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J Mallet

Publications and source records attributed to J Mallet.

At least 289 records · Page 16Linked to original sources
PubMed1987

The use of a tyrosine-hydroxylase cDNA probe to study the neurotransmitter plasticity of rat sympathetic neurons in culture.

We have compared quantitatively the effects of muscle-conditioned medium (CM) and elevated K+ concentration (40 mM) on the enzymatic activity of tyrosine hydroxylase (TH) and on TH-mRNA levels in primary cultures of rat sympathetic neurons. Northern blot analysis of RNA from cultured neurons with a 32P-labeled rat TH-cDNA probe was performed. The probe hybridized strongly with a single RNA species of 1.9 kb, similar in size to the TH-mRNA from PC12 pheochromocytoma cells. In agreement with earlier data both CM and a partially purified factor from CM increased choline acetyltransferase activity up to 200-fold and depressed TH activity by 2- to 7-fold in cultured sympathetic neurons. These effects were accompanied by a decrease in TH-mRNA level, which correlated with the decrease in TH activity. On the other hand, a culture medium supplemented with 40 mM KCl caused a 1.5- to 5-fold increase in TH activity, which was accompanied by an increase in TH-mRNA level of the same order of magnitude. As a working hypothesis, we suggest that CM and neuronal depolarization control the transcription of the TH gene in an antagonistic manner.

Animals↗
PubMed1987

Molecular genetics of catecholamines as an approach to the biochemistry of manic-depression.

Manic depressive illness has been clearly established to exhibit a strong genetic component and is therefore amenable to linkage analysis using random DNA markers. In view of the catecholamine hypothesis of this disorder, the gene encoding tyrosine hydroxylase (TH) the limiting enzyme in catecholamines is a good candidate to investigate. This gene has been localized to chromosome 11 in close linkage with Harvey-ras-1. The various transcriptional and post-transcriptional mechanisms that modulate short and long-term TH activity are discussed. Human tyrosine hydroxylase is coded by at least three distinct mRNAs derived from a single gene. This variation has clear functional consequences and could represent a novel mode of regulating catecholamines levels in normal and pathological neurons.

Bipolar Disorder↗
PubMed1987

cDNA cloning and complete sequence of porcine choline acetyltransferase: in vitro translation of the corresponding RNA yields an active protein.

A cDNA clone encoding the complete sequence of porcine choline acetyltransferase (ChoAcTase; acetyl-CoA: choline O-acetyltransferase, EC 2.3.1.6.) has been identified. A cDNA library, constructed from poly(A)+ RNA of ventral spinal cord, was screened with a mixture of eight oligonucleotides corresponding to the N-terminal sequence of pig brain ChoAcTase. Among five positive clones, one, pChAT-1, was identified as a ChoAcTase cDNA clone based on the following criteria. (i) This clone has an open reading frame coding for a protein of the size expected for ChoAcTase (640 amino acids). (ii) The amino acid composition deduced from the nucleotide sequence of this open reading frame matches that of purified porcine ChoAcTase. (iii) When subcloned in the T7 expression system, the corresponding RNA directs the synthesis in the rabbit reticulocyte lysate of a protein that is specifically immunoprecipitated by antibodies raised against ChoAcTase. (iv) Finally and most important, this corresponding RNA, when translated in the reticulocyte lysate, as well as in the Xenopus oocyte system, directs the synthesis of a protein displaying ChoAcTase activity. This activity is inhibited by the specific ChoAcTase inhibitor 4-(1-naphthylvinyl)pyridine. Comparison of porcine ChoAcTase sequence with that of Drosophila reveals 32% identity between these proteins, when the sequences are suitably aligned. pChAT-1 probe hybridizes with a porcine mRNA species that is at least 7000 nucleotides long, whereas the equivalent rat mRNA species is 3700 nucleotides long.

Amino Acid Sequence↗
PubMed1987

Modulation of tyrosine hydroxylase gene expression in the central nervous system visualized by in situ hybridization.

