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W G Thilly

Publications and source records attributed to W G Thilly.

At least 73 records · Page 4Linked to original sources
PubMed1993

Analysis of large X-ray-induced mutant populations by denaturing gradient gel electrophoresis.

To increase the precision by which predominant point mutations can be observed, hypoxanthine guanine phosphoribosyl transferase (HPRT)-deficient mutants selected en masse from large X-irradiated cultures of human lymphoblastoid cells (line TK6) were analyzed by denaturing gradient gel electrophoresis (DGGE). Four independent experiments yielded approximately 7 x 10(3) and 3.2 x 10(3) initial surviving 6-thioguanine-resistant (6-TGr) mutants in X-ray-treated and untreated cultures, respectively. The hprt exon 3 fragments were amplified from DNA extracted from these mixed 6-TGr cell populations by employing the polymerase chain reaction using modified T7 DNA polymerase. DGGE was used to separate the mutant sequences from the wild-type as mutant/wild-type heteroduplexes. The X-irradiated populations contained several mutant bands in the 104-bp low-melting region of exon 3 that were not observed in the untreated cultures. Two exon 3 specific mutations were observed in more than one treated culture and various tests for potential biases suggested that these were radiation-specific mutational hotspots. These two recurring mutations were specific 1-bp deletions in either a run of four T:A's (bp 294-297) or a run of 3 A:T's (bp 247-249). Several other "sporadic" signals observed in X-irradiated cultures were caused by small deletions ranging from 2 to 25 bp in length.

Base Sequence↗
PubMed1992

Mutational spectra in human B-cells. Spontaneous, oxygen and hydrogen peroxide-induced mutations at the hprt gene.

To test the hypothesis that reactive species in the oxygen cascade are responsible for spontaneous mutation, we examined the spectra of oxygen and hydrogen peroxide-induced mutations at the hprt locus in a human B-lymphoblastoid cell line. We compared these spectra with the spontaneous mutational spectrum. Large gene alterations were studied by Southern analysis of individual TGR clones. A combination of high fidelity polymerase chain reaction, denaturing gradient gel electrophoresis and direct DNA sequencing were used to detect and identify point mutations in exon 3 of hprt. With regard to spontaneous mutations, a previous study showed that 39% of the spontaneous TGR clones had large gene alterations. In the present study, the analysis of spontaneous point mutations within exon 3 revealed two hotspots. A one base-pair deletion (-A) at base-pair 256 or 257 and a two base-pair deletion (-GG) at base-pair 237 and 238, were detected in triplicate cultures. Each of the hotspots comprised about 1% of the TGR mutants. The analysis of individual oxygen-induced TGR clones (48 h, 910 microM-O2) showed 43% had large gene alterations similar to the spontaneous TGR clones. However, none of the spontaneous point mutation hotspots was found among triplicate oxygen-treated cultures. Two point mutations in common with H2O2-treated cultures were found in one of the three oxygen-treated cultures. Hydrogen peroxide-induced mutations (1 h, 20 microM) also differed from spontaneous mutations. Only 24% of the hydrogen peroxide-induced TGR clones had large gene alterations. The analysis of point mutations showed three hotspots within exon 3 of hprt. An AT to TA transversion at base-pair 259 had an average frequency of 3% of all TGR mutants (present in all of 3 H2O2-treated cultures). Two GC to CG transversions at base-pairs 243 and 202 were present at a frequency of 0.6% and 0.4%, respectively. A five base-pair deletion (base-pair 274 to 278) was present at an average frequency of 0.3%. The latter three mutations were detected in two of three H2O2-treated cultures. Thus, the point mutation spectra of both oxygen and hydrogen peroxide were significantly different from the spontaneous spectrum. The oxygen and hydrogen peroxide-induced spectra shared some features, suggesting that oxygen and hydrogen peroxide share some but not all pathways for induction of mutations within the DNA sequence studied here.(ABSTRACT TRUNCATED AT 400 WORDS)

B-Lymphocytes↗
PubMed1992

Mutational spectrometry: a general approach for hot-spot point mutations in selectable genes.

