Deoxyribonucleic Acid Polymerase of Trout Liver Nuclei

1970 ◽  
Vol 27 (1) ◽  
pp. 117-123 ◽  
Author(s):  
H. L. A. Tarr ◽  
Linda Gardner
Keyword(s):  
Dna Polymerase ◽  
Specific Activity ◽  
Polymerase Activity ◽  
Freezing And Thawing ◽  
Low Concentrations ◽  
Mandatory Requirement ◽  
Mammalian Tissues ◽  
Liver Nuclei ◽  
Deoxynucleoside Triphosphate ◽  
High Concentrations

The DNA polymerase of intact trout liver nuclei behaved similarly to that present in mammalian tissues in its mandatory requirement for four deoxynucleoside triphosphates, Mg2+, and DNA primer. It was inhibited by DNase and pyrophosphate in comparatively low concentrations and by RNase and Actinomycin D in comparatively high concentrations. Soluble DNA polymerase was extracted from buffered suspensions of the nuclei by centrifugation at 100,000 g. It possessed properties very similar to those exhibited by the enzyme in intact nuclei. The polymerase activity of intact nuclei was increased by freezing and thawing, and the soluble enzyme was unstable at 30 C and lost most of its activity in 1 day at 0 C. The specific activity of the enzyme was 10–29 picomoles of deoxynucleoside triphosphate incorporated into DNA by 1 mg of protein in 1 hr at 25 C.

DNA Polymerases of Newborn Rat Brain and Liver

1971 ◽  
Vol 49 (8) ◽  
pp. 978-986 ◽  
Author(s):  
A. D. Bharucha ◽  
M. R. V. Murthy
Keyword(s):  
Rat Brain ◽  
Specific Activity ◽  
Hydrolytic Enzymes ◽  
Polymerase Activity ◽  
Newborn Rat ◽  
Newborn Brain ◽  
Rat Brain And Liver ◽  
Mammalian Tissues ◽  
Optimum Ph ◽  
Sulfhydryl Compounds

DNA polymerase activity was found to be present in appreciable quantities in the extracts of whole tissue (TS) as well as of nuclei (NS) isolated from newborn rat brain and liver. The NS fractions of either of the two tissues exhibited a higher specific activity per unit protein than the corresponding TS fractions. The optimum pH requirements as well as the ability to support DNA synthesis over a long period indicate that the NS fractions were also comparatively less contaminated by interfering substances than the TS fractions.The reaction requirements for the incorporation of TMP residues into DNA by the NS fractions of newborn rat brain and liver and the effect of various inhibitors and hydrolytic enzymes on this reaction were also investigated. These extracts resembled preparations from other mammalian tissues in that they exhibited absolute requirements for the primer DNA, the four complimentary deoxynucleoside triphosphates, and Mg2+ ions. When three of the four deoxynucleoside triphosphates were omitted and only TTP-2-14C was added to the reaction mixture, a limited incorporation of TMP-2-14C into DNA occurred. Other investigations such as the effect of actinomycin and of sulfhydryl compounds revealed that a large part of incorporation by the TS and NS fractions of newborn brain and liver was due to the replicative DNA nucleotidyltransferase enzyme.

Effect of benzo[a]pyrene on DNA synthesis and DNA polymerase activity of rat liver nuclei

1985 ◽  
Vol 34 (6) ◽  
pp. 755-762 ◽  
Author(s):  
Inés Salazar ◽  
Laura Tarragó-Litvak ◽  
Simón Litvak ◽  
Lionel Gil
Keyword(s):  
Dna Synthesis ◽  
Dna Polymerase ◽  
Rat Liver ◽  
Polymerase Activity ◽  
Rat Liver Nuclei ◽  
Liver Nuclei

scholarly journals Poly(adenosine diphosphate ribose) polymerase activity and nicotinamide adenine dinucleotide in differentiating cardiac muscle

