Luc Construct Research Articles

Isolation and characterization of maize ZmPP2C26 gene promoter in drought-response.

The clade A members of serine/threonine protein phosphatase 2Cs (PP2Cs) play crucial roles in plant growth, development, and stress response via theABA signaling pathway. But little is known about other PP2C clades in plants. Our previous study showed that maizethe ZmPP2C26, a clade B member of ZmPP2Cs, negatively regulated drought tolerance in transgenic Arabidopsis. However, the upstream regulatory mechanism of ZmPP2C26 remains unclear. In the present study, the expression of ZmPP2C26 gene in maize was analyzed by quantitative real time PCR (qRT-PCR). The results showed that the expression of ZmPP2C26 in shoot and root was both significantly inhibited by drought stress. Subsequently, a 2175bp promoter of ZmPP2C26 was isolated from maize genome (P 2175). To validate whether the promoter possess some key cis-element and negatively drive ZmPP2C26 expression in drought stress, three 5´-deletion fragments of 1505, 1084 and 215bp was amplified from P 2175 and were fused to β-glucuronidase (GUS) and luciferase gene (LUC) to produce promoter::GUS and promoter::LUC constructs, and transformed into tobacco, respectively. Transient expression assays indicated that all promoters could drive GUS and LUC expression. The GUS and LUC activity were both significantly inhibited by PEG-6000 treatment. Notably, the - 1084 to - 215bp promoter possess one MBS element and inhibits the expression of GUS and LUC under drought stress. Meanwhile, we found that the 215bp length is enough to drive ZmPP2C26 expression. These findings will provide insights into understanding the transcription-regulatory mechanism of ZmPP2C26 negatively regulating drought tolerance.

Microrna-150 Regulates STAT5b Levels in Juvenile Myelomonocytic Leukemia (JMML)

IntroductionJMML is a rare leukemia characterized by aberrant myeloid proliferation and hypersensitivity to GM-CSF. Mutually exclusive mutations in PTPN11, N/K-RAS, CBL, or NF1 are found in ~90% of patients. These mutations cause the disease at least in part by activating STAT5 through phosphorylation and promoting cell growth. MicroRNAs (miRs) are small (~22 nucleotides) noncoding RNAs, regulating gene expression and often deregulated in leukemia. We investigated whether miRs are deregulated in JMML and whether miRs target critical proteins involved in the disease pathogenesis.Patients and MethodsMiRs expression profile of 40 bone marrow (BM) samples from untreated JMML patients and 8 BM samples from healthy controls was performed using the Ncounter Human v2 miRNA Expression Assay (nanostring). MiR150-5p and STAT5b mRNA expression were assessed using quantitative (q)RT-PCR. Selected miRs and target expression were measured in BM and spleen from a JMML (PTPN11) murine model (D61Y mutation). We overexpressed miR-150-5p in AML cell lines (K562, OCI-AML-3, KG1a) using a miR-150-5p precursor plasmid and STAT5b mRNA was assessed by qRT-PCR. STAT5 and phospho-STAT5 protein levels were assessed by Western Blot in miR-150-5p transfected AML cell lines, human JMML and mice BM cells. STAT5b 3'UTR sequence was cloned in a Firefly/Renilla Luc construct and co-transfected with miR-150-5p miRNA mimic into 293T cell line.ResultsWe found that 25 miRs were differentially expressed in whole BM cells from JMML patients respect to healthy controls (Table 1). MiR-150-5p was the most downregulated miR in JMML. We focused on miR-150-5p, since it has been described to be downregulated in AML cases and is predicted to target STAT5b, a critical gene in JMML biology. We validated that miR-150-5p was down-regulated in JMML cases respect to controls performing qRT-PCR on 38 BM samples from JMML patients. Likewise, miR-150-5p was downregulated in BM and spleen samples from PTPN11 mutated mice respect to controls (0.35 and 0.27 Average Fold Change decrease respectively). STAT5 protein, a predicted target for miR-150-5p, was highly expressed in the JMML patients and mice BM samples respect to their controls (4.3 and 1.3 Average Fold Change increase respectively). MiR-150-5p overexpression in K562, OCI-AML-3 and KG1a cell lines led to decrease of STAT5 protein levels and phosphorilation at 48 hours. Direct interaction of STAT5b 3'UTR with miR-150-5p was demonstrated by luciferase assay (~50% Luc activity inhibition, P<0.001). Last, overexpression of miR-150-5p in primary JMML samples using a GFP tagged lentivirus significantly decreased cell growth respect to controls (empty vector) in response to GM-CSF (P<0.01).DiscussionWe showed that miR-150-5p is downregulated in JMML samples and in JMML animal models. Functionally, miR-150-5p directly inhibits the translation of STAT5b mRNA resulting also in a decrease of phosphorylation of STAT5 total protein. These findings identify an alternative mechanism that supports STAT5 deregulation in JMML and that could be therapeutically targeted.Table 1Deregulated miRNAs in JMMLGeneAccession #P valueFold-Change (Log2)miR-150-5pMIMAT00004510,001-2,382let-7g-5pMIMAT00004140,002-1,660miR-1260aMIMAT00059110,009-1,592let-7a-5pMIMAT00000620,010-1,574miR-4454MIMAT00189760,021-1,399miR-148a-3pMIMAT00002430,030-1,211miR-146b-5pMIMAT00028090,009-1,084miR-342-3pMIMAT00007530,010-1,075let-7f-5pMIMAT00000670,021-1,022miR-26a-5pMIMAT00000820,034-1,008let-7d-5pMIMAT00000650,038-1,005miR-30b-5pMIMAT00004200,019-0,972miR-29b-3pMIMAT00001000,044-0,956miR-29a-3pMIMAT00000860,024-0,761miR-338-3pMIMAT00007630,0420,654miR-23a-3pMIMAT00000780,0400,689miR-222-3pMIMAT00002790,0180,756miR-548aiMIMAT00189890,0090,993miR-494MIMAT00028160,0231,021miR-320eMIMAT00150720,0071,175miR-224-5pMIMAT00002810,0241,227miR-4508MIMAT00190450,0161,369miR-575MIMAT00032400,0141,429miR-3195MIMAT00150790,0141,432miR-630MIMAT00032990,0012,272 [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Tumour suppressor protein p53 regulates the stress activated bilirubin oxidase cytochrome P450 2A6

