Haploinsufficiency-based large-scale forward genetic analysis of filamentous growth in the diploid human fungal pathogen C.albicans
2003; Springer Nature; Volume: 22; Issue: 11 Linguagem: Inglês
10.1093/emboj/cdg256
ISSN1460-2075
Autores Tópico(s)Plant tissue culture and regeneration
ResumoArticle1 June 2003free access Haploinsufficiency-based large-scale forward genetic analysis of filamentous growth in the diploid human fungal pathogen C.albicans M.Andrew Uhl M.Andrew Uhl Department of Microbiology and Immunology, University of California at San Francisco, 513 Parnassus Avenue, S-410, San Francisco, CA, 94143-0414 USA Search for more papers by this author Matt Biery Matt Biery Howard Hughes Medical Institute, Department of Molecular Biology and Genetics, Johns Hopkins University School of Medicine, Baltimore, MD, 21205 USA Search for more papers by this author Nancy Craig Nancy Craig Howard Hughes Medical Institute, Department of Molecular Biology and Genetics, Johns Hopkins University School of Medicine, Baltimore, MD, 21205 USA Search for more papers by this author Alexander D. Johnson Corresponding Author Alexander D. Johnson Department of Microbiology and Immunology, University of California at San Francisco, 513 Parnassus Avenue, S-410, San Francisco, CA, 94143-0414 USA Department of Biochemistry and Biophysics, University of California at San Francisco, 513 Parnassus Avenue, S-410, San Francisco, CA, 94143-0414 USA Search for more papers by this author M.Andrew Uhl M.Andrew Uhl Department of Microbiology and Immunology, University of California at San Francisco, 513 Parnassus Avenue, S-410, San Francisco, CA, 94143-0414 USA Search for more papers by this author Matt Biery Matt Biery Howard Hughes Medical Institute, Department of Molecular Biology and Genetics, Johns Hopkins University School of Medicine, Baltimore, MD, 21205 USA Search for more papers by this author Nancy Craig Nancy Craig Howard Hughes Medical Institute, Department of Molecular Biology and Genetics, Johns Hopkins University School of Medicine, Baltimore, MD, 21205 USA Search for more papers by this author Alexander D. Johnson Corresponding Author Alexander D. Johnson Department of Microbiology and Immunology, University of California at San Francisco, 513 Parnassus Avenue, S-410, San Francisco, CA, 94143-0414 USA Department of Biochemistry and Biophysics, University of California at San Francisco, 513 Parnassus Avenue, S-410, San Francisco, CA, 94143-0414 USA Search for more papers by this author Author Information M.Andrew Uhl1, Matt Biery2, Nancy Craig2 and Alexander D. Johnson 1,3 1Department of Microbiology and Immunology, University of California at San Francisco, 513 Parnassus Avenue, S-410, San Francisco, CA, 94143-0414 USA 2Howard Hughes Medical Institute, Department of Molecular Biology and Genetics, Johns Hopkins University School of Medicine, Baltimore, MD, 21205 USA 3Department of Biochemistry and Biophysics, University of California at San Francisco, 513 Parnassus Avenue, S-410, San Francisco, CA, 94143-0414 USA *Corresponding author. E-mail: [email protected] The EMBO Journal (2003)22:2668-2678https://doi.org/10.1093/emboj/cdg256 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Candida albicans is the most prevalent human fungal pathogen. Here, we take advantage of haploinsufficiency and transposon mutagenesis to perform large-scale loss-of-function genetic screen in this organism. We identified mutations in 146 genes that affect the switch between its single-cell (yeast) form and filamentous forms of growth; this switch appears central to the virulence of C.albicans. The encoded proteins include those involved in nutrient sensing, signal transduction, transcriptional control, cytoskeletal organization and cell wall construction. Approxim ately one-third of the genes identified in the screen lack homologs in Saccharomyces cerevisiae and other model organisms and thus constitute candidate antifungal drug targets. These results illustrate the value of performing forward genetic studies in bona fide pathogens. Introduction Candida albicans is a commensal of humans and other warm-blooded animals that can cause mucosal infections in immunocompetent individuals as well as a broad spectrum of symptoms in immunocompromised patients, including serious disseminated infections (for reviews see Odds, 1988; San-Blas et al., 2000; Calderone and Fonzi, 2001; Haynes, 2001). Progress in understanding many aspects of the biology of C.albicans has been hindered by the inability to carry out simple, large-scale genetic screens. Such screens are highly effective ways of gaining access to and ultimately understanding biological problems, as evidenced by their widespread utility in the ‘model’ yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe. The lack of traditional genetic approaches in C.albicans has been due largely to the absence of a well characterized sexual cycle in this organism. The lack of