Once bound to methylated CpG sites, methyl-CpG-binding protein 2 (MeCP2) is thought to silence transcription of downstream genes by recruiting a histone deacetylase (HDAC). Mutations within the MeCP2 gene have been found to cause Rett syndrome, a disorder of arrested neuronal development. Using immunohistochemistry, we found that Mecp2, as well as the methyl-CpG-binding protein MBD1, were significantly induced in normal adult rat brain after repeated injections of fluoxetine or cocaine for 10 days (one injection per day). Mecp2 was not induced by repeated injections of 1-(2-bis(4-fluorphenyl)-methoxy)-ethyl)-4-(3-phenyl-propyl)piperazine (GBR-12909) or nortriptyline. Together, the data indicate that the serotonergic system is predominantly involved. Using real-time reverse transcription-polymerase chain reaction experiments, MBD1 mRNA and both Mecp2_e1 and Mecp2_e2 transcripts were found to be induced by fluoxetine. Induction of the methylbinding proteins was accompanied with enhanced HDAC2 labeling intensity and mRNA synthesis in response to fluoxetine. In tandem, acetylated forms of histone H3 were found to be decreased. The effect was characterized in three serotonin projection areas, the caudate-putamen, the frontal cortex, and the dentate gyrus subregion of hippocampus. Our data highlight GABAergic neurons as major target cells expressing Mecp2 in response to the serotonin-elevating agents and suggest that serotonin signaling enhances gene silencing in postmitotic neurons.
Injection of the histone deacetylases inhibitor trichostatin A to rats has been shown to decrease the reinforcing properties of cocaine. In the present study, we investigated alterations in gene expression patterns in the anterior cingulate cortex, caudate-putamen and nucleus accumbens of rats self-administering cocaine and treated with trichostatin A. As recent studies highlighted the importance of chromatin remodelling in the regulation of gene transcription in neurons, we studied the expression of Mecp2 and of several histone deacetylases. Cocaine self-administration was accompanied by an increased synthesis of Mecp2, HDAC2 and HDAC11 and by a decreased nuclear localization of HDAC5 and of the phospho-form of HDAC5, suggesting a nuclear export of this protein in response to the drug. The latter mechanism was further addressed by the demonstration of an enhanced expression of MEF2C transcription factor. Among the genes we examined, treatment with trichostatin A before each cocaine self-administration session was found to mostly affect Mecp2 and HDAC11 expression. A correlation was found between the modification of Mecp2 and MEF2C gene expression and the reinforcing property of cocaine. The two factors known to regulate gene transcription are likely to play a role in the neurobiological mechanism underlying a decrease in the reinforcing properties of cocaine.
The complete genome sequence of the hyperthermophilic archaeon Pyrococcus abyssi revealed the presence of a family B DNA polymerase (Pol I) and a family D DNA polymerase (Pol II). To extend our knowledge about euryarchaeal DNA polymerases, we cloned the genes encoding these two enzymes and expressed them in Escherichia coli. The DNA polymerases (Pol I and Pol II) were purified to homogeneity and characterized. Pol I had a molecular mass of < 90 kDa, as estimated by SDS/PAGE. The optimum pH and Mg 21 concentration of Pol I were 8.5-9.0 and 3 mM, respectively. Pol II is composed of two subunits that are encoded by two genes arranged in tandem on the P. abyssi genome. We cloned these genes and purified the Pol II DNA polymerase from an E. coli strain coexpressing the cloned genes. The optimum pH and Mg 21 concentration of Pol II were 6.5 and 15-20 mM, respectively. Both P. abyssi Pol I and Pol II have associated 3 0 !5 0 exonuclease activity although the exonuclease motifs usually found in DNA polymerases are absent in the archaeal family D DNA polymerase sequences. Sequence analysis has revealed that the small subunit of family D DNA polymerase and the Mre11 nucleases belong to the calcineurin-like phosphoesterase superfamily and that residues involved in catalysis and metal coordination in the Mre11 nuclease three-dimensional structure are strictly conserved in both families. One hypothesis is that the phosphoesterase domain of the small subunit is responsible for the 3 0 !5 0 exonuclease activity of family D DNA polymerase. These results increase our understanding of euryarchaeal DNA polymerases