Osteoblasts can synthesize the insulin-like growth factors (IGFs) and the IGF-binding proteins (IGFBPs), which may either enhance or attenuate IGF-stimulated bone cell proliferation. Since estrogen induced osteoblastic differentiation and proliferation through an estrogen-responsive gene in target cells, we investigated the effects of estrogen on IGFBP-6 expression in the human osteoblastic-like cell line SaOS-2. Expressions of IGFBP-6 protein and mRNA increased 2.8 and 2-fold, respectively, in the presence of 17-beta-estradiol (E2) (0.01 to 1 micrometer) and estrogen receptor (ER) in SaOS-2 cells. On the other hand, E2 induced a 2-fold increase in SaOS-2 cell proliferation. To identify genomic sequences associated with estrogen responsiveness, the 5'-promoter region (-44 to +118) of the IGFBP-6 gene was cloned into a chloramphenicol acetyltransferase (CAT) reporter vector. E2 induced a 3-fold increase in CAT activity in SaOS-2 cells transiently transfected with this construct. Identification of the estrogen-responsive element (ERE) [5'-CCTTCA CCTG-3'] (-9 to +1) in this IGFBP-6 gene promoter region was confirmed using electromobility shift assays and deletion analysis. This functional ERE was important for E2-induced trans-activation of the IGFBP-6 gene. These results demonstrate that E2 exhibits a positive effect on IGFBP-6 gene transcription through estrogen-liganded ER binding to the functional ERE in the IGFBP-6 gene promoter in SaOS-2 cells.
Blotting, Western ; Cell Proliferation ; Chloramphenicol O-Acetyltransferase/metabolism ; Electrophoretic Mobility Shift Assay ; Estradiol/*pharmacology ; Estrogen Receptor alpha/genetics/metabolism ; Estrogens/pharmacology ; Humans ; Insulin-Like Growth Factor Binding Protein 6/*genetics/metabolism ; Osteoblasts/*drug effects/metabolism ; Promoter Regions, Genetic/*genetics ; RNA, Messenger/genetics/metabolism ; Response Elements ; Reverse Transcriptase Polymerase Chain Reaction ; *Transcriptional Activation ; Tumor Cells, Cultured

OBJECTIVETo analyze the upstream region of radiation-induced junB gene.

METHODSFour plasmids containing 250 bp, 590 bp, 900 bp and 1650 bp, and CAT reporter gene were constructed separately and introduced to L8704 cells. The cells were irradiated with 2 Gy X-rays and incubated at different intervals. Total RNA was extracted from the cells and fluctuation of the CAT mRNA level was assessed by the RNA ratio of CAT/beta-actin measured by quantitative Northern blot hybridization.

RESULTSCAT mRNA expression containing 900 bp and 1560 bp junB promoter remarkably and rapidly increased, and reached its peak 30 min after 2 Gy X-ray irradiation.

CONCLUSIONS590-900 bp fragments located in the upstream region of junB gene play an important role in the early process of cells against radiation.

Actins ; analysis ; metabolism ; Animals ; Blotting, Northern ; Cells, Cultured ; Chloramphenicol O-Acetyltransferase ; genetics ; Gene Expression Regulation ; radiation effects ; Genes, Reporter ; Genes, jun ; genetics ; radiation effects ; In Situ Hybridization ; Mice ; Mice, Inbred BALB C ; Plasmids ; analysis ; genetics ; RNA ; isolation & purification ; metabolism ; RNA, Messenger ; analysis ; metabolism ; Time Factors ; X-Rays

OBJECTIVETo analyze the transcription activation and possible regulation mechanism of human X-box binding protein 1(XBP1)gene 5'upstream DNA sequence in different cell lines.