Human cytochrome P450 (CYP) 2A6 enzyme has been proposed to play a role in cellular defence against chemical-induced oxidative stress. The encoding gene is regulated by various stress activated transcription factors. This paper demonstrates that p53 is a novel transcriptional regulator of the gene. Sequence analysis of the CYP2A6 promoter revealed six putative p53 binding sites in a 3kb proximate promoter region. The site closest to transcription start site (TSS) is highly homologous with the p53 consensus sequence. Transfection with various stepwise deletions of CYP2A6-5′-Luc constructs – down to −160bp from the TSS – showed p53 responsiveness in p53 overexpressed C3A cells. However, a further deletion from −160 to −74bp, including the putative p53 binding site, totally abolished the p53 responsiveness. Electrophoretic mobility shift assay with a probe containing the putative binding site showed specific binding of p53. A point mutation at the binding site abolished both the binding and responsiveness of the recombinant gene to p53. Up-regulation of the endogenous p53 with benzo[α]pyrene – a well-known p53 activator – increased the expression of the p53 responsive positive control and the CYP2A6-5′-Luc construct containing the intact p53 binding site but not the mutated CYP2A6-5′-Luc construct. Finally, inducibility of the native CYP2A6 gene by benzo[α]pyrene was demonstrated by dose-dependent increases in CYP2A6 mRNA and protein levels along with increased p53 levels in the nucleus. Collectively, the results indicate that p53 protein is a regulator of the CYP2A6 gene in C3A cells and further support the putative cytoprotective role of CYP2A6.

