Insertional mutagenesis in Coprinus cinereus: use of a dominant selectable marker to generate tagged, sporulation-defective mutants

Curr Genet. 1999 Dec;36(6):371-82. doi: 10.1007/s002940050512.

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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Coprinus / genetics*
  • Coprinus / physiology
  • Genes, Dominant
  • Genetic Markers*
  • Genetic Techniques
  • Meiosis
  • Mutagenesis, Insertional*
  • Mutation
  • Phosphotransferases (Alcohol Group Acceptor) / genetics
  • Recombinant Proteins / genetics
  • Recombinant Proteins / metabolism
  • Selection, Genetic
  • Spores, Fungal / genetics*
  • Transformation, Genetic*
  • Tubulin / genetics
  • Tubulin / metabolism

Substances

  • Genetic Markers
  • Recombinant Proteins
  • Tubulin
  • Phosphotransferases (Alcohol Group Acceptor)
  • hygromycin-B kinase