Identification of a single nucleotide polymorphism in the choline acetyltransferase gene associated with Alzheimer's disease.

The dysfunction of the cholinergic system in Alzheimer's disease (AD) supports the hypothesis that a decline in choline acetyltransferase (ChAT) activity in memory as well as in cognitive functions in AD might be functionally linked. To assess the physiological relevance of an allelic variation in the ChAT gene we investigated the presence of a possible polymorphism in AD patients and in elderly non-demented subjects as controls. By using polymerase chain reaction, single stranded conformation polymorphism or the LightCycler analysis we detected a single nucleotide polymorphism in the first common coding exon of the ChAT gene.

Synergistic activation of the human choline acetyltransferase gene by c-Myb and C/EBPbeta.

To elucidate regulatory mechanisms at the transcriptional level of the human choline acetyltransferase gene (hChAT) we performed cotransfections assays in NG108-15 and SN56 cells using ChAT-CAT reporter plasmids with c-Myb and C/EBPbeta expression plasmids. The hChAT gene has several promoters, one of which (promoter P2 or M-type) is both c-Myb and C/EBPbeta inducible as 3-4-fold trans-activation was obtained in both cell lines when using either c-Myb or C/EBPbeta expression vectors alone. The simultaneous expression of c-Myb and C/EBPbeta in the absence or presence of NGFI-C (egr4) leads respectively to a 15-fold and 32-fold synergistic transcriptional activation of promoter P2.

A novel untranslated 'exon H' of the human choline acetyltransferase gene in placenta.

To investigate the existence of 5'-region(s) of human choline acetyltransferase (hChAT) mRNA in placenta we analyzed the presence or absence of ChAT 5'-untranslated regions (UTR) in human neuronal and non-neuronal cells. Total RNA from human spinal cord, placenta, cultured choriocarcinoma JEG-3 and neuroblastoma CHP126 and MC-IXC cells was reverse transcribed and used for polymerase chain reaction amplification (RT-PCR). We used a sense primer located in the 5'-flanking region, in the previously defined intronic sequence and an anti-sense primer located in the common coding exon 2 of the hChAT gene.

Production and functional expression of an epitope-tagged human choline acetyltransferase.

cDNA containing the entire coding region of the human choline acetyltransferase gene (hChAT) was fused to the influenza virus hemagglutinin (HA) epitope preceded by a Kozak sequence. The recombinant HA-hChAT was then inserted into an expression vector under the transcriptional control of the cytomegalovirus (CMV) promoter. After transient transfection into COS-1 cells, expression was assayed by Northern and Western blot analysis and immunofluorescence.

Transcriptional activation of human choline acetyltransferase by AP2- and NGF-induced factors.

ChAT (choline acetyltransferase) is the enzyme responsible for acetylcholine synthesis and is specifically expressed in cholinergic neurons. To further characterize the transcriptional regulation of the hCHAT (human ChAT) gene by NGF, we examined the effects upon ChAT promoter activity of a family of transcription factors which are activated by NGF and several extracellular stimuli and encoded by immediate-early genes. These include NGFI-A (Egr1, zif268), NGFI-C (Egr2), Krox-20 and NGFI-B (Nurr77).

Positive and negative effects of nuclear receptors on transcription activation by AP-1 of the human choline acetyltransferase proximal promoter.

We have examined the 5'-flanking region (944 bp) of the human choline acetyltransferase (hChAT) gene for sequences that modulate its transcriptional activity and identified a sequence 5'-TGACCCA-3' which confers c-Jun/c-Fos (AP-1) inducibility of homologous and heterologous promoters. Using transient transfections in neuroblastoma NE-1-115 and COS-1 cells, we show that ligand-activated estrogen receptor (HEGo) represses the transcriptional activation by c-Fos/c-Jun. Testing HEGo mutants in transfection assays reveals that the ligand-binding domain is crucial for this repression, whereas the N-terminal (A/B) region and the DNA-binding domain are not essential.

A previously unidentified choline acetyltransferase transcript in the human foetal brain.

To identify choline acetyltransferase (ChAT) transcript(s) in human foetal brain we used the reverse polymerase chain reaction including different couples of oligonucleotide primers. The analysis indicated the presence of a new choline acetyltransferase transcript containing at its 5' end an untranslated exon (E1A) of 57 nucleotides in length extending from Nt 3771 to Nt 3828. It differs in the 5'-non coding region from the M-N-R type mRNA previously described in mouse and rat.

Trans-activation by thyroid hormone receptors of the 5' flanking region of the human ChAT gene.

Fusion gene constructs containing the human choline acetyltransferase 5' flanking region are stimulated by thyroid hormone (T3) in neuronal NG108-15 and NE1-115 cells but not in non neuronal COS-1 and JEG-3 cells. To identify potential T3 receptor binding elements (T3RE), chimeric plasmids containing various lengths of the 5' end of the hChAT gene linked to the CAT reporter gene were assayed by transient transfections into NG108-15, NE1-115 and COS-1 cells. We show that regulation is T3 specific as estrogen, dexamethasone, dihydrotestosterone, all-trans-retinoic acid and 9-cis-retinoic acid have no effect.

