Hsd Genes Research Articles

Arabidopsis 3β-Hydroxysteroid Dehydrogenases/C4-Decarboxylases Are Essential for the Pollen and Embryonic Development.

The biosynthesis of C27-29 sterols from their C30 precursor squalene involves C24-alkylation and the removal of three methyl groups, including two at the C4 position. The two C4 demethylation reactions require a bifunctional enzyme known as 3β-hydroxysteroid dehydrogenase/C4-decarboxylase (3βHSD/D), which removes an oxidized methyl (carboxylic) group at C4 while simultaneously catalyzing the 3β-hydroxyl→3-keto oxidation. Its loss-of-function mutations cause ergosterol-dependent growth in yeast and congenital hemidysplasia with ichthyosiform erythroderma and limb defect (CHILD) syndrome in humans. Although plant 3βHSD/D enzymes were well studied enzymatically, their developmental functions remain unknown. Here we employed a CRISPR/Cas9-based genome-editing approach to generate knockout mutants for two Arabidopsis 3βHSD/D genes, HSD1 and HSD2, and discovered the male gametophytic lethality for the hsd1 hsd2 double mutation. Pollen-specific expression of HSD2 in the heterozygous hsd1 hsd2/+ mutant not only rescued the pollen lethality but also revealed the critical roles of the two HSD genes in embryogenesis. Our study thus demonstrated the essential functions of the two Arabidopsis 3βHSD/D genes in male gametogenesis and embryogenesis.

Comparative Analyses Reveal the Genetic Mechanism of Ambergris Production in the Sperm Whale Based on the Chromosome-Level Genome.

Sperm whales are a marine mammal famous for the aromatic substance, the ambergris, produced from its colon. Little is known about the biological processes of ambergris production, and this study aims to investigate the genetic mechanism of ambergris production in the sperm whale based on its chromosome-level genome. Comparative genomics analyses found 1207 expanded gene families and 321 positive selected genes (PSGs) in the sperm whale, and functional enrichment analyses suggested revelatory pathways and terms related to the metabolism of steroids, terpenoids, and aldosterone, as well as microbiota interaction and immune network in the intestine. Furthermore, two sperm-whale-specific missense mutations (Tyr393His and Leu567Val) were detected in the PSG LIPE, which has been reported to play vital roles in lipid and cholesterol metabolism. In total, 46 CYP genes and 22 HSD genes were annotated, and then mapped to sperm whale chromosomes. Furthermore, phylogenetic analysis of CYP genes in six mammals found that CYP2E1, CYP51A and CYP8 subfamilies exhibited relative expansion in the sperm whale. Our results could help understand the genetic mechanism of ambergris production, and further reveal the convergent evolution pattern among animals that produce similar odorants.

Glucocorticoid Hormones Regulate Pendrin Abundance and Subcellular Distribution

IntroductionFollowing a low salt diet or aldosterone administration, total and apical plasma membrane pendrin abundance increase through a mechanism thought to involve aldosterone binding to the mineralocorticoid receptor (MR). However, in other cells, aldosterone binding to the MR depends on 11 b Hydroxysteroid Dehydrogenase 2 (11 b HSD2) to oxidize and inactivate glucocorticoids, which are also MR ligands. Although 11 b HSD2 abundance is low or possibly absent in intercalated cells (ICs), it’s abundance might be sufficient to inactivate glucocorticoids, making aldosterone the preferred ligand. If so, 11 b HSD2 gene ablation should reduce pendrin expression. Alternatively, the IC MR might preferentially bind another ligand such as glucocorticoids. To test these possibilities, we examined the effect of 11 b HSD2 gene ablation as well as glucocorticoid administration on pendrin abundance and subcellular distribution.Methods11 b HSD2 KO rats and age‐, and sex matched wild type rats on the same background (controls) were given either a diet with a low or high NaCl content for 10 days. Wild type mice were adrenalectomized and given a diet with either zero, normal, or high (i.e. stress level) doses of corticosterone or dexamethasone for 10 days. Pendrin total protein abundance and subcellular distribution were determined in rats and mice from each group by immunoblot and immunohistochemistry.ResultsBoth pendrin total protein abundance and pendrin’s relative abundance in the most apical region of the cell were greater in the 11 b HSD2 null than in control rats following either a NaCl‐replete or ‐restricted diet. Moreover, we observed a dose‐dependent increase in both total pendrin abundance and pendrin abundance in the most apical region of the cell following either dexamethasone or corticosterone administration, although the increase appeared greater in mice given corticosterone than dexamethasone.ConclusionsGlucocorticoids increase pendrin total protein abundance and its relative abundance in the most apical region of the cell. These results might explain, in part, why pendrin is upregulated in the known absence of 11 b HSD2.

