Targeted gene deletion in Saccharomyces cerevisiae and Schizosaccharomyces pombe.

Gene deletion is an important element in the functional characterization of gene and protein function. Efficient tools for gene deletion have been developed in the model yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe, all of which rely on the replacement of the endogenous gene of interest with a selectable marker gene by homologous recombination. In order to minimize incidental recombination events between DNA sequences within the marker gene and a chromosomal sequence, gene deletion cassettes consisting entirely of heterologous DNA sequences are preferred. The gene deletion cassettes, which are composed of the marker gene flanked by short DNA segments homologous to the chromosomal sequences lying to the left and right of the gene to be deleted, are generated by PCR and mediate highly efficient one-step gene deletion events. Incorporation of loxP sites flanking the marker gene allows Cre recombinase-mediated rescue, so that the marker can be reused for the next gene deletion. This is particularly useful for the characterization of gene families in S. cerevisiae. The one-step gene deletion method is not limited to the elimination of individual genes, but can also be used for the removal of chromosomal segments exceeding 100 kbp in length. Here we describe a comprehensive set of gene deletion cassettes and outline their use in S. cerevisiae and S. pombe.

Delete and repeat: a comprehensive toolkit for sequential gene knockout in the budding yeast Saccharomyces cerevisiae.

Gene inactivation is an essential step in the molecular dissection of gene function. In the yeast Saccharomyces cerevisiae, many tools for gene disruption are available. Gene disruption cassettes comprising completely heterologous marker genes flanked by short DNA segments homologous to the regions to the left and right of the gene to be deleted mediate highly efficient one-step gene disruption events. Routinely, in more than 50% of analyzed clones, the marker cassette is integrated in the targeted location. The inclusion of loxP sites flanking the disruption marker gene allows sequence-specific Cre recombinase-mediated marker rescue so that the marker can be reused to disrupt another gene. Here, we describe a comprehensive toolbox for multiple gene disruptions comprising a set of seven heterologous marker genes including four dominant resistance markers for gene disruption, plus a set of Cre expression plasmids carrying eight different selection markers, four of them dominant.

Missense variants in hMLH1 identified in patients from the German HNPCC consortium and functional studies.

Missense mutations of the DNA mismatch repair gene MLH1 are found in a significant fraction of patients with Lynch syndrome (hereditary nonpolyposis colorectal cancer, HNPCC) and their pathogenicity often remains unclear. We report here all 88 MLH1 missense variants identified in families from the German HNPCC consortium with clinical details of these patients/families. We investigated 23 MLH1 missense variants by two functional in vivo assays in yeast; seven map to the ATPase and 16 to the protein interaction domain. In the yeast-2-hybrid (Y2H) assay three variants in the ATPase and twelve variants in the interaction domain showed no or a reduced interaction with PMS2; seven showed a normal and one a significantly higher interaction. Using the Lys2A (14) reporter system to study the dominant negative mutator effect (DNE), 16 variants showed no or a low mutator effect, suggesting that these are nonfunctional, three were intermediate and four wild type in this assay. The DNE and Y2H results were concordant for all variants in the interaction domain, whereas slightly divergent results were obtained for variants in the ATPase domain. Analysis of the stability of the missense proteins in yeast and human embryonic kidney cells (293T) revealed a very low expression for seven of the variants in yeast and for nine in human cells. In total 15 variants were classified as deleterious, five were classified as variants of unclassified significance (VUS) and three were basically normal in the functional assays, P603R, K618R, Q689R, suggesting that these are neutral.