Mutagenesis Analysis of ABCB8 Gene Promoter of Danio rerio

The ABCB8 is one of the members under the ABCB subfamily of ATP-Binding Cassette (ABC) transporter which possess the ability in regulating the intracellular iron and heme transport. The loss of function mutation of ABCB8 gene leads to iron and heme accumulation in the cell which is highly toxic to human. However, the information regarding the expression regulation of this gene remains scarce. Hence, the objectives of this project are to determine the transcription factors binding site (TFBS) of ABCB8 and to identify the transcriptional roles of the cis-elements through mutagenesis analysis. To examine this, total genomic DNA was extracted from Danio rerio and the promoter sequence was isolated by using specific pair of primers through polymerase chain reaction (PCR). The sample was sent for DNA sequencing and the result showed 98% similarities to the zebrafish DNA sequence from clone DKEYP-87A6 in linkage group 24. Besides, the TFBS was studied in aspect of TFBS abundance, TFBS composition and TFBS distribution. The two most abundant TFBSs based on liver-specific profile were HNF-3β and C/EBPβ, with 38 and 39 binding sites, respectively. The sequence of ABCB8 promoter gene was mutated through substitution of the AP-1 binding site at location 535 with other nucleotides by using a pair of mutagenic primers (forward primer: 5’-TGGGGGTTTAGATATTGAAAC-3’; reverse primer: 5’-AACTCGC ATACATTTCAGTCATC-3’). This result may benefit the development of new diagnostics and therapeutics for iron-associated disorder.

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Arabidopsis thaliana Myb59 Gene Is Involved in the Response to Heterodera schachtii Infestation, and Its Overexpression Disturbs Regular Development of Nematode-Induced Syncytia

International Journal of Molecular Sciences ◽

10.3390/ijms22126450 ◽

2021 ◽

Vol 22 (12) ◽

pp. 6450

Author(s):

Anita Wiśniewska ◽

Kamila Wojszko ◽

Elżbieta Różańska ◽

Klaudia Lenarczyk ◽

Karol Kuczerski ◽

...

Keyword(s):

Cell Metabolism ◽

Gene Promoter ◽

Promoter Sequence ◽

Regulatory Elements ◽

Heterodera Schachtii ◽

Myb Transcription Factor ◽

Parasitic Nematodes ◽

Regulatory Sequences ◽

Gene Encoding ◽

Constitutive Overexpression

Transcription factors are proteins that directly bind to regulatory sequences of genes to modulate and adjust plants’ responses to different stimuli including biotic and abiotic stresses. Sedentary plant parasitic nematodes, such as beet cyst nematode, Heterodera schachtii, have developed molecular tools to reprogram plant cell metabolism via the sophisticated manipulation of genes expression, to allow root invasion and the induction of a sequence of structural and physiological changes in plant tissues, leading to the formation of permanent feeding sites composed of modified plant cells (commonly called a syncytium). Here, we report on the AtMYB59 gene encoding putative MYB transcription factor that is downregulated in syncytia, as confirmed by RT-PCR and a promoter pMyb59::GUS activity assays. The constitutive overexpression of AtMYB59 led to the reduction in A. thaliana susceptibility, as indicated by decreased numbers of developed females, and to the disturbed development of nematode-induced syncytia. In contrast, mutant lines with a silenced expression of AtMYB59 were more susceptible to this parasite. The involvement of ABA in the modulation of AtMYB59 gene transcription appears feasible by several ABA-responsive cis regulatory elements, which were identified in silico in the gene promoter sequence, and experimental assays showed the induction of AtMYB59 transcription after ABA treatment. Based on these results, we suggest that AtMYB59 plays an important role in the successful parasitism of H. schachtii on A. thaliana roots.

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Embryonal Carcinoma P19 Cells Produce Erythropoietin Constitutively But Express Lactate Dehydrogenase in an Oxygen-Dependent Manner

Blood ◽

10.1182/blood.v91.4.1185 ◽

1998 ◽

Vol 91 (4) ◽

pp. 1185-1195 ◽

Cited By ~ 12

Author(s):

Taiho Kambe ◽

Junko Tada ◽

Mariko Chikuma ◽

Seiji Masuda ◽

Masaya Nagao ◽

...

Keyword(s):

Lactate Dehydrogenase ◽

Binding Site ◽

Embryonal Carcinoma ◽

Hypoxia Inducible Factor ◽

Embryonic Stem ◽

Gene Promoter ◽

Dependent Manner ◽

P19 Cells ◽

Independent Manner ◽

Hep3b Cells

Abstract Embryonic stem cells and embryonal carcinoma P19 cells produce erythropoietin (Epo) in an oxygen-independent manner, although lactate dehydrogenase A (LDHA) is hypoxia-inducible. To explore this paradox, we studied the operation of cis-acting sequences from these genes in P19 and Hep3B cells. The Epo gene promoter and 3′ enhancer from P19 cells conveyed hypoxia-inducible responses in Hep3B cells but not in P19 cells. Together with DNA sequencing and the normal transcription start site of P19 Epo gene, this excluded the possibility that the noninducibility of Epo gene in P19 cells was due to mutation in these sequences or unusual initiation of transcription. In contrast, reporter constructs containing LDHA enhancer and promoter were hypoxia inducible in P19 and Hep3B cells, and mutation of a hypoxia- inducible factor 1 (HIF-1) binding site abolished the hypoxic inducibility in both cells, indicating that HIF-1 activation operates normally in P19 cells. Neither forced expression of hepatocyte nuclear factor 4 in P19 cells nor deletion of its binding site from the Epo enhancer was effective in restoring Epo enhancer function. P19 cells may lack an unidentified regulator(s) required for interaction of the Epo enhancer with Epo and LDHA promoters.

