Isolation and characterization of temperature-sensitive respiratory mutants of Neurospora crassa

Abstract

Filtration-enrichment and inositol-less death methods of mutant isolation, coupled with a screen for cyanide-insensitive respiration, proved to be highly efficient methods for isolating temperature-sensitive (ts) nuclear Neurospora mutants having defective respiration. Eighteen different ts respiratory mutants have been isolated. Most of them are pleiotropic and defective in one or more of the following phenotypes: cytochrome aa3, b, and c (individual or multiple defects); oligomycin inhibition of ATPase activity; respiration and its inhibition by KCN and salicyl hydroxamic acid; and growth rates in liquid and solid media at 25 degrees and 38 degrees. Among these mutants are the first cytochrome c mutant of Neurospora and an extranuclear ts ATPase mutant. An added bonus was the fact that over half of the mutants were affected either in ribosome assembly or in protein synthesis in the mitochondrion. We have yet to find any mutants completely lacking activities associated with the respiratory chain. However, the wide spectrum of mutants isolated here, along with those currently available, constitutes a considerable resource for investigating respiration in obligate aerobes.