| When the cytochrome mediated electron transport chain in the fungus Neurospora crassa is disrupted, an alternative oxidase, encoded by the nuclear gene aod-1, is induced. The alternative oxidase donates electrons directly to oxygen from ubiquinone, bypassing two proton pumping sites of the normal electron transport chain.; Little is known about the control of aod-1 expression. To facilitate isolation of mutations affecting the regulation of the gene, a tyrosinase based reporter system was constructed. The reporter construct contains the region upstream of the aod-1 gene fused to the coding sequence of the N. crassa enzyme tyrosinase, so that expression of the enzyme is controlled by signals that normally affect aod-1. Expression of tyrosinase is easily monitored, as its activity turns colonies brown when they are overlaid with a tyrosine solution. The reporter construct was integrated into the genome of a strain shown to carry a null allele of the endogenous gene encoding tyrosinase.; When cells containing the reporter system were mutagenized and assayed for tyrosinase activity under conditions that normally induce alternative oxidase, eighteen mutant strains were isolated that affected induction of both the reporter and endogenous alternative oxidase. Complementation analysis revealed that four novel loci involved in aod-1 regulation, named aod-4, aod-5, aod-6, and aod-7, had been isolated. Mutations in these genes were shown to prevent induction of aod-1 mRNA and protein. An aod-4 strain also exhibited a temperature-sensitive growth defect at 30°C and was deficient in cytochromes aa3 and b. The aod-4 gene maps to the right arm of linkage group V.; A chloramphenicol resistant mutation (chl-1) was also isolated. Chloramphenicol prevents mitochondrial translation, thereby disrupting oxidative phosphorylation and inducing alternative oxidase. Unlike wild-type strains grown in the presence of chloramphenicol, cells carrying the chl-1 mutation have normal levels of cytochromes aa 3 and b, and do not induce alternative oxidase activity. However, growth of the chl-1 strain in the presence of the complex III inhibitor antimycin A does induce alternative oxidase activity. The chl-1 gene maps to the right arm of linkage group II, and is the only chloramphenicol resistance marker reported for N. crassa to date. |