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

Molecular Endocrinology ◽

10.1210/mend.11.11.9971 ◽

1997 ◽

Vol 11 (11) ◽

pp. 1651-1658 ◽

Cited By ~ 1

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.

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Characterization of an iron-regulated promoter involved in desferrioxamine B synthesis in Streptomyces pilosus: repressor-binding site and homology to the diphtheria toxin gene promoter.

Journal of Bacteriology ◽

10.1128/jb.175.11.3295-3302.1993 ◽

1993 ◽

Vol 175 (11) ◽

pp. 3295-3302 ◽

Cited By ~ 38

Author(s):

K Günter ◽

C Toupet ◽

T Schupp

Keyword(s):

Binding Site ◽

Diphtheria Toxin ◽

Gene Promoter ◽

Toxin Gene ◽

Desferrioxamine B ◽

Repressor Binding Site

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pGR71 plasmid promotor sequence temporally regulated in Bacillus subtilis

Canadian Journal of Microbiology ◽

10.1139/w98-121 ◽

1998 ◽

Vol 44 (12) ◽

pp. 1186-1192

Author(s):

Guy Daxhelet ◽

Philippe Gilot ◽

Etienne Nyssen ◽

Philippe Hoet

Keyword(s):

Bacillus Subtilis ◽

Binding Site ◽

Transcription Initiation ◽

Promoter Sequence ◽

Initiation Site ◽

Chloramphenicol Acetyltransferase ◽

Ribosome Binding Site ◽

Chloramphenicol Resistance ◽

E Coli ◽

Ribosome Binding

pGR71, a composite of plasmids pUB110 and pBR322, replicates in Escherichia coli and in Bacillus subtilis. It carries the chloramphenicol resistance gene (cat) from Tn9, which is not transcribed in either host by lack of a promoter. The cat gene is preceded by a Shine-Dalgarno sequence functional in E. coli but not in B. subtilis. Deleted pGR71 plasmids were obtained in B. subtilis when cloning foreign viral DNA upstream of this cat sequence, as well as by BAL31 exonuclease deletions extending upstream from the cat into the pUB110 moiety. These mutant plasmids expressed chloramphenicol acetyltransferase (CAT), conferring on B. subtilis resistance to high chloramphenicol concentrations. CAT expression peaked at the early postexponential phase of B. subtilis growth. The transcription initiation site of cat, determined by primer extension, was located downstream of a putative promoter sequence within the pUB110 moiety. N-terminal amino acid sequencing showed that native CAT was produced by these mutant plasmids. The cat ribosome-binding site, functional in E. coli, was repositioned within the pUB110 moiety and had consequently an extended homology with B. subtilis 16S rRNA, explaining the production of native enzyme.Key words: chloramphenicol acetyltransferase, Bacillus subtilis, postexponential gene expression, plasmid pUB110, ribosome-binding site, transcriptional promoter.

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Negative-feedback regulation of FGF signalling by DUSP6/MKP-3 is driven by ERK1/2 and mediated by Ets factor binding to a conserved site within the DUSP6/MKP-3 gene promoter

Biochemical Journal ◽

10.1042/bj20071512 ◽

2008 ◽

Vol 412 (2) ◽

pp. 287-298 ◽

Cited By ~ 122

Author(s):

Maria Ekerot ◽

Marios P. Stavridis ◽

Laurent Delavaine ◽

Michael P. Mitchell ◽

Christopher Staples ◽

...

Keyword(s):