1976 ◽  
Vol 154 (2) ◽  
pp. 387-393 ◽  
Author(s):  
W C. Claycomb
Keyword(s):  
Cyclic Amp ◽  
Dna Synthesis ◽  
Cardiac Muscle ◽  
Dna Polymerase ◽  
Specific Activity ◽  
Polymerase Activity ◽  
Neonatal Rat ◽  
Dibutyryl Cyclic Amp ◽  
Fresh Tissue ◽  
Dna Polymerase Α

Poly(ADP-ribose) polymerase activity in nuclei isolated from differentiating cardiac muscle of the rat has been characterized and its activity measured during development. Optimum enzyme activity is observed at pH 8.5. Poly(ADP-ribose) polymerase is inhibited by ATP, thymidine, nicotinamide, theophylline, 3-isobutyl-1-methylxanthine and caffeine and stimulated by actinomycin D. The activity measured under optimal assay conditions increases during differentiation of cardiac muscle and is inversely related to the rate of DNA synthesis and to the activities of DNA polymerase α and thymidine kinase. When DNA synthesis and the activity of DNA polymerase α are inhibited in cardiac muscle of the 1-day-old neonatal rat by dibutyryl cyclic AMP or isoproterenol, the specific activity of poly(ADP-ribose) polymerase measured in isolated nuclei is increased. The concentration of NAD+ in cardiac muscle increases during postnatal development. In the adult compared with the 1-day-old neonatal rat the concentration of NAD+ relative to fresh tissue weight, DNA or protein increased 1.7-fold, 5.2-fold or 1.4-fold respectively. The concentration of NAD+ in cardiac muscle of the 1-day-old neonatal rat can be increased by approx. 20% by dibutyryl cyclic AMP. These data suggest that NAD+ and poly(ADP-ribose) polymerase may be involved with the repression of DNA synthesis and cell proliferation in differentiating cardiac muscle.

scholarly journals The DNA Polymerase Gamma R953C Mutant Is Associated with Antiretroviral Therapy-Induced Mitochondrial Toxicity

2016 ◽  
Vol 60 (9) ◽  
pp. 5608-5611 ◽  
Author(s):  
Min Li ◽  
Andrea C. Mislak ◽  
Yram Foli ◽  
Esinam Agbosu ◽  
Vivek Bose ◽  
...  
Keyword(s):  
Antiretroviral Therapy ◽  
Dna Polymerase ◽  
Infected Patient ◽  
Polymerase Activity ◽  
Mitochondrial Toxicity ◽  
Wild Type ◽  
Polymerase Gamma ◽  
Dna Polymerase Gamma ◽  
Deoxynucleoside Triphosphate ◽  
Dntp Binding

ABSTRACTWe found a heterozygous C2857T mutation (R953C) in polymerase gamma (Pol-γ) in an HIV-infected patient with mitochondrial toxicity. The R953C Pol-γ mutant binding affinity for dCTP is 8-fold less than that of the wild type. The R953C mutant shows a 4-fold decrease in discrimination of analog nucleotides relative to the wild type. R953 is located on the “O-helix” that forms the substrate deoxynucleoside triphosphate (dNTP) binding site; the interactions of R953 with E1056 and Y986 may stabilize the O-helix and affect polymerase activity.

Increase in DNA polymerase α activity associated with DNA synthesis due to FSH or oestrogen in ovaries of immature rats

1986 ◽  
Vol 110 (2) ◽  
pp. 353-360 ◽  
Author(s):  
S. Usuki ◽  
M. Shioda
Keyword(s):  
Dna Synthesis ◽  
Dna Polymerase ◽  
Dna Content ◽  
Specific Activity ◽  
Polymerase Activity ◽  
Dna Polymerase Α ◽  
Immature Rats ◽  
Hypophysectomized Rats ◽  
Α Activity ◽  
Intact Rats

