The metabolic network in the yeast Saccharomyces cerevisiae was reconstructed using currently available genomic, biochemical, and physiological information. The metabolic reactions were compartmentalized between the cytosol and the mitochondria, and transport steps between the compartments and the environment were included. A total of 708 structural open reading frames (ORFs) were accounted for in the reconstructed network, corresponding to 1035 metabolic reactions. Further, 140 reactions were included on the basis of biochemical evidence resulting in a genome-scale reconstructed metabolic network containing 1175 metabolic reactions and 584 metabolites. The number of gene functions included in the reconstructed network corresponds to approximately 16% of all characterized ORFs in S. cerevisiae. Using the reconstructed network, the metabolic capabilities of S. cerevisiae were calculated and compared with Escherichia coli. The reconstructed metabolic network is the first comprehensive network for a eukaryotic organism, and it may be used as the basis for in silico analysis of phenotypic functions.

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Genome-Scale Reconstruction of the Saccharomyces cerevisiae Metabolic Network

Expression cloning of TMEM16A as a calcium-activated chloride channel subunit.

Heterologous markers are important tools required for the molecular dissection of gene function in many organisms, including Saccharomyces cerevisiae. Moreover, the presence of gene families and isoenzymes often makes it necessary to delete more than one gene. We recently introduced a new and efficient gene disruption cassette for repeated use in budding yeast, which combines the heterologous dominant kanr resistance marker with a Cre/loxP-mediated marker removal procedure. Here we describe an additional set of four completely heterologous loxP-flanked marker cassettes carrying the genes URA3 and LEU2 from Kluyveromyces lactis, his5+ from Schizosaccharomyces pombe and the dominant resistance marker bler from the bacterial transposon Tn5, which confers resistance to the antibiotic phleomycin. All five loxP–marker gene–loxP gene disruption cassettes can be generated using the same pair of oligonucleotides and all can be used for gene disruption with high efficiency. For marker rescue we have created three additional Cre expression vectors carrying HIS3, TRP1 or bler as the yeast selection marker. The set of disruption cassettes and Cre expression plasmids described here represents a significant further development of the marker rescue system, which is ideally suited to functional analysis of the yeast genome.

A second set of loxP marker cassettes for Cre-mediated multiple gene knockouts in budding yeast.

Eukaryotic DNA mismatch repair.

■ Abstract Mismatch repair (MMR) systems play a central role in promoting genetic stability by repairing DNA replication errors, inhibiting recombination between non-identical DNA sequences and participating in responses to DNA damage. The discovery of a link between human cancer and MMR defects has led to an explosion of research on eukaryotic MMR. The key proteins in MMR are highly conserved from bacteria to mammals, and this conservation has been critical for defining the components of eukaryotic MMR systems. In eukaryotes, there are multiple homologs of the key bacterial MutS and MutL MMR proteins, and these homologs form heterodimers that have discrete roles in MMR-related processes. This review describes the genetic and biochemical approaches used to study MMR, and summarizes the diverse roles that MMR proteins play in maintaining genetic stability.

DNA Mismatch Repair and Genetic Instability

In a systematic approach to the study of Saccharomyces cerevisiae genes of unknown function, 150 deletion mutants were constructed (1 double, 149 single mutants) and phenotypically analysed. Twenty percent of all genes examined were essential. The viable deletion mutants were subjected to 20 different test systems, ranging from high throughput to highly specific test systems. Phenotypes were obtained for two-thirds of the mutants tested. During the course of this investigation, mutants for 26 of the genes were described by others. For 18 of these the reported data were in accordance with our results. Surprisingly, for seven genes, additional, unexpected phenotypes were found in our tests. This suggests that the type of analysis presented here provides a more complete description of gene function.