Glucocorticoid-induced S-Adenosylmethionine Enhances the Interferon Signaling Pathway by Restoring STAT1 Protein Methylation in Hepatitis B Virus-infected Cells

Patients with chronic hepatitis B usually exhibit a low response to treatment with interferon α (IFN-α). An alternative approach to increase the response rate of IFN-α might be to immunologically stimulate the host with glucocorticoids (GCs) before treatment with IFN-α, but the underlying mechanism remains unclear. We hypothesized that the GCs enhance IFN signaling by inducing S-adenosylmethionine (AdoMet) when hepatitis B virus (HBV) replication was effectively suppressed by IFN-α. Here, we investigated the effect of GCs and IFN-α on AdoMet production and methionine adenosyltransferase 1A (MAT1A) expression in vitro. Furthermore, we determined whether post-transcriptional regulation is involved in HBV-repressed MAT1A expression and AdoMet production induced by dexamethasone (Dex). We found that AdoMet homeostasis was disrupted by Dex and that Dex directly regulated MAT1A expression by enhancing the binding of the glucocorticoid receptor (GR) to the glucocorticoid-response element (GRE) of the MAT1A promoter. HBV reduced AdoMet production by increasing methylation at GRE sites within the MAT1A promoter. The X protein of hepatitis B virus led to hypermethylation in the MAT1A promoter by recruiting DNA methyltransferase 1, and it inhibited GR binding to the GRE in the MAT1A promoter. Dex could increase an antiviral effect by inducing AdoMet production via a positive feedback loop when HBV is effectively suppressed by IFN-α, and the mechanism that involves Dex-induced AdoMet could increase STAT1 methylation rather than STAT1 phosphorylation. These findings provide a possible mechanism by which GC-induced AdoMet enhances the antiviral activity of IFN-α by restoring STAT1 methylation in HBV-infected cells.

Ceramide synthases CerS4 and CerS5 are upregulated by 17β-estradiol and GPER1 via AP-1 in human breast cancer cells

Ceramide synthases (CerS) are important enzymes of the sphingolipid pathway, responsible for the production of ceramides with distinct chain lengths. In human breast cancer tissue, we detected a significant increase in CerS4 and CerS6 mRNA in estrogen receptor positive (ER+) cancer tissue. To clarify the molecular mechanism of this upregulation, we cloned CerS2, -4, -5 and CerS6 promoter and 3′-UTR fragments into luciferase reporter gene plasmids and determined luciferase activity in MCF-7 (ERα/β) and MDA-MB-231 (ERβ) cells after 17β-estradiol treatment. Only the activities of CerS4 and CerS5 promoter Luc constructs, as well as CerS2- and CerS5-3′-UTR Luc constructs increased after estradiol treatment in MCF-7 cells, and this could be inhibited by the anti-estrogen fulvestrant. Co-transfection with the G protein-coupled estrogen receptor 1 (GPER1) also enhanced CerS2, CerS4 and CerS6 promoter activity whereas CerS5 promoter activity was inhibited in both cell lines. Promoter deletion and mutation constructs from CerS4 and CerS5 promoters revealed that estradiol and GPER1 mediate their effects on both promoters by activating AP-1, most likely through dimerization of c-Jun and c-Fos. At least we could show, that cell proliferation induced by estradiol could be blocked by co-treatment with Fumonisin B1, indicating that upregulation of CerS in breast cancer cells by estrogen is important for cell proliferation and possibly tumor development. In conclusion, our data highlight transcriptional and posttranscriptional mechanisms regulating CerS expression in human cells which provide the basis for further studies investigating the regulation of CerS expression and ceramide synthesis after diverse stimuli in physiological and pathophysiological processess.

OsWRKY42 represses OsMT1d and induces reactive oxygen species and leaf senescence in rice.

We isolated a rice (Oryza sativa L.) WRKY gene which is highly upregulated in senescent leaves, denoted OsWRKY42. Analysis of OsWRKY42-GFP expression and its effects on transcriptional activation in maize protoplasts suggested that the OsWRKY42 protein functions as a nuclear transcriptional repressor. OsWRKY42-overexpressing (OsWR KY42OX) transgenic rice plants exhibited an early leaf senescence phenotype with accumulation of the reactive oxygen species (ROS) hydrogen peroxide and a reduced chlorophyll content. Expression analysis of ROS producing and scavenging genes revealed that the metallothionein genes clustered on chromosome 12, especially OsMT1d, were strongly repressed in OsWRKY42OX plants. An OsMT1d promoter:LUC construct was found to be repressed by OsWRKY42 overexpression in rice protoplasts. Finally, chromatin immunoprecipitation analysis demonstrated that OsWRKY42 binds to the W-box of the OsMT1d promoter. Our results thus suggest that OsWRKY42 represses OsMT1d-mediated ROS scavenging and thereby promotes leaf senescence in rice.

