Terminal deoxyribonucleotidyltransferase from nuclei of rat intestinal mucosa

1970 ◽  
Vol 48 (5) ◽  
pp. 537-540 ◽  
Author(s):  
F. Y. T. Leung ◽  
S. H. Zbarsky
Keyword(s):  
Dna Polymerase ◽  
Snake Venom ◽  
Intestinal Mucosa ◽  
Deae Cellulose ◽  
Nuclear Extract ◽  
Polymerase Activity ◽  
Snake Venom Phosphodiesterase ◽  
Deoxyribonucleoside Triphosphate

A terminal deoxyribonucleotidyltransferase has been isolated from extracts of nuclei of rat intestinal mucosa. The enzyme was obtained in partially purified form by rechromatography of material with DNA polymerase activity isolated previously by chromatography of the nuclear extract on DEAE-cellulose. The enzyme, which required heat-denatured DNA as a primer, catalyzed the incorporation of radioactivity from a single labelled deoxyribonucleoside triphosphate into the DNA product. The incorporation was inhibited up to 70% in the presence of all four complementary deoxyribonucleoside triphosphates. Evidence has been presented based on the release of radioactivity from the DNA product during its hydrolysis by snake venom phosphodiesterase which indicates that the enzyme catalyzes the addition of deoxyribonucleotides to the 3′-OH terminus of heat-denatured DNA. The properties of the enzyme are those of a terminal deoxyribonucleotidyltransferase as distinct from the replicative transferase, DNA polymerase.

Deoxyribonucleic acid polymerase from nuclei of rat intestinal mucosa

1970 ◽  
Vol 48 (5) ◽  
pp. 529-536 ◽  
Author(s):  
F. Y. T. Leung ◽  
S. H. Zbarsky
Keyword(s):  
Dna Polymerase ◽  
Intestinal Mucosa ◽  
Gradient Centrifugation ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Molecular Size ◽  
Sucrose Density Gradient ◽  
Polymerase Activity ◽  
Sucrose Density Gradient Centrifugation ◽  
Dna Template

An extract with DNA polymerase activity was prepared from nuclei of intestinal mucosa of the rat. Chromatography of the crude extract on DEAE-cellulose yielded three enzymically active fractions: I, II, and III. Each fraction could be resolved further into two components with DNA polymerase activity by rechromatography on smaller columns of DEAE-cellulose. A similar result was obtained by gel filtration of fraction II material on Sephadex G-150. The result of sucrose density gradient centrifugation of the fractions obtained by gel filtration suggested that each still consisted of a mixture of proteins with DNA polymerase activity. The approximate molecular weights of the active proteins, estimated by comparison with marker proteins, ranged from 25 000 to 300 000. Partially purified DNA polymerase (fraction II) required for activity the four deoxyribonucleoside triphosphates, Mg2+, 2-mercaptoethanol, and DNA template. The optimum pH for activity was 8.0 in Tris–HCl buffer and 7.4 in phosphate buffer. The two components obtained by gel filtration of fraction II differed in their requirements for DNA template. The one of smaller molecular size was more active with native DNA whereas the larger was equally active with either native or heat-denatured DNA.

Effect of ribonucleotides on substrate availability for DNA polymerase assays in cytoplasmic fractions of rat intestinal mucosa

1977 ◽  
Vol 55 (4) ◽  
pp. 489-492
Author(s):  
S. H. Zbarsky ◽  
S. Chevalier
Keyword(s):  
Dna Synthesis ◽  
Dna Polymerase ◽  
Intestinal Mucosa ◽  
Inorganic Phosphate ◽  
Enzymatic Degradation ◽  
Polymerase Activity ◽  
Subcellular Fractions ◽  
Substrate Availability ◽  
Thymidine Triphosphate ◽  
Deoxyribonucleoside Triphosphate

The deoxyribonucleoside triphosphate substrates for DNA synthesis were hydrolysed during the DNA polymerase (EC 2.7.7.7) assay with cytoplasmic subcellular fractions of rat intestinal mucosa. Presumably because of phosphatase (EC 3.1.3.2) activity in these fractions, inorganic phosphate was liberated from the nucleotides, and radioactive thymidine triphosphate was shown to be degraded to thymidine di- and mono-phosphate, thymidine, and thymine. Addition of ATP to the postmicrosomal supernatant increased its DNA polymerase activity by sparing the deoxyribonucleotide precursors from enzymatic degradation.

