title: Insertional mutagenesis in Coprinus cinereus: use of a dominant selectable marker to generate tagged, sporulation-defective mutants.
abstract: We have constructed a dominant selectable marker, PHT1, for transformation of the basidiomycete Coprinus cinereus. PHT1 consists of a bacterial hygromycin B resistance gene fused to the promoter and terminator regions of the C. cinereus beta-tubulin gene. We found in transformation experiments that PHT1 confers hygromycin B resistance to all strains of C. cinereus tested, that it integrates without apparent bias into the genome, and that it is stable through meiotic crosses. We used a plasmid containing this marker, pPHT1, for restriction enzyme-mediated integration (REMI) and found that this technique could increase transformation efficiencies more than seven-fold. In REMI experiments using KpnI, the integrated DNA was flanked by intact KpnI sites in 53% of the cases examined, single-copy insertions represented 60% of the integration events, and most multicopy insertions were oriented head-to-tail. A screen of REMI-generated transformants yielded sporulation-defective mutants at a frequency of 1.2%. Genetic analysis showed that in six of nine mutants examined, the defect in spore formation is most likely a direct result of the pPHT1 insertion, and in three of these mutants a single pPHT1 locus was shown to cosegregate with the sporulation defect. We used semi-random PCR to isolate the genomic DNA adjacent to one pPHT1 insertion in a sporulation-defective mutant and found that we had disrupted the C. cinereus spo11 gene. Thus, REMI, in combination with pPHT1, is a powerful tool for the dissection of the meiotic process in C. cinereus.
authors.affiliation: Department of Biology, Indiana University, Bloomington 47405, USA.
authors.forename: W J
authors.initials: WJ
authors.lastname: Cummings
authors.forename: M
authors.initials: M
authors.lastname: Celerin
authors.forename: J
authors.initials: J
authors.lastname: Crodian
authors.forename: L K
authors.initials: LK
authors.lastname: Brunick
authors.forename: M E
authors.initials: ME
authors.lastname: Zolan
mesh_terms: D003302:Coprinus / Q000235:genetics* / Q000502:physiology; D005799:Genes, Dominant; D005819:Genetic Markers; D005821:Genetic Techniques; D008540:Meiosis; D016254:Mutagenesis, Insertional; D009154:Mutation; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012641:Selection, Genetic; D013172:Spores, Fungal / Q000235:genetics*; D014170:Transformation, Genetic; D014404:Tubulin / Q000235:genetics / Q000378:metabolism
chemical_list: D005819:Genetic Markers; D011994:Recombinant Proteins; D014404:Tubulin; D017853:Phosphotransferases (Alcohol Group Acceptor); C063242:hygromycin-B kinase