Paxilline negative mutants of Penicillium paxilli generated by heterologous and homologous plasmid integration : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Molecular Genetics at Massey University, Palmerston North, New Zealand

Abstract

Using a monoclonal antibody-based ELISA, 600 pAN7-1 plasmid-tagged mutants of Penicillium paxilli were screened for paxilline accumulation and one paxilline negative mutant, YI-20, was identified (Itoh, unpublished data). A molecular analysis of this mutant showed that pAN7-1 was inserted at a single site but was present as 4-6 copies arranged in a head-to tail tandem repeat. Rescue of flanking sequences and analysis of the corresponding genomic region revealed that YI-20 has an extensive deletion at the site of pAN7-1 integration. Probing of a CHEF gel with the same sequences showed that associated with the deletion is a rearrangement of chromosome Va. Targeted gene disruption of wild-type sequences adjacent to the site where pAN7-1 inserted, resulted in the generation of two additional paxilline-negative mutants; both were single crossovers with deletions extending outside the region mapped. Neither of these new mutants had a rearrangement of chromosome Va, suggesting that deletion of genes on this chromosome is responsible for the paxilline-negative phenotype