OBJECTIVEClone of sucrose synthase of Dendribium officinale and expression analysis, to provide the theory basis for research the relationship between polysaccharide synthesis of D. officinale and sucrose synthase activity.

METHODAccording to homologous sequence of sucrose synthase gene on GenBank, application the technology of RT-PCR and RACE, clone the full length of D. officinale. Target gene amplified with T vector was transformed into competent E. coli. BL21, IPTG induced expression, SDS-PAGE analysis.

RESULTA full length cDNA encoding sucrose synthase was isolated from the D. officinale, named DOSS1, the GenBank accession number is HQ856835, the cDNA is 2781 bp in length containing an open reading frame of 2424 bp encoding 807 amino acids with a predicted molecular mass of 92.3 x 10(3), the deduced amino acid sequence of D. officinale sucrose synthase shares 95% identity with Mokara yellow (AF530568); shares 90% identity with Oncidium goldiana (AF530567); shares more than 80% with other monocotyledonous plants.

CONCLUSIONCloned the sucrose synthase gene and induced an obvious band successfully.

3' Untranslated Regions ; genetics ; 5' Untranslated Regions ; genetics ; Cloning, Molecular ; Dendrobium ; enzymology ; genetics ; metabolism ; Escherichia coli ; genetics ; Gene Expression Regulation, Plant ; Glucosyltransferases ; genetics ; metabolism ; Phylogeny ; Polysaccharides ; biosynthesis

The special nucleic acid fragments, 5' untranslated region (5' UTR) and internal ribosome entry site (IRES) of foot-and-mouth disease virus (FMDV), which interact with the capsid proteins, were selected as scaffolds to investigate the assembly efficiency of foot-and-mouth disease (FMD) virus-like particles (VLPs). The assembled product was characterized by evaluation of particle size, surface potential, gel retardation assay, nuclease digestion experiments, size-exclusion chromatography, transmission electron microscopy and circular dichroism analysis. The results confirmed that the 5' UTR and IRES of FMDV co-assembled with the FMD VLPs and facilitated the assembly efficiency of FMD-VLPs. It demonstrates that the assembly efficiency of 75S particles of VLPs-5'UTR was significantly higher than those of the VLPs (P<0.001) and VLPs-IRES group (P<0.01). Comparatively the assembly efficiency of 12S particles of VLPs-IRES was significantly higher than those of the VLPs (P<0.000 1) and VLPs-5'UTR (P<0.000 1). It showed that the 5' UTR represented more effective in facilitating the assembly of VLPs. This study proposes an optimized strategy for improving the assembly efficiency of VLPs for the development of VLPs vaccine.
5' Untranslated Regions ; Capsid Proteins/metabolism* ; Foot-and-Mouth Disease Virus/physiology* ; Internal Ribosome Entry Sites ; Nucleic Acids/metabolism* ; Virus Assembly

We report here, that a vector constructed based on ppET-1 gene promoter and 5' untranslated region induced a high level of gene expression in endothelial cells and the specificity is even further enhanced under hypoxia-mimic conditions due to a natural hypoxia responsive element within the promoter region. A naked DNA vector that confers endothelial cell specific gene expression as well as efficient levels of gene expression was constructed with an endothelial cell specific naked DNA vector, pETlong, by using the full length promoter of the preproendothelin-1 gene and the entire 5' untranslated region upstream from the start codon. Inclusion of the entire 5' untranslated region in pETlong increased gene expression 2.96 fold as compared with that from pETshort, which contains only the promoter sequences. Reporter gene expression from pETlong was 7.9 fold higher as compared with that from CMV-driven promoter based vector in calf pulmonary endothelial cells. However, in nonendothelial COS cells, luciferase activity from pETlong was only 0.3 fold as compared with that of CMV-based vector. Similar results were observed in other nonendothelial cells. These results demonstrate that the pETlong drives gene expression in endothelial cells with high efficacy and specificity. We have examined hypoxia responsiveness of pETlong as the promoter region of the preproendothelin-1 gene contains hypoxia responsive elements. The activity of the pETlong vector was increased 1.6 fold under hypoxia-mimic conditions using cobalt chloride. The high levels of hypoxia-inducible expression in endothelial cells relative to the low levels of background expression in other cells shows that pETlong could be a useful tool for vascular targeting of vascular disease and cancer gene therapy.
*5' Untranslated Regions ; Animals ; Anoxia/metabolism ; Cattle ; Endothelial Cells/*metabolism ; Endothelin-1/*genetics/metabolism ; Endothelium, Vascular/metabolism ; Gene Transfer Techniques ; *Genetic Vectors ; Human ; *Promoter Regions (Genetics)

