Large-scale phenotypic analysis reveals identical contributions to cell functions of known and unknown yeast genes

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1 Yeast Yeast 2001; 18: DOI: /yea.784 Yeast Functional Analysis Report Large-scale phenotypic analysis reveals identical contributions to cell functions of known and unknown yeast genes Michele M. Bianchi 1 *, Saravuth Ngo 2, Micheline Vandenbol 3, Geppo Sartori 4, Alessandro Morlupi 1, Carlo Ricci 1, Stefania Stefani 4, Giovanni B. Morlino 1, François Hilger 3, Giovanna Carignani 4, Piotr P. Slonimski 2 and Laura Frontali 1 1 Department of Cell and Developmental Biology, University of Rome La Sapienza, I-00185Rome, Italy 2 Centre de Génétique Moléculaire du CNRS, Gif-sur-Yvette, France 3 Animal and Microbial Biology Unit, Gembloux Agricultural University, B-5030 Gembloux, Belgium 4 Department of Biological Chemistry, University of Padova, I Padova, Italy * Correspondence to: M. M. Bianchi, Department of Cell and Developmental Biology, University of Rome La Sapienza, P.le Aldo Moro 5, I Rome, Italy. Michele.Bianchi@uniromal.it Received: 26 March 2001 Accepted: 16 June 2001 Abstract Sequencing of the yeast genome has shown that about one-third of the yeast ORFs code for unknown proteins. Many other have similarity to known genes, but still the cellular functions of the gene products are unknown. The aim of the B1 Consortium of the EUROFAN project was to perform a qualitative phenotypic analysis on yeast strains deleted for functionally orphan genes. To this end we set up a simple approach to detect growth defects of a relatively large number of strains in the presence of osmolytes, ethanol, high temperature, inhibitory compounds or drugs affecting protein biosynthesis, phosphorylation level or nucleic acids biosynthesis. We have now developed this procedure to a semi-quantitative level, we have included new inhibitors, such as hygromycin B, benomyl, metals and additional drugs interfering with synthesis of nucleic acids, and we have performed phenotypic analysis on the deleted strains of 564 genes poorly characterized in respect to their cellular functions. About 30% of the deleted strains showed at least one phenotype: many of them were pleiotropic. For many gene deletions, the linkage between the deletion marker and the observed phenotype(s) was studied by tetrad analysis and their co-segregation was demonstrated. Co-segregation was found in about two-thirds of the analysed strains showing phenotype(s). Copyright # 2001 John Wiley & Sons, Ltd. Keywords: Saccharomyces cerevisiae; genome; functional analysis; growth inhibitors Introduction The sequence of the Saccharomyces cerevisiae genome was completed few years ago (Goffeau et al., 1996). Since that date, the entire genomic sequences of many prokaryotic and eukaryotic organisms have been released or are being completed. During the systematic sequencing of the yeast genome, many of the newly identified ORFs showed no putative function or had no similarity with known genes and they were termed orphan genes (Oliver, 1996). Due to the rapidly increasing amount of data from the sequencing of other organisms, a large proportion of the orphan genes have now been found to have similarities with other genes. Yeast databases, such as MIPS (Mewes et al., 1997; YPD (Costanzo et al., 2000; com/databases/ypd/) or SGD (Cherry et al., genome- are continuously updated with this respect. However, the functions of the proteins encoded by many of these genes still remain unknown. In the post-genomic era, one of the important challenges is the assignment of functions to unknown genes and the establishment of possible roles of known and unknown genes in functional networks. S. cerevisiae ORFs have been annotated on the Copyright # 2001 John Wiley & Sons,Ltd.

2 1398 M. M. Bianchi et al. basis of the presence of start and stop codons, assigned function, existence of homologues or similarity to known genes and proteins, codon bias, codon adaptation index and ORF length. Some simple and reasonable criteria were adopted when overlapping unknown ORFs were considered. Following these criteria, 6357 protein-encoding sequences have been annotated and they have been listed at the MIPS database, divided into six classes of similarity. In the framework of the EUROFAN project, the B0 Consortium has systematically deleted unknown genes or poorly characterized genes and a large collection of haploid and diploid deleted strains has been produced and deposited at EUROSCARF ( FB/fb15/mikro/euroscarf/). These strains have been distributed to the nodes of the EUROFAN project for studies on specific cell activities. The B1 Consortium of EUROFAN was devoted to a functional analysis of deleted strains showing no evident phenotype. The scope of the B1 Consortium was to test the growth of the deleted strains on a large number of compounds and conditions, affecting various aspects of the cell activities, with the aim of assigning a biological role to the proteins encoded by unknown genes. Pilot projects and first release of data on a relatively small number of genes have been recently published (Rieger et al., 1997, 1999; Bianchi et al., 1999). In this work, we report a phenotypic analysis concerning 564 genes, not including those previously studied. The corresponding deleted strains have been tested for growth phenotypes related to osmotic, ethanol and thermal stresses, protein glycosylation and phosphorylation, protein and nucleic acids synthesis and metal resistance. Cosegregation of the phenotypes with the deletion marker has been studied in a conspicuous number of cases by analysing tetrads from diploid heterozygous strains. Materials and methods Strains and media All strains were derived from the standard strain FY1679 (MATa/MATa; ura3-52/ura3-52; leu2d1/+; trp1d63/+; his3d200/+). Haploid MATa and MATa deleted strains and diploid heterozygous deletiontstrains were obtained from EURO- SCARF. A complete list of the 564 genes analysed is available upon request to the corresponding author or to MIPS. The genotypes of the haploid deleted strains are accessible at EUROSCARF. The haploid wild-type strains used as controls were obtained from the diploid FY1679 standard strain. Basic media were 2% w/vdifco BACTO 2 peptone and 1% w/vdifco yeast extract (YP); 2% w/vdifco BACTO 2 peptone, 1% w/vdifco yeast extract and 2% w/vglucose (YPD); 0.67% w/vdifco yeast nitrogen base without amino acids, 2% w/vglucose and auxotrophic requirements as needed (SD). 2% w/vdifco BiTEK 2 agar was added for solid media. Geneticin was added to a final concentration of 200 mg/ml for the selection of the deleted strains. Growth and test conditions Strains were grown on liquid YPD into the wells of microtitre plates at 28uC. Cultures were then sequentially diluted with sterile water and replicaplated to the test media as described in Bianchi et al. (1999). This procedure allowed the growth of isolated colonies in the most diluted samples. The plates were then incubated at 28uC. Some of the tests were performed at both 28uC and 37uC (see Table 1 for details). Table 1 shows the inhibitors and their concentrations used in our phenotypic analyses. The concentrations of the inhibitors have been chosen at the threshold of sensitivity of the wild-type strain, in order to easily detect sensitive or resistant growth phenotypes. Tests were performed on MATa and MATa strains of each deletion. Growth was scored after 2 5 days, depending on the test. Sensitivity (S) to the test conditions corresponded to reduced size or number of colonies. Absence of growth or highly reduced colony number or size in the test conditions as compared to the wild-type were indicated as highly sensitive (HS) growth phenotype. When the mutant strains grew better than the wild-type strain in the same conditions, the phenotype was scored as resistant (R). In some cases S and HS phenotypes have been found, depending on the test conditions, when two concentrations of the inhibitory compound were tested, or depending on different growth rates of the MATa and MATa deleted strains in the same test condition. In very few cases, the MATa and MATa deleted strains gave S and R phenotypes. Only gene deletions yielding at least one phenotype both in the MATa and MATa genetic backgrounds were considered as test positives. More than single phenotypic tests have been performed in the screening on MATa and MATa deleted strains.