Improved transformation efficiency of group A Streptococcus by inactivation of a type I restriction modification system.

Streptococcus pyogenes or group A Streptococcus (GAS) is a leading cause of bacterial pharyngitis, skin and soft tissue infections, life-threatening invasive infections, and the post-infectious autoimmune syndromes of acute rheumatic fever and post-streptococcal glomerulonephritis. Genetic manipulation of this important pathogen is complicated by resistance of the organism to genetic transformation. Very low transformation efficiency is attributed to recognition and degradation of introduced foreign DNA by a type I restriction-modification system encoded by the hsdRSM locus. DNA sequence analysis of this locus in ten GAS strains that had been previously transformed with an unrelated plasmid revealed that six of the ten harbored a spontaneous mutation in hsdR, S, or M. The mutations were all different, and at least five of the six were predicted to result in loss of function of the respective hsd gene product. The unexpected occurrence of such mutations in previously transformed isolates suggested that the process of transformation selects for spontaneous inactivating mutations in the Hsd system. We investigated the possibility of exploiting the increased transformability of hsd mutants by constructing a deletion mutation in hsdM in GAS strain 854, a clinical isolate representative of the globally dominant M1T1 clonal group. Mutant strain 854ΔhsdM exhibited a 5-fold increase in electrotransformation efficiency compared to the wild type parent strain and no obvious change in growth or off-target gene expression. We conclude that genetic transformation of GAS selects for spontaneous mutants in the hsdRSM restriction modification system. We propose that use of a defined hsdM mutant as a parent strain for genetic manipulation of GAS will enhance transformation efficiency and reduce the likelihood of selecting spontaneous hsd mutants with uncharacterized genotypes.

Probiotics as a Complementary Therapy in the Model of Cadmium Chloride Toxicity: Crosstalk of β-Catenin, BDNF, and StAR Signaling Pathways.

Cadmium chloride (CdCl2) is a ubiquitous environmental toxicant that causes a variety of disturbances in biological systems, including brain dysfunction and testicular tissue degeneration. On the other hand, it is supposed that beneficial properties of probiotic bacteria (Lactobacillus and Acidobacillus) are related to their capacity to adhere or bind different targets, thus leading to improved intestinal microbial balance and other benefits to the host. Bearing aforementioned in mind, the present study was undertaken to investigate the protective effect of probiotic supplementation against cadmium chloride-induced brain and testis toxicity in mice model. Animals received Lactobacillus and Acidobacillus either alone or added to folic acid for 1week before CdCl2 intoxication in a dose of 20mg/kg BW followed by probiotics (5 × 109) and/or folic acid (12mg/kg) treatment for 3weeks. The levels of malondialdehyde (MDA), butyrl choline esterase (BCHE), reduced glutathione (GSH), and total superoxide dismutase (SOD) activities were investigated. Finally, cadmium neurotoxicity was determined by estimating the gene expression of β-catenin and brain-derived neurotrophic factor (BDNF), as well as estimating the alterations in testicular function by determining acid phosphatase level in addition to steroidogenic acute regulatory protein (StAR) and 17-hydroxy steroid dehydrogenase (17-β HSD) gene expression. Based on our results, we can conclude that exposure of mice to cadmium chloride resulted in a significant elevation in MDA, BCHE levels accompanied with a significant reduction in GSH and SOD activities compared to the control value. CdCl2 also downregulated the gene expression of β-catenin and BDNF, as well as acid phosphatase level, in addition to StAR and 17-β HSD gene expression. These deviated parameters were significantly modulated in the co-treated animals with probiotics compared with the cadmium-treated group. In conclusion, Lactobacillus and folic acid in a mixture with cadmium acted beneficially to an organism, increasing the cadmium excretion in feces, and consequently increasing β-catenin and BDNF in brain tissue and StAR and 17-β HSD in testis and improving their functions. Histoarchitecture analysis confirmed these results.

