Isolation and Molecular Characterization of a cDNA Encoding the 23-kDa Subunit of Human RNA Polymerase II

U K Pati,S M Weissman

doi:10.1016/s0021-9258(18)51603-3

Abstract

We have shown that antibodies against native calf thymus RNA polymerase II and antibodies against its 23-kDa subunit cross-reacted with the 23-kDa subunit of human RNA polymerase II. Immunoglobin G (IgG) against the 23-kDa subunit of calf thymus RNA polymerase II inhibited transcription in vitro from the adenovirus major late promoter. By immunoscreening of a human placenta lambda gt11 cDNA library with IgG against native CT RNA polymerase II and with IgG against its 23-kDa subunit, we isolated and characterized a full length 1.2-kilobase cDNA. We also generated oligonucleotide probes from a sequence of amino acid residues obtained by a modified peptide microsequencing procedure. The cDNAs isolated both from oligoscreening and immunoscreening were identical. The amino acid sequence deduced from the nucleotide sequence analysis indicates a polypeptide of 197 amino acid (23 kDa). The in vitro translation product of human cDNA HP-23 was precipitated by IgG against the 23-kDa subunit of CT RNA polymerase II. The amino acid sequence deduced from HP-23 showed no obvious homology with Escherichia coli RNA polymerase subunits or with any of its sigma factors.

Highlights

Polymerase I1 (1.5 X lo4 kDa) have been cloned from yeast [13],Drosophila [14], and bovine cells.’ The immunologicalrelationship of RNA polymerase I1from different eukaryotic organisms has been well documented [15]
When >2 kgof calf thymus were used for protein purification, the eluted material from the phosphocellulose column was passed through another DE52 column followed by a heparin-agarose column
Immuno-cross-reactivity of Calf Thymus Antibodieswith Human RNA Polymerase11Subunits-Calf thymus was used for large scale purification of RNA polymerase I1 because it was easilyavailable

Summary

Restriction enzymes were purchased from New England Biolabs

This article must and theprotoblot immunoscreening system were obtained from Protherefore be hereby marked “advertisement” in accordance with 18 mega. Oligolabeling kits were bought from Pharmacia LKB Biotechnology Inc. The composition of all solutions and buffers are as follows: TBE: 30 mM Tris, 45 mM boric acid, and 2 mM EDTA, adjusted to pH 9; PBS: 5.8 mM phosphate, 140 mM NaCl, and 3 mM KCl, pH 7.9; RNA transfer formaldehyde gel buffer: 40 mM MOPS: pH 7.2,lO mM sodium acetate, 1mM EDTA; 2 X SSPE: 0.36 M NaC1, 10 mM sodium phosphate, pH7.7,l mM EDTA; TBST buffer: 10 mM Tris-HC1, pH 8.0,150 mM NaC1, 0.05% Tween-20; Transcription reaction mixture: 10 mM Tris, pH 7.9,7.5 mMMgC12, 50 mM KCl, 10% glycerol, 0.25 mM dithiothreitol, 500 p~ ATP, GTP, UTP, 10 p~ [cx-~'P]CTP(800 Ci/mmol), 80 p~ dCTP, 50 pg of DNA template (Ad major late promoter -680 to +197), and SlOO extract (10 pl); Luria broth (LB) was made with 10 g/liter of tryptone, 5 g/liter of sodium chloride, and 10 g/liter of yeast extract with addition of 15 g/ liter of agarose for plates and 100 mg/liter of ampicillin for drug selection

Methods

RESULTS

CT control

Molecular Cloning of cDNfRoANr A