Functional characterization of the S. cerevisiae genome by gene deletion and parallel analysis.

The functions of many open reading frames (ORFs) identified in genome-sequencing projects are unknown. New, whole-genome approaches are required to systematically determine their function. A total of 6925 Saccharomyces cerevisiae strains were constructed, by a high-throughput strategy, each with a precise deletion of one of 2026 ORFs (more than one-third of the ORFs in the genome). Of the deleted ORFs, 17 percent were essential for viability in rich medium. The phenotypes of more than 500 deletion strains were assayed in parallel. Of the deletion strains, 40 percent showed quantitative growth defects in either rich or minimal medium.

Functional assessment of surface loops: deletion of eukaryote-specific peptide inserts in thymidylate synthase of Saccharomyces cerevisiae.

The primary structure is known for at least 29 thymidylate synthases and the crystal structure is known for several from both prokaryotes and eukaryotes. All these are markedly similar making thymidylate synthase one of the most highly conserved enzymes known. There are, however, two surface loops, one near the active site and the other near the dimer interface, which exist in distinctly prokaryotic and eukaryotic versions. Specifically, in eukaryotes these two surface loops have small peptide inserts conserved in size and partly conserved in sequence, that are not present in the prokaryotic thymidylate synthases. To address the possibility that these inserts provide eukaryote-specific functions the Saccharomyces cerevisiae loops were individually modified to mimic their prokaryotic counterparts. Altering the surface loop near the active site increased Km for the nucleotide substrate and decreased apparent Vmax. Mutant variants with alterations in the other surface loop were unable to dimerize. Therefore these surface loops have acquired, perhaps by way of the eukaryotic inserts, characteristics that are important for catalytic activity and quaternary structure respectively.

Molecular cloning of chromosome I DNA from Saccharomyces cerevisiae: characterization of the 54 kb right terminal CDC15-FLO1-PHO11 region.

Gene density near the ends of Saccharomyces cerevisiae chromosomes is much lower than on the rest of the chromosome. Non-functional gene-fragments are common and a high proportion of the sequences are repeated elsewhere in the genome. This sequence arrangement suggests that the ends of chromosomes play a structural rather than a coding role and may be analogous to the highly repeated heterochromatic DNA of higher organisms. In order to evaluate the function of the ends of S. cerevisiae chromosomes, the rightmost 54-kb of DNA from chromosome I was investigated. The region contains 16 open reading frames (ORFs) and two tRNA genes. Gene-disruption studies indicated that none of these genes are essential for growth on rich or minimal medium, mating or sporulation. In contrast to the central region where 80% of the genes are transcribed when cells are grown on rich medium, only seven ORFs and the two tRNA genes appeared to produce transcripts. Six of the transcribed ORFs were from the centromere-proximal part of the region, leaving the rightmost 35-kb with only a single sequence that is transcribed during vegetative growth. Two genes located 3 and 10-kb from the chromosome I telomere are almost identical to two genes located somewhat further from the chromosome VIII telomere. Surprisingly, the chromosome VIII copies were transcribed while the chromosome I genes were not. These results suggest that the chromosome I genes may be repressed by a natural telomere position effect. The low level of transcription, absence of essential genes as well as the repetitive nature of these sequences are consistent with their having a structural role in chromosome function.

The nucleotide sequence of Saccharomyces cerevisiae chromosome XVI.

The nucleotide sequence of the 948,061 base pairs of chromosome XVI has been determined, completing the sequence of the yeast genome. Chromosome XVI was the last yeast chromosome identified, and some of the genes mapped early to it, such as GAL4, PEP4 and RAD1 (ref. 2) have played important roles in the development of yeast biology. The architecture of this final chromosome seems to be typical of the large yeast chromosomes, and shows large duplications with other yeast chromosomes. Chromosome XVI contains 487 potential protein-encoding genes, 17 tRNA genes and two small nuclear RNA genes; 27% of the genes have significant similarities to human gene products, and 48% are new and of unknown biological function. Systematic efforts to explore gene function have begun.

Completion of the Saccharomyces cerevisiae genome sequence allows identification of KTR5, KTR6 and KTR7 and definition of the nine-membered KRE2/MNT1 mannosyltransferase gene family in this organism.

The KRE2/MNT1 mannosyltransferase gene family of Saccharomyces cerevisiae currently consists of the KRE2, YUR1, KTR1, KTR2, KTR3 and KTR4 genes. All six encode putative type II membrane proteins with a short cytoplasmic N-terminus, a membrane-spanning region and a highly conserved catalytic lumenal domain. Here we report the identification of the three remaining members of this family in the yeast genome. KTR5 corresponds to an open reading frame (ORF) of the left arm of chromosome XIV, and KTR6 and KTR7 to ORFs on the left arms of chromosomes XVI and IX respectively. The KTR5, KTR6 and KTR7 gene products are highly similar to the Kre2p/Mnt1p family members. Initial functional characterization revealed that some mutant yeast strains containing null copies of these genes displayed cell wall phenotypes. None was K1 killer toxin resistant but ktr6 and ktr7 null mutants were found to be hypersensitive and resistant, respectively, to the drug Calcofluor White.