Role of an estradiol regulatory factor-hydroxysteroid dehydrogenase (HSD) in Toxoplasma gondii infection and pathogenicity

Toxoplasma gondii is an apicomplexan parasite that infects most species of warm-blooded animals, including humans, and causes abortions and severe damage to the fetal central nervous system. During pregnancy, the prevalence of toxoplasmosis increases throughout the second and third quarter of gestation, while the hormones progesterone and estradiol simultaneously increase. Thus, it has been suggested that these hormones could affect parasite reproduction. This study was mainly focused on an estradiol regulatory factor-Hydroxysteroid dehydrogenase (HSD) gene in T. gondii.Our data showed that estradiol promoted Pru (Type II) and VEG (Type III) infection and thus significantly contributed to the pathogenicity of T. gondii in mice. Subsequently, we found that this phenomenon may relate to the interplay of T. gondii and estradiol. We reported that estradiol can enter T. gondii tachyzoites. Bioinformatics analysis showed that T. gondii may have a residual estradiol metabolism-related gene HSD. To verify the gene function, HEK293T cells were transiently transfected with Tg-HSD and gene expression was induced. Then, HPLC (high-performance liquid chromatography) analysis showed that Tg-HSD can efficiently transform estrone into estradiol. Moreover, Tg-HSD −overexpressing parasites showed significantly enhanced pathogenicity and upregulation of estradiol levels in mice. In conclusion, estradiol can promote T. gondii infection in vitro and in vivo, and this may be related to its Tg- HSD gene.

A Case with late onset of ambiguous genitalia

Ambiguous genitalia is an uncommon situation that happens between 1 and 2 per every 1000 live births and falls under the umbrella diagnosis of disorders of sexual development. In this article, we report a case of male pseudohermaphroditism with ambiguous genitalia. The proband was a 12 yr old girl without any uterus or ovarian tissues. Karyotype of the case is 46, XY. Genes involved in sexual differentiation such as AR, SRD5A2, LH, LHR, FSH, 17 B HSD and SRY genes were sequenced in both directions. No mutations were found in these genes either. It seems advisable to be cautious in similar cases, and revise protocol for tracing the genes involved in the patients.

Alteration of sex hormone levels and steroidogenic pathway by several low molecular weight phthalates and their metabolites in male zebrafish (Danio rerio) and/or human adrenal cell (H295R) line

Low molecular weight phthalates, such as diethyl phthalate (DEP), benzyl butyl phthalate (BBzP), or diisobutyl phthalate (DiBP), are suspected to disrupt endocrine system. However, their adverse effects on sex steroid hormones and underlying mechanisms are not well-documented. The aim of this study is to investigate the effects of major low molecular weight phthalates (LMWPs), i.e., DEP, BBzP, and DiBP, and their hydrolytic metabolites, on sex steroid hormone system, employing male zebrafish and/or a human adrenocortical carcinoma (H295R) cell. In male zebrafish, 14-day exposure to DEP, BBzP, or DiBP significantly decreased testosterone (T) concentrations. All test compounds significantly up-regulated cyp19a gene expression, and down-regulated star and 3β hsd genes in the male fish. In H295R cell, all test compounds except monoisobutyl phthalate (MiBP) reduced T concentrations and increased E2/T ratio. Gene expression changes in H295R cell, e.g., significant down-regulation of StAR gene and up-regulation of CYP19A gene, supported depressed synthesis of sex hormones in the adrenal cell. Our results show that not only DEP, BBzP, and DiBP, but also their hydrolytic metabolites disrupt sex hormone balances through modulating key steroidogenic genes in the human adrenal cells and in zebrafish.

