Achim Wach

Bio: Achim Wach is an academic researcher from University of Basel. The author has contributed to research in topics: Gene & Saccharomyces cerevisiae. The author has an hindex of 12, co-authored 13 publications receiving 11422 citations. Previous affiliations of Achim Wach include University of North Carolina at Chapel Hill.

Additional modules for versatile and economical PCR-based gene deletion and modification in Saccharomyces cerevisiae

Abstract: An important recent advance in the functional analysis of Saccharomyces cerevisiae genes is the development of the one-step PCR-mediated technique for deletion and modification of chromosomal genes This method allows very rapid gene manipulations without requiring plasmid clones of the gene of interest We describe here a new set of plasmids that serve as templates for the PCR synthesis of fragments that allow a variety of gene modifications Using as selectable marker the S cerevisiae TRP1 gene or modules containing the heterologous Schizosaccharomyces pombe his5 + or Escherichia coli kan r gene, these plasmids allow gene deletion, gene overexpression (using the regulatable GAL1 promoter), C- or N-terminal protein tagging [with GFP(S65T), GST, or the 3HA or 13Myc epitope], and partial N- or C-terminal deletions (with or without concomitant protein tagging) Because of the modular nature of the plasmids, they allow eYcient and economical use of a small number of PCR primers for a wide variety of gene manipulations Thus, these plasmids should further facilitate the rapid analysis of gene function in S cerevisiae ? 1998 John Wiley & Sons, Ltd

New heterologous modules for classical or PCR‐based gene disruptions in Saccharomyces cerevisiae

Abstract: We have constructed and tested a dominant resistance module, for selection of S. cerevisiae transformants, which entirely consists of heterologous DNA. This kanMX module contains the known kanr open reading-frame of the E. coli transposon Tn903 fused to transcriptional and translational control sequences of the TEF gene of the filamentous fungus Ashbya gossypii. This hybrid module permits efficient selection of transformants resistant against geneticin (G418). We also constructed a lacZMT reporter module in which the open reading-frame of the E. coli lacZ gene (lacking the first 9 codons) is fused at its 3' end to the S. cerevisiae ADH1 terminator. KanMX and the lacZMT module, or both modules together, were cloned in the center of a new multiple cloning sequence comprising 18 unique restriction sites flanked by Not I sites. Using the double module for constructions of in-frame substitutions of genes, only one transformation experiment is necessary to test the activity of the promotor and to search for phenotypes due to inactivation of this gene. To allow for repeated use of the G418 selection some kanMX modules are flanked by 470 bp direct repeats, promoting in vivo excision with frequencies of 10(-3)-10(-4). The 1.4 kb kanMX module was also shown to be very useful for PCR based gene disruptions. In an experiment in which a gene disruption was done with DNA molecules carrying PCR-added terminal sequences of only 35 bases homology to each target site, all twelve tested geneticin-resistant colonies carried the correctly integrated kanMX module.

Heterologous modules for efficient and versatile PCR-based gene targeting in Schizosaccharomyces pombe

Abstract: We describe a straightforward PCR-based approach to the deletion, tagging, and overexpression of genes in their normal chromosomal locations in the fission yeast Schizosaccharomyces pombe. Using this approach and the S. pombe ura4+ gene as a marker, nine genes were deleted with efficiencies of homologous integration ranging from 6 to 63%. We also constructed a series of plasmids containing the kanMX6 module, which allows selection of G418-resistant cells and thus provides a new heterologous marker for use in S. pombe. The modular nature of these constructs allows a small number of PCR primers to be used for a wide variety of gene manipulations, including deletion, overexpression (using the regulatable nmt1 promoter), C- or N-terminal protein tagging (with HA, Myc, GST, or GFP), and partial C- or N-terminal deletions with or without tagging. Nine genes were manipulated using these kanMX6 constructs as templates for PCR. The PCR primers included 60 to 80 bp of flanking sequences homologous to target sequences in the genome. Transformants were screened for homologous integration by PCR. In most cases, the efficiency of homologous integration was > or = 50%, and the lowest efficiency encountered was 17%. The methodology and constructs described here should greatly facilitate analysis of gene function in S. pombe.

PCR‐synthesis of marker cassettes with long flanking homology regions for gene disruptions in S. cerevisiae

Abstract: A PCR-method for fast production of disruption cassettes is introduced, that allows the addition of long flanking homology regions of several hundred base pairs (LFH-PCR) to a marker module. Such a disruption cassette was made by linking two PCR fragments produced from genomic DNA to kanMX6, a modification of dominant resistance marker making S. cerevisiae resistant to geneticin (G418). In a first step, two several hundred base pairs long DNA fragments from the 5'- and 3'- region of a S. cerevisiae gene were amplified in such a way that 26 base pairs extensions homologous to the kanMX6 marker were added to one of their end. In a second step, one strand of each of these molecules then served as a long primer in a PCR using kanMX6 as template. When such a LFH-PCR-generated disruption cassette was used instead of a PCR-made disruption cassette flanked by short homology regions, transformation efficiencies were increased by at least a factor of thirty. This modification will therefore also help to apply PCR-mediated gene manipulations to strains with decreased transformability and/or unpredictable sequence deviations.

