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Transcripts Encoding (transcript + encoding)
Selected AbstractsTemporal coupling of cyclic AMP and Ca2+/calmodulin-stimulated adenylyl cyclase to the circadian clock in chick retinal photoreceptor cellsJOURNAL OF NEUROCHEMISTRY, Issue 4 2006Shyam S. Chaurasia Abstract cAMP signaling pathways play crucial roles in photoreceptor cells and other retinal cell types. Previous studies demonstrated a circadian rhythm of cAMP level in chick photoreceptor cell cultures that drives the rhythm of activity of the melatonin synthesizing enzyme arylalkylamine N -acetyltransferase and the rhythm of affinity of the cyclic nucleotide-gated channel for cGMP. Here, we report that the photoreceptor circadian clock generates a rhythm in Ca2+/calmodulin-stimulated adenylyl cyclase activity, which accounts for the temporal changes in the cAMP levels in the photoreceptors. The circadian rhythm of cAMP in photoreceptor cell cultures is abolished by treatment with the l -type Ca2+ channel antagonist nitrendipine, while the Ca2+ channel agonist, Bay K 8644, increased cAMP levels with continued circadian rhythmicity in constant darkness. These results indicate that the circadian rhythm of cAMP is dependent, in part, on Ca2+ influx. Photoreceptor cell cultures exhibit a circadian rhythm in Ca2+/calmodulin-stimulated adenylyl cyclase enzyme activity with high levels at night and low levels during the day, correlating with the temporal changes of cAMP in these cells. Transcripts encoding two of the Ca2+/calmodulin-stimulated adenylyl cyclases, type 1 and type 8 (Adcy1 and Adcy8), displayed significant daily rhythms of mRNA expression under a light,dark cycle, but only the Adcy1 transcript rhythm persisted in constant darkness. Similar rhythms of Adcy1 mRNA level and Ca2+/calmodulin-stimulated adenylyl cyclase activity were observed in retinas of 2-week-old chickens. These results indicate that a circadian clock controls the expression of Adcy1 mRNA and Ca2+/calmodulin-stimulated adenylyl cyclase activity; and calcium influx into these cells gates the circadian rhythm of cAMP, a key component in the regulation of photoreceptor function. [source] A family of stage-specific alanine-rich proteins on the surface of epimastigote forms of Trypanosoma bruceiMOLECULAR MICROBIOLOGY, Issue 1 2007Simon Urwyler Summary A ,two coat' model of the life cycle of Trypanosoma brucei has prevailed for more than 15 years. Metacyclic forms transmitted by infected tsetse flies and mammalian bloodstream forms are covered by variant surface glycoproteins. All other life cycle stages were believed to have a procyclin coat, until it was shown recently that epimastigote forms in tsetse salivary glands express procyclin mRNAs without translating them. As epimastigote forms cannot be cultured, a procedure was devised to compare the transcriptomes of parasites in different fly tissues. Transcripts encoding a family of glycosylphosphatidyl inositol-anchored proteins, BARPs (previously called bloodstream alanine-rich proteins), were 20-fold more abundant in salivary gland than midgut (procyclic) trypanosomes. Anti-BARP antisera reacted strongly and exclusively with salivary gland parasites and a BARP 3, flanking region directed epimastigote-specific expression of reporter genes in the fly, but inhibited expression in bloodstream and procyclic forms. In contrast to an earlier report, we could not detect BARPs in bloodstream forms. We propose that BARPs form a stage-specific coat for epimastigote forms and suggest renaming them brucei alanine-rich proteins. [source] DLEU2 encodes an antisense RNA for the putative bicistronic RFP2/LEU5 gene in humans and mouseGENES, CHROMOSOMES AND CANCER, Issue 4 2004Martin M. Corcoran Our group previously identified two novel genes, RFP2/LEU5 and DLEU2, within a 13q14.3 genomic region of loss seen in various malignancies. However, no specific inactivating mutations were found in these or other genes in the vicinity of the deletion, suggesting that a nonclassical tumor-suppressor mechanism may be involved. Here, we present data showing that the DLEU2 gene encodes a putative