Analysis of O2′-Methylnucleoside 5′-Phosphates in Snake Venom Hydrolysates of RNA: Identification of O2′-Methyl-5-Carboxymethyluridine as a Constituent of Yeast Transfer RNA

1975 ◽  
Vol 53 (7) ◽  
pp. 735-746 ◽  
Author(s):  
M. W. Gray
Keyword(s):  
Snake Venom ◽  
Deae Cellulose ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Assay Method ◽  
Blocking Group ◽  
Wheat Embryo ◽  
Yeast Trna ◽  
Snake Venom Phosphodiesterase ◽  
Hydrolysis Of

Snake venom phosphodiesterase liberates the O2′-methylnucleoside (Nm) constituents of RNA as the corresponding 5′-nucleotides (pNm), which, in contrast to normal 5′-nucleotides (pN), are resistant to dephosphorylation by venom 5′-nucleotidase. This property provides the basis of a convenient and highly reproducible quantitative assay for Nm residues in RNA. The assay method involves: (1) hydrolysis of RNA with whole or partially-purified snake venom; (2) isolation of the pNm derivatives, as a group, by anion-exchange chromatography on DEAE-cellulose; (3) resolution of the individual pNm compounds by two-dimensional paper chromatography; (4) identification and quantitative measurement of pNm derivatives by ultraviolet absorption spectrophotometry. Using this procedure, the molar proportions of the Nm constituents of wheat embryo, yeast, and Escherichia coli tRNA have been determined. The close correspondence between the values measured by venom hydrolysis and those obtained by analysis of alkali-stable dinucleotide (Nm-Np) sequences attests to the validity of the venom assay, and further indicates that alkali-stable sequences larger than dinucleotides are not present in significant amounts in the tRNA of the above three organisms.During the present investigation, several ultraviolet-absorbing components, not immediately identifiable as ribose-methylated nucleotides, were isolated along with the expected O2′-methylnucleoside 5′-phosphates. Preliminary characterization of one of these compounds suggests that it is a derivative of a novel nucleoside, O2′-methyl-5-carboxymethyluridine (cm5Um). Venom hydrolysis of yeast tRNA liberates the 5′-nucleotide of cm5Um in the form of a carboxyl-blocked derivative (pU-2). During alkaline hydrolysis of yeast tRNA, the blocking group in U-2 is labilized and cm5Um is released as part of an alkali-stable dinucleotide, cm5Um-Ap. The proportion of pU-2 in venom hydrolysates of yeast tRNA (0.02 mol%, the same as the content of cm5Um-Ap in alkaline hydrolysates) suggests that O2′-methyl-5-carboxymethyluridine may be confined to a single isoaccepting species of tRNA in yeast.In an allied study, reinvestigation of the alkali-stable dinucleotide sequences of baker's yeast tRNA has confirmed previous results concerning the sequence distribution of O2′-methylribose in yeast tRNA (Gray, M. W. &Lane, B. G. (1967) Biochim. Biophys. Acta 134, 243–257).

scholarly journals The activity of deoxyribonucleic acid polymerase in some species of algae

1972 ◽  
Vol 129 (2) ◽  
pp. 285-290 ◽  
Author(s):  
O. Th. Schönherr ◽  
H. M. Keir
Keyword(s):  
Dna Polymerase ◽  
Green Algae ◽  
Dna Polymerases ◽  
Deae Cellulose ◽  
Chlorella Pyrenoidosa ◽  
Nuclease Activity ◽  
Polymerase Activity ◽  
Ph Optimum ◽  
Blue Green Algae ◽  
Wide Range

1. The activities of DNA polymerase preparations from the algae Euglena gracilis, Chlamydomonas reinhardtii, Chlorella pyrenoidosa, Anabaena variabilis and Anacystis nidulans were measured. The blue–green algae Anabaena and Anacystis contain a 5–20-fold higher activity of the enzyme than do the green algae. DNA polymerases from the blue–green algae show a pH optimum of 9 and prefer a relatively low Mg2+concentration (1–3mm). DNA polymerases from the green algae, however, display a pH optimum between 7.5 and 8.5 and an optimum Mg2+concentration of 8mm. With all algae, a higher polymerase activity was obtained with denatured salmon sperm DNA as template than with native DNA. All four deoxyribonucleoside 5′-triphosphates must be present for full activity of the polymerases. 2. With one exception, the deoxyribonuclease activities in the preparations, measured under conditions of the DNA polymerase assay, are low compared with corresponding preparations from Escherichia coli. Chlamydomonas extracts contain a high deoxyribonuclease activity. 3. After purification on columns of DEAE-cellulose, the polymerase activity was linear over a wide range of protein concentrations, except for Chlamydomonas preparations, where the observed deviation from linearity was probably attributable to the high nuclease activity. 4. DNA polymerases from all these algae bind strongly to DNA–cellulose; 6–40-fold purifications of the enzyme were obtained by chromatography on columns of DNA–cellulose. 5. The partially purified polymerases of Euglena and Anacystis are heat-labile but become much more heat-stable when tested in the presence of DNA.

