In excitable and endocrine organs, calcium influxes through the L-type voltage-gated calcium channel (VGCC) which is composed of four (alpha 1, alpha 2, beta, and gamma) subunits. Temporal and spatial expression of calcium channel activity is regulated by the transcription of alpha 1 subunit. To elucidate the genomic organization of the VGCC alpha 1D subunit gene, a genomic clone was isolated from the human genomic library and its sequence was analyzed. A 12 kb genomic clone contained the 5'-flanking regulatory region and first two exons was selected and the initiation site for alpha 1D mRNA synthesis was examined by primer extension analysis. The major initiation site was found at the -523 NT position in the translation initiation site. The TATA box could not be found above the transcription initiation site. The CAT vector construct containing the 2.5 kb upstream region had high CAT activity on transfection to NG108-15 and PC12 cells, which confers the neuronal expression of the alpha 1D gene.
Amino Acid Sequence ; Base Sequence ; Calcium Channels/genetics* ; Chloramphenicol O-Acetyltransferase/genetics ; Cloning, Molecular ; Gene Library ; Genetic Vectors ; Human ; Molecular Sequence Data ; Regulatory Sequences, Nucleic Acid* ; Restriction Mapping

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

American ginseng (AG) has been demonstrated to inhibit breast cancer cell growth in vitro. p21 protein, a universal cell cycle inhibitor, binds cyclin-CDK complexes, an important mechanism in cell cycle regulation. The purpose of this investigation was to determine if AG induces p21 gene expression in hormone sensitive (MCF-7) and insensitive (MDA-MB-231) breast cancer cell lines. Cells grown in steroid stripped medium (SSM) were treated with AG, 17-beta-estradiol (E2), genistein or cycloheximide (CHX). Northern blot analyses were performed using human p21Cip1 and 36B4 cDNA probes. Cell lines were transiently transfected with select mouse p21 CAT reporter constructs, including those lacking a p53 binding site. Cell cycle analyses was performed by FACScan. The results revealed that AG induced p21 mRNA expression in MCF-7 and MDA-MB-231 cells (p=0.0004; p< or =0.0001, respectively). Neither E2 nor genistein alter p21 mRNA expression. CHX, a protein synthesis inhibitor, did not block p21 mRNA expression induced by AG, indicating that p21 is induced as an immediate early gene. AG activated p21 reporter constructs in transfected cells, independent of p53 binding sites. The cell cycle proliferative phase was significantly decreased by AG and increased by E2 (p< or =0.0001). AG may inhibit breast cancer cell growth by transcriptional activation of the p21 gene, independent of p53.
Animal ; Binding Sites ; Breast Neoplasms ; Cell Division/drug effects ; Chloramphenicol O-Acetyltransferase/genetics ; Cyclins/*genetics ; Female ; Genes, Reporter ; HT29 Cells ; Human ; Mice ; *Panax ; Plant Extracts/pharmacology ; Protein p53/metabolism ; *RNA, Messenger ; *Trans-Activation (Genetics) ; Tumor Cells, Cultured

The equilibrium between deposition and degradation of extracellular matrix(ECM) is essential to normal tissue development and repair of wound or inflammatory responses. It has recently become apparent that several cytokines and growth factors are capable of modulating fibroblast proliferation and biosynthetic activity. To understand the role of these factors in connective tissue regulation, we examined the effect of interferon-gamma (IFN-gamma) on stromelysin-1 gene expression in cultured human dermal fibroblasts. The steady-state levels of stromelysin-1 mRNA were increased in IFN-gamma treated cultured dermal fibroblasts. In the CAT assay, the stromelysin-1 promoter activity was increased 2.8-fold compared with untreated control. Therefore IFN-gamma stimulates the stromelysin-1 promoter activity, resulting in transcriptional enhancement of gene expression. Transforming growth factor-beta (TGF-beta) showed the antagonistic action to the effects of IFN-gamma in cultured dermal fibroblasts. Furthermore, gel mobility shift assays demonstrated enhanced AP-1 binding activities in nuclear extracts from cells incubated with IFN-gamma. These data suggest that IFN-gamma is an up-regulator and TGF-beta is a down regulator on the stromelysin-1 gene expression, respectively, and the AP-1 binding site may be necessary for gene response.
Cell Nucleus ; Cells, Cultured ; Chloramphenicol O-Acetyltransferase/metabolism ; Chloramphenicol O-Acetyltransferase/genetics ; Collagenases/genetics ; Collagenases/drug effects ; Fibroblasts/metabolism ; Fibroblasts/drug effects* ; Gene Expression Regulation/drug effects ; Human ; Interferon Type II/pharmacology* ; Promoter Regions (Genetics) ; Recombinant Proteins/metabolism ; Recombinant Proteins/genetics ; Skin/cytology* ; Stromelysin 1/metabolism* ; Stromelysin 1/genetics* ; Stromelysin 1/drug effects ; Transcription Factor AP-1/metabolism ; Transcription, Genetic ; Transforming Growth Factor beta/pharmacology ; Up-Regulation (Physiology)

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 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

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 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

ATP-citrate lyase (ACL), an enzyme catalyzing the first step in biosynthesis of fatty acids, is induced during the lipogenesis and cholesterologenesis. We demonstrate that the region -213 to -128 of human ACL promoter is responsible for conferring glucose-mediated transcription. This region in the ACL promoter contains Sp1 binding sites determined by DNase I foot-printing assay. Gel retardation assay using oligonucleotides from -179 to -141 and -140 to -110 showed two specific DNA-protein complexes postulated to be formed by transcription factor Sp1. Competition gel shift and supershift assays have confirmed that these DNA-protein complexes were the result of induced Sp1 as well as another Sp1-related proteins. Western blot analysis also demonstrated that transcription factor Sp1 was slightly increased in the nuclear proteins extracted from Alexander cells following supplementation of glucose. In addition, expression of 110 kDa protein reacting with antibody against Sp3 was dramatically increased by glucose supplementation, while isoforms of Sp3, about 80 kDa in size was decreased in its amounts. Our results suggest that changes in the expression of Sp1 family proteins play an important role in activation of the ACL promoter by glucose.
ATP Citrate (pro-S)-Lyase/metabolism ; ATP Citrate (pro-S)-Lyase/genetics* ; Binding Sites ; Cells, Cultured ; Chloramphenicol O-Acetyltransferase/genetics ; DNA Footprinting/methods ; Deoxyribonuclease I/metabolism ; Electrophoresis, Polyacrylamide Gel ; Gene Expression Regulation, Enzymologic* ; Glucose/pharmacology ; Glucose/metabolism* ; Human ; Immunoblotting ; Promoter Regions (Genetics)* ; Transcription Factor, Sp1/metabolism* ; Transcription, Genetic* ; Transfection