Abstract
A 43-kDa DNA binding protein which recognizes the TGACGTCA element of the rat somatostatin promoter has been purified from rat brain. Purification of the protein involved initial separation of three sequence-specific binding activities, b1-b3, from each other using DEAE-Sepharose chromatography. The protein corresponding to the b2 complex was further purified to apparent homogeneity by two cycles of sequence-specific DNA affinity chromatography, yielding a single species with an apparent mass of 43,000 daltons on a silver-stained polyacrylamide gel. Sequence-specific DNA binding of this purified protein was demonstrated by Southwestern blotting, renaturation, and DNase I footprinting studies. The 43-kDa protein was phosphorylated on serine residue(s) by the catalytic subunit of cAMP-dependent protein kinase, as shown by phosphoamino acid analysis. Furthermore, the purified protein specifically stimulated transcription from the rat somatostatin promoter in an in vitro transcription system. These results indicate that this 43-kDa protein is a transcription factor required for somatostatin gene expression.
Highlights
A 43-kDa DNA binding protein which recognizes the TGACGTCA element of the rat somatostatin promoter has been purified from rat brain
The purified protein stimulated transcription from the rat somatostatin promoter in an in vitro transcription system. These results indicate that this 43-kDa protein is a transcription factor required for somatostatin gene expression
Purification of the 43-kDa Protein-Previous studies have identified three sequence-specific DNA-protein complexes formed when extracts of CA-77, HeLa, or rat brain cells were incubated with the -70 to -29 region of the ratsomatostatin promoter [10].The availability of large quantities of rat brain made it thteissue of choice in purification of the DNA binding proteins
Summary
‘One activity unit (unit) is arbitrarilydefined as theDNA binding activity of 43-kDa protein from 1mg of the (NH4)2S04-fractionatedrat brain extract. Renaturation of the 43-kDa Protein from SDS-PAGE-The affinity column-purified material was mixed with 1%SDS andheated a t 54°C for 15 min before being subjected to electrophoresis. After the extract was precipitated with 5 volumes of acetone, the precipitates were dissolved in 6 M guanidine HCl. The mixtures were incubated a t room temperature for 20 min and dialyzed overnight against 20 mM Hepes buffer, pH 7.9, containing 20% glycerol, 0.1 mM EDTA, 5 mMMgC12, 2 mM DTT, and 100 mM KCl. The renatured samples were tested for the DNA binding activity by DNase I footprinting assay. In Vitro Transcription-Preparation of HeLa cell extracts and somatostatin promoter transcription activity-depleted extracts will be described elsewhere [19]. The TGACGTCA binding activity depleted extract was prepared by passing the HeLa extract through a DNA affinity column. The adenovirus major late promoter was used as aninternal control
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