To observe point mutational spectra with a high degree of precision, independent large cultures of human lymphoblastoid cells were treated with a mutagen, benzo[a]pyrene diol epoxide, and mutants at the HPRT gene were selected en masse by 6-thioguanine resistance. An average of 1.6 x 10(4) 6-thioguanine-resistant mutants were created per experiment and the kinds, positions, and numbers of the most frequent mutations were examined in exon 3 of the HPRT gene by using a high-fidelity polymerase chain reaction and denaturing gradient gel electrophoresis. Sixteen exon 3-specific mutations were found to be predominantly G----T transversions and corresponded to an average of 3500 induced mutants per experiment. Of these mutations, 6 occurred within a run of 6 guanines and 5 occurred in the sequence 5'-GAAGAG-3'. The variation among independent experiments is consistent with the numerical expectation that all 16 mutations fulfill reasonable statistical criteria for mutational hot spots. The agreement with data from various systems using clone-by-clone analysis shows that the protocol reported herein can be a useful tool to study hot-spot point mutational spectra for DNA sequences for which phenotypic selection systems exist.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗
PubMed1992

Mismatch amplification mutation assay (MAMA): application to the c-H-ras gene.

We have found that under appropriate conditions, an allele-specific polymerase chain reaction (PCR) can achieve a sensitivity suitable for measuring specific, infrequent mutations in single cell systems or in animal tissues. Using the 12th codon GC-to-AT mutation in the rat c-Ha-ras gene as a model system, we have defined conditions that allow for measurement of mutations present at frequencies as low as one in 10(5) gene copies. Our approach involved the use of PCR primers that created a single mismatch with the mutated allele (GAA) but created a double mismatch with the wild-type allele (GGA). Five out of the six such double-mismatch primers we tested permitted amplification of the mutant allele (GAA) with a high degree of specificity. The specificity of the assay was further enhanced by using a two-step PCR cycle consisting of a denaturation step (1 min incubation at 94 degrees C) and an annealing/extension step (1 min incubation at 50 degrees C) in the presence of 10% (vol/vol) glycerol. Reconstruction experiments using genomic DNA demonstrate that this procedure cna measure the presence of 30 copies of the transforming ras allele present amongst 3 x 10(6) copies of the wild-type allele.

Alleles↗
PubMed1992

Determination of point mutational spectra of benzo[a]pyrene-diol epoxide in human cells.

The primary goal of our research consists of developing means sufficiently sensitive to allow assessment of human exposure to environmental carcinogens. We describe here a new approach for analyzing point mutational spectra and a test for its validity and precision using cultured human cells exposed to high doses of environmental carcinogens. The approach in its present form includes a) treatment of independent large cultures of human cells with a carcinogen, b) selection of mutant cells en masse by 6-thioguanine resistance, c) amplification of sequences of interest directly from 6TGR cells using high-fidelity polymerase chain reaction, and d) separation of mutant sequences from nonmutant sequences using denaturing gradient gel electrophoresis. We report use of this protocol to observe induced mutational spectra in exon 3 of the hprt gene in cultured human cells by benzo[a]pyrene-diol epoxide (BPDE), an active form of the widely distributed environmental carcinogen benzo[a]pyrene. BPDE induced predominantly G to T transversions within this target sequence. The variation of the frequency of the mutations among independent cultures is consistent with the interpretation that each of them corresponds to a hotspot.

Benzopyrenes↗
PubMed1991

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.

Base Sequence↗
PubMed1991

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.

Azacitidine↗
PubMed1991

Analysis of point mutations induced by ultraviolet light in human cells.

Mutations induced in cultured human cells by 254-nm UV light were analyzed within exon 3 of the hypoxanthine guanine phosphoribosyl transferase (HPRT) gene. Five large independent cultures of human lymphoblastoid cells, line TK6, were exposed to 4 J/m2 of 254-nm UV light and mutants at the HPRT locus were selected en masse by 6-thioguanine (6TG) resistance. Exon 3 of the HPRT gene was amplified from the mutant cells by polymerase chain reaction (PCR) using modified T7 DNA polymerase. Denaturing gradient gel electrophoresis (DGGE) was used to separate the mutant sequences from the wild type as mutant/wild-type heteroduplexes. Individual mutant bands were isolated from the gel and the nature of the mutations was determined by direct sequencing. Eight predominant mutations were detected in the 184-bp exon 3 sequence. Of these, 3 transition, including 2 G-C to A-T and 1 A-T to G-C and 2 A-T to C-G transversions, appeared in all 5 UV-treated cultures but not in untreated cultures and were thus considered to be mutational hotspots. These observations are similar in nature to those previously reported in bacterial and rodent cells. A single G deletion, a tandem substitution of CpT for TpA, and a tandem triple substitution of GpGpA for ApApG were also observed but in only 2, 2 and 3 of the 5 UV-treated cultures, respectively. Numerical analysis of the mutant fractions of these 8 mutations indicated that each of them was distributed as a set of non-random and independent events, i.e., a mutational hotspot.

Alleles↗
PubMed1991

Long-term low-dose mutation studies in human cells: metanil yellow and orange II.

