Mercury vapor concentrations inside buildings.

The concentrations of elemental gaseous mercury in several homes, offices, and laboratories in the Dallas area have been measured. The values are substantially higher than the ambient natural background concentration, about 3 nanograms of mercury per cubic meter of air in San Francisco, Dallas, and Washington, D.C.

High-level expression of the cloned ada gene of Escherichia coli by deletion of its regulatory sequence.

The Ada protein, a methyltransferase for repair of several alkyl adducts in DNA, was expressed in its native form at a high level in Escherichia coli from a pUC9 recombinant plasmid carrying ada gene from which the sequence controlling the Ada induction was deleted. The regulatory sequence appears to act as a terminator of transcription initiated from the lac promoter of the vector. However, deletion of the regulatory sequence resulted in elimination of ada induction by alkylating agents, providing confirmation of its role in activation of ada expression.

Efficiency and fidelity of cell-free protein synthesis by transfer RNA from aged mice.

Transfer RNAs (tRNAs) from heart, kidney, liver, and spleen of mature (10-12 months old) and aged (29 months old) C57BL/6 mice were tested for their ability to translate encephalomyocarditis viral RNA in a tRNA-dependent cell-free system derived from mouse ascites tumor cells. The rates of in vitro protein synthesis were compared as a function of tRNA concentration, and the fidelity of translation was examined by sodium dodecyl sulfate gel electrophoresis and isoelectric focusing of the viral polypeptides synthesized in vitro. No significant age-related differences in either the efficiency or fidelity of synthesis were discovered, indicating that alterations in tRNAs are probably not involved in the cellular aging of these tissues.

Age-dependent modulation of tissue-specific repair activity for 3-methyladenine and O6-methylguanine in DNA in inbred mice.

3-Methyladenine-DNA N-glycosylase (MAG) and O6-methylguanine-DNA methyltransferase (MGMT) activities were assayed in liver, lungs, brain and ovaries of female mice of two inbred stocks, C3Hf and C57BL/E, as a function of age. In addition to differences in the enzyme levels between the two stocks for each organ, the suckling animals (9-day-old) have consistently lower levels of both MAG and MGMT than young adults (7- or 8-week-old). While the MGMT levels in adults did not decrease with age, the MAG levels in 15- to 17-month-old animals were, in general, significantly lower than those in young adults. These results raise the possibility that the older animals are at a higher risk than young adults following exposure to alkylating mutagens.

Novel microfabricated device for electrokinetically induced pressure flow and electrospray ionization mass spectrometry.

A novel microchip device for electrospray ionization has been fabricated and interfaced to a time-of-flight mass spectrometer. Fluid is electrokinetically transported through the chip to a fine fused-silica capillary inserted directly into a channel at the edge of the device. Electrospray is established at the tip of the capillary, which assures a stable, efficient spray. The electric potential necessary for electrospray generation and the voltage drop for electroosmotic pumping are supplied through an electrically permeable glass membrane contacting the fluidic channel holding the capillary. The membrane is fabricated on the microchip using standard photolithographic and wet chemical etching techniques. Performance relative to other microchip electrospray sources has been evaluated and the device tested for potential use as a platform for on-line electrophoretic detection. Sensitivity was found to be approximately three orders of magnitude better than spraying from the flat edge of the chip. The effect of the capillary on electroosmotic flow was examined both experimentally and theoretically.

Quantitation of O6-methylguanine-DNA methyltransferase in HeLa cells.

A synthetic DNA polymer containing [8-3H]O6-methylguanine (m6G) was used as a substrate to assay the in situ demethylation of the alkylated base by an activity in HeLa cell extracts. The repair activity appears to be similar to the O6-methylguanine-DNA methyltransferase of E. coli and to be inactivated by reaction with the substrate. Extracts of a methylation-repair proficient (Mer+) cell strain, HeLa CCL2, were found to contain m6G repair activity equivalent to approx. 100 000 molecules of methyltransferase per cell, assuming that each molecule can demethylate one m6G residue. No activity could be detected in the extract of a repair deficient (Mer-) cell strain, HeLa S3, and there is no evidence of an inhibitor of repair activity in this strain.

