) The goal of this application is to analyze the molecular, genetic, cytogenetic and cellular mechanisms that initiate and perpetuate genomic instability after cellular exposure to radiations commonly encountered in space. This goal will be addressed by means of five specific aims.
Specific aim 1 will test the hypothesis that genomic instability, as measured by delayed chromosomal instability, can be induced by high-energy protons, high-Z and high-energy (HZE) iron ions, and high LET alpha particles in both log-phase and confluence arrested cells.
Specific aim 2 will test the hypothesis that the critical cellular target for high LET-induced genomic instability is located in both the nuclear and extranuclear cellular compartments. This will be achieved by selectively targeting the cell nucleus and/or the plasma membrane and cytoplasm to more than 1 alpha particle, or to the decay of 125iodine incorporated into the DNA as 125IUdR or bound to the plasma membrane as 125I-conjugated concanavalin A.
Specific aim 3 will test the hypothesis that cells showing one point of genomic instability, e.g., chromosomal instability, demonstrate a """"""""mutator"""""""" phenotype and display other endpoints of instability, specifically, increased mutation at the HPRT and APRT loci, gene amplification at the DHFR and CAD loci, increased SCE levels, and delayed reproductive cell death.
Specific aim 4 will test the hypothesis that high LET radiation-induced genomic instability is increased in isogenic repair-deficient cell lines as a function of known mutations in DNA doublestrand-break repair.
Specific aim 5 will test the hypothesis that there is a molecular basis for induced genomic instability that can be observed cytogenetically. The applicant will monitor instability of the (TTAGGG)n interstitial telomere-like repeat sequence and use comparative genomic hybridization to identify chromosomal regions that are consistently different between unstable and stable clones.
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