a well-behaved plasmid system and the fact that C.albicans is diploid have also been impediments to this type of approach. A critical feature of C.albicans—one that has attracted researchers for decades—is its ability to switch between different morphological forms. Candida albicans can grow as single-celled, budding yeast forms (blastospores) or as filamentous forms (including both pseudohyphae and true hyphae) in which cells remain joined end-to-end following cell division (Odds, 1988). The three primary morphological forms, blastospores, pseudohyphae and hyphae, are all found in infected tissues, and work to date indicates that the transition between these forms is critical for pathogenesis. The transition between the morphological forms can also be manipulated in the laboratory, by altering the growth medium. For example, on rich media [such as yeast extract with peptone (YEP) supplemented with 2% glucose (YEPD)] at 30°C, C.albicans grows primarily in the blastospore form; addition of fetal calf serum (FCS) rapidly induces filamentous (pseudohyphal and hyphal) growth of nearly every cell. Other environmental factors, including pH, temperature, oxygen availability, nitrogen availability and carbon source, also affect the distribution of cells among the three primary morphological forms. To date, molecular genetic studies of the C.albicans morphological transitions have largely relied on S.cerevisiae as an experimental and conceptual model. In response to limitation of specific nutrients, S.cerevisiae undergoes a transition from the single-celled budding form to a pseudohyphal form (Gimeno et al., 1992; for a recent review see Palecek et al., 2002). Based on this overall resemblance to the transition in C.albicans, S.cerevisiae has been used as a recipient to screen gene libraries from C.albicans for their ability to affect pseudohyphal growth (see for example Stoldt et al., 1997; Feng et al., 1999; Kadosh and Johnson, 2001). Alternatively, C.albicans genes with close similarity to S.cerevisiae genes involved in pseudohyphal growth have been identified and disrupted in Candida and the resultant phenotype studied (see for example Liu et al., 1994; Leberer et al., 1996). While these strategies have been successful for identifying certain aspects of filamentous growth regulation in C.albicans, there are important differences between S.cerevisiae and C.albicans with regard to filamentous growth. For example, S.cerevisiae does not exhibit true hyphal growth (in which cells are joined end-to-end with no visible constrictions at cell junctions), whereas this is the predominant morphological form of C.albicans under a number of growth conditions (Odds, 1988). Moreover, certain environmental signals, such as serum, are very strong inducers of filamentous growth in C.albicans but have little or no effect on S.cerevisiae. In addition, a comparison of their genome sequences has revealed that only about two-thirds of Candida genes appear to have clear orthologs in S.cerevisiae. All of these considerations raise the possibility that strategies that rely strictly on similarities between S.cerevisiae and C.albicans are likely to miss crucial features of Candida biology, especially those specific to its pathogenesis. Here, we describe a large-scale genetic screen in C.albicans designed to identify genes that affect the transition between blastospore and filamentous forms of the yeast. Of particular significance, this strategy makes no prior assumptions about the similarities or differences between C.albicans and S.cerevisiae. This effort led to the identification of 146 different genes that affect the blastospore–filament transition. Only six of these genes had been identified from previous work; the majority, including 39 genes that lack close relatives in S.cerevisiae, were not predicted from previous studies and provide new insights into the mechanism of the blastospore–filament transition. The results of this screen provide a framework for understanding the complex control of this morphological transition. Results Transposon mutagenesis has been used in many bacterial and yeast genetic screens (Berg and Howe, 1989; Cormack et al., 1999; Ross-Macdonald et al., 1999). To carry out a large-scale transposon-based screen in C.albicans, we constructed a library of 18 000 strains, each containing an independent Tn7-based transposon insertion. These insertion strains were constructed by first transposing Tn7 into Candida genomic DNA in vitro and then transforming a large population of C.albicans with this DNA (Figure 1). Individual transformants were picked and arranged in microtiter dishes; this library of 18 000 strains represents an insertion, on average, every 2.5 kb per haploid genome. Because Candida is diploid, we relied on haploinsufficiency combined with sensitive indicator plates (discussed below) to identify insertion mutants altered in the blastospore–filament transition. Since the