Probing yeast RNA polymerase A subunits with monospecific antibodies

Monoclonal antibodies were raised in mouse against native RNA polymerase A from Saccharomyces cerevisiae. After screening with the spot‐immunodetection technique, 14 hybridomas were selected and the antibodies produced in mice. Their specificity, analyzed by blot‐immunodetection, was found to be mar...

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Bibliographic Details
Published in:The EMBO journal 1982-01, Vol.1 (10), p.1193-1198
Main Authors: Huet, J., Phalente, L., Buttin, G., Sentenac, A., Fromageot, P.
Format: Article
Language:English
Subjects:
Animals
Antibodies, Monoclonal
Antigen-Antibody Complex
DNA Polymerase II - analysis
DNA Polymerase II - metabolism
Electrophoresis, Polyacrylamide Gel
Epitopes - analysis
Hybridomas - immunology
Macromolecular Substances
Mice
Mice, Inbred BALB C
Plasmacytoma - immunology
Saccharomyces cerevisiae
Saccharomyces cerevisiae - enzymology
Templates, Genetic
Transcription, Genetic
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Summary:Monoclonal antibodies were raised in mouse against native RNA polymerase A from Saccharomyces cerevisiae. After screening with the spot‐immunodetection technique, 14 hybridomas were selected and the antibodies produced in mice. Their specificity, analyzed by blot‐immunodetection, was found to be markedly biased towards a few RNA polymerase subunits: A135, A49, A43, and A14.5. A different monoclonal antibody directed against the largest subunit, A190, was obtained by immunizing a mouse with RNA polymerase A dissociated into its subunits with SDS. Two antibodies, which probably recognized the same antigenic determinant on subunit A135, inhibited in vitro RNA synthesis. Inhibition was prevented by preincubation of the enzyme with DNA, suggesting a role for the A135 subunit in template binding. The antibody directed against A14.5 interacted with the A14.5 kd subunit present in all three forms of the yeast nuclear RNA polymerases but did not interfere with RNA polymerase activity. These antibody probes will be useful to study subunit function in reconstituted transcription systems.
ISSN:0261-4189
1460-2075
DOI:10.1002/j.1460-2075.1982.tb00012.x