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M C Darmon

Publications and source records attributed to M C Darmon.

11 recordsLinked to original sources
PubMed1995

The human tryptophan hydroxylase gene. An unusual splicing complexity in the 5'-untranslated region.

We report the isolation and the organization of the gene encoding human tryptophan hydroxylase (TPH) and an analysis of the corresponding mRNAs. The gene spans a region of 29 kilobases, which contains at least 11 exons and a variably spliced 5'-untranslated region (5'-UTR). The sequence of the coding region and the majority of the positions of the intron-exon boundaries of human TPH gene are very similar to those encoding human tyrosine hydroxylase and phenylalanine hydroxylase, the other members of the aromatic amino acid hydroxylase family. Phylogenetic analysis evidences the early divergence and the independent evolution of the three hydroxylase types. TPH cDNA cloning and anchored polymerase chain reaction revealed a diversity of the TPH mRNA, which is restricted to the 5'-UTR. Four TPH mRNA species were detected by Northern blot with pineal gland and carcinoid tumor RNAs. These messengers are transcribed from a single transcriptional initiation site, and their diversity results from differential splicing of three intron-like regions and of three exons located in the 5'-UTR. Analysis by S1 nuclease protection revealed that the intron-like regions in the 5'-UTR are mostly unspliced and that TPH mRNA species where the three intron-like regions are eliminated are present at low level in pineal gland and not detectable in carcinoid tumors.

Animals↗
PubMed1995

Characterization of the human tryptophan hydroxylase gene promoter. Transcriptional regulation by cAMP requires a new motif distinct from the cAMP-responsive element.

We isolated and sequenced 2,117 nucleotides of the promoter region of the human tryptophan hydroxylase (TPH) gene. Transient transfection in pinealocyte cultures and PC12 cells was used to investigate the human TPH (hTPH) gene promoter activity and its regulation by the cAMP signaling pathway. A region of 2,117 base pairs upstream of the transcription initiation site of the hTPH gene efficiently directed the transcription of a luciferase reporter gene but not in a cell-specific manner. The hTPH promoter activity was significantly enhanced by a cyclic AMP analog in the two cell types. Deletion analysis showed that the promoter region from -73 to +2 is sufficient to direct cAMP-dependent transcription, although it does not contain a motif exhibiting a significant identity to the cAMP-responsive element (CRE) or AP-2 binding site. Following site-directed mutagenesis of the region between -73 and -51, an inverted CCAAT box motif was identified as essential for cAMP inducibility of the hTPH promoter. This sequence between -73 and -51 alone allowed cAMP enhancement of transcription when fused to a heterologous promoter. Additionally, electrophoretic mobility shift assays showed that a specific protein-DNA complex is formed between an oligonucleotide corresponding to the inverted CCAAT box motif and nuclear proteins from pinealocytes treated or not treated with cAMP. Thus cAMP responsiveness of hTPH gene expression is mediated by a cis-acting element, which shares strong identity with an inverted CCAAT box and which binds to a constitutively produced nuclear factor.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1989

An efficient strategy for cloning 5' extremities of rare transcripts permits isolation of multiple 5'-untranslated regions of rat tryptophan hydroxylase mRNA.

The 5' end mapping of rat tryptophan hydroxylase (TPH) mRNA indicated a diversity in 5'-untranslated regions. Corresponding sequences were isolated by a variant of the Polymerase Chain Reaction, recently designated as 'anchor PCR', and a 'cRNA enrichment' procedure. The latter circumvents the limitations of 'anchor PCR', which failed to yield minor TPH sequences: this novel strategy allows purification of specific DNA fragments by elimination of the unspecific products, generated by the PCR, which prevent further amplification. Analysis of TPH sequences strongly suggests that TPH mRNAs are synthesized from at least two promoters, the proximal one exhibiting two 'CCAAT homologies'.

Animals↗
PubMed1989

Differential control of tryptophan hydroxylase expression in raphe and in pineal gland: evidence for a role of translation efficiency.

