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W G Thilly

Publications and source records attributed to W G Thilly.

At least 55 records · Page 3Linked to original sources
PubMed1995

Multi-copy nuclear pseudogenes of mitochondrial DNA reveal recent acute genetic changes in the human genome.

Four nuclear pseudogenes homologous to the 10031-10195-bp region of the human mitochondrial genome were detected by constant denaturant capillary electrophoresis. Among them, one pseudogene is present as at least five copies in each cell, in accordance with our previous observations of multi-copy mitochondrial DNA pseudogenes. The presence of multiple identical copies of pseudogenes suggests that the human genome underwent a series of genetic changes, including gene amplifications, very recently in evolutionary history, i.e., within the last 390000 years.

Base Sequence↗
PubMed1995

Separation of transforming amino acid-substituting mutations in codons 12, 13 and 61 the N-ras gene by constant denaturant capillary electrophoresis (CDCE).

We used high fidelity PCR and constant denaturant capillary electrophoresis (CDCE) [Khrapko et al. (1994) Nucleic Acids Res., 22, 364-369] to separate wild type and different mutant N-ras exon 1 and 2 sequences. The set of plasmids containing N-ras cDNA, wild type or mutant sequences representing all transforming amino acid-substituting single base pair changes in codons 12, 13 (exon 1) and 61 (exon 2), were amplified using Pfu polymerase in a limited cycle polymerase chain reaction. One of the primers used for the amplification of each exon included a 40 nucleotide GC rich sequence that created high and low melting domains. The amplified fragments 151 bp (exon 1) and 150 bp (exon 2) were run on the CDCE with the 'denaturant zone' temperature of the capillary corresponding to the melting temperature of 111 bp (exon 1) and 110 bp (exon 2) low melting domains. The separation was achieved between wild type and mutant sequences as homoduplexes in 15 out of 19 cases, as a single base substitution alters the electrophoretic mobility of a partially melted double stranded fragment. The denaturation and reannealing of wild type and mutant fragments together created wild type/mutant heteroduplexes. All the heteroduplexes were well resolved from wild type homoduplex. In the current form mutant sequences were detected at a frequency of 10(-3) in the presence of wild type. This study has resulted in obtaining electrophoretic spectrum of different N-ras mutants on CDCE as homoduplexes as well as heteroduplexes.

Base Sequence↗
PubMed1995

Elevated incidence of childhood leukemia in Woburn, Massachusetts: NIEHS Superfund Basic Research Program searches for causes.

Between 1966 and 1986, the childhood leukemia rate in Woburn, Massachusetts, was 4-fold higher than the national average. A multidisciplinary research team from MIT, which is being supported by the NIEHS Superfund Basic Research Program, has explored the possible importance of a temporal correlation between the period of elevated leukemia and a previously unrecognized mobilization of toxic metals from a waste disposal site in north Woburn. Residents of Woburn may have been exposed to arsenic (70 micrograms/l) and chromium (240 micrograms/l) at levels in excess of federal drinking water standards (50 micrograms/l for each metal) by consuming municipal groundwater contaminated with these metals. Research is currently underway a) to elucidate the mechanisms and the pathways by which these metals were transported from the waste disposal site to the drinking water supply; b) to determine the identity of the principal human cell mutagens in samples of aquifer materials collected from the site of the municipal supply wells; and c) to measure the extent of exposure and genetic change in residents who consumed the contaminated well water.

Arsenic Poisoning↗
PubMed1994

Evolutionary trail of the mitochondrial genome as based on human 16S rDNA pseudogenes.

In the course of studies on mutations in human mitochondrial (mt) DNA, we have uncovered and sequenced four new nuclear pseudogenes corresponding to bp 2457-2657 of the mt 16S rDNA. The four genes and their homologies with human mtDNA are E2 (62.4%), K10 (74.4%), E1 (84.6%) and LE6 (93.2%). When these five pseudogene sequences and another previously reported pseudogene sequence are compared with each other, they display what appears to be an ordered series of steps from a hypothetical common ancestor. The sequence of the hypothetical ancestor closely resembles that found in a wide variety of present-day mammalian mt genomes. The pseudogene sequences suggest an evolutionary trail of mt mutation dominated by base pair transitions punctuated by integration into the nuclear genome. Once integrated into the nuclear genome, the pseudogenes appear to follow the distinctive nuclear mutational pathway in which GC to AT transitions predominate and CpG sequences are preferentially eliminated.

