Increased RNase H (hybridase) activity in the integument of blowfly larvae during development and under the influence of β‐ecdysone

Doenecke, Detlef; Marmaras, Vassilis J.; Sékeris, Constantin E.

doi:10.1016/0014-5793(72)80245-x

Cited by 25 publications

(4 citation statements)

References 10 publications

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“…The changes in hybridase activity evoked during hormonal induces changes in transcription [7], the effects of hormones on the amount of DNA * RNA hybrids [31] and the effects of hybridase on transcription in vitro [32] are in accord with this hypothesis.…”

Section: Discussionmentioning

confidence: 96%

Purification and Characteristics of Hybridase (Ribonuclease H) from Rat‐Liver Cytosol

Roewekamp¹,

Sékeris²

1974

European Journal of Biochemistry

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Ribonuclease H or hybridase, the enzyme cleaving the RNA moiety of DNA -RNA hybrids has been extensively purified from rat-liver cytosol by ammonium sulphate precipitation, DEAEcellulose chromatography, Sephadex 0-200 and G-150 superfine gel filtration, isoeIectric focusing, phosphocellulose and hydroxyapatite chromatography. Neither native or denatured DNS, nor single or double-stranded natural or synthetic ribopolynucleotides are degraded by the enzyme. The pH optimum of the reaction is 7.9. Enzyme activity is totally dependent on the divalent cations Mg2+ and Mn2+, Mga+ being optimal for the reaction. The enzyme acts on the hybrid as an endonuclease resulting in end products with a chain length less than 15, having a 5'-phosphate and a free 3'-hydroxyl end group. Synthetic homopolymer hybrids are digeated only if they are composed of purine bases. The enzyme has a molecular weight of 120000-125000 and seems to be composed of two subunits with molecular weights of 85000 and 43000. MaterialsMale Wistar B R I I rats, weighing 120-18Og obtained commercially were used. Ribonucleoside triphosphates and poly(deoxyribonuc1eotides) were supplied by Boehringer Mannheim GmbH (Mannheim, Germany). 3H-Labelled uridine triphosphate was purchased from New England Nuclear Corporation and 3H-labelled polyribonucleotides from Schwarz/ Mann.Biogel A-1.5 m was obtained from Bio-Rad (Munich), Sephadex products from Pharmacia (Uppsala) and ion-exchange cellulose from Whatman. Hydroxyapatite was prepared according to [lo]. PEI-cellulose sheets were supplied by Schleicher & Schull. Double-stranded 3H-labelled M12 phage RNA and E . coli [3H]DNA were kind donations of Prof. M. Hofschneider (Max-Planck-Institut fur Biochemie, Martinsried) and of Dr Radzak (Hygiene Institut, Marburg), respectively. All other reagents were of analyticaI grade. SubstratesSynthetic hybrids were prepared by mixing equimolar quantities of a [3H]ribohomopolymer and the respective deoxyhomopolymer (determined by using the appropriate absorption coefficients, see below), in 0.2 M NaC1, 30 mM Tris-HC1 pH 7.9 and then heating the mixtures for 5 min at 45-50 "C. After annealing, the solutions were dialyzed against water and lyophylized to give the sodium salts of the complexes. The lyophylized products were dissolved in 0.1 M Eur. J. Biochem. 43 (1974)

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Section: Discussionmentioning

confidence: 96%

Purification and Characteristics of Hybridase (Ribonuclease H) from Rat‐Liver Cytosol

Roewekamp¹,

Sékeris²

1974

European Journal of Biochemistry

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“…Recent evidence implicates eukaryotic cell ribonuclease H in processes leading to RNA transcription stimulation in certain cells (Hausen &Stein, 1970;Doenecke et al, 1972) or to stimulation of replicative and consequently proliferative activity in other cells (Sawai et al, 1982;Sawai & Tsukada, 1983). This and the following evidence prompted us to begin investigation of the enzyme in leukaemia cells and explore its role in human leukaemias: It has been shown that the retroviral ribonuclease H is physically as well as functionally linked to the virus-coded enzyme reverse transcriptase (Mollingetal, 1971;Grandgenettetal, 1972Grandgenettetal, ,1973Bergeret al, 1983) and that reverse transcriptase activity has been demonstrated in human leukaemic cells, but not in phytohaemagglutinin-stimulated normal lymphocytes (Gallo et al 1970).…”

