| dc.description.abstract | Radiation sensitivity can be caused by many factors. A mutant, rad-4, of the nematode Caenorhabditis elegans was isolated in 1982 that possesses interesting properties in addition to conferring sensitivity to UV radiation.The purpose of this study was to use observable recombinational events to more precisely locate the mutation on the genetic map. Once the mutation is located candidate genes can then be sequenced in order to molecularly identify rad-4. First, kill curves were performed (comparing rad-4 to the wild-type strain N2) to confirm the radiation hypersensitivity of rad-4.Most experiments employed two-hour embryos; however, the sensitivity of rad-4 was also testedat later stages in development, four and twenty-four hours. There was no increased sensitivity observed in rad-4 in later stages in development. Once the dosage of a one second was shown to be sufficient to distinguish rad-4 from rad-4+ in two hour embryos, the mapping experiments began.Three factor crosses were employed in which rad-4 was position relative to two other mutations that conferred visible phenotypes.The results from the original study were first confirmed by the dpy-11 unc-60/ rad-4 cross. The next cross performed necessitated a creation of a new double homozygote, lin-40 unc-60. A triple heterozygote (rad-4/lin-40 unc-60) was then generated and Lin, Non-Unc recombinants were selected.One third of these recombinants observed included rad-4. This allowed the possible genes to be narrowed to a much smaller range centered around -11.75 map units on the fifth chromosome. Finally, WormBase was used to locate the possible genes and NIH pBLAST analysis was employed to determine possible functions for genes on the list with unknown functions. The list was then limited to seven sequences, six of them having high homology suggesting that they were the same gene with different splice sites. Several mutant strains of each gene were ordered and will be tested for radiation sensitivity. After this confirmation rad-4 will be able to be sequenced and studied further. | |