Placental HSD2 Expression and Activity Is Unaffected by Maternal Protein Consumption or Gender in C57BL/6 Mice

The placenta acts as a physiological barrier, preventing the transfer of maternal glucocorticoids to the developing fetus. This is accomplished via the oxidation, and subsequent inactivation, of endogenous glucocorticoids by the 11-β hydroxysteroid dehydrogenase type 2 enzyme (HSD2). Maternal protein restriction during pregnancy has been shown to result in a decrease in placental HSD2 expression and fetal glucocorticoid overexposure, especially late in gestation, resulting in low birth weight and “fetal programming” of the offspring. This dietary intervention impairs fetal growth and cardiovascular function in adult C57BL/6 offspring, but the impact on placental HSD2 has not been defined. The goal of the current study was to examine the effects of a maternal low-protein diet (18% versus 9% protein) on placental HSD2 gene expression and enzyme activity in mice during late gestation. In contrast to previous studies in rats, a maternal low-protein diet did not affect HSD2 protein or enzyme activity levels in the placentas of C57BL/6 mice and this was irrespective of the gender of the offspring. These data suggest that the effects of maternal protein restriction on adult phenotypes in C57BL/6 mice depend upon a mechanism that may be independent of placental HSD2 or possibly occurs earlier in gestation.

18β-Glycyrrhetinic acid inhibits periodontitis via glucocorticoid-independent nuclear factor-κB inactivation in interleukin-10-deficient mice

18β-Glycyrrhetinic acid (GA) is a natural anti-inflammatory compound derived from licorice root extract (Glycyrrhiza glabra). The effect of GA on experimental periodontitis and its mechanism of action were determined in the present study. Periodontitis was induced by oral infection with Porphyromonas gingivalis W83 in interleukin-10-deficient mice. The effect of GA, which was delivered by subcutaneous injections in either prophylactic or therapeutic regimens, on alveolar bone loss and gingival gene expressions was determined on day 42 after initial infection. The effect of GA on lipopolysaccharide (LPS)-stimulated macrophages, T cell proliferation and osteoclastogenesis was also examined in vitro. 18β-Glycyrrhetinic acid administered either prophylactically or therapeutically resulted in a dramatic reduction of infection-induced bone loss in interleukin-10-deficient mice, which are highly disease susceptible. Although GA has been reported to exert its anti-inflammatory activity via downregulation of 11β-hydroxysteroid dehydrogenase-2 (HSD2), which converts active glucocorticoids to their inactive forms, GA did not reduce HSD2 gene expression in gingival tissue. Rather, in glucocorticoid-free conditions, GA potently inhibited LPS-stimulated proinflammatory cytokine production and RANKL-stimulated osteoclastogenesis, both of which are dependent on nuclear factor-κB. Furthermore, GA suppressed LPS- and RANKL-stimulated phosphorylation of nuclear factor-κB p105 in vitro. These findings indicate that GA inhibits periodontitis by inactivation of nuclear factor-κB in an interleukin-10- and glucocorticoid-independent fashion.