3 Role of unknown genes in yeast 1399 Table 1. Assayed inhibitors and test conditions Acronym Inhibitor Concentration Analysis on tetrads Basic medium Aec S-2-Aminoethyl-L-cysteine 0.15 mg/ml SD Alg DL-C-allyl-glycine mg/ml SD Azs Azaserine 0.53 mg/ml SD Be Benomyl 40 mg/ml YPD Ca CaCl M YPD Caf 1 Caffeine 0.1% w/v YPD Cd CdCl 2 55 mm YPD Co CoCl mm YPD Cor Cordycepin mg/ml SD Cs CsCl 0.1 M YPD Cyc Cycloheximide 0.18 mg/ml YPD Dau Daunomycin 0.05 mg/ml YPD DE Ethanol (with glucose) 8 10% v/v YPD Don Diazo-oxo-norleucine 0.1 mg/ml SD E Ethanol (without glucose) 4 6% v/v YP F 1 NaF 35 mm YPD Fcy 5-Fluoro-cytosine 0.02 mg/ml SD Fpa DL-p-Fluorophenyl-alanine mg/ml SD Ftr 5-Fluoro-tryptophan 0.1 mg/ml SD Fty m-fluoro-dl-tyrosine mg/ml SD Fur 5-Fluoro-uracil 0.02 mg/ml SD Hg HgCl mm YPD Hy Hygromycin B mg/ml YPD K KCl M YPD Kp 2 KCl M YPD N NaCl M YPD Np 2 NaCl M YPD OHq 8-Hydroxyquinoline 26 mg/ml YPD Pat 3 Patulin 0.22 mg/ml SD So Sorbitol M YPD Sop 2 Sorbitol M YPD T 4 Heat (waterbath at 52uC) min YPD Tal b-2-thienyl-dl-alanine mg/ml SD Tfp 1 Trifluoperazine 15 mm YPD Tp 4,5 Heat (waterbath at 52uC) min YPD V 1 Vanadate 3 5 mm YPD Ver Verrucarin A 2.45 mg/ml YPD This table alphabetically lists the 32 assayed inhibitors (31 chemicals + heat) together with their acronyms, concentrations and growth media. 1 These compounds were tested both at 28uCand 37uC. Caffeine and fluoride sensitivity phenotypes were found only at 37uC. 2 Np, Kp and Sop are the acronyms for the salt preconditioned tests. In these assays, the cells were precultivated on YPD containing 0.3 M NaCl before being transferred onto the test plates. 3 This compound was tested only with EUROSCARF strains Temperature and incubation times of the heat tolerance tests have been indicated in the second and third columns, respectively. 5 Tp is the acronym for the heat preconditioned test. In this assay the test plates were preincubated at 38uCfor 90 min before being treated at 52uC. The diploid heterozygous deletiontstrains were induced to sporulate following the standard procedures used for the EUROFAN B0 project and the spores dissected. For each gene deletion, 10 complete tetrads of haploid segregant strains showing a 2 : 2 segregation of all the genetic markers were chosen for further analysis. The haploid segregant strains were tested as described above for the MATa and MATa strains. Only cases in which a clear co-segregation of the phenotype(s) with the deletion marker phenotype was found were considered to test positive for tetrad analysis. A total of 293 phenotypic tests have been performed on tetrads issued from the 75 diploid strains selected for this second round of assays, yielding more than further single tests. Correlation between phenotypes The frequency of simultaneous occurrence of two independent phenotypes, a and b, in the same strain should be the product of the frequency of occurrence of the single phenotype: (n a /n t )r(n b /n t ), where n a and n b are the numbers of strains showing phenotypes a and b, respectively, and n t is the total number of analysed strains. When the actual number of strains exhibiting both phenotypes is much higher than (n a rn b )/n t, a positive correlation between the two phenotypes can be deduced; when the actual number of strains exhibiting both phenotypes is much lower than (n a rn b )/n t, a negative correlation can be inferred. Results Genes analysed and choice of tests In a previous work (Bianchi et al., 1999) we described procedures for the detection of phenotypes related to osmotolerance, resistance to ethanol, thermotolerance and sensitivity to compounds affecting protein phosphorylation level, protein synthesis or nucleic acids synthesis. At that time, deletions in 66 genes were analysed. In the present work, we have included tests of sensitivity to hygromycin B, benomyl, and metals (calcium, cadmium, cobalt, caesium and mercury) and we have added further compounds affecting nucleic acid synthesis (azaserine, diazo-oxo-norleucine, cordycepin, 5-fluorouracil, 5-fluorotryptophan, 8- hydroxyquinoline, patulin) and amino acid analogues (S-2-aminoethyl-L-cysteine and M-fluoro-DL-tyrosine). The complete list of the tested inhibitors (31 chemicals + heat) is reported in Table 1. A total

