1.The upstream sequence of Mycobacterium leprae 18-kDa gene confers transcription repression activity in orientation-independent manner.
Experimental & Molecular Medicine 2004;36(6):510-514
In order to understand the role of the upstream region of the Mycobacterium leprae 18-kDa gene on the gene regulation, the region was divided into two at the -50 position from the first start codon of the gene and their effect on transcription was examined by using a LacZ transcriptional reporter gene assay. The presence of each of these two regions conferred transcription repression not only on its cognate M. lepraerae 18-kDa gene promoter, but also on a heterologous promoter such as the Mycobacterium bovis BCG hsp65 gene promoter. Moreover, it was found that these regions could confer transcription repression activity in both cases in an orientation-independent manner. Thus, these results indicate that the upstream region of the M. leprae 18-kDa gene harbors transcription repression responsive element(s) acting as an operator and can be further divided into two separately functional regions, suggesting a bipartite structure of the element(s). The identification of transcription repression activity of the upstream region in the M. leprae 18-kDa gene will contribute greatly for the understanding of the 18-kDa gene regulation mechanism, and provide also useful information for the manipulation of mycobacterium gene expression.
Bacterial Proteins/*genetics
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Down-Regulation/*genetics
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*Gene Expression Regulation, Bacterial
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Mycobacterium leprae/*genetics
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Response Elements/*genetics
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Transcription, Genetic
2.Identification of a mutation in the human raloxifene response element of the transforming growth factor-3 gene.
Ki Ok HAN ; Young Soon KANG ; Chang Sun HWANG ; In Gul MOON ; Chang Hoon YIM ; Ho Yeun CHUNG ; Hak Chul JANG ; Hyun Koo YOON ; In Kwon HAN ; Young Kil CHOI
Journal of Korean Medical Science 2001;16(5):549-552
The human transforming growth factor-3 (TGF-3) is an important cytokine to maintain bone mass by inhibiting osteoclast differentiation. Recently raloxifene response element (RRE), a new enhancer with a polypurine sequence for estrogen receptor (ER)-mediated gene activation, was identified on the TGF-3 gene. Functional analysis of the RRE-mediated pathway has shown that this would be an important pathway for bone preserving effect. We found a novel mutation in the RRE sequence by single-strand conformational polymorphism analysis in one of 200 Korean women. Cloning and sequencing revealed a heterozygote in which one allele had an insertion of 20 nucleotides (AGAGAGGGAGAGGGAGA GGG) between nucleotide +71 and +72 and a point mutation at nucleotide +75 (G-A transition), and the other allele had normal sequence. The insertion was a nearly perfect tandem duplication of the wild type DNA sequence. The bone mineral density of the affected woman was not much lower than that of age-matched controls. Transient transfection of the mutant allele showed no significantly different activity compared with that of the wild type allele. These observations suggest that the heterozygote variation of the RRE sequence seems not to be operative in determination of bone mass.
Estrogen Antagonists/*pharmacology
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Female
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Human
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Middle Age
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*Mutation
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Raloxifene/*pharmacology
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*Response Elements
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Transfection
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Transforming Growth Factor beta/*genetics
3.Establishment of Transgenic cell model based on anti-oxidative response element.
Hairong XU ; Ping BO ; Xiangming LI
Journal of Biomedical Engineering 2010;27(3):631-635
To establish a transgenic cell model based on anti-oxidative response element (ARE) and green fluorescence protein(GFP) reporter gene, the TK minimal promoter was amplified by PCR and cloned into pEGFP-N1 for constructing reporter vector pTK-GFP/Neo. Four synthetic oligonucleotide ARE motifs were annealed and purified and then were inserted into pTK-GFP/Neo one by one to construct the eukaryotic reporter vector p4ARE-TK-GFP/Neo. Two reconstruct eukaryotic reporter vectors were transfected into HepG2 cells mediated by lipofectamine. The positive clones were obtained by the screen of G418. The cell model was tested with PDTC and tBHQ, well known inducers of phase II enzymes, by determining GFP activity. The results showed that the expression level of GFP was significantly increased by PDTC and tBHQ, and a transgenic cell model based on ARE was established successfully.