ABSTRACT DNA polymerase activities and DNA content of ovaries from immature intact rats (4–29 days after birth), hypophysectomized rats and hormone-treated hypophysectomized rats were measured. During normal ovarian growth DNA polymerase α activity and DNA content of ovaries increased. The polymerase activity decreased gradually after hypophysectomy without any alteration in the DNA content. Administration of ovine FSH (2 μg/day) or oestradiol-17β (1 mg/day) to hypophysectomized rats enhanced ovarian DNA content and DNA polymerase α activity, whereas DNA polymerase β activity did not change significantly. These results suggest that DNA polymerase α participates in DNA synthesis in these ovaries. The specific activity of DNA polymerase α (the activity per μg DNA) in the ovaries increased between 4 and 14 days after birth, and then remained almost constant; the specific activity declined gradually after hypophysectomy. Administration of FSH or oestradiol-17β but not of ovine LH, progesterone or testosterone to hypophysectomized rats restored the specific activity. Mixing experiments with different kinds of ovarian extracts suggested that no activators of DNA polymerase α were present in the extracts. These results suggest that FSH or oestrogen causes the induction of DNA polymerase α accompanied by DNA synthesis during cell proliferation in ovaries of immature rats. J. Endocr. (1986) 110, 353–360

scholarly journals Characteristics of deoxyribonucleic acid polymerase activity in nuclear and supernatant fractions of cultured mouse cells

1970 ◽  
Vol 119 (5) ◽  
pp. 839-848 ◽  
Author(s):  
J. G. Lindsay ◽  
S. Berryman ◽  
R. L. P. Adams
Keyword(s):  
Dna Polymerase ◽  
Deoxyribonucleic Acid ◽  
Specific Activity ◽  
Polymerase Activity ◽  
Partial Purification ◽  
Isolated Nuclei ◽  
Exonuclease I ◽  
Mouse Cells ◽  
Dna Template ◽  
L929 Mouse

1. DNA polymerase activity is present in both nuclear and supernatant fractions prepared from rapidly dividing L929 mouse cells. 2. Nuclear preparations are 2–5 times more active with added native DNA as template and the supernatant fractions show an equivalent preference for heat-denatured DNA. 3. Isolated nuclei can carry on limited DNA synthesis in the absence of added template but are stimulated five- to ten-fold by addition of 50μg of native DNA per assay. 4. DNA polymerase activity can be released from intact nuclei by ultrasonic treatment or by extraction with 1.5m-potassium chloride. 5. The activities in nuclear and supernatant fractions, with their preferred templates, respond similarly to changes in pH and Mg2+ and K+ concentrations. 6. Maximal enzyme activity is approached with 40μg of DNA per assay and activation of the DNA template by treatment with deoxyribonuclease does not decrease the amount of DNA required to reach saturation. 7. The nuclear enzyme, incubated with native DNA, is markedly inhibited by the addition of heat-denatured DNA to the assay. In contrast, the supernatant DNA polymerase activity on denatured templates is not affected by the presence of native DNA. 8. The nuclear enzyme exhibits high activity in the absence of one or more deoxyribonucleoside triphosphates but this is much diminished after partial purification of the enzyme by precipitation at pH5 and fractionation on Sephadex G-200 columns. 9. The 3H-labelled DNA products formed by Sephadex-purified nuclear and supernatant fractions, with their preferred templates, were found to be resistant to treatment with exonuclease I. Alkali-denaturation of the 3H-labelled DNA products rendered them susceptible to attack by exonuclease I. 10. Analysis of the products on alkaline sucrose density gradients suggests that the newly synthesized material may not be covalently bound to the original DNA template. 11. By using their preferred templates the specific activity of supernatant fractions varies markedly with the position of the cells in the cell-cycle, but the specific activity of nuclear fractions varies only slightly.

scholarly journals Purification of Autographa californica nucleopolyhedrovirus DNA polymerase from infected insect cells

1999 ◽  
Vol 80 (9) ◽  
pp. 2519-2526 ◽  
Author(s):  
Xin Hang ◽  
Linda A. Guarino
Keyword(s):  
Enzymatic Activity ◽  
Dna Polymerase ◽  
Specific Activity ◽  
Intrinsic Property ◽  
Nuclease Activity ◽  
Polymerase Activity ◽  
Autographa Californica ◽  
Exonuclease Activity ◽  
Coomassie Blue ◽  
Autographa Californica Nucleopolyhedrovirus