OBJECTIVETo try to find the ways to enhance the expression of ADS gene encoding amorpha-4,11-diene synthase, a key enzyme in artemisinin biosynthesis pathway catalyzing the formation of amorpha-4,11-diene from farnesyl diphosphate, and accelerate the artemisinin synthesis, the promoter of ADS was isolated and characterized.

METHOD5' untranslated regions of ADS were isolated from Artemisia annua with PCR. For functional characterization, the isolated fragment was fused with GUS reporter gene and introduced into Nicotiana tabacum by Agrobacterium-mediated transformation. The GUS expression regulated by 5' untranslated regions of ADS in transgenic N. tabacum under the normal or stressed conditions were detected by histochemical staining and quantitative spectrophotometry assay.

RESULTThe 2 448 bp DNA fragment upstream of ADS coding sequence was isolated from A. annua and introduced into N. tabacum. Histochemical staining showed that the isolated fragment conferred stable GUS expression in transgenic plants. The quantitative results showed that the GUS activity in transgenic tobacco plants treated by low-temperature (4 degrees C) and ultraviolet irradiation were 1. 6 and 2.2 folds higher than that in the controls.

CONCLUSIONIt was suggested that the isolated fragment had promoter activity and maybe responsive to adverse environmental stresses.

5' Untranslated Regions ; genetics ; Alkyl and Aryl Transferases ; genetics ; metabolism ; Artemisia annua ; enzymology ; genetics ; Gene Expression Regulation, Plant ; Genetic Vectors ; genetics ; Molecular Sequence Data ; Promoter Regions, Genetic ; genetics

OBJECTIVETo study the promoter activity in HepG2 cells of the DNA sequence corresponding to the HCV 5'UTR.

METHODSPlasmids, 5'UTR-Luc(+) and 5'UTR-Luc(-) carrying the forward and reverse DNA sequences corresponding to the HCV 5'UTR respectively were constructed, and subsequently transfected into HepaG2 cells. The luciferase activity and the mRNA of the luciferase gene were then detected. The 5'UTR sequence was cloned into a GFP vector to make 5'UTR-EGFP, and then the GFP expression was confirmed by fluorescence microscopy.

RESULTS5'UTR-Luc(+) had an obvious luciferase activity whereas 5'UTR-Luc(-) had nearly no luciferase activity. The former had a high level of luciferase mRNA while the latter could not be detected. An intense green fluorescence expression was observed in the cells transfected with the plasmid of 5'UTR-EGFP.

CONCLUSIONThe forward DNA sequence corresponding to HCV 5'-UTR had an obvious promoter activity in hepG2 cells. It may play an important role in the replication of HCV.

5' Untranslated Regions ; genetics ; Carcinoma, Hepatocellular ; virology ; DNA, Viral ; genetics ; Hepacivirus ; genetics ; isolation & purification ; Humans ; Liver Neoplasms ; virology ; Luciferases ; metabolism ; Promoter Regions, Genetic ; genetics ; Sequence Analysis, DNA

OBJECTIVETo assess the telomerase activity inhibitory effect of ham merhead ribozyme targeting the 5'-end of human telomerase reverse transcriptase mRNA (hTERT-5'RZ), to compare it with the effect of another ribozyme teloRZ, and the combine the applications of the two ribozymes.

METHODShTERT-5'RZ gene was synthesized and cloned into pcDNA3.1(+); the ribozyme was produced by in vitro transcription. The teloRZ ribozyme was produced in the same way by in vitro transcription of p(SPT19-teloRZ) which had been constructed by the present authors. The ribozymes were transiently transfected into HeLa cells by liposome every 24 hours. After 72 hours, the cells were collected and their telomerase activities were assayed.

RESULTSThe ribozyme targeting the 5'-end of hTERT mRNA exhibited a very strong telomerase-inhibitory activity, the combined use of hTERT-5'RZ and teloRZ also showed clear inhibitory activity, but the inhibitory effect of teloRZ used alone was not so strong.

