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A Maxam Gilbert sequencing (G/A) reaction was used as a size marker ( sequences −299/−137, whereas footprints 1–4 were removed individually by further 5′-deletions.

The 3′-end of these deletion mutants was maintained at nt 7 relative to the transcription start site (Fig.3).

The regulation of SHBG production has been studied using human Hep G2 hepatoblastoma cells (11, 12), but little is known about the molecular control of have been introduced into the mouse genome (13, 14), and the temporal and spatial expression of these transgenes generally reflects the species-specific patterns of expression associated with these genes.

In essence, a rat transgene, which included an ∼1.5-kb sequence flanking the major transcription start site in the rat testis, was expressed in the mouse testis at high levels, but no expression could be detected in the livers of adult transgenic mice (14). The Nuclear proteins were isolated from adult mouse liver (16), and the protein content of the extracts was determined by Bradford protein assay using bovine serum albumin as standard (17).

Human Hep G2 hepatoblastoma cells were transfected transiently with each promoter reporter plasmid together with a p CMVlac Z plasmid as a control for transfection efficiency. E.; ( promoter deletion fragment was also analyzed in the context of a luciferase reporter gene in He La cells to assess the cellular specificity of its transcriptional activity (not shown).

Although the promoter was active in this cell line, the reporter gene expression was lower than in Hep G2 cells and regulation at each of the footprinted regions was not observed.

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Little, if any, expression of this human proximal promoter sequences that flank the transcription units for human SHBG in the liver and rat androgen-binding protein in the testis, and we used mouse liver nuclear protein extracts and human hepatoblastoma cells to identify possible targets for transcriptional regulators within the human promoter-luciferase reporter construct. DNase I (Amersham Pharmacia Biotech, FPLC pure) was used at a dilution (0.45 units/20-μl reaction) to digest approximately 20,000 cpm of end-labeled probe in an footprinting assay (16).When this sequence was replaced with an idealized HNF-4-binding site, the transcriptional activity of the promoter increased in Hep G2 cells.