Helicobacter pylori-induced posttranscriptional regulation of H-K-ATPase α-subunit gene expression by miRNA

Acute Helicobacter pylori infection of gastric epithelial cells induces CagA oncoprotein- and peptidoglycan (SLT)-dependent mobilization of NF-κB p50 homodimers that bind to H-K-ATPase α-subunit (HKα) promoter and repress HKα gene transcription. This process may facilitate gastric H. pylori colonization by induction of transient hypochlorhydria. We hypothesized that H. pylori also regulates HKα expression posttranscriptionally by miRNA interaction with HKα mRNA. In silico analysis of the HKα 3' untranslated region (UTR) identified miR-1289 as a highly conserved putative HKα-regulatory miRNA. H. pylori infection of AGS cells transfected with HKα 3' UTR-Luc reporter construct repressed luciferase activity by 70%, whereas ΔcagA or Δslt H. pylori infections partially abrogated repression. Transfection of AGS cells expressing HKα 3' UTR-Luc construct with an oligoribonucleotide mimetic of miR-1289 induced maximal repression (54%) of UTR activity within 30 min; UTR activity was unchanged by nontargeting siRNA transfection. Gastric biopsies from patients infected with cagA(+) H. pylori showed a significant increase in miR-1289 expression compared with uninfected patients or those infected with cagA(-) H. pylori. Finally, miR-1289 expression was necessary and sufficient to attenuate biopsy HKα protein expression in the absence of infection. Taken together, these data indicate that miR-1289 is upregulated by H. pylori in a CagA- and SLT-dependent manner and targets HKα 3' UTR, affecting HKα mRNA translation. The sensitivity of HKα mRNA 3' UTR to binding of miR-1289 identifies a novel regulatory mechanism of gastric acid secretion and offers new insights into mechanisms underlying transient H. pylori-induced hypochlorhydria.

Characterization of Circadian Rhythms Through a Bioluminescence Reporter Assay in Lactuca sativa L.

Circadian rhythms are observed in many physiological events in plant, and clock genes orchestrate the rhythms of expression of many genes. Precise environmental control of circadian rhythms provides a key technology for enhancing plant growth in artificial environments. In this study, we investigated the basic properties of a circadian rhythm for establishment of its control engineering in lettuce (Lactuca sativa L.). Bioluminescence of transgenic lettuce carrying an AtCCA1::LUC construct as a reporter of circadian gene expression was measured in young lettuce seedlings. We observed three basic properties; free-running circadian rhythms under constant conditions without day-night cycles, entrainment to red and blue light cycles with 12 h light-12 h dark period, and temperature compensation of a free-running period in three lettuce cultivars (Cisco, Cos, Greenwave). In addition, a light-quality dependence of the free-running period and sensitivity to blue-light cycles with small amplitude (20% of variance in average light intensity) and non-24 h period were also observed. The results in this study indicated that it is possible to control lettuce circadian rhythms by non-24 h period light cycles in LED illumination. It will play an important role in the research of control engineering for the circadian clock of lettuce in closed plant factories with artificial lighting.

Transcriptional regulation of the human bone sialoprotein gene by fibroblast growth factor 2

Fibroblast growth factor 2 (FGF2), a member of the FGF family, positively regulates bone formation and osteoblast differentiation. Bone sialoprotein (BSP) is highly expressed during early bone formation and may play a role in primary mineralization of bone. In the present study, FGF2 (10 ng/mL) was found to increase the levels of Runx2 and BSP mRNA at 3 and 12 h in human osteoblast-like Saos2 cells. Transient transfection assays were performed using chimeric constructs of the human BSP gene promoter ligated with a luciferase reporter gene. FGF2 (10 ng/mL, 12 h) induced the luciferase activities of the -84LUC and -927LUC constructs in Saos2 cells. The results of gel shift assays showed that FGF2 (10 ng/mL) increased the binding of nuclear protein to the FGF2 response element (FRE) and the activator protein 1 (AP1) binding site. Antibodies against Dlx5, Msx2, Runx2 and Smad1 blocked FRE-protein complex formation, and antibodies against CREB1, c-Jun and Fra2 interrupted AP1-protein complex formation. These results indicate that FGF2 increases BSP transcription by targeting the FRE and AP1 elements in the proximal promoter of the human BSP gene. Moreover, the transcription factors Dlx5, Msx2, Runx2, Smad1, CREB1, c-Jun and Fra2 could be key regulators of the effects of FGF2 on human BSP transcription.