We tested the mutagenic effects of two commonly used fold colors, metanil yellow and orange II, in AHH-1 human lymphoblast cells. The cell line, which is competent for oxidative metabolism of various chemicals, was exposed to both compounds in high-dose x short-term (3 day) or high-dose x long-term (10-day) and low-dose x long-term (20-day) treatments. Concentrations of metanil yellow and orange II as low as 22 nM and 12 nM, respectively, were sufficient to induce mutation rates which were equal to twice the spontaneous mutation rate at the HPRT locus in AHH-1 cells.

Azo Compounds↗
PubMed1991

Optimization of the polymerase chain reaction with regard to fidelity: modified T7, Taq, and vent DNA polymerases.

The fidelity of DNA polymerases used in the polymerase chain reaction (PCR) can be influenced by many factors in the reaction mixture. To maximize the fidelity of DNA polymerases in the PCR, pH, concentrations of deoxynucleoside triphosphates, and magnesium ion were varied. Denaturing gradient gel electrophoresis was used to separate the polymerase-induced mutants from wild-type DNA sequences. Thermolabile modified T7 DNA polymerase, thermostable Taq, and Vent DNA polymerases were studied. Fidelity of all three DNA polymerases was sensitive to concentrations of deoxynucleoside triphosphates, magnesium ion, and pH. Within conditions that permitted efficient amplification, optimization with regard to these three factors yielded an average error rate in error/base pair incorporated of 7.2 x 10(-5) for Taq, 4.5 x 10(-5) for Vent, and 4.4 x 10(-5) for modified T7 (Sequenase) DNA polymerases.

Base Sequence↗
PubMed1991

A high-yield sampler for toxicological characterization of complex mixtures in combustion effluents.

Combustion sampling for toxicological assessment often requires that large (greater than 100 mg) lots of complex organic mixtures of wide volatility range be rapidly recovered from high temperature gases without contamination. A new sampler, meeting these criteria for studies of public health interest, has been developed and demonstrated. The device provides high sampling rates and intimate contacting of the samples stream with large volumes of a well-cooled, liquid solvent, dichloromethane (DCM). This promotes rapid organics dissolution from carrier gas and particulates and prompt dilution and quenching of the resulting solution, resulting in high organics collection efficiencies with minimal DCM losses. Solvent separation then remits large quantities of concentrated organics for chemical analysis and toxicological testing. One- to seven-hour interrogations of in-flame, post-flame, and flue gas regions gave 50- to 250-mg yields of complex organic mixtures. In side-by-side sampling of combustion exhaust, the DCM sampler provided higher yields of DCM solubles (identified with complex organic mixtures) and of S. typhimuirim mutagens (active without exogenous metabolizing agents) than did a filter/polymeric sorbent bed sampling train. The new sampler also collects polar and high volatile hydrocarbons such as benzaheyde, pentadiyne, m- and p-diethynyl-benzene, and 1-hexen-3,5-diyne. Nitration of naphthalene and pyrene in DCM solution (1 mg/mL each) was less than 1 part in 10(7) after a 345-min exposure to a bubbling flow of moist N2/air mixture (1:1 v/v) containing 107 ppm NO and 1.5 ppm NO2, indicating that for these condition a DCM sampler should resist artifactual nitration of aromatics. However, because of the very high bacterial mutagenicity of some nitroaromatics and the wide range of sampling conditions of environmental interest, nitration and all artifacts must still be scrutinized when using the DCM sampler. The DCM sampler is expected to contribute to public health impact assessments by facilitating detailed determinations of the identities, compositions, concentrations, sources, formation mechanisms, and biological activity of environmental toxicants in gaseous atmospheres.

Atmospheric Pressure↗
PubMed1991

Assay of mutation induced in human lymphoblastoid cells by combustion-generated soot particles.

A human lymphoblastoid cell line has been used to test for mutations caused by combustion-generated soot particles and their constituent components, which are substrate carbon-black and adsorbed condensate, principally in the form of polycyclic aromatic hydrocarbons (PAH). It was found that the mutagenicity of the PAH fraction is higher when it is contacted with cells as a liquid extract than when it is supplied as a coating on soot particles. The substrate particles were found to be nonmutagenic. The rate of transfer of mutagens from the surface of particles, combined with the retention time of respirable aerosol particles, are deemed to define their mutagenic potential.

Adsorption↗
PubMed1990

Molecular analysis of complex human cell populations: mutational spectra of MNNG and ICR-191.

