Characterization of revertants of the CHO EM9 mutant arising during DNA transfection.
We have studied reversion in DNA repair deficient EM9 cells, by selection for ethylmethanesulfonate (EMS) resistance. EM9 is a mutant CHO cell line that is hypersensitive to killing by EMS and X-rays and deficient in DNA single-strand break (SSB) repair. EM9 cells were transfected with DNA from a cosmid library, and transfectants resistant to EMS were isolated. Four revertant lines were obtained, which varied in their sensitivity to killing by EMS, ionizing radiation and other genotoxic agents. When the cell lines were analyzed for resistance to killing by chlorodeoxyuridine (CldUrd) or N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), a different relative ranking among the cell lines was observed. The recently cloned human XRCC1 gene is capable of correcting the deficiencies of the EM9 cell line. Using the human XRCC1 cDNA (pXR1-30) as a probe, we determined that the resistant-transfectant cell lines contained only the endogenous hamster XRCC1 gene, implying that a hamster XRCC1 gene was altered during the transfection/selection procedure and was responsible for the EMS resistance. In these cells the levels of XRCC1 mRNA corresponded roughly to the degrees of resistance of the reverted cell lines to killing by EMS or X-rays. The degree of increased resistance to killing by EMS or X-rays also roughly correlated with increased SSB repair. These results suggest that increased cellular levels of the endogenous XRCC1 gene mRNA may largely, though not completely, explain the phenotypes of revertant, EMS-resistant EM9 cell lines.
Barrows
L R
LR
University of Utah, Department of Pharmacology and Toxicology, Salt Lake City, UT 84112.
Paxton
M B
MB
Kennedy
K A
KA
Thompson
L H
LH
D000818:Animals; D015152:Blotting, Northern; D015139:Blotting, Southern; D002273:Carcinogens; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects / Q000528:radiation effects; D006224:Cricetinae; D003412:Cricetulus; D004247:DNA / Q000235:genetics*; D015342:DNA Probes; D004260:DNA Repair; D005786:Gene Expression Regulation; D009154:Mutation; D012333:RNA, Messenger / Q000032:analysis; D014162:Transfection
D002273:Carcinogens; D015342:DNA Probes; D012333:RNA, Messenger; D004247:DNA