Spatiotemporal dynamics of circadian clock in lettuce

Abstract Plant circadian clock controls many physiological events by synchronization with environmental changes. The clock can be controlled artificially by imposing various light conditions in a closed system for plant cultivation in plant factory. The plant circadian rhythm is formed by enormous self-sustained cellular circadian oscillators, so that the synchronization of circadian oscillators is of great importance to the formation of individual circadian rhythms. Therefore, the study of synchronization phenomena is important for precise control of the circadian rhythm. In this study, we have investigated the spatiotemporal dynamics of circadian oscillators in lettuce (Lactuca sativa L.). Bioluminescence of transgenic lettuce carrying a CCA1::LUC construct as a reporter of a circadian gene expression was measured in the leaf and root. We observed phase wave propagation in the leaf with a phase delay in the region of the primary vein, with the primary vein region showing lower amplitude than the other regions. Wave propagation occurred from the edge of the leaf inward. In addition, a striped wave traveled from the base to the tip along the roots, which were grown under continuous dark or light conditions. The features observed in the lettuce plants could be explained by phase oscillator models established in the study of Arabidopsis thaliana. The results of this study show the possibility of applying circadian clock control in a model plant for plant production.

Replication of Hepatitis C Virus Genotype 3a in Cultured Cells

Hepatitis C virus (HCV) genotype 3a is widespread worldwide, but no replication system exists for its study. We describe a subgenomic replicon system for HCV genotype 3a. We determined the consensus sequence of an HCV genome isolated from a patient, and constructed a subgenomic replicon using this clone. The replicon was transfected into HuH-7 cells and RNA replication was confirmed. We identified cell culture-adaptive mutations that increased colony formation multiple-fold. We have therefore established a genotype 3a replicon system that can be used to study this HCV genotype.

A mimic of phosphorylated prolactin induces apoptosis by activating AP-1 and upregulating p21/waf1 in human prostate cancer PC3 cells

A mimic of phosphorylated prolactin (S179D PRL) has been demonstrated to inhibit prostate cancer cell growth in vitro and in vivo; however, the mechanisms involved in this process remain unknown. In this study, we identified that a four-day treatment of S179D PRL (1 μg/ml) in human prostate PC3 cancer cells activated JNK, c-fos and c-jun, and led to apoptosis. We also demonstrated that p21/waf1 was upregulated in cells transfected with the human PRL receptor (S1b) following a four-day incubation with S179D PRL (1 μg/ml). Once the cells were cotransfected with S1b and either c-fos, c-jun or the c-fos/c-jun constructs for 24 h, S17D PRL activated JNK, c-fos and c-jun, and induced apoptosis in the c-fos/c-jun transfected cells. Additionally, S179D PRL upregulated p21 luciferase activity in the cells transfected with the S1b, activating protein-1 (AP-1) (7x) Luc or p21 Luc constructs. SP600125 (25 μM), a JNK blocker, inhibited the upregulation of AP-1 Luc and p21 Luc in the c-fos/c-jun transfected cells. These results demonstrate that S179D PRL activates JNK and AP-1, which leads to p21 upregulation and apoptosis in human prostate PC3 cancer cells.

Bioluminescence imaging of bone formation using hairless osteocalcin-luciferase transgenic mice

Osteocalcin is a major noncollagenous protein component of bone extracellular matrix, synthesized and secreted exclusively by osteoblastic cells during the late stage of maturation. We introduced a 10kb human osteocalcin enhancer/promoter (OC)-luciferase (Luc) construct into a hairless mouse line. Examination of tissue RNAs from these transgenic mice showed a predominant restriction of Luc mRNA expression to bone-associated tissues. Immunohistochemical staining of calvaria tissue sections revealed the localization of Luc protein to osteoblasts. Utilizing in vivo bioluminescence imaging, supplementation of 1α,25-dihydroxyvitamin D3 increased Luc activity throughout the skeleton, consistent with in vitro transient transfection studies in osteoblast-like cells. Moreover, we observed an abrupt decrease in bioluminescence activity as the mice reached puberty, and a further decrease gradually thereafter. Using a radius skeletal repair model, we observed enhanced bioluminescence at the fracture site in both young (14–22weeks old) and aged (50–66weeks old) mice. However, peak bioluminescence was delayed in aged mice compared with young mice, suggesting retarded osteocalcin expression with aging. Our in vivo imaging system may contribute to the therapy and prevention of various bone metabolic disorders through its effective monitoring of the bone formation process.

