Biomedical subjects

Horace H Loh

Publications and source records attributed to Horace H Loh.

At least 19 recordsLinked to original sources

PubMed2007

Role of mu-opioid receptor in modulation of preproenkephalin mRNA expression and opioid and dopamine receptor binding in methamphetamine-sensitized mice.

We examined mRNA expression of preproenkephalin (PPE), a precursor of the endogenous opioid peptide enkephalin, and ligand binding to opioid and dopamine receptors in the striatum and nucleus accumbens in methamphetamine (METH)-sensitized mu-opioid receptor (mu-OR) knockout mice and their wild-type controls. Animals received daily intraperitoneal (i.p.) injections of METH (0, 0.625, 2.5, or 10 mg/kg) for 7 consecutive days to induce sensitization. Brain tissues were taken for biochemical analysis on experimental day 11 (4 days after the last injection). Expression of PPE mRNA and ligand binding were determined by in situ hybridization and autoradiography, respectively. Results indicate that there is an increase in PPE mRNA expression and a decrease in mu-OR ligand binding in METH-sensitized wild-type mice. These changes were not detected in METH-sensitized mu-OR knockout mice. A significant increase in delta-opioid receptor (delta-OR) ligand binding was found in mu-OR knockout mice. After repeated METH exposure, striatal and nucleus accumbal dopamine D1 receptor binding was decreased in mu-OR knockout mice but was not changed in wild-type mice. D2 receptor ligand binding was increased in wild-type mice and exhibited a biphasic change, with a decrease at 0.625 and 2.5 mg/kg doses of METH and an increase with 10 mg/kg of METH, in mu-OR knockout mice. These findings suggest that the mu-OR is involved in the regulation of METH-induced changes in an endogenous opioid peptide and dopamine receptors.

Animals↗

PubMed2006

Axonal mRNA transport and localized translational regulation of kappa-opioid receptor in primary neurons of dorsal root ganglia.

kappa-opioid receptor (KOR) is detected pre- and postsynaptically, but the subcellular localization, translation, and regulation of kor mRNA in presynaptic compartments of sensory neurons remain elusive. In situ hybridization detected axonal distribution of kor mRNA in primary neurons of dorsal root ganglia (DRG). The MS2-fused GFP tracked kor mRNA transport from DRG neuronal soma to axons, requiring its 5' and 3' UTRs. In Campenot chambers, axonal translation of kor mRNA was demonstrated for DRG neurons, which depended on its 5' UTR and was stimulated by KCl depolarization. KCl depolarization of DRG neurons rendered redistribution of kor mRNA from the postpolysomal fraction to the translationally active polysomal fraction. This study provided evidence for mRNA transport and regulation of presynaptic protein synthesis of nonstructural proteins like KOR in primary sensory neurons and demonstrated a mechanism of KCl depolarization-stimulated axonal mRNA redistribution for localized translational regulation.

Animals↗

PubMed2006

Action of NF-kappaB on the delta opioid receptor gene promoter.

The G protein-coupled delta opioid receptor gene (dor) is temporally and spatially expressed during development. The DOR receptor plays important roles in diverse biological processes, including pain control, immune functions, and cell survival. We previously found that PI3K/Akt/NF-kappaB signaling is important in the regulation of dor gene expression during nerve growth factor (NGF)-induced differentiation of PC12h cells, which prompted us to examine whether NF-kappaB p65 is directly or indirectly involved in the regulation of dor promoter activity. In this study, deletional and functional analysis of the dor promoter revealed a 94-bp NGF-responsive fragment upstream of the dor promoter region and involvement of NF-kappaB in regulating the promoter activity. Chromatin immunoprecipitation assays demonstrated that NF-kappaB p65 is directly bound to the dor promoter and such binding is related to NGF/PI3K signaling. Together, the results show that direct association of p65 with the promoter is important in NGF-induced dor promoter activity.

Animals↗

PubMed2006

Evidence of the neuron-restrictive silencer factor (NRSF) interaction with Sp3 and its synergic repression to the mu opioid receptor (MOR) gene.

