Insertional mutagenesis based on illegitimate recombination in Schizosaccharomyces pombe.
An efficient insertional mutagenesis system has been developed for Schizosaccharomyces pombe based on linear PCR-generated cassettes containing selectable markers. It depends upon illegitimate recombination for integration into the genome. Various selectable markers of different sizes can be used to obtain sufficiently high transformation and integration frequencies. Based on Southern blotting, a single insertion is found in each strain and integration sites are broadly distributed in the genome. Sequence analysis of the insert junctions frequently reveals small regions of homology (4-10 bp) between the ends of the integrated cassette and the disrupted gene. The system has been used for simple genetic screens of various types and as a promoter trap for in-frame GFP fusions.
Chua
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G
Department of Biology, Biosciences Complex, Queen's University, Kingston, Ontario K7L 3N6, Canada.
Taricani
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L
Stangle
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Young
P G
PG
D002455:Cell Division / Q000235:genetics; D004271:DNA, Fungal; D017353:Gene Deletion; D005819:Genetic Markers; D016681:Genome, Fungal; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000235:genetics; D016254:Mutagenesis, Insertional; D016133:Polymerase Chain Reaction; D011995:Recombination, Genetic; D012568:Schizosaccharomyces / Q000235:genetics*; D012689:Sequence Homology, Nucleic Acid; D014170:Transformation, Genetic
D004271:DNA, Fungal; D005819:Genetic Markers; D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins