Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.7.7 (DNA polymerase)
17,007 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Anti-DNA polymerase alpha and Ki-67 are monoclonal antibodies that recognize nuclear antigens expressed in proliferating cells. In this study, we evaluated various methods of embedding and fixing brain tumor specimens to optimize staining with these antibodies. In fresh frozen sections, postfixation with 4% paraformaldehyde, 100% methanol, 95% ethanol and 10% buffered formalin were tested; also tested were prefixation with 4% paraformaldehyde followed by freezing and fixation with 100% methanol, 95% ethanol, or 10% buffered formalin followed by embedding in paraffin. For both antibodies, postfixation of fresh frozen sections with 4% paraformaldehyde at 4 C gave the most intense staining and lowest background activity while preserving histological features. This technique can be used in routine clinical practice to predict the growth potential of tumors.
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PMID:Embedding and fixation techniques for immunohistochemical staining with anti-DNA polymerase alpha and Ki-67 monoclonal antibodies to analyze the proliferative potential of tumors. 137 8

Plasmid DNA released from bacteria by boiling in the presence of lysozyme and Triton x-100 and without further purification can be sequenced by the dideoxy method using T7 DNA polymerase, when conditions during alkali denaturation and subsequent ethanol precipitation are adjusted to remove contaminants. The samples remain in the same microcentrifuge tubes from the harvesting of the bacteria until the splitting of the sample into four aliquots for the termination reactions. Less background label is observed with end-labelled primers (radioactivity or fluorescence), but even when radioactive nucleotides are incorporated during the sequencing reactions, 250 bases or more can be read from template prepared from 1.5 ml bacterial culture. The DNA can also be cut by restriction enzymes; the purification procedure described thus provides the rapid preparation of plasmids for a variety of purposes.
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PMID:Rapid and simple preparation of plasmids suitable for dideoxy DNA sequencing and other purposes. 180 39

Polysomal poly(A)+-RNA prepared from isolated calf liver polysomes by deproteinization and affinity chromatography on oligo(dT)-Sepharose at pH 6 contains low molecular weight peptides (between 600-1500 daltons) bound noncovalently. These peptides were extracted from the poly(A)+-RNA-peptides complex by precipitation of the nucleic acids with 80% (v/v) ethanol at alkaline pH (9.5) and purified on Sephadex G-25 and G-15 columns. Further fractionation was performed by silica gel chromatography and high performance liquid chromatography (h.p.l.c.). The amino acid composition of the isolated peptidic fraction was compared with similar peptides obtained from rat liver, rabbit reticulocyte and calf thymus polysomes. Effluent (ribosomal) RNA contains only negligible amount of peptides. Isolated polysomal RNA peptides were named "deprimerones" (from Latin "deprimere"), since they have a general depressing effect on gene expression in vitro (Hillar & Przyjemski, 1979). Isolated deprimerones not only inhibit DNA transcription, RNA translation in reconstituted cell-free systems, but also DNA replication by DNA polymerase beta with single- and double-stranded DNA template and synthetic deoxyribonucleotide polymers. The inhibitory effect on replication was correlated with the inhibition of [3H]-deoxyribonucleotide incorporation in isolated chromatin and in stimulated lymphocyte cell cultures. The isolated deprimerones are characterized by similar amino acid compositions in various species.
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PMID:Small peptides bound to polysomal RNA inhibit gene expression in cell-free systems, replication of stimulated lymphocytes and DNA repair in isolated chromatin. 241 32

A method for nucleic acid sequencing has been developed based on the observation that phosphorothioate diesters are hydrolysed by treatment with 2-iodoethanol in a solution of aqueous ethanol. For DNA sequencing, primed single-stranded M13 DNA is polymerised with the Klenow fragment of DNA polymerase I in the presence of the three normal deoxyribonucleotide triphosphates and one alpha-phosphorothioate derivative. This is followed by treatment with 2-iodoethanol, precipitation of the DNA fragments and analysis by polyacrylamide electrophoresis. RNA transcribed from plasmids containing the SP6 RNA polymerase promoter is sequenced by including the alpha-phosphorothioate derivative of the ribonucleotide triphosphates in the polymerisation and treating the product with iodoethane. The cleavage reaction involves alkylation of the sulfur atom to form the phosphorothioate triester and hydrolysis catalysed by an adjacent hydroxyl group.
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PMID:DNA and RNA sequencing utilizing phosphorothioate chemistry. 244 67

DNA strand break in HeLa cells induced by radiation was detected using the in situ nick translation method. The cells were exposed to radiation of 3, 6, 12, 18, and 24 Gy in Lab-Tek tissue culture chamber/slides and were fixed with ethanol/acetic acid on the slide glass. The break sites in DNA were translated artificially in the presence of Escherichia coli DNA polymerase I and [3H]-labeled dTTP. Autoradiographic observation was made of the level of break sites in the DNA. The DNA strand break appeared even with a 3 Gy exposure, increased 8.6 times at 24 Gy compared with the control cells, and this level correlated reciprocally to change in cell viability. This nick translation method provides a rapid in situ assay for determining radiation-induced DNA damage of cultured cells, in a semi-quantitative manner.
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PMID:Nick translation detection in situ of cellular DNA strand break induced by radiation. 264 85

Metabolic activation of cyclophosphamide (CP) by microsomal mixed function hydroxylases yields 4-hydroxycyclophosphamide and aldophosphamide defined as activated CP. Activated CP shows relatively high cancerotoxic selectivity in vivo and cytotoxic specificity in vitro and can be trapped rapidly by reversible reaction of hemiaminal group of the oxazaphosphorine ring with protein thiols to form protein bound activated CP (protein-S-CP). Protein-S-CP stores activated CP in a highly stable form. From pharmacokinetics of activated CP in mice after the injection of cyclophosphamide, it was calculated that about 17% of the CP dose given was stored in a pool of protein bound activated CP lasting for several days. From therapy studies with 4-(S-ethanol)-sulfido-CP in combination with excess of cysteine, it was concluded that the protein-S-CP pool may be that form of activated CP which is mainly responsible for the specific cytotoxic effects in the tumor cells. On the other hand free unbound 4-OH-CP was shown to contribute mainly to overall toxicity. No spontaneous toxicogenation of activated CP was noted under in vivo conditions. 3'-5' Exonucleases were found to hydrolyze 4-OH-CP, yielding phosphoramide mustard and acrolein as split products. Because of the low affinity of 4-OH-CP for plain 3'-5' exonucleases, it seems however unlikely that these enzymes play a major role in the antitumor effect of CP in vivo. 3'-5' Exonucleases associated to DNA polymerase like in DNA polymerase delta from rabbit bone marrow or in DNA polymerase I from E. coli are more likely candidates for 4-OH-CP toxicogenation because of the much higher specific activity with 4-OH-CP as substrate. In experiments with DNA polymerase I from E. coli, 4-OH-CP was shown to inhibit DNA polymerase activity after toxicogenation by the 3'-5' exonuclease subsite of the enzyme. This suggests an enzyme mechanism based suicide inactivation of the DNA polymerase. Because of the close spatial cooperation of the DNA polymerase and 3'-5' exonuclease subsites with primer/template a site-specific alkylation of DNA is also postulated. Thus we raised the hypothesis that cytotoxic specificity of activated CP is based on the interaction of protein-S-CP (protein bound activated CP) with DNA polymerase/3'-5' exonuclease as the target. In a P 815 mouse mast-cell tumor we determined by means of 5' AMP agarose affinity chromatography two/third of total DNA polymerase to be associated with 3'-5' exonuclease.
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PMID:The enzymatic basis of cyclophosphamide specificity. 302 54

DNA strand break in HeLa cells induced by heat was detected using the in situ nick translation method. The cells were incubated at 43 degrees C for various times (15, 30, 45, 60, 90 or 120 min) in Lab-Tek tissue culture chamber/slides and were fixed with ethanol/acetic acid on the slide glass. The break sites in DNA were translated artificially in the presence of Escherichia coli DNA polymerase I and 3H-labeled dTTP. The level of break sites in the DNA was visualized by autoradiographic observation of the grains. The DNA strand break appeared as early as 15 min, increased to 10.3-fold at 45 min of 43 degrees C treatment and this level related reciprocally to clonogenicity of the cell. The nick translation method thus provides a rapid in situ assay for determining heat-induced DNA damage of cultured cells, in a semi-quantitative manner.
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PMID:In situ nick translation method reveals DNA strand scission in HeLa cells following heat treatment. 337 Jun 28

