). Thus, the increase in the relative synthesis of Snz1p during stationary phase may also reflect a change in SNZ1 mRNA translatability. Because SNZ1 mRNA accumulated during growth to stationary phase, we wanted to determine whether it was regulated similarly to other postexponentially expressed genes. Many postexponential genes, such as SSA3, HSP26, and CTT1, are induced by heat shock (49, 50, 62, 74; see references 19 and 48 for reviews). Consistent with the unique pattern of SNZ1 mRNA accumulation, SNZ1 mRNA did not accumulate in response to heat shock (data not shown), and neither heat shock elements (19) nor the cAMP-repressed STRE element (49, 50) was found upstream of the SNZ1 gene. To determine whether SNZ1 mRNA accumulation is regulated by glucose repression, cells were grown in glucose-containing medium and transferred to low-glucose (0.05%) medium for 1 h (14). Under these conditions, accumulation of the SNZ1 mRNA increased (data not shown), although to a significantly lower level than during entry into stationary phase (Fig. 3). SNZ1 mRNA did not accumulate in cells growing with acetate as the sole carbon source (data not shown), providing evidence that the increased accumulation of the SNZ1 mRNA observed in low-glucose medium is likely to have resulted from perceived carbon starvation rather than the relief of glucose repression. These results support the hypothesis that SNZ1 mRNA accumulation in stationary-phase S. cerevisiae is unique and does not occur in response to previously characterized signals. Phenotypic analysis of snz1 mutants. To determine whether SNZ1 has a role in the starvation response, strains carrying a snz1 disruption-deletion mutation (snz1D1) and a snz1 deletion mutation (snz1D2) were constructed in the W303 strain background (see Materials and Methods). However, both snz1 mutants grown to stationary phase in rich, glucose-based medium exhibited saturation densities and viabilities identical to those of isogenic wild-type cells (data not shown). Neither mutant exhibited impaired growth at either 30 or 378C on rich media containing glucose, galactose, or acetate as a sole carbon source. Homozygous diploid snz1D1 and snz1D2 mutants were capable of sporulation. Similar results were obtained with snz1D1 mutants in a different strain background (DS10). Despite these results, Snz1p may play an important role during starvation, with the defect caused by the absence of Snz1p being complemented by SNZ2/3, since Snz1p, Snz2p, and Snz3p show a high degree of sequence identity (Table 1) and may show some functional redundancy (71). Despite the potential for functional redundance, it is still surprising that deleting such a highly conserved gene does not result in a strong phenotype, since it is generally thought that the functional importance of proteins limits the rate at which they evolve, with the most functionally important proteins evolving the most slowly (42, 43, 44; but see reference 35). Because most yeast proteins (approximately 85%) are maintained despite being nonessential for viability (13, 32) and because a majority of yeast proteins (approximately 60%) are maintained despite the fact that they can be mutated without causing a readily detectable phenotype (13, 32), it may be that the selective forces responsible for the maintenance of many genes are relatively small and difficult to detect (39, 82). This Downloaded from http://jb.asm.org/ on February 21, 2016 by guest FIG. 3. SNZ1 mRNA exhibits increased accumulation during growth to stationary phase, later than other postexponential mRNAs. Accumulation of the SNZ1 mRNA during the growth of wild-type S. cerevisiae (S288C) to stationary phase is compared with that of SSA3, BCY1, and SUC2. The ethidium bromidestained gel is shown below the autoradiographs. Time points are as follows: A, early exponential phase (optical density at 600 nm [OD600] 5 1); B, middle exponential phase (OD600 5 2.8); C, late exponential phase (OD600 5 7); D, diauxic shift (immediately after glucose exhaustion, OD600 5 8); E, early postdiauxic phase (2 h after glucose exhaustion); F, middle postdiauxic phase (16 h after glucose exhaustion); G, late postdiauxic phase (40 h after glucose exhaustion); H, early stationary phase (4 days after inoculation, 72 h after glucose exhaustion); I, stationary phase (7 days after inoculation); J, stationary phase (10 days after inoculation); K, stationary phase (14 days after inoculation). Autoradiographs were exposed for either 7 days (BCY1 and SSA3) or 9 days (SNZ1 and SUC2). J. BACTERIOL. VOL. 178, 1996 A CONSERVED STATIONARY-PHASE GENE IN S. CEREVISIAE 13. 14. 15. 16. 17. 18. 19. 20. 21. ACKNOWLEDGMENTS We thank Ken Cline, Gerry Johnston, Don Natvig, Mary Anne Nelson, Vickie Peck, Rebecca Kimball, and Matthew Crawford for carefully reading the manuscript and participating in helpful discussions and Sal Pietromonico for helpful discussions during the purification of Snz1p. We also thank Don Natvig and Mary Anne Nelson for discussing unpublished work with us and Alena Gallegos for expert technical assistance. We are grateful to Shawn Ahmed, W. Ford Doolittle, Tudor Jones, Ken Keegstra, Rebecca Kimball, Tokio Kogoma, Jordan Konisky, Mary Anne Nelson, David Prescott, and Paul Young for generously providing DNA samples, to Jacqueline Segall for generously providing a strain, and to Dan Gietz for generously providing plasmids. This work was supported by grants HRD-9253051, MCB-9057514 (Presidential Young Investigator) and DCB-9000556 to M.W.-W. from the National Science Foundation and by a Patricia Roberts Harris fellowship to P.A.P. 22. 23. 24. 25. 26. 27. REFERENCES 1. 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