Melatonin synthesis in retina: cAMP‐dependent transcriptional regulation of chicken arylalkylamineN‐acetyltransferase by a CRE‐like sequence and a TTATT repeat motif in the proximal promoter

Arylalkylamine N-acetyltransferase (AANAT) is the key regulatory enzyme controlling the daily rhythm of melatonin biosynthesis. In chicken retinal photoreceptor cells, Aanat transcription and AANAT activity are regulated in part by cAMP-dependent mechanisms. The purpose of this study was to identify regulatory elements within the chicken Aanat promoter responsible for cAMP-dependent induction. Photoreceptor-enriched retinal cell cultures were transfected with a luciferase reporter construct containing up to 4 kb of 5'-flanking region and the first exon of Aanat. Forskolin treatment stimulated luciferase activity driven by the ∼4 kb promoter construct and by all 5'-deletion constructs except the smallest, Aanat (-217 to +120)luc. Maximal basal and forskolin-stimulated expression levels were generated by the Aanat (-484 to +120)luc construct. This construct lacks a canonical cyclic AMP-response element (CRE), but contains two other potentially important elements in its sequence: an eight times TTATT repeat (TTATT₈) and a CRE-like sequence. Electrophoretic mobility shift assays, luciferase reporter assays, chromatin immunoprecipitation, and siRNA experiments provide evidence that these elements bind c-Fos, JunD, and CREB to enhance basal and forskolin-stimulated Aanat transcription. We propose that the CRE-like sequence and TTATT₈ elements in the 484 bp proximal promoter interact to mediate cAMP-dependent transcriptional regulation of Aanat.

Distinctive Properties of the Hyaluronan-binding Domain in the Lymphatic Endothelial Receptor Lyve-1 and Their Implications for Receptor Function

The lymphatic endothelial hyaluronan (HA) receptor Lyve-1 is a member of the Link protein superfamily most similar to the leukocyte HA receptor CD44. However, the structure of Lyve-1 and the nature of its interaction with ligand are obscure. Here we present new evidence that Lyve-1 is functionally distinct from CD44. Using truncation mutagenesis we confirm that Lyve-1 in common with CD44 contains an extended HA-binding unit, comprising elements flanking the N and C termini of the consensus lectin-like Link module, bridged by a third conserved disulfide linkage that is critical for HA binding. In addition, we identify six essential residues Tyr-87, Ile-97, Arg-99, Asn-103, Lys-105, and Lys-108 that define a compact HA-binding surface on Lyve-1, encompassing the epitope for an adhesion-blocking monoclonal antibody 3A, in an analogous position to the HA-binding surface in CD44. The overtly electrostatic character of HA binding in Lyve-1 and its sensitivity to ionic strength (IC(50) of 150 mm NaCl) contrast markedly with CD44 (IC(50) > 2 m NaCl) in which HA binding is mediated by hydrogen bonding and hydrophobic interactions. In addition, unlike the extended Link module in CD44, which binds HA efficiently when expressed as a soluble monomer (K(d) = 65.7 mum), that of Lyve-1 requires artificial dimerization, although the full ectodomain is active as a monomer (K(d) = 35.6 mum). Finally, full-length Lyve-1 did not form stable dimers in binding-competent 293T transfectants when assessed using bioluminescent resonance energy transfer. These results reveal that elements additional to the extended Link module are required to stabilize HA binding in Lyve-1 and indicate important structural and functional differences with CD44.

