Production of monoclonal antibodies against RNA polymerase I from nonimmunized autoimmune MRL/lpr mice and their use in rDNA transcription analysis.

A L Barsoum,M L Webb,C D Balaban,S T Jacob

doi:10.1016/s0021-9258(18)45271-4

Abstract

A hybridoma secreting monoclonal antibodies against RNA polymerase I was produced by the fusion of myeloma cells with spleen cells from a nonimmunized MRL/lpr mouse which is known to produce autoantibodies to RNA polymerase I. The antibodies (McAb-2D11) belong to the IgG2b subclass, reacted specifically with the second largest (120 kDa) subunit of RNA polymerase I, and inhibited accurate transcription of cloned rat rDNA in a fractionated cell extract following immunoprecipitation of RNA polymerase I. McAb-2D11 did not inhibit RNA polymerase II-mediated transcription of the mouse metallothionein-I gene. Immunocytochemical procedures with biotinylated second antibody demonstrated specific immunolocalization of RNA polymerase I in the nucleus. These studies have (a) presented direct evidence that autoantibodies to functional RNA polymerase I are produced in a murine model of systemic lupus erythematosus, (b) demonstrated specificity of the monoclonal antibody for RNA polymerase I, and (c) provided a useful tool for the purification of RNA polymerase I and/or transcription factor(s) associated with RNA polymerase I.

Highlights

(McAb-2D11) belong to the IgG2b subclass, reacted with the second largest (120 kDa) subunit of RNA polymerase I, and inhibited accurate transcription of cloned rat rDNA in a fractionated cell extract following immunoprecipitation of RNA polymerase I
Hybridoma Screening anSdubtype-Hybridoma 2D11 from bodies to functional RNA polymerase I are produced in a single MRL/lpr spleenfusion experiment reactedpositively a murine model of systemic lupus erythematosus, ( b ) to purified RNApolymerase I in an enzyme-linked immunodemonstrated specificity of the monoclonal antibody sorbent assay analysis(see “Experimental Procedures”)
Western Blot Analysis-Extensively purified RNA polymerase I from Morris hepatoma3924A (Rose etal., 1981) or the Eukaryotic RNA polymerase I, the enzyme responsible for enzyme partially purified by chromatography on a DEAE

Summary

RESULTS

Zation of RNApolymerase I in the nucleus. These studies have (a)presented direct evidence that autoanti-. Hybridoma Screening anSdubtype-Hybridoma 2D11 from bodies to functional RNA polymerase I are produced in a single MRL/lpr spleenfusion experiment reactedpositively a murine model of systemic lupus erythematosus, ( b ) to purified RNApolymerase I in an enzyme-linked immunodemonstrated specificity of the monoclonal antibody sorbent assay analysis(see “Experimental Procedures”). RNA polymerase I purified analysis (Fig. 1).In either case, only one major band correfrom a rat hepatoma (Rose eatl., 1981) and other sources Deline- polymerase I preparations (data not shown) These polypepation of subunit function(s) of this enzyme and eukaryotic tides are probably degradation producotsf the 120-kDapoly-. One way toaddressthis issue isto produce monoclonal higher than kDa, the third largest subunoift RNA polymantibodies against individual subunits anudse them as probes erase I, and freshly prepared highly purified enzyme prepain functional analysis.

Present address

Findings

DISCUSSION