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-cell-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 library screenings and polymerase chain reaction amplification. Isoforms Pax-5b and Pax-5e have spliced out their second exon, resulting in proteins with only a partial DNA-binding domain. Isoforms Pax-5d and Pax-5e have deleted the 3'-region, which encodes the transactivating domain, and replaced it with a novel sequence. The existence of alternative Pax-5 transcripts was confirmed using RNase protection assays. Furthermore, Pax-5a and Pax-5b proteins were detected using Western blot analysis. Pax-5a was detectable in pro-, pre-, and mature B-cell lines, but not in two plasmacytomas; Pax-5b was shown to be present at low levels 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 that in vitro translated Pax-5a and Pax-5d, but not Pax-5b or Pax-5e, could interact with a B-cell-specific activator protein-binding site on the blk promoter. Using this assay, we also showed that Pax-5d was present in nuclear extracts of some (but not all) B-lymphoid lines and interacts with the B-cell-specific activator protein-binding site. The pattern of differential expression of alternatively spliced Pax-5 isoforms suggests that they may be important regulators of transcription during B-cell maturation.
Zwollo
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Department of Biology, Occidental College, Los Angeles, California 90041, USA. Zwollop@bengal.oxy.edu
Arrieta
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Ede
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Molinder
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Desiderio
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Pollock
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D017398:Alternative Splicing; D000595:Amino Acid Sequence; D000818:Animals; D001402:B-Lymphocytes / Q000166:cytology*; D001483:Base Sequence; D015153:Blotting, Western; D002454:Cell Differentiation; D018076:DNA, Complementary / Q000737:chemistry; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000235:genetics* / Q000378:metabolism; D051757:PAX5 Transcription Factor; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000378:metabolism; D011817:Rabbits; D012260:Ribonucleases / Q000378:metabolism; D014157:Transcription Factors / Q000235:genetics* / Q000378:metabolism
D018076:DNA, Complementary; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D051757:PAX5 Transcription Factor; C497281:PAX5 protein, human; D012333:RNA, Messenger; D014157:Transcription Factors; D012260:Ribonucleases