5-Azacytidine inhibits the induction of transient TK-deficient cells by 5-bromodeoxyuridine. A novel hypothesis for the facilitation of hypermethylation by 5-bromodeoxyuridine.

This paper examines the mechanism by which 5-bromodeoxyuridine (BrdUrd) induces a high frequency of transient trifluorothymidine (F3TdR)-resistant variants in the TK6 human lymphoblast cell line (a TK +/- heterozygote). This phenomenon has previously been termed 'pseudomutation' (Liber et al., 1985). We now report that 5-azacytidine (5-AzaC), an inhibitor of DNA methylation, reverses BrdUrd-induced pseudomutation in a dose-dependent manner. The inhibition by 5-AzaC is highly specific and does not appear to involve nucleotide pool perturbations. 5-AzaC inhibits the pseudomutagenic effect (transient trifluorothymidine resistance in a thymidine kinase heterozygote), but not the stable mutagenic effect (stable 6-thioguanine resistance or trifluorothymidine resistance in a hypoxanthine-guanine phosphoribosyltransferase-proficient cell) induced by BrdUrd. 5-AzaC did not affect the induction nor expression of mutation induced by several other chemical mutagens at either the tk or hgprt loci. Inhibition of pseudomutation by 5-AzaC did not appear to be caused by a number of potential confounding factors. Although significant changes in the levels of DNA methylation were detected by HPLC analysis in BrdUrd-treated cells, the dose response for inhibition of pseudomutation by 5-AzaC was correlated with a significant decrease in 5-methylcytidine levels. These results and additional data in the literature have led us to postulate a novel mechanism in which the substitution of BrdUrd in a TpG dinucleotide(s) may serve as a substrate for non-heritable methylation and hence transiently inactivate tk gene expression.     

Deletion mutagenesis during polymerase chain reaction: dependence on DNA polymerase

Polymerase chain reaction (PCR) was performed with two polymerases. Thermus aquaticus DNA polymerase (Taq), and modified T7 DNA polymerase (Sequenase). Both polymerases were used to amplify the same portion of the human 18S rRNA gene. We report a PCR artifact, namely a deletion of 54 bp, when Taq polymerase was used to amplify a portion of the human 18S rRNA gene. PCR performed with Sequenase did not produce this artifact. The deletion eliminated a potentially stable hairpin loop. Our data are consistent with the following model for generation of the deletion: (i) the formation of an intrastrand hairpin, and (ii) polymerization across the base of the hairpin, thus deleting the nucleotide sequence in the hairpin. Furthermore, we show that the deletion occurs mainly during synthesis of the (-)DNA strand. Our observations suggest that similar artifacts may occur in other sequences containing stable secondary structures.     

Assay for gene mutation in a human lymphoblast line, AHH-1, competent for xenobiotic metabolism

A novel quantitative gene-locus mutation assay has been developed using a line of human lymphoblast cells, designated AHH-1, competent in oxidative xenobiotic metabolism. AHH-1 cells are sensitive to the mutagenic action of both chemically reactive mutagens and mutagens which require oxidative metabolism to exert their mutagenicity. These cells are readily mutated by direct exposure to ethyl methanesulfonate, ICR-191, 2-acetoaminofluorene, aflatoxin B1, benzo[a]pyrene (BP), cyclopenta[c, d]pyrene, dimethylnitrosamine, lasiocarpine, and 1-methylphenanthrene.     

Comparison of toxicity and mutagenicity of butyl methanesulfonate among human lymphoblast lines.

The toxic and mutagenic effects of butyl methanesulfonate (BMS) were compared among four diploid human lymphoblast lines, MIT-2, WI-L2, MGL8B-2 and GM 130. The toxic and mutagenic effects of 24-h exposure to BMS were similar for the MIT-2, WI-L2 and MGL8B-2 lines, while the GM 130 line was strikingly resistant to the toxic and mutagenic effects of BMS.     

Cell-by-cell scanning of whole mitochondrial genomes in aged human heart reveals a significant fraction of myocytes with clonally expanded deletions

Quantitative information on the cell-to-cell distribution of all possible mitochondrial DNA (mtDNA) mutations in young and aged tissues is needed to assess the relevance of these mutations to the aging process. In the present study, we used PCR amplification of full-length mitochondrial genomes from single cells to scan human cardiomyocytes for all possible large deletions in mtDNA. Analysis of more than 350 individual cells that were derived from three middle-aged and four centenarian donors demonstrates that while most of the cells contain no deletions, in certain cardiomyocytes a significant portion of the mtDNA molecules carried one particular deletion. Different affected cells contained different deletions. Although similar numbers of cells were screened for each donor, these deletion-rich cells were found only in the hearts of old donors, where they occurred at a frequency of up to one in seven cells. These initial observations demonstrate the efficiency of the method and indicate that mitochondrial mutations have the potential to play an important role in human myocardial aging.     

Detection of low-frequency mutations in exon 8 of the TP53 gene by constant denaturant capillary electrophoresis (CDCE).

We extended our development of the means to measure point mutations at the DNA level to the problem of detecting TP53 gene variants in the area around tumors where mutant fractions are expected to be as low as one mutant per 1000 wild-type (WT) sequences. We met this criterion by using the following methods. The TP53 exon 8 sequence was amplified from genomic DNA samples under conditions of high polymerase fidelity using a fluorescein-labeled primer. This mixture of WT and mutant sequences was converted to heteroduplexes of mutant and WT sequences by melting and re-annealing. The mixture was resolved by capillary gel electrophoresis under appropriate constant denaturing conditions. Using laser-induced fluorescence, we found that fractions as low as 1/1000 could be detected without any prior enrichment for mutant sequences, and that the melting properties of the amplified DNA will leave "fingerprints" in the electropherogram that can be used to identify the specific mutation. This method is fast and robust and should be applicable to clinical analyses in which rapid scanning for any mutation in an exonic sequence is important.     

Constant denaturant capillary electrophoresis (CDCE): a high resolution approach to mutational analysis.

Using a zone of constant temperature and denaturant concentration in capillary electrophoresis, we have devised a simple, rapid, and reproducible system for separating mutant from wild type DNA sequences with high resolution. Important to the success of this method, which we call Constant Denaturant Capillary Electrophoresis (CDCE), has been the use of linear polyacrylamide at viscosity levels that permit facile replacement of the matrix after each run. For a typical 100 bp fragment, point mutation-containing heteroduplexes are separated from wild type homoduplexes in less than 30 minutes. Using laser-induced fluorescence to detect fluorescent-tagged DNA, the system has an absolute limit of detection of 3 x 10(4) molecules with a linear dynamic range of six orders of magnitude. The relative limit of detection at present is 3 x 10(-4), i.e. 10(5) mutant sequences are recognized among 3 x 10(8) wild type sequences. The new approach should be applicable to the identification of low frequency mutations, to mutational spectrometry and to genetic screening of pooled samples for detection of rare variants.     

Contents Volume 93 (1982)

A thymidine kinase heterozygote was isolated from a diploid human lymphoblast line which forms colonies with high efficiency in microtiter dishes. We show that this cell line, called TK6, can be mutated from a tK^+/? to TK^?/? state by diverse mutagens, including ethyl methanesulfonate, butyl methanesulfonate, nitrosomethylurea, UV light, ICR-191, 4-nitroquinoline oxide, fluorodeoxyuridine, benzo[a]pyrene and aflatoxin B₁. We report here the experiments required to demonstrate the applicability of this new line in quantitative assays of mutation in human cells.Mitotic recombination between the centromere and the tk locus could not be induced by either dimethylsulfoxide or phorbol-12-myristate-13-acetate.