Previously, we reported that the neuron-restrictive silencer element (NRSE) of mu opioid receptor (MOR) functions as a critical regulator to repress the MOR transcription in specific neuronal cells, depending on neuron-restriction silence factor (NRSF) expression levels [C.S.Kim, C.K.Hwang, H.S.Choi, K.Y.Song, P.Y.Law, L.N. Wei and H.H.Loh (2004) J. Biol. Chem., 279, 46464-46473]. Herein, we identify a conserved GC sequence next to NRSE region in the mouse MOR gene. The inhibition of Sp family factors binding to this GC box by mithramycin A led to a significant increase in the endogenous MOR transcription. In the co-immunoprecipitation experiment, NRSF interacted with the full-length Sp3 factor, but not with Sp1 or two short Sp3 isoforms. The sequence specific and functional binding by Sp3 at this GC box was confirmed by in vitro gel-shift assays using either in vitro translated proteins or nuclear extract, and by in vivo chromatin immunoprecipitation assays. Transient transfection assays showed that Sp3-binding site of the MOR gene is a functionally synergic repressor element with NRSE in NS20Y cells, but not in the NRSF negative PC12 cells. The results suggest that the synergic interaction between NRSF and Sp3 is required to negatively regulate MOR gene transcription and that transcription of MOR gene would be governed by the context of available transcription factors rather than by a master regulator.

Animals↗

PubMed2006

Agonist-dependent postsynaptic effects of opioids on miniature excitatory postsynaptic currents in cultured hippocampal neurons.

Although chronic treatment with morphine is known to alter the function and morphology of excitatory synapses, the effects of other opioids on these synapses are not clear. Here we report distinct effects of several opioids (morphine, [d-ala(2),me-phe(4),gly(5)-ol]enkephalin (DAMGO), and etorphine) on miniature excitatory postsynaptic currents (mEPSCs) in cultured hippocampal neurons: 1) chronic treatment with morphine for >3 days decreased the amplitude, frequency, rise time and decay time of mEPSCs. In contrast, "internalizing" opioids such as etorphine and DAMGO increased the frequency of mEPSCs and had no significant effect on the amplitude and kinetics of mEPSCs. These results demonstrate that different opioids can have distinct effects on the function of excitatory synapses. 2) mu opioid receptor fused with green fluorescence protein (MOR-GFP) is clustered in dendritic spines in most hippocampal neurons but is concentrated in axon-like processes in striatal and corticostriatal nonspiny neurons. It suggests that MORs might mediate pre- or postsynaptic effects depending on cell types. 3) Neurons were cultured from MOR knock-out mice and were exogenously transfected with MOR-GFP. Chronic treatment with morphine suppressed mEPSCs only in neurons that contained postsynaptic MOR-GFP, indicating that opioids can modulate excitatory synaptic transmission postsynaptically. 4) Morphine acutely decreased mEPSC amplitude in neurons expressing exogenous MOR-GFP but had no effect on neurons expressing GFP. It indicates that the low level of endogenous MORs could only allow slow opioid-induced plasticity of excitatory synapses under normal conditions. 5) A theoretical model suggests that morphine might affect the function of spines by decreasing the electrotonic distance from synaptic inputs to the soma.

Analgesics, Opioid↗

PubMed2006

Netrin-1 signaling regulates de novo protein synthesis of kappa opioid receptor by facilitating polysomal partition of its mRNA.

The expression of kappa opioid receptor (KOR) is subjected to both transcriptional and posttranscriptional controls. We report that KOR translation is regulated by netrin-1 in primary neurons of dorsal root ganglion (DRG) and in P19 embryonal carcinoma cells. Without stimulation, a significant portion of KOR mRNA is maintained in a dormant state and partitions in the translationally inactive, post-polysomal fraction. During netrin-1 stimulation, which activates its downstream target focal adhesion kinase (FAK), KOR mRNA rapidly partitions to the translationally active polysomal fraction. Functionally, the newly synthesized KOR proteins in DRG neurons are able to bind to specific ligands. This report describes the first example of netrin-1 signaling in the translational control of a drug receptor KOR, which involves the mediator of netrin-1, FAK, and a novel mechanism that enhances the association of target mRNA with polysomes for translational activation.

Animals↗

PubMed2006

Molecular basis of cellular localization of poly C binding protein 1 in neuronal cells.

