We previously characterized a clone of CHO cells, clone B, that displayed tolerance to the cytotoxic effects of N-methylnitrosourea (MNU) and 6-thioguanine (6-TG). To determine whether this phenotype affected the mutagenic response of the cells, MNU-induced mutation to 8-azaadenine resistance (8-AAr) was measured in the parental and clone B cells. Comparable mutation frequencies were found in the two cell lines up to 0.5 mM MNU, while at higher MNU concentrations mutations could be reproducibly measured only in clone B cells. Similar amounts of DNA methylated bases were found in the two cell lines after a 30 min treatment with different concentrations of [3H]MNU and the same linear relationship was observed when mutation induction by MNU was plotted as a function of the amount of O6-methylguanine (O6-MeGua) in DNA, indicating that mutation induction in both cell lines was related to the presence of this methylated base. Fifteen MNU-induced 8-AAr mutants were isolated from each cell line and the sequences of the adenine phosphoribosyltransferase (aprt) mutations determined. The type (in 90% of the cases, GC to AT transitions), the sequence context and the strand localization of the mutations indicated that all mutations were targeted at O6-MeGua in DNA and no difference was found between the two lines. These results are consistent with a mechanism of tolerance of O6-MeGua that does not alter the processing of this methylated base into a mutation. Growth in 6-TG induced point mutations in clone B but not in the parental cells. A model is proposed in which the alkylation tolerant variant is altered in a mismatch correction pathway responsible for the cytotoxicity of the methylated base.
The distribution of six genetic loci analyzed by PCR using the commercial AmpliType® PM (PolyMarker) kit (Perkin Elmer, Norwalk, CT) was evaluated in 200 unrelated Italian individuals. The examined loci included: Group-specific component (Gc) (1), D7S8 (2), hemoglobin G gammaglobin (HBGG) (3), glycophorin A (GYPA) (4), low density lipoprotein receptor (LDLR) (5), and HLA DQ-alpha (6). The AmpliType PM Kit analysis is based on the reverse dot blot format and the results are interpreted by reading the pattern of blue dots which determine the alleles present at each locus. The population data collected allow the implementation of AmpliType PM into routine casework.
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