Histone H1 in Saccharomyces cerevisiae.

The existence of histone H1 in the yeast, Saccharomyces cerevisiae, has long been debated. In this report we describe the presence of histone H1 in yeast. YPL127c, a gene encoding a protein with a high degree of similarity to histone H1 from other species was sequenced as part of the contribution of the Montreal Yeast Genome Sequencing Group to chromosome XVI. To reflect this similarity, the gene designation has been changed HHO1 (Histone H One). The HHO1 gene is highly expressed as poly A+ RNA in yeast. Although deletion of this gene had no detectable effect on cell growth, viability or mating, it significantly altered the expression of beta-galactosidase from a CYC1-lacZ reporter. Fluorescence observed in cells expressing a histone H1-GFP protein fusion indicated that histone H1 is localized to the nucleus.

Molecular characterization of GCV3, the Saccharomyces cerevisiae gene coding for the glycine cleavage system hydrogen carrier protein.

YAL044, a gene on the left arm of Saccharomyces cerevisiae chromosome one, is shown to code for the H-protein subunit of the multienzyme glycine cleavage system. The gene designation has therefore been changed to GCV3, reflecting its role in the glycine cleavage system. GCV3 encodes a 177-residue protein with a putative mitochondrial targeting signal at its amino terminus. Targeted gene replacement shows that GCV3 is not required for growth on minimal medium; however, it is essential when glycine serves as the sole nitrogen source. Studies of GCV3 expression revealed that it is highly regulated. Supplementation of minimal medium with glycine, the glycine cleavage system's substrate, induced expression at least 30-fold. In contrast, and consistent with the cleavage of glycine providing activated single-carbon units, the addition of the metabolic end products that require activated single-carbon units repressed expression about 10-fold. Finally, like many amino acid biosynthetic genes, GCV3 is subject to regulation by the general amino acid control system.

Analysis of a 103 kbp cluster homology region from the left end of Saccharomyces cerevisiae chromosome I.

The DNA sequence and preliminary functional analysis of a 103-kbp section of the left arm of yeast chromosome I is presented. This region, from the left telomere to the LTE1 gene, can be divided into two distinct portions. One portion, the telomeric 29 kbp, has a very low gene density (only five potential genes and 21 kbp of noncoding sequence), does not encode any "functionally important" genes, and is rich in sequences repeated several times within the yeast genome. The other portion, with 37 genes and only 14.5 kbp of noncoding sequence, is gene rich and codes for at least 16 "functionally important" genes. The entire gene-rich portion is apparently duplicated on chromosome XV as an extensive region of partial gene synteney called a cluster homology region. A function can be assigned with varying degrees of precision to 23 of the 42 potential genes in this region; however, the precise function is know for only eight genes. Nineteen genes encode products presently novel to yeast, although five of these have homologs elsewhere in the yeast genome.

Functional analysis of a 38 kilobase region on chromosome XVI in Saccharomyces cerevisiae.

In this report we assess the functional importance of 16 open reading frames (ORFs) contained within a 38 780 base-pair region immediately adjacent to the centromere on the right arm of chromosome XVI in Saccharomyces cerevisiae. This analysis involved replacing one copy of each ORF in a diploid strain with a cassette encoding the green fluorescent protein from the jellyfish Aequorea victoria and HIS3. Each replacement cassette was generated by PCR using oligonucleotide pairs with 45-base extensions complementary to sequences immediately upstream and downstream of the target gene's coding region. After replacement of the targeted genes, each gene-replacement strain was subjected to a series of genetic and phenotypic tests to assess the functional importance of the deleted gene. This analysis showed that two ORFs were essential, one for spores to germinate and another for vegetative growth. A third gene encoded a copper-fist-like transcription factor that was required for proper bud-site selection. One of the 16 ORFs was duplicated, a situation not observed in the strain used to sequence the yeast genome (S288C). RNA analysis showed 11 of the 16 ORFs in this region expressed steady-state poly(A+) RNA levels that were greater than or equal to 2% of the level expressed from the yeast actin gene, ACT1.

Molecular analysis of UFE1, a Saccharomyces cerevisiae gene essential for spore formation and vegetative growth.