O6-Methylguanine-DNA methyltransferase in human cells.

O6-Methylguanine-DNA methyltransferase activity was measured in extracts of human tumor cells and was partially purified from human placenta. Repair of O6-methylguanine in DNA inactivated the methyltransferase, and treatment of cells with MNNG, which produces this alkylated base in DNA, depleted the cells of active methyltransferase. RNA and protein synthesis were required for restoration of methyltransferase activity, which transiently exceeded the original levels by 50% 48 h after treatment. One species of methyltransferase of Mr = 22 kd was present in human tumor cells and human placenta.

Lack of induction of O6-methylguanine-DNA methyltransferase in mammalian cells treated with N-methyl-N'-nitro-N-nitrosoguanidine.

A synthetic DNA substrate containing O6-methyl[8-3H]-guanine was used to assay demethylation of the premutagenic base by O6-methylguanine-DNA methyltransferase in extracts of HeLa cells, Chinese hamster ovary cells and normal rat kidney cells which had been treated with multiple doses of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). No induction of methyltransferase activity was observed in any of the cell lines tested. Constitutive levels of methyltransferase in cell lines proficient (Mex+) in O6-methylguanine repair were decreased in a dose-dependent fashion by either single or multiple treatments with MNNG over a broad range of dose levels. Recovery of constitutive levels of activity required 24- to 48-h incubation periods. Repair deficient (Mex-) cell lines lacked both constitutive and inducible methyltransferase activity.

Inhibition of O6-methylguanine-DNA methyltransferase in HeLa S3 cells by antisense oligodeoxynucleotides.

There is an increasing interest in using selected antisense oligodeoxynucleotides to control the ribosomal translational process in cells. The oligonucleotide hybridizes to a target RNA to inhibit its translation. In order to prevent the degradation of oligonucleotides by residual and constitutive ribonucleases, various molecular alterations to antisense oligos have been attempted. In this report, evidence is offered that a phosphorothioate-modified 16-mer synthetic oligodeoxynucleotide inhibits the synthesis of O6-methylguanine-DNA methyltransferase (MGMT) in cultured HeLa S3 cells within six hours after introduction.

Adaptive response of Bacillus subtilis to N-methyl-N'-nitro-N-nitrosoguanidine.

Cell extracts of Bacillus subtilis contain a methyltransferase that appears to remove the O6-methyl group from O6-methylguanine in DNA in situ. This reaction proceeds in a stoichiometric fashion, as in Escherichia coli. However, the basal level of the enzyme (approximately 240 molecules per cell) is significantly higher in B. subtilis than in E. coli. In addition, the methyltransferase level increases by an order of magnitude as a result of de novo protein synthesis after adaptive treatment with a low concentration of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), as in E. coli. Concomitant with adaptation, B. subtilis cells become more resistant to both killing and mutagenesis by a challenge dose of N-methyl-N'-nitro-N-nitrosoguanidine. We present evidence to support the hypothesis that the majority of N-methyl-N'-nitro-N-nitrosoguanidine-induced mutations in B. subtilis are of the guanine-to-adenine transition type.

Base-pairing properties of O6-methylguanine in template DNA during in vitro DNA replication.

The kinetics of incorporation of deoxynucleotide precursors directed by the promutagenic base, O6-methylguanine (m6Gua), was analyzed during in vitro replication of m6Gua-containing synthetic polydeoxynucleotides by T4 and T5 phage DNA polymerases and Escherichia coli DNA polymerase I. When poly(dT,m6dG) and poly(dC,m6dG) with covalently attached primers were replicated, O6-methylguanine paired with either thymine or cytosine but with a much higher preference for thymine. dCTP and dTTP acted as competitive inhibitors of each other during DNA synthesis. O6-Methylguanine also directed incorporation of dAMP by T5 DNA polymerase. This dAMP incorporation was not inhibited by dTTP. Contrary to theoretical predictions that the m6dG X dT pair should be comparable to the dA X dT pair, the presence of m6dG in the template inhibited DNA synthesis. Based on Kappm values, E. coli DNA polymerase I showed a much higher preference for dTMP incorporation over dCMP opposite m6dG in the template than T4 and T5 DNA polymerases. At the same time, there was a higher turnover of dCTP than of dTTP by the E. coli enzyme. However, in all cases, the turnover of deoxynucleotides during replication of m6Gua-containing templates was more than that observed with templates without the alkylated base.

