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The Isolation, Purification and Characterization of Three RNA Polymerases from Novikoff Hepatoma Ascites Tumor

Citation

Froehner, Stanley Charles (1973) The Isolation, Purification and Characterization of Three RNA Polymerases from Novikoff Hepatoma Ascites Tumor. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/M8Q4-DB66. https://resolver.caltech.edu/CaltechTHESIS:07062018-115304742

Abstract

DNA-dependent RNA polymerase has been isolated from nuclei of Novikoff hepatoma ascites tumor cells and resolved into three activities, designated Ia, Ib, and II, by a combination of phosphocellulose and DEAE cellulose chromatography. Ia and Ib have been further purified by sucrose density centrifugation. Both gradient profiles exhibit coincidence of the polymerase activity and protein peaks, suggesting that the two may be homogeneous enzymes. Ia migrates as a single species on non-denaturing polyacrylamide gel electrophoresis. SDS polyacrylamide gel electrophoresis indicates that Ia contains subunits of 170,000, 125,000, 69,000, 49,000, 44,000 and 37,000 molecular weights in equimolar ratios except for the 69,000 and 37,000 dalton subunits which may be present in two copies per enzyme molecule. A molecular weight of 600,000 for the enzyme calculated from the molecular weights of the subunits is in good agreement with that determined by exclusion chromatography. The probable molecular structure of Ib is subunits of 190,000 and 135,000 daltons, each present twice per enzyme molecule. The enzymological characterization of these three enzymes suggests that Ia and Ib are the nucleolar polymerases while II is nucleoplasmic. Ia and Ib are most active at low ionic strength with Mg++ on native DNA and are insensitive to α-amanitin. II prefers Mn++, high ionic strength, a denatured template and is inhibited by low concentrations of α-amanitin. A factor present in the material which does not bind to the DEAE cellulose column used in the purification scheme, stimulates the activity of all three of the enzymes. Ia and Ib are inactive at low enzyme concentrations in the absence of this factor. The active agent in the factor is probably a protein, since it is heat sensitive, and may be a subunit of the enzyme.

Item Type:Thesis (Dissertation (Ph.D.))
Subject Keywords:Biochemistry; Neurophysiology
Degree Grantor:California Institute of Technology
Division:Biology
Major Option:Biochemistry
Minor Option:Neurobiology
Thesis Availability:Public (worldwide access)
Research Advisor(s):
  • Bonner, James Frederick
Thesis Committee:
  • Bonner, James Frederick (chair)
  • Davidson, Norman R.
  • Dreyer, William J.
  • Mitchell, Herschel K.
  • Strumwasser, Felix
Defense Date:23 June 1972
Funders:
Funding AgencyGrant Number
United States Public Health ServiceUNSPECIFIED
Record Number:CaltechTHESIS:07062018-115304742
Persistent URL:https://resolver.caltech.edu/CaltechTHESIS:07062018-115304742
DOI:10.7907/M8Q4-DB66
Default Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:11101
Collection:CaltechTHESIS
Deposited By: Lisa Fischelis
Deposited On:13 Jul 2018 19:48
Last Modified:21 Dec 2019 01:41

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