The long term goal of this project is to find out which of the gamma-ray induced lesions in DNA are responsible for the biological effects; lethality, mutagenesis and chromosomal rearrangements.
The specific aim of this proposal is to determine which role each kind of gamma-ray induced DNA-damage plays in causing lethality, mutagenesis and recombination in an E. coli- plasmid DNA system. The plasmid DNA will be irradiated in vitro under controlled conditions so that the amounts and ratios of the kinds of damage can be changed. The amounts of damage will be determined by measuring the conversion of the form I to forms II and III DNA under neutral conditions and after alkaline hydrolysis. The biological effects will be measured by transfecting the irradiated plasmid and the separated DNA forms into calcium treated E. coli host cells. The irradiated plasmid will be separated into forms I, II and III by agarose gel electrophoresis and sucrose gradient sedimentation. Lethality will be determined from the loss of tetracycline or ampicillin resistant colonies depending on which plasmid is used. Mutagenesis will be measured as forward mutations of the Lac gene in plasmid PBRPI which contains the E. coli Lac operon cloned into PBR322. The host cells in this case will be delta lac. Recombination will be measured by counting the number of Lac-colonies produced when this plasmid is transfected into an E. coli host containing a mutant (Lac Y1) allele. The recombinations will be confirmed by plasmid isolation and restriction enzyme analysis to locate the Pst I and Pvu II sites associated with the Lac Y1 mutation. The structure of the Lac genes in E. coli host DNA will be checked by Southern blotting of restriction fragments separated on agarose gels. Using particular combinations of Lac+ and Lac- hosts with plasmids pBRM4 and pBRM5, which contain subcloned segments of the Lac Y gene, the number of reciprocal and non-reciprocal recombinations from host-to-plasmid and plasmid-to-host will be determined. In the long term this system will be used to measure and/or predict the biological effects of radio-protective or sensitizing agents which may be used in radiation therapy or for protection of populations likely to be exposed to gamma-rays or other highly ionizing radiation.
| Mudgett, J S; Buckholt, M; Taylor, W D (1991) Ultraviolet light-induced plasmid-chromosome recombination in Escherichia coli: the role of recB and recF. Gene 97:131-6 |
| Mudgett, J S; Taylor, W D (1990) Recombination between irradiated shuttle vector DNA and chromosomal DNA in African green monkey kidney cells. Mol Cell Biol 10:37-46 |
| Mudgett, J S; Manzella, J M; Taylor, W D (1990) Homologous recombination and mutagenesis of gamma-irradiated plasmid DNA in Escherichia coli host cells. Radiat Res 124:57-61 |
