Isolation of Structural Genes for Yeast RNA Polymerases by Immunological Screening

• Published in: Proceedings of the National Academy of Sciences - PNAS 1986-03, Vol.83 (6), p.1554-1558
• Main Authors: Riva, Michel, Memet, Sylvie, Micouin, Jean-Yves, Huet, Janine, Treich, Isabelle, Dassa, Janie, Young, Richard, Buhler, Jean-Marie, Sentenac, André, Fromageot, Pierre
• Format: Article
• Language: English
• Subjects: Antibodies; Antibodies, Fungal - immunology; Bacteriophage lambda - genetics; Bacteriophages; Biological and medical sciences; Cloning, Molecular; DNA; DNA probes; DNA, Recombinant - analysis; DNA-Directed RNA Polymerases - genetics; DNA-Directed RNA Polymerases - immunology; Drosophila melanogaster - genetics; Escherichia coli - genetics; Fundamental and applied biological sciences. Psychology; Fungal Proteins - genetics; Fungal Proteins - immunology; Genes; Genes, Fungal; Genes. Genome; Genetic hybridization; Genetic Markers; Genetic screening; Immunologic Techniques; Immunology; Messenger RNA; Molecular and cellular biology; Molecular genetics; Nucleic Acid Hybridization; Recombinant Proteins - genetics; Recombinant Proteins - immunology; RNA; RNA, Fungal - analysis; RNA, Messenger - analysis; Saccharomyces cerevisiae - genetics; Saccharomyces cerevisiae - immunology; Yeasts
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.83.6.1554

A λ gt11 yeast genomic library was screened with antibodies directed against yeast RNA polymerases A, B, and C. Thirty-five individual recombinant phages that expressed proteins in Escherichia coli that were antigenically related to RNA polymerases A, B, or C were isolated by using 22 distinct antisera. Thus, all 22 genes for the RNA polymerase subunits were potentially cloned. In three cases (λ A-43, λ A-40, and λ A-34.5), an antigenic protein was expressed in E. coli with the same molecular weight as the corresponding subunit. When λ A-40 DNA was used to hybrid-select yeast mRNA, the protein translated in vitro was the expected size for the A-40 subunit, further supporting our isolation of the A-40 gene. However, mRNA hybrid selected by λ A-27 DNA did not code for a protein of the correct size. The lengths of the mRNA that hybridized to phage λ A-190 or λ C-160 DNA on RNA blots were in agreement with the predicted sizes of the coding regions of the corresponding genes. As predicted by our previous immunological results, yeast DNA inserts of the λ A-190 and λ C-160 clones cross-hybridized to the B-220 subunit gene. The cloned genes for the RNA polymerase subunits will prove to be valuable tools for the study of the function, regulation, and genetics of the yeast RNA polymerases.