Transcriptional activators are required to turn on the expression of genes in a eukaryotic cell. Activators bound to the enhancer can facilitate either the recruitment of RNA polymerase II to the promoter or its elongation. This article examines a few selected issues in understanding activator functions and activation mechanisms.
Animals ; Humans ; Trans-Activators ; genetics ; metabolism ; Transcription Factors ; genetics ; metabolism ; Transcription, Genetic ; Transcriptional Activation

Cell Differentiation ; Cell Line, Tumor ; metabolism ; Cell Proliferation ; Humans ; Trans-Activators ; biosynthesis ; genetics ; Transfection

To determine the feasibility of a nonradioactive electrophoresis mobility shift assay for detecting nuclear transcription factor, double-stranded oligonucleotides encoding the consensus target sequence of NF-kappa B were labelled with DIG by terminal transferase. After nuclear protein stimulated with phorbol 12-myristate 13-acetate (PMA) or PMA and pyrrolidine dithiocarbamate (PDTC) electrophoresed on 8% nondenaturing poliacrylamide gel together with oligeonucleotide probe, they were electro-blotted nylon membrane positively charged. Anti-DIG-AP antibody catalyzed chemiluminescent substrate CSPD to image on X-film. The results showed that nuclear proteins binded specifically to the NF-kappa B consensus sequence in the EMSA by chemiluminescent technique method and the activity of NF-kappa B in PMA group was more than that in PMA + PDTC group. It is suggested that detection of NF-kappa B by EMSA with chemiluminescent technique is feasible and simple, which can be performed in ordinary laboratories.
Chemiluminescent Measurements ; DNA-Binding Proteins/*analysis ; DNA-Binding Proteins/genetics ; Electrophoretic Mobility Shift Assay ; NF-kappa B/*analysis ; NF-kappa B/genetics ; NF-kappa B/metabolism ; Rats, Sprague-Dawley ; *Trans-Activation (Genetics) ; Trans-Activators/analysis ; Trans-Activators/genetics ; *Transcription, Genetic

OBJECTIVETo construct pEGFP-HBx eukaryotic expression plasmid and establish stable and effective transfected rat oval cell (LE/6) strain expressing EGFP-HBx fusion protein to explore the roles of HBx gene and oval cell in carcinogenesis of hepatocellular carcinoma (HCC).

METHODSHBx gene with EcoRI and Hind III endo-enzyme sites was obtained by using PCR from plasmid pcDNA3.1-HBx. The purified HBx gene fragment was inserted into pEGFP-N1 expression vector, and the recombinant plasmid pEGFP-HBx was identified by restriction endonuclease and DNA sequencing analysis. LE/6 cells were transfected with recombinant pEGFP-HBx by lipofectamine reagent. Resistant to G418 clones were selected, and expression of EGFP-HBx fusion protein in clones were examined directly with fluorescence microscope, and these clones were isolated and proliferated. The expression of HBx was detected by RT-PCR analysis and immunocytochemistry.

RESULTSPlasmid pEGFP-HBx has whole HBx gene base and correct reading frame as indicated by restriction endonuclease and DNA sequencing analysis. After transfecting with pEGFP-HBx plasmid, LE/6 cell clones expressing EGFP-HBx fusion protein were obtained. RT-PCR analysis and immunocytochemistry showed that HBx gene was only expression in transfected pEGFP-HBx cells.

CONCLUSIONSThe pEGFP-HBx recombinant expression vector was successfully constructed, and the stable transfected LE/6 strain expressing EGFP-HBx fusion protein was successfully established. It will be helpful in the further study on the roles of HBx and liver oval cell in carcinogenesis of HCC.

