Regulation of renal EGF receptor expression is normal in Denys-Drash syndrome.

In patients with Denys-Drash syndrome, mutations of the Wilms' tumor suppressor gene are associated with nephroblastomas and developmental abnormalities of the genital tract and renal glomerulus. Normally, the Wilms' tumor gene product (WT1) is expressed at high levels in visceral glomerular epithelial cells (VGEC) of the emerging fetal glomerulus. We demonstrate that WT1 could normally serve to suppress EGF receptor expression in VGEC, since immunoreactive EGF receptor is strikingly absent compared to epithelial cells of the emerging proximal and distal tubule, which lack WT1. When HEK293 cells were co-transfected with plasmids containing EGFR enhancer/promoter elements linked to a CAT reporter and plasmids containing WT1 cDNA, EGFR enhancer/promoter activity was suppressed by all wild-type WT1 isoforms, but not by deletion mutants of WT1 lacking normal zinc-finger or N-terminal domains. Surprisingly, plasmids expressing a Denys-Drash WT1 mutant (R394W) retained the ability to suppress EGFR promoter activity in this system. Furthermore, we found that immunoreactive EGFR was appropriately undetectable in glomeruli from a three-year-old girl with Denys-Drash syndrome and in sections of her Wilm's tumor. These data suggest that faulty suppression of EGFR cannot account for the abnormalities of glomerulogenesis seen in Denys-Drash patients.

Cloning of a cDNA for short/branched chain acyl-Coenzyme A dehydrogenase from rat and characterization of its tissue expression and substrate specificity.

The acyl-CoA dehydrogenases are a family of related enzymes which catalyze the alpha,beta-dehydrogenation of acyl-CoA esters, transferring electrons to electron-transferring flavoprotein. A cDNA for human short/branched chain acyl-CoA dehydrogenase has recently been cloned, and it has been suggested that this enzyme represents the human homolog for the previously reported 2-methyl branched chain acyl-CoA dehydrogenase purified from rat liver. We now report the cloning and expression of rat short/branched chain acyl-CoA dehydrogenase and characterization of its substrate specificity. The rat enzyme is more active toward longer carbon side chains than its human counterpart, while the human enzyme can utilize substrates with longer primary carbon chains. In addition, short/branched chain acyl-CoA dehydrogenase can utilize valproyl-CoA as a substrate. Northern blotting of mRNA shows ubiquitous tissue expression of both the rat and human enzyme. Further study of these enzymes will be helpful in understanding structure/function relationships in this gene family.

Isolation and expression of a cDNA encoding the precursor for a novel member (ACADSB) of the acyl-CoA dehydrogenase gene family.

The acyl-CoA dehydrogenases (ACDs) are a family of mitochondrial enzymes that oxidize straight chain or branched chain acyl-CoAs in the metabolism of fatty acids or branched chain amino acids. Deficiencies in members of this gene family are important causes of human disease. A cDNA encoding the human precursor for a novel member (gene symbol ACADSB) of the ACD gene family has been isolated and characterized. The open reading frame of 1.3 kb encodes a precursor protein of 431 amino acids, which is processed in vitro to yield a mature protein of 399 amino acids. The cDNA has significant sequence similarity to other members of the acyl-CoA dehydrogenase family, with the greatest homology (38%) to the short chain acyl-CoA dehydrogenase. The cDNA was expressed in eukaryotic (COS) and prokaryotic (Escherichia coli) cells, producing a protein of the expected size, with activity toward the short branched chain acyl-CoA derivatives ((S)-2-methylbutyryl-CoA, isobutyryl-CoA, and 2-methylhexanoyl-CoA), as well as toward the short straight chain acyl-CoAs (butyryl-CoA and hexanoyl-CoA).