Antioxidants
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metabolism
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Base Sequence
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Enhancer Elements, Genetic
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genetics
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Genetic Vectors
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genetics
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Green Fluorescent Proteins
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biosynthesis
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genetics
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Hep G2 Cells
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Humans
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Molecular Sequence Data
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Response Elements
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genetics
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Thymidine Kinase
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biosynthesis
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genetics
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Transfection
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Transgenes
4.Killing effect improved by fusion gene HRE1.Egr-1. yCDglyTK on gene-radio therapy of nasopharyngeal cancer in vitro.
Yu ZHONG ; Yao-yun TANG ; Chang-ning XIE ; Su-ping ZHAO
Journal of Central South University(Medical Sciences) 2008;33(2):110-114
OBJECTIVE:
To construct hypoxia/radiation inducible promotor HRE1.Egr-1, and to observe its promotive effect on the expression of yCDglyTK gene in nasopharyngeal cancer HNE-1 cells and the anti-tumor effect of yCDglyTK and to lay an experimental foundation for further exploration of new gene-radio therapy of nasopharyngeal cancer.
METHODS:
pcDNA3.1(-)HRE1.Egr-1.yCDglyTK was constructed by gene recombination technique. Stable yCDglyTK-expressing HNE-1 cells were generated by transfecting the recombinant plasmid into the target cells with liposome. The expression of yCDglyTK was detected by Western blot in 4 groups: a normoxia group, a radiation group, a hypoxia group, and a hypoxia and radiation group. The killing effect of 5-FC in different circumstances was determined by MTT.
RESULTS:
The expression of yCDglyTK/5-FC gene in all the groups was significantly different(P<0.01),especially in the hypoxia and radiation group. The killing effect of 5-FC on HNE1 cells varied under different conditions, especially in the hypoxia and radiation group.
CONCLUSION
Hypoxia and radiation can induce the activity of fusion promoter HRE1.Egr-1, and obviously promote the anti-tumor effect of yCDglyTK/5-FC system, suggesting that yCDglyTK may be a candidate suicide gene for gene-radio therapy of NPC.
Early Growth Response Protein 1
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genetics
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Flucytosine
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pharmacology
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Gene Fusion
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physiology
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Genetic Therapy
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methods
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Humans
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Hypoxia-Inducible Factor 1, alpha Subunit
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genetics
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Nasopharyngeal Neoplasms
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genetics
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radiotherapy
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therapy
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Response Elements
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genetics
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Thymidine Kinase
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genetics
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metabolism
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Tumor Cells, Cultured
5.Identification of a highly conserved domain that suppresses the DNA-binding domain-DNA interactions in the androgen receptor.
Guo-zhen LIU ; Hua WANG ; Zheng-xing WANG
Journal of Central South University(Medical Sciences) 2005;30(4):394-398
OBJECTIVE:
To determine the gene regulation of androgen receptor (AR).
METHODS:
Gel shift assay, protein-protein pull down assay, western blot and technique of site-directed mutagenesis were used to study the gene regulation of AR.
RESULTS:
The N terminal of AR contained an inhibitory domain located in an 81-amino acid segment lying upstream of the DNA-binding domain (DBD). The inhibitory domain interacted directly with DBD and repressed DBD binding to the ARE. Mutations of the conserved amino acid residues (K520E and R538E) within the inhibitory domain decreased its inhibiting ability in vitro and increased AR trans-activation in vivo.
CONCLUSION
These data demonstrated the existence of a novel inhibitory domain in the N-terminal part of AR, which might play important role in the regulation of AR trans-activation.