and are of importance to push forward the complete understanding of the DNA replication in P. abyssi.Keywords: Pyrococcus abyssi; archaea; DNA polymerase; DNA replication; exonuclease activity.DNA polymerases play a leading role in the replication and maintenance of the genome and are central to the accurate transmission of genetic information from generation to generation. While our knowledge about DNA replication in eukarya and bacteria is quite advanced [1], limited information is available on the replication mechanism in archaea, the third major domain of life [2]. Recently, comparative genomics revealed that most archaeal proteins involved in DNA replication, transcription and translation are similar to those in eukarya, although the cellular appearance and organization of archaea are more similar to bacteria. Recent investigations [3,4] revealed that within the archaeota, euryarchaeota and crenarchaeota, the two major subdomains differ in their DNA replication mechanisms. The analysis of genome sequences indicated that many euryarchaea [5][6][7] [13] indicates that several B-type DNA polymerases exist in the crenarchaeotal genomes. These findings confirm that the DNA replication mechanism of the euryarchaeal and crenarchaeal subdomains of archaea differs, and therefore opens the discussion of the evolution of DNA polymerases, a group of indispensable proteins that are central to the replication process. However, fo...
This work reports the first isolation and characterization of an alkaline phosphatase (AP) from a hyperthermophilic archaeon. An AP gene from Pyrococcus abyssi, a euryarchaeon isolated from a deep-sea hydrothermal vent, was cloned and the enzyme expressed in Escherichia coli. Analysis of the sequence showed conservation of the active site and structural elements of the E. coli AP. The recombinant AP was purified and characterized. Monomeric and homodimeric active forms were detected, with a monomer molecular mass of 54 kDa. Apparent optimum pH and temperature were estimated at 11.0 and 70°C, respectively. Thus far, P. abyssi AP has been demonstrated to be the most thermostable AP, with half-lives at 100 and 105°C of 18 and 5 h, respectively. Enzyme activity was found to be dependent on divalent cations: metal ion chelators inhibited activity, whereas the addition of exogenous Mg(II), Zn(II), and Co(II) increased activity. The enzyme was inhibited by inorganic phosphate, but not by molybdate and vanadate. Strong inhibitory effects were observed in the presence of thiolreducing agents, although cysteine residues of the P. abyssi AP were not found to be incorporated within intraor interchain disulfide bonds. In addition, P. abyssi AP was demonstrated to dephosphorylate linear DNA fragments with dephosphorylation efficiencies of 93.8 and 84.1% with regard to cohesive and blunt ends, respectively.
Kinetics of condensation of ribonucleotides to dinucleotides, leading to trinucleotide products formation, have been studied using wheat germ RNA polymerase II and poly(dAT). Assay conditions can be selected under which both ApUpA and UpApU are formed in catalytic amounts. The kinetic parameters associated with these reactions indicate that the rate of trinucleotide formation might be affected by DNA sequence, as reported for E.coli RNA polymerase. Kinetics of disappearance of ApUpA and UpApU were studied under experimental conditions allowing poly(rAU) synthesis. The results can be interpreted as if after formation of a phosphodiester bond, a slow isomerisation step of the ternary transcription complex could occur. During this step, transcription complexes could dissociate with a finite probability, releasing trinucleotides in an abortive pathway. The above results are discussed in the view that, under these experimental conditions, wheat germ RNA polymerase II catalyses poly(rAU) synthesis, as if it is a non-processive enzyme. Cordycepin triphosphate can be condensed to a dinucleotide primer, yielding ApUpA. However the ATP analogue cannot be incorporated into longer products than a trinucleotide. On the other hand 3'-dATP behaves as a very potent inhibitor of translocation, with an inhibition constant of 0.15 microM, a value which is two orders of magnitude smaller than the Km value corresponding to ATP utilization in poly(rAU) synthesis. Simple models are proposed which allow a comparison with E.coli RNA polymerase, for which the results are well documented.
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