Repair-deficient mutants of Chinese hamster ovary (CHO) cells are being used to identify human genes that correct the repair defects and to study mechanisms of DNA repair and mutagenesis. Five independent tertiary DNA transformants were obtained from the EM9 mutant, which is noted for its very high sister-chromatid exchange frequencies. In these clones a human DNA sequence was identified that correlated with the resistance of the cells to chlorodeoxyuridine (CldUrd). After EcoRI digestion, Southern transfer, and hybridization of transformant DNAs with the BLUR-8 Alu family sequence, a common fragment of 25–30 kilobases (kb) was present. Since the DNA molecules used to produce these transformants were sheared to <50kb in size, the correcting gene should be small enough to clone in a cosmid vector.
Using drug-resistance markers to select for hybrids after fusion, we have done complementation experiments with ultraviolet light (u.v.)-sensitive mutants and have identified a sixth complementation group, line UV61. Additionally, CHO mutants UV27-1 and MMC-2, isolated in other laboratories, were found to belong to UV group 3, which is represented by line UV24.
To study the behaviour of transfected DNA molecules in repair-deficient cells, we treated plasmid pSV2gpt with either u.v. radiation or cis-diamminedichloroplatinum(II) (cis-DDP) and introduced the damaged DNA into normal CHO cells (AA8) and mutants UV4 and UV5. Unrepaired damage to the plasmid was indicated by loss of colony-forming ability of the transfected cells in selective medium containing mycophenolic acid. With u.v. damage, the differential survival of the cell lines was similar to that seen when whole cells are treated with u.v. However, with cis-DDP damage, mutant UV4 did not exhibit the extreme hypersensitivity (50-fold) that occurs when cells are treated. This result suggests that UV4 cells may be able to repair cross-links in transfected DNA.
- © The Company of Biologists Limited 1987