scholarly journals Mutagenesis Analysis of ABCB4 Gene Promoter of Danio rerio

2020 ◽  
Vol 3 (2) ◽  
pp. a44-52
Author(s):  
ZI XUAN YEAW ◽  
LEONARD WHYE KIT LIM ◽  
HUNG HUI CHUNG
Keyword(s):  
Promoter Region ◽  
Gene Expression Regulation ◽  
Consensus Sequence ◽  
Gene Promoter ◽  
Chemotherapeutic Agents ◽  
Site Directed Mutagenesis ◽  
Activator Protein 1 ◽  
Functional Roles ◽  
Abcb4 Gene ◽  
Reverse Primer

Zebrafish abcb4 gene (ortholog to human ABCB1 gene) serves primarily in multidrug resistance (MDR) mechanism by effluxing chemotherapeutic agents, chemicals, xenobiotics, and numerous anti-cancer drugs out of the cells. This study aims to identify the specific transcription factor binding sites (TFBS) within the promoter region of zebrafish abcb4 gene and determine the functional roles of these factors in abcb4 gene expression regulation via mutagenesis analysis. First, primers were designed to target and amplify the promoter region of zebrafish abcb4 gene through gradient PCR. The zebrafish abcb4 gene promoter was then cloned into pGL3.0 vector and sent for sequencing. The sequencing results revealed high similarity to zebrafish DNA sequence from clone DKEY-24I24 in linkage group 16, indicating a successful cloning of targeted gene. Thereafter, consensus sequence of zebrafish abcb4 gene promoter was generated with the length of 1,392 bp which was close to its expected size during primer design (1,500 bp). Using MATCH tool, 155 TFBSs were found within zebrafish abcb4 gene promoter region. Activator protein 1 (AP-1) TFBS at 1,255 bp was chosen to be mutated through site-directed mutagenesis. Mutagenic primers (forward primer: 5’ GGG CAA GGC AGT ATA AAC GTG 3’ and reverse primer: 5’ TTA TGT TTC TAG GGA TTA CGT CAC 3’) were designed to substitute AGT with GGG to remove the AP-1 TFBS. By mutating the zebrafish abcb4 gene promoter, the MDR phenomenon driven by zebrafish abcb4 gene can be elucidated and this might provide clues to the development of tumor and malignancy in human. The results from this study may enrich the knowledge in chemotherapy and cancer treatments.

scholarly journals Characterization of the Autographa californica Nucleopolyhedrovirus Ubiquitin Gene Promoter

2008 ◽  
Vol 63 (3-4) ◽  
pp. 277-283 ◽  
Author(s):  
Xu Ai Lin ◽  
Yin Chen ◽  
Wei Hua Xu ◽  
Yong Zhu Yi ◽  
Zhi Fang Zhang
Keyword(s):  
Promoter Region ◽  
Promoter Activity ◽  
Gene Promoter ◽  
Active Regions ◽  
Autographa Californica ◽  
Negative Regulator ◽  
Site Directed Mutagenesis ◽  
Promoter Regions ◽  
Transient Expression Assay ◽  
Native Promoter

Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) encodes an ubiquitin protein, which may be involved in virus infection. Functional analysis of the AcMNPV ubiquitin promoter was performed by progressive deletion of sequence or mutation of putative cis-activating motifs in the promoter region. In the presence of viral factors, a transient expression assay demonstrated that the active regions responsive to promoter transcription are mainly located within the range of -595 to -382 bp upstream of ATG. A 196-bp fragment (-383 to -187 bp), consisting of the distal TAAG, CAAT motif and TATA box, could also drive the expression of a reporter gene. Site-directed mutagenesis analyses indicated that mutations of TATA boxes and TAAG motifs reduce the promoter activity remarkably, while CAAT mutations enhance the promoter activity by about 3- or 4-fold as compared to the native promoter. All the results suggested that two continuous promoter regions are involved in the transcription of the ubiquitin gene and the cis-activating motifs corresponding to viral factors are mainly present within the 5′ region of the promoter. In addition, CAAT motifs in the promoter region function as negative regulator(s) binding sites

EPIGENETIC ALTERATIONS ASSOCIATED WITH PREMATURE OVARIAN FAILURE

2008 ◽  
Vol 31 (4) ◽  
pp. 11
Author(s):  
Manda Ghahremani ◽  
Courtney W Hannah ◽  
Maria Peneherrera ◽  
Karla L Bretherick ◽  
Margo R Fluker ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Premature Ovarian Failure ◽  
Promoter Region ◽  
Gene Promoter ◽  
Ovarian Failure ◽  
P Value ◽  
Epigenetic Alterations ◽  
Methylation Array ◽  
Cpg Sites ◽  
Deficient Mice

