No abstract

Mutant lines of Chinese hamster ovary cells that show hypersensitivity to killing and mutagenesis by UV light were analyzed by genetic complementation analysis to determine whether defects in different gene loci might underlie a common cellular phenotype. To facilitate rapid screening of mutant clones, a procedure was devised that allowed presumptive complementation to be assessed on the basis of the frequency of UV-resistant cells after fusion by polyethylene glycol. Four classes were identified among 44 clones tested. By using drug-resistance markers for selection ofhybrid cells in crosses between UV mutant and wild type, a mutant from each of the four classes was shown to behave as phenotypically recessive. Hybrids were also isolated from crosses between each ofthe pair combinations ofthe four mutants. All such hybrids were relatively resistant to UV killing, providing confirmation of the complementation classes. When mutants representing the four UV-complementation classes were tested with the polyaromatic hydrocarbon 7-bromomethylbenz(a)anthracene, complementation was again seen for all pair combinations. These results suggest that each class ofmutants represents a biochemical defect that plays a common role in the repair of both UV-induced and chemically induced lesions in the DNA. The study ofmutant cell lines having defects in enzymatic pathways that repair damaged DNA is an important approach to understanding how cells recover from genetic injury and to elucidating the mechanisms of mutagenesis. Of the human mutations that are known to involve repair deficiencies, xeroderma pigmentosum (XP) has been the most intensively studied. XP fibroblasts and lymphoblasts are defective in the excision repair that operates on damage resulting from exposure to UV and many chemical mutagens and carcinogens (1, 2). The defects in XP appear to lie at or prior to the incision step (24) and may involve factors that play a role in enzymatic recognition ofdamaged DNA within chromatin (3, 4). Seven complementation classes of XP have been reported (5)(6)(7)(8).Recently, two techniques were introduced for isolating UVsensitive mutant clones from the Chinese hamster ovary (CHO) cell line (9, 10). Assay of DNA-repair replication indicated that these mutants, like XP mutants, have low or undetectable repair synthesis in response to UV (9, 10). These UV mutants of CHO cells, as well as alkylation-sensitive-mutants (9), provide an alternative to human mutant strains for studying the role of DNA repair in genetic damage. CHO cells can be readily manipulated in a quantitative manner, and they may allow isolation of mutants having repair deficiencies not represented among human mutants. In the.-present-study, we examined 44 UV mutants having pronounced sensitivity. We show here that they are genetically recessive and can be classified into four complementation groups on the basis of their responses to either UV or the chemical mutagen 7-bromomethylbenz(a)anthracene (7-BrMeBA). These results show that different genetic defects ...

Five UV-sensitive mutant strains of CHO cells representing different genetic complementation groups were analyzed for their ability to perform the incision step of nucleotide excision repair after UV exposure. The assay utilized inhibitors of DNA synthesis to accumulate the short-lived strand breaks resulting from repair incisions. After 6 J/m2, each of the mutants showed less than 10% of the incision rate of the parental AA8 cells. After 50 J/m2, the rate in AA8 was similar to that at 6 J/m2, but the rates in the mutants were significantly higher (approximately 20% of the rate of AA8). Thus by this incision assay the mutants were phenotypically indistinguishable. Each of the mutants were hypersensitive to mutation induction at both the hprt and aprt loci by a factor of 10, and in the one strain tested ouabain resistance was induced sevenfold more efficiently than in AA8 cells. Sister chromatid exchange was also induced with sevenfold increased efficiency in the two mutant strains examined. Thus, these CHO mutants resemble xeroderma pigmentosum cells in terms of their incision defects and their hypersensitivity to DNA damage by UV.

The effects of phagocytosis and killing on microbial structural integrity have not been well defined. Recently we have presented evidence suggesting that rapid killing of Escherichia coli in vitro by intact or disrupted rabbit polymorphonuclear leukocytes occurs without major structural disorganization (1, 2). This conclusion was based on the findings that: (a) degradation of structural constituents such as lipids and peptidoglycans was limited, (b) macromolecular synthesis, including the induction of/~-galactosidase continued after more than 95 % of the E.coli could no longer divide, (c) the killed E.coli population retained its ability to "step-up" macromolecular synthesis in response to enrichment of the medium with simple nutrients (3), and (d) by contrast, under identical conditions other microorganisms, such as gram-positive Micrococcus lysodeikticus and Bacillus megaterium, underwent more rapid and extensive destruction of envelope constituents and within minutes lost biosynthetic activity (i).This report concerns further studies on the effects of granulocytes on the envelope of E.coli. The results support our contention that killing of E.coli by granulocytes is not linked to general structural disruption. However, killing by granulocytes and purified fractions prepared from granulocytes is temporally closely related to an increase in permeability. Some of the properties of the activity in disrupted granulocytes that causes this permeability change in E.coli and certain other gram-negative microorganisms will be described.

A UV-sensitive mutant line of CHO cells, UV20, was shown to be phenotypically corrected to resistance by fusion with human lymphocytes or fibroblasts. Only human chromosome 19 correlated with the DNA repair phenotype of resistant hybrid clones and their resistant or sensitive subclones. This study demonstrates the mapping of a human repair gene by direct selection of complementing hybrids in the presence of a DNA-damaging agent (mitomycin C).