Evaluation and application of a luciferase fusion system for rapidin vivoanalysis of RNAi targets and constructs in plants

The practical use of RNA-mediated approaches including antisense RNA, ribozymes and siRNAs for specific inhibition of gene expression is limited by lack of simple quantitative methods to rapidly test efficacy in vivo. There have been indications that cotransfer of target::reporter gene fusions with constructs designed against the target sequence, followed by quantification of transient reporter gene activity might be effective. Here, we report detailed testing of the approach in plants, using diverse target::luciferase fusions and antisense or ribozyme constructs. We used quantitative transient luciferase activity (Luc) assays to test antisense constructs against beta-glucuronidase, PR glucanase, vacuolar invertase and cucumber mosaic virus, as well as ribozymes against watermelon mosaic virus and tobacco anionic peroxidase. For constructs previously tested in transgenic plants, the results correspond well with those from the transient expression assay. Target susceptibility was generally not strongly influenced by luciferase fusion, and the assay was not highly dependent on target sequence length. Some sequences reduced Luc activity below the level for reliable quantification, but suitable alternative fusions were readily produced. Transcriptional and translation fusions were effective for 5' target::luc constructs. Translational fusions were more reliable for luc::target 3' constructs. With minimal preliminary work to prepare suitable target::luciferase fusions, the approach appears generally applicable for rapid in vivo validation of effectiveness and specificity of constructs designed for RNA-mediated down-regulation of plant genes.

Identification of an Activator Protein-1-Like Sequence as the Glucocorticoid Response Element in the Rat Tyrosine Hydroxylase Gene

Glucocorticoids (GCs) generally stimulate gene transcription via consensus glucocorticoid response elements (GREs) located in the promoter region. To identify the GRE in the rat tyrosine hydroxylase (TH) gene promoter, we transiently transfected PC12 cells with a 9-kilobase (kb) TH promoter-luciferase (Luc) construct. Dexamethasone (Dex) stimulated Luc activity, which was abolished by mifepristone (RU486). Serial deletion mutations revealed a Dex-responsive 7-base pair (bp) sequence, TGACTAA, located at -5734 to -5728. Deletion of just these seven nucleotides from the 9-kb promoter completely abolished the Dex response and partially reduced the response to phorbol ester but not to forskolin. The Dex response was fully retained in a construct in which most of the 9-kb promoter was deleted, except for 100 bp around the -5.7-kb region, clearly identifying this 7-bp sequence as solely responsible for GC responsiveness. Conversely, deletion of the proximal cAMP-response element (-45/-38) or activator protein-1 (AP-1) (-207/-201) sites in the 9-kb promoter did not affect Dex and phorbol ester responses. A radiolabeled 25-bp promoter fragment bearing the 7-bp TH-GRE/AP-1 showed specific binding to PC12 nuclear proteins. Using antibodies against the glucocorticoid receptors and AP-1 family of proteins and primers for the TH-GRE/AP-1 region, we detected a specific DNA amplicon in a chromatin immunoprecipitation assay. This 7-bp TH-GRE/AP-1 sequence (TGACTAA) does not bear similarity to any known GRE but closely resembles the consensus AP-1 binding site, TGACTCA. Our studies describe for the first time a novel GRE/AP-1 site present in the TH gene promoter that is critical for glucocorticoid regulation of the TH gene.

