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Chromosomal promoter replacement in Saccharomyces cerevisiae: Construction of conditional lethal strains for the cloning of glycosyltransferases from various organisms

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Abstract

Heterologous complementation in yeast has been a successful tool for cloning and characterisation of genes from various organisms. Therefore we constructed conditionally lethal Saccharomyces cerevisiae strains by replacing the endogenous promoter from the genes of interest (glycosyltransferases) by the stringently regulated GAL1-promoter, by a technique called chromosomal promoter replacement. Such yeast strains were constructed for the genes Alg 1, Alg7, Sec59, Wbp1 involved in N-Glycosylation, the genes Gpi2, Gpi3/Spt14, Gaal, Pis1, involved in GPI-anchor biosynthesis and Dpm involved in both pathways. All strains show the expected conditionally lethal phenotype on glucose-containing medium when expression of the respective gene is turned off.

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Author notes

  1. Dominik Mumberg

    Present address: Department of Pathology, University of Chicago, 5418 South Maryland Avenue, Chicago, Illinois, 60637

  2. Martin Funk

    Present address: MediGene, Lochhammerstr. 11, 82152, Martinsried/Muenchen, Germany

Authors and Affiliations

  1. Institut für Medizinische Mikrobiologie und Hygiene, Philipps-Universität-Marburg, Robert-Koch Strasse 17, 35037 Marburg, Germany

    Ramin Mazhari-Tabrizi, Michaela Blank, Ralph T. Schwarz & Volker Eckert

  2. Institut fuer Molekularbiologie und Tumorforschung, Philipps-Universitaet-Marburg, Emil Mankopff Strasse 2, 35037, Marburg, Germany and

    Dominik Mumberg & Martin Funk

Authors
  1. Ramin Mazhari-Tabrizi
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  2. Michaela Blank
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  3. Dominik Mumberg
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  4. Martin Funk
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  5. Ralph T. Schwarz
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  6. Volker Eckert
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Mazhari-Tabrizi, R., Blank, M., Mumberg, D. et al. Chromosomal promoter replacement in Saccharomyces cerevisiae: Construction of conditional lethal strains for the cloning of glycosyltransferases from various organisms. Glycoconj J 16, 673–679 (1999). https://doi.org/10.1023/A:1007132907235

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  • DOI: https://doi.org/10.1023/A:1007132907235

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