Transcriptional control of human steroid sulfatase.

Steroid sulfatase (STS) is a membrane-bound microsomal enzyme that hydrolyzes various alkyl and aryl steroid sulfates, leading to the in situ formation of biologically active hormones. The entire human STS gene spans over approximately 200kbp of which the first 100kbp include the regulatory region, while the STS-coding region is located downstream. Previous studies indicated that STS expression, in different human tissues, could be regulated by at least six different promoters associated with alternative first exons. Here, we describe two new splicing patterns: the first, found in the prostatic cell line PC3, is based upon a partially coding new first exon (0d) that is spliced to a new second exon (1e). The second variant was found in the ovary and it is characterized by the novel splicing of the untranslated exon 0b to exon 0c, which is then spliced to the common exon 1b. We also report the results of a multiplex ligation-dependent probe amplification (RT-MLPA) analysis for the simultaneous detection, in qualitative and/or semi-quantitative terms, of the transcription patterns of STS in different tissues.

The expression of the human steroid sulfatase-encoding gene is driven by alternative first exons.

We have analyzed steroid sulfatase (STS) gene transcription in 10 human tissues: ovary, adrenal cortex, uterus, thyroid, liver, pancreas, colon, mammary gland, dermal papilla of the hair follicle, and peripheral mononuclear leukocytes. Overall, six different promoters were found to drive STS expression, giving rise to transcripts with unique first exons that were labeled 0a, 0b, 0c, 1a, 1c, and 1d, of which the last two and 0c are newly reported. All of them, except exon 1d, vary in length owing to the occurrence of multiple transcriptional start sites. While placental exon 1a is partially coding, the other five first exons are all untranslated. Three of these (0a, 0b, and 0c) are spliced to the common partially coding exon 1b, whereas the other two (1c and 1d) are spliced to the coding exon 2, which occurs in all transcripts. Whatever the ATG actually used, the differences are restricted to the signal peptide which is post-transcriptionally cleaved. Transcripts with exons 0a and 0b have the broadest tissue distribution, occurring, in 6 out of the 12 tissues so far investigated, while the other first exons are restricted to one or two tissues. The proximal promoter of each first exon was devoid of TATA box or initiator element and lacked consensus elements for transcription factors related to steroidogenesis, suggesting that regulatory sequences are probably placed at greater distance. In conclusion, the regulation of STS transcription appears to be more complex than previously thought, suggesting that this enzyme plays a substantial role in intercellular integration.

Beta-keratins of differentiating epidermis of snake comprise glycine-proline-serine-rich proteins with an avian-like gene organization.

Beta-keratins of reptilian scales have been recently cloned and characterized in some lizards. Here we report for the first time the sequence of some beta-keratins from the snake Elaphe guttata. Five different cDNAs were obtained using 5'- and 3'-RACE analyses. Four sequences differ by only few nucleotides in the coding region, whereas the last cDNA shows, in this region, only 84% of identity. The gene corresponding to one of the cDNA sequences has a single intron present in the 5'-untranslated region. This genomic organization is similar to that of birds' beta-keratins. Cloning and Southern blotting analysis suggest that snake beta-keratins belong to a family of high-related genes as for geckos. PCR analysis suggests a head-to-tail orientation of genes in the same chromosome. In situ hybridization detected beta-keratin transcripts almost exclusively in differentiating oberhautchen and beta-cells of the snake epidermis in renewal phase. This is confirmed by Northern blotting that showed, in this phase, a high expression of two different transcripts whereas only the longer transcript is expressed at a much lower level in resting skin. The cDNA coding sequences encoded putative glycine-proline-serine rich proteins containing 137-139 amino acids, with apparent isoelectric point at 7.5 and 8.2. A central region, rich in proline, shows over 50% homology with avian scale, claw, and feather keratins. The prediction of secondary structure shows mainly a random coil conformation and few beta-strand regions in the central region, likely involved in the formation of a fibrous framework of beta-keratins. This region was possibly present in basic reptiles that originated reptiles and birds.

Tissue-specific transcriptional initiation of the CYP19 genes in rainbow trout, with analysis of splicing patterns and promoter sequences.