리포트 시스템을 이용한 살리실산 생합성 유전자 SID2의 발현 해석

SA는 천연 페놀 화합물로써 식물체가 생성하는 호르몬 중의 하나이다. SA는 특히 병저항성, 생물학적, 비생물학적 스트레스로 인해 합성이 촉진되며 식물 방어 기작을 일으킨다고 알려져 있다. 식물의 방어 기작은 바로 식물에서 얻어지는 생산량에 영향을 미치기 때문에 SA에 대한 연구가 많이 되어져 왔다. 하지만 SA를 이해하기에는 아직까지 많은 연구가 필요 되어 지고 있다. 따라서 본 연구는 애기장대에서 SA 생합성하는데 중요한 효소인 SID2가 병저항성이 강한 siz1-2 돌연변이체와 야생형에서 어떠한 조절의 차이를 보이는 지를 SID2 promoter에 의해서 조절되는 GUS와 LUC를 가진 각각의 형질전환 식물체를 통하여 관찰하였다. GUS의 발현을 GUS histochemical assay, GUS enzyme assay 그리고 LUC의 발현을 CCD 카메라를 이용한 이미지 촬영과 Luciferase enzyme assay 수행한 결과, siz1-2를 사용한 형질전환 식물체에서 야생형에 비해 발현이 높게 일어났다. 이것을 바탕으로 SA에 반응하는 유전자들의 발현이 siz1-2 돌연변이체에서는 높은 이유가 SID2의 발현이 높게 조절 받기 때문이라는 것을 SID2 promoter:GUS::LUC/siz1-2 형질전환 식물체를 통해 알 수 있었다. Salicylic acid(SA) is a phytohormone that is related to plant defense mechanism. The SA accumulation is triggered by abiotic and biotic stresses. SA acts as a signal molecular compound mediating systemic acquired resistance and hypersensitive response in plant. Although the role of SA has been studied extensively, an understanding of the SA regulatory mechanism is still lacking in plants. In order to comprehend SA regulatory mechanism, we have been transformed with a SID2 promoter:GUS::LUC fusion construct into siz1-2 mutant and wild plant(Col-0). SIZ1 encodes SUMO E3 ligase and negatively regulates SA accumulation in plants. SID2(SALICYLIC ACID INDUCTION DEFICIENT2) is a crucial enzyme of SA biosynthesis. The Arabidopsis SID2 gene encodes isochorismate synthase(ICS) that controls SA level by conversion of chorismate to isochorismate. We compared the regulation of SID2 in wild-type and siz1-2 transgenic plants that express SID2 promoter:GUS::LUC constructs respectively. The expressions of <TEX>$\beta$</TEX>-GLUCURONIDASE and LUCIFERASE were higher in siz 1-2 transgenic plant without any stress treatment. SID2 promoter:GUS::LUC/siz1-2 transgenic plant will be used as a starting material for isolation of siz1-2 suppressor mutants and genes involved in SA-mediated stress signaling pathway.

Drd2 expression in the high alcohol-preferring and low alcohol-preferring mice

The high alcohol-preferring (HAP) and low alcohol-preferring (LAP) mice were selectively bred for differences in alcohol preference and consumption. Recently, a large-effect QTL was identified on chromosome 9. The peak for this QTL is near the Drd2 (dopamine receptor 2) locus. The present study examined Drd2 mRNA expression differences between the HAP1 and LAP1 mice in brain regions important in the dopaminergic-reward pathway, including the nucleus accumbens, hippocampus, amygdala, and septum. Results show that alcohol-naïve HAP1 mice exhibited lower levels of Drd2 mRNA expression in the nucleus accumbens and the hippocampus compared to LAP1 mice. No differences were found in the amygdala or septum. To determine if a sequence difference might underlie the expression difference, the Drd2 cDNA was sequenced in each line and one single nucleotide polymorphism (SNP) was identified in the 3' UTR. Both HAP and LAP 3' UTR were cloned in the luc-pGL3-promoter-luc vector. The polymorphism in the Drd2 3' UTR was assessed to determine its functional significance in modulating expression. In vitro expression analysis using neuroblastoma SK-N-SH cells resulted in a significant decrease in expression of the HAP 3' UTR luc construct compared with the LAP 3' UTR construct. This decreased expression is consistent with lower levels of Drd2 expression in the nucleus accumbens and the hippocampus as evidenced by qRT-PCR. These results suggest that the SNP may play a role in the differential expression of Drd2 between the HAP and LAP mice and that the polymorphism in Drd2 may contribute to alcohol preference.

Assignment of UVB-responsive cis-element and Protoplastization- (Dilution-) and Elicitor-responsive Ones in the Promoter Region of a Carrot Phenylalanine Ammonia-lyase Gene (gDcPAL1)¶

Expression of a carrot phenylalanine ammonia-lyase (PAL) gene (gDcPAL1) in suspension-cultured carrot cells is induced by dilution of the culture or by application of a fungal elicitor, as well as by ultraviolet B (UVB) irradiation. We demonstrated that among its upstream cis-elements (Takeda et al.[1997] Photochem. Photobiol. 66, 464–470), L4 is UVB responsive, and L1 is protoplastization- (dilution-) and elicitor responsive, from studies with transiently transformed mutated or truncated gDcPAL1 promoter–luc constructs. This conclusion is consistent with our observation that PAL activities induced by UVB and by protoplastization (dilution) or elicitor are additive.