SEQUENCE REPEAT INDUCED DISRUPTION OF THE MAJOR HEAT INDUCIBLE HSP70 GENE OF NEUROSPORA-CRASSA

The process of repeat-induced point mutation (RIP) was used to disrupt hsps-1, the gene encoding the major heat-inducible member of the HSP7 0 family of Neurospora crassa. A plasmid DNA, containing an incomplete copy of hsps-1 and the selectable marker qa-2(+), was introduced into germinated conidia. The sexual progeny of transformants with ectopica lly integrated hsps-1 DNA was examined for RIP by Southern-blot analys is of MboI- and Sau3A-digested genomic DNA. Progeny strains, showing R IP, were tested for heat shock-responsive expression of hsps-1, by RNA -blot hybridization and Western-blot analysis, as well as for thermoto lerance. Isolates with RIP showed low levels of hsps-1 mRNA and a lack of induction of HSP70 protein by heat shock, accompanied by only a ma rginal decrease in the acquisition of thermotolerance.