A homokaryotic derivative of a Phanerochaete chrysosporium strain and its use in genomic analysis of repetitive elements

Analysis of complex gene families in the lignin degrading basidiomycete Pha nerochaete chrysosporium has been hampered by the dikaryotic nuclear condit ion. To facilitate genetic investigations in P. chrysosporium strain BKM3-F -1767, we isolated a homokaryon from regenerated protoplasts. The nuclear c ondition was established by PCR amplification of five unlinked genes follow ed by probing with allele-specific oligonucleotides. Under standard nitroge n-limited culture conditions, lignin peroxidase, manganese peroxidase, and glyoxal oxidase activities of the homokaryon mere equivalent to those of th e parental dikaryon. We used the homokaryon to determine the genomic organi zation and to assess transcriptional effects of a family of repetitive elem ents. Previous studies had identified an insertional mutation, Pce1, within lignin peroxidase allele lip12. The element resembled nonautonomous class II transposons and was present in multiple copies in strain BKM-F-1767. In the present study, three additional copies of the Peel-like element were cl oned and sequenced. The distribution of elements was nonrandom; all localiz ed to the same 3.7-Mb chromosome, as assessed by segregation analysis and S outhern blot analysis of the homokaryon. Reverse transcription-PCR (RT-PCR) showed that Pec1 was not spliced from the lip12 transcript in either the h omokaryon or the parental dikaryon. However, both strains had equivalent li gnin peroxidase activity, suggesting that some lip genes may be redundant.