CONCLUSIONThese observations suggest that the use of hTERT-5'RZ and the combined use of hTERT-5'RZ and teloRZ are more effective in telomerase inhibition as compare with the use of teloRZ alone. They may find applications in cancer therapy.

5' Untranslated Regions ; Cloning, Molecular ; DNA-Binding Proteins ; HeLa Cells ; Humans ; RNA, Catalytic ; metabolism ; RNA, Messenger ; Telomerase ; antagonists & inhibitors ; genetics ; Transcription, Genetic

OBJECTIVETo examine the role of domain II of hepatitis C virus (HCV) 5' noncoding region (5' NCR) in its translation initiation activity.

METHODSThe fragment of HCV 5' NCR with deletions of 5'-proximal 118 nucleotides were amplified with PCR, then was used to substitute for the full length HCV 5' NCR of plasmid pCMVNCRluc, a firefly luciferase (Fluc) eukaryotic expression plasmid regulated by HCV 5' NCR, to generate recombinant plasmid pCNl-d3. pCMVNCRluc, pCNl-d2 (modified pCMVNCRluc with deletions of HCV 5' NCR ntl-43) and pCNl-d3 were transfected into HepG2 cells with liposome transfection protocol, respectively, and the relative luciferase activity was measured to analyze the regulatory effect of the truncated 5'NCR on Flue gene expression. Meanwhile the Flue mRNA levels were detected by RT-PCR.

RESULTSThe recombinant plasmid was successfully constructed. The Flue mRNA levels of the 3 plasmids were not significantly different (P > 0.05), and there were also no significant difference between the relative luciferase activity of plasmids pCNl-d2 and pCMVNCRluc (P > 0.05). However, that of pCNl-d3 was decreased significantly (P < 0.01, compared with pCNl-d2 or pCMVNCRluc).

CONCLUSIONStructural domain II of HCV 5' NCR plays an important role in its translation initiation activity.

5' Untranslated Regions ; Hep G2 Cells ; Hepacivirus ; chemistry ; genetics ; metabolism ; Hepatitis C ; Humans ; Nucleic Acid Conformation ; Peptide Chain Initiation, Translational ; RNA, Viral ; chemistry ; genetics

Through PCR amplification, 1.3 kb of 5'-proximal promoter (TA, 1.3 kb) of the beta-actin gene of white cloud mountain minnow Tanichthys albonubes was obtained. Using Genome Walker, a 1.7 kb 5'-upstream sequence from the proximal promoter of the beta-actin gene was isolated, and a further promoter (3.0 kb in size) was amplified according to the isolated 5'-proximal and upstream sequences (TLA, 3.0 kb). Both the 1.3 kb and 3.0 kb promoters contain elements that were critical to the transcription activity of other species, including the CCAAT Box (-89 approximately -85), CArG Box (-59 approximately -49), TATA Box (-26 approximately -20). Results of putative transcription binding sites analysis of the promoters by software TRANSFAC 6.0 revealed the presence of E-box, several transcript binding sites NF-Y, SP1 (Stimulating Protein 1), AP1 (Activator Protein 1), and some more transcription binding sites existing in the further promoter. The two promoter sequences were inserted into the expression vector to construct the recombinant expression vector, pTA-DsRed and pTLA-DsRed, respectively. The vectors were microinjected into the fertilized eggs of Tanichthys albonubes and higher positive rate was obtained and stronger red fluorescence was observed in pTLA-DsRed transgenic fish. RT-PCR analysis showed that RFP (Red fluorescent protein) mRNA level in pTLA-DsRed transgenic fish was 35.7% higher than that of the pTA-DsRed transgenic fish of 15-days-post-hatched. The present study showed that both the proximal and further promoter sequences have effective transcription activities and the 3.0 kb promoter possesses higher potent activity than that of the 1.3 kb promoter.
5' Untranslated Regions ; genetics ; Actins ; genetics ; metabolism ; Animals ; Animals, Genetically Modified ; genetics ; Base Sequence ; Cyprinidae ; genetics ; Genetic Vectors ; Molecular Sequence Data ; Promoter Regions, Genetic ; RNA, Messenger ; genetics ; metabolism ; Recombinant Proteins ; genetics ; metabolism