Background/Purpose: Premature ovarian failure (POF) affects 1% of women with a largely idiopathic and poorly understood etiology. The objective of this study was to identify specific epigenetic alterations by measuring DNA methylation of gene regulatory regions in women with POF vs. controls. Methods: Blood samples were collected from idiopathic POFpatients (Amenorrhea for at least 3 months and 2 serum FSH levels of > 40mIU/ml obtained > 1 month apart prior to age 40) and control women (CW) (healthy pregnancy after age 37 with out a pregnancy loss). Genomic DNA was extracted from EDTA anticoagulated blood and bisulfite converted for analysis using the Illumina Golden Gate Methylation Panel which measures DNA methylation at 1506 CpG sites in the promoter regions of 807 genes in 10 POF and 12 CW. Candidate genes with altered epigenetic marks between POF and CW at a nominal P-value < 0.05 were identified using a t-testcomparison within the Illumina bead studio software. Genes of interest were further analyzed for quantitative methylation at specific CpG sites using pyrosequencing in 30 POF and 30 CW. Results: Comparison of DNA methylation profiles of our initial POF and CW groups identified several genes with statistically significanthyper- or hypo- methylation in the POF group (P < 0.05), including the Androgen Receptor (AR)promoter region, which was significantly hypermethylated. To further validate these results, DNA methylation of the AR gene promoter was quantified bypryosequencing in a larger group of POF and CW. Pyrosequencing further confirmed a significantly higher DNA methylation of the AR promoter region inPOF vs. CW (P=0.007). Conclusions: This is a novel study identifying epigenetic alterations in POF. The hypermethylation of the AR gene in POF patients may cause decreased level of the AR in these women. This is especially interesting given a recent report of induced POF in AR deficient mice^1. Specific epigenetic markers, as identified by DNA methylation array profiling in blood, may serve as useful biomarkers for POF and other fertility disorders. However, it will need to be determined if these methylation changes are present prior to diagnosis, or are a consequence of menopause itself. Reference: 1.Hiroko S. et al. Premature ovarian failure in androgenreceptor deficient mice. PNAS;103:224-9

scholarly journals Characterization of the human pleiotrophin gene. Promoter region and chromosomal localization.

1992 ◽  
Vol 267 (36) ◽  
pp. 26011-26016 ◽  
Author(s):  
Y.S. Li ◽  
R.M. Hoffman ◽  
M.M. Le Beau ◽  
R Espinosa ◽  
N.A. Jenkins ◽  
...  
Keyword(s):  
Promoter Region ◽  
Chromosomal Localization ◽  
Gene Promoter ◽  
Gene Promoter Region

scholarly journals Silencing of the Gene for the α-Subunit of Human Chorionic Gonadotropin by the Embryonic Transcription Factor Oct-3/4

1997 ◽  
Vol 11 (11) ◽  
pp. 1651-1658 ◽  
Author(s):  
Limin Liu ◽  
Douglas Leaman ◽  
Michel Villalta ◽  
R. Michael Roberts
Keyword(s):  
Transcription Factor ◽  
Binding Site ◽  
Gene Promoter ◽  
Site Directed Mutagenesis ◽  
Mobility Shift ◽  
Α Subunit ◽  
Choriocarcinoma Cells ◽  
Human Choriocarcinoma Cells ◽  
Electrophoretic Mobility Shift Assays ◽  
Jar Cells

Abstract CG is required for maintenance of the corpus luteum during pregnancy in higher primates. As CG is a heterodimeric molecule, some form of coordinated control must be maintained over the transcription of its two subunit genes. We recently found that expression of human CG β-subunit (hCGβ) in JAr human choriocarcinoma cells was almost completely silenced by the embryonic transcription factor Oct-3/4, which bound to a unique ACAATAATCA octameric sequence in the hCGβ gene promoter. Here we report that Oct-3/4 is also a potent inhibitor of hCG α-subunit (hCGα) expression in JAr cells. Oct-3/4 reduced human GH reporter expression from the −170 hCGα promoter in either the presence or absence of cAMP by about 70% in transient cotransfection assays, but had no effect on expression from either the −148 hCGα or the −99 hCGα promoter. Unexpectedly, no Oct-3/4-binding site was identified within the −170 to −148 region of the hCGα promoter, although one was found around position −115 by both methylation interference footprinting and electrophoretic mobility shift assays. Site-directed mutagenesis of this binding site destroyed the affinity of the promoter for Oct-3/4, but did not affect repression of the promoter. Therefore, inhibition of hCGα gene transcription by Oct-3/4 appears not to involve direct binding of this factor to the site responsible for silencing. When stably transfected into JAr cells, Oct-3/4 reduced the amounts of both endogenous hCGα mRNA and protein by 70–80%. Oct-3/4 is therefore capable of silencing both hCGα and hCGβ gene expression. We suggest that as the trophoblast begins to form, reduction of Oct-3/4 expression permits the coordinated onset of transcription from the hCGα and hCGβ genes.