Kinetics of incorporation of O6-methyldeoxyguanosine monophosphate during in vitro DNA synthesis.

O6-Methyldeoxyguanosine triphosphate (m6dGTP), known to be produced in vivo by methylation of deoxyguanosine triphosphate with simple methylating mutagens, is utilized by prokaryotic DNA polymerases during in vitro replication of synthetic and natural DNA template-primers. A study of the kinetic behavior of m6dGTP during DNA replication in vitro and of its effect on DNA replication indicates that m6dGTP acts as an analogue of dATP with Kappm of about 6 microM for Escherichia coli DNA polymerase I (Klenow fragment) compared to the Kappm of about 0.8 microM for dATP. m6dGTP is not incorporated in the complete absence of dATP (a competitive inhibitor). m6dGTP also inhibits in vitro DNA synthesis. Different DNA polymerases behave differently in utilization and turnover of m6dGTP. T4 DNA polymerase shows stronger discrimination against m6dGMP incorporation than either T5 DNA polymerase or E. coli DNA polymerase I. The possibility that m6dGTP is unlikely to contribute significantly to in vivo mutation is discussed.

O6-methylguanine-DNA methyltransferase in wild-type and ada mutants of Escherichia coli.

O(6)-Methylguanine-DNA methyltransferase is induced in Escherichia coli during growth in low levels of N-methyl-N'-nitro-N-nitrosoguanidine. We have developed a sensitive assay for quantitating low levels of this activity with a synthetic DNA substrate containing 3H-labeled O(6)-methylguanine as the only modified base. Although both wild-type and adaptation-deficient (ada) mutants of E. coli contained low but comparable numbers (from 13 to 60) of the enzyme molecules per cell, adaptation treatment caused a significant increase of the enzyme in the wild type but not in the ada mutants, suggesting that the ada mutation is in a regulatory locus and not in the structural gene for the methyltransferase.

Site-directed mutation of the Escherichia coli ada gene: effects of substitution of methyl acceptor cysteine-321 by histidine in Ada protein.

Oligodeoxynucleotide-mediated mutagenesis of the ada gene of Escherichia coli was used to produce two mutant Ada proteins. In mutant I the methyl acceptor Cys-321 for O6-methylguanine was replaced by histidine; and in mutant II the positions of Cys-321 and His-322 of the wild-type protein were inverted. Neither mutant protein had O6-methylguanine-DNA methyltransferase activity, but both retained the phosphotriester-DNA methyltransferase activity involving methyl group transfer to Cys-69. Under the control of the endogenous promoter, synthesis of mutant I protein was undetectable before or after adaptation treatment with promoter, synthesis of mutant I protein was undetectable before or after adaptation treatment with N-methyl-N'-nitro-N-nitrosoguanidine. This appeared to be due to both inhibition of transcription of the mutant gene and degradation of the synthesized protein. On the other hand, mutant II protein was inducible by N-methyl-N'-nitro-N-nitrosoguanidine, although to a smaller extent than the wild-type protein was, and the phosphotriester-DNA methyltransferase activity appeared to reside in 24- to 30-kilodalton cleavage products. Mutant I protein could be produced under lac promoter control, and its cleavage products, unlike those of mutant II protein, tended to aggregate. These results indicate that (i) Cys-321 cannot be replaced or transposed with the nucleophilic amino acid histidine for O6-methylguanine-DNA methyltransferase function, (ii) single amino acid replacement or transposition at the O6-methylguanine methyl acceptor site can have a profound effect on the in vivo stability and regulatory function of the Ada protein, and (iii) the integrity of the protein may not be absolutely needed for its transcription-activation function.