The rainbow trout (Oncorhynchus mykiss Walbaum) genome contains three separate CYP19 genes for distinct isoforms of cytochrome P450arom: CYP19A encoding the prevalently ovarian isoform P450aromA, and CYP19B-I and II, encoding forms I and II of the mainly cerebral variant P450aromB. RNA Ligase-Mediated 5'-Rapid Amplification of cDNA Ends analysis was used to determine the 5'-untranslated terminal regions (5'-UTRs) of the corresponding mRNAs, which are actually all expressed in the ovary, brain and gills. CYP19A is transcribed at different transcription start sites (TSSs) in each tissue, the most distal TSS being found in the brain, the intermediate one in the gills, and the proximal one in the ovary. CYP19B-I also displays tissue-specific TSSs, but transcripts undergo three distinct splicing patterns: the same pattern as previously reported for the brain and occurring also in the gills, and two novel patterns, established in the ovary and brain, which include two cryptic 3'-splice sites in intron 1, leading to the inclusion of intronic sequences of 92/94 and 66 b in the 5'-UTRs. Lastly, the CYP19B-II transcript in the ovary shows the same splicing pattern previously described for the brain. A PCR-based gene walking strategy was used to explore the promoter regions of the rainbow trout CYP19 genes, which were found to contain potential binding sites for a variety of transcription factors.

Characterisation of three variants of estrogen receptor beta mRNA in the common sole, Solea solea L. (Teleostei).

In all vertebrates, estrogen action is mediated by cognate nuclear receptors. In this study, we cloned the different transcripts of the estrogen receptor beta (ERbeta) gene of the common sole, Solea solea. 5'-RLM-RACE (RNA Ligase-Mediated 5'-Rapid Amplification of cDNA Ends) and 3'-RACE analyses revealed three isoforms of different length, called Long, Intermediate and Short isoforms, consisting of 2212, 1531 and 1207 b, respectively. The Long isoform is characterised by an open reading frame (ORF) encoding 589aa, with an estimated molecular weight of 65kDa. Phylogenetic analysis established that it belongs to the teleost ERbeta1 or ERbetaa cluster. The Intermediate isoform encodes a 490-aa protein, which lacks the first 99aa of the Long isoform, but still retains a complete DNA-binding domain (DBD). In the Short variant (363aa-long), all the N-terminal region, down to the two zinc fingers included, is missing, thus crippling DBD. ERbeta transcription was analysed by semiquantitative RT-PCR with specific primers, common to the Long and Intermediate isoforms, in various sole tissues, such as brain, gills, muscle, stomach, intestine, spleen, head kidney, kidney, liver and gonads. This analysis revealed that ERbeta displays a widespread or ubiquitous pattern of transcription, with the highest levels being found in the gonads and liver.

Phylogeny of betanodaviruses and molecular evolution of their RNA polymerase and coat proteins.

The betanodaviruses are the causative agent of the disease viral nervous necrosis in fishes. Betanodavirus genome consists of two single-stranded positive-sense RNA molecules (RNA1 and RNA2). RNA1 gene encodes the RNA polymerase, named also protein A, while RNA2 encodes the coat protein precursor, the CPp protein. We investigated the evolutionary relationships among betanodaviruses working on partial sequences of both RNA1 and RNA2. Phylogenetic analyses were performed by applying a maximum likelihood approach. The phylogenetic relationships among the major betanodavirus clades SJNNV-IV, TPNNV-III, BFNNV-II and RGNNV-I were resolved differently in the trees obtained, respectively, from RNA1 and RNA2 multiple alignments. The alternative topologies were corroborated by strong bootstrap values. The molecular evolution of proteins A and CPp was also investigated. Protein A appeared to have evolved under strong purifying selection while the CPp protein was subject to both purifying and neutral selection in different amino acid residues. Intragenic recombination in RNA1 and RNA2 genes was investigated by applying several methods and was not detected. Conversely reassortment of RNA1 and RNA2 genes was demonstrated in some isolates. Finally RNA1 and RNA2 genes substitution rates do not follow a clock-like behavior thus impeding estimation of a possible origin time for Betanodavirus genus.

RETRACTED: Occurrence of steroid-converting enzymes in the gonads of the Manila clam, Ruditapes philippinarum (Adams and Reeve, 1850), and correlation with the gametogenic cycle.

