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 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.
Chakraborty
B N
BN
Cellular, Molecular, and Microbial Biology Division, Department of Biological Sciences, The University of Calgary, Calgary, Alberta T 2N 1N4, Canada.
Ouimet
P M
PM
Sreenivasan
G M
GM
Curle
C A
CA
Kapoor
M
M
D015152:Blotting, Northern; D015139:Blotting, Southern; D018840:HSP70 Heat-Shock Proteins / Q000235:genetics*; D016296:Mutagenesis; D009492:Neurospora crassa / Q000235:genetics*; D010957:Plasmids; D017354:Point Mutation; D012091:Repetitive Sequences, Nucleic Acid; D013696:Temperature; D014170:Transformation, Genetic
D018840:HSP70 Heat-Shock Proteins