Animals↗
PubMed1994

N-nitroso-N-methylurea-induced rat mammary tumors arise from cells with preexisting oncogenic Hras1 gene mutations.

GGA to GAA mutations in the 12th codon of the Hras gene are frequently observed in rat mammary tumors induced by N-nitroso-N-methylurea (NMU). We developed an assay to measure point mutations present in tissues at a frequency of 10(-5) and have now applied this assay to measure the specific G to A transition of the Hras gene in rat mammary epithelium. We find that (i) 70% of untreated rats contain detectable levels of Hras mutants; (ii) these mutants are clustered within the gland as sectors in a manner consistent with their origin as a mutation arising during early organ development; and (iii) treatment with a carcinogenic dose of NMU did not result in a significant increase in the number of such mutants, the fraction of organ sectors with mutant cells, or the fraction of animals containing detectable levels of ras mutants. We conclude that the NMU-induced mammary tumors carrying the G to A transition at the 12th codon of the Hras gene arose from preexisting ras mutants and that an independent effect of NMU was directly or indirectly responsible for tumor formation.

Animals↗
PubMed1994

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.

Base Sequence↗
PubMed1994

Mutagenicity of mono- and dinitropyrenes in the Salmonella typhimurium TM677 forward mutation assay.

Nitropyrenes are a group of widespread environmental pollutants, some of which are highly potent as bacterial and mammalian cell mutagens and as animal carcinogens. A quantitative bacterial forward mutation assay, based on resistance to 8-azaguanine (8-AG) in Salmonella typhimurium TM677, was employed as an alternative to reversion assays to reexamine the mutagenicity of 1-, 2-, and 4-nitropyrene (1-, 2-, and 4-NP) and 1,3-, 1,6-, and 1,8-dinitropyrene (1,3-, 1,6-, and 1,8-DNP) in the presence and absence of rat liver postmitochondrial supernatant (PMS). The major finding is that 2-NP, reported as a potent mutagen in the absence of PMS in bacterial reversion assays, was inactive in the absence of PMS in this assay. However, 2-NP was mutagenic in the presence of PMS. The implications of this observation with respect to sample purity and the metabolism of 2-NP are discussed. Without PMS the following minimum detectable mutagen concentration (MDMC) potency series expressed as nmol/ml was obtained: 1,8-DNP (0.5 x 10(-3)), 1,6-DNP (1.2 x 10(-3)), 1,3-DNP (2.3 x 10(-3)), 4-NP (0.2), 1-NP (0.2), 2-NP (> 1200), pyrene (> 1500). With PMS the potency series was: 1,6-DNP (0.7), 1,8-DNP (2.1), 4-NP (2.2), 2-NP (2.6), 1,3-DNP (3.7), 1-NP (4.6), pyrene (> 1500). With the exception of 2-NP, all the nitropyrenes were more mutagenic without PMS than with PMS. The greatest difference was observed with the dinitropyrenes, which were three orders of magnitude less potent in the presence of PMS. Pyrene, often reported as a bacterial mutagen in the presence of PMS, was nonmutagenic in this assay when a purified sample was tested.

Animals↗
PubMed1994

Resolution and conservation of mismatches in DNA end joining.