noncoding antisense RNA, with one exon directly overlapping the first exon of the RFP2/LEU5 gene in the opposite orientation. In addition, the RFP2/LEU5 transcript can be alternatively spliced to produce either several monocistronic transcripts or a putative bicistronic transcript encoding two separate open-reading frames, adding to the complexity of the locus. The finding that these gene structures are conserved in the mouse, including the putative bicistronic RFP2/LEU5 transcript as well as the antisense relationship with DLEU2, further underlines the significance of this unusual organization and suggests a biological function for DLEU2 in the regulation of RFP2/LEU5. © 2004 Wiley-Liss, Inc. [source] Increased expression of GluR2-flip in the hippocampus of the Wistar audiogenic rat strain after acute and kindled seizuresHIPPOCAMPUS, Issue 1 2010Daniel Leite Góes Gitaí Abstract The Wistar Audiogenic Rat (WAR) is an epileptic-prone strain developed by genetic selection from a Wistar progenitor based on the pattern of behavioral response to sound stimulation. Chronic acoustic stimulation protocols of WARs (audiogenic kindling) generate limbic epileptogenesis, confirmed by ictal semiology, amygdale, and hippocampal EEG, accompanied by hippocampal and amygdala cell loss, as well as neurogenesis in the dentate gyrus (DG). In an effort to identify genes involved in molecular mechanisms underlying epileptic process, we used suppression-subtractive hybridization to construct normalized cDNA library enriched for transcripts expressed in the hippocampus of WARs. The most represented gene among the 133 clones sequenced was the ionotropic glutamate receptor subunit II (GluR2), a member of the ,-amino-3-hydroxy-5-methyl-4-isoxazoleopropionic acid (AMPA) receptor. Although semiquantitative RT-PCR analysis shows that the hippocampal levels of the GluR2 subunits do not differ between naïve WARs and their Wistar counterparts, we observed that the expression of the transcript encoding the splice-variant GluR2-flip is increased in the hippocampus of WARs submitted to both acute and kindled audiogenic seizures. Moreover, using in situ hybridization, we verified upregulation of GluR2-flip mainly in the CA1 region, among the hippocampal subfields of audiogenic kindled WARs. Our findings on differential upregulation of GluR2-flip isoform in the hippocampus of WARs displaying audiogenic seizures is original and agree with and extend previous immunohistochemical for GluR2 data obtained in the Chinese P77PMC audiogenic rat strain, reinforcing the association of limbic AMPA alterations with epileptic seizures. © 2009 Wiley-Liss, Inc. [source] Molecular cloning and immunoglobulin E reactivity of a natural rubber latex lecithinase homologue, the major allergenic component of Hev b 4CLINICAL & EXPERIMENTAL ALLERGY, Issue 11 2005E. Sunderasan Summary Background Hev b 4 is an allergenic natural rubber latex (NRL) protein complex that is reactive in skin prick tests and in vitro immunoassays. On SDS-polyacrylamide gel electrophoresis (SDS-PAGE), Hev b 4 is discerned predominantly at 53,55 kDa together with a 57 kDa minor component previously identified as a cyanogenic glucosidase. Of the 13 NRL allergens recognized by the International Union of Immunological Societies, the 53,55 kDa Hev b 4 major protein is the only candidate that lacks complete cDNA and protein sequence information. Objective We sought to clone the transcript encoding the Hev b 4 major protein, and characterize the native protein and its recombinant form in relation to IgE binding. Methods The 5,/3, rapid amplification of cDNA ends method was employed to obtain the complete cDNA of the Hev b 4 major protein. A recombinant form of the protein was over-expressed in Escherichia coli. The native Hev b 4 major protein was deglycosylated by trifluoromethane sulphonic acid. Western immunoblots of the native, deglycosylated and recombinant proteins were performed using both polyclonal antibodies and sera from latex-allergic patients. Results The cDNA encoding the Hev b 4 major protein was cloned. Its open reading frame matched lecithinases in the conserved domain database