The long-term goal of this research program is to gain knowledge concerning the mechanisms involved in the response of mammalian cells to ionizing radiation, with particular reference to its carcinogenic and mutagenic effects. The approach is a multi-faceted one, and the endpoints under investigation include mutagenesis, malignant transformation, cell survival and the induction of chromosomal abnormalities. The program has the overall purpose of further defining the risks of low-level radiation exposure by gaining a better understanding of the cellular and molecular mechanisms for its effects. A particular focus is on the induction of genetic instability by radiation, and the role that homologous recombinational events may play in this process.
The Specific Aims are: 1) To identity oncogenes activated or expressed during the process of malignant transformation induced by radiation, by use of denaturing gradient gel electrophoresis blots and subtraction cloning by the representational differences analysis (RDA) technique; 2) To examine the role of genome-wide instability in malignant transformation, in particular to seek evidence for the involvement of homologous recombination in this process; genetic rearrangements will be examined in minisatellite loci as well as random DNA sequences. 3) To examine the recombinogenic potential of several classes of agents in an assay for measuring interallelic homologous recombination in human cells developed during the prior grant period, including the phorbol ester tumor promoter TPA and radiation; 4) To seek evidence for coincident or second site mutations in a group of random, non-selected microsatellite loci in cells selected for mutations at the tk locus; 5) To better characterize the delayed mutagenic effects of radiation by examining the molecular structure of late-arising mutations and identifying the gene(s) that may be induced or activated by radiation in mutator clones; 6) To examine further the mechanism for the effect of SV40 large T antigen on radiosensitivity, by use of a large panel of T-antigen mutants containing single-base point mutations in specific sequences of the various functional domain of the gene; and 7) To determine whether specific mRNAs are expressed in alpha irradiated cells by use of the differential display technique, in order to identity genes that may be turned on in response specifically to alpha radiation.
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