DNA end joining is a major pathway for the elimination of double-strand breaks from chromosomal DNA of higher eucaryotic cells. Extracts of Xenopus laevis eggs rejoin such breaks even when their short single-stranded termini are expected to form imperfectly matched overlaps. However, end-joined products cloned in Escherichia coli, necessarily give rise to perfectly matched products. Therefore it has not been possible to determine whether the end joining process creates mismatched products, perfectly matched (resolved) products or both. To investigate whether mismatch resolution was the result of the X. laevis end joining process or of activities of the bacterial host we used denaturing gradient gel electrophoresis to analyse joined products. We found that the end joining process does include mismatch resolution, the degree of which varies with regard to the nature of the original overlap structure. Mismatches 3' to a gap are completely resolved, mismatches 3' to a nick and 5' to a nick or gap are resolved to some extent but are generally conserved. Mismatches between base matches are always conserved. These findings suggest competing processes of ligation, DNA fill-in synthesis or exonucleolytic excision of mismatched bases next to a gap or nick. At mismatches 3' to a nick the probability of ligation is greater than that of excision while at mismatches 3' to a gap the probability of excision is greater than elongation of a given mismatch. At mismatches 5' to nicks or gaps it appears that ligation or elongation and ligation, respectively, are the most probable pathways but products resulting from mismatch excision, elongation and ligation are also detected.

Animals↗
PubMed1994

Bacterial mutagenicity of pyrolysis tars produced from chloro-organic fuels.

Droplets of toluene and three chlorinated organics, ortho-dichlorobenzene, 1,2-dichloroethane, and trichloroethylene, were pyrolyzed in pure nitrogen. The composition and bacterial mutagenicity of the product tars were measured. The presence of organic chlorine was found to affect both pyrolysis product tar composition and total tar mutagenicity. Pyrolysis in the absence of chlorine produced tars whose bacterial mutagenicity was found to be largely due to the presence of cyclopenta[cd]pyrene, fluoranthene, and benzo[a]pyrene. Small amounts of chlorine in the fuel (i.e., Cl/H molar ratios of less than 0.3) enhanced the formation of highly condensed polycyclic aromatic hydrocarbons (including cyclopenta[cd]pyrene) and increased tar mutagenicity. Larger amounts of organic chlorine (Cl/H ratios of between 0.3 and 0.6) resulted in significant yields of mono- and dichlorinated aromatics and higher levels of tar mutagenicity, which could not be accounted for by the presence of mutagens produced by pyrolysis in the absence of chlorine. Furthermore, unlike tars containing little or no chlorine, tars containing aryl chlorine were more mutagenic in the absence of added enzymes (intended to mimic in vivo mammalian metabolism) than in their presence. We hypothesize that at least one of the chlorinated aromatic products is strongly mutagenic. Two specific conditions that gave notably different results were a) the low-temperature (i.e., below 1400 K) pyrolysis of ortho-dichlorobenzene, which produced tri- and tetrachlorinated biphenyls almost exclusively; and b) the chlorine-rich pyrolysis of trichloroethylene, during which mostly perchloroaromatics were formed. Neither of these tars was found to mutate bacteria.

Hydrocarbons, Chlorinated↗
PubMed1994

Use of denaturing-gradient gel electrophoresis to study chromium-induced point mutations in human cells.

A large number of hprt-mutants were obtained by treating human lymphoblast cells (TK6) with 5 microM K2Cr2O7 for 5 hr and selecting by growth in 6-thioguanine. A combination of high fidelity polymerase chain reaction (PCR) and denaturing gradient gel electrophoresis (DGGE) allowed us to measure mutant frequencies as a function of DNA sequence. Chromium(VI) induced four hotspots in a 104 bp domain of hprt exon 3. Substitutions at G:C base pairs were the predominant mutations. One of the chromium-induced hotspots was located at the same position as previously determined hydrogen peroxide and benzo(a)pyrene diol epoxide hotspots.

Chromium↗
PubMed1993

Quantitative differential effects of rhodamine 123 on normal cells and human colon cancer cells by magnetic resonance spectroscopy.

