Partial Purification of a DNA-polymerase from the Non-histone Chromatin Proteins of Rat Liver

Patel, Gordhan L.; Howk, Richard; Wang, T. Y.

doi:10.1038/2151488a0

Cited by 31 publications

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“…Previous studies in this laboratory have shown that the nonhistone chromatin proteins from rat liver and calf thymus contain a replicative type D N A polymerase (1,2). It was subsequently found that isolated cell nuclei from solid Walker tumor are especially rich in this enzymic activity.…”

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Partial Purification of Chromosomal DNA Polymerase from Rat Walker Tumor

Wang

1968

Experimental Biology and Medicine

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Previous studies in this laboratory have shown that the nonhistone chromatin proteins from rat liver and calf thymus contain a replicative type D N A polymerase (1, 2). It was subsequently found that isolated cell nuclei from solid Walker tumor are especially rich in this enzymic activity. Investigation of the acidic chromosomal proteins from Walker 256 carcinosarcoma has resulted in the partial purification of a DNA polymerase. The method of the enzyme purification is described in the present report. I t will be shown that the purified DNA polymerase requires only native D N A as template and a cumplete supplement of all four deoxyriboside triphosphates as substrates. The enzymic reaction also depends on the presence of M e + , monovalent cations being inhibitory. Materials and Methods. Walker 256 carcinosarcomas were developed for 7 days after tumor cells were injected intramuscularly into the hind legs of rats. The tumor cells had a mitotic cycle of 22-25 days and * Supported by grants from the American Cancer Society (E-44) and the Public Health Service (GM-11698).showed well-organized endoplasmic reticulum network. All the following operations to be described were carried out at 2 4 ' .Cell nuclei were isolated from the tumor tissue by the method of Chauveau et al. ( ) .The tumor nuclei were extracted with 100 vol of 0.05 M Tris-HC1, pH 7.4, containing 5 mM MgC12, followed by repeating the buffer extraction three times. The nonhistone chromosomal proteins were prepared from the washed nuclei according to previously described procedure (4).The isolated acidic chromosomal proteins were dialyzed overnight against 0.05 M Tris-HC1, pH 8.5, containing 1 mM 2-mercaptoethanol. To the dialyzed acidic protein solution, saturated ammonium sulfate (AS) solution (adjusted to pH 8.5 with NH,OH) was added slowly with stirring to 40% saturation with respect to ammonium sulfate. After standing for 15 min, the mixture was centrifuged at 10,OOg for 15 min and the precipitate was discarded. To the clear supernate enough ammonium sulfate was added to a final 607% saturation of ammonium sulfate. The precipitate (4&60% AS) thus formed at UNSW Library on July 11, 2015 ebm.sagepub.com Downloaded from

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“…The incorporation of radioactivity into acid-insoluble material was measured as described previously ( 1). The reaction mixture, in a total volume of 0.50 ml, was incubated at 37' for 1 hr.…”

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Partial Purification of Chromosomal DNA Polymerase from Rat Walker Tumor

Wang

1968

Experimental Biology and Medicine

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“…The bacteria were grown in iron-limited medium and harvested after 72 hr., and extracts were prepared as described by Winder & Coughlan (1967a,b). DNA polymerase was determined by ising [3H]TTP in the assay mixture of Lehman (1963) but by employing the procedure of Patel, Howk & Wang (1967).…”

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Partial purification of deoxyribonucleic acid polymerase from Mycobacterium smegmatis

1968

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lOr PROCEEDINGS OF THE BIOCHEMICAL SOCIETY differential centrifugation as described by Falcone & Nickerson (1956). Defatted cell wall gave a typical analysis of carbohydrate, 85%; N, 1.3%; P, 0.6%; mannose :glucose ratio, 1-05:1. Pronase digestion of this material solubilized 54% of the total carbohydrate and 38% of the total phosphorus. The mannose:glucose ratio of the residue was 1:2. The soluble fraction from the Pronase digest was fractionated on DEAE-cellulose to give phosphoglycopeptide material (250 mg. from lg. defatted cell wall; carbohydrate, 94%; mannose :glucose ratio, 4*6 :1; N, 0.8%; P, 0.9%).

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“…Enhanced levels of chromatin-RNA polymerase activity were found in soybean hypocotyls treated with 2,4-D (14); furthermore, the products of chromatin-directed RNA polymerization differed when soybean hypocotyls were treated with auxin or ethylene (5). Chromatin-RNA polymerase activity of dwarf pea seedlings was enhanced by treatment of the plants with GA (13 (15) and mitochondria (8), sea urchin embryos (12), and cultured human cells (4). Soluble Data are pooled from several experiments in which the activity observed in the normal reaction mixture was about 40 pmoles of TMP incorporated per 100 lOg of DNA.…”

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Enhanced Deoxyribonucleic Acid Polymerase Activity of Chromatin from Soybean Hypocotyls Treated with 2,4-Dichlorophenoxyacetic Acid

1971

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Chromatin isolated from soybean (Glycine max L., var. Wayne) hypocotyls was capable of catalyzing the polymerization of labeled deoxyribonucleoside triphosphate in the presence of the three other deoxyribonucleoside triphosphates into a trichloroacetic acid-insoluble product. This product was insensitive to base hydrolysis and ribonuclease, but was sensitive to acid hydrolysis and deoxyribonuclease. Chromatin

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Partial Purification of a DNA-polymerase from the Non-histone Chromatin Proteins of Rat Liver

Cited by 31 publications

References 11 publications

Partial Purification of Chromosomal DNA Polymerase from Rat Walker Tumor

Partial Purification of Chromosomal DNA Polymerase from Rat Walker Tumor

Partial purification of deoxyribonucleic acid polymerase from Mycobacterium smegmatis

Enhanced Deoxyribonucleic Acid Polymerase Activity of Chromatin from Soybean Hypocotyls Treated with 2,4-Dichlorophenoxyacetic Acid

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