Heterologous and homologous plasmid integration at a spore-pigment locus in Penicillium paxilli generates large deletions

Abstract

Mutations in a spore pigmentation locus (brs; brown spore) in Penicillium paxilli were isolated at a relatively-high frequency (0.17%) following integrative transformation of the hygromycin-resistance plasmid pAN7-1. A molecular analysis of four independently-isolated Brs^- mutants showed that all contained pAN7-1 integrated at a single-site that was unique for each mutant. A previously-described Brs^- mutant, YI-34 (Itoh et al. 1994), was a two-site integration. Three of the mutants had multiple copies of pAN7-1 arranged in head-to-tail tandem arrays. A 9.6-kb BamHI junction fragment was cloned from one of these, YI-33, by plasmid rescue and used to isolate two overlap** lambda clones, λWB33-1 and λWB33-2, that span about 30 kb in the region of the wild-type locus. When genomic digests of the five Brs^- mutants were probed with these lambda clones all of them were found to contain an extensive deletion through a common region of the P. paxilli genome. Subsequent attempts to generate one-step gene replacements within a 4.5-kb EcoRI fragment at the wild-type locus resulted in the isolation of Brs^- mutants at a frequency of 1.6%, but all mutants with this phenotype were also found to contain an extensive genomic deletion. Therefore, a common outcome of both heterologous and homologous plasmid integration at this locus is deletion formation.