scholarly journals The Presence and Characterization of a virF Gene on Agrobacterium vitis Ti Plasmids

1998 ◽  
Vol 11 (5) ◽  
pp. 429-433 ◽  
Author(s):  
B. Schrammeijer ◽  
J. Hemelaar ◽  
P. J. J. Hooykaas
Keyword(s):  
Amino Acids ◽  
Agrobacterium Tumefaciens ◽  
Molecular Mass ◽  
Promoter Region ◽  
Consensus Sequence ◽  
Tumor Formation ◽  
Nicotiana Glauca ◽  
Agrobacterium Vitis ◽  
Ti Plasmids

Octopine and nopaline strains of Agrobacterium tumefaciens differ in their ability to induce tumors on Nicotiana glauca. The presence of a virF locus on the octopine Ti plasmid makes N. glauca a host plant for these strains, indicating that the VirF protein is a host-range determinant. Here we show the presence of a virF locus not only on the Agrobacterium vitis octopine/cucumopine plasmids pTiAg57 and pTiTm4, but also on the nopaline Ti plas-mids pTiAT1, pTiAT66a, and pTiAT66b. On the octopine Ti plasmids from A. tumefaciens the virF gene is located between the virE locus and the left border of the T-region. In contrast, the virF gene on Ti plasmids of A. vitis is located at the very left end of the vir-region near the virA locus. The virF gene of pTiAg57 has been sequenced and codes for a protein of 202 amino acids with a molecular mass of 22,280 Da. Comparison showed that the virF gene from A. vitis strain Ag57 is almost identical to that from A. tumefaciens octopine strains. The transcription of the pTiAg57 virF is inducible by the plant phenolic compound acetosyringone through the presence of a vir-box consensus sequence in its promoter region. The VirF protein from pTiAg57 can complement octopine A. tumefaciens strains deleted for virF as shown by tumor formation on N. glauca.

scholarly journals Genetic analysis of the human thymidine kinase gene promoter.

1986 ◽  
Vol 6 (8) ◽  
pp. 2903-2909 ◽  
Author(s):  
J A Kreidberg ◽  
T J Kelly
Keyword(s):  
Thymidine Kinase ◽  
Base Pair ◽  
Promoter Region ◽  
Genomic Sequence ◽  
Gene Promoter ◽  
Thymidine Kinase Gene ◽  
Rich Region ◽  
Kinase Gene ◽  
Inverted Orientation ◽  
Pair Sequence

The promoter of the human thymidine kinase gene was defined by DNA sequence and genetic analyses. Mutant plasmids with deletions extending into the promoter region from both the 5' and 3' directions were constructed. The mutants were tested in a gene transfer system for the ability to transform TK- cells to the TK+ phenotype. This analysis delimited the functional promoter to within an 83-base-pair region upstream of the mRNA cap site. This region contains sequences common to other eucaryotic promoters including G X C-rich hexanucleotides, a CAAT box, and an A X T-rich region. The CAAT box is in an inverted orientation and is part of a 9-base-pair sequence repeated twice in the promoter region. Comparison of the genomic sequence with the cDNA sequence defined the first exon of the thymidine kinase gene.

Genetic analysis of the human thymidine kinase gene promoter

1986 ◽  
Vol 6 (8) ◽  
pp. 2903-2909
Author(s):  
J A Kreidberg ◽  
T J Kelly
Keyword(s):  
Thymidine Kinase ◽  
Base Pair ◽  
Promoter Region ◽  
Genomic Sequence ◽  
Gene Promoter ◽  
Thymidine Kinase Gene ◽  
Rich Region ◽  
Kinase Gene ◽  
Inverted Orientation ◽  
Pair Sequence

The promoter of the human thymidine kinase gene was defined by DNA sequence and genetic analyses. Mutant plasmids with deletions extending into the promoter region from both the 5' and 3' directions were constructed. The mutants were tested in a gene transfer system for the ability to transform TK- cells to the TK+ phenotype. This analysis delimited the functional promoter to within an 83-base-pair region upstream of the mRNA cap site. This region contains sequences common to other eucaryotic promoters including G X C-rich hexanucleotides, a CAAT box, and an A X T-rich region. The CAAT box is in an inverted orientation and is part of a 9-base-pair sequence repeated twice in the promoter region. Comparison of the genomic sequence with the cDNA sequence defined the first exon of the thymidine kinase gene.

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