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confidence: 99%

Activity of ribonuclease H in cells of chronic B‐lymphocytic leukaemia: correlation with clinical stage

et al. 1988

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Peripheral blood mononuclear cells (PBMNC) from 23 healthy subjects and 39 patients with B-cell chronic leukaemia (B-CLL) were assayed for ribonuclease H activity using as substrate the filter-immobilized synthetic homopolymer hybrid 3H-poly(rA):poly(dT). In 69% of the leukaemia patients examined enzyme activities were above those estimated in cells from the healthy controls. The mean enzyme levels for the normal and the leukaemic samples group were (per cent substrate hydrolysis): 8.1 +/- 8.9 and 58.7 +/- 40.8, respectively, their difference being statistically highly significant (P less than 0.0001). This result does not represent homogeneity within the cells but is due to a subclass of cells within the leukaemic clone containing the enzyme, thus contributing through pool size fluctuation to the wide variations of enzyme activity observed among the patients. These cells containing high activity could not be identified with either the prolymphocytes or the large lymphocytes. The activity of ribonuclease H in the examined CLL patients correlated with disease stage (Binet) (P = 0.011) and appeared to serve as a sensitive indicator of disease progression when compared with a number of other known prognostic parameters.

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“…In certain eukaryotic cell systems evi dence indicates that stimulation of transcrip tional activity [6] or of proliferative activity [8] in cells may result in or be dependent on the elevation above basal (resting state of the unstimulated cell) levels of the activity of ribonucléase H. To further explore the role of eukaryotic cell ribonucléase H, a compar ative study was designed to investigate the enzyme in normal human leukocytes and in leukocytes of leukemic patients. We report here the results of the study of ribonucléase H in 37 patients of acute and chronic my elogenous leukemia and in 23 normal indi viduals.…”

Section: Introductionmentioning

confidence: 99%

Activity and Function of Hybrid Ribonuclease in Cells of Acute and Chronic Myelogenous Leukemia

Papaphilis¹,

Kamper²,

Pangalis³

et al. 1987

Pathobiology

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The activity of hybrid ribonuclease (ribonuclease H) has been determined in mononuclear blood cells (lymphocytes plus monocytes) from 23 normal individuals and cells (pool of immature granulocytes, metamyelocytes and lymphocytes) from 35 untreated acute and chronic myelogenous leukemia cases. It was found that in 86% of the leukemic samples the activity of ribonuclease H was above two standard deviations from the mean activity level drawn for the group of normal samples along the 0–100% substrate hydrolysis scale. The activity of the enzyme in leukemic cells correlated linearly with the DNA-synthesizing activity of the cells in vitro and in the examined CML cases it paralleled the inverse relationship of the incorporation of tritiated thymidine into DNA to the size of the pool of immature granulocytes. In one CML patient who received chemotherapy with Myleran, the activity of ribonuclease H, high at the initiation of drug therapy, was reduced to a normal level at remission, but increased again at the stage of subsequent relapse. These findings indicate that the levels of ribonuclease H in leukemic cells reflect the proliferative activity of the population in the cases of untreated myelogenous leukemias.

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Increased RNase H (hybridase) activity in the integument of blowfly larvae during development and under the influence of β‐ecdysone

Cited by 25 publications

References 10 publications

Purification and Characteristics of Hybridase (Ribonuclease H) from Rat‐Liver Cytosol

Purification and Characteristics of Hybridase (Ribonuclease H) from Rat‐Liver Cytosol

Activity of ribonuclease H in cells of chronic B‐lymphocytic leukaemia: correlation with clinical stage

Activity and Function of Hybrid Ribonuclease in Cells of Acute and Chronic Myelogenous Leukemia

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