Functional analysis of 150 deletion mutants in Saccharomyces cerevisiae by a systematic approach

We have analysed the correction of defined mismatches in wild-type and msh2, msh3, msh6 and msh3 msh6 mutants of Saccharomyces cerevisiae in two different yeast strain backgrounds by transformation with plasmid heteroduplex DNA constructs. Ten different base/base mismatches, two single-nucleotide loops and a 38-nucleotide loop were tested. Repair of all types of mismatches was severely impaired in msh2 and msh3 msh6 mutants. In msh6 mutants, repair efficiency of most base/base mismatches was reduced to a similar extent as in msh3 msh6 double mutants. G/T and A/C mismatches, however, displayed residual repair in msh6 mutants in one strain background, implying a role for Msh3p in recognition of base/base mismatches. Furthermore, the efficiency of repair of base/base mismatches was considerably reduced in msh3 mutants in one strain background, indicating a requirement for MSH3 for fully efficient mismatch correction. Also the efficiency of repair of the 38-nucleotide loop was reduced in msh3 mutants, and to a lesser extent in msh6 mutants. The single-nucleotide loop with an unpaired A was less efficiently repaired in msh3 mutants and that with an unpaired T was less efficiently corrected in msh6 mutants, indicating non-redundant functions for the two proteins in the recognition of single-nucleotide loops.

Analysis of in vivo correction of defined mismatches in the DNA mismatch repair mutants msh2, msh3 and msh6 of Saccharomyces cerevisiae.

Transcription of theSaccharomyces cerevisiae DNA mismatch repair genesPMS1, MSH2, andMSH6, a recently discovered homolog of theEscherichia coli mutS gene, was shown to be cell cycle regulated. In contrast, transcription of theMSH1, MSH3 andMLH1 genes was not regulated during the cell cycle. TheMSH1 gene, which is thought to be involved in DNA mismatch repair in mitochondria, was also not induced under aerobic growth conditions. Regulation of thePMS1 gene was dependent on intactMluI cell cycle boxes, as demonstrated by analysis of a promoter mutant. Both reduced and increased expression ofPMS1 resulted in a mitotic mutator phenotype. Analysis of mRNA levels was performed with a newly developed reverse transcription-PCR (polymerase chain reaction) approach using fluorescently labeled primers and an automated DNA sequencer for detection of PCR products.

Transcription of mutS and mutL-homologous genes in Saccharomyces cerevisiae during the cell cycle.

We have analysed the levels of mRNA transcripts of the mutS- and mutL-homologous genes of the yeast Saccharomyces cerevisiae during the course of meiosis, by quantitative RT-PCR. We found that all mutS homologues (MSH1–6) were induced during meiosis, whereas no evidence for regulation of the mutL homologues (PMS1, MLH1–3) was obtained. Temporal expression patterns indicative of co-regulation were observed for the gene pairs MSH4/MSH5 and MSH2/SPO11. Sequence comparisons of the 5′ flanking regions revealed similar sequence stretches in the respective gene pairs, which may constitute regulatory elements. Similar sequences were also found in the 5′ flanking regions of the pairs MSH1/MSH3 and MSH1/MSH6. Upstream of MSH2 three closely spaced sequences similar to UASH elements were found, which – surprisingly – are located within the coding region of SPO21. Deletion of these elements resulted in loss of meiotic induction of MSH2. Genetic analysis of homozygous deletion mutants did not reveal any differences from wild type with respect to genetic distance estimates, aberrant segregation, or suppression of homoeologus recombination in an interspecies cross with Saccharomyces paradoxus.

Transcription of mutS- and mutL-homologous genes during meiosis in Saccharomyces cerevisiae and identification of a regulatory cis-element for meiotic induction of MSH2

W. Kramer

Papers

Functional analysis of 150 deletion mutants in Saccharomyces cerevisiae by a systematic approach

Analysis of in vivo correction of defined mismatches in the DNA mismatch repair mutants msh2, msh3 and msh6 of Saccharomyces cerevisiae.

Transcription of mutS and mutL-homologous genes in Saccharomyces cerevisiae during the cell cycle.

Transcription of mutS- and mutL-homologous genes during meiosis in Saccharomyces cerevisiae and identification of a regulatory cis-element for meiotic induction of MSH2