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. 1992 Mar 25;20(6):1355–1361. doi: 10.1093/nar/20.6.1355

An alternatively spliced Pit-1 isoform altered in its ability to trans-activate.

A E Morris 1, B Kloss 1, R E McChesney 1, C Bancroft 1, L A Chasin 1
PMCID: PMC312183  PMID: 1561093

Abstract

Although alternative splicing has been shown to give rise to isoforms of a number of transcription factors, such isoforms have not previously been detected for the POU homeodomain protein Pit-1. Screening of a rat pituitary GH3 cell cDNA expression library yielded a clone, termed pCMVPit-1a, encoding a 35.8 kD protein (Pit-1a) containing a 26 amino acid insert in the Pit-1 trans-activation domain. The position of the insert, plus Southern blot analysis, implied that Pit-1a mRNA arises by alternative splicing of the Pit-1 gene transcript. Pit-1a mRNA was detected in GH3 rat pituitary tumor cells at levels about 1/7 that of Pit-1 mRNA. Pit-1a mRNA-specific sequences were also detected in rat and mouse pituitary, and in mouse thyrotropic tumor TtT cells. DNA mobility shift assays showed that Pit-1a binds specifically to Pit-1 binding sites in the proximal prolactin promoter, but produces DNA-protein complexes of markedly different mobilities than Pit-1. In stably transfected CHO cells which accumulated approximately equal levels of either of the two proteins, Pit-1 trans-activated a prolactin promoter-driven CAT construct, while Pit-1a yielded no detectable transactivation, implying a trans-activation ratio for Pit-1a/Pit-1 of less than 0.05. Thus, the insertion of 26 amino acids of similar composition into the activation domain of Pit-1 has at once affected both the mode of binding of this protein and its ability to function as a trans-activator.

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