Using modern technology, it is now sometimes possible to repair damaged genes. We will define genes responsible for radio-sensitivity and insert normal DNA to attempt to correct or partially correct the unknown defect to restore a more "normal"" radio-resistant cell phenotype.

Project Goals:

1. Identify new genes controlling radio-sensitivity.
2. Obtain information regarding the frequencies of polymorphisms of these and other such genes.
3. Assess their biological significance with respect to risk assessment for low doses of low- LET ionizing radiation.

Experimental Approach:

The criterion for assessing differences in radio-sensitivity involves measurement of radiation-induced chromosomal aberrations, since these figure prominently in carcinogenesis; the main hazard of interest for radiation protection. The focus concentrates particularly on chromosomal radio-sensitivity following low-doses of gamma radiation, in the 0 to 10 cGy range, and for continuous low dose-rate exposures. We have human cell cultures available from some 33 different individuals that differ by a factor of about two in sensitivity for cell killing after high-dose, high dose-rate irradiation. Among these individuals, there is no known defect in any currently known DNA repair or damage processing genes. We will attempt to correct or partially correct the unknown defect to restore a more ""normal"" radio-resistant phenotype by well established procedures of DNA library gene transfection. For acute high dose-rate exposures, the difference in killing for hypersensitive and normal cells is unlikely to be great enough for an efficient selection to isolate the gene-corrected cells. We will therefore employ a low dose-rate (LDR) strategy we previously used to isolate a radiosensitive CHO mutant to map and identify the gene and the mutation involved. We will then isolate DNA from partially or fully corrected human clones by selective PCR amplification of the unique primer-flanked sequence from the human library, then sequence and compare the DNA with sequences of known or unknown function. Radiation hypersensitivity for cell killing correlates with hypersensitivity for chromosomal aberration induction in every instance where it has been examined, but we will examine this for the cells from this study with whole chromosome painting and mFISH techniques, to compare chromosomal radio-sensitivities at low doses and dose-rates. The focus here would be on stable aberrations of the general kinds known to be relevant to cancer. A few similar comparisons of low dose and low dose-rate chromosomal radio-sensitivities would be made using cells from individuals who are heterozygous or homozygous for genes known to involve ""repair defects"" for ionizing radiation such as Nijmegen Breakage Syndrome (NBS), BRCA1 and 2, or Ataxia-Telangiectasia. In some instances, genetic complementation studies with cell hybrids may be necessary.

Expected Outcomes:

Current estimates of the risks of radiation exposure to humans are based largely on the probability of an effect to the "average" individual in an irradiated population. By better identification of genetic factors underlying the control of radio-sensitivity it may be possible to tailor risk estimates to individuals based on their genotype.