METHODSSix kinds of XBP1 promoter deletion mutants were cloned into pGEM-Teasy vector, which included XBP1 gene 5' upstream -1039 to 66 bp,-859 to 66 bp,-623 to 66 bp,-351 to 66 bp,-227 to 66 bp,-227 to -45 bp respectively. Every deletion mutant sequence was cut from Teasy-XBP1p by KpnI and Xho I, and subcloned into pCAT3-Basic to produce a set of constructs termed as p1-XBP1p, p2-XBP1p, p3-XBP1p, p4-XBP1p, p5-XBP1p, p6-XBP1p, respectively. The transcription activity of each construct was detected after transiently transfecting K562, HepG2,NIH-3T3 and L0(2)cell with FuGENE 6 transfection reagent. Cells transfected by pCAT3-Basic or pCAT3-Promoter were used as negative and positive controls. The activity of chloramphenicol acetyltransferase(CAT), which reflects the transcription activation of the XBP1 gene promoter, was detected by ELISA after 48 hours of transfection.

RESULTSThe reporter vectors of six kinds of XBP1 promoter deletion mutants were successfully constructed, as confirmed by restriction enzyme digestion and sequencing. The activities of p4-XBP1p and p5-XBP1p were higher than the other deletion mutants in K562 and HepG2. And the activity of p5-XBP1p was the highest in HepG2. There was no activity detected from any transfected NIH-3T3.

CONCLUSIONThe XBP1 gene promoter can transactivate its downstream gene to transcription. The core sequence of XBP1 promoter was implied between -227 bp and 66 bp. This sequence was connected with the transcriptional activity of XBP1 promoter closely. Its transcription activity varies with different cell lines. XBP1 promoter might drive gene expression with cell-type specificity.

3T3 Cells ; 5' Flanking Region ; genetics ; Animals ; Base Sequence ; Cell Line ; Chloramphenicol O-Acetyltransferase ; metabolism ; DNA ; analysis ; DNA-Binding Proteins ; genetics ; Gene Deletion ; Gene Expression Regulation ; physiology ; Genes, Reporter ; Humans ; K562 Cells ; Mice ; Molecular Sequence Data ; Nuclear Proteins ; genetics ; Promoter Regions, Genetic ; genetics ; Regulatory Factor X Transcription Factors ; Transcription Factors ; Transcription, Genetic ; physiology ; Transcriptional Activation ; Transfection ; Tumor Cells, Cultured ; X-Box Binding Protein 1

BACKGROUNDIt is still unclear whether viral genetic variability influences response to interferon (IFN)-alpha treatment. Recent reports suggest that IFN-alpha effects may be associated with hepatitis B virus (HBV) post-transcriptional regulation. This study was designed to explore the heterogeneity of HBV post-transcriptional regulatory elements (HPRE) and the relationship between the diversity of HPRE and the response to IFN-alpha treatment.

METHODSThe HPRE sequences from 31 Chinese patients infected with HBV were determined by directly sequencing of polymerase chain reaction (PCR) product, and comparing them to those from Caucasian patients. Subsequently, eukaryotic expression vectors containing HPRE at various points were constructed and transfected into HepG2 cells, which were then exposed to recombinant human cytokines.

RESULTSThe T to C point mutation at nt 1504 and the C to T (G) at nt 1508 in HPRE were found in 21 and 19 patients with chronic hepatitis B, respectively; the C to T point mutation at nt 1509 was found in 17 patients. These point mutations did not exist in the HPRE of the Caucasian patients. The activity of the CAT gene obviously increased in the case of T to C point mutation at nt 1504, but did not change in the case of the C to T (G) mutations at nt 1508 and 1509. The activity of the CAT gene at these point mutations of HPRE could be inhibited by IFN-alpha/gamma and tumor necrosis factor (TNF)-alpha except for the point mutations at nt 1508 of HPRE which may escape the suppression role of IFN-alpha on HPRE.

CONCLUSIONSThere are point mutations between the HPRE of Chinese and Caucasian HBV patients, which might be correlated with response to IFN-alpha. The variation of HPRE might affect the function of HPRE and influence the regulative function of IFN-alpha other than that of IFN-gamma or TNF-alpha on HPRE.

