Molecular cloning, cDNA structure and predicted amino acid sequence of bovine 3 beta-hydroxy-5-ene steroid dehydrogenase/delta 5-delta 4 isomerase

We have used our recently characterized human 3 beta-hydroxy-5-ene steroid dehydrogenase/delta 5-delta 4-isomerase (3 beta-HSD) cDNA as probe to isolate cDNAs encoding bovine 3 beta-HSD from a bovine ovary lambda gtll cDNA library. Nucleotide sequence analysis of two overlapping cDNA clones of 1362 bp and 1536 bp in length predicts a protein of 372 amino acids with a calculated molecular mass of 42,093 (excluding the first Met). The deduced amino acid sequence of bovine 3 beta-HSD displays 79% homology with human 3 beta-HSD while the nucleotide sequence of the coding region shares 82% interspecies similarity. Hybridization of cloned cDNAs to bovine ovary poly(A)+ RNA shows the presence of an approximately 1.7 kb mRNA species.     

Steroid hormone synthesis by a vaccinia enzyme: a new type of virus virulence factor.

Vaccinia virus open reading frame (ORF) SalF7L has 31% amino acid identity to human 3 beta-hydroxysteroid dehydrogenase/delta 5-delta 4 isomerase (3 beta-HSD). Here we show that SalF7L encodes an active 3 beta-HSD, by the conversion of pregnenolone to the steroid hormone progesterone. The gene is transcribed early during infection into a 1.4 kb mRNA from an initiation site 12 bp upstream of the ORF. An antiserum raised against bacterially expressed SalF7L immunoprecipitated a 38 kDa polypeptide from infected cells, but not from mock infected cells or from cells infected with a mutant virus from which the SalF7L ORF had been removed. Deletion of the gene had no effect on virus replication in CV-1 cells in culture, yet the deletion mutant was attenuated when intranasally inoculated into mice. This steroid hormone synthesizing enzyme is a novel type of virus virulence factor.     

Structural analysis of the gene encoding human 3 beta-hydroxysteroid dehydrogenase/delta 5----4-isomerase

The structural gene encoding human 3 beta-hydroxysteroid dehydrogenase/delta 5----4-isomerase (3 beta HSD) was isolated from a human EMBL3 genomic library. The gene encompasses approximately 8 kilobases of DNA and is comprised of two large introns and three exons encoding amino acid residues 1-48, 49-103, and 104-373, respectively. The exonic sequence is identical to that of the cDNA that we previously isolated and expressed in COS 1 cells. DNA sequence analysis reveals a putative TATA (TATATAA) motif 26 basepairs up-stream of the beginning of exon I, as determined by S1 nuclease protection analysis. However, primer extension analysis using poly(A)+ RNA isolated from both placenta and corpora lutea indicates that the RNA initiates up-stream of the putative TATA motif, and that an additional 53-basepair exon, which is untranslated, is present 5' to the first coding exon. Southern hybridization analysis of genomic DNA using a single exon probe suggests that there may be more than one copy of the gene in the human genome. In addition, we confirm from Southern analysis of genomic DNA isolated from human x hamster somatic cell hybrids that the gene is located on human chromosome 1. These findings will provide a foundation for the characterization of apparent 3 beta HSD clinical deficiencies when these are due to a mutation in the structural gene.     

Superoxide dismutase as a regulatory switch in mammalian testicular steroidogenesis

The delta 4-pathway of testosterone biosynthesis in leydig cells, widely believed to proceed through pregnenolone--greater than pregnenedione--greater than progesterone route catalyzed by 5 delta-3 beta-hydroxysteroid dehydrogenase and delta 5-delta 4-isomerase respectively is shown to pass through an alternate pathway mediated by superoxide dismutase and peroxidase. A built-in regulatory switch is incorporated in this route, with the superoxide dismutase inducible upon LH-stimulation of the leydig cells.     

Characterization of the structure-activity relationships of rat types I and II 3 beta-hydroxysteroid dehydrogenase/delta 5 -delta 4 isomerase by site-directed mutagenesis and expression in HeLa cells

The membrane-bound enzyme 3 beta-hydroxysteroid dehydrogenase/delta 5 -delta 4 isomerase (3 beta-HSD) catalyzes the conversion of delta 5 -3 beta-hydroxysteroid precursors into delta 4-ketosteroids, thus representing an essential step in the biosynthesis of all classes of hormonal steroids. We have recently characterized two types of cDNA clones encoding rat 3 beta-HSD proteins, the rat type I protein being much more active than type II. In order to characterize further the functional difference between these two 3 beta-HSD types, transient expression of type I and type II 3 beta-HSD cDNAs was performed in HeLa human cervical carcinoma cells. The present study demonstrates that the type I 3 beta-HSD protein has a relative specificity 64- and 46-fold higher than type II protein for pregnenolone (PREG) and dehydroepiandrosterone (DHEA) as substrates, respectively. The Km values of type I and type II enzymes were calculated at 0.74 and 14.3 microM, respectively, using PREG as substrate whereas the respective Km values were 0.68 and 12.9 microM when DHEA was used, thus showing that their different relative specificity results largely from a different affinity for substrates. Since the change of 4 amino acid residues in type II could prevent the formation of a putative membrane-spanning domain (MSD) predicted between amino acid residues 75 and 91, chimeric cDNAs containing either type I MSD in type II (II + MSD) or an absence of this MSD in type I (I-MSD) were constructed and transiently expressed. The addition of MSD intype II 3 beta-HSD markedly increased the affinity leading to Km values similar to those found in type I 3 beta-HSD, namely 0.36 and 0.40 microM for PREG and DHEA, respectively. II + MSD chimera thus encodes a protein having a relative specificity for PREG and DHEA of 58 and 73%, respectively, to that of native type I 3 beta-HSD. Moreover, removal of MSD in the type I protein (I-MSD chimera) decreased the relative specificity of type I 3 beta-HSD protein for PREG and DHEA to only 0.37 and 0.48%, with respective Km values of 11.7 and 11.0 microM, thus strongly indicating the functional importance of this putative MSD which is predicted in wild type rat type I as well as in macaque and human 3 beta-HSD proteins.     

Plant expression signals of the Agrobacterium T-cyt gene.

Within the 5' and 3' non-coding regions of the T-cyt gene from the octopine T-DNA of Agrobacterium tumefaciens sequences required for expression of this gene in plant cells were identified by deletion mutagenesis. The results show that 184 bp of the 5' non-coding region and 270 bp of the 3' non-coding region are sufficient for wild-type expression. Within the 5' non-coding region two essential expression signals were identified: (1.) an activator element located between -185 and -129 with respect to the ATG start codon and (2.) one out of two TATA boxes. Deletions of the activator element or the two TATA boxes resulted in nonfunctional genes. Deletion of the upstream TATA box and both putative CAAT boxes did not significantly affect expression. Within the 3' non-coding region, the polyadenylation box most distal to the stop codon was not essential for expression, but sequences more upstream, including a second polyadenylation box were found to be required for wild-type expression.