Chromosomal promoter replacement in Saccharomyces cerevisiae: construction of conditional lethal strains for the cloning of glycosyltransferases from various organisms.
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.
Mazhari-Tabrizi
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Institut für Medizinische Mikrobiologie und Hygiene, Philipps-Universität-Marburg, Germany. mazhari@wehi.edu.au
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Mumberg
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Funk
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Schwarz
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Eckert
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D015825:Chromosomes, Fungal; D003001:Cloning, Molecular / Q000379:methods*; D005816:Genetic Complementation Test; D006031:Glycosylation; D016695:Glycosyltransferases / Q000235:genetics*; D011401:Promoter Regions, Genetic; D012441:Saccharomyces cerevisiae / Q000235:genetics*
D016695:Glycosyltransferases