Paxilline-negative mutants of Penicillium paxilli generated by heterologous and homologous plasmid integration.
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. 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 array. 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. Telomeric fingerprinting of genomic digests of P. paxilli, combined with pulsed-field gel electrophoresis of chromosomal DNA, established that there are a minimum of eight chromosomes in this fungus.
Young
C
C
Institute of Molecular BioSciences, Massey University, Palmerston North, New Zealand.
Itoh
Y
Y
Johnson
R
R
Garthwaite
I
I
Miles
C O
CO
Munday-Finch
S C
SC
Scott
B
B
D002874:Chromosome Mapping; D015825:Chromosomes, Fungal; D003434:Crossing Over, Genetic; D004926:Escherichia coli / Q000235:genetics; D017353:Gene Deletion; D015698:Genomic Library; D007211:Indoles / Q000032:analysis*; D009183:Mycotoxins / Q000235:genetics; D010407:Penicillium / Q000235:genetics* / Q000378:metabolism; D010957:Plasmids; D012091:Repetitive Sequences, Nucleic Acid; D014170:Transformation, Genetic
D007211:Indoles; D009183:Mycotoxins; C048220:paxilline