4 1400 M. M. Bianchi et al. number of 49 growth conditions have been tested, differing by nature and/or concentration of the inhibitor. The new set of tests has been performed on a large number of deleted strains. 564 genes of chromosomes II, IV, VII, X, XII, XIV and XV deleted, with very few exceptions, in both MATa and MATa strains have been analysed. The complete list of these genes is available in the web pages of the B1 Consortium at the MIPS site ( mips.gsf.de/proj/eurofan/eurofan_2/b1/allgenestested. html). When this work was started, most of the genes included in the study coded for unknown proteins or for proteins with similarities to proteins of unknown function in yeast. Data about these genes must be frequently updated because of the continuous addition of information to database from the systematic sequencing of other organisms and functional genomics. In order to facilitate interpretation of the results and on the basis of the MIPS classification of yeast ORFs (as of January 2001), we have grouped the 564 genes into four functional classes, called U, SU, SK and K. The U class contained five questionable ORFs and 113 genes coding for proteins of unknown function; the SU class contained 120 genes coding for proteins similar to proteins of unknown function from yeast or other organisms; the SK class was composed of 165 genes coding for proteins showing various degrees of similarity to proteins of known function; and the K class contained 161 genes coding for proteins of known function. It must be noted that proteins encoded by genes of class K, although they are of known or partially characterized function, very often still do not have a known cellular role or, alternatively, they participate to well-defined cell activities but they are unknown proteins or have been very poorly characterized. Gene deletions yielding phenotype(s) in MATa and MATa strains In our analysis, we have found 163 single-gene deleted strains, which corresponds to about onethird of the 564 analysed genes that exhibited at least one phenotype in both MATa and MATa strains. The list of genes and the phenotypes of the corresponding deleted strains are reported in Table 2. The tested phenotypes, which relate to different aspects of cell functioning, can be grouped into 10 categories, depending on the general biological activities affected by the inhibitory compounds, as follows: phenotypes related to stress conditions, such as thermotolerance (T, Tp), osmotolerance (K, Kp, N, Np, S, Sp) and ethanol resistance (DE, E); phenotypes derived from defects in DNA/mRNA/ trna formation (Azs, Dau, Don, Fcy, Fur, OHq, Pat) or protein synthesis (Cor, Cyc, Ver); phenotypes related to altered levels of protein phosphorylation (Caf, F, Tfp, V); resistance or sensitivity to metals (Ca, Cd, Co, Cs and Hg) and to amino acid analogues (Aec, Alg, Fpa, Fty, Ftr, Tal). We included in our analysis benomyl (Be) and hygromycin B (Hy). Benomyl is a benzimidazole derivative affecting cytoskeletal functions (Thomas et al., 1985). Altered sensitivity to the aminoglycoside antibiotic hygromycin B can frequently be correlated to glycosylation defects (Dean, 1995). This antibiotic can also be used to detect translational defects (Hampsey, 1997). We have detected 664 phenotypes, distributed among the 163 test-positive deleted strains. The found phenotypes, numerically reported as segments of histograms representing the above categories, are reported in Figure 1. The most commonly found phenotypes were responses to metals; in particular, sensitivity or resistance to cadmium and cobalt. Other very frequent phenotypes were sensitivity to compounds that affect protein phosphorylation/ dephosphorylation, especially to caffeine, to the protein synthesis inhibitor cycloheximide, sensitivity to hygromycin B, and sensitivity or resistance to benomyl. Sensitivity to individual inhibitors of nucleic acids or protein synthesis and phenotypes related to specific cellular aspects, i.e. resistance to ethanol, were less frequent. The results of the analysis of phenotype distribution among the deleted strains is reported in Figure 2, in which we have arbitrarily considered and grouped as highly pleiotropic the 13 single-gene deletions showing more than 10 phenotypes. A large proportion of the deletion mutants exhibiting phenotypes were monotropic or showed sensitivity to few inhibitors, suggesting a very restricted role in cellular activities. Benomyl sensitivity or resistance were often found as a monotropic phenotype of strains deleted of genes encoding unknown proteins, such as Ybl010p, Ybl029p, Ybl031p (She1p), Yll032p, Yll049p and Ynr009p, indicating that a conspicuous number of new proteins might be specifically involved in cytoskeletal functions. Moderately and highly pleiotropic deleted strains were a considerable proportion of test-positive