Heterologous HIS3 marker and GFP reporter modules for PCR-targeting in Saccharomyces cerevisiae.

Abstract: We have fused the open reading frames of his3-complementing genes from Saccharomyces kluyveri and Schizosac-charomyces pombe to the strong TEF gene promotor of the filamentous fungus Ashbya gossypii. Both chimeric modules and the cognate S. kluyveri HIS3 gene were tested in transformations of his3 S. cerevisiae strains using PCR fragments flanked by 40 bp target guide sequences. The 1.4 kb chimeric Sz. pombe module (HIS3MX6) performed best. With less than 5% incorrectly targeted transformants, it functions as reliably as the widely used geniticin resistance marker kanMX. The rare false-positive His+ transformants seem to be due to non-homologous recombination rather than to gene conversion of the mutated endogenous his3 allele. We also cloned the green fluorescent protein gene from Aequorea victoria into our pFA-plasmids with HIS3MX6 and kanMX markers. The 0.9 kb GFP reporters consist of wild-type GFP or GFP-S65T coding sequences, lacking the ATG, fused to the S. cerevisiae ADH1 terminator. PCR-synthesized 2.4 kb-long double modules flanked by 40-45 bp-long guide sequences were successfully targeted to the carboxy-terminus of a number of S. cerevisiae genes. We could estimate that only about 10% of the transformants carried inactivating mutations in the GFP reporter.

Additional modules for versatile and economical PCR-based gene deletion and modification in Saccharomyces cerevisiae

Abstract: An important recent advance in the functional analysis of Saccharomyces cerevisiae genes is the development of the one-step PCR-mediated technique for deletion and modification of chromosomal genes This method allows very rapid gene manipulations without requiring plasmid clones of the gene of interest We describe here a new set of plasmids that serve as templates for the PCR synthesis of fragments that allow a variety of gene modifications Using as selectable marker the S cerevisiae TRP1 gene or modules containing the heterologous Schizosaccharomyces pombe his5 + or Escherichia coli kan r gene, these plasmids allow gene deletion, gene overexpression (using the regulatable GAL1 promoter), C- or N-terminal protein tagging [with GFP(S65T), GST, or the 3HA or 13Myc epitope], and partial N- or C-terminal deletions (with or without concomitant protein tagging) Because of the modular nature of the plasmids, they allow eYcient and economical use of a small number of PCR primers for a wide variety of gene manipulations Thus, these plasmids should further facilitate the rapid analysis of gene function in S cerevisiae ? 1998 John Wiley & Sons, Ltd

Functional profiling of the Saccharomyces cerevisiae genome.

Abstract: Determining the effect of gene deletion is a fundamental approach to understanding gene function. Conventional genetic screens exhibit biases, and genes contributing to a phenotype are often missed. We systematically constructed a nearly complete collection of gene-deletion mutants (96% of annotated open reading frames, or ORFs) of the yeast Saccharomyces cerevisiae. DNA sequences dubbed 'molecular bar codes' uniquely identify each strain, enabling their growth to be analysed in parallel and the fitness contribution of each gene to be quantitatively assessed by hybridization to high-density oligonucleotide arrays. We show that previously known and new genes are necessary for optimal growth under six well-studied conditions: high salt, sorbitol, galactose, pH 8, minimal medium and nystatin treatment. Less than 7% of genes that exhibit a significant increase in messenger RNA expression are also required for optimal growth in four of the tested conditions. Our results validate the yeast gene-deletion collection as a valuable resource for functional genomics.

Global analysis of protein localization in budding yeast

Abstract: A fundamental goal of cell biology is to define the functions of proteins in the context of compartments that organize them in the cellular environment. Here we describe the construction and analysis of a collection of yeast strains expressing full-length, chromosomally tagged green fluorescent protein fusion proteins. We classify these proteins, representing 75% of the yeast proteome, into 22 distinct subcellular localization categories, and provide localization information for 70% of previously unlocalized proteins. Analysis of this high-resolution, high-coverage localization data set in the context of transcriptional, genetic, and protein-protein interaction data helps reveal the logic of transcriptional co-regulation, and provides a comprehensive view of interactions within and between organelles in eukaryotic cells.

Life with 6000 Genes

Abstract: The genome of the yeast Saccharomyces cerevisiae has been completely sequenced through a worldwide collaboration. The sequence of 12,068 kilobases defines 5885 potential protein-encoding genes, approximately 140 genes specifying ribosomal RNA, 40 genes for small nuclear RNA molecules, and 275 transfer RNA genes. In addition, the complete sequence provides information about the higher order organization of yeast's 16 chromosomes and allows some insight into their evolutionary history. The genome shows a considerable amount of apparent genetic redundancy, and one of the major problems to be tackled during the next stage of the yeast genome project is to elucidate the biological functions of all of these genes.

Functional Characterization of the S. cerevisiae Genome by Gene Deletion and Parallel Analysis

Abstract: 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.