Genetic resources of nodule bacteria

Nodule bacteria (rhizobia) form highly specific symbiosis with leguminous plants. The efficiency of accumulation of biological nitrogen depends on molecular-genetic interaction between the host plant and rhizobia. Genetic characteristics of microsymbiotic strains are crucial in developing highly productive and stress-resistant symbiotic pairs: rhizobium strain-host plant cultivar (species). The present review considers the issue of studying genetic resources of nodule bacteria to identify genes and their blocks, responsible for the ability of rhizobia to form highly effective symbiosis in various agroecological conditions. The main approaches to investigation of intraspecific and interspecific genetic and genomic diversity of nodule bacteria are considered, from MLEE analysis to the recent methods of genomic DNA analysis using biochips. The data are presented showing that gene centers of host plants are centers of genetic diversification of nodule bacteria, because the intraspecific polymorphism of genetic markers of the core and the accessory rhizobial genomes is extremely high in them. Genotypic features of trapped and nodule subpopulations of alfalfa nodule bacteria are discussed. A survey of literature showed that the genomes of natural strains in alfalfa gene centers exhibit significant differences in genes involved in control of metabolism, replication, recombination, and the formation of defense response (hsd genes). Natural populations of rhizobia are regarded as a huge gene pool serving as a source of evolutionary innovations.

Phosphorylation of Type IA restriction-modification complex enzyme EcoKI on the HsdR subunit

Phosphorylation of Type I restriction-modification (R-M) enzymes EcoKI, EcoAI, and EcoR124I - representatives of IA, IB, and IC families, respectively - was analysed in vivo by immunoblotting of endogenous phosphoproteins isolated from Escherichia coli strains harbouring the corresponding hsd genes, and in vitro by a phosphorylation assay using protein kinase present in subcellular fractions of E. coli. From all three R-M enzymes, the HsdR subunit of EcoKI system was the only subunit that was phosphorylated. Further, evidence is presented that HsdR is phosphorylated in vivo only when coproduced with HsdM and HsdS subunits - as part of assembled EcoKI restriction endonuclease, while the individually produced HsdR subunit is not phosphorylated. In vitro phosphorylation of the HsdR subunit of purified EcoKI endonuclease occurs on Thr, and is strictly dependent on the addition of a catalytic amount of cytoplasmic fraction isolated from E. coli. So far this is the first case of phosphorylation of a Type I R-M enzyme reported.

An estrogen metabolism-related polymorphism of the 17-α HSD gene is associated with perimenopausal body mass index

In a cross-sectional study of 2,802 perimenopausal caucasian women, carriage of at least one mutated allele of the 17-alpha-hydroxysteroid dehydrogenase type 1 (17-alpha HSD) vlV A-->C single nucleotide polymorphism (SNP) was associated with a significantly increased body mass index (mean 24.3 +/- 4.4 kg/m(2) vs. 23.5 +/- 4.2 kg/m(2); P<.001), and obesity was more frequent among mutant allele carriers (P=.06; odds ratio 1.38; 95% confidence interval 0.97-1.95), providing evidence of 17-alpha HSD as a candidate gene of perimenopausal obesity.

Cellular localization of Type I restriction–modification enzymes is family dependent

Cellular localization of Type I restriction–modification enzymes EcoKI, EcoAI, and EcoR124I—the most frequently studied representatives of IA, IB, and IC families—was analyzed by immunoblotting of subcellular fractions isolated from Escherichia coli strains harboring the corresponding hsd genes. EcoR124I shows characteristics similar to those of EcoKI. The complex enzymes are associated with the cytoplasmic membrane via DNA interaction as documented by the release of the Hsd subunits from the membrane into the soluble fraction following benzonase treatment. HsdR subunits of the membrane-bound enzymes EcoKI and EcoR124I are accessible, though to a different extent, at the external surface of cytoplasmic membrane as shown by trypsinization of intact spheroplasts. EcoAI strongly differs from EcoKI and EcoR124I, since neither benzonase nor trypsin affects its association with the cytoplasmic membrane. Possible reasons for such a different organization are discussed in relation of the control of the restriction–modification activities in vivo.

Deletion mapping of chromosome 16q24 in hepatocellular carcinoma in Taiwan and mutational analysis of the 17-beta-HSD gene localized to the region.