transposon introduces unique DNA sequences into the C.albicans genome, the sites of insertion of identified mutants could readily be determined by direct DNA sequencing and comparison with the Stanford genome sequence (http://www-sequence.stanford.edu/group/candida). Details of the library construction and screening conditions are given in Materials and methods. Here, we discuss several general issues that pertain to the screen. Figure 1.Method for transposon mutagenesis of C.albicans. Linearized C.albicans genomic DNA fragments generated by restriction enzyme digestion were added to the donor plasmid containing a modified Tn7 transposon and Tn7 transposase. The modified transposon contains a promoterless Streptococcus thermophilus lacZ (Uhl and Johnson, 2001), C.albicans URA3 (Gillum et al., 1984) and the ampicillin resistance gene (bla) and origin of replication from pBluescriptKS+ (Stratagene). The sacB gene located on the donor plasmid external to the Tn7 repeats allows for selection against the donor plasmid (see Materials and methods). Following the transposition reaction (Biery et al., 2000), mutagenized genomic DNA was ligated and transformed into E.coli. The library was amplified, linearized by digestion with BsrGI and transformed in batch into C.albicans strain CAI4 (ura3/ura3). The transformed DNA was allowed to integrate into the C.albicans genome by homologous recombination, and successful integrants were selected as URA+ transformants. Download figure Download PowerPoint First, we consider the use of haploinsufficiency to screen directly for loss-of-function mutations in a diploid organism. Although C.albicans is indeed diploid, loss of one functional copy of a gene often produces a noticeable phenotype. For example, haploinsufficiency has been reported for a number of genes, including CPH1, INT1, TUP1, CLN1, STE7 and STE20, involved in the regulation of filamentous growth (Liu et al., 1994; Köhler and Fink, 1996; Leberer et al., 1996; Braun and Johnson, 1997; Gale et al., 1998; Loeb et al., 1999). In these studies, one allele of a gene of interest was specifically disrupted by transformation and homologous recombination, and the resultant phenotype was analyzed. In most cases, loss of one allele caused a less severe form of the phenotype produced by disrupting both alleles. To test the feasibility of utilizing such haploinsufficiency for a general genetic screen, we experimented with three C.albicans strains hemizygous for disruptions of genes known to be involved in filamentous growth: TUP1 (a negative regulator of filamentous growth; Braun and Johnson, 1997), CLN1 (a positive regulator of filamentous growth; Loeb et al., 1999) and EFG1 (a positive regulator of filamentous growth; Stoldt et al., 1997). Consistent with published reports, the TUP1/tup1, CLN1/cln1 and EFG1/efg1 strains each showed a clear difference in colony appearance when compared with the ‘wild-type’ parent strain, CAF2-1 (CAF2-1 is a clinical isolate of C.albicans with one copy of URA3 disrupted; Fonzi and Irwin, 1993). Moreover, microscopic examination of the cells in these colonies confirmed published reports that macroscopic differences in colony morphology could be used to monitor microscopic differences in filamentous growth (see Materials and methods). We used these three strains to choose two screening conditions, one based on serum induction of filamentous growth and one based on nutrient limitation as the inducer. Serum and starvation are both potent inducers of filamentous growth and appear to represent different but overlapping responses. For example, some gene knockouts affect induction by one environmental condition but not the other, whereas others affect the response to both (for reviews see Brown and Gow, 1999; Ernst, 2000; Whiteway, 2000). Although 10–15% FCS is most commonly utilized for induction of filamentous growth, exposure of the EFG1/efg1, CLN1/cln1 and TUP1/tup1 test strains to a range of serum concentrations indicated that 1% FCS was a more sensitive condition for detecting changes in filamentous growth caused by deletion of a single allele. For nutrient limitation, we used Spider medium, as it revealed clear differences among the three test strains and between each test strain and the parent strain (Liu et al., 1994). Based on these preliminary experiments, the mutant screen was performed twice, once on YEPD + 1% FCS and once on Spider medium. We next consider the overall number of mutants obtained in the screen. We replica-plated 18 000 independent insertion mutants on each type of medium and examined them over the course of 2 weeks. Colonies that displayed altered filamentous growth (either increased or decreased) were rescreened, and only those with reproducible phenotypes were considered further. A total of 340 (∼2%) of the insertion strains exhibited altered patterns of filamentous growth as deduced from changes in the pattern of wrinkling of the central portion of