Cellular Responses to Superoxide-Mediated Stress in N. crassa

本研究的目的是研究真菌神经孢子菌对超氧化物介导的应激的遗传反应的性质和程度。研究的重点将放在超氧化物歧化酶基因和响应超氧化物生成剂诱导的匿名基因上。具体目标是：(1)继续分析我们最近克隆和测序的稻草胞质超氧化物歧化酶（sod-1）基因的功能和调控。研究人员将对最近构建的SOD -1突变体进行研究，以确定CuZn SOD对N. crassa好氧生长和繁殖的重要性。调控研究将检查该基因和MnSOD基因（见下文）在氧化应激条件下被诱导的程度。此外，体外和体内研究将探讨假设对sod-1表达重要的序列的意义。(2)构建带N. crassa MnSOD基因的重组质粒亚粒（目前在40 Kbp的cosmid克隆中鉴定为6 Kbp的HindIII片段），并利用rip介导的中断创建该基因的突变并表征。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学研究。(3)进行初步实验，以评估子囊菌中是否存在全局超氧化物（Sox）反应，类似于最近提出的大肠杆菌。我们将利用诱导抗超氧胁迫的生理研究和匿名超氧诱导基因的遗传研究。，通过标记的cdna进行鉴别筛选。

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.