ISOLATION AND CHARACTERIZATION OF MITOCHONDRIAL RNA POLYMERASE FROM DROSOPHILA MELANOGASTER.
Item
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Title
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ISOLATION AND CHARACTERIZATION OF MITOCHONDRIAL RNA POLYMERASE FROM DROSOPHILA MELANOGASTER.
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Identifier
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AAI8123025
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identifier
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8123025
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Creator
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GOLDENTHAL, MICHAEL JOSEPH.
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Contributor
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Dr. J. Nishiura
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Date
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1981
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Language
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English
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Publisher
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City University of New York.
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Subject
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Biology, General
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Abstract
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A DNA-dependent RNA polymerase was solubilized from sucrose-gradient fractionated, DNAse-treated mitochondria isolated from Drosophila melanogaster. These fractionated mitochondria were shown to be free of nuclear or bacterial contamination by the analysis of the DNA of these organelles by CsCl buoyant density gradient centrifugation, and PLK chromatography. The polymerase is solubilized most efficiently by non-ionic detergents (0.2% NP-40 or 3% Triton X-100). The same concentration of detergent also solubilized cytochrome c oxidase which suggests an inner membrane site for the mitochondrial RNA polymerase. This RNA polymerase has the following properties: 40-50% inhibition by 50 ug/ml rifampicin, an (NH(,4))(,2)SO(,4) optima of 0.025 M, Mg('++) optima of 15 mM, and a Mn('++) optima of 2 mM. Stimulation of polymerase activity by Mg('++) is roughly 2 fold that with Mn('++). There is no significant inhibition of enzyme activity by 100 ug/ml alpha-amanitin.;Gel filtration of the mitochondrial RNA polymerase on Sephacryl S-200 columns resolves multiple peaks of apparent molecular weights of 60,000, 110,000, 160,000, 210,000, and a peak > 250,000. These activities share most of the properties listed above with some differences in the extent of Mn('++) stimulation, and rifampicin sensitivity. The lowest apparent molecular weight activity is greatly enriched when the Sephacryl column is eluted in high salt. These results suggest that the peak of lowest apparent molecular weight is capable of aggregation, more so in low than in high salt to form dimers, trimers, etc. This is further supported by glycerol gradient centrifugation of the low molecular weight activity which yields multiple slow and fast sedimenting polymerase activities, the distribution of which depends on salt concentration. Alternative explanations involving endogenous DNA-binding, and associated protein factors can not be entirely precluded at present, although they are made less probable by the following results. DNAse treatment, PEG precipitation or pre-chromatography of the mitochondrial extracts on heparin-Sepharose have no effect on the heterogenous profile from Sephacryl S-200 columns. In addition, SDS gel electrophoresis shows that the fastest and slowest sedimenting RNA polymerase activities have a single major protein component of 60,000 d.;A similar heterogeneity is observed with ion-exchange chromatography of the mitochondrial RNA polymerase. A correspondence is noted between distinct ion-exchanges peaks from DEAE Sephadex and phosophocellulose columns, and various sized activities as resolved on Sephacryl S-200. One explanation of this data is that the different sized molecular aggregates of mitochondrial RNA polymerase have different charge properties, which may in turn have physiological significance, e.g., in the binding of polymerase to the membrane, or DNA template.;Two preparative schemes for purifying mitochondrial RNA polymerase from isolated mitochondria are presented which are similar in enzyme yields (15-20%), specific activity (800-1000 units/mg protein) and amount of enzyme protein recovered (25-60 ug/200 g Flies).;This enzyme can also be prepared from whole fly homogenates without mitochondrial isolation. This procedure involves PEG precipitation, (NH(,4))(,2)SO(,4) fractionation, batch phosphocellulose, gel filtration and heparin-Sepharose chromatography resulting in a low molecular weight RNA polymerase with the same catalytic properties and single subunit structure as that found with the enzyme from purified mitochondria.;In summary, a low molecular weight RNA polymerase can be isolated from mitochondria with demonstrably different properties from the nuclear or bacterial RNA polymerases.
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Type
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dissertation
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Source
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PQT Legacy CUNY.xlsx
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degree
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Ph.D.
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Program
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Biology