5 Role of unknown genes in yeast 1401 Table 2. Gene deletions and corresponding phenotypes Systematic gene name Euroscarf number Class YPD 28uC HS phenotypes S phenotypes R phenotypes YBL006c U Be Be, Cor, Fcy, Hg, K, Kp, N, Np, Tfp YBL010c U Be YBL024w K T, Ver Co, Hg YBL025w K S Cd, Cyc Be, Ca, Co, Cs, Hg, OHq YBL029w U Be YBL031w U Be Be YBL047c SK Azs, Caf, V Be, Cd, Co, Cs, Cyc Hy YBL048w U K, E K, Kp, OHq Cyc, Co YBL051c SU Hy, Ver Aec, Be, Caf, Cd, Co, Cs, Cyc, Dau, Tp YBR032w U Caf YBR043c SK Ca, Co YBR076w K T YBR078w K Caf, Hy Hg, Hy, T YBR131w K Caf, Fcy Ca, Cyc, N YBR175w SK Caf, Cd, Hy YBR187w SK E YBR204c SK T, Tp YBR216c SU Cd YBR223c U V YBR225w U V YBR229c K Hy Cd, Cs, F, K V YBR255w U Hy YBR266c U 1 S Ca, Cd, DE, Hy, K, Kp, N, Np, So, Sop, T, Tp YBR283c K S Hy, Cd YDL005c K Co, Cyc, Hy, V Ca, Caf, Cd, Cs, E, Hg, Hy, K, Kp, OHq, T, Tp YDL025c SK Hy YDL059c K Azs YDL063c SK Hy Hy, T YDL065c K Cor YDL074c SK Azs, Cor, Pat Caf, Cd, Co, Cyc, Hy, T Be YDL082w K Hy, K, Kp, So, Sop Be YDL100c SK F, Hy Cd, Hy YDL110c U Cd Cd YDL115c U S Fur Be, Ca, Cs, Cyc, Fcy, Hg, Kp, Kp, Np, Sop Np, OHq, Sop YDL117w SU Hy Ca, Cd, Co, Cs, K, So, Sop YDL119c SK K, Kp, So, Sop, T, Tp YDL120w K Cd, OHq Ca, Co, Cs, Cyc, Hg, OHq YDL131w K Ver YDL142c K T YDL202w K Cor, Fpa OHq YDL219w SU S Hy Aec, E YDL225w K Caf Co YDL231c U Cd, Co, Hy Cd, Co, Cs YDL234c SU Hy YDL243c K Be YDR067c SU Caf, Cor, Fty, Hg YDR071c SK T T YDR072c K Tfp YGL064c SU S E Fpa, K Kp, OHq, So, Sop YGL078c K Fcy Caf, Fur YGL094c K Be, Pat, Tal

6 1402 M. M. Bianchi et al. Table 2. Continued Systematic gene name Euroscarf number Class YPD 28uC HS phenotypes S phenotypes R phenotypes YGL099w SK S Cd, Co, Cs, Cyc, Hg Be, Ca, Hg, OHq YGL100w K DE, E, Hy, T YGL124c SU Fcy, Hy Ca, Co, Hg, Hy YGL128c SK Be, Hg, Ohq Be, Ca, Co, Cs, Hg, OHq YGL129c SU Cs, DE Cyc YGL131c SU Ver Azs, DE, E, Hy, Pat Be YGL133w SU Cs, Hy, T T YGL134w K Cd YGL138c U Ver Caf, Cd YGL186c SK Be, Cd, Cs, Cyc, Hg, OHq YGL194c K Cor YGL231c U Cd, Hy YGR187c K Dau YGR196c SK Ohq YGR216c K Co YGR226c U Tp YGR237c SU Hy Cs, N, Np YGR260w SK Fty YGR262c SK S Be, Ca, Co, Cs, Hg, OHq Be, Ca, Caf, Cd, Co, Cyc, F, Ohq, Tfp, V YJL004c K Caf, F Cd, Hy YJL006c K S Be, Ca, Caf, Cs, Cyc, Ca, Hg, OHq, So, Sop, T, Tp F, Hg, Hy, K, Kp, N, Np, T, Tfp, V YJL018w U 1 Be YJL019w U Be, Caf Be, Hy, T, Tfp YJL020c SK Caf Cyc YJL029c K S Cd, Co, Cs, Cyc, F, V Ca, Cd, Cyc, E, Hg, K, Kp, N, Np, OHq YJL047c SK Azs, Hy, Ver Co, V YJL056c K Co, Cyc, Hy YJL071w K Co YJL094c K Cd Cd, Hy YJL124c K Azs, Cyc, Fcy, Fpa Co, Fur, Hg Alg YJL144w U Hg YJL148w K S K, Kp K, Kp, N, Np, So, Sop YJL204c SK Azs, Cor, Don, Cd, Cyc, K, Kp, N, Np, Cd Fcy, Hy So, T, Tp, Ver, YJL207c SK Cd, K, Kp, Tfp YJR013w SU Be, Caf, F Be, Cs, Hg, Tfp, V YJR044c SU Ver Cd YJR056c U Caf YJR059w K Caf, Cd YJR074w K S 37u Cd, Cyc, Ver Be, Cd, Co, Cyc, Hg, Kp, N, Np YJR075w K Hy Co, Cyc, Hg, Ver Alg, V YLL029w SK Co YLL030c U Co Co YLL032c U Be YLL033w U S Caf Cd, DE, E, Fpa, Hy, K, Kp, Hy Np, OHq, So, Sop, T YLL049w U Be YLR015w SU Caf Cd Be YLR018c K Be YLR021w U Caf

