Isolation of Uv-Sensitive and Temperature Sensitive Conditionally Lethal Mutants and Adaptation of the Chromosome Mediated Gene Transfer System in a Haploid Cell Line Derived from the Frog Rana Pipiens

摘要

This study was carried out to examine the DNA repair pathways present in animal cells through the isolation of mutant cell lines sensitive to uv light. In addition, the chromosome mediated gene transfer system was investigated as to its mechanism and possible relationship with a pathway involved in DNA repair. An enrichment scheme was also developed for the isolation of temperature--sensitive conditionally--lethal mutants. Five clones were isolated that expressed greater sensitivity to uv light as compared to the parental cell line. One of these clones proved to be more sensitive when tested at 27.5 exp 0 C than when tested at 20 exp 0 C and thus displayed a temperature--sensitive phenotype. Three of these clones were deficient in their ability to carry out repair replication while the two other clones also exhibited a defect most likely associated with DNA synthesis. No alteration in postreplication repair or mutagenesis was observed in these uv-sensitive cell lines. The chromosome mediated gene transfer system was adapted to the ICR 2A cell line and transfer of the TT exp + , TK exp + , and oua/sup res/ phenotypes was carried out. The notation TT exp + indicates the presence of a thymidine--specific transport system, TK exp + denotes production of the deoxypyrimidine kinase, and oua/sup res/ indicates an ability to grow in medium containing 5 x 10 exp -5 M ouabain. It appeared through these studies that the integration of the transferred piece of genetic material was not restricted to the homologous site of the particular gene in the recipient cell but may have occurred at several locations. It is suggested that the enzyme systems altered in the uv-sensitive clones or involved in sister chromatid exchange production are not associated with the chromosome transfer or integration process. The enrichment scheme used to isolate uv-sensitive mutants was modified for use in the isolation of a temperature-sensitive conditionally lethal mutant. (ERA citation 04:014610)