Adult
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DNA
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metabolism
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DNA-Binding Proteins
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chemistry
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genetics
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Gene Expression Regulation
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Humans
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Male
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Mutation
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Receptors, Androgen
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chemistry
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genetics
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Response Elements
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Transcriptional Activation
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Transfection
6.Cloning and expression of androgen response elements of prostate specific antigen promoter.
Yu HU ; Xue-jun SHANG ; Jing-ping GE ; Yi SUN ; Yu-feng HUANG
National Journal of Andrology 2005;11(12):930-932
OBJECTIVETo provide a possible targeted gene therapy scheme for prostate cancer, and explore the expression efficiency and tissue-specific expression of prostate specific antigen (PSA) promoter.
METHODSThree plasmids with egfp, pa-EGFP(including ARI, ARII), pba-EGFP (including ARI, ARII, ARIII) and pdeltaba-EGFP (including ARI, ARII and mutated ARIII) were designed, and the expression status was observed by transfecting into HepG2, SMMC-7721, Hela and PC-3.
RESULTSIn prostate cancer cell PC-3, pba-EGFP expressed more GFP than pa-EGFP and pdeltaba-EGFP, which showed that ARIII could notably increase the transcription efficiency of PSA promoter. Further, there was no GFP expression in HepG2, SMMC-7721 and Hela transfected with pa-EGFP, p deltaba-EGFP and pba-EGFP.
CONCLUSIONAn expression vector based on elements of the PSA gene regulatory sequences has been developed and shown to be tightly regulated in a panel of cells from tissues of various origins. With the tissue-specific functional protein, it should provide a solid platform for clinical studies.
Androgens ; metabolism ; Cell Line, Tumor ; Cloning, Molecular ; Humans ; Male ; Promoter Regions, Genetic ; Prostate ; metabolism ; Prostate-Specific Antigen ; biosynthesis ; genetics ; Response Elements ; Transfection
7.Identification of a NFB responsive element within the dimethylarginine dimethylaminohydrolase 2 gene promoter.
Yinghui LI ; Jiaqi LI ; Lu SUN ; Guoming CHU ; Yanyan ZHAO
Chinese Journal of Medical Genetics 2014;31(3):322-326
OBJECTIVESTo identify a NFB responsive element within the dimethylarginine dimethylaminohydrolase 2 gene (DDAH) promoter and demonstrate its role in DDAH2 transactivation.
METHODSDDAH2 promoter was analyzed with software to identify potential binding sites of transcription factors. A series of truncated DDAH2 promoter luciferase reporter plasmids were constructed and transfected into human embryonic kidney derived HEK293 cells. Luciferase assays were carried out to analyze the activity of the promoter. Electrophoresis mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP) were used to identify the NFB responsive element in vitro and in vivo. DDAH2 promoter luciferase reporter plasmid with mutated NFB site was constructed and transfected into cells, and its activity was compared with that of the wild-type plasmid.
RESULTSPotential bindings sites of many transcription factors were found within the DDAH2 promoter. The transcription activity of the DDAH2 promoter was high, and -530 to -437 was a positive regulating region. -476 to -469 of the DDAH2 promoter was a NFB responsive element, to which NFB can specifically bind. Mutation of the NFB element could significantly decrease the DDAH2 promoter activity.
CONCLUSION-476 to -469 of the DDAH2 promoter was a NFB responsive element and is important for the transactivation of DDAH2.
Amidohydrolases ; genetics ; metabolism ; Base Sequence ; Gene Expression Regulation, Enzymologic ; Humans ; Molecular Sequence Data ; NF-kappa B ; metabolism ; Promoter Regions, Genetic ; Protein Binding ; Response Elements
8.Role of the interferon-stimulated response elements I/II in expression regulation of the retinoic acid induced gene G.
Ye-jiang LOU ; Xiao-rong PAN ; Pei-min JIA ; Zhang-lin ZHANG ; Gui-ping XU ; Dong LI ; Jian-hua TONG
Chinese Journal of Medical Genetics 2010;27(3):255-258
OBJECTIVETo study the regulatory role of interferon-stimulated response elements (ISREs) located on the retinoic acid-induced gene G (RIG-G) promoter in RIG-G expression.
