Heterologous and homologous plasmid integration at a spore-pigment locus in Penicillium paxilli generates large deletions.
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 overlapping lambda clones, lambda WB33-1 and lambda WB33-2, 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.
Itoh
Y
Y
Department of Plant Pathology, Tottori University, Japan.
Scott
B
B
D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers; D004271:DNA, Fungal / Q000032:analysis; D004926:Escherichia coli; D017353:Gene Deletion; D018014:Gene Transfer Techniques; D016681:Genome, Fungal; D008969:Molecular Sequence Data; D010407:Penicillium / Q000235:genetics* / Q000502:physiology; D010957:Plasmids; D016133:Polymerase Chain Reaction; D015183:Restriction Mapping; D013172:Spores, Fungal
D017931:DNA Primers; D004271:DNA, Fungal