Chloramphenicol O-Acetyltransferase ; metabolism ; Genes, Regulator ; Hepatitis B virus ; drug effects ; genetics ; Hepatitis B, Chronic ; drug therapy ; virology ; Humans ; Interferon-alpha ; pharmacology ; Interferon-gamma ; pharmacology ; Plasmids ; Point Mutation ; Tumor Necrosis Factor-alpha ; pharmacology

OBJECTIVETo investigate the mechanism of cytokeratin 13 (CK13) gene expression control and the effects of different motifs of CK13 gene 5' flanking region on its transcriptional activity.

METHODSThe molecular clone technique and reporter gene analysis were used to assay the effects of different motifs of 513 bp of CK13 gene 5' flanking region on its transcriptional activity. The pCAT enhancer vectors with different motifs of CK13 gene 5' flanking region were constructed and transferred to HeLa cells with the help of lipofectin. The instant CAT expression of different clones was detected and the effects of different motifs of the CK13 gene 5' flanking region on its transcriptional activity were evaluated.

RESULTS119 bp from -nt.325 to -nt.207 upstream of the first ATG of CK13 gene 5' flanking region included a silent element. 113 bp region from -nt.206 to -nt.94 included an enhanced element.

CONCLUSION513 bp of CK13 gene 5' flanking region includes a silent element and an enhanced element. Further locating these cis elements and detecting the related trans reaction factors may unveil some important clues to the details of the mechanisms for the CK13 gene expression and tissue-specific expression.

5' Flanking Region ; genetics ; Base Sequence ; Chloramphenicol O-Acetyltransferase ; genetics ; metabolism ; Enhancer Elements, Genetic ; genetics ; HeLa Cells ; Humans ; Keratins ; genetics ; Molecular Sequence Data ; Recombinant Fusion Proteins ; genetics ; metabolism ; Regulatory Sequences, Nucleic Acid ; genetics ; Transcription, Genetic ; genetics ; Transfection ; methods

BACKGROUNDTo establish a sandwich ELISA method for detection of reporter chloramphenicol acetyltransferase (CAT) gene.

METHODSThe full length sequence of CAT gene was amplified with PCR using plasmid pBLCAT6 as template, and inserted into the prokaryotic expression plasmid Pgex-2T. The purified fusion protein was emulsified with complete or incomplete Freund adjuvant and injected subcutaneously into rabbits. The antibody was labeled with biotin, and a sandwich ELISA technique with biotin streptavidin amplify system was established. Several CAT reporter plasmids containing different HPV 16 LCR sequences were generated and transfected transiently to monolayer cells in vitro. The cytoplasm proteins were extracted and the expressions of CAT were evaluated with the newly established ELISA assay.

RESULTSSDS-PAGE displayed that the molecular weight of the expressed fusion protein was about 54,000. The prepared antiserum was able to recognize the CAT protein expressed by mammalian cells or prokaryote cells. Under the control of different promoters and their regulate sequences,two to eight folds CAT expression increased were evaluated in transiently transfected mammalian cells by the newly established sandwich ELISA method.

CONCLUSIONSThe established method could sensitively reflect the activities of the upstream promoters, as well as the influence of exchanges of nucleotides within the regulate region on the promoter activities. Therefore, it proposes a convenient assay for the studies using CAT as the reporter gene.

Animals ; Antibodies ; analysis ; Cells, Cultured ; Chloramphenicol O-Acetyltransferase ; analysis ; genetics ; immunology ; Enzyme-Linked Immunosorbent Assay ; methods ; Genes, Reporter ; Male ; Papillomaviridae ; genetics ; Plasmids ; genetics ; Rabbits ; Recombinant Fusion Proteins ; analysis ; biosynthesis ; immunology

OBJECTIVETo identify the type of CTGAATCA from -nt.199 to -nt.192 of the cytokeratin 13(CK13) gene 5' flanking region and determine its transcriptional effect on CK13 gene expression.