7 Role of unknown genes in yeast 1403 Table 2. Continued Systematic gene name Euroscarf number Class YPD 28uC HS phenotypes S phenotypes R phenotypes YLR024c SK Co, Hy, T Co, Cs, Hy, T, Tp YLR034c SK Tp Tp YLR114c SU Hy YLR143w SU Fpa YNL021w K Cd, Co YNL040w SK Co YNL044w K Hy YNL051w U Caf, Co, F Be, Co, Hy YNL054w K Caf, Cd Cd, Cyc YNL056w SU Caf, Dau YNL059c K S Be, Cd Ca, Cyc, E, Hg, K, Kp, N, Np, OHq, So, Sop, YNL080c U S Cor, E, Hy, Ver Be, Ca, Cd, DE, Fur, Hy, K, Kp, N, Np, OHq, So, Sop YNL081c SK S E Cor, DE, Fpa, Hy, K, Kp, N, Np, OHq, So, Sop YNL091w SK Co, Cs, Cyc, Hy YNL097c K Cyc Be YNL099c SU Caf Cd Co, Ver YNL101w SU N, Np Fpa Alg, Cd, Co, Hg, V YNL107w SK Be, Cs, Hy Aec, Cd, Cs, Cyc, F, Hg, Hy, K, N, Np, Tfp YNL119w SU Caf, Hg, N, Np, V YNL148c SK Be YNL206c SK Hy Cyc, N, Np Alg YNL213c U Cd Co YNL214w K Cor YNL215w SU S Cd, Cs, Cyc YNL219c K Cd YNL224c U Cyc YNL227c SK T Cd, Hy, K, Kp, So, Sop, T, Tp YNL231c K Be Be, Co YNL273w K Azs, Hg, OHq YNL288w SU Caf, Cd YNL293w SK Cd YNL294c U Caf, N, Np Cd YNL295w U Tfp YNL297c SU Cd Cd, Hy, N, Np YNL306w K Co, Cyc YNL310c SU Be YNL311c U Azs YNL325c SK Caf YNR004w SK Be, Cd YNR009w U Be YNR020c U Cs Co YNR051c SK Caf, Don Aec, Be, Ca, Cd, Fpa Ver YOL018c K Hy Fpa, N, Np YOL072w U Be, Cd, Cyc, Hg Cd, Hg YOL087c SU Caf Caf, Hg, Hy Alg YOL088c K Cd YOL095c K S 37uC E Cs, DE, E YOL098c SU Fcy, Fur YOL111c SK DE, E YOL115w K Pat Cd, DE, E, Ftr, Hy, K, Kp, N, Np, T, Tp

8 1404 M. M. Bianchi et al. Table 2. Continued Systematic gene name Euroscarf number Class YPD 28uC HS phenotypes S phenotypes R phenotypes YOL124c SU Hy, N, Np Hy Alg, Cd, V YOL138c U Hg Hg, Hy Alg YOL141w SU Cs, OHq Co YOL151w SK Hy, N, Np, T Cyc, Hy, T Cd, V YOR007c SK Hy YOR109w K F, Hy Cd YOR154w SU Cd YOR162c K Caf YOR164c U Hy Hy YOR267c SK Caf, Cd Hy YOR275c SK Ca, N, Np Cd YOR279c U V Caf, Cd, Co, Cs, Cyc, K, Kp, T, Tp YOR311c SU Cd YOR322c SU Caf, Cd, V Ca, Cd, Co, F 1 Questionable ORFs (included in the U class). See text and Table 1 for acronyms and abbreviations. strains. While this is a result to be expected for known genes involved in general cellular mechanisms, it is worthwhile noting that also unknown genes (YDL115c, YLL033w, and YNL080c), genes with similarity to an unknown gene (YBL051c) and a questionable ORF (YBR266c) had highly pleiotropic phenotypes, suggesting participation in signal cascades or other general functions. Within the highly pleiotropic strains, the deletion of genes YBR266c, YDL115c, YJL006c, YJL029c, YLL033w, YNL059c, YNL080c and YNL081c yielded mutant strains that grew slower than wild-type on YPD, even in the absence of inhibitors. Reduced growth rate on YPD was rarely found in non-pleiotropic mutant strains. Co-segregation of phenotypes and with the deletion marker The finding of the same phenotype in two strains, MATa and MATa, carrying the same gene deletion is a good indication, but it is not a demonstration, that phenotype and deletion are linked. Confirmatory experiments can be performed by recovery of the wild-type phenotype after transformation with the cognate gene or by analysis of tetrads issued from heterozygous deletedtstrains and verification of co-segregation of the phenotype with the deletion marker in the haploid segregant strains. These experimental procedures, although not completely equivalent, are interchangeable in cases of poor sporulation or low transformation efficiency. However, transformation with the cognate gene might have drawbacks such as plasmid loss, copy number or gene expression effects, or possible mutations introduced by cell treatments. Therefore, we preferred tetrad analysis as a systematic approach and we performed this procedure on 75 out of the 163 genes that exhibited phenotype(s) in MATa and MATa genetic backgrounds of the deleted strains. The analysed genes were representatives of the four functional classes and the corresponding MATa and MATa deleted strains exhibited monotropic to highly pleiotropic phenotypes. The phenotypes tested for each gene deletion by tetrad analysis are reported in Table 3. Examples of analysis on tetrads are shown in Figure 3. Co-segregation with the deletion marker of at least one of the tested phenotypes was demonstrated for 52 gene deletions, which corresponded to more than two-thirds of the genes analysed. The percentage of co-segregation varied considerably from test to test. The detailed results of the percentages of phenotype co-segregation relative to a selected subset of inhibitors are shown in Table 4. Sensitivity or resistance to many inhibitors, such as calcium, caffeine, caesium, fluoride, hygromycin B and hydroxyquinoline, were found to co-segregate at high frequency, while a few other compounds, such as cobalt, mercury, osmolytes and verrucarin, showed a low frequency of co-segregation with the deletion marker, suggesting that strain variability, either spontaneous or induced by manipulations, might more frequently affect growth on these inhibitors. No conclusive inferences could be

