DNA binding and transcription activation specificity of hepatocyte nuclear factor 4.

Hepatocyte nuclear factor 4 (HNF-4) is an orphan intracellular receptor that appears to be a key factor in the regulation of energy metabolism. In order to gain greater understanding of the binding and activation requirements of HNF-4, we performed genetic analysis of the apoCIII promoter, a promoter that has previously been shown to be highly sensitive to HNF-4-induced transcription. We identified two elements within the apoCIII promoter that bind HNF-4, either of which are sufficient to confer promoter induction in response to HNF-4.

Utilization of recombinant adenovirus and dominant negative mutants to characterize hepatocyte nuclear factor 4-regulated apolipoprotein AI and CIII expression.

Using recombinant adenoviral vectors and a dominant negative mutant of HNF-4, we have examined the contribution of hepatocyte nuclear factor 4 (HNF-4) to endogenous apolipoprotein AI and CIII mRNA expression. Overexpression of HNF-4 leads to a 7.4-fold increase in apolipoprotein CIII expression, while infection with the dominant negative mutant of HNF-4 reduces the level of apolipoprotein CIII mRNA by 80%, demonstrating that endogenous HNF-4 is necessary for apolipoprotein CIII expression.

Expression purification and characterization of recombinant human inducible prostaglandin G/H synthase from baculovirus-infected insect cells.

The inducible isoform of human prostaglandin G/H synthase (human cyclooxygenase; hCOX2) has been produced in Sf21 insect cells using the baculovirus expression system. The full-length gene for hCOX2 was placed under the control of the hybrid pCap/PolH promoter and recombinant virus generated by homologous recombination. Insect cells infected with recombinant virus synthesized active hCOX2 at levels exceeding 5% of total cellular protein 72 h postinfection.

Mutations in the -10 TATAAT sequence of the gyrA promoter affect both promoter strength and sensitivity to DNA supercoiling.

Transcription of the gyrA and gyrB genes, which encode the subunits of DNA gyrase, increases in response to DNA relaxation. Previous studies have shown that a small segment of DNA extending from the -10 consensus hexamer to the start of transcription encodes the sequence determinants for this response. In this study, we examined the role of the -10 region in relaxation-stimulated transcription (RST).

Expression and functional characterization of recombinant human vascular cell adhesion molecule-1 (VCAM1) synthesized by baculovirus-infected insect cells.

Vascular cell adhesion molecule-1 (VCAM1) is a cell surface glycoprotein produced by the vascular endothelium, as well as on macrophage-like and dendritic cell types, in response to certain inflammatory stimuli. VCAM1 interacts with the integrin VLA4 present on mononuclear leukocytes. We have isolated the cDNA for VCAM1 using RT-PCR by screening a cDNA library from IL-1 beta-activated human endothelial cells.