Abstract
Expression of gastrin, a gut hormone and growth factor, has tissue-specific transcriptional regulation and can be induced in some tumors. Previous studies have shown that a CACC cis-regulatory element is important for transcriptional activation in pancreatic insulinoma cells. To identify CACC-binding proteins, a lambda phage cDNA library derived from a rat insulinoma cell line, RIN 38A, was screened by a Southwestern method. A novel member of the Cys2-His2 zinc finger gene family was cloned and designated RIN ZF, having a cDNA sequence of 3.8 kilobases. One full-length and a shorter splice variant were sequenced and had predicted protein masses of 91.6 and 88.7 kDa. Expression of both splice forms were ubiquitous in fetal and adult rat tissues. Recombinant RIN ZF protein exhibited sequence-specific binding to the gastrin CACC element in a gel mobility shift assay. In transient transfections, both splice variants appeared to have only weak activating effects on gastrin-luciferase reporter gene transcription. Furthermore, RIN ZF coexpression with Sp1 appeared to block the strongly activating effects of Sp1 mediated through the CACC element. These findings suggest that a novel set of zinc finger proteins may help regulate gastrin gene expression by interfering with Sp1 transactivation.
Highlights
Gastrin, a peptide hormone and growth factor of the gastrointestinal tract, is expressed in a developmental and tissuespecific manner
Several DNA-binding proteins demonstrating sequence-specific binding to the cotransfected into SL2 cells with a (CACC) element in the gastrin promoter were detected in nuclear extracts from RIN 38A cells
All of the cDNA inserts identified in this assay for CACC binding possessed zinc finger domains of the Cys2-His2 type, including one, clone 25A, which otherwise had no close homology with known genes
Summary
A peptide hormone and growth factor of the gastrointestinal tract, is expressed in a developmental and tissuespecific manner. Mutational analysis of the CACC element showed a close correlation between DNA binding by protein complexes and transcriptional activation of gastrin-luciferase reporter genes in insulinoma cell lines. Transfection of SL 2 Cells and Recombinant Protein Expression—The full-length coding region and 3Ј-untranslated sequences of RIN ZF and RIN ZFsv were excised and ligated in-frame with a start codon into an actin promoter-driven expression vector, pPac [19].
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