METHODSThe CAT systems were used to assess the effects of different motifs of CK13 gene 5' flanking region on transcription. The clones of pCAT-enhancer with the total length, -nt.207 to +nt.63 and the same length of -nt.207 to +nt.63, but the T, G of -nt.198, -nt.197 being changed to A, T of the CK13 gene 5' flanking region, were constructed and transferred to HeLa cells with the help of lipofectin. Then work was done to detect the instant CAT expression of different clones and evaluate the effects of CTGAATCA of the 5' flanking region on CK13 gene expression. The type of the cis-element of CTGAATCA was identified with electrophoretic mobility shift assay (EMSA) and competition-EMSA.

RESULTSCTGAATCA in the CK13 gene 5' flanking region is an AP1 cis-element by EMSA and competition-EMSA, it promotes CK13 gene expression.

CONCLUSIONCTGAATCA from -nt.199 to nt.192 of the CK13 gene 5' flanking region is an AP1 reaction element, not a cAMP reaction element. It promotes transcriptional activity of CK13 gene 5' flanking region.

5' Flanking Region ; genetics ; Base Sequence ; Binding Sites ; genetics ; Binding, Competitive ; Chloramphenicol O-Acetyltransferase ; genetics ; metabolism ; DNA ; genetics ; metabolism ; Electrophoretic Mobility Shift Assay ; Gene Expression Regulation ; HeLa Cells ; Humans ; Keratins ; genetics ; Mutation ; Recombinant Fusion Proteins ; genetics ; metabolism ; Transcription Factor AP-1 ; metabolism ; Transcription, Genetic ; genetics ; Transfection

Direct injection of the vascular endothelial growth factor (VEGF) gene plasmid DNA into the myocardium was shown to induce development of new blood vessels to increase the circulation in the heart of patients with coronary artery diseases. However, such angiogenic gene therapy (via naked DNA) was limited by low level of gene expression. Furthermore, the temporal and spatial characteristics of VEGF gene transfer in the heart are not known. In this study, we demonstrated that a plasmid vector, containing the human cytomegalovirus immediate early (HCMV IE) promoter and enhancer, induces greater expression of gene in the rat heart monitored by gene fused to the chloramphenicol acetyl transferase (CAT) reporter, than four different viral and cellular promoters. Interestingly, expression of VEGF121 protein showed an earlier peak, a shorter duration, and a wider distribution than that of CAT only. Therefore, a plasmid vector with an HCMV IE promoter/enhancer provides clear advantages over other previously developed plasmids. Furthermore, expression profile of VEGF121 gene may provide useful information in the design of angiogenic gene therapy in the heart
Animals ; Chloramphenicol O-Acetyltransferase/analysis/genetics ; Comparative Study ; Cytomegalovirus/*genetics ; DNA, Viral/*administration & dosage/*genetics ; Endothelial Growth Factors/analysis/*genetics ; *Enhancer Elements (Genetics) ; Gene Expression Regulation, Viral ; Gene Fusion ; *Gene Transfer Techniques ; Genes, Viral ; Genetic Vectors ; Intercellular Signaling Peptides and Proteins/analysis/*genetics ; Lymphokines/analysis/*genetics ; Male ; Myocardium/*metabolism ; Plasmids/genetics ; *Promoter Regions (Genetics) ; Rats ; Rats, Sprague-Dawley ; Time Factors ; Vascular Endothelial Growth Factor A ; Vascular Endothelial Growth Factors