Rhodamine 123 is a lipophilic cationic compound that is selectively taken up by cancer cell mitochondria. This compound is toxic to epithelial cancer cells in vitro and displays significant anticancer activity in vivo. However, the mechanism of action of rhodamine 123 in intact, actively metabolizing cell preparations is unknown. We have used 31P- and 13C-nuclear magnetic resonance spectroscopy to quantitatively characterize how rhodamine 123 affects the energetics of human colon cancer cells (HCT-116) and spontaneously immortalized normal epithelial cells (CV-1). Rhodamine 123 differentially altered the phosphorus and glucose metabolism of HCT-116 and CV-1 cells. 31P-nuclear magnetic resonance detected mitochondrial poisoning in the HCT-116 human colon cancer cell line in its early stages after selective uptake of rhodamine 123. When we compared administration of rhodamine 123 and [1-C13]glucose to administration of [1-C13]glucose alone in the HCT-116 cells, we noted a marked decrease in intracellular pH to 6.7 +/- 0.06 (mean +/- SD) units, a 2.2-fold increase in lactate production, and a 1.8-fold increase in glucose consumption after 10 h. In addition, we found a 2-fold rise in intracellular free magnesium 12 h after rhodamine 123 administration. These results suggest that when rhodamine 123 inhibits mitochondrial ATP production, it initially stimulates cytoplasmic glycolysis in an attempt to maintain cellular energy demands. The marked fall in intracellular pH and rise in intracellular free magnesium after administration of rhodamine 123 may inhibit activity of several glycolytic enzymes: this effect would inhibit cytoplasmic ATP generation and interfere with multiple cell enzymatic processes, leading to cell death. The CV-1 cells showed no change in intracellular pH, intracellular free magnesium, or magnesium-bound ATP levels over the 24-h period following rhodamine 123 administration. Rhodamine 123 also failed to alter glucose utilization and lactate production levels significantly in the CV-1 cells. These results prove the usefulness of 31P- and 13C-nuclear magnetic resonance spectroscopy for quantifying differing effects of rhodamine 123 on the high energy phosphate metabolism and glucose metabolism of HCT-116 and CV-1 cells.

Adenosine Triphosphate↗
PubMed1993

An alkylation-tolerant, mutator human cell line is deficient in strand-specific mismatch repair.

The human lymphoblastoid MT1 B-cell line was previously isolated as one of a series of mutant cells able to survive the cytotoxic effects of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). MT1 cells nevertheless remain sensitive to mutagenesis by MNNG and display a mutator phenotype. These phenotypes have been attributed to a single genetic alteration postulated to confer a defect in strand-specific mismatch repair, a proposal that attributes the cytotoxic effect of DNA alkylation in wild-type cells to futile attempts to correct mispairs that arise during replication of alkylated template strands. Our results support this view. MNNG-induced mutations in the HPRT gene of MT1 cells are almost exclusively G.C-->A.T transitions, while spontaneous mutations observed in this mutator cell line are single-nucleotide insertions, transversions, and A.T-->G.C transitions. In vitro assay has demonstrated that the MT1 line is in fact deficient in strand-specific correction of all eight base-base mispairs. This defect, which is manifest at or prior to the excision stage of the reaction, is due to simple deficiency of a required activity because MT1 nuclear extracts can be complemented by a partially purified HeLa fraction to restore in vitro repair. These findings substantiate the idea that strand-specific mismatch repair contributes to alkylation-induced cytotoxicity and imply that this process serves as a barrier to spontaneous transition, transversion, and insertion/deletion mutations in mammalian cells.

Alkylation↗
PubMed1993

13C- and 31P-NMR studies of human colon cancer in-vitro and in-vivo.

We report comparative 31P-NMR studies in-vivo and in-vitro of the human adenocarcinoma cell line HCT-116 in a high-density, perfused microcarrier culture and as a tumour from the same cell line grown in three different immune-suppressed animal models (NIH triple deficient, Nude, SCID). The phosphate metabolite ratios, pHNMR and intracellular free magnesium, derived from the 31P-NMR spectra, were compared for the in-vivo and in-vitro systems. Results obtained with HCT-116 cells on microcarrier beads are quantitatively similar to that of small (122 mm3), tumours in-vivo derived from the same cell line in any of the immune-suppressed animal systems studied. This suggests that in-vitro microcarrier cell culture serves as a useful model system for deriving information about metabolism of small, tumours in-vivo. It offers the additional advantages of allowing for precise control of substrate milieu, perfusion and oxygenation. The microcarrier system was also used to measure flux through glycolysis and the pentose cycle. In particular, we measured glucose utilization and the production of lactate, alanine, glutamine and glycogen in proton-decoupled 13C-NMR experiments following administration of [1-13C]glucose. We found that (63% +/- 6%) of the glucose utilized was released as [3-13C] lactate in the presence of oxygen, indicating that the HCT-116 cells have a high level of aerobic glycolysis. Serial labelling experiments with [1-13C] glucose and [6-13C] glucose reveal that at least (11.6% +/- 1.3%) of the glucose utilized enters the pentose cycle. We determined that (6.9% +/- 1.2%) of the glucose utilized is recycled to glucose via the pentose cycle while (4.7% +/- 1.4%) of the glucose utilized enters the pentose cycle to form lactate. The high rate of recycling via the pentose cycle suggests that a significant fraction of cellular NADPH is generated by the pentose cycle as opposed to generation by the malate-pyruvate shuttle.