A rat tyrosine hydroxylase [TyrOHase; tyrosine 3-monooxygenase; L-tyrosine, tetrahydropteridine:oxygen oxidoreductase (3-hydroxylating); EC 1.14.16.2] cDNA probe was used for in situ hybridization studies on histological sections through the locus coeruleus, substantia nigra, and the ventral tegmental area of the rat brain. Experimental conditions were established that yielded no background and no signal when pBR322 was used as a control probe. Using the tyrosine hydroxylase probe, we ascertained the specificity of the labeling over catecholaminergic cells by denervation experiments and comparison of the hybridization pattern with that of immunoreactivity. The use of 35S-labeled probe enabled the hybridization signal to be resolved at the cellular level. A single injection of reserpine into the rat led to an increase of the intensity of the autoradiographic signal over the locus coeruleus area, confirming an RNA gel blot analysis. The potential of in situ hybridization to analyze patterns of modulation of gene activity as a result of nervous activity is discussed.

Animals↗
PubMed1986

Linkage of tyrosine hydroxylase to four other markers on the short arm of chromosome 11.

Tyrosine hydroxylase is the rate-limiting enzyme in catecholamine synthesis; the gene has previously been cloned and localised to the short arm of chromosome 11. Because of the interest in tyrosine hydroxylase as a candidate gene for manic-depressive psychosis and other affective disorders, we carried out family studies to determine the linkage of tyrosine hydroxylase with insulin, beta-globin, D11S12 and Harvey-ras 1, members of a linkage group which has previously been localised to 11p. Using DNA from the Centre d'Etude du Polymorphisme Humain (CEPH) and from two large British pedigrees, we show that tyrosine hydroxylase is closely linked to these four loci (z = 7.36, theta = 0.04 for linkage to insulin) and suggest a gene order based on multipoint mapping.

Alleles↗
PubMed1986

Synthesis of catalytically active choline acetyltransferase in Xenopus oocytes injected with messenger RNA from rat central nervous system.

Choline acetyltransferase (ChAT) mRNA and ChAT enzymatic activity have been compared in different regions of the rat central nervous system. mRNA was assayed by exploiting the Xenopus oocyte system which was first tested by measuring the electrophysiological response to glycine after injection of mRNA derived from the ventral part of the spinal cord (VSC). This tissue was found to contain the highest ChAT mRNA level. The striatum, which yielded the maximal enzymatic activity, contained 10 times less ChAT mRNA than the VSC. These results are discussed in terms of the neuroanatomical differences between the two structures.

Animals↗
PubMed1986

A single RNA species injected in Xenopus oocyte directs the synthesis of active tyrosine hydroxylase.

Tyrosine hydroxylase, the rate limiting enzyme in the biosynthesis of catecholamine, is a tetramer composed of four subunits of the same molecular mass. A full length cDNA clone encoding tyrosine hydroxylase has been inserted into the SP6 expression system. Translation of the corresponding RNA in Xenopus oocyte results in enzymatic activity, demonstrating that a single gene contains all the necessary genetic information to code for a functional enzyme. The potential of this system in the analysis of posttranslational tyrosine hydroxylase modifications is discussed.

Animals↗
PubMed1986

Time course of the changes of TH mRNA in rat brain and adrenal medulla after a single injection of reserpine.

A single injection of reserpine causes a long lasting enhancement of the activity of tyrosine hydroxylase (TH), the enzyme catalyzing the rate-limiting step in the biosynthesis of catecholamines. A sensitive method has been developed to assay both TH mRNA level and enzyme activity in tissue from a single rat. The time course of the induction was analysed in adrenals, locus coeruleus and substantia nigra. In both locus coeruleus and adrenals reserpine caused respectively 4.2- and 4.5-fold increase of TH mRNA which was maximal 2 days after drug injection. This increase is about twice that of the enzyme activity. No change was observed in substantia nigra. The effect lasted longer in locus coeruleus than in adrenal. In the latter, TH mRNA had almost returned to initial values at day 4 whereas at this time it is 3-fold higher in locus coeruleus and still significant at day 18. This result suggests that induction of TH results from an enhanced transcription of the TH gene. The time course difference between locus coeruleus and adrenals is most likely to result from a difference in the stability of TH mRNA in the two structures.

Adrenal Medulla↗
PubMed1986

The primary structure of bovine chromogranin A: a representative of a class of acidic secretory proteins common to a variety of peptidergic cells.

