The goal of the present study is to investigate the nature and magnitude of the genetic response to superoxide-,mediated stress in the fungus Neurospora crassa. The emphasis of the study will be on genes for superoxide dismutase and anonymous genes that are induced in response to superoxide generating agents. Specific goals are to:(1) Continue analysis of the function and regulation of the gene for N. crassa cytosolic CuAn superoxide dismutase (sod-1), which we have recently cloned and sequenced. Recently constructed sod-1 mutants will be studied to determine the importance of CuZn SOD for the aerobic growth and reproduction of N. crassa . Regulation studies will examine the extent to which this gene and the gene (s) for MnSOD (see below) are induced in response to conditions of oxidative stress. In addition, in vitro and in vivo studies will investigate the significance of sequences hypothesized to be important for sod-1 expression. (2) Construct sublcones of the recombinant plasmid carrying the gene for N. crassa MnSOD (currently identified as a 6 Kbp HindIII fragment in a 40 kbp cosmid clone) and create and characterize mutations in this gene using RIP-mediated disruption. These mutant strains, when considered along with sod-1 mutants, will permit genetic and physiological studies of the importance, function and evolution of superoxide dismutases. (3) Perform preliminary experiments toward assessing whether there exists a global superoxide (Sox) response in ascomycetous fungi, similar to that recently proposed for E. coli. We will employ physiological studies of induced resistance to superoxide stress and genetic studies of anonymous superoxide-induced genes in N. crassa., identified by differential screening with labeled cDNAs.