the patch or from differences in the appearance of peripheral hyphae (Figure 2; see also Figure 3). Some mutant strains displayed altered filamentous growth on both types of medium, but many showed differences from the parent strain on only one medium (Table I). Overall, roughly half the mutants showed enhanced filamentous growth on at least one type of medium, and half showed reduced filamentous growth. Figure 2.Examples of mutants identified in the screen. (A) A portion of a replica plate containing nine independent members of the insertion library grown on Spider medium for 4 days at 30°C. The center patch has a significantly less wrinkled appearance relative to its neighbors and was scored as a mutant reduced for filamentous growth. At this time point, the patches have not yet produced peripheral hyphae. (B) Examples of three other types of mutants obtained from the screen. From left to right, wild-type strain CAF2-1 (URA3/ura3:: imm434) (Fonzi and Irwin, 1993), a mutant reduced for filamentous growth, a mutant reduced for filamentous growth in the center of the patch but increased for production of peripheral hyphae, and a strongly hyperfilamentous mutant. Strains were grown for 4 days on Spider medium. Download figure Download PowerPoint Figure 3.Comparison of phenotypes of original transposon insertion mutants with those of reconstructed hemizygous deletion mutants. In each case, the deletion mutant was constructed in a fresh strain (RM1000) by deleting one copy of the indicated gene. These genes were identified by the site of transposon insertion in the original mutants. Colonies were grown on the indicated media for the indicated length of time and photographed at 2.5× magnification. Note the complex and individualistic colony morphologies, particularly those of the ZAP1/zap1 and FGR22/fgr22 mutants. Download figure Download PowerPoint Table 1. Categories of mutants obtained from the screen Category Description of mutant Total obtained I Less filamentous only on Spider 117 II Less filamentous only on serum 1 III Less filamentous on both media 12 IV More filamentous only on Spider 57 V More filamentous only on serum 44 VI More filamentous on both media 49 VII Hyperfilamentous with growth defect 45 Seven general categories are defined, based on the phenotypes on Spider medium (Liu et al., 1994) and YEPD (yeast extract with peptone and 2% glucose) with 1% FCS. After restreaking and verifying the original mutant phenotypes, the transposon insertion points were determined for a subset of the mutants; this analysis revealed 163 unambiguous insertion points (see Materials and methods). Eighty-eight insertion points were within structural genes, 47 were within the upstream regions of structural genes (within 500 nucleotide pairs of the presumptive translation startpoint) and 28 were downstream of structural genes (within 500 nucleotide pairs of the presumptive stop codon). Ten insertions were in unique sequence but were located >500 nucleotide pairs from the nearest structural gene. We have assigned the transposon insertion point to the nearest open reading frame (ORF), with the understanding that these assignments become less certain the further the insertion point is from the assigned ORF (Supplementary table 1 available at The EMBO Journal Online). When an identified gene had a close relative in S.cerevisiae, we used the same name for the C.albicans gene. The C.albicans genes identified in the screen that did not have close relatives in S.cerevisiae or other model organisms (see Materials and methods) were denoted FGR (filamentous growth regulator) and arbitrarily designated FGR1, FGR2, etc. A critical issue in the study of filamentous growth in C.albicans concerns position effects on URA3 expression. Diminished URA3 expression (caused by, for example, insertion of URA3 into a transcriptionally ‘cold’ region of the genome) can have a direct effect on filamentous growth, as recently described by Sundstrom et al. (2002) for a URA3 insertion in the HWP1 gene. In this case, the filamentous defect could be reversed by adding uridine to the growth medium. In our library of transposon mutants, the only source of URA3 is the transposon; therefore, it was important to demonstrate that the filamentous growth effects exhibited by the different mutants were not due simply to differences in URA3 expression that might arise from different genomic positions of the transposon. To this end, the entire collection of mutants was re-tested on plates containing uridine; all of the mutant phenotypes were reproduced on this medium, and it is therefore highly unlikely that differences in URA3 expression could account for the mutant phenotypes. In this regard, we note that the URA3 gene in our transposon is flanked by several kilobases of DNA, and this may serve to buffer URA3 from position effects. We next addressed whether the transposon insertions themselves (and not additional, unlinked genetic