Animals ; Cell Line ; Genetic Vectors ; Hepatocytes ; cytology ; metabolism ; Plasmids ; genetics ; Rats ; Stem Cells ; cytology ; metabolism ; Trans-Activators ; genetics ; Transfection

Pdx-1, an important transcription factor highlighting in the early pancreatic development, islet functions and pancreatic disorders, needs to be more investigated in zebrafish, and siRNA is still seldom applied in zebrafish embryo-related research. Our aim was to explore the role of pdx-1 in pancreatic development of zebrafish embryos by using siRNA approach. Microinjection, reverse transcriptase-PCR (RT-PCR), in situ hybridization and immunofluorescent staining were used in this research, and the morphology of the islet in normal zebrafish embryos, and in those treated with the siRNA specific to pdx-1 (siPDX-1) or siGFP was observed and compared. The expression of pdx-1 was detected in the stages of 1-cell, 2-cell, 4-cell, 8-cell, 16-cell, 16-hour by RT-PCT. The in situ hybridization and immunofluorescent staining results showed that siPDX-1 disturbed the formation of the islet in zebrafish embryos. Pdx-1 played multiple roles in maintaining the phenotype of the islet during embryogenesis in zebrafish.
Embryo, Nonmammalian ; Homeodomain Proteins/genetics ; Homeodomain Proteins/*metabolism ; Islets of Langerhans/cytology ; Islets of Langerhans/*embryology ; Islets of Langerhans/metabolism ; RNA Interference ; RNA, Small Interfering/*genetics ; Trans-Activators/genetics ; Trans-Activators/*metabolism ; Zebrafish

To investigate the protein and mRNA expression of pancreas/duodenal homeobox-1 (PDX-1), a transcription factor as a marker for pancreatic stem cells, in pancreatic ductal cells of rats after partial (90%) pancreatectomy and evaluated the significance of the PDX-1 expression. Western blot and Reverse transcriptase-polymerase chain reaction (RT-PCR) were used to detect the expression of PDX-1 protein and mRNA respectively. PDX-1 protein was only faintly detected in pancreatic ductal cells on the day 1 after partial pancreatectomy. On the day 2 and 3 after operation in operation group, a 2-3 fold increased PDX-1 protein was observed, corresponding to the characteristic 42-kD protein in Western blot. There was significant difference between operation group and sham-operation group (P<0.05). PDX-1 protein expression on the day 5 and 7 after operation had already been no difference from control group (P>0.05). RT-PCR revealed the PDX-1 mRNA expression showed no significant difference between operation group at various time points and sham-operation group (P> 0.05). These results indicate that there was overexpression of PDX-1 in the cells of pancreatic epithelium during the regeneration of remnant pancreas after partial pancreatectomy in adult rats, suggesting the pancreatic stem cells in pancreatic ductal epithelial cells are involved in the regeneration of remnant pancreas and the expression of PDX-1 in ductal cells was regulated posttranscription.
Epithelial Cells/metabolism ; Homeodomain Proteins/*biosynthesis ; Homeodomain Proteins/genetics ; Pancreatectomy/methods ; Pancreatic Ducts/cytology ; Pancreatic Ducts/*metabolism ; RNA, Messenger/biosynthesis ; RNA, Messenger/genetics ; Rats, Sprague-Dawley ; Trans-Activators/*biosynthesis ; Trans-Activators/genetics

The HBV X gene was amplified by PCR according to the pecob6 containing the whole fragment of adw subtype of HBV, then the fragment was inserted into the multiple clone site of pAdTrack-CMV. The linearized shuttle plasmid was homogenously recombined with AdEasy-1 in BJ5183 and the recombinant adenoviral plasmid pAd-X was generated. Then plasmid pAd-X was digested with Pac I and transfected into 293 cells for packaging and amplifying. Infection titer and rate were monitored by green fluorescent protein (GFP) expression. With restriction endonuclease analysis and PCR methods, it has been confirmed that HBV X gene was cloned into the adenovirus vector successfully. The expression of X protein in HepG2 cells was detected by Western-blot. The recombined adenovirus Ad-X was constructed successfully, which would contribute to the advanced functional study of HBV X protein.
Adenoviridae ; genetics ; metabolism ; Genetic Vectors ; genetics ; metabolism ; Green Fluorescent Proteins ; metabolism ; Hep G2 Cells ; Humans ; Recombinant Proteins ; biosynthesis ; genetics ; Trans-Activators ; biosynthesis ; genetics ; Transfection

OBJECTIVETo explore the effect of a non-lethal heat shock, in comparison with the treatment of interferon-gamma (IFN gamma), on the expression of major histocompatibility complex transactivator (CTA) and its downstream target gene of the human leukocyte antigens (HLA)-DR in Jurkat cells.