and contained 10 predicted glycosylation sites. Detection of glycans on the Hev b 4 lecithinase homologue confirmed it to be a glycoprotein. The deglycosylated lecithinase homologue was discerned at 40 kDa on SDS-PAGE, this being comparable to the 38.53 kDa mass predicted by its cDNA. Deglycosylation of the lecithinase homologue resulted in the loss of IgE recognition, although reactivity to polyclonal rabbit anti-Hev b 4 was retained. IgE from latex-allergic patients also failed to recognize the non-glycosylated E. coli recombinant lecithinase homologue. Conclusion The IgE epitopes of the Hev b 4 lecithinase homologue reside mainly in its carbohydrate moiety, which also account for the discrepancy between the observed molecular weight of the protein and the value calculated from its cDNA. [source] Interleukin-5 does not influence differential transcription of transmembrane and soluble isoforms of IL-5R, in vivoEUROPEAN JOURNAL OF HAEMATOLOGY, Issue 3 2006Jonas Byström Abstract:, Interleukin-5 (IL-5) promotes signal transduction and expansion of eosinophil colonies in bone marrow via interactions with its heterodimeric receptor (IL-5R). Two variants encoding soluble forms of the alpha subunit (sIL-5R,) have been described, although the signals promoting and/or limiting differential transcription remain to be clarified. Objectives:,Our intent was to explore the role of IL-5 in regulating differential transcription of these splice variants in vivo. Methods:,We have designed a quantitative reverse transcriptase-polymerase chain reaction assay to detect transcripts encoding the transmembrane, soluble 1 and 2 forms of IL-5R, in two strains of wild-type (BALB/c and C57BL/6) and corresponding IL-5 gene-deleted mice. Wild-type mice respond to S. mansoni infection with a gradual increase in serum IL-5 and eosinophilia, which is not observed in IL-5 gene-deleted mice. Results and conclusions:,We find that IL-5 is not necessary for differential splicing to occur in vivo, as all three forms of the IL-5R, are detected in both strains of IL-5 gene-deleted mice, with ratios of transcript expression (transmembrane : soluble 1 : soluble 2) that were indistinguishable from their wild-type counterparts. Differential splicing does vary markedly between strains, potentially because of local effects of strain-specific polymorphisms. [source] Transcriptional Regulation of 2,,3,-Cyclic Nucleotide 3,-Phosphodiesterase Gene Expression by Cyclic AMP in C6 CellsJOURNAL OF NEUROCHEMISTRY, Issue 5 2000M. Gravel Abstract: It was recently shown that the two transcripts encoding the isoforms of 2,,3,-cyclic nucleotide 3,-phosphodiesterase (CNP1 and CNP2) are differentially regulated during the process of oligodendrocyte maturation. In oligodendrocyte precursors, only CNP2 mRNA is present, whereas in differentiating oligodendrocytes, both CNP1 and CNP2 mRNAs are expressed. This pattern of CNP expression is likely due to stage-specific transcriptional regulation of the two CNP promoters during the process of oligodendrocyte differentiation. Here, we report the influence of increased intracellular cyclic AMP (cAMP) levels on the transcription of both CNP1 and CNP2 mRNAs in rat C6 glioma cells. We found that the transcription of CNP1 mRNA was significantly increased in comparison with that of CNP2 mRNA in cells treated with cAMP analogues to elevate intracellular cAMP levels. This up-regulation of CNP1 expression (a) is due to an increase of transcription, (b) requires de novo protein synthesis, and (c) requires the activity of protein kinase A. These results are physiologically significant and support the idea that a cAMP-mediated pathway is part of the molecular mechanisms regulating the expression of CNP1 in oligodendrocytes. The regulation of CNP1 promoter activity by cAMP was then investigated in stably transfected C6 cell lines containing various deletions of the CNP promoter directing the bacterial chloramphenicol acetyltransferase gene. We showed that the sequence between nucleotides -126 and -102 was essential for the cAMP-dependent induction of CNP1 expression. Gel retardation analysis showed that two protein-DNA