Human chromosome band 16q24 commonly undergoes loss of heterozygosity (LOH) in human hepatocellular carcinoma (HCC). To further localize the region of deletion on 16q24 and to evaluate the genetic role of 17-beta-HSD, which is near 16q24, in HCC, we examined the pattern of loss of heterozygosity in 88 HCC patients. DNAs from 88 pairs of HCCs and corresponding non-tumor parts were prepared. Loss of heterozygosity on chromosomes 16q24 was investigated by 11 sets of microsatellite markers. Mutation analysis of type II 17-beta-HSD was performed by automatic sequencing. LOH on 16q24 for at least 1 locus was found in 43 of the 88 tumor DNAs (49%). Three non-overlapping regions of frequent LOH were defined in these 43 tumors with partial deletions. The first region was between D16S516 loci and D16S507, encompassed by a 1-cM region, defined by the D16S504. The second region was defined by the 17HSDB2 locus between D16S505 and D16S422, encompassed approximately by a 1-cM region. The third region was between D16S520 and D16S413, defined by D16S3048, encompassed approximately by a 4-cM region. Homozygous deletions of any exons in 17HSDB2 gene were identified in 7 of 27 cases (26%). Automated sequencing analysis of 17HSDB2 failed to demonstrate mutations in any of these specimens. Our data suggest that the 17HSDB2 locus is a frequent target of deletion in HCC but the inactivation of 17HSDB2 may not involve sequence mutations. Furthermore, the presence of the other 2 frequent LOH regions suggest that the putative tumor suppressor genes at these locations might be involved in the development of HCC.

Expression of the 11β-hydroxysteroid dehydrogenase 2 gene in the mouse

Aldosterone acts via mineralocorticoid receptors (MRs) to control salt and water flux in epithelial organs such as the kidney and colon to maintain circulatory homeostasis. Inappropriate glucocorticoid-mediated activation of MRs in aldosterone-target tissues is prevented by the glucocorticoid-metabolizing enzyme 11beta-hydroxysteroid dehydrogenase type 2 (HSD2). We have studied HSD2 expression in the mouse at the level of gene transcription and further analyzed, with HSD1, its pattern of tissue-restricted gene expression. The mouse HSD2 gene, including upstream regulatory regions, has been cloned, and its transcription start site has been mapped in colon and kidney. A 2.5 kb upstream region has been sequenced, and its proximal promoter region has been analyzed. We have compared the relative expression of HSD1 and HSD2 in a variety of tissues from male mice using ribonuclease protection analysis. HSD1 was expressed in liver, kidney, adrenal, lung, spleen, thymus, fat, small intestine, stomach, heart, skin, and epididymis. HSD2 was expressed in kidney, colon, small intestine, stomach, and epididymus. No expression of either HSD1 and HSD2 was detected in bladder, testis, seminal vesicles, vas deferens, prostate, or skeletal muscle. Finally, to investigate the specific roles of HSD2 in vivo, we have created "floxed" HSD2 alleles using gene targeting in mouse embryonic stem cells with the aim to create tissue-specific ablation of HSD2 in mice via Cre recombinase mediated gene targeting.

A family of phase-variable restriction enzymes with differing specificities generated by high-frequency gene rearrangements.

The hsd genes of Mycoplasma pulmonis encode restriction and modification enzymes exhibiting a high degree of sequence similarity to the type I enzymes of enteric bacteria. The S subunits of type I systems dictate the DNA sequence specificity of the holoenzyme and are required for both the restriction and the modification reactions. The M. pulmonis chromosome has two hsd loci, both of which contain two hsdS genes each and are complex, site-specific DNA inversion systems. Embedded within the coding region of each hsdS gene are a minimum of three sites at which DNA inversions occur to generate extensive amino acid sequence variations in the predicted S subunits. We show that the polymorphic hsdS genes produced by gene rearrangement encode a family of functional S subunits with differing DNA sequence specificities. In addition to creating polymorphisms in hsdS sequences, DNA inversions regulate the phase-variable production of restriction activity because the other genes required for restriction activity (hsdR and hsdM) are expressed only from loci that are oriented appropriately in the chromosome relative to the hsd promoter. These data cast doubt on the prevailing paradigms that restriction systems are either selfish or function to confer protection from invasion by foreign DNA.

The effect of recA mutation on the expression of EcoKI and EcoR124I hsd genes cloned in a multicopy plasmid.

