Repeat-induced point mutations of HSP80 gene of Neurospora crassa: methylation of duplicated DNA sequences in the vegetative state.

The process of repeat-induced point mutations (RIP) was used to disrupt the gene encoding the 80-kDa heat-inducible protein of Neurospora crassa. Germinated conidia of the wild-type recipient strain were electrotransformed with a plasmid containing a 7-kb fragment harbouring the complete hsp80 gene sequence. Some of the transformants with a duplication of hsp80 gene sequence showed extensive methylation of these sequences even in vegetatively growing cells. The presence of an extra gene copy in transformants of this type resulted in a marked reduction in the expression of this gene. Progeny of a cross of one such transformant, showing methylation of hsp80, was analyzed by Southern blot and Northern blot hybridization to examine the relationship between methylation and the accumulation of hsp80 mRNA under hyperthermia. In addition, HSP80 polypeptide levels were monitored in stressed and unstressed cells by immunoblot analysis using polyclonal anti-HSP80 IgG preparations. A correlation between the extent of RIP and expression of this gene was observed in the progeny isolates.