Differential in vitro transcription from the promoter of a rat alpha 2u globulin gene in liver and spleen nuclear extracts.

When used in an in vitro transcription assay, the promoter of a cloned alpha 2u globulin gene is much more active in liver than in spleen nuclear extracts. Promoter deletion experiments suggest that both positive and negative regulatory mechanisms may be involved in the differential in vitro transcription from the alpha 2u globulin promoter in these two nuclear extracts. Interestingly, removal of promoter elements upstream from position -74 results in a significant increase of in vitro transcription in spleen but not in liver nuclear extracts, and thus reduces the difference in transcription observed with longer alpha 2u promoters in these two extracts. Deletion of additional nucleotides to position -43 strongly reduces the in vitro transcription efficiency of the promoter in extracts from both tissues. None of the examined promoters containing between 3000 and 22 nucleotides of 5' flanking regions are differentially transcribed in liver nuclear extracts from either male or female rats. Thus, in contrast to cell-type specificity, sex-specificity could not be observed in our in vitro transcription experiments. DNase I protection experiments with crude nuclear extracts and partially or highly purified nuclear proteins suggests the presence of six recognition sites for DNA-binding factors between the TATA element and position -210. Some of these factors could be identified as proteins that also bind to elements within the albumin gene promoter.

Rapid identification of DNA fragments containing promoters for RNA polymerase II.

We describe a direct procedure for screening genomic recombinant DNA libraries or restriction fragments of cloned DNA regions for RNA polymerase II promoters. Cellular polyadenylated mRNA is chemically de-capped by beta-elimination reaction and enzymatically re-capped with [alpha-32P]GTP by vaccinia guanylyl transferase. Since this enzyme only accepts di- or triphosphorylated 5' termini as a substrate, the mRNAs are labeled exclusively at the first nucleotide, irrespective of whether the mRNA was intact or fragmented before in vitro capping. By using in vitro-capped mRNA as a hybridization probe, recombinant DNA molecules or restriction fragments that carry a cap site (and thus likely an RNA polymerase II promoter) can directly be identified. Here, we demonstrate the applicability of this procedure by the isolation and characterization of several genomic DNA clones containing RNA polymerase II promoter sequences, that are highly active in liver.