Vania Toffolo

Isolation of a mRNA encoding a glycine‐proline–rich β‐keratin expressed in the regenerating epidermis of lizard

During scale regeneration in lizard tail, an active differentiation of β‐keratin synthesizing cells occurs. The cDNA and amino acid sequence of a lizard β‐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 β‐layers of the regenerating scales. The synthesis of β‐keratin from its mRNA occurs among ribosomes or is associated with the surface of β‐keratin filaments. Developmental Dynamics 234:934–947, 2005.

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

The β‐keratins constitute the hard epidermis and adhesive setae of gecko lizards. Nucleotide and amino acid sequences of β‐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 β‐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 (β‐pleated fold) likely responsible for the formation of β‐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 β‐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 β‐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 β‐keratins. Developmental Dynamics 236:374–388, 2007.

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.

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.