In a previous study, we characterized two tryptophan hydroxylase mRNAs (TPH mRNAs) in the pineal gland. However, we failed to detect these species in the raphe by Northern blot experiments. Here, we report by S1 nuclease analysis and in situ hybridization that these two TPH mRNAs, as well as a third species, are expressed both in pineal gland and in raphe. In both tissues, the three mRNAs are transcribed predominantly from the same promoter. Strikingly, from the results of S1 maping analysis, it was observed that the total level of TPH mRNA per tissue is at least 150 times lower in the raphe than in the pineal gland. In contrast, TPH antigen as quantified by immunoblot experiments is about threefold more abundant per raphe than per pineal gland. TPH mRNA from one raphe and one pineal gland yield in vitro about the same amount of TPH antigen, suggesting that the discrepancy in the ratios of TPH mRNA and TPH antigen between the raphe and the pineal gland results, at least in part, from a difference in the translation efficiency of TPH mRNAs in the two structures.

Animals↗
PubMed1988

Sequence of two mRNAs encoding active rat tryptophan hydroxylase.

Two full-length cDNA clones that encode functional rat tryptophan hydroxylase (EC 1.14.16.4), the key enzyme in serotonin synthesis, have been isolated from a rat pineal gland library. These two clones correspond to the 1.8- and 4-kilobase mRNA species, respectively. They contain the same coding sequence corresponding to a 51,010-dalton protein and differ in the length of their 3' untranslated regions.

Amino Acid Sequence↗
PubMed1987

The primary structure of human dopamine-beta-hydroxylase: insights into the relationship between the soluble and the membrane-bound forms of the enzyme.

A full length dopamine-beta-hydroxylase (DBH) cDNA clone was isolated from a human pheochromocytoma lambda gt11 library. Both structural and functional evidence confirms the authenticity of the clone: (i) antibodies selected with fusion proteins generated by positive clones precipitate DBH activity, (ii) the sequence of three internal DBH tryptic peptides are included in the deduced DBH sequence, (iii) the previously reported N-terminal 15 amino acids of bovine DBH exhibits a nearly complete identity with that predicted for human DBH. The polypeptide chain of DBH comprises 578 amino acids corresponding to an unmodified protein of 64 862 daltons and is preceded by a cleaved signal peptide of 25 residues. DBH exists in both membrane-bound and soluble forms. The hydropathy plot reveals no obvious hydrophobic segment, except the signal peptide. S1 mapping analysis indicates no diversity in the 5' and 3' extremities of the DBH mRNA. Taken together with available biochemical data, these observations suggest that the membrane attachment of DBH probably results from a post-translational modification, glypiation being the most likely candidate. Comparative amino acid sequence analysis establishes that DBH shares no homology with the other catecholamine synthesizing enzymes, tyrosine hydroxylase and phenylethanolamine-N-methyl transferase.

Adrenal Gland Neoplasms↗
PubMed1987

Sequence of a cDNA encoding human keratin No 10 selected according to structural homologies of keratins and their tissue-specific expression.

We present here the nucleotide sequence of a 1700 bp-long cDNA encoding human epidermal keratin No. 10 (56.5 kDa). cDNA clones of the acidic keratin family were first isolated from a pBR322 human epidermal cDNA library by hybridization with a probe coding for keratin No. 14. Differential hybridization using total cDNA probes prepared from poly(A)+ RNA extracted either from epidermis (which contains keratin No. 10) and from squamous carcinoma or hepatoma cell lines (which do not express keratin No. 10) made possible the selection of clones potentially coding for keratin No. 10. The 1.7 kb sequence exhibits the characteristic features of an acidic keratin with a constant central rod domain and C-terminal variable structures. Moreover, the sequence shows extensive homologies with the cDNA of murine keratin No. 10.

Amino Acid Sequence↗
PubMed1985

Translational activity of mRNA coding for cytoskeletal brain proteins in newborn and adult mice: a comparative study.

Translational activity of mRNA coding for cytoskeletal brain proteins was used to determine the relative abundance of the mRNA in the brains of newborn and adult mice. mRNA was translated in a cell-free system containing rabbit reticulocyte factors. The products of translation were analyzed by two-dimensional gel electrophoresis and characterized by peptide map analysis. Comparison of the products of translation from newborn and from adult brain mRNA shows a 50% decrease in actin and tubulin from newborn to the adult stage. In contrast, the 70 kd neurofilament protein and glial fibrillary acidic protein show a twofold increase in the adult stage. The heat-shock protein HSP70 increases slightly (30%) whereas the brain isozyme of creatine kinase and the heat-shock protein HSP90 are three times as high in adult subjects as in newborns.

Age Factors↗