We describe a method to identify and enumerate mutants at the nucleotide level in complex cell populations. Several thousand different mutants were induced at the HPRT locus in human lymphoblastoid cultures by either MNNG, an alkylating agent, or by ICR-191, a substituted acridine. HPRT mutants were selected en masse by resistance to 6-thioguanine. The most frequent mutations (hotspots) in HPRT exon 3 were determined by a combination of denaturing gradient gel electrophoresis and polymerase chain reaction. MNNG predominantly produced GC----AT transitions at nucleotides in a GGGGGG sequence, while ICR-191 produced both +1 frameshifts in the same GGGGGG sequence and +1 frameshifts in a CCC sequence.

Aminacrine↗
PubMed1990

Mutational spectrometry in animal toxicity testing.

The microbial geneticists who pioneered the study of mutational spectra have shown us how rich a source of mutagen-specific information it can be for the pharmacologist and toxicologist. Contributions by physical chemists and molecular biologists have led to a practical means to observe mutation spectra directly from the DNA of cultured human cells and this review suggests that the same methodology may be applied successfully to the study of mutations and mutational spectra in tissues of experimental animals and humans. In toxicological testing, the new field of mutational spectrometry offers a previously unattainable level of sensitivity and specificity as well as new tools for dissecting mechanisms of pharmacologic and toxic action.

Animals↗
PubMed1989

Long-term, low-dose benzo[a]pyrene-induced mutation in human lymphoblasts competent in xenobiotic metabolism.

We have studied the mutagenic effects of benzo[a]pyrene (BP) administered in a long-term, low-dose fashion to metabolically competent human lymphoblastoid cells. A continuous dose as low as 0.02 microM for 20 days produced a significant increase in mutant fraction at the 6TG-resistance (HGPRT) locus. The slope of the mutant fraction over time in the 0.02 microM BP-treated culture was twice that observed in the untreated concurrent control; 0.02 microM therefore represents the doubling dose of BP for gene mutation in this cell line. For higher doses of 0.1, 0.5 or 1 microM BP, the rate (or efficiency) of induced mutation was considerably higher for the first 5 or 6 days of exposure than for the last 14-15. This did not appear to be due to a growth disadvantage against early-arising mutants. Comparison to previously published data in the same cell system (Crespi and Thilly, 1984) revealed that the long-term , low-dose protocol (0-1 microM for up to 20 days) was significantly more efficient at inducing mutations than a short-term, high-dose protocol (0-10 microM for 1 day).

Benzo(a)pyrene↗
PubMed1989

A statistical model to estimate variance in long term-low dose mutation assays: testing of the model in a human lymphoblastoid mutation assay.

Long term-low dose mutation assays offer a means to study the genetic effects of environmental mutagens at concentrations relevant to human exposure. These assays involve continuous induction of mutants, serial dilution of cultures and sampling to determine the mutant fraction as a function of time and mutagen concentration. An arithmetic model for the expected variance among identically treated cultures is presented. This model provides means to calculate a predicted variance of the mutant fractions and mutation rates in typical long term-low dose experiments. We have calculated the expected variances of the mutant fraction with this model and compared them to the observed variances among 4 independent experiments in which human lymphoblastoid cells were treated for 5, 10, 15 and 20 days with a non-toxic concentration of the mutagen 4-aminobiphenyl. Mutations at the HPRT locus were measured by determining the 6-thioguanine-resistant mutant fraction. The expected and observed variances of the mutant fractions are in close agreement. This model is adequate to predict the variance of the mutant fraction and should be useful in experimental design and objective evaluation of long term-low dose mutation assays.

Cells, Cultured↗
PubMed1989

Fidelity of DNA polymerases in DNA amplification.

Denaturing gradient gel electrophoresis (DGGE) was used to separate and isolate the products of DNA amplification by polymerase chain reaction (PCR). The strategy permitted direct enumeration and identification of point mutations created by T4, modified T7, Klenow fragment of polymerase I, and Thermus aquaticus (Taq) DNA polymerases. Incorrectly synthesized sequences were separated from the wild type by DGGE as mutant/wild-type heteroduplexes and the heteroduplex fraction was used to calculate the average error rate (mutations per base duplication). The error rate induced in the 104-base-pair low-temperature melting domain of exon 3 of the human hypoxanthine/guanine phosphoribosyltransferase (HPRT) gene was approximately 3.4 x 10(-5) for modified T7, 1.3 x 10(-4) for Klenow fragment, and 2.1 x 10(-4) for Taq polymerases after a 10(6)-fold amplification. The error rate for T4 DNA polymerase was not more than 3 x 10(-6) error per base duplication. The predominant mutations were sequenced and found to be transitions of G.C to A.T for T4 and modified T7 DNA polymerases, and A.T to G.C for Taq polymerase. Klenow fragment induced both possible transitions and deletions of 2 and 4 base pairs.

Base Sequence↗