changes caused by the transformation or other manipulations) were the causes of the phenotypes. This question can be answered easily by simple backcrosses in organisms with well characterized sexual cycles, but is problematic in Candida. This issue is particularly worrisome since C.albicans is known to undergo spontaneous chromosomal rearrangements at relatively high frequencies. Although we cannot yet be certain for every one of the insertion mutants, we believe that the transposon insertion is nearly always the direct cause of the mutant phenotype. This belief is supported by several lines of evidence. First, six genes previously known to affect filamentous growth in C.albicans were identified in the screen (Table II), and the phenotypes of the newly isolated insertion mutations matched those described in the published studies. Secondly, genes closely related to those that affect filamentous growth in other organisms [for example, kin1 in Ustilago maydis (Lehmler et al., 1997) and PDE2 in S.cerevisiae (Lorenz and Heitman, 1997)] were identified in the screen. Thirdly, for 18 genes identified in the screen, we isolated at least two independent mutants; we know they are independent because the transposon insertion points within the ORF are different. In all 18 cases, the phenotypes produced by independent insertions in the same coding region were identical. Considering that 98% of the transposon insertion mutations did not show any filamentous phenotype, the isolation of independent insertion mutations in the same gene that cause the same phenotype argues very strongly that the screen is converging on a specific set of genes, and that the phenotypes are caused directly by the insertions. Table 2. Known regulators of filamentous growth identified by the screen Name Nearest homolog Protein function Organism Reference TUP1 TUP1 General repressor of transcription S.cerevisiae Braun and Johnson (1997) PDE2 PDE2 cAMP phosphodiesterase S.cerevisiae Lorenz and Heitman (1997) CBK1 cot-1 Ser/thr kinase N.crassa Yarden et al. (1992) NRG1 NRG1 DNA-binding protein S.cerevisiae Braun et al. (2001); Murad et al. (2001) RFG1 ROX1 DNA-binding protein S.cerevisiae Kadosh and Johnson (2001) CZF1 DNA-binding protein C.albicans Brown et al. (1999) Names of genes are indicated, along with their closest homolog as defined by BLAST searches (Altschul et al., 1997). Finally, we performed a direct test of the idea that the transposon insertions were the cause of the mutant phenotypes. For this analysis, we chose 18 genes representing a variety of different phenotypes observed among the original insertion mutants. Using a PCR-based knockout strategy (Wilson et al., 2000), we directly inactivated one copy of each of the 18 genes in a wild-type strain and compared them, on several types of media, with the original transposon insertion strains. Of the 18 genes tested, disruption of 16 exactly reproduced the phenotype of the original transposon insertion, despite the fact that the phenotypes were often complex and highly specific to each mutant (Figure 3; Table III). Although this correlation is very strong, it is instructive to consider the two exceptions. A knockout of one allele of FGR32 gave a similar but more severe phenotype (hyperfilamentous) than that displayed by the original transposon insertion. Because the original transposon insertion is located in the C-terminal third of the predicted ORF, the transposon insertion may have generated an allele with a partial function. In the case of ERG5, the recreated deletion strain had a wild-type appearance. The transposon insertion in this mutant was ∼400 bases downstream of the ORF, and may affect ERG5 expression or localization or even influence a neighboring gene. The overall analysis indicates there is a very high probability that the insertion is the cause of the phenotype, and suggests that the majority of the phenotypes observed in the original mutants are due to the loss of function of one allele caused by the transposon insertion. Table 3. Recreation of phenotypes Gene Function Tn location Transposon phenotype Recreated phenotype Phenotype match FGR3 ??? ORF LF Spider LF Spider Yes LIP2-3 Lipase Upstream LF Spider LF Spider Yes PDE2 cAMP response ORF HF Spider/serum HF Spider/serum Yes CBK1 Ser/thr kinase ORF LF Spider LF Spider Yes FGR13 ??? ORF Variable morphology Variable morphology Yes ERG5 Lipid synthesis Downstream HF Spider/serum Wild type No FGR5 ??? ORF LF Spider LF Spider Yes ZAP1 DNA binding ORF LF Spider LF Spider Yes FGR32 Transport ORF HF serum HF Spider/LF serum No ESC4 Silencing ORF LF Spider LF Spider Yes FGR22 ??? Downstream HF Spider/LF serum HF Spider/LF serum Yes FGR17 DNA binding ORF LF Spider LF Spider Yes SHE3 ORF HF Spider/serum HF Spider/serum Yes FGR27 DNA binding Upstream HF Spider HF Spider/HF serum Yes RDI1 Cell polarity ORF HF Spider/serum HF Spider/serum Yes TUP1 Repression ORF HF Spider/serum HF Spider/serum Yes NRG2 