complexes are formed between this sequence and nuclear factors from C6 cells treated or not treated with cAMP. This suggests that the induction of CNP1 mRNA transcription is not mediated by changes in binding of nuclear factors that interact directly with the -126/-102 sequence. Sequence analysis of this region revealed the presence of a putative activator protein-2 (AP-2) binding site. It is interesting that mutagenesis of this region resulted in a significant reduction in transcriptional responses to cAMP, implying a possible role for the AP-2 factor in the expression of CNP1. In addition, we have shown that putative binding sites for activator protein-4 and nuclear factor-1 adjacent to the AP-2 site are required for efficient induction of CNP1 expression by cAMP. Taken together, our results show that the cAMP-dependent accumulation of CNP1 mRNA appears to depend on the synergistic interaction of several regulatory elements. [source] Global H3K9 dimethylation status is not affected by transcription, translation, or DNA replication in porcine zygotesMOLECULAR REPRODUCTION & DEVELOPMENT, Issue 5 2010Ki-Eun Park Methylation of the lysine 9 residue of histone H3 (H3K9) is linked to transcriptional repression. The observed structure of chromatin in porcine and murine embryos is different with regard to H3K9 dimethylation status, leading to our hypothesis that the intracellular mechanisms responsible for H3K9 methylation would also differ between these two species. The objectives of this study were: (1) to determine the extent that DNA, mRNA, and protein synthesis serve in maintaining the asymmetrical distribution of dimethylated H3K9 in porcine zygotes, (2) determine the extent to which the intracellular localization of individual pronuclei correlated with H3K9 dimethylation status, and (3) to determine the abundance of transcripts encoding the histone methyltransferases, with H3K9 methylation activity, in porcine oocytes and embryos. Our findings are that (1) H3K9 dimethylation status is not affected by DNA replication, transcription, or protein synthesis, (2) the location of a pronucleus does not significantly affect the H3K9 dimethylation status of the chromatin within that pronucleus, and (3) the histone methyltransferases with activity for H3K9 differ in transcript abundance in porcine oocytes and cleavage stage embyros. These results support our hypothesis that there is a difference in intracellular mechanisms affecting dimethylation status of H3K9 between porcine and murine embryos. Mol. Reprod. Dev. 77: 420,429, 2010. © 2010 Wiley-Liss, Inc. [source] Contribution of Kv4 channels toward the A-type potassium current in murine colonic myocytesTHE JOURNAL OF PHYSIOLOGY, Issue 2 2002Gregory C. Amberg A rapidly inactivating K+ current (A-type current; IA) present in murine colonic myocytes is important in maintaining physiological patterns of slow wave electrical activity. The kinetic profile of colonic IA resembles that of Kv4-derived currents. We examined the contribution of Kv4 ,-subunits to IA in the murine colon using pharmacological, molecular and immunohistochemical approaches. The divalent cation Cd2+ decreased peak IA and shifted the voltage dependence of activation and inactivation to more depolarized potentials. Similar results were observed with La3+. Colonic IA was sensitive to low micromolar concentrations of flecainide (IC50= 11 ,M). Quantitative PCR indicated that in colonic and jejunal tissue, Kv4.3 transcripts demonstrate greater relative abundance than transcripts encoding Kv4.1 or Kv4.2. Antibodies revealed greater Kv4.3-like immunoreactivity than Kv4.2-like immunoreactivity in colonic myocytes. Kv4-like immunoreactivity was less evident in jejunal myocytes. To address this finding, we examined the expression of K+ channel-interacting proteins (KChIPs), which act as positive modulators of Kv4-mediated currents. Qualitative PCR identified transcripts encoding the four known members of the KChIP family in isolated colonic and jejunal myocytes. However, the relative abundance of KChIP transcript was 2.6-fold greater in colon tissue than in jejunum, as assessed by quantitative PCR, with