Poly C binding protein 1 (PCBP) is involved in the transcriptional regulation of neuronal mu-opioid receptor gene. In this study, we examined the molecular basis of PCBP cellular/nuclear localization in neuronal cells using EGFP fusion protein. PCBP, containing three KH domains and a variable domain, distributed in cytoplasm and nucleus with a preferential nuclear expression. Domain-deletional analyses suggested the requirement of variable and KH3 domains for strong PCBP nuclear expression. Within the nucleus, a low nucleolar PCBP expression was observed, and PCBP variable domain contributed to this restricted nucleolar expression. Furthermore, the punctate nuclear pattern of PCBP was correlated to its single-stranded (ss) DNA binding ability, with both requiring cooperativity of at least three sequential domains. Collectively, certain PCBP domains thus govern its nuclear distribution and transcriptional regulatory activity in the nucleus of neurons, whereas the low nucleolar expression implicates the disengagement of PCBP in the ribosomal RNA synthesis.

Animals↗

PubMed2006

Molecular basis underlying the poly C binding protein 1 as a regulator of the proximal promoter of mouse mu-opioid receptor gene.

Previous studies showed poly C binding protein 1 (PCBP) participating in the mu-opioid receptor (MOR) gene regulation via binding to a single-stranded (ss) DNA element. In this report, we therefore investigate the molecular basis of PCBP regulating the MOR gene expression. Various truncated PCBPs, including one domain (KH1, KH2, variable or KH3), two- (K12, K2v or Kv3) or three-sequential domains (K12v or K2v3), were constructed. The MOR ssDNA binding abilities of these truncated PCBPs were examined using electrophoretic mobility shift assay (EMSA). KH1 domain possessed a strong MOR ssDNA binding activity. Variable domain displayed no binding, and KH2 or KH3 domain possessed a weak MOR ssDNA binding activity. Binding of two-domain PCBPs indicated an additive effect of two-domain combinations. Interestingly, K2v3, a three-domain PCBP, displayed as strong ssDNA binding as that of K12v, suggesting synergism of KH2, KH3 and variable domains for the binding activity. Functional analysis demonstrated one-domain PCBPs exhibiting no transactivation on the MOR proximal promoter. Two-domain PCBPs displayed approximately 20% activity, while three-domain PCBPs displayed 70%-85% of full-length PCBP activity. Taken together, these results suggested that no single domain possessed sufficient functional activity to serve as an independent transactivation domain, and the combination of three sequential domains was necessary for its optimal activity to activate the MOR proximal promoter. In summary, our data suggested that cooperativity of three sequential domains is essential for PCBP functioning as a MOR gene regulator. Various ways in which this cooperativity could occur are discussed.

Animals↗

PubMed2006

Interplay of Sps and poly(C) binding protein 1 on the mu-opioid receptor gene expression.

The proximal promoter of mouse mu-opioid receptor (MOR) gene is the dominant promoter for directing MOR-1 gene expression in brain. Sp1/Sp3 (Sps) and poly(C) binding protein 1 (PCBP) bind to a cis-element of MOR proximal promoter. Functional interaction between Sps and PCBP and their individual roles on MOR proximal core promoter were investigated using SL2 cells, devoid of Sps and PCBP. Each factor contributed differentially to the promoter, with a rank order of activity Sp1>Sp3>PCBP. Functional analysis suggested the interplay of Sps and PCBP in an additive manner. The in vivo binding of individual Sps or PCBP to MOR proximal promoter was demonstrated using chromatin immunoprecipitation (ChIP). Re-ChIP assays further suggested simultaneous bindings of Sps and PCBP to the proximal promoter, indicating physiologically relevant communication between Sps and PCBP. Collectively, results documented that a functional coordination between Sps and PCBP contributed to cell-specific MOR gene expression.

Animals↗

PubMed2006

The opioid ligand binding of human mu-opioid receptor is modulated by novel splice variants of the receptor.

The pharmacological actions of morphine and morphine-like drugs, such as heroin, mediate primarily through the mu-opioid receptor (MOR). It has been proposed that the functional diversity of MOR may be related to alternative splicing of the MOR gene. Although a number of MOR mRNA splice variants have been reported, their biological function has been controversial. In this study, two novel splice variants of the human MOR gene were discovered. Splice variants 1 and 2 (here called the SV1 and SV2) retain different portions of intron I. In vitro translation of SV1 and SV2 produced proteins with the predicted molecular weights. The splice variant proteins were identical to the wild-type MOR-1 up to the first transmembrane domains, but were different after the first intracellular loop domains. SV1 and SV2 of hMOR were present in human neuroblastoma NMB cells and human whole brain confirmed by RT-PCR. In a receptor binding assay, cells expressing the SV1 and SV2 do not exhibit binding to [(3)H]diprenorphine. The formations of MOR.SV1 and MOR.SV2 heterodimers were demonstrated by co-immunoprecipitation and bioluminescence resonance energy transfer between MOR and splice variants. Co-transfection of MOR-GFP and SV-DsRed gene showed that MOR and SV protein co-localized at the cytoplasmic membrane. In NMB cells expressing human MOR gene, transfection of SV1 or SV2 reduced binding activity of the endogenous MOR. These data support a potential role of SV1 and SV2 proteins as possible biological modulator of human mu-opioid receptor.