Identification and analysis of the human choline acetyltransferase gene promoter.

Choline acetyltransferase (ChAT) is the key enzyme responsible for the synthesis of the neurotransmitter acetylcholine and is reduced in various central neurodegenerative diseases. From a previously selected 12.6 kb human choline acetyltransferase (hChAT) genomic clone, we have identified and characterized a promoter region of 895 bp.

A limiting factor mediates the differential activation of promoters by the human progesterone receptor isoforms.

The two transcription activation functions (TAFs) of the human progesterone receptor (hPR) have been characterized. TAF-1, located in the N-terminal region A/B, has been narrowed down to a 91-amino acid sequence, which is sufficient for transcription activation in chimeras with the GAL4 DNA binding domain. Both hPR TAF-1 and TAF-2 activate a minimal promoter in yeast.

Expression of active hormone and DNA-binding domains of the chicken progesterone receptor in E. coli.

Bacterially-expressed fusion proteins containing the DNA-(region C) or hormone-binding (region E) domains of the chicken progesterone receptor (cPR) fused to the C terminus of Escherichia coli beta-galactosidase were analysed for the specificity of interaction with natural and synthetic hormone-responsive elements (HREs) and progestins, respectively. The purified fusion protein containing the progestin-binding domain bound progesterone with an apparent Kd of 1.0-1.

The chicken progesterone receptor: sequence, expression and functional analysis.

The complete mRNA sequence of the chicken progesterone receptor (cPR) has been determined. Expression of the cloned cDNA both in vivo and in vitro produces a protein that has the same apparent mol. wt on SDS--polyacrylamide gels as the 'natural' cPR form B (109 kd) as determined by immunoblotting and photoaffinity labelling.

Cloning of the chicken progesterone receptor.

Monospecific antibodies directed against the chicken progesterone receptor (PR) form B were used to screen a randomly primed phage lambda gt11 cDNA expression library prepared from size-fractionated chicken oviduct mRNA. Two independent immunoreactive clones, lambda cPR1 and lambda cPR2, were isolated. Antibodies selected from anti-PR form B antiserum on matrices of lambda cPR1 and lambda cPR2 fusion proteins detected two proteins on electrophoretic immunoblots of crude and purified PR preparations.

Histone H1 kinase from mouse plasmacytoma. Further characterization and molecular structure.

A cAMP-independent protein kinase which phosphorylates histone H1 to a high level and which may correspond to the mitotic H1 kinase has been partially purified and characterized from mouse plasmacytoma microsomes [Quirin-Stricker, C., and Schmitt, M. (1981) Eur.

Microsomal cAMP-independent histone H1 kinase activity in plasmacytoma, Morris hepatoma and normal liver.

A protein kinase activity with high specificity for histone H1 was isolated from mouse plasmacytoma, Morris hepatoma and normal mouse liver and compared by ion exchange chromatography after DEAE-cellulose, hydroxylapatite and Sephadex G-200 chromatography. This cAMP-independent histone H1 kinase is not affected by the heat-stable cAMP-dependent protein kinase inhibitor. It has the following particular properties: it prefers GTP to ATP as substrate and was found to be present with a great activity only in neoplastic tissues.

Purification and characterization of a specific histone H1 protein kinase from mouse plasmacytoma.

A protein kinase with high specificity for histone H1 was purified from a plasmacytoma microsomal fraction by a high-salt wash, ammonium sulfate precipitation, chromatography on DEAE-cellulose, hydroxyapatite and Sephadex G-200 columns, and the main properties of this kinase were studied. A sulfhydryl compound, such as 2-mercaptoethanol or dithiothreitol, was necessary for full activity. The optimum pH was 7.

Messenger RNA binding of a ribosome-associated protein kinase and a ribosomal phosphoprotein(s) in mouse plasmacytoma.

A specific ribosome-associated protein kinase (protein kinase II) and phosphoprotein(s) from the ribosomal KCl wash fraction termed "PPx" have been isolated from plasmacytoma, and tested for their ability to bind to poly(A) and to different plasmacytoma polynucleotides. The nitrocellulose filter binding technique was used to measure RNA-protein interaction. Protein kinase II and PPx preferentially bound mRNA compared to poly(A).

Resolution and general properties of different types of ribosomal protein kinases in mouse plasmocytoma.

Three different types of protein kinases (ATP: protein phosphotransferase, EC 2.7.1.

A plasmocytoma ribosome-associated protein kinase which phosphorylates a specific protein of the ribosomal KCl wash.

One ribosomal protein kinase activity and 3 soluble protein kinase activities have been identified in plasma cell tumors by DEAE-cellulose chromatography. We have shown phosphorylation in vivo and in vitro of a protein fraction from the ribosomal KCl wash which we have termed 'PPx fraction'. Phosphorylation of this protein fraction has been obtained in vitro with the ribosome-associated protein kinase.

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We have shown the existence in rat liver of free light cytoplasmic particles containing a stable, rapidly labelled RNA. These particles differ from the 45 S ribosomal fraction by several characters.The buoyant density and sedimentation properties suggest that these particles are similar to dRNA ribonucleoprotein particles released from polysomes and to those present in rat liver nuclei.