The UFE1 gene of Saccharomyces cerevisiae was cloned, sequenced and characterized. The coding region of UFE1 is separated from the TMP1 gene on chromosome XV by 624 bp. Gene-disruption experiments demonstrated that UFE1 is essential for both the germination of ascospores and for vegetative growth. Translation of the UFE1 coding region generates a protein with significant similarity to cytokeratin and to the coiled-coil region of SED5, USO1 and restin, suggesting that it is involved in the secretory pathway and may also be related to intermediate filament-associated proteins.

Mutations of iso-1-cytochrome c at positions 13 and 90. Separate effects on physical and functional properties.

Residues at positions 13 (lysine or arginine) and 90 (glutamate or aspartate) of eukaryotic cytochromes c have been conserved during evolution; Cys102, however, is found only in yeast cytochrome c. The positively charged residue at position 13 and the negatively charged residue at position 90 are close together in those cytochromes c for which three-dimensional structures are available. We have replaced the amino acids at these two positions by cysteine in Saccharomyces cerevisiae iso-1-cytochrome c; in an earlier study, Cys102 was replaced by threonine without negatively influencing the physical or enzymic properties of the protein. The mutated proteins [R13C, C102T]cytochrome c (iso-1-cytochrome c containing Arg13-->Cys and Cys102-->Thr mutations), [D90C, C102T]cytochrome c (iso-1-cytochrome c containing Asp90-->Cys and Cys102-->Thr mutations) and [R13C, D90C, C102T]cytochrome c (iso-1-cytochrome c containing Arg13-->Cys, Asp90-->Cys, and Cys102-->Thr mutations) are functional in vivo. Free sulfhydryl titration shows that the doubly mutated forms each contain one sulfhydryl group while the triple mutant contains two sulfhydryl groups. The stability of mutant [R13C, C102T]cytochrome c resembles that of [C102T] cytochrome c, whereas the stability of [D90C, C102T]cytochrome c resembles the stability of [R13C, D90C, C102T]cytochrome c. The activity of cytochrome-c oxidase using cytochrome c was monitored polarographically. Compared to the wild-type or [C102T]cytochrome c, which shows two kinetic phases with cytochrome-c oxidase, [D90C, C102T]cytochrome c has much the same profile; [R13C, C102T]cytochrome c and [R13C, D90C, C102T]cytochrome c exhibit one kinetic phase with decreased activity. Electron-transfer activity of the mutant cytochromes c is inhibited by Hg2+. The inhibition is highest for the triple mutant, less for [R13C, C102T]cytochrome c, even less for [D90C, C102T]cytochrome c and insignificant for the wild type. It would appear as though the stability of the triple mutant follows the changes that result from the Asp90-->Cys mutation while the activity changes follow those of the Arg13-->Cys mutation.

LTE1 of Saccharomyces cerevisiae is a 1435 codon open reading frame that has sequence similarities to guanine nucleotide releasing factors.

The DNA sequence of the LTE1 gene on the left arm of chromosome I of Saccharomyces cerevisiae has been determined. The LTE1 open reading frame comprises 4305 bp that can be translated into 1435 amino acid residues. The position of this open reading frame corresponds well to that of a 4.7 kb transcript that has been mapped to this position. The derived amino acid sequence has significant similarities to the amino acid sequence of the guanine nucleotide releasing factor isolated from a rat brain library. The carboxy-terminus of the LTE1 protein also shows similarities to other guanine nucleotide exchange factors of the S. cerevisiae CDC25 family.

Sequencing of chromosome I of Saccharomyces cerevisiae: analysis of the 42 kbp SPO7-CENI-CDC15 region.

Determination of the DNA sequence and preliminary functional analysis of a 42 kbp centromeric section of chromosome I have been completed. The section spans the SPO7-CEN1-CDC15 loci and contains 19 open reading frames (ORFs). They include an apparently inactive Ty1 retrotransposon and eight new ORFs with no known homologs or function. The remaining ten genes have been previously characterized since this part of the yeast genome has been studied in an unusually intensive manner. Our directed sequencing allows a complete ordering of the region.

Thymidylate synthase is localized to the nuclear periphery in the yeast Saccharomyces cerevisiae.

To date, the organization of DNA precursor synthesis within eukaryotic cells remains unresolved. Previous studies have suggested the existence of a multienzyme complex that is responsible for DNA precursor synthesis and is associated with sites of replication within the nucleus. Contrasting this, other studies have proposed that DNA precursor synthesis occurs outside the nucleus. To further these studies, we have addressed the location where thymidylate synthase resides in yeast. Subcellular fractionation experiments indicate thymidylate synthase is associated with purified nuclei. Consistent with this, immunofluorescence analysis suggests that thymidylate synthase is situated at the nuclear periphery.

The YAL017 gene on the left arm of chromosome I of Saccharomyces cerevisiae encodes a putative serine/threonine protein kinase.

