THE PAX-5 GENE IS ALTERNATIVELY SPLICED DURING B-CELL DEVELOPMENT

The transcription factor Pax-5 is expressed during the early stages of B-cell differentiation and influences the expression of several B-cel l-specific genes, In addition to the existing isoform (Pax-5, which we have named Pax-5a), we have isolated three new isoforms, Pax-5b, Pax- 5d, and Pax-5e, from murine spleen and B-lymphoid cell lines using lib rary screenings and polymerase chain reaction amplification. Isoforms Pax-5b and Pax-5e have spliced out their second exon, resulting in pro teins with only a partial DNA binding domain, Isoforms Pax-5d and Pax- 5e have deleted the 3'-region, which encodes the transactivating domai n, and replaced it with a novel sequence. The existence of alternative Pax-5 transcripts was confirmed using RNase protection assays, Furthe rmore, Pax-5a and Pax-5b proteins were detected using Western blot ana lysis, Pax-5a was detectable in pro-, pre-, and mature B cell lines, b ut not in two plasmacytomas; Pax-5b was shown to be present at low lev els in mature B-cell lines and, unexpectedly, in one plasma cell line, but not in pro-B cell or T-cell lines. Mobility shift assays showed t hat in vitro translated Pax-5a and Pax-5d, but not Pax-5b or Pax-5e, c ould interact with a B-cell-specific activator protein-binding site on the blk promoter, Using this assay, we also showed that Pax-5d was pr esent in nuclear extracts of some (but not all) B-lymphoid lines and i nteracts with the B-cell-specific activator protein-binding site. The pattern of differential expression of alternatively spliced Pax-5 isof orms suggests that they may be important regulators of transcription d uring B-cell maturation.