Alternative Splicing↗

PubMed2006

Short- and long-term regulation of adenylyl cyclase activity by delta-opioid receptor are mediated by Galphai2 in neuroblastoma N2A cells.

Activation of the opioid receptor results in short-term inhibition of intracellular cAMP levels followed by receptor desensitization and subsequent increase of cAMP above the control level (adenylyl cyclase superactivation). Using adenovirus to deliver pertussis toxin-insensitive mutants of the alpha-subunits of G(i/o) that are expressed in neuroblastoma Neuro2A cells (Galpha(i2), Galpha(i3), and Galpha(o)), we examined the identities of the G proteins involved in the short- and long-term action of the delta-opioid receptor (DOR). Pertussis toxin pretreatment completely abolished the ability of [d-Pen(2), d-Pen(5)]-enkephalin (DPDPE) to inhibit forskolin-stimulated intracellular cAMP production. Expression of the C352L mutant of Galpha(i2), and not the C351L mutants of Galpha(i3) or Galpha(o), rescued the short-term effect of DPDPE after pertussis toxin treatment. The ability of Galpha(i2) in mediating DOR inhibition of adenylyl cyclase activity was also reflected in the ability of Galpha(i2), not Galpha(i3) or Galpha(o), to coimmunoprecipitate with DOR. Coincidently, after long-term DPDPE treatment, pertussis toxin treatment eliminated the antagonist naloxone-induced superactivation of adenylyl cyclase activity. Again, only the C352L mutant of Galpha(i2) restored the adenylyl cyclase superactivation after pertussis toxin treatment. More importantly, the C352L mutant of Galpha(i2) remained associated with DOR after long-term agonist and pertussis toxin treatment whereas the wild-type Galpha(i2) did not. These data suggest that Galpha(i2) serves as the signaling molecule in both DOR-mediated short- and long-term regulation of adenylyl cyclase activity.

Adenoviridae↗

PubMed2006

Adenylyl cyclase superactivation induced by long-term treatment with opioid agonist is dependent on receptor localized within lipid rafts and is independent of receptor internalization.

Long-term opioid agonist treatment results in adenylyl cyclase superactivation. A recent "RAVE" theory implicates a direct correlation between the ability of agonist to induce receptor internalization and the magnitude of adenylyl cyclase superactivation. We decided to test such a theory by examining the adenylyl cyclase superactivation after long-term activation of mu-opioid receptor (MOR) in an EcR293 cell model. We examined the magnitudes of adenylyl cyclase superactivation in the presence of naloxone after long-term treatment with morphine, etorphine, and methadone, three agonists reported to have differential activities in promoting MOR internalization. It can be shown that the magnitudes of adenylyl cyclase superactivation after treating with these three agonists, although different, were dependent on MOR density. Blunting MOR internalization with the dominant-negative mutant of dynamin, K44E, did not alter the magnitude of either morphine- or etorphine-induced adenylyl cyclase superactivation. In the presence of diprenorphine, the magnitude of adenylyl cyclase superactivation after etorphine treatment was identical to that observed with morphine. It could be demonstrated further that adenylyl cyclase superactivation is dependent on the cell surface-located MOR. Sucrose gradient fractionation demonstrated the colocalization of MOR and adenylyl cyclase V/VI with caveolin-1, a marker for lipid rafts. After long-term agonist treatment, the majority of MOR remained at the lipid rafts. Methyl-beta-cyclodextrin (MbetaCD) completely blunted the adenylyl cyclase superactivation and agonist-induced receptor internalization. These MbetaCD actions were reversed by incubating the cells with cholesterol. Thus, the adenylyl cyclase superactivation is not dependent on agonist-induced receptor internalization. Rather, the location of MOR at lipid rafts is an absolute requirement for the observed adenylyl cyclase superactivation.

Adenylyl Cyclases↗

PubMed2005

Sustained activation of phosphatidylinositol 3-kinase/Akt/nuclear factor kappaB signaling mediates G protein-coupled delta-opioid receptor gene expression.