Autographa californica nucleopolyhedrovirus (AcMNPV) DNA polymerase was purified from virus-infected cells using conventional chromatographic methods. The enzymatic activity of fractions eluting from single-stranded agarose gels was found to exactly coincide with a single polypeptide with an apparent molecular mass of approximately 110000 Da on denaturing polyacrylamide gels stained with Coomassie blue. This purification scheme resulted in a 228-fold purification of AcMNPV DNA polymerase with recovery of 3·5% of the initial activity. The specific activity of the most purified fraction of DNA polymerase was 5000 units/mg, which is sufficiently high to eliminate the possibility that contaminants significantly contribute to the polymerase activity. Preparations of purified DNA polymerase had 3′–5′ exonuclease activity, but no 5′–3′ exonuclease activity. The proofreading activity was apparently an intrinsic property of the enzyme as the ratio of nuclease activity to polymerase activity was constant throughout purification. Using a singly-primed M13 DNA template, RF-II DNA was detected within 3 min, indicating a polymerization rate of 40 nt/s. The effects of several DNA polymerase inhibitors on the enzymatic activity of purified DNA polymerase were also determined.

Isolation and preservation of cell nuclei for studies on RNA polymerase activity

1970 ◽  
Vol 48 (5) ◽  
pp. 559-565 ◽  
Author(s):  
R. S. D. Read ◽  
C. M. Mauritzen
Keyword(s):  
Rna Polymerase ◽  
Mammalian Cell ◽  
Cell Types ◽  
Polymerase Activity ◽  
Cell Nuclei ◽  
Low Concentrations ◽  
Nonionic Detergent ◽  
Rna Polymerase Activity ◽  
Liver Nuclei ◽  
Nonionic Detergents

The suitability of saponin for the isolation and of glycerol for the preservation of mammalian cell nuclei has been investigated. The nonionic detergent saponin was found to be a useful cell lytic agent in a procedure for the isolation of nuclei from several mammalian cell types. The RNA polymerase activity of rat liver nuclei was not affected by treatment with saponin or with some other nonionic detergents that were tested. Low concentrations of ionic agents also did not affect the activity of the enzyme though higher concentrations caused lysis of the nuclei. The preservation of structure and enzyme activity in the isolated nuclei was achieved by storage at low temperature in a medium containing 70% glycerol together with a suitable concentration of a divalent metal and phosphate buffer.

XV.—The metabolism of DNA in the liver during precocious induction of metamorphosis in Rana catesbeiana

1969 ◽  
Vol 70 (4) ◽  
pp. 295-310 ◽  
Author(s):  
Ailsa M. Campbell ◽  
Martita H. Corrance ◽  
J. N. Davidson ◽  
H. M. Keir
Keyword(s):  
Dna Polymerase ◽  
Nuclear Dna ◽  
Rana Catesbeiana ◽  
Specific Activity ◽  
Mitochondrial Fraction ◽  
Polymerase Activity ◽  
Nucleic Acid Metabolism ◽  
Cell Extracts

SynopsisStudies have been made on the incorporation of [8H]-deoxythymidine into the DNA of the livers of Rana catesbeiana tadpoles. When metamorphosis was induced with tri-iodothyronine, the specific activity of the nuclear DNA rose 5 days after administration of the hormone. In contrast, the specific activity of the DNA from the mitochondrial fraction rose grave–2 days after hormone administration.In order to determine whether the in vivo change was due to alterations in the pool sizes of the DNA precursors, in vitro studies on DNA polymerase were carried out. It was found that under conditions where the enzyme activity was not limited by availability of template or substrates, there was a rise in the DNA polymerase activity in crude cell extracts from the tadpole liver. Fractionation of the cell components showed that little of this increment in activity appeared to be located in the nucleus, but that a large percentage alteration in activity occurred in the mitochondrial and cell sap fractions.A possible interpretation of these results is that an increase in the mitochondrial DNA polymerase is one of the early effects of thyroid hormone. This possibility is discussed in relation to the other known effects of thyroid hormones in tadpoles, with particular reference to nucleic acid metabolism and also to mitochondrial hyperplasia.

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