Cellular Responses to Superoxide-Mediated Stress in N. crassa

本研究的目的是研究粗糙脉孢菌对超氧化物介导的胁迫的遗传反应的性质和大小。这项研究的重点将放在超氧化物歧化酶基因和超氧化物生成剂诱导的匿名基因上。具体的目标是：(1)继续分析我们最近克隆和测序的粗面拟青霉胞质Cuan超氧化物歧化酶(SOD-1)基因的功能和调控。最近构建的sod-1突变体将被研究以确定铜锌超氧化物歧化酶对粗毛拟青霉有氧生长和繁殖的重要性。调节研究将检查该基因和MnSOD的基因(S)在多大程度上被诱导，以响应氧化应激条件。此外，体外和体内研究将调查假设对sod-1表达重要的序列的意义。(2)构建携带粗面乳杆菌MnSOD基因(目前鉴定为40kbP粘粒克隆中的6kbpHindIII片段)的重组质粒的亚克隆，并利用RIP介导的干扰产生该基因的突变并鉴定其特征。这些突变株，当与sod-1突变株一起考虑时，将允许对超氧化物歧化酶的重要性、功能和进化进行遗传和生理研究。(3)进行初步实验，以评估子囊菌是否存在全球超氧化物(SOX)反应，类似于最近对大肠杆菌提出的反应。我们将使用诱导抗超氧胁迫的生理学研究和匿名的超氧化物诱导基因的遗传学研究，通过标记的cDNA的差异筛选来鉴定。

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.