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D K Dube

Publications and source records attributed to D K Dube.

At least 73 records · Page 4Linked to original sources
PubMed1981

On the fidelity of DNA replication. Nucleoside monophosphate generation during polymerization.

During catalysis by homogeneous procaryotic DNA polymerases, nucleoside monophosphates are generated by a 3' leads to 5'-exonucleolytic activity. Using Escherichia coli DNA polymerase I and poly[d(A-T)] as a template, the contribution of this activity to the fidelity of DNA synthesis has been evaluated by three different criteria. 1) The ratio between the rates of monophosphate generation and incorporation of the noncomplementary nucleotide with Mg2+ as an activating cation was 0.6 +/- 0.6, which is insufficient to account for the high fidelity of polymerization. 2) Inhibition of polymerization by pyrophosphate fails to diminish fidelity, although some kinetic models suggest that optimal error correction via monophosphate release requires the polymerization reaction to be strongly driven by pyrophosphate release. 3) The addition of deoxynucleoside monophosphates in concentrations as great as 10 mM to the reaction mixture does not alter the fidelity of DNA synthesis. These observations argue against the kinetic proofreading mode to account for the fidelity of E. coli DNA polymerase I when copying poly[d(A-T)] in a Mg2+-activated reaction. Furthermore, they suggest that the polymerase may enhance specificity at the base-selection step. However, the 3' leads to 5' exonuclease plays a larger role when the polymerase is activated with Mn2+ and may also be important in copying natural DNA where lower error rates are observed in vitro.

DNA Polymerase I↗
PubMed1981

In situ transcription analysis of chromatin template activity of the S-chromosome of Drosophila following high molar NaCl treatment.

The chromatin template activity of the polytene chromosomes in larval salivary glands of Drosophila hydei has been assayed by in situ transcription, on the fixed chromatin using E. coli RNA polymerase holoenzyme and 3H-UTP as the monitoring substrate, both with optimal salt and high salt concentration (1 M NaCl). Results reveal an increase in the net transcription of all chromosomes in the high salt treatment series in comparison with the control. The X-chromosomes of the male larval gland also shows an increase in the labelling following the high salt treatment, but the increase is significantly less than that of the autosomes of the same nucleus. On the basis of these findings it has been suggested that the X-chromosomal hyperactivity of the male, as normally known to exist, might be guided by an inherent modulation of the structure of the X-chromatin.

Animals↗
PubMed1981

Induction of carboxymethylcellulase in Macrophomina phaseolina.

Macrophomina phaseolina, the well-known jute pathogenic fungus produces very low levels of both extra- and intracellular carboxymethylcellulase even in the absence of any cellulose as carbon source in the medium. However, the production of these enzymes is greatly induced by soluble carboxymethylcellulose. The carboxymethylcellulase in M. phaseolina is repressed by glucose.

Carboxymethylcellulose Sodium↗
PubMed1981

Studies on the biosynthesis of the variant surface glycoproteins of Trypanosoma brucei: sequence of glycosylation.

The variant surface glycoprotein (VSG) of several Trypanosoma brucei clones has been metabolically labeled with [35S]methionine in parasites grown in the presence or in the absence of tunicamycin. Pulse and chase experiments followed by immunoprecipiation with anti-VSG sera and polyacrylamide gel electrophoresis of the immunoprecipitates, have helped to elucidate two steps of the sequence of glycosylation of VSG. Immediately after, or concomitantly with the synthesis of the protein, a first type of oligosaccharide side chain is also synthesized. Tunicamycin, a specific inhibitor of asparagine glycosylation, completely inhibits this reaction. The total amount of VSG found in trypanosomes grown in the presence of tunicamycin is less than in controls. The unglycosylated molecule has an apparent molecular weight 5-10% smaller than the mature form. A subsequent step, occurring after translation, is the synthesis of carbohydrate side chains which contain a cross-reacting antigenic determinant detected in all VSGs so far studied by immunoprecipitations with heterologous antisera. The percentage of total VSG bearing sugars involved in cross-reactions is variable in different clones, increases with time and subsequently decreases, suggesting that some of the carbohydrate might undergo trimming. Polyacrylamide gel electrophoresis of immunoprecipitates suggests that the absence or the incomplete synthesis of this oligosaccharide does not alter the apparent molecular weight of VSG. In four out of the five clones studied, the sugar(s) responsible for cross-reactions seem to be located within oligosaccharides linked to the protein through both N-glycosidic and other unidentified types of linkages. This is suggested by the partial effect of tunicamycin on the extent of VSGs cross-reactivities measured by immunoprecipitations with heterologous antiserum.

Animals↗
PubMed1980

The effect of nitrofurantoin on deoxyribonucleic acid synthesis in plant mitochondria.