METHODSBy using point mutation technique, the authors constructed the wide type and site mutant reporter gene plasmids according to the ISRE sequence on RIG-G promoter, and detected the functional activities by luciferase reporter assay.
RESULTSMutation in ISRE II alone had no obvious effect on the expression of the reporter gene, whereas mutation in ISRE I dramatically inhibited the transactivity of RIG-G promoter. Mutation in both ISRE I and ISRE II resulted in complete loss of its response to the transcription factors for the reporter gene.
CONCLUSIONBoth ISRE I and ISRE II on the RIG-G promoter are the binding sites for the complex of transcription factors. They are required for RIG-G expression, and ISRE I has a preferential role over ISRE II.
Cell Line, Tumor ; Humans ; Interferon Regulatory Factor-1 ; genetics ; metabolism ; Interferons ; genetics ; metabolism ; Intracellular Signaling Peptides and Proteins ; genetics ; Mutation ; Promoter Regions, Genetic ; genetics ; Response Elements ; genetics ; STAT2 Transcription Factor ; genetics ; metabolism
9.Establishment of a reporter gene-based cell screening model for discovering new agonists of estrogen receptor beta subtype.
Li-min CHEN ; Qiu-jun LÜ ; Inoue SATOSHI ; Guang-xing BIAN ; Zhen-hua CHEN ; Li-qing WEN
Acta Pharmaceutica Sinica 2006;41(8):721-726
AIMTo establish a sensitive and efficient reporter gene-based screening model for finding agonists of estrogen receptor beta subtype.
METHODSA recombinant vector pTAL-ERE-SEAP was constructed by inserting a synthetic sequence composed of five estrogen responsive elements in front of promoter of pTAL-SEAP vector. pTAL-ERE-SEAP was then transfected into human embryonic kidney (HEK293) cells. G418 (200 microg x mL(-1)) was added to select positive clones that can be induced by E2 to express reporter gene SEAP. The speciality was tested by several ligands of relative nuclear receptors of the same family. The stability of the model, the time-effect relationship, the dose-response relationship, and the immunocytochemistry staining of ERbeta expression after transfection were observed. 2 622 compounds were screened by using this model.
RESULTSStably transfected clones were obtained. The expression levels of reporter gene SEAP of positive clones was induced by E2 in a dose-response and time-effect relationship manners. The Z' factor value was 0.7. The expression levels of dexamethasone and other ligands were low. The result of immunocytochemistry staining showed the expression of ERbeta. E2 had no proliferating effects on stably transfected clones.
CONCLUSIONStably transfected positive clones transfected with recombinant vector pTAL-ERE-SEAP were obtained. The positive clones may be used to screen for agonists of estrogen receptor beta subtype by measurement of luminescent value of expressed SEAP in wells of microlitre plate.
Alkaline Phosphatase ; genetics ; metabolism ; Cell Line ; Estradiol ; pharmacology ; Estrogen Receptor beta ; agonists ; genetics ; metabolism ; Gene Expression Regulation ; drug effects ; Genetic Vectors ; Humans ; Immunohistochemistry ; Promoter Regions, Genetic ; genetics ; Recombinant Fusion Proteins ; genetics ; metabolism ; Response Elements ; genetics ; Transfection
10.Down-regulation of Nrf2-ARE signaling pathway.
Xiu-jun WANG ; Jia-guo WU ; Xiu-wen TANG
Journal of Zhejiang University. Medical sciences 2010;39(1):1-5
Antioxidants
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metabolism
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pharmacology
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Down-Regulation
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Gene Expression Regulation
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Humans
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NF-E2-Related Factor 2
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genetics
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metabolism
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physiology
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Neoplasms
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genetics
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metabolism
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Oxidative Stress
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genetics
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physiology
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Response Elements
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physiology
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Signal Transduction
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physiology