We have determined the primary structure of bovine chromogranin A as a first step in the elucidation of the function of this widespread protein. After oligonucleotide screening of a cDNA library of bovine adrenal medulla, a clone (insert length 1.9 kb) containing the entire coding region for chromogranin A was isolated and sequenced. The authenticity of the sequence was verified by comparison with N-terminal, several internal, and C-terminal amino acid sequences as well as the amino acid composition of chromogranin A. The cDNA clone hybridized to an mRNA of 2.1 kb and, after in vitro transcription-translation, yielded a polypeptide with a similar electrophoretic mobility in SDS gels to chromogranin A. The polypeptide chain of chromogranin A comprises 431 amino acid residues, corresponding to an unmodified protein of 48 kd, and is preceded by a cleaved signal peptide of 18 amino acid residues. Interesting features of the chromogranin A structure include repeated clusters of glutamic acid residues, the occurrence of eight potential dibasic cleavage sites, six of which are located in the C-terminal domain, and the presence, in the N-terminal domain, of -Arg-Gly-Asp- (RGD), a three amino acid sequence involved in the binding of several constitutively secreted proteins to cell membranes.

Adrenal Medulla↗
PubMed1986

An efficient approach for the selective isolation of specific transcripts from complex brain mRNA populations.

As a consequence of the complexity of the nervous system, many characteristic proteins are only expressed at low levels, and their detection and purification often represents a formidable task. A strategy based on liquid hybridizations, which greatly facilitates the identification of the mRNA transcript for this kind of protein, is presented here. A ten-fold-enriched recombinant library was generated from cDNA transcribed from forebrain mRNA after subtraction of cerebellum sequences. Clones specific to forebrain could then be revealed with cerebellum subtracted probes, by colony hybridization. The use of selected cDNA populations greatly enhanced the sensitivity of the screening procedure; clones corresponding to transcripts present at an abundance as low as 0.0005% could still be detected. About 5% of specific clones were recognized with an enriched forebrain probe. Additional clones were revealed with subtracted probes from restricted areas such as cerebral cortex, brainstem, and hippocampus. The important features and potential applications of this approach are discussed.

Animals↗
PubMed1986

Localization of the human tyrosine hydroxylase gene to 11p15: gene duplication and evolution of metabolic pathways.

Phenylalanine hydroxylase (PAH) and tyrosine hydroxylase (TH) are consecutive enzymes in the metabolic pathway leading to the production of catecholamine neurotransmitters. A comparison of recently available sequence data of these enzymes in the rat indicates about 70% homology in the 3' coding regions. We have localized TH by in situ hybridization to human chromosome region 11p15. Consideration of this assignment and that of PAH to chromosome 12, together with the known distribution of other pairs of related genes on these two chromosomes, provides convincing evidence of their ancestral relationship and suggests a role for gene duplication in the diversification of metabolic pathways in the vertebrate ancestors of mammals.

Animals↗
PubMed1985

Cloning and sequence analysis of the cDNA encoding a snake neurotoxin precursor.

A recombinant plasmid has been constructed containing a sequence of 186 nucleotides encoding a potent neurotoxin found in the venom of the sea-snake Laticauda semifasciata and designated as erabutoxin a. This sequence is flanked, in the upstream region, by a sequence of 60 nucleotides encoding a hydrophobic peptide fragment presumably involved in the secretion process of the neurotoxin. The sequence coding for the toxin ends with a termination codon which is followed by a 3'-untranslated sequence of approximately 240 nucleotides (excluding the poly(A) tract).

Amino Acid Sequence↗
PubMed1985

Complete coding sequence of rat tyrosine hydroxylase mRNA.

Several clones specific for tyrosine hydroxylase [tyrosine 3-monooxygenase, L-tyrosine, tetrahydropteridine:oxygen oxidoreductase (3-hydroxylating), EC 1.14.16.2] have been identified from a rat PC12 library by using the previously characterized clone pTH-1. The most complete of these, pTH-51, is 1758 base pairs long and covers most of the length of the mRNA, including the entire coding and 3' untranslated region. The polypeptide has an estimated molecular weight of 55,903 and some of its characteristic features are discussed.

Amino Acid Sequence↗