DNA binding ORF HF Spider/serum HF Spider Yes RFG1 DNA binding ORF HF Spider/serum HF Spider/serum Yes Phenotypes are shown for transposon insertions and the recreated strains containing a deletion of the respective ORF. The location of the transposon insertion (upstream of an ORF, in an ORF and downstream of an ORF) is shown, as well as the phenotypes on Spider medium and YEPD with 1% FCS (HF, hyperfilamentous; LF, less filamentous). Of the 18 genes tested, 16 of the deletion strains matched the original transposon mutant phenotype. Discussion We identified 146 genes that affect the blastospore–filament transition in C.albicans. As discussed in the Introduction, this transition is believed to be central to the virulence of this human fungal pathogen. General features of the blastospore–filament transition In this section, we discuss several broad generalizations based on the results of the screen. First, 40% of the genes identified do not have close relatives in S.cerevisiae and other well-studied organisms (Figure 4A). Because they lack close relatives in S.cerevisiae, it seems likely that these genes control aspects of filamentous growth that are specific to the ability of C.albicans to colonize and proliferate in warm-blooded animals, the only known hosts for this organism. Because these genes also lack close relatives in humans, they may identify useful targets for antifungal drugs. Some of these genes are members of much larger gene families in the Candida genome: for example, the FGR6 family has ∼10 members, and the FGR33/FGR38 family has >20 members. If the C.albicans genome as a whole is considered, approximately one-third of all C.albicans genes do not have close relatives in S.cerevisiae (Figure 4B). Thus the filamentous growth program in C.albicans is made up of both conserved and non-conserved genes in approximately the same proportion as those of the genome as a whole. Figure 4.Similarity of C.albicans genes that affect filamentous growth to genes in S.cerevisiae. (A) Roughly two-thirds of the C.albicans genes identified in the screen for filamentous growth mutants have closely related genes in S.cerevisiae. Nearly 30% lack significant similarity (as indicated by a BLAST score less than −15) to genes in any available databases. (B) The overall distribution of C.albicans genes relative to the S. cerevisiae genome. (C) Putative functions for the C.albicans genes that affect filamentous growth and have a close relative in S.cerevisiae. Each of these C.albicans genes was assigned a function based on the S.cerevisiae counterpart (see Supplementary table 1 for individual assignments). Categories are derived from the Saccharomyces Genome Database (http://genome-www.stanford.edu/Saccharomyces/). Download figure Download PowerPoint Approximately 60% of the 146 genes identified in the screen have close relatives in S.cerevisiae, and most of these can be assigned a general function (see Figure 4C; Tables II and IV) The largest class of these genes is involved with transcription and its regulation, indicating that the blastospore–filament transition is based on a large transcriptional program. In this class are 11 putative sequence-specific DNA-binding proteins (encoded by ADR1, CZF1, FCR1, FGR17, FGR27, GAT2, RFG1, NRG1, TEA1, YBR239c and ZAP1), three of which were known from previous work. It is known that filamentous growth can be induced by a variety of different environmental signals, including serum, starvation, contact, pH and temperature, and it seems likely that these gene-regulatory proteins are involved in coordinating these different responses. Consistent with this idea, mutations in five of the genes encoding DNA-binding proteins affect filamentous growth only on Spider (nutrient-poor) medium, one affects filamentous growth only on serum, and five affect filamentous growth on both types of media. Table 4. Categories of genes identified by screen Category Examples Transcription/chromatin structure RPO21, SIN3, SPT5, SPT6, SRB9, ESC4, CDC39, CDC73, POB3, RPC40, TFG1, YTA6 Cell wall biosynthesis, lipid metabolism ECM4, ECM17, CDS1, ECM29, ECI1, ERG5, CHO1, FAS1, CSI2, SUR1 Cell polarity/cytoskeletal structure SHE3, RSR1, RDI1, ARC40, CDC25, BNI4 Nutrient acquisition/metabolism CDC19, FGR2, FCY2, CHA1, CDC25, ZAP1, RNR1, TFI1, ADR1, QNS1 Filamentous growth TUP1, RFG1, PDE2, CZF1, NRG1, NOT1 Other signaling pathways FUS3, STE13, STE23, CBK1 Unknown function without related genes FGR1–FGR51 Categories are defined based upon examples from S.cerevisiae and the Saccharomyces Genome Database (http://genome-www.stanford.edu/Saccharomyces). Genes involved in more general aspects of transcriptional regulation were also identified in the screen. For example, we isolated mutations in TUP1 (which encodes a general transcriptional repressor) and mutations in components and associated proteins of the large CCR4/NOT1 complex (i
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