METHODSThe changes of CTA mRNA in Jurkat cells before and after the treatment of heat shock or IFN gamma were detected using real time RT-PCR. The changes of CTA protein were detected with Western blot. The expression of HLA-DR was detected with flow cytometry. : CTA mRNA and protein were induced in Jurkat cells under heat shock, but not with IFN-gamma. The expression of HLA-DR gene significantly increased after recovery (P<0.01).

CONCLUSIONThe expressions of CTA and HLA-DR in Jurkat cells remarkably increase after heat shock, indicating that heat shock may help reconstruct relevant genes in cells with immunologic gene deficiencies.

HLA-DR Antigens ; metabolism ; Heat-Shock Response ; physiology ; Humans ; Jurkat Cells ; Nuclear Proteins ; genetics ; metabolism ; RNA, Messenger ; genetics ; Trans-Activators ; genetics ; metabolism

Confocal laser scanning microscopy was used to observe the spatio-temporal expression of the pathway-specific gene redD during S. coelicolor cell cultivation. The corresponding mutant S. coelicolor lyqRY1522 carrying redD::eyfp in the chromosome was constructed. The temporal expression results of the fusion protein during submerged cultivation demonstrated that expression of redD began in the transition phase, continuing through the exponential growth phase to the stationary phase, and reached maximum in the stationary phase. On the other hand, redD was expressed only in substrate mycelia during solid-state culture, while aerial mycelia remained essentially non-fluorescent throughout culture. Results demonstrated that the expression pattern of redD coincides with that of the biosynthesis of the antibiotics during culture, revealing a direct correlation between the spatio-temporal distribution of regulatory gene expression and second metabolism.
Anti-Bacterial Agents ; biosynthesis ; Bacterial Proteins ; genetics ; metabolism ; Gene Expression Profiling ; Gene Expression Regulation, Bacterial ; physiology ; Mutation ; Signal Transduction ; physiology ; Streptomyces coelicolor ; genetics ; metabolism ; Trans-Activators ; genetics ; metabolism

To observe the alteration in the expression of DNA repair enzymes hOGG1 and hMYHalpha and the change in 8-OHdG levels in the HBx gene-transfected cells HepG2/HBx and to explore the mechanisms of the HBV-associated hepatocellular carcinoma, the gene-transfected cells HepG2/HBx which stably expressed HBx was established, and the effect of HBx on the cell cycle and proliferation of HepG2 was examined. By using the beta-actin as the interior control, real-time polymerase chain reaction (Real-time qPCR) was employed to quantitatively detect the expression of DNA repair enzymes hOGG1 and hMYHalpha in the HepG2/HBx, the control cells HepG2 and HepG2 transfected with pcDNA3.1 vector (HepG2/pDNA3.1). The 8-OHdG levels were determined by HPLC/ECD in the established gene-transfected cells HepG2/HBx and the control cells HepG2 and HepG2/pcDNA3.1. Our results showed that the expression of DNA repair enzyme hMYHalpha in the HepG2/HBx (0.021+/-0.007) was significantly lower than that of HepG2 (0.099+/-0.041) (P<0.05) and HepG2/pDNA3.1 (0.121+/-0.005) (P<0.05). However, the no significant differences existed in the expression of DNA repair enzyme hOGG1 among the three cell strains (P>0.05). The 8-OHdG level in the HepG2/HBx was significantly higher than that in HepG2 and HepG2/pcDNA3.1 (P<0.05). It is concluded that HBx gene may inhibit the expression of DNA repair enzyme hMYHalpha mRNA to impair the ability to repair the intracellular DNA oxidative damage, to increase the oxidative DNA-adduct 8-OHdG and to affect the nucleotide excision repair function, thus participate in the occurrence and development of hepatocellular carcinoma.
DNA Glycosylases/genetics ; DNA Glycosylases/*metabolism ; Deoxyguanosine/analogs & derivatives ; Deoxyguanosine/metabolism ; Hep G2 Cells ; RNA, Messenger/genetics ; RNA, Messenger/metabolism ; Trans-Activators/*genetics