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The yeast gene encoding the 95-kDa subunit of the class III gene transcription factor TFIIIC was cloned. This gene, termed TFC1 (transcription factor C, gene 1), was isolated by screening a lambda gt11 yeast cDNA expression library using a polyclonal antiserum preparation which was previously shown to specifically recognize the 95-kDa subunit of yeast TFIIIC (Parsons, M. C., and Weil, P. A. (1990) J. Biol. Chem. 265, 5095-5103). TFC1 was found to be a single copy gene which contained a continuous open reading frame about 2 kilobases in length. TFC1 was shown to encode the 95-kDa subunit of TFIIIC by several criteria. Like the authentic yeast protein, the protein encoded by TFC1 had an apparent molecular weight of 95,000. In addition, the protein encoded by the TFC1 gene bound to the same antibody species as the yeast 95-kDa subunit of TFIIIC. Last, the sizes of the cleavage products of the Escherichia coli-expressed protein were indistinguishable from those of the cleavage products of the bona fide yeast 95-kDa protein.