Adenocarcinoma↗
PubMed1993

Predominant mutations induced by the Thermococcus litoralis, vent DNA polymerase during DNA amplification in vitro.

We have analyzed the predominant mutations created during DNA amplification by PCR utilizing a DNA polymerase isolated from the Thermococcus litoralis (Vent DNA polymerase). Exon 3 of the human hypoxanthine guanine phosphoribosyl transferase (HPRT) gene was amplified using conditions optimized for efficiency of DNA amplification. The resulting PCR product was subjected to denaturing gradient gel electrophoresis (DGGE) to separate polymerase-induced mutant sequences from correctly amplified sequences. The nature of induced mutations was determined by isolating and sequencing the mutant sequences from the gel. Eighteen predominant mutations were found in the 104-bp low temperature melting domain of exon 3 and consisted of 16 A/T to G/C transitions, a G/C to T/A transversion and a complex 4-bp deletion. Thus, the Vent exonuclease proofreading activity seems to affect all misincorporation events with apparently equal probability (i.e., by a factor of five). The comparison of the error rates between analogues of Vent DNA polymerase proficient and deficient in the proofreading 3'-->5' exonuclease activity indicates that the lack of proofreading resulting in an approximate five-fold increase in induced error rate. However, the similarity of the patterns of the mutant sequences observed in DGGE suggested that both enzymes created predominantly the same kinds of mutations and at the same positions in this DNA template under the in vitro reaction conditions studied. This predominance of A/T to G/C transition is also a characteristic of the Taq DNA polymerase, although the positions of most errors induced by both enzymes are not identical.

Bacteria↗
PubMed1993

Bacterial and human cell mutagenicity study of some C18H10 cyclopenta-fused polycyclic aromatic hydrocarbons associated with fossil fuels combustion.

A number of isomeric C18H10 polycyclic aromatic hydrocarbons (PAHs), thought to be primarily cyclopenta-fused PAHs, are produced during the combustion and pyrolysis of fossil fuels. To determine the importance of their contributions to the total mutagenic activity of combustion and pyrolysis samples in which they are found, we characterized reference quantities of four C18H10 CP-PAHs: benzo[ghi]fluoranthene (BF), cyclopenta[cd]pyrene (CPP), cyclopent[hi]acephenanthrylene (CPAP), and cyclopent[hi]aceanthrylene (CPAA). Synthesis of CPAA and CPAP is described. The availability of reference samples of these isomers also proved to be an essential aid in the identification of the C18H10 species often found in combustion and pyrolysis samples. Chemical analysis of selected combustion and pyrolysis samples showed that CPP was generally the most abundant C18H10 isomer, followed by CPAP and BF. CPAA was detected only in pyrolysis products from pure PAHs. We tested the four C18H10 PAHs for mutagenicity in a forward mutation assay using S. typhimurium. CPP, BF, and CPAA were roughly twice as mutagenic as benzo[a]pyrene (BaP), whereas CPAP was only slightly active. These PAHs were also tested for mutagenic activity in human cells. In this assay, CPP and CPAA were strongly mutagenic but less active than BaP, whereas CPAP and BF were inactive at the dose levels tested. Also, the bacterial and human cell mutagenicity of CPAA and CPAP were compared with the mutagenicity of their monocyclopenta-fused analogs, aceanthrylene and acephenanthyrlene. Although the mutagenicities of CPAP and acephenanthrylene are similar, the mutagenic activity of CPAA is an order of magnitude greater than that of aceanthyrlene.

Cell Line↗