Binding Site ◽

Negative Feedback ◽

Fluorescent Protein ◽

Gene Promoter ◽

Negative Regulator ◽

Mitogen Activated Protein Kinase ◽

Negative Feedback Regulation ◽

Fgf Signalling

DUSP6 (dual-specificity phosphatase 6), also known as MKP-3 [MAPK (mitogen-activated protein kinase) phosphatase-3] specifically inactivates ERK1/2 (extracellular-signal-regulated kinase 1/2) in vitro and in vivo. DUSP6/MKP-3 is inducible by FGF (fibroblast growth factor) signalling and acts as a negative regulator of ERK activity in key and discrete signalling centres that direct outgrowth and patterning in early vertebrate embryos. However, the molecular mechanism by which FGFs induce DUSP6/MKP-3 expression and hence help to set ERK1/2 signalling levels is unknown. In the present study, we demonstrate, using pharmacological inhibitors and analysis of the murine DUSP6/MKP-3 gene promoter, that the ERK pathway is critical for FGF-induced DUSP6/MKP-3 transcription. Furthermore, we show that this response is mediated by a conserved binding site for the Ets (E twenty-six) family of transcriptional regulators and that the Ets2 protein, a known target of ERK signalling, binds to the endogenous DUSP6/MKP-3 promoter. Finally, the murine DUSP6/MKP-3 promoter coupled to EGFP (enhanced green fluorescent protein) recapitulates the specific pattern of endogenous DUSP6/MKP-3 mRNA expression in the chicken neural plate, where its activity depends on FGFR (FGF receptor) and MAPK signalling and an intact Ets-binding site. These findings identify a conserved Ets-factor-dependent mechanism by which ERK signalling activates DUSP6/MKP-3 transcription to deliver ERK1/2-specific negative-feedback control of FGF signalling.

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Brain and muscle creatine kinase genes contain common TA-rich recognition protein-binding regulatory elements.

Molecular and Cellular Biology ◽

10.1128/mcb.10.9.4826 ◽

1990 ◽

Vol 10 (9) ◽

pp. 4826-4836 ◽

Cited By ~ 29

Author(s):

R A Horlick ◽

G M Hobson ◽

J H Patterson ◽

M T Mitchell ◽

P A Benfield

Keyword(s):

Creatine Kinase ◽

Binding Site ◽

Regulatory Element ◽

Gene Promoter ◽

Regulatory Elements ◽

Muscle Creatine Kinase ◽

Enhancer Activity ◽

L6 Myotubes ◽

Recognition Protein ◽

Muscle Creatine

We have previously reported that the rat brain creatine kinase (ckb) gene promoter contains an AT-rich sequence that is a binding site for a protein called TARP (TA-rich recognition protein). This AT-rich segment is a positively acting regulatory element for the ckb promoter. A similar AT-rich DNA segment is found at the 3' end of the 5' muscle-specific enhancer of the rat muscle creatine kinase (ckm) gene and has been shown to be necessary for full muscle-specific enhancer activity. In this report, we show that TARP binds not only to the ckb promoter but also to the AT-rich segment at the 3' end of the muscle-specific ckm enhancer. A second, weaker TARP-binding site was identified in the ckm enhancer and lies at the 5' end of the minimal enhancer segment. TARP was found in both muscle cells (C2 and L6 myotubes) and nonmuscle (HeLa) cells and appeared to be indistinguishable from both sources, as judged by gel retardation and footprinting assays. The TARP-binding sites in the ckm enhancer and the ckb promoter were found to be functionally interchangeable. We propose that TARP is active in both muscle and nonmuscle cells and that it is one of many potential activators that may interact with muscle-specific regulators to determine the myogenic phenotype.

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Chromosome Localization and Structure of the Murine Cyclin G1 Gene Promoter Sequence

Genomics ◽

10.1006/geno.1997.4947 ◽

1997 ◽

Vol 45 (2) ◽

pp. 297-303 ◽

Cited By ~ 8

Author(s):

Michael R. Jensen ◽

Valentina M. Factor ◽

Drazen B. Zimonjic ◽

Mark J. Miller ◽

Catherine L. Keck ◽

...

Keyword(s):

Gene Promoter ◽

Promoter Sequence ◽

Chromosome Localization ◽

Cyclin G1

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Doc2b Ca2+ binding site mutants enhance synaptic release at rest at the expense of sustained synaptic strength

Scientific Reports ◽

10.1038/s41598-019-50684-1 ◽

2019 ◽

Vol 9 (1) ◽

Author(s):

Quentin Bourgeois-Jaarsma ◽

Matthijs Verhage ◽

Alexander J. Groffen

Keyword(s):

Binding Site ◽

Neurotransmitter Release ◽

Action Potentials ◽

Synaptic Strength ◽

Cultured Neurons ◽

Loss Of Function ◽

Short Term ◽

Synaptic Release ◽

Activated State ◽

Primary Cultured Neurons

Abstract Communication between neurons involves presynaptic neurotransmitter release which can be evoked by action potentials or occur spontaneously as a result of stochastic vesicle fusion. The Ca2+-binding double C2 proteins Doc2a and –b were implicated in spontaneous and asynchronous evoked release, but the mechanism remains unclear. Here, we compared wildtype Doc2b with two Ca2+ binding site mutants named DN and 6A, previously classified as gain- and loss-of-function mutants. They carry the substitutions D218,220N or D163,218,220,303,357,359A respectively. We found that both mutants bound phospholipids at low Ca2+ concentrations and were membrane-associated in resting neurons, thus mimicking a Ca2+-activated state. Their overexpression in hippocampal primary cultured neurons had similar effects on spontaneous and evoked release, inducing high mEPSC frequencies and increased short-term depression. Together, these data suggest that the DN and 6A mutants both act as gain-of-function mutants at resting conditions.

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