Nitrofurantoin (1-([(5-nitrofuran-2-yl)methylene]amino)imidazolidine-2,4-dione), a widely used drug and also a well-known bacterial mutagen, inhibits DNA synthesis in mitochondria from 48 h etiolated seedlings of Vigna sinensis (Linn.) Savi (snake bean). The effect appears at the level of the uptake of radioactive deoxynucleoside triphosphates by the plant mitochondria. Nitrofurantoin does not inhibit DNA synthesis in vitro by homogeneous Escherichia coli DNA polymerase I and DNA polymerase from avian-myeloblastosis virus. No specific nitroreductase activity could be detected in mitochondria.

Avian Myeloblastosis Virus↗
PubMed1979

Distinctive properties of mammalian DNA polymerases.

DNA polymerase-alpha and -beta can be distinguished from one another by the differential effects of N-ethylmaleimide, KCl, ara-CTP and temperature, as well as on the basis of sedimentation. The sensitivity of DNA polymerase-beta to elevated temperatures as compared to DNA polymerase-alpha provides a new means of distinguishing between these two enzymes even in crude extracts and a possible probe for determining their function. DNA polymerase-alpha and -beta share several properties in common, including the ability to readily incorporate dUTP in place of dTTP. The Km for dUTP varies from 10 to 30 micron with different preparations of DNA polymerase-alpha and -beta. Thus, in mammalian cells, dUMP could be incorporated into DNA, and if excised by an endonuclease, would lead to discontinuities. Initial analyses of fidelity in direct comparative studies indicate that beta-class DNA polymerases are highly accurate in base selection when copying poly[d(A-T)]. Less than one molecule of dGMP is incorporated for every 12 000-45 000 molecules of dAMP and dTMP polymerized. DNA polymerase-alpha is somewhat less accurate, making one mistake for every 4000-10 000 correct nucleotides incorporated. Since both polymerases lack an exonucleolytic activity, this accuracy must be the result of selectivity for the complementary nucleotide by the polymerase.

Animals↗
PubMed1979

Detection and characterization of DNA polymerase from Trypanosoma brucei.

The predominant DNA polymerase activity has been isolated from the parasitic flagellated protozoan, Trypanosoma brucei. Like mammalian DNA polymerase-alpha the trypanosome DNA polymerase is of large molecular weight (S, 6--8), is resistant to thermal denaturation, is sensitive to N-ethylmaleimide, and is inhibited by high ionic strength. However, specific antisera that cross-react with mammalian DNA polymerase-alpha from different species fail to cross-react with the trypanosome polymerase.

Animals↗
PubMed1977

Infidelity of DNA synthesis as related to mutagenesis and carcinogenesis.

An assay system has been developed for measuring the fidelity of DNA synthesis in vitro by using synthetic polynucleotide templates and purified DNA polymerases. Nearest-neighbor analysis of the synthesized product indicates that noncomplementary nucleotides are incorporated as single base substitutions. The accuracy of DNA synthesis can be decreased by (1) prior alkylation of the template, (2) increasing the relative concentration of incorrect nucleotides, and (3) addition of specific metal salts to the reaction mixture. As an initial evaluation of the utility of this system, the effects of 31 metal salts on the fidelity of DNA synthesis have been determined. The results indicate that potential metal mutagens and/or carcinogens may be detected by measuring alterations in the fidelity of DNA synthesis.

Avian Myeloblastosis Virus↗
PubMed1976

On the association of reverse transcriptase with polynucleotide templates during catalysis.

The association of avian myeloblastosis virus (AMV) DNA polymerase with polynucleotide templates during catalysis has been studied. During the course of polymerization, different template-primer complexes were added and the ability of the enzyme to switch from one polynucleotide template to another was determined. At 37 degrees C as well as at 4 degrees C, the polymerase is able to switch from certain template-primer complexes to others. For example, the addition of poly(A)-oligo(dT) during the course of synthesis with poly(C)-oligo(dG) results in the immediate cessation of dGMP polymerization and the start of dTMP polymerization without any lag. Early during the course of polymerization, the size of the product, as determined by alkaline sucrose gradient centrifugation, is, in part, a function of the ratio of the template-primer complex to the enzyme. These cumulative experiments indicate that catalysis on polynucleotide templates with avian myeloblastosis virus DNA polymerase under the conditions tested is not processive in a classical sense. Similar to cellular DNA polymerases the enzyme can shift from one template-primer to another. Using autoradiography after gel electrophoresis to estimate the product size, it can be calculated that the enzyme switches from one template to another within 0.25 min at 37 degrees C which corresponds to the incorporation of greater than 25 nucleotides. At 4 degrees C, switching can be calculated to occur in less than three nucleotide addition steps. Thus, with certain homopolymers, conditions can be found by which AMV DNA polymerase can switch from one template-primer complex to another, perhaps after each nucleotide addition step.

Avian Leukosis Virus↗