Substrate kinetics of the alternative oxidase of Neurospora crassa.

The kinetics of the succinate oxidation by cyanide-sensitive and cyanide-insensitive submitochondrial particles of Neurospora crassa cells suggest that both respiratory pathways use the same complex II. This is confirmed by comparing the kinetics of the reductase activities of the isolated succinate-ubiquinone oxidoreductase (complex II) of cyanide-sensitive and cyanide-insensitive cells respectively. No alternative-oxidase activity was found to be associated with the isolated complex II of cyanide-insensitive cells.     

Immunological identification of the alternative oxidase of Neurospora crassa mitochondria.

Neurospora crassa mitochondria use a branched electron transport system in which one branch is a conventional cytochrome system and the other is an alternative cyanide-resistant, hydroxamic acid-sensitive oxidase that is induced when the cytochrome system is impaired. We used a monoclonal antibody to the alternative oxidase of the higher plant Sauromatum guttatum to identify a similar set of related polypeptides (Mr, 36,500 and 37,000) that was associated with the alternative oxidase activity of N. crassa mitochondria. These polypeptides were not present constitutively in the mitochondria of a wild-type N. crassa strain, but were produced in high amounts under conditions that induced alternative oxidase activity. Under the same conditions, mutants in the aod-1 gene, with one exception, produced apparently inactive alternative oxidase polypeptides, whereas mutants in the aod-2 gene failed to produce these polypeptides. The latter findings support the hypothesis that aod-1 is a structural gene for the alternative oxidase and that the aod-2 gene encodes a component that is required for induction of alternative oxidase activity. Finally, our results indicate that the alternative oxidase is highly conserved, even between plant and fungal species.     

Genetic evidence for a regulatory pathway controlling alternative oxidase production in Neurospora crassa

When the cytochrome-mediated mitochondrial electron transport chain of Neurospora crassa is disrupted, an alternative oxidase encoded by the nuclear aod-1 gene is induced. The alternative oxidase donates electrons directly to oxygen from the ubiquininol pool and is insensitive to chemicals such as antimycin A and KCN that affect the standard electron transport chain. To facilitate isolation of mutants affecting regulation of aod-1, a reporter system containing the region upstream of the aod-1 coding sequence fused to the coding sequence of the N. crassa tyrosinase gene (T) was transformed into a strain carrying a null allele of the endogenous T gene. In the resulting reporter strain, growth in the presence of chloramphenicol, an inhibitor of mitochondrial translation whose action decreases the level of mitochondrial translation products resulting in impaired cytochrome-mediated respiration, caused induction of both alternative oxidase and tyrosinase. Conidia from the reporter strain were mutagenized, plated on medium containing chloramphenicol, and colonies that did not express tyrosinase were identified as potential regulatory mutants. After further characterization, 15 strains were found that were unable to induce both the reporter and the alternative oxidase. Complementation analysis revealed that four novel loci involved in aod-1 regulation had been isolated. The discovery that several genes are required for regulation of aod-1 suggests the existence of a complex pathway for signaling from the mitochondria to the nucleus and/or for expression of the gene.     

Regulation of biosynthesis of l-amino acid oxidase by Neurospora crassa

A temperature gradient device has been used to produce pH-temperature gradient plates. The response of Salmonella typhimurium and Bacillus cereus to continuous gradients of pH and temperature has been mapped. A third variable, sodium chloride, was investigated by incorporating it at different concentrations in a series of gradient plates. Both organisms exhibited an optimum temperature and pH range for maximum salt tolerance.     

Mutation induction in Neurospora crassa incubated in mice and rats

Summary When Neurospora crassa conidia were injected into the peritoneal cavity of untreated mice or rats and kept there for more than 24 hours, the ad-3 mutation frequency among the surviving conidia increased sharply, more so in rats than in mice. This increase in the ad-3 mutation frequency was attributed both to direct cellular contact between conidia and mammalian cells and to macromolecules already present in untreated animals. Conidia enclosed in dialysis tubing or in diffusion chambers placed surgically in the peritoneal cavity had a much lower frequency of ad-3 mutations than conidia injected into the peritoneal cavity as a suspension. This was interpreted as indicating that the major fraction of mutations are mediated through a cellular contact.To determine whether the dialysis bags were permeable to mutagens, a comparison was made between the mutation frequencies obtained with conidia placed in dialysis bags and with conidia distributed at random throughout the peritoneal cavity in host animals treated with methyl methanesulfonate (MMS). MMS (100 mg/kg) was injected into the tail vein 8 hours after the conidia were placed in the animals. Ten hours after the injection of the mutagen, the conidia were recovered and analyzed for the induction of ad-3 mutations. The MMS-induced mutation frequency was the same among both groups of conidia, demonstrating that the dialysis bags did not become impermeable to small molecules during the time of incubation in the animal.In the host-mediated assay an indiactor organism is injected into the peritoneal cavity of an animal which is then treated with a chemical or its metabolites, to assay for mutagenicity. The present experiments show that the increased mutation frequency induced in the indicator organism after intraperitoneal injection and incubation may give false positive results in the host-mediated assay unless a comparison is made with suitable untreated controls.Autopsies of animals 24 days after intraperitoneal inoculation with Neurospora conidia, and sectioning and staining of various organs (Malling and Cosgrove, 1970) showed that some conidia were still localized in various organs, even though essentially all of them had been inactivated 96 hours after injection. The inactivation of these fungal spores may result from an enzymatic degradation of their DNA, mediated by the host, and halting this process prematurely may result in the induction of recessive-lethal mutations. Thus these studies also suggest that one of the important defense mechanisms of higher animals against infectious organisms may be the induction of mutations.Research sponsored by the National Cancer Institute, National Institutes of Health, and by the U. S. Atomic Energy Commission under contract with the Union Carbide Corporation.     

Transient kinetic studies of Neurospora crassa cytochrome c oxidase.

The reaction of Neurospora crassa cytochrome c oxidase with CO was studied by flash-photolysis and rapid-mixing experiments, leading to the determination of the association and dissociation rate constants (7 X 10(4) M-1 X s-1 and 0.02s-1 respectively). Pre-steady-state kinetic investigations of the catalytic properties of the enzyme showed that under proper conditions Neurospora cytochrome c oxidase can be 'pulsed', i.e. activated, like the mammalian enzyme. The 'pulsed' species is spectroscopically different from the 'resting' one, and the decay into the 'resting' state is fast (t1/2 approx. 3 min).