Expression of the delta-opioid receptor gene (dor) is tightly controlled during neuronal differentiation and developmental stages. Such distinct temporal and spatial expression of dor during development suggests a role for the delta-opioid receptor in early developmental events. However, little is known about intracellular signaling pathways that control dor expression. A well established cell line model for the study of gene expression during neuronal differentiation is the rat adrenal pheochromocytoma PC12 cell line. Here we found that the constitutively activated TrkA/phosphatidylinositol 3-kinase/Akt (protein kinase B)/NF-kappaB survival cascade mediates dor expression during nerve growth factor (NGF)-induced differentiation of PC12h cells. Biochemical experiments showed that constitutive phosphorylation of Akt and IkappaBalpha correlates with NGF-induced dor expression. Overexpression of the transcriptional activator NF-kappaB/p65 increased dor promoter activity. Overexpression of the NF-kappaB signaling super inhibitor mutant IkappaBalpha (S32A/S36A) abolished the effect of p65 and blocked NGF-induced activation of NF-kappaB signaling, resulting in a significant reduction in dor promoter activity. Treatment with SN50, an NF-kappaB-specific nuclear translocation peptide inhibitor, inhibited the translocation of NF-kappaB, resulting in a reduction of dor mRNA. The gel shift assay supported the fact that there exists an NF-kappaB-binding site on the dor promoter. RNA interference experiments using NF-kappaB/p65 small interfering RNA confirmed that NF-kappaB signaling is required for dor expression. Our findings not only provide a new mechanistic explanation for NGF-induced dor expression but also shed some light on the molecular mechanism of the temporal and spatial expression of dor and the roles of the delta-opioid receptor during neuronal differentiation.

Active Transport, Cell Nucleus↗

PubMed2005

Differential effects of morphine- and cocaine-induced nNOS immunoreactivity in the dentate gyrus of hippocampus of mice lacking mu-opioid receptors.

This study investigated the expression of nNOS after repeated morphine or cocaine administration in order to determine if nNOS (neuronal nitric oxide synthase) is involved in the morphine- or cocaine-induced behavioral sensitization in mu-opioid receptor knockout (MOR(-/-)) mice. Higher numbers of nNOS-positive cells were observed in the dentate gyrus of the hippocampus (DG) of the wild-type (MOR(+/+)) mice repeatedly treated with either morphine or cocaine than in the saline treated MOR(+/+) mice (morphine, +122%; cocaine, +82%). Moreover, the MOR(-/-) mice also showed significantly higher morphine- or cocaine-induced nNOS expression levels in the DG than in the saline treated MOR(+/+) mice (morphine, +234%; cocaine, +54%). The MOR(-/-) mice showed a significantly higher morphine-induced nNOS expression level (+103%) or a lower cocaine-induced nNOS expression level (+38%) in the DG than in the morphine- or cocaine-treated MOR(+/+) mice. These results suggest that morphine and cocaine sensitization is differentially regulated by the mu-opioid receptors in MOR(-/-) mice via the nNOS systems in the DG.

Animals↗

PubMed2005

Morphine induces CD4+ T cell IL-4 expression through an adenylyl cyclase mechanism independent of the protein kinase A pathway.

Impaired host defense mechanisms after major operative procedures and trauma are recognized as important factors in the development of infectious complication. Trauma is associated with impaired cellular immunity and CD4+ T cell Th2 differentiation. We have previously implicated morphine treatment as a possible mechanism for Th2 differentiation after injury. In this investigation we first establish that morphine treatment in vivo results in Th2 differentiation and that this effect is mediated through a naltrexone-sensitive opioid receptor. We investigated the intracellular mechanism by which morphine controls CD4+ T cell differentiation and demonstrate that morphine treatment in vitro 1) increases anti CD3/CD28 Ab-induced CD4+ T cell IL-4 protein synthesis, IL-4 mRNA, and GATA-3 mRNA accumulation through a pertussis toxin-sensitive receptor; 2) results in a dose-dependent increase in anti-CD3/CD28 Ab-induced CD4+ T cell cytoplasmic cAMP concentration; and 3) increases the forskolin-stimulated cytoplasmic cAMP level through a pertussis toxin-sensitive receptor. We also demonstrate that chronic morphine treatment increases anti-CD3/CD28 Ab-induced IL-4 promoter activity and IL-4 immunoprotein expression through a p38 MAPK-dependent, but protein kinase A- and Erk1/Erk2-independent, mechanism.