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Isolation of a mRNA encoding a glycine-proline-rich beta-keratin expressed in the regenerating epidermis of lizard.

During scale regeneration in lizard tail, an active differentiation of beta-keratin synthesizing cells occurs. The cDNA and amino acid sequence of a lizard beta-keratin has been obtained from mRNA isolated from regenerating epidermis. Degenerate oligonucleotides, selected from the translated amino acid sequence of a lizard claw protein, were used to amplify a specific lizard keratin cDNA fragment from the mRNA after reverse transcription with poly dT primer and subsequent polymerase chain reaction (3'-rapid amplification of cDNA ends analysis, 3'-RACE). The new sequence was used to design specific primers to obtain the complete cDNA sequence by 5'-RACE. The 835-nucleotide cDNA sequence encodes a glycine-proline-rich protein containing 163 amino acids with a molecular mass of 15.5 kDa; 4.3% of its amino acids is represented by cysteine, 4.9% by tyrosine, 8.0% by proline, and 29.4% by glycine. Tyrosine is linked to glycine, and proline is present mainly in the central region of the protein. Repeated glycine-glycine-X and glycine-X amino acid sequences are localized near the N-amino and C-terminal regions. The protein has the central amino acid region similar to that of claw-feather, whereas the head and tail regions are similar to glycine-tyrosine-rich proteins of mammalian hairs. In situ hybridization analysis at light and electron microscope reveals that the corresponding mRNA is expressed in cells of the differentiating beta-layers of the regenerating scales. The synthesis of beta-keratin from its mRNA occurs among ribosomes or is associated with the surface of beta-keratin filaments.

Expression of cytochrome P450c17 and other steroid-converting enzymes in the rat kidney throughout the life-span.

We have investigated the metabolism of [14C]-labelled progesterone (P4) and dehydroepiandrosterone (DHEA) by kidney tissues of newborn and 7-, 15-, 30-, 60- and 365-day-old rats of both sexes. The following enzymes were revealed at all ages by radiochemical identification of the corresponding products: 5alpha-reductase, cytochromes P450c17 and P450c21, 3beta-hydroxysteroid dehydrogenase (HSD)/delta5-delta4 isomerase, and 17beta- and 20alpha-HSDs, catalyzing reductive reactions. The major P4 metabolites were 5alpha-reduced C21 steroids, whose formation was almost completely suppressed by the 5alpha-reductase 4-azasteroid inhibitor, PNU 156765. Androstenedione and testosterone were also formed via 17alpha-hydroxyprogesterone, together with 11-deoxycorticosterone and 20alpha-dihydroprogesterone. DHEA was mainly converted to androst-5-ene-3beta,17beta-diol, with smaller amounts of the above androgens. Cytochrome P450c17 mRNA and protein were demonstrated by Northern blotting and Western blotting analyses, respectively. P450c17 mRNA, assessed by Northern blotting, protein and catalytic activity all peaked in the kidney samples at 15 days of life and declined thereafter. Cytochrome P450arom was below the level of detection of semi-quantitative RT-PCR. Since the rat kidney has been previously shown to contain cytochrome P450scc as well as androgen and estrogen receptors, it is suggested that it is capable of autonomous hormonal steroidogenesis and that renal steroids, or nephrosteroids, may act locally, in a paracrine or autocrine fashion.

Expression of cytochrome P450scc mRNA and protein in the rat kidney from birth to adulthood.