KChIP1 showing predominance. This observation is in accordance with the amplitude of the A-type current present in these two tissues, where colonic myocytes possess densities twice that of jejunal myocytes. From this we conclude that Kv4.3, in association with KChIP1, is the major molecular determinant of IA in murine colonic myocytes. [source] mRNA metabolism of flowering-time regulators in wild-type Arabidopsis revealed by a nuclear cap binding protein mutant, abh1THE PLANT JOURNAL, Issue 6 2007Josef M. Kuhn Summary The precise regulation of RNA metabolism has crucial roles in numerous developmental and physiological processes such as the induction of flowering in plants. Here we report the identification of processes associated with mRNA metabolism of flowering-time regulators in wild-type Arabidopsis plants, which were revealed by an early flowering mutation, abh1, in an Arabidopsis nuclear mRNA cap-binding protein. By using abh1 as an enhancer of mRNA metabolism events, we identify non-coding polyadenylated cis natural antisense transcripts (cis-NATs) at the CONSTANS locus in wild-type plants. Our analyses also reveal a regulatory function of FLC intron 1 during transcript maturation in wild type. Moreover, transcripts encoding the FLM MADS box transcription factor are subject to premature intronic polyadenylation in wild type. In each case, abh1 showed altered patterns in RNA metabolism in these events compared with wild type. Together, abh1 enhances steps in the RNA metabolism that allowed us to identify novel molecular events of three key flowering-time regulators in wild-type plants, delivering important insights for further dissecting RNA-based mechanisms regulating flowering time in Arabidopsis. [source] Phototropin involvement in the expression of genes encoding chlorophyll and carotenoid biosynthesis enzymes and LHC apoproteins in Chlamydomonas reinhardtiiTHE PLANT JOURNAL, Issue 1 2006Chung-Soon Im Summary Phototropin (PHOT) is a photoreceptor involved in a variety of blue-light-elicited physiological processes including phototropism, chloroplast movement and stomatal opening in plants. The work presented here tests whether PHOT is involved in expression of light-regulated genes in Chlamydomonas reinhardtii. When C. reinhardtii was transferred from the dark to very low-fluence rate white light, there was a substantial increase in the level of transcripts encoding glutamate-1-semialdehyde aminotransferase (GSAT), phytoene desaturase (PDS) and light-harvesting polypeptides (e.g. LHCBM6). Increased levels of these transcripts were also elicited by low-intensity blue light, and this blue-light stimulation was suppressed in three different RNAi strains that synthesize low levels of PHOT. The levels of GSAT and LHCBM6 transcripts also increased following exposure of algal cells to low-intensity red light (RL). The red-light-dependent increase in transcript abundance was not affected by the electron transport inhibitor 3-(3,4-dichlorophenyl)-1,1-dimethylurea, implying that the influence of RL on transcript accumulation was not controlled by cytoplasmic redox conditions, and that a red-light photoreceptor(s) may be involved in regulating the levels of transcripts from specific photosynthesis-related genes in C. reinhardtii. Interestingly, elevated GSAT and LHCBM6 transcript levels in RL were significantly reduced in the PHOT RNAi strains, which raises the possibility of co-action between blue and RL signaling pathways. Microarray experiments indicated that the levels of several transcripts for photosystem (PS) I and II polypeptides were also modulated by PHOT. These data suggest that, in C. reinhardtii, (i) PHOT is involved in blue-light-mediated changes in transcript accumulation, (ii) synchronization of the synthesis of chlorophylls (Chl), carotenoids, Chl-binding proteins and other components of the photosynthetic apparatus is achieved, at least in part, through PHOT-mediated signaling, and (iii) a red-light photoreceptor can also influence levels of certain transcripts associated with photosynthetic function, although its action requires normal levels of PHOT. [source] | |||||||||||||