Isolation of structural genes for yeast RNA polymerases by immunological screening.
A lambda 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 (lambda A-43, lambda A-40, and lambda A-34.5), an antigenic protein was expressed in E. coli with the same molecular weight as the corresponding subunit. When lambda 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 lambda A-27 DNA did not code for a protein of the correct size. The lengths of the mRNA that hybridized to phage lambda A-190 or lambda 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 lambda A-190 and lambda 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.
D000908:Antibodies, Fungal / Q000276:immunology; D010582:Bacteriophage lambda / Q000235:genetics; D003001:Cloning, Molecular; D004274:DNA, Recombinant / Q000032:analysis; D012321:DNA-Directed RNA Polymerases / Q000235:genetics* / Q000276:immunology; D004331:Drosophila melanogaster / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D005656:Fungal Proteins / Q000235:genetics* / Q000276:immunology; D005796:Genes; D005800:Genes, Fungal; D005819:Genetic Markers; D007158:Immunologic Techniques; D009693:Nucleic Acid Hybridization; D012331:RNA, Fungal / Q000032:analysis; D012333:RNA, Messenger / Q000032:analysis; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000276:immunology
D000908:Antibodies, Fungal; D004274:DNA, Recombinant; D005656:Fungal Proteins; D005819:Genetic Markers; D012331:RNA, Fungal; D012333:RNA, Messenger; D011994:Recombinant Proteins; D012321:DNA-Directed RNA Polymerases