Objective To detect the single nucleotide polymorphisms ( SNPs) in the upstream promoter region of chemokine like factor ( CKLF) gene and analyze their possible associations with asthma and asthma-related phenotypes. Methods Direct Sequence of the 1553bp upstream promoter region of CKLF gene was performed in 245 Chinese Han human genomic DNAs ( 119 asthmatics and 126 controls) .The frequencies of alleles, genotypes, and haplotypes were determined and the association of these SNPs with asthma were further analyzed. Results Four novel SNPs, SNP88 ( T gt; C) , SNP196 ( T gt; C) , SNP568 ( C gt;G) , and SNP1047 ( C gt; G) were found in the promoter region of CKLF. The frequency of rare allele was 0. 168 ( SNP88C) , 0. 168 ( SNP196C) , 0. 352 ( SNP568G) and 0. 167 ( SNP1047G) , respectively.Haplotypes, their frequencies and the linkage disequilibrium coefficients between SNPs were constructed.Complete linkage disequilibrium( LDs) were observed between SNP88 and SNP196, SNP88 and SNP1047,as well as SNP196 and SNP1047, respectively ( D′=1. 000, r2 = 1. 000) . SNP568 was in partial LD with the other three SNPs ( r2 = 0. 366) . No association between asthma and the SNPs was observed. Conclusions Four SNPs in the regulatory region of CKLF in Chinese Han population were firstly identified. Although no significant correlation with asthma was revealed, the SNP and haplotype information is useful for other disease association studies in the future.
OBJECTIVE: To investigate the effects of basic fibroblast growth factor (bFGF) on the promoter activities of human alpha 1(I) procollagen gene and the interaction between bFGF and transforming growth factor-beta 1 (TGF-beta 1). METHODS: Fibroblasts of the hypertrophic scar and normal skin from a 3-year-old patient were primarily cultured and subcultured in vitro. Both of the fibroblasts were transient transfected with phCOL 2.5, containing -2.5 kb of 5’f lank sequence of human alpha 1(I) procollagen gene and CAT reporter gene by FuGENE transfection reagent; and treated thereafter by 16 ng/ml bFGF, 2 ng/ml TGF-beta 1 and 16 ng/ml bFGF + 2 ng/ml TGF beta 1 for 24 hours. The relative CAT expression values were determined by CAT-ELISA. RESULTS: TGF-beta 1 bly induced the CAT expression level, however, bFGF not only inhibited the basal CAT expression but also reduced the CAT expression up-regulated by TGF-beta 1 in normal skin and hypertrophic scar fibroblasts (P lt; 0.05). CONCLUSION: bFGF can reduce the promoter activities of human alpha 1(I) procollagen gene and antagonize the role of TGF-beta 1 in up-regulating the promoter activities of human alpha 1(I) procollagen gene in normal skin and hyertrophic scar fibroblasts.
ObjectiveTo construct a luciferase reporter fusion containing the human connective tissue growth factor (CTGF) gene promoter.MethodsThe promoter region of the human CTGF gene (-835/+214) was amplified by polymerase chain reaction (PCR) using specially-designed primers, and subsequently cloned into the pGL3.0-Basic vector. Following screening and verification by single colony PCR, double digestion, and sequencing, the resulting pGL3.0-Basic-CTGF was used to transfect the human embryonic kidney cells 293T, human bronchial epithelial cells HBE and human lung epithelial cells A549, and its function in each cell line was determined by luciferase assay.ResultsSequence alignment showed 99.5% identity, suggesting successful construction of the pGL3.0-Basic-CTGF reporter fusion. Promoter activities were detected 48 hours after transfection of pGL3.0-Basic-CTGF into the 293T, HBE, and A549 cells, and the promoter activities were 2.416, 0.027, and 0.121, respectively (P<0.01). Moreover, the luciferase activity in the A549 cells was statistically higher than that in the HBE cells (P<0.01).ConclusionsThe human pGL3.0-Basic-CTGF luciferase reporter fusion has been successfully constructed. The construct exhibits promoter activity in the bronchial epithelial cells HBE and the lung epithelial cells A549, and can therefore serve as a useful tool for future research in transcriptional regulation.
ObjectiveTo lay a theoretical foundation for the research of regulation of Hyperpolarization activated cyclic nucleotide gated channel 1 (HCN1) gene expression and its involvement in the pathogenesis of Mesio-temporal lobe epilepsy (MTLE) and other related diseases, the bioinformatics methods were used to analyze sequence characteristic, transcription factors and their binding sites in the promoter region of human HCN1 gene, and the physicochemical properties, signal peptides, hydrophobicity, transmembrane regions, protein structure, interacting proteins and functions of HCN1 proteins.MethodBiological software and website, such as Protparam, Protscale, MHMM, SignalP 5.0, NetPhos 3.1, Swiss-Model, Promoter 2.0, AliBaba2.1 and EMBOSS were used to analyze and predict physicochemical properties, structural functions, localized expression, phylogenetic relationships and protein interactions with human HCN1 protein, and promoter, CpG island and transcription factor characteristics of HCN1 gene.ResultsThe evolutionary analysis of HCN1 protein showed that the genetic distance between human and Pongo abelii was the smallest, indicating the closest genetic relationship between human and Pongo abelii. Human HCN1 protein was an unstable hydrophilic protein located on the plasma membrane, which contained two transmembrane structure. However, the predicted results showed that there was no signal peptide and nuclear localization sequence in this protein. The secondary structure of HCN1 protein was mostly random coil and alpha helix, and it contained multiple potential phosphorylation sites. The ontology analysis results of HCN1 protein were showed as follows. The cellular component of HCN1 protein was located in the plasma membrane (GO:0005886); the molecular functionof HCN1 protein were cyclic adenosine monophosphate binding (GO:0030552) and voltage-gated ion channel activity (GO:0005244); the biological process of this protein were reacting to cAMP (GO:0071320) and transmembrane transport of potassium (GO:0071805). The analysis results of String database showed that the proteins that had close interaction with human HCN1 protein mainly included the ten proteins (HCN2, HCN4, PEX5L, MARCH7, KCTD3, GNAT3, SHKBP1, KCNQ2, FLNA and NEDD4L). These proteins were mainly involved in regulation of ion transport and transmembrane transport of potassium (GO:0071805). The HCN1 gene was located at 5p12 and contained 8 exons and 7 introns.There were at least three promoter regions in the nucleotide sequence of 2 000 bp from the upstream of the HCN1 gene to the 5 'flanks, and contained a 158 bp CpG island in the promoter region and one TATA boxes and one CAAT boxes in the 5' regulation region ofHCN1 gene; niceteen transcription factors, including NF-κB, NF-1, AP-1, TBP, IRF-1, c-Ets-1, Elf-1, HNF-3, HNF-1, YY1, GATA-1, RXR-α, GR, AP-2αA, ENKTF-1, C/EBPβ, C/EBPα, c-Fos and c-Jun, binding in the promoter region of the HCN1 gene were predicted by both softwares (AliBaba2.1 and PROMO2).ConclusionThe analysis results provide important information for further studies on the role of HCN1. Bioinformatics analysis of the promoter region can improve the research efficiency of gene promoters, and provide theoretical basis for subsequent experiments to construct expression vectors of HCN1 gene promoters and identify their functions.