Direct injection of the vascular endothelial growth factor (VEGF) gene plasmid DNA into the myocardium was shown to induce development of new blood vessels to increase the circulation in the heart of patients with coronary artery diseases. However, such angiogenic gene therapy (via naked DNA) was limited by low level of gene expression. Furthermore, the temporal and spatial characteristics of VEGF gene transfer in the heart are not known. In this study, we demonstrated that a plasmid vector, containing the human cytomegalovirus immediate early (HCMV IE) promoter and enhancer, induces greater expression of gene in the rat heart monitored by gene fused to the chloramphenicol acetyl transferase (CAT) reporter, than four different viral and cellular promoters. Interestingly, expression of VEGF121 protein showed an earlier peak, a shorter duration, and a wider distribution than that of CAT only. Therefore, a plasmid vector with an HCMV IE promoter/enhancer provides clear advantages over other previously developed plasmids. Furthermore, expression profile of VEGF121 gene may provide useful information in the design of angiogenic gene therapy in the heart
Animals ; Chloramphenicol O-Acetyltransferase/analysis/genetics ; Comparative Study ; Cytomegalovirus/*genetics ; DNA, Viral/*administration & dosage/*genetics ; Endothelial Growth Factors/analysis/*genetics ; *Enhancer Elements (Genetics) ; Gene Expression Regulation, Viral ; Gene Fusion ; *Gene Transfer Techniques ; Genes, Viral ; Genetic Vectors ; Intercellular Signaling Peptides and Proteins/analysis/*genetics ; Lymphokines/analysis/*genetics ; Male ; Myocardium/*metabolism ; Plasmids/genetics ; *Promoter Regions (Genetics) ; Rats ; Rats, Sprague-Dawley ; Time Factors ; Vascular Endothelial Growth Factor A ; Vascular Endothelial Growth Factors

OBJECTIVETo screen the 5' regulatory region of the aldose reductase (AR) gene for genetic variabilities causing changes in protein expression and affecting the promoter function.

METHODSThe screenings were carried out by polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP). All SSCP variants were submitted for DNA sequencing and inserted into the plasmid chloromycetin acetyl transferase (CAT) enhancer vector. The constructs were used to transfect Hela cells, and CAT assays were performed to assess promoter activity. Gel mobility shift and footprinting assays were also performed to determine the interaction between the DNA and nuclear proteins.

RESULTSTwo polymorphisms, C (-106) T and C (-12) G, were identified in the regulatory region in 123 Chinese control subjects and 145 patients with type 2 diabetes mellitus. The frequencies of genotypes WT/WT, WT/C (-12) G and WT/C (-106) T were not significantly different between the subjects and patients. In the patients with and without retinopathy, frequencies of WT/C (-106) T were 31.5% and 17.5% (P < 0.05) respectively, and the frequencies of WT/C (-12) G were 10.5% and 2.5% (P > 0.05) respectively. The total frequency of WT/C (-12) G and WT/C (-106) T in patients with retinopathy was 41.8%, significantly higher than that (20.0%) in patients without retinopathy (P < 0.025). The relative transcription activities of the wild-type, the C (-12) G and the C (-106) T were 15.7%, 31.0% and 32.2%, respectively. The results of DNA-protein interaction assays showed that these variations did not change the binding site of DNA with trans-acting factors.

CONCLUSIONThe polymorphisms C (-12) G and C (-106) T strongly associated with diabetic retinopathy in the Chinese population have been identified in the regulatory region of the aldose reductase gene.

5' Flanking Region ; genetics ; Adult ; Aldehyde Reductase ; genetics ; metabolism ; Binding Sites ; genetics ; China ; Chloramphenicol O-Acetyltransferase ; genetics ; metabolism ; DNA ; chemistry ; genetics ; DNA Footprinting ; Diabetes Mellitus, Type 2 ; enzymology ; genetics ; Electrophoretic Mobility Shift Assay ; Female ; HeLa Cells ; Humans ; Male ; Middle Aged ; Mutation ; Polymerase Chain Reaction ; Polymorphism, Genetic ; Polymorphism, Single-Stranded Conformational ; Recombinant Fusion Proteins ; genetics ; metabolism ; Regulatory Sequences, Nucleic Acid ; genetics ; Sequence Analysis, DNA ; Transcription, Genetic