The main properties of the duck hepatitis B virus (DHBV) DNA polymerase have been studied and compared with those of the human hepatitis B virus (HBV) and of the woodchuck hepatitis virus (WHV) DNA polymerases. All 3 enzymes are active under high salt conditions in the presence of high magnesium concentration. DHBV DNA polymerase was found less sensitive to ethanol and to operate at higher optimal pH than the HBV and WHV DNA polymerases. Like the other two viral endogenous DNA polymerases, the DHBV enzyme was strongly inhibited by phosphonoformic acid but not by aphidicolin, sulfhydryl group blockers or phosphonoacetic acid. Inhibition of DHBV DNA polymerase by the triphosphate derivatives of several nucleoside analogs appeared similar to that reported for HBV or WHV endogenous polymerase. FIACTP was the most, and ACVTP the least effective inhibitor; BVdUTP was of intermediary potency; araCTP and araTTP had a greater inhibitory effect on DHBV DNA polymerase than HBV or WHV DNA polymerase. The similarities in the properties of DHBV and HBV DNA polymerase justify the use of the duck hepatitis B polymerase model for screening and evaluation of potentially active drugs against HBV infection.
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PMID:Main properties of duck hepatitis B virus DNA polymerase: comparison with the human and woodchuck hepatitis B virus DNA polymerases. 344 17

Chronokinetic synergism, a holistic and extremely sensitive experimental design, has shown in the mouse embryo that site-specific epigenetic forces differentially regulate genesis of the palate (cleft palate) and limb bud organogenesis (shortened stature). Acute exposures of 11-day pregnant mice to minimally effective doses of thymidine or ethanol followed 5 or 8 hr later by minimal exposure to retinoic acid have enabled quantitative and qualitative assay for genomic-epigenetic interactions. These site-specific morphogenetic regulations occurred during palatal genesis from the maxillary prominence of the first pharyngeal arch and during limb bud prechondrogenesis. Thymidine is presumed to induce its response by inhibition of DNA polymerase and hence by transitory cytostatic block. (Embryo size was not detectably changed). Ethanol is interpreted, guilt by associated response, indirectly to interfere with histone regulation of transcription. Two central findings have demonstrated the coordinated regulation of genomic and epigenetic positional information. First, thymidine or ethanol as epigenetic probes for limb prechondrogenesis and palatal precursor cells have activated distinctive site-specific responses. Second, responses to chronokinetic synergisms have indicated that epigenetic regulators for limb and palate dysmorphogenesis may affect distinctly different phases of the cell division cycle and hence induce differential DNA expressions. Although each of palate and limb is concurrently susceptible to epigenetic regulation, their differential intrinsic genomic capabilities appear to have been uncoupled. The putative homeostatic balance of genomic expressions in the palate precursor and the prechondrogenic limb bud cells of the 11-day mouse embryo has been characterized as epigenetically regulated, alternatively expressed, and positionally restricted. We propose that the chronokinetic synergisms have disclosed the existence of distinctive palate-determining genes and stature-determining genes.
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PMID:Epigenetically regulated genomic expressions for shortened stature and cleft palate are regionally specific in the 11-day mouse embryo. 349 Nov 8

The nick-translation procedure without external addition of DNase was performed in situ on sections of various rat organs to detect possible DNA single-strand breaks (nicks) in normal tissues. The freshly frozen sections were briefly fixed in ethanol/acetone and nick-translated in the presence of E. coli DNA polymerase I. A significant difference in the amount of nuclear reaction was found among the different cell populations as detected by autoradiography following incorporation of tritiated TTP as well as by histochemical staining following incorporation of biotin-dUTP into nuclei. Such incorporation of triphosphates was localized in the DNA and was entirely dependent on E. coli DNA polymerase I. The nuclei with the highest reactivity were found in skeletal muscle cells, lymphocytes in various lymphatic organs, the proliferative cells in the gastrointestinal tract, stratified squamous epithelial cells, duct epithelial cells of salivary gland and the maturing spermatids in the seminiferous tubules. These results suggest that, under the conditions adopted, the cells in various tissues reveal different chromatin structures resulting in varying rates of nick translation reaction. Such difference(s) in chromatin structure, presumably including that in the number of DNA single-strand breaks or in the level of endogenous nuclease activity, may be associated with the mechanisms involved in cell growth and differentiation.
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PMID:DNA strand breaks in rat tissues as detected by in situ nick translation. 377 92


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