Multiple genetic changes are required for the development of a malignant tumor cells and many environmentally induced cancers show a delayed onset of more than 20 years following exposure. The frequency of such changes found in cancer cells is higher than can be explained through random mutation and it was proposed that a sub-population of cells develop a mutator phenotype. Such a persistent elevated level of genetic instability is also a major contributor to the progressive, multistage development of malignant disease. This phenotype, ionizing radiation but the mechanism has not been defined. We have observed a similar genomic instability more than 50 cell divisions after exposure to ionizing radiation in the yeast Saccharomyces cerevisiae. We observed a persistently decreased plating efficiency These effects cannot be due to initial damage because of their persistence over many generations. Mutations in a single gene leading to an elevated level of genetic instability also cannot account for these effects because they occur in up to 70% of the exposed cells. It is thus more likely that a difference in gene expression accounts for the high frequency of deletions (HFD) phenotype. This proposal is designed to investigate the mechanism of these delayed inheritable changes. We propose to further characterize the phenotype of clones showing an HFD phenotype in terms of sensitivity to carcinogens, cis- versus trans-acting effects, levels of DNA strand breaks and the involvement of oxidative stress. Furthermore we will determine whether illegitimate DNA integration is elevated in HFD clones and if so, we will define the sequence specificity and the genomic distribution of the target sites of such integration events. We will also develop a complete gene expression profile (6200 genes) for yeast HFD cultures and control cultures to identify genes which may be involved in the maintenance or destabilization of genetic integrity. Finally, we will alter the expression of genes that are up or down regulated in HFD clones, and determine the effect of this altered gene expression on the initiation and/or inheritance of the HFD phenotype. This project should characterize the phenomenon of persistently elevated genetic instability, give insights into its mechanism and might also provide molecular targets for intervention to reverse the phenotype.
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