Characterization of revertants of the CHO EM9 mutant arising during DNA transfection

L. R. Barrows, M. B. Paxton, K. A. Kennedy, L. H. Thompson

Research output: Contribution to journalArticle

26 Citations (Scopus)

Abstract

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.

Original languageEnglish (US)
Pages (from-to)805-811
Number of pages7
JournalCarcinogenesis
Volume12
Issue number5
StatePublished - May 1991
Externally publishedYes

Fingerprint

Mutant
Transfection
DNA
Cells
Cell Line
Cell
Genes
Line
Repair
X rays
X-Rays
Cricetinae
Gene
DNA Repair-Deficiency Disorders
Messenger RNA
Single-Stranded DNA Breaks
Ionizing radiation
Methylnitronitrosoguanidine
Cosmids
CHO Cells

ASJC Scopus subject areas

  • Cancer Research
  • Statistics, Probability and Uncertainty
  • Applied Mathematics
  • Physiology (medical)
  • Physiology
  • Behavioral Neuroscience

Cite this

Barrows, L. R., Paxton, M. B., Kennedy, K. A., & Thompson, L. H. (1991). Characterization of revertants of the CHO EM9 mutant arising during DNA transfection. Carcinogenesis, 12(5), 805-811.

Characterization of revertants of the CHO EM9 mutant arising during DNA transfection. / Barrows, L. R.; Paxton, M. B.; Kennedy, K. A.; Thompson, L. H.

In: Carcinogenesis, Vol. 12, No. 5, 05.1991, p. 805-811.

Research output: Contribution to journalArticle

Barrows, LR, Paxton, MB, Kennedy, KA & Thompson, LH 1991, 'Characterization of revertants of the CHO EM9 mutant arising during DNA transfection', Carcinogenesis, vol. 12, no. 5, pp. 805-811.
Barrows LR, Paxton MB, Kennedy KA, Thompson LH. Characterization of revertants of the CHO EM9 mutant arising during DNA transfection. Carcinogenesis. 1991 May;12(5):805-811.
Barrows, L. R. ; Paxton, M. B. ; Kennedy, K. A. ; Thompson, L. H. / Characterization of revertants of the CHO EM9 mutant arising during DNA transfection. In: Carcinogenesis. 1991 ; Vol. 12, No. 5. pp. 805-811.
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