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The Chinese hamster ovary (CHO) mutant strain EM9 was previously shown to be hypersensitive to killing by ethyl methanesulfonate (EMS) and methyl methanesulfonate (MMS), to have a 12-fold increased baseline incidence of sister-chromatid exchanges (SCE), and to be defective in rejoining DNA strand breaks after treatment with EMS, MMS, or X-rays. A study was performed to determine if the primary biochemical defect might be a DNA ligase. DNA-ligase activities were assayed and compared after separation of the multiple forms of ligase by AcA 34 gel-filtration chromatography of total cellular extracts. In EM9 cells the levels of the presumptive replicative forms, DNA ligase Ia (480 kd) and ligase Ib (240 kd) were about 50% and 60%, respectively, of those in the parental AA8 cells, whereas DNA ligase II (80 kd) was unaltered in EM9 . In a phenotypic revertant line ( 9R1 ) ligases Ia, Ib and II levels were 35%, 37% and 100%, respectively, of those in AA8 . The reduced levels of ligases Ia and Ib in EM9 and 9R1 cells are apparently not related directly to the mutant phenotype and may be attributable to the somewhat slower growth rates of these strains compared with those of AA8 . To determine if the repair defect in EM9 might reside in the ability to induce DNA-ligase activity after treatment with a DNA-damaging agent, AA8 and EM9 cells were treated with MMS at 30 micrograms/ml for 60 min before preparing fractions for ligase assays. Under these conditions the activities of ligases Ia and Ib decreases 70-80% in both cell lines, but ligase II increased 2.0- and 2.6-fold, respectively, in AA8 and EM9 . As a further test of defective ligase activities in EM9 , assays were performed in the presence of 0.1 M NaCl or after heating the fractions for 10 min at 50 degrees C. Although all 3 forms of ligase showed altered activity under both of these conditions, there were no significant differences between EM9 and AA8 cells. These data combined with the above results provide strong evidence that the site of the primary defect in EM9 is not in either of the DNA ligases .

Two mutant lines (US31, US46) of mouse lymphoma cells that are hypersensitive to ultraviolet (UV) radiation were previously found to belong to different complementation groups. The mutants were tested for their ability to complement the six known complementation groups of UV-sensitive Chinese hamster ovary (CHO) cells, which are defective in nucleotide excision repair, as well as a seventh group represented by a V79 mutant. Hybrid cells were produced by fusion with polyethylene glycol and tested in situ for UV resistance. The mouse mutant US46 complemented all CHO mutants except UV61. Therefore, US46 is assigned to the same complementation group as UV61, and it is probably defective in the same locus. The mouse mutant US31 produced UV-resistant hybrid cells in each of the seven crosses, indicating that it forms an eighth complementation group among the rodent mutants. Thus, at least eight genes are likely required to repair UV damage in rodent cells.

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 less than 50 kb 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.

Procedures are described for the isolation of conditional lethal mutants of mouse L-60T cells. The mutant lines were temperature sensitive by the following criteria: (a) colony-forming ability, (b) growth in suspension culture, and (c) rate of uptake of tritiated-thymidine.

A cell mutant of the Chinese hamster ovary line, which is temperature sensitive for protein synthesis, is specifically defective in vivo in its ability to charge tRNA with leucine. Cytoplasmic extracts exhibited temperature-sensitive leucyl-tRNA synthetase activity. It is, therefore, highly likely that the mutant has a structural alteration in leucyl-tRNA synthetase. The low leakiness and low reversion rate of this mutant, combined with the specificity of the defect in its protein-synthesizing machinery, make it an appealing tool for investigating regulatory mechanisms in animal cells.