The DNA sequence of a region between the LTE1 and CYS3 genes on the left arm of chromosome I from Saccharomyces cerevisiae contains an open reading frame (ORF), YAL017, corresponding to the 5.0 kb FUN31 (Function Unknown Now) transcribed region. The predicted protein from this ORF contains 1358 amino acid residues with a molecular weight of 152,531, and an identifiable serine/threonine protein kinase catalytic domain. When compared with other yeast protein kinases, the Yal017p kinase most resembles the SNF1 serine/threonine protein kinase which is involved in regulating sucrose fermentation genes. The Yal017p kinase shows highest amino acid identities with two mammalian carcinoma-related serine/threonine protein kinases; PIM-1, which shows induced expression in T-cell lymphomas; and p78A1, whose expression is lost in human pancreatic carcinomas. Gene disruption of YAL017 indicates that it is non-essential for growth on glucose.

Physical localization of yeast CYS3, a gene whose product resembles the rat gamma-cystathionase and Escherichia coli cystathionine gamma-synthase enzymes.

We have cloned, sequenced and physically mapped the CYS3 gene of Saccharomyces cerevisiae. This gene can complement the cys3-1 allele, and disruptions at this locus lead to cysteine auxotrophy. The predicted CYS3 product is closely related (46% identical) to the rat cystathionine gamma-lyase (Erickson et al., 1990), but differs in lacking cysteine residues. These results provide further evidence that the S288C strain of yeast resembles mammals in synthesizing cysteine solely via a trans-sulfuration pathway. The CYS3 product was found to have strong homology to three other enzymes involved in cysteine metabolism: the Escherichia coli metB and metC products and the S. cerevisiae MET25 gene product. The trans-sulfuration enzymes appear to form a diverged family and carry out related functions from bacteria to mammals.

Sequencing of chromosome I from Saccharomyces cerevisiae: analysis of a 32 kb region between the LTE1 and SPO7 genes.

The DNA sequencing and preliminary functional analysis of a 32 kb section of yeast chromosome I has been completed. This region lies on the left arm of the chromosome between the LTE1 and SPO7 genes and contains 14 open reading frames (ORFs) positioned closely together, with an average spacing of approximately 350 nucleotides between coding regions. Three of these ORFs correspond to previously identified genes, a further three show significant homology with other proteins, while the remaining eight ORFs share no significant homology to genes in the databases.

Identification of a Saccharomyces cerevisiae homolog of the SNF2 transcriptional regulator in the DNA sequence of an 8.6 kb region in the LTE1-CYS1 interval on the left arm of chromosome I.

The DNA sequence of an 8.6 kb region of the left arm of chromosome I has been determined. This region, between the LTE1 and CYS1 loci, is approximately 40 kb from the centromere. There are six potential open-reading frames (ORFs), provisionally named YAL001-006 within this fragment of chromosome I. Four of these ORFs can be aligned with previously identified FUN transcripts: FUN28 with YAL006, FUN29 with YAL004, FUN30 with YAL001 and FUN31 with YAL002. The YAL001 ORF shows significant homology to the SNF2 transcriptional regulator. A region of the DNA contains an extensive repeat of the bases C-A-T positioned in the 5' terminus of the YAL004 promoter region.

The periodically expressed TMP1 gene of Saccharomyces cerevisiae is subject to START-dependent and START-independent regulation.

The rate of transcription of the Saccharomyces cerevisiae gene encoding thymidylate synthase (TMP1) fluctuates periodically during the cell cycle. The simplest explanation for this pattern of expression is that transcription occurs during the late G1 and early S phases and does not occur during other stages of the cell cycle. In this report, however, we show that TMP1 is subject to regulation that results in at least three different levels of expression: essentially nondetectable expression during stationary phase (G0), moderate level expression in START-arrested growing cells (START-independent), and a high level of expression in proliferating cells (START-dependent). Our analysis also shows that upstream elements important for START-independent expression and required for START-dependent expression are located within a 37-base region.

Ubiquitin gene expression: response to environmental changes.

It has previously been shown that the yeast ubiquitin genes UBI1, 2 and 3 are strongly expressed during the log-phase of batch culture growth, whereas the UBI4 gene is weakly expressed. We found that heat shock, treatment with DNA-damaging agents, starvation, and the feeding of starved cells all transiently induced UBI4. These results suggest that UBI4 is induced whenever a change in culture conditions dictates a dramatic shift in cellular metabolism, and that UBI4 expression returns to lower levels once cellular metabolism has adapted to the new conditions. In contrast, all of the treatments tested, except starvation, transiently repressed the UBI1, 2 and 3 genes. Although starvation also repressed UBI1, 2 and 3 its effect was not transient, and expression only recovered upon the addition of fresh media. These results, together with others presented here, suggest that high levels of UBI1, 2 and 3 expression are dependant upon ongoing cell growth, and that treatments which slow or stop growth repress their expression.