Accurate Initiation of Rat Ribosomal RNA Gene Transcription Using a Fractionated Nuclear Extract from Normal Liver and a Hepatoma

Overview

Journal Proc Natl Acad Sci U S A

Specialty Science

Date 1985 Feb 1

PMID 3856247

Citations 7

Authors

R N Kurl

S T Jacob

Affiliations

Soon will be listed here.

Abstract

Nuclear extracts obtained from normal rat liver and from Morris hepatoma 3924A were fractionated by DEAE-Sephadex chromatography. The fraction eluted with 175 mM (NH4)2SO4 (DE-B), which contains greater than 90% of RNA polymerase I activity, supported accurate transcription of cloned rat rDNA. A similar fraction obtained from the cytosol had all of the factors required for rDNA transcription. However, its transcriptional activity was at most one-sixth that of the corresponding nuclear fraction, as determined by the amount of protein needed to produce a similar quantity of the transcript. Unfractionated nuclear or cytosol preparations did not yield an accurate transcript. Optimal KCl and magnesium concentrations for rDNA transcription were 60 mM and 5-7.5 mM, respectively. The extent of transcriptional activity was in the following order: hepatoma nuclear fraction DE-B greater than whole cell extract derived from rat mammary adenocarcinoma cells much greater than normal liver fraction DE-B. The hepatoma preparation produced at least 10 times the amount of transcript produced by the corresponding liver nuclear preparation. Transcriptional activity was proportional to the levels of RNA polymerase I and to the rate of rRNA synthesis in these tissues.

Citing Articles

Role of de novo DNA methyltransferases and methyl CpG-binding proteins in gene silencing in a rat hepatoma.

Majumder S, Ghoshal K, Datta J, Bai S, Dong X, Quan N J Biol Chem. 2002; 277(18):16048-58.

PMID: 11844796 PMC: 2241740. DOI: 10.1074/jbc.M111662200.

Enhancer 1 binding factor, a Ku-related protein, is a positive regulator of RNA polymerase I transcription initiation.

Hoff C, Ghosh A, Prabhakar B, Jacob S Proc Natl Acad Sci U S A. 1994; 91(2):762-6.

PMID: 8290597 PMC: 43029. DOI: 10.1073/pnas.91.2.762.

Isolation, fractionation and reconstitution of a nuclear extract capable of transcribing ribosomal DNA.

Haglund R, Rothblum L Mol Cell Biochem. 1987; 73(1):11-20.

PMID: 3807897 DOI: 10.1007/BF00229371.

Role of DNA topoisomerase I in the transcription of supercoiled rRNA gene.

Garg L, Diangelo S, Jacob S Proc Natl Acad Sci U S A. 1987; 84(10):3185-8.

PMID: 3033639 PMC: 304833. DOI: 10.1073/pnas.84.10.3185.

Transcription of eukaryotic ribosomal RNA gene.

Jacob S Mol Cell Biochem. 1986; 70(1):11-20.

PMID: 3012322 DOI: 10.1007/BF00233800.

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