Date of Award
1994
Degree Type
Dissertation
Degree Name
Doctor of Philosophy
Abstract
Nucleotide excision repair of DNA, demonstrated by the removal of cyclobutane pyrimidine dimers (CPDs), proceeds much more rapidly in genes undergoing transcription than in inactive genes and this rapid repair is confined to the strand being transcribed. The phenomenon is referred to as transcription-repair coupling and it has been demonstrated to occur in the chromosomes of mammalian cells, yeast cells and Escherichia coli. It was of interest to examine the repair characteristics of plasmids both in E. coli and Chinese hamster (CHO) cells.;Some of the findings obtained with the E. coli system were the following: The Rec A function is necessary for the efficient repair of pGA293 in E. coli. Unexpectedly, both strands of the 3.5 Kbp and 2.4 Kbp domains of pGA293 were repaired with the same kinetics. An examination of the sequences of the designated non-transcribed strands revealed the presence of potential promoter sequences which left open the possibility that these strands were in fact being transcribed. The rates of repair of both strands of the 3.5 Kbp and 2.4 Kbp domains were slower than that reported for the transcribed strand of the E. coli chromosomal lacZ gene. However, there were 10 copies of the pGA293 genes but only 1 copy of the chromosomal gene, and this along with the greater number of CPDs in the plasmid DNA, could account for the slower rate of repair. These considerations made it likely that for both strands in the 3.5 Kbp and 2.4 Kbp domains transcription repair coupling was being observed. For the 3.0 Kbp domain, the repair rate for the non-transcribed strand was slower than that for the transcribed strand and was the same as the repair rate reported for the non-transcribed strand of the E. coli chromosomal lacZ gene. This suggests that the transcribed strand of the 3.0 Kbp domain was undergoing coupled repair while the other strand was not.;Using the CHO system, the following information was obtained: Expression of {dollar}\beta{dollar}-galactosidase from indicated pGA293 recovered readily after transformation, indicating that repair had taken place. However, it was not possible to demonstrate removal of CPDs from the 3.0 Kbp domain of pGA293 which contains the transcribed {dollar}\beta{dollar}-galactosidase gene. (Abstract shortened by UMI.)
Recommended Citation
Baig, Masood-ur-rehman, "Repair Of Uv-irradiated Plasmids In Escherichia Coli And Chinese Hamster Cells" (1994). Digitized Theses. 2355.
https://ir.lib.uwo.ca/digitizedtheses/2355