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 disrup t the gene encoding the 80-kDa heat-inducible protein of Neurospora cr assa. Germinated conidia of the wild-type recipient strain were electr otransformed with a plasmid containing a 7-kb fragment harbouring the complete hsp80 gene sequence. Some of the transformants with a duplica tion of hsp80 gene sequence showed extensive methylation of these sequ ences even in vegetatively growing cells. The presence of an extra gen e copy in transformants of this: type resulted in a marked reduction i n the expression of this gene. Progeny of a cross of one such transfor mant, showing methylation of hsp80, was analyzed by Southern blot and Northern blot hybridization to examine thr relationship between methyl ation and the accumulation of hsp80 mRNA under hyperthermia. In additi on, HSP80 polypeptide levels were monitored in stressed and unstressed cells by immunoblot analysis using polyclonal anti-HSP80 IgG preparat ions. A correlation between the extent of RIP and expression of this g ene was observed in the progeny isolates.