Recent evidence shows that transcriptional silencing as a consequence of hypermethylation of CpG islands is an important mechanism in the inactivation of p16INK4 tumor suppressor gene. This study is designed to clarify the significance of p16INK4 hypermethylation in 23 cases of glioblastomas (GBMs) by methylation-specific polymerase chain reaction (PCR) and p16 immunostaining. Fourteen cases (60.9%) out of 23 GBMs revealed hypermethylation on p16. p16 immunostaining revealed that 13 (93%) of these 14 hypermethylation cases showed complete loss of immunoreactivity and only one (7%) case retained immunoreactivity. Among 9 methylation-negative cases, 4 were immunonegative, which might be related to mutations or deletions other than hypermethylation. The most significant finding was that of 17 cases with immunonegativity, 13 cases (76.5%) showed hypermethylation. We reconfirmed that p16 hypermethylation may be one of the major mechanisms of tumorigenesis of GBMs and the results between the methylation specific-PCR study and p16 immunostaining had a good correlation.
5' Untranslated Regions/metabolism* ; 5' Untranslated Regions/genetics ; Adult ; Antisense Elements (Genetics) ; Brain Neoplasms/pathology ; Brain Neoplasms/genetics* ; Brain Neoplasms/chemistry ; CpG Islands/physiology* ; DNA Methylation* ; Female ; Gene Silencing/physiology ; Glioblastoma/pathology ; Glioblastoma/genetics* ; Glioblastoma/chemistry ; Human ; Male ; Middle Age ; Polymerase Chain Reaction ; Protein p16/genetics* ; Protein p16/analysis

OBJECTIVETo analyze the cleavage activity of two deoxyribozymes targeting at hepatitis C virus (HCV) RNA in vitro and evaluate their prospects of antiviral therapy.

METHODSTwo specific sequences containing 5' ...A / U... 3' in HCV 5'-noncoding region and 5'-fragment of C region (5'-NCR-C) were selected as the target sites, and with the active region of 5'GGCTAGCTACAACGA3', two phosphorothioate deoxyribozymes (TDRz) named as TDRz-127 and TDRz1 were synthesized. HCV RNA 5'-NCR-C was transcribed in vitro from plasmid pHCV-neo which was completely linearized with restriction endonuclease Nar I, and its 5'-end phosphoric acid was deleted by calf intestinal alkaline phosphatase (CIP), then radiolabelled with T4 polynucleotide kinase and gamma-32P-ATP. Under the conditions such as pH 7.5 and a 10 mmol/L Mg2+ concentration, TDRz-127 and TDRz1 were separately (a 5 micromol/L final concentration) or combinedly (each 2.5 micromol/L) mixed with the substrate RNA (200 nmol/L). After denaturation and then renaturation, the reaction systems were incubated in 37 degrees C, and aliquots were removed to terminate the reaction at intended time points. The cleavage products were separated with 8% denaturated polyacrylamide gel electrophoresis and displayed by autoradiography. Finally, the optical density of each product band was measured with Gel Documentation-Analyzing Systems for calculating the percentages of cleaved HCV 5'-NCR-C.

RESULTSAfter reaction for 15, 30, 45, 60, 75 and 90 min under the adopted conditions, about 8.3%, 16.1%, 24.3%, 26.2%, 29.4% and 31.1% of HCV 5'-NCR-C was cleaved by TDRz-127 respectively; 7.4%, 13.0%, 15.6%, 18.7%, 19.4% and 20.3% by TDRz1; and 15.1%, 29.6%, 37.8%, 39.1%, 41.5%, 42.6% by combining the two TDRzs.

CONCLUSIONSCleavage percentage of both TDRz-127 and TDRz1 increases with the time, and the effect of combining the two TDRzs is better than that of anyone.

5' Untranslated Regions ; metabolism ; Base Sequence ; DNA, Catalytic ; genetics ; metabolism ; Hepacivirus ; enzymology ; genetics ; Humans ; Molecular Sequence Data ; RNA Processing, Post-Transcriptional ; RNA, Catalytic ; metabolism ; RNA, Viral ; metabolism ; mRNA Cleavage and Polyadenylation Factors ; genetics ; metabolism