The expression of cytochrome P450scc, encoded by the CYP11A gene, was investigated in the rat kidney from birth to adulthood. In the male and female rat kidneys, the corresponding mRNA was detected by semi-quantitative reverse transcriptase and polymerase chain reaction (RT-PCR) analysis with specific primers, resulting in higher levels of expression during the first 15 days from birth. RT-PCR and sequence analysis showed that the P450scc mRNA coding region was the same for both kidney and testis, whereas 5'-RACE analysis (rapid amplification of cDNA ends) demonstrated that the renal transcription utilizes a distal transcription start site (TSS) located 76 b upstream of that used in ovarian and testicular P450scc mRNA expression, which is placed 43 b upstream of the first ATG. The 5'-UTR sequence of renal P450scc cDNA exactly matched the contiguous upstream untranslated region of the gene, suggesting that alternative splicing was not involved in the synthesis of this transcript. Northern hybridization detected a specific transcript only in the newborn male, but not in adult rat kidney, confirming the higher levels of expression in the first days of the rat's life. Positive immunodetections of cytochrome P450scc were found in renal cortical distal tubules and the results were confirmed by Western blotting analysis. As demonstrated by semi-quantitative RT-PCR, the male kidney also expresses the messengers corresponding to the steroidogenic acute regulatory (StAR) and steroidogenic factor 1 (SF-1) proteins, which are normally required for steroidogenesis in steroidogenic tissues, such as gonads and adrenal cortex. These studies suggest that the rat kidney has the capability for local steroid hormone production, although the physiological significance of the pregnenolone eventually produced remains to be established.

Cloning of two mRNA variants of brain aromatase cytochrome P450 in rainbow trout (Oncorhynchus mykiss Walbaum).

This work describes the molecular cloning of the cDNA encoding the rainbow trout (Oncorhynchus mykiss Walbaum) brain cytochrome P450arom by means of reverse transcriptase and polymerase chain reaction (RT-PCR) and 5'- and 3'-rapid amplification of cDNA ends (RACE) analyses. The results obtained demonstrate that, as in other teleost fishes, the trout genome contains, besides the gene previously identified in the ovary, a second CYP19 gene (CYP19B) expressed at high level in the brain. Moreover, two P450aromB mRNAs, forms I and II, were found to be transcribed in trout. Form I (1816 sequenced nt) contains an open reading frame (ORF) of 1464b, a 5'-untranslated terminal region (UTR) of 124b and at least 228b in the 3'-UTR (incomplete, as the polyadenylation signal was not determined). Form II (1930 sequenced nt) contains an ORF of 1362b, a 5'-UTR of 340b and the same 3'-UTR as form I. Form II lacks the first 34 amino acids of form I, corresponding to the membrane-anchoring segment, whereas the sequence of the remaining coding region is almost the same in the two forms, resulting in proteins of 454 and 488 amino acids, respectively. Whether the two transcripts derive from the same gene by alternative splicing or are encoded by different CYP19B genes remains to be clarified. On Northern blot analyses with brain and ovary specific ORF probes and poly(A)(+)-enriched RNAs from trout ovary and brain, a transcript of about 2.6kb was identified in the ovary, as expected, whereas the full-length mRNA of brain P450arom is about 3.8kb. The brain form is expressed in the brain and gonads, whereas expression in peripheral tissues is limited mostly to the gills. The two trout CYP19 genes are not equivalent in tissue-specific expression, indicating the possibility of distinct promoters and regulatory mechanisms.

Rat cytochrome P450c17 gene transcription is initiated at different start sites in extraglandular and glandular tissues.

In the male rat, the mRNA of the steroidogenic cytochrome P450c17 is expressed extraglandularly in the stomach, duodenum, kidney and liver, throughout the animal's lifespan, as demonstrated by reverse transcriptase and polymerase chain reaction (RT-PCR) analysis with specific primers. Northern analysis indicated that all tissues, except the kidney, contain high levels of such mRNA, but the relative mobility of liver mRNA is slightly less than that of the testis and other tissues. Thus, we analysed their 5'- and 3'-untranslated terminal regions (UTRs) by means of 5'- and 3'-rapid amplification of cDNA ends (RACE) techniques. All tissues utilised the same polyadenylation site as the testis. In the 5'-UTR of liver mRNA, however, we found a distal transcription start site (TSS) located 252b upstream of that used in testicular P450c17 mRNA, which is placed 41b upstream of the first ATG. The 5'-UTR sequence of liver P450c17 cDNA exactly matched the contiguous upstream untranslated region of the gene, suggesting that alternative splicing was not involved in the synthesis of liver P450c17 transcript. The other tissues used the same TSS present in the testis. Nevertheless, a second TSS located 125b upstream of the first ATG was found in the stomach and duodenum. These results show that the transcriptional regulation of the CYP17 gene in the rat is complex and differs between tissues in the use of TSSs.