Adenylyl Cyclases↗

PubMed2005

Ethanol-induced FOS immunoreactivity in the brain of mu-opioid receptor knockout mice.

Using mu-opioid receptor knockout (MKO) mice, we examined ethanol-induced FOS immunoreactivity (FOSir) in the brain as an indicator of neuronal activation to assess the role of the mu-opioid receptor in modulating ethanol's actions in the central nervous system (CNS). Saline stimulated FOSir in the paraventricular thalamic nucleus (PVA) and the dorsal hypothalamic area (DA) in MKO mice, but not in wild-type (WT), suggesting that MKO homozygotes may differ responsively from WT. Treatment with ethanol (4 g/kg, i.p.) induced FOSir in the PVA, DA, supraoptic (SO), paraventricular hypothalamic (PVN), lateral parabrachial (LPB), locus coeruleus (LC) and Edinger-Westphal (EW) nuclei in both MKO and WT mice. However, ethanol stimulated modest FOSir in the lateral septal division (LSV), suprachiasmatic nucleus (SCh) and the dorsal and ventral lateral geniculate nuclei (DLG and VLG) in WT mice, but not in MKO mice. In contrast, higher levels of ethanol-induced FOSir were observed in the ventral pallidum (VP) and globus pallidus (GP) of MKO mice as compared to WT. These data suggest that ethanol continues to activate several brain regions, even without the mu-opioid receptor pathway. However, the mu-opioid receptor may be significant in mediating ethanol's effects in some restricted areas of the brain.

Animals↗

PubMed2005

Poly(C) binding protein family is a transcription factor in mu-opioid receptor gene expression.

The mouse mu-opioid receptor (MOR) gene has two promoters, referred to as distal and proximal promoter. Previously, our colleagues reported that a 26-base pair (bp) cis-acting element of the mouse MOR gene activates MOR gene expression. Here, we report the cloning of four members of the poly(C) binding protein (PCBP) family and show that the 26-bp polypyrimidine stretch in MOR proximal promoter interacts with these PCBPs and activates MOR transcription. The PCBPs bind not only to single-stranded but also to double-stranded DNA. The nuclear run-off assay and semiquantitative RT-PCR shows that PCBPs enhance the transcription rate of MOR gene. Furthermore, we performed refined mapping to elucidate the core region (-317/-304) involved in mediating the PCBP-induced MOR promoter activity. Decoy oligonucleotides against the polypyrimidine stretch inhibit the PCBP-induced MOR promoter activity, thereby reconfirming the role of this element in regulating MOR promoter activity. Chromatin immunoprecipitation assay confirmed the interaction of PCBPs with MOR promoter in vivo. In conclusion, we demonstrate that PCBPs act as a transcription factor and positively regulate MOR gene expression in NMB cells.

Animals↗

PubMed2005

A major species of mouse mu-opioid receptor mRNA and its promoter-dependent functional polyadenylation signal.

The pharmacological effects of opioid drugs are mediated mainly by the mu-opioid receptor (MOR), which is encoded by an mRNA transcript named MOR1. Although several MOR mRNA splice variants have been reported, their biological relevance has been debated. In this study, we found that probes of regions essential for the production of functional MOR, as well as that of the 3'-downstream region of the MOR gene coding region, detected by Northern blot analyses, a major species of mature transcript MOR1 from mouse brain of approximately 11.5 kilobases (kb). Although exon 3 probe detected an additional 3.7-kb transcript, this transcript was not detected by other probes, ruling out its ability to produce functional MOR. The 3'-untranslated region (UTR) of MOR1 is contiguously extended from the end of the coding region, and uses a single polyadenylation [poly (A)] signal (located 10,179 bp downstream of the MOR1 stop codon). The poly (A) signal (AAUAAA) is located 26 bp upstream of the poly (A) site. Transient transfection using luciferase reporters verified the functionality of this poly (A) signal, in particular on a reporter driven by the MOR promoter. This poly (A) is much less effective for a heterologous promoter, such as simian virus 40, indicating a functional coupling of MOR promoter and its own poly (A). This report verifies MOR1 as the major mature MOR gene transcript that has the full capacity to produce functional MOR protein, identifies the 3'-UTR of MOR1 transcript, and uncovers functional coupling of the MOR gene promoter and its polyadenylation signal.

Animals↗