Type I restriction-modification (R-M) endonucleases are composed of three subunits--HsdR, required for restriction, and HsdM and HsdS which can produce a separate DNA methyltransferase. The HsdS subunit is required for DNA recognition. In this paper we describe the effect of cloned EcoKI and EcoR124I hsd genes on the resulting R-M phenotype. The variability in the expression of the wild type (wt) restriction phenotype after cloning of the wt hsd genes in a multicopy plasmid in Escherichia coli recA+ background suggests that the increased production of the restriction endonuclease from pBR322 is detrimental to the cell and this leads to the deletion of the cloned hsd genes from the hybrid plasmid and/or inactivation of the enzyme. The effect of a mutation in E. coli recA gene on the expression of R-M phenotype is described and discussed in relation to the role of the cell surface and the localization of the restriction endonuclease in the cell.

The hsd loci of Mycoplasma pulmonis: organization, rearrangements and expression of genes.

Two paralogous, site-specific invertible loci, designated hsd1 and hsd2 (host specificity determinant), have been identified in the Mycoplasma pulmonis genome. They encode putative type I restriction and modification (R-M) systems with maximum sequence homology to the type IC family, which includes EcoR124II and EcoDXXI. Each locus encodes an endonuclease subunit (HsdR), a methylase subunit (HsdM) and two DNA specificity subunits (HsdS). The gene organization at each locus is such that hsdR and hsdM are flanked by two hsdS genes. Within each locus, one of the hsdS genes, hsdR and hsdM, is encoded in tandem by the same DNA strand, while the second hsdS gene is encoded by the complementary strand but without overlap with the other three hsd genes. The hsdR and hsdM sequences of one locus are almost identical to their counterparts in the other. The four hsdS genes (two per locus) are highly homologous at their 5' ends and also share sequence similarities in the 3' ends of their corresponding coding regions. Owing to the disposition of and sequence similarities among the hsdS genes, they form inverted repeats at each locus. Analysis by polymerase chain reaction (PCR) has shown that both loci behave as site-specific DNA invertible elements with multiple inversion sites, termed 'vipareetus', occurring within the hsdS genes. The inversions lead to a reassortment of hsdS sequences, generating an array of recombinant genes that probably encode S subunits possessing alternative DNA-binding specificities. Sequence information obtained from the analysis of hsd2 transcripts by 5' RACE (rapid amplification of cDNA ends) indicates that inversion induces the transcription of alternative hsdS genes by the relocation of coding sequences downstream of a promoter and ribosome-binding site (RBS) situated at one end of each locus.

A new Comamonas testosteroni steroid-inducible gene: Cloning and sequence analysis

Comamonas testosteroni can grow on a variety of steroid compounds as the sole carbon and energy source. In a previous study, we cloned and sequenced the testosterone-inducible β hsd gene from C. testosteroni (Genti-Raimondi, S., Tolmasky, M., Patrito, L., Flury, A. and Actis, L., Molecular cloning and expression of the β-hydroxysteroid dehydrogenase gene from Pseudomonas testosteroni. Gene, 1991, 105, 43–49.). Herein we report the cloning and characterization of another steroid-inducible gene ( stdC), located 2400 bp upstream of β hsd. Nucleotide sequencing of a region encompassing the stdC gene revealed an open reading frame 546 bp long including the stop codon TGA with significant similarity to the orf4, orf1 and orf4 of unknown function described in the polyhydroxyalkanoic acid (PHA) cluster of Chromatium vinosum, Rhizobium meliloti and Thiocystis violacea, respectively. The aminoacid sequence deduced from the nucleotide sequence predicts a putative protein of 181 amino acids with a molecular weight of 20 715 Da. Northern blot experiments indicate that the stdC gene was transcribed as a monocistronic mRNA with an apparent molecular size of 670 nt. The stdC transcript was abundant in C. testosteroni cells grown with different steroid carbon sources harvested in the exponential phase and was found to be under catabolite repression.