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.

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.

Cloning and characterization of scale beta-keratins in the differentiating epidermis of geckoes show they are glycine-proline-serine-rich proteins with a central motif homologous to avian beta-keratins.

The beta-keratins constitute the hard epidermis and adhesive setae of gecko lizards. Nucleotide and amino acid sequences of beta-keratins in epidermis of gecko lizards were cloned from mRNAs. Specific oligonucleotides were used to amplify by 3'- and 5'-rapid amplification of cDNA ends analyses five specific gecko beta-keratin cDNA sequences. The cDNA coding sequences encoded putative glycine-proline-serine-rich proteins of 16.8-18 kDa containing 169-191 amino acids, especially 17.8-23% glycine, 8.4-14.8% proline, 14.2-18.1% serine. Glycine-rich repeats are localized toward the initial and end regions of the protein, while a central region, rich in proline, has a strand conformation (beta-pleated fold) likely responsible for the formation of beta-keratin filaments. It shows high homology with a core region of other lizard keratins, avian scale, and feather keratins. Northern blotting and reverse transcriptase-polymerase chain reaction (RT-PCR) analysis show a higher beta-keratin gene expression in regenerating epidermis compared with normal epidermis. In situ hybridization confirms that mRNAs for these proteins are expressed in cells of the differentiating oberhautchen cells and beta-cells. Expression in adhesive setae of climbing lamellae was shown by RT-PCR. Southern blotting analysis revealed that the proteins are encoded by a multigene family. PCR analysis showed that the genes are presumably located in tandem along the DNA and are transcribed from the same DNA strand like in avian beta-keratins.

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.

Scale keratin in lizard epidermis reveals amino acid regions homologous with avian and mammalian epidermal proteins.

Small proteins termed beta-keratins constitute the hard corneous material of reptilian scales. In order to study the cell site of synthesis of beta-keratin, an antiserum against a lizard beta-keratin of 15-16 kDa has been produced. The antiserum recognizes beta-cells of lizard epidermis and labels beta-keratin filaments using immunocytochemistry and immunoblotting. In situ hybridization using a cDNA-probe for a lizard beta-keratin mRNA labels beta-cells of the regenerating and embryonic epidermis of lizard. The mRNA is localized free in the cytoplasm or is associated with keratin filaments of beta-cells. The immunolabeling and in situ labeling suggest that synthesis and accumulation of beta-keratin are closely associated. Nuclear localization of the cDNA probe suggests that the primary transcript is similar to the cytoplasmic mRNA coding for the protein. The latter comprises a glycine-proline-rich protein of 15.5 kDa that contains 163 amino acids, in which the central amino acid region is similar to that of chick claw/feather while the head and tail regions resemble glycine-tyrosine-rich proteins of mammalian hairs. This is also confirmed by phylogenetic analysis comparing reptilian glycine-rich proteins with cytokeratins, hair keratin-associated proteins, and claw/feather keratins. It is suggested that different small glycine-rich proteins evolved from progenitor proteins present in basic (reptilian) amniotes. The evolution of these proteins originated glycine-rich proteins in scales, claws, beak of reptiles and birds, and in feathers. Some evidence suggests that at least some proteins contained within beta-keratin filaments are rich in glycine and resemble some keratin-associated proteins present in mammalian corneous derivatives. It is suggested that glycine-rich proteins with the chemical composition, immunological characteristics, and molecular weight of beta-keratins may represent the reptilian counterpart of keratin-associated proteins present in hairs, nails, hooves, and horns of mammals. These small proteins produce a hard type of corneous material due to their dense packing among cytokeratin filaments.

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.