9 Role of unknown genes in yeast 1405 Figure 1. The histograms represent the 10 categories into which the phenotypic tests can be grouped. An additional histogram indicates the general phenotype of slow growth in standard YPD medium without inhibitory compounds. Each histogram is composed of sections relative to the individual tests included in the category. Individual tests are marked as follows,upwards from bottom. Stippled,Cd,Np,Caf,Cyc,Hy,OHq,Be,T,E,Fpa and YPD; chequered,co,n,v,ver,azs, Tp,DE and Alg; horizontal stripes,hg,k,f,cor,fcy and Aec; oblique stripes,cs,kp,tfp,fur and Fty; vertical stripes,ca, So,Pat and Ftr; white,sop,dau and Tal; black,don drawn for compounds for which tetrad analysis was performed on only a few deleted genes. We have also found that auxotrophy for tryptophan, which was heterozygous in most of the dissected diploid strains (trp1d63t), frequently increased the sensitivity to cobalt and mercury, thereby blurring the analysis of co-segregation with the deletion marker. Sporulation and asci dissection for tetrad analysis showed that eight heterozygous diploid strains yielded only two viable spores. These strains are reported in Table 5. In four cases (ygl064cdt, yjl018wdt, yjl019wdt and yjr013wdt), the spore non-viability was associated with geneticin resistance, suggesting a linkage with the deleted gene. Since haploid deleted strains of these genes do exist, they are supposed not to yield lethal deletions. The

10 1406 M. M. Bianchi et al. Figure 2. Each histogram represent a group of gene deletions exhibiting the same number of phenotypes,1 10. The eleventh histogram collects the gene deletions showing more than 10 phenotypes (pleiotropic strains). The number of individuals that constitute the groups are indicated at the tops of the histograms causes of failure of spore germination and/or cell multiplication remain to be explained. The genes whose deletions were linked to spore non-viability belonged to the U and SU classes. Discussion Role of unknown proteins in cellular functions A phenotypic analysis, set up to be performed on a large number of yeast strains and successfully evaluated in a previous pilot project, has been carried out on 564 gene deletions in both MATa and MATa genetic backgrounds of the standard strain FY1679. About one-third of the gene deletions tested showed growth phenotype(s) in both strains. Our analysis included unknown genes (class U), unknown genes having interspecific or intraspecific similarity to other unknown genes (class SU), genes showing different degrees of similarity to known genes or to families of genes (class SK) and

11 Role of unknown genes in yeast 1407 Table 3. Segregation of phenotypes and deletion marker in tetrads Systematic gene name Euroscarf number Class Co-segregating phenotypes Non-co-segregating phenotypes YBL006c U Be, Cor, Fcy YBL010c U Be YBL024w K Ver YBL047c SK Be, Hy R, Azs, Cyc, Cd, Caf, Tfp, V Co, Cs YBL048w U Cyc R,Co R, E, K, Kp, OHq YBL051c SU Hy, Tp, Cd, Cs, Caf Dau, Aec, Ver, Cyc, Co, Be YBR078w K Hg YBR216c SU Cd YBR223c U V R YBR229c K Cd, Cs YBR255w U Hy YBR283c K Cd YDL025c SK Hy R YDL100c SK F YDL110c U Cd YDL115c U Be, Ca, Cs, Fur, Fcy, Cyc, Cd, OHq Hg YDL117w SU So, Sop, Hy K, Ca, Cd, Co, Cs YDL120w K Be, Ca, Cs, Cyc, Cd, Co, Hg, OHq YDL202w K Cs, OHq YDL219w SU E, Hy Aec YDL231c U Cd, Cs, Hy Co YDR067c SU Cor, Caf Fty YDR072c K Tfp YGL124c SU Ca, Cs, Fcy, Caf, Tfp Co, Hg YGL128c SK Be, Ca, Cyc, Cd, Cs, OHq Co, Hg YGL131c SU DE, E, Hy, Azs, Pat, Ver YGL134w K Cd YGL186c SK Be, Ca, Cyc, Cd, Cs, OHq Hg YGL231c U Cd YGR187c K Dau YGR237c SU Cs, OHq YGR260w SK Fty YGR262c SK Be, Ca, Cd, Co, Cs, Cyc, Hg, OHq YJL004c K Cd, Caf, F YJL006c K Be, Ca, Cs, Cyc, Co, Cd, Hg, OHq YJL020c SK Caf Cyc YJL029c K Be, Cyc, Cd, Co, Ca, Cs, Hg, K, Kp, N, Np, OHq, Caf, F, Tfp, V YJL047c SK Azs, Ver, V YJL124c K Be, Cyc, Hg Co YJL144w U Hg YJL204c SK Co, Cs, N, Np, K, Kp, Hy, Azs, F, Tfp, Ver, Cd Fcy, Don, Cor, Cyc YJL207c SK Cd, K, Kp, Tfp YJR074w K Ca, Cd, Co, Cs, Cyc, OHq Be, Hg YLL029w SK Co YLL033w U Cd, Cs, OHq YLL049w U Be YLR018c K Be R YLR024c SK Co, Cs, Hy YNL021w K Cd YNL051w U Be, Co YNL059c K Be, Cyc, Cd, Ca, Cs, OHq Hg YNL080c U Be, Ca, Cd, Co, Cs, Cyc, Hg, OHq YNL097c K Be R,Cyc YNL099c SU Cd, Caf Co R, Ver R

