Inhibition Of Luciferase Expression Research Articles

Effect of Polyethylene Glycol Conjugated to DNA-Transfecting Complexes Targeted at the Transferrin Receptor of HeLa Cells

Modification of streptavidin (biotinylated transferrin)-biotinylated polylysine conjugates for DNA delivery by covalent addition of polyethylene glycol chains was investigated. It was found that addition of two to four monomethoxy-polyethylene glycol chains to bio20-polylysine100, a component of the transfecting conjugate, increased luciferase activity approximately four- to five-fold in a HeLa cell system. The increase occurred only in the presence of the lysosomotropic agent chloroquine and was inhibited by free transferrin. The latter result indicating that DNA uptake was via transferrin receptor-mediated endocytosis. Titration of cell cultures with transfecting complexes, with and without attached polyethylene glycol, showed luciferase activity to decrease at higher concentrations of the complexes. This was more marked with complexes containing polyethylene glycol. Complexes containing higher concentrations of attached polyethylene glycol inhibited luciferase expression strongly. Free polyethylene glycol at equivalent concentrations was without effect. It is suggested that at higher complex concentrations, the larger amounts of polyethylene glycol present occlude transferrin receptors, thus preventing uptake of DNA-containing complexes. The results indicate that transfecting complexes containing small amounts of polyethylene glycol function more efficiently than those without.

Liver-specific Enhancer of the Glucokinase Gene

Glucokinase gene regions that are important for liver specific expression of the enzyme have been functionally identified using transient transfection of rat hepatocytes. Maximal luciferase activity was elicited by a reporter plasmid with 3.4 kilobase pairs of genomic DNA flanking the liver glucokinase promoter. Deletion of a gene fragment between -1000 and -600 with respect to the start of transcription resulted in a 60% decrease in luciferase activity. Further reduction, close to background level, occurred upon deletion of a 90-base pair sequence between -123 and -34. Reporter plasmids with the liver glucokinase promoter and any length of flanking sequence were minimally active in INS-1 insulinoma cells, and conversely reporters with the beta-cell-specific promoter were ineffective in primary hepatocytes. In FTO-2B hepatoma cells, a differentiated line expressing many liver-specific traits but not the endogenous glucokinase gene, the promoter proximal region between -123 and -34 markedly stimulated the expression of transfected plasmids above background. However, addition of the flanking region up to -1000 inhibited luciferase expression. The gene fragment from -1003 to -707 was shown to be a bona fide, hepatocyte-specific enhancer by the following criteria: 1) it stimulated reporter expression by more than 10- and 5-fold when inserted directly upstream of the glucokinase TATA box or complete promoter, respectively, regardless of orientation; 2) it stimulated gene expression from the heterologous SV 40 promoter 4-fold; 3) it was also effective from a downstream position; and 4) in contrast to the enhancer effect in primary hepatocytes, the sequence acted as a silencer in FTO-2B cells and was neutral in INS-1 cells. Both the promoter proximal and the enhancer regions were marked by DNase I hypersensitive sites in the chromatin of primary hepatocytes but not hepatoma or insulinoma cells. Seven footprinted elements termed A through G were mapped in the enhancer by the in vitro DNase I protection assay. Elements A-C may bind liver enriched factors, because they were not protected by spleen nuclear extract. In hepatocyte transfection, the downstream half of the enhancer containing elements A-C was about half as effective as the complete enhancer in stimulating glucokinase promoter activity. Site-directed mutagenesis of element A virtually abrogated the activity of the half-enhancer, whereas mutation of element C had a more moderate effect. The sequence between -732 and -578 upstream of the liver start of transcription in the human glucokinase gene displays 79% sequence identity with the downstream half of the rat enhancer. The human gene fragment ligated to the minimal rat liver glucokinase promoter was shown to work as an enhancer in the hepatocyte transfection system.

Characterization of the Mouse Epidermal Growth Factor Promoter and 5′-Flanking Region: ROLE FOR AN ATYPICAL TATA SEQUENCE

As a step toward delineating mechanisms that regulate its activity, we have characterized the mouse epidermal growth factor (EGF) promoter. Primer extension and S1 nuclease analyses identified prominent (+1/+2) and minor (+28) transcription start sites, with the dominant +1/+2 site located 33 bases downstream from a TTTAAA sequence. A restriction fragment that spanned these start sites and contained 390 base pairs of 5'-flanking sequence directed transcription from the +1/+2 site in vitro in the presence of HeLa cell nuclear extracts. Additionally, it promoted expression of a coupled luciferase reporter gene in transfected cell lines. The inclusion of additional 5'-flanking sequence either stimulated or inhibited luciferase expression depending on the cell line. Approximately 2 kilobases of EGF 5'-flanking sequence was determined and found to contain several motifs with partial homology to steroid hormone response elements. Despite this fact and evidence that EGF expression might be regulated by androgens in vivo, EGF promoter-luciferase constructs were not steroid-responsive in cells cotransfected with steroid receptor expression vectors. An oligonucleotide containing the aforementioned TTTAAA sequence specifically bound TATA-binding protein and TFIIA in gel shift assays, and an EGF promoter-luciferase construct in which the core TA dinucleotide was mutated to CG was not active in transfected cells. These data suggest that the TTTAAA sequence functions as an atypical TATA box.