The long term goals of this research are to understand the molecular basis for oxygen toxicity in eukaryotes. Oxygen radicals such as superoxide anion (O2-) are generated during respiration and through the exposure to environmental oxidants. These reactive species are thought to cause serious damage to biomolecules and have been associated with a large number of human disorders including atherosclerosis, cancer and aging. Our hypothesis is that oxygen toxicity can be mediated through the O2- inactivation of factors critical to cell growth and development. To address this, a genetic approach was developed to identify critical molecular targets and determinants of oxidative damage. These studies will employ mutants of the yeast S. cerevisiae that lack superoxide dismutase (SOD), an O2-scavenging enzyme. Our recent studies have indicated that these mutants are not only hyper-sensitive to the cytotoxic effects of oxygen, but are defective in meiosis and in a limiting step in lysine biosynthesis. Moreover, the aerobic growth defects noted with these mutants can be bypassed through additional mutations in one of two genes that we have identified as BSD1 and BSD2 ('Bypass SOD defect'). The BSD1 gene has recently been isolated. Our current objectives are to understand the role of the BSD factors as determinants of oxygen toxicity and furthermore, to identify the targets of oxidative damage in lysine biosynthesis and meiosis.
The specific aims designed to meet these goals are as follows:
(AIM 1) To clone the BSD2 gene: This gene will be isolated from a yeast genomic library through functional complementation.
(AIM 2) To characterize the BSD1 and BSD2 genes: The BSD genes will be subject to meiotic mapping and the identity of the encoded proteins will be established through gene sequence analyses. The role of the BSD factors in cell growth and viability will be investigated through gene deletion studies in yeast (AIM 3) To understand the biochemical basis for the oxygen-resistance conferred by BSD gene mutations: Tests for measuring oxygen consumption and O2- production will assess whether the bsd mutations affect cellular levels of O2-.
(AIM 4) To understand the role of SOD in lysine biosynthesis: The lysine biosynthetic defect of SOD mutants will be reconstructed in vitro. Secondly, the biosynthetic factor inactivated in SOD mutants will be identified by isolating genes that reverse the lysine auxotrophy of these cells.
(AIM 5) To understand the role of SOD in meiosis: The meiotic step blocked in SOD mutants will be identified through studies on premeiotic DNA replication and DNA reeombination. Furthermore, the meiosis factor inactivated in SOD mutants will be identified by isolating genes that specifically bypass the sporulation defect of these cells. Collectively, these studies should provide valuable new information regarding the molecular basis of oxygen toxicity. Elucidation of the BSD factors would reveal important determinants of oxidative damage and identifying the oxygen-sensitive component of lysine biosynthesis and meiosis would expose critical targets of oxygen toxicity. Conceivably, these findings could ultimately foster the development of new pharmacological methods for treating the various disorders attributed to oxygen free radicals.
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