12 1408 M. M. Bianchi et al. Table 3. Continued Systematic gene name Euroscarf number Class Co-segregating phenotypes Non-co-segregating phenotypes YNL101w SU Cd R,Co R,Hg R, N, Np, V R YNL107w SK Aec, Be, Cyc, Cd, Cs, OHq, Hy, Tfp Hg, K, N, Np, F YNL224c U Cyc YNL227c SK Cd, Cs YNL231c K Be, Co YNL273w K Azs YNL311c U Azs YNR051c SK Be, Ca, Cd, Cyc, Don, Fpa, Aec, Ver R OHq, Caf, V YOL072w U Cyc, Ca, Cs, Hg R, OHq Be, Cd YOL098c SU Fur, Fcy YOL111c SK Hy DE, E YOL124c SU Alg R,Cd R, Hy, N, Np, V R YOL141w SU Co R,Cs YOL151w SK Cyc, Cd R YOR109w K Cd, F YOR154w SU Cd YOR164c U Hy YOR267c SK Hy R YOR275c SK Cd R,Caf YOR279c U Cyc, Co, Cd, Cs YOR322c SU Caf, F, V See text and Table 1 for acronyms and abbreviations. All the reported phenotypes were of sensitivity or high sensitivity to the inhibitor, except when specified by R (resistance). known genes (class K) in most cases poorly characterized. These functional categories were similarly represented in the present phenotypic analysis. The first relevant output of our data is that gene deletions affecting growth in the presence of the inhibitors were equally distributed among the four functional classes, indicating that known and unknown proteins contribute similarly to the investigated cellular functions (Table 6). The frequent occurrence of monotropic phenotypes in deleted strains of unknown genes indicates that a large proportion of these genes are involved in very specific cell activities, especially cytoskeletal functions (benomyl sensitivity and/or resistance). On the other hand, unknown genes might also occupy relevant positions in functional networks or regulatory cascades that control different cell activities, as suggested by the finding of highly pleiotropic phenotypes in deleted strains of other unknown genes. The contribution of unknown genes to cellular functions could not be foreseen and points to the relevance in the cell of genes discovered by genome sequencing and not detected by classical approaches of biochemistry, physiology and genetics. Co-segregation of growth defects and gene deletions have been demonstrated by phenotypic analysis on several haploid segregant strains issued from heterozygous diploid strains. Tetrad analysis showed co-segregation for about two-thirds of the 75 gene deletions tested. Deleted strains exhibiting at least one co-segregating phenotype have been found in all the four functional classes, as reported in Table 6, confirming the importance of the unknown proteins in the cellular activities explored in this analysis. Five questionable ORFs have been included in our analysis and in two cases phenotypes have been observed. ORF YBR266c, encoding a hypothetical membrane protein (Doignon et al., 1993), overlaps with the unknown gene YBR267w, which contains a typical zinc finger domain. Our results indicate a pleiotropic sensitivity to metals, osmolytes, high temperature and hygromycin B of ybr266c-deleted strains, suggesting a general cellular role for Ybr266p. Since no particular growth defect has been reported to date for ybr267w-deleted strains (see web databases), which were not included in our phenotypic investigations, more detailed analyses

13 Role of unknown genes in yeast 1409 Figure 3. Examples of co-segregations of the gene deletion phenotype (resistance to geneticin) and the sensitivities to growth inhibitors in tetrads issued from some deletiontheterozygous strains are reported. One tetrad is shown for each example. Growth on standard YPD medium and on YPD medium supplemented with geneticin or with the inhibitor is shown in first,second and third columns,respectively. Inhibitors are shown the fourth column and the gene deletions subjected to the phenotypic tests are shown in the fifth column should be performed to assign individual functions to these genes. YJL018w is a 315 nt ORF that overlaps, by 130 nucleotides and on the same strand, the 3k end of the 1863 nt ORF of the unknown gene YJL019w. Recently, Yjl019p has been assigned to the functional category of proteins involved in chromatin/chromosome structure, on the basis of physical interactions with a putative component of telomerase (Est1p) and with a histone-related protein (Htz1p; Schwikowski et al., 2000). In our functional analysis, yjl018w- and yjl019w-deleted strains shared benomyl sensitivity and a spore germination defect; yjl019w-deleted strains also exhibited some additional phenotypes (Table 2). These results suggest that an activity involved in cytoskeleton-related functions might be located the C-terminal portion, i.e. the 82 nucleotides that are deleted in yjl018wd strains, of Yjl019p. Alternatively, involvement in cytoskeleton and spore germination functions might

14 1410 M. M. Bianchi et al. Table 4. Percentages of co-segregation of selected phenotypes 1 Phenotype (acronym) Number of tested gene deletions Number of gene deletions showing co-segregation Percentage of co-segregation 2 Azs (t 19) Be (t 10) C a (t 7) Caf (t 0) C d (t 7) Co (t 10) C s (t 8) Cyc (t 9) F (t 14) Fcy (t 0) Hg (t 12) Hy (t 10) OHq (t 6) Osmolytes (t 10) Tfp (t 0) V (t 0) Ver (t 13) 1 This table lists phenotypic tests that have been performed on more than five heterozygous deleted strains. 2 Errors reported in parentheses have been calculated by [x(1 x)/n] 1/2, where x is the ratio between the number of gene deletions showing co-segregation (values reported in column 3) and the number of tested gene deletions (values reported in column 2), and n is the number of tested gene deletions (column 2). 3 This group includes phenotypes N, Np, K, Kp, So and Sop. be attributed to Yjl018p and only the additional phenotypes to the deletion of YJL019w. Confirmatory experiments, such as transformation of the deleted strains with YJL018w and detection of the recovered phenotypes, should be performed. Table 5. Formation of non-viable spores from heterozygous diploid strains Strain Euroscarf number Class ybr131wd/+* K No ydl063cd/ SK No ydl074cd/ SK No ygl064cd/+* SU Yes yjl018wd/ U Yes yjl019wd/ U Yes yjr013wd/ SU Yes ylr015wd/ SU No Linkage with the deletion marker *Very reduced sporulation phenotype was reported for homozygous diploid deleted strains of genes YBR131w and YGL064c by the EUROFAN Development Node 8. Table 6. Distribution of phenotypes among functional classes of genes Gene classes Gene deletions with phenotype(s) in MATa and MATa strains The same set of deleted genes has been analysed by other nodes of the EUROFAN project for defects in specific aspects of cellular activities. A detailed analysis of possible correlation between detected phenotypes is still to be performed and could be very useful. In a preliminary comparison we have found a positive correlation between sensitivity to hygromycin B, which correlates with glycosylation defects, and phenotypes studied by the Secretion Node 5, such as altered CPY secretion or lucifer yellow accumulation (endocytosis assay), but not with the UPR (unfolded protein response) assay. A positive correlation has also been found between sensitivity to the cytoskeletal drug benomyl and UV light- or c-ray-sensitive phenotypes detected by Node 1 (DNA metabolism and meiosis). Integrating functional approaches Gene deletions with phenotype(s) co-segregating with marker gene (n/n tot ) (%) (n/n tot ) (%) K 53/ / SK 41/ / SU 32/ / U 38/ / K+SK+SU+U 163/ / The comparison and integration of results from different genome-wide functional approaches can obviously help in the definition of roles and mechanisms of action of poorly characterized proteins. The following example will illustrate an integration of results from our phenotypic analysis and a two-hybrid protein protein interaction search (Uetz et al., 2000). The ynl311cd strains were monotropic and showed co-segregation with the deletion marker of sensitivity to azaserine, a glutamine analogue that inhibits purine biosynthetic steps requiring glutamine as donor of amino groups. In the two-hybrid database, the Ynl311p is reported to interact with Yil074p (Ser33p) and Ykl001p (Met14p), which are involved in serine biosynthesis and sulphur assimilation, respectively (Masselot et al., 1975; Melcher and Entian, 1992). The first interacting protein is an isozyme of 3-phosphoglycerate dehydrogenase. We

