While classical 3 beta-hydroxysteroid dehydrogenase/delta 5-delta 4 isomerase deficiency (3 beta-HSD) is a known cause of adrenal hyperplasia resulting in ambiguous genitalia and adrenal insufficiency at birth, nonclassical or late-onset 3 beta-HSD deficiency is found in an important proportion of women with androgen excess. We have previously isolated and sequenced the cDNA and gene for the human type I 3 beta-HSD, which represents the main species expressed in the placenta and skin. Recently, we isolated, sequenced, and expressed the functional cDNA encoding type II 3 beta-HSD, which is the predominant 3 beta-HSD expressed in human adrenals and gonads. The present study describes the isolation and complete sequence of the corresponding type II 3 beta-HSD gene, which is the form most likely responsible for human 3 beta-HSD deficiency. The structural gene contains four exons of 57, 231, 165, and 1,214 bp, respectively, separated by introns of 128, 3,383, and 2,162 bp. DNA sequence analysis of the 5'-flanking region reveals the existence of two putative TATA boxes situated 28 and 140 nucleotides upstream from the transcription start site whereas two putative CAAT boxes are located 57 and 38 nucleotides upstream from the TATA boxes, respectively. A restriction fragment length pattern specific for each gene has been characterized. The present findings should provide the tools required for detailed analysis of the molecular basis of 3 beta-HSD deficiency as well as of normal sex steroid biosynthesis.

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High levels of expression for the gene encoding human type I 3 beta-hydroxysteroid dehydrogenase (3 beta-HSDI) have been detected in placenta and skin but not in adrenals, which, however, express high levels of type II 3 beta-HSD. In this study, we addressed the issue of whether the differential pattern of cell-specific expression for type I 3 beta-HSD can be explained by the differential utilization of cis-acting regulatory elements present in the 3 beta-HSDI gene regulatory sequences. Deletion analyses indicated that removal of intron 1 strongly impaired the transcriptional activity directed by the 3 beta-HSDI basal promoter. Consequently, we focused our attention to the characterization of the 128 base pair first intronic sequence from the 3 beta-HSDI gene. A single protected region, designated the 3 beta I-A element, was identified by DNase I footprinting. Gel mobility shift assays indicated that at least four nuclear proteins with distinct biochemical and binding properties possess the ability to bind the 3 beta I-A element to produce four DNA-protein complexes (R1 to R4). However, the one producing R1, a 37-kilodalton protein that has been found in both human choriocarcinoma JEG-3 and adrenal cortex adenocarcinoma SW13 cells, as well as in all tested tissue culture cells, clearly accounts for the major 3 beta I-A-binding species. Site-directed mutagenesis provided the evidence that the 3 beta I-A element acts positively on the 3 beta-HSD-I gene promoter-mediated transcriptional activity upon transient transfection of both JEG-3 and SW13 cells. No homology has been found between the 3 beta I-A element and target sequences for other known transcription factors. In addition, of the four proteins binding the 3 beta I-A element, that producing R2 was identified as the positive transcription factor Sp1, whereas the identity of the remaining factors is still unknown. This is consistent with the presence of an Sp1 motif overlapping the 3 beta I-A element in intron 1, therefore pointing toward an important function played by this particular region in 3 beta-HSDI basal, but not cell-specific, gene expression.

Escherichia coli TB1 was transformed with pUC9 containing fragmented DNA (4–10 kilobases (kb)) from Corynebacterium sepedonicum. The resulting genomic bank was screened by a dot blot assay to identify clones specifically hydridizing to C. sepedonicum DNA and not to the DNA of several other Gram-positive and Gram-negative bacteria. Two clones (III24 and III31) were selected because of their ability to strongly hybridize to C. sepedonicum DNA and weakly hybridize to the DNA of C. michiganense, Erwinia carotovora, Agrobacterium tumefaciens, Bacillus subtilis, Pseudomonas solanacearum, Micrococcus luteus, and Arthrobacter globiformis. These two clones were also specific for C. sepedonicum DNA when tested against the DNA from 30 isolates of soil bacteria. Restriction enzyme analysis has shown that the two clones have an insert of 8 kb (III24) and 4 kb (III31). On the basis of restriction enzyme patterns, one clone (III24) does not correspond to plasmid pCL 50, a cryptic plasmid found in several C. sepedonicum isolates. Because purified III24 and III31 DNA can be used to detect approximately 1 ng of C. sepedonicum genomic DNA, the two clones can complement serological or biological detection methods. This could be useful, especially when a high degree of specificity is required for detection or identification of this plant pathogen.