scholarly journals Multiple forms of nuclear deoxyribonucleic acid polymerases and their relationship with the soluble enzyme

1973 ◽  
Vol 131 (2) ◽  
pp. 237-246 ◽  
Author(s):  
R. L. P. Adams ◽  
M. A. L. Henderson ◽  
W. Wood ◽  
J. G. Lindsay
Keyword(s):  
Molecular Weight ◽  
Dna Synthesis ◽  
High Molecular Weight ◽  
Dna Polymerase ◽  
Deae Cellulose ◽  
Polymerase Activity ◽  
Enzymic Activity ◽  
Supernatant Fraction ◽  
Molecular Weights ◽  
Molecular Weight Peak

1. DNA polymerase from nuclear and supernatant fractions of cultured mouse L929 cells was fractionated on columns of Sephadex G-200, Sepharose 4B and of DEAE-cellulose. Several peaks of activity are found on Sephadex chromatography and the distribution of activity between these depends on: (a) the source of the enzyme, i.e. nuclear or supernatant fraction; (b) the mode of extraction of the enzyme from the nucleus; (c) the amount of enzyme applied to the column. 2. The DNA polymerase activity in the lower-molecular-weight peaks (approximate molecular weights are 35000, 70000 and 140000) is firmly bound within the cell nucleus and shows a preference for native DNA as template, whereas the high-molecular-weight peak (peak I, molecular weight 250000 or greater) is found in supernatant fractions and shows greater activity with a denatured DNA template. 3. During periods of DNA synthesis the high-molecular-weight enzyme becomes more firmly bound within the nucleus. 4. Peak I enzymic activity is relatively unstable and is inhibited by thiol-blocking reagents and deoxycholate, but it is stimulated by univalent cations. 5. Very little endonuclease is present in the polymerase preparations, but a very active exonuclease and nucleoside diphosphokinase are present. On Sephadex chromatography, however, it was shown that the immediate precursors for DNA synthesis, at least by peak I enzyme, are the deoxyribonucleoside triphosphates. 6. Attempts to decrease the molecular weight of the peak I enzyme while still retaining activity failed.

Facile and Sensitive Fluorescence Assay of DNA Polymerase Activity Using Cu2+ and Ascorbate as Signal Developers

2019 ◽  
Vol 4 (8) ◽  
pp. 2398-2403 ◽  
Author(s):  
Xingxing Zhang ◽  
Qiang Liu ◽  
Yan Jin ◽  
Baoxin Li
Keyword(s):  
Dna Polymerase ◽  
Polymerase Activity ◽  
Fluorescence Assay

Suggestive Evidence for an Oncorna-Virus-Specific DNA Polymerase from C-Type Particles of Bovine Leukosis

1974 ◽  
Vol 29 (1-2) ◽  
pp. 72-75 ◽  
Author(s):  
B. Dietzschold ◽  
O.R. Kaaden ◽  
S. Ueberschaer ◽  
F. Weiland ◽  
O. C. Straub
Keyword(s):  
Dna Polymerase ◽  
Polymerase Activity ◽  
Friend Virus ◽  
Leukaemia Virus ◽  
Virus Particles ◽  
Virus Rna ◽  
Feline Leukaemia Virus ◽  
Bovine Leukosis ◽  
Bovine Lymphocytes ◽  
Viral Preparation

Abstract Typical C-type oncorna virus particles as shown by electron microscopy have been purified from the supernatant of cultured lymphocytes from bovine leukosis. In the purified C-particle fraction a DNA-polymerase activity was detected. Using several synthetic RNA-or DNA-homopolymers and 70S Friend virus RNA the template response of this bovine leukosis cell particle DNA polymerase was compared with those of feline leukaemia virus DNA polymerase and DNA polymerase from normal bovine lymphocytes. The DNA polymerase detected in the viral preparation of bovine leukosis is suggested to be an oncorna-virus-specific enzyme.

scholarly journals Stereochemistry of hydrolysis by snake venom phosphodiesterase.

1979 ◽  
Vol 254 (16) ◽  
pp. 7476-7478 ◽  
Author(s):  
P.M. Burgers ◽  
F. Eckstein ◽  
D.H. Hunneman
Keyword(s):  
Snake Venom ◽  
Snake Venom Phosphodiesterase
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