15 Role of unknown genes in yeast 1411 suggest that Ynl311p might be an intermetabolic regulator of serine and purine biosynthetic pathways, and possibly also of sulphur assimilation. Ynl311p might act by regulating cellular levels of glutamine and glutamate, the latter of which is the amino group donor of the 3-phosphoserine biosynthetic step catalysed by Ser1p. Interestingly, SER1 has been demonstrated to be allelic to ade9, which is a mutation formerly isolated as defective for purine synthesis (Buc and Rolfes, 1999). Further development of phenotypic analysis The examples discussed above suggest that even an integrated analysis of the available genomic functional approaches will not, in the vast majority of cases, be conclusive for determination of the single protein function. Specific and dedicated analyses to single cases will be necessary, including cellular localization, gene overexpression and multiple deletions analysis. In this respect, the finding of clear and easily selectable phenotypes and the demonstration of the linkage between gene deletion and phenotype(s) by tetrad analysis are preliminary conditions to be fulfilled for the elucidation of protein functions by classical genetic approaches such as isolation of suppressors. Many proteins belonging to the class of known proteins are actually very poorly characterized: the vesicular protein Vps53p (Yjl029p) is a component of the complex Vps52p Vps53p Vps54p, involved in retrograde protein transport of the Golgi (Conibear and Stevens, 2000). Vps53p is classified as a known protein but its role in the complex and its mechanism of action are unknown. In addition, yjl029c mutant phenotypes partial missorting of CPY and mislocalization of Golgi membrane proteins are not easily selectable. Our phenotypic analysis showed that yjl029c-deleted strains are hypersensitive to many growth inhibitors, such as cyclohexymide, cadmium, cobalt, caesium, fluoride and vanadate, and that sensitivity cosegregates with the deletion marker. These phenotypes might provide an alternative procedure for the selection of various kinds of extragenic suppressors, homologous and/or heterologous, that will help to shed light on the details of its cellular role. Conclusions The characterization of new yeast genes has greatly increased in the last years, thanks to the recently development of mass analyses, computational approaches and sequencing of new genomes, which decreases the number of orphan genes. This trend might suggest that, in a few years, all the yeast genes will have an assigned role in the cell machine. However, there are still one-third of the unknown genes and of the genes similar to unknown genes from other organisms analysed by the EUROFAN network, whose deleted strains did not exhibit any phenotype in the EUROFAN phenotypic analysis. These hyperunknown genes might be functionally redundant, they might be associated with very subtle phenotypes or they might be involved in cellular mechanisms not covered by the EURO- FAN project. Their involvement in completely new and unexpected cellular activities is also not excluded. These genes are the next challenge for functional analysis. Acknowledgement This work was supported by BIO4-CT (Eurofan II). References Bianchi MM, Sartori G, Vandenbol M, et al How to bring orphan genes into functional families. Yeast 15: Buc PS, Rolfes RJ ade9 is an allele of SER1 and plays an indirect role in purine biosynthesis. Yeast 15: Cherry JM, Ball C, Dolinski K, Dwight S, et al. Saccharomyces Genome Database; web site: saccharomyces/. Conibear E, Stevens TH Vps52p, Vps53p and Vps54p form a novel subunit complex required for protein sorting at the yeast late Golgi. Mol Biol Cell 11: Costanzo MC, Hogan JD, Cusick ME, et al The Yeast Proteome Database (YPD) and Caenorhabditis elegans Proteome Database (WormPD): comprehensive resources for the organization and comparison of model organism protein information. Nucleic Acids Res 28: Dean N Yeast glycosylation mutants are sensitive to aminoglycosides. Proc Natl Acad Sci U S A 95: Doignon F, Biteau N, Crouzet M, Aigle M The complete sequence of a bp segment located on the right arm of chromosome II from Saccharomyces cerevisiae. Yeast 9: Goffeau A, et al Life with 6000 genes. Science 274: Hampsey M A review of phenotypes in Saccharomyces cerevisiae. Yeast 13: Masselot M, De Robichon-Szulmajster H Methionine biosynthesis in Saccharomyces cerevisiae. I. Genetical analysis of auxotrophic mutants. Mol Gen Genet 139: Melcher K, Entian KD Genetic analysis of serine biosynthesis and glucose repression in yeast. Curr Genet 21:

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