Sequence repeat-induced disruption of the major heat-inducible HSP70 gene of Neurospora crassa

Abstract

The process of repeat-induced point mutation (RIP) was used to disrupt hsps-1, the gene encoding the major heat-inducible member of the HSP70 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 ectopically integrated hsps-1 DNA was examined for RIP by Southern-blot analysis of MboI- and Sau3A-digested genomic DNA. Progeny strains, showing RIP, were tested for heat shock-responsive expression of hsps-1, by RNA-blot hybridization and Western-blot analysis, as well as for thermotolerance. 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 marginal decrease in the acquisition of thermotolerance.

Heat Shock Protein 80 of Neurospora crassa, a Cytosolic Molecular Chaperone of the Eukaryotic Stress 90 Family, Interacts Directly with Heat Shock Protein 70

“…Furthermore, there may be a functional overlap in the action of major molecular chaperones. The results of our previous experi- ments demonstrating that inactivation of either Hsp70 or Hsp80 alone, by means of the RIP (repeat-induced point mutations) process, resulted in slower-than-normal growth but did not altogether eliminate the capacity of the cells to develop thermotolerance (Chakraborty et al, 1995;Vijayaraghavan & Kapoor, 1996) support the hypothesis of a functional overlap.…”

“…Antibody Preparation and Protein Determination. The preparation of anti-Hsp80 IgG and that of anti-Hsp70 antiserum has been described previously (Roychowdhury et al, 1992a;Chakraborty et al, 1995). The protein content of different fractions was estimated employing the Bio-Rad micro assay using bovine serum albumin as a calibration standard (Bradford, 1976).…”

Electroporation Mediated DNA Transformation of Filamentous Fungi

“…Approximately 50 and 60 % of the progeny from crosses of E-26 and E-43 were qa 2+ , respectively. Southern blot analysis of various progeny strains, digested with Mbo I and Sau 3A both recognizing the sequence GATC, while methylation of the C residue blocks Sau 3A to cleave at such sites, whereas MboI digestion is unaffected (Chakraborty et al 1995 ). However, a GC-AT transition will render this sequence unrecognizable by either enzyme.…”

High constitutive peroxidase activity and constitutive thermotolerance in Neurospora crassa

“…When these transformants, with hsp70 DNA integrated at ectopic chromosomal locations, were used as parental strains in sexual crosses, a majority of the isolates showed evidence of RIP in the duplicated hsp70 DNA in the progeny (Chakraborty et al 1995). However, one exceptional transformant (designated E-45) was recovered, which in contrast with other transformants did not yield the expected progeny with methylation of cytosine residues associated with RIP in crosses with the wild-type strain.…”

“…The strain E-45, isolated according to the procedure described previously (Chakraborty et al 1995), was examined for some phenotypic attributes including growth rate, conidiation, and response to variations in temperature. For determination of the optimal growth temperature, conidial suspensions were plated on sorbose medium (Vm-1% sorbose-0.1% sucrose-1.5% agar) and incubated at 28° and 30°C; after 5 days, the colonies were counted.…”