The use of monoclonal antibodies for the characterization of a 5 S gene-specific transcription factor (IIIA) from Xenopus laevis.

Monoclonal antibodies were raised against a 5 S gene-specific transcription factor (TFIIIA) from Xenopus laevis oocytes. Seven stable hybridoma lines were established and shown to secrete antibodies directed against different antigenic structures present on distinct peptides of the factor (generated by cleavage of TFIIIA with cyanogen bromide). All antibodies react with denatured TFIIIA in nitrocellulose blots, but only five are able to bind to the native protein. These antibodies recognize the transcription factor not only in its purified (RNA-free) form, but also when it is complexed with 5 S RNA in 7 S ribonucleoprotein complexes. None of the monoclonal antibodies inhibits transcription of 5 S genes in vitro, although an interaction of the antibodies with the factor in a transcription reaction could be demonstrated by a depletion assay. Thus, binding of the antibodies to TFIIIA does not interfere with the function of the factor during transcription initiation. Additionally, it was found that transcriptionally active TFIIIA could be purified to greater than 95% homogeneity from ovarian extracts using an antibody coupled to Sepharose.