scholarly journals Architecture of the Sporulation-Specific Cdc14 Promoter from the Oomycete Phytophthora infestans

2007 ◽  
Vol 6 (12) ◽  
pp. 2222-2230 ◽  
Author(s):  
Audrey M. V. Ah-Fong ◽  
Qijun Xiang ◽  
Howard S. Judelson
Keyword(s):  
Phytophthora Infestans ◽  
Transcription Initiation ◽  
Start Site ◽  
Mobility Shift ◽  
Specific Expression ◽  
Upstream Site ◽  
Core Promoters ◽  
Nuclear Extracts ◽  
Minor Site ◽  
Electrophoretic Mobility Shift Assays

ABSTRACT The Cdc14 gene of Phytophthora infestans is transcribed specifically during sporulation, with no mRNA detectable in vegetative hyphae, and is required for sporangium development. To unravel the mechanisms regulating its transcription, mutated Cdc14 promoters plus chimeras of selected Cdc14 sequences and a minimal promoter were tested in stable transformants. This revealed that a tandem repeat of three copies of the motif CTYAAC, located between 67 and 90 nucleotides (nt) upstream of the major transcription start site, is sufficient to determine sporulation-specific expression. All three repeats need to be present for activity, suggesting that they bind a transcription factor through a cooperative mechanism. Electrophoretic mobility shift assays indicated that the CTYAAC repeats are specifically bound by a protein in nuclear extracts. Evidence was also obtained for a second region within the promoter that activates Cdc14 transcription during sporulation which does not involve those repeats. The CTYAAC motif also affects the specificity of transcription initiation. Wild-type Cdc14 is transcribed from a major start site and minor site(s) located about 100 nt upstream of the major site. However, stepwise mutations through the CTYAAC triad caused a graded shift to the upstream sites, as did mutating bases surrounding the major start site; transcripts initiated from the upstream site remained sporulation specific. Replacing the Cdc14 initiation region with the Inr-like region of the constitutive Piexo1 gene had no apparent effect on the pattern of transcription. Therefore, this study reports the first motif determining sporulation-induced transcription in oomycetes and helps define oomycete core promoters.

A G-string positive cis-regulatory element in the LpS1 promoter binds two distinct nuclear factors distributed non-uniformly in Lytechinus pictus embryos

Development ◽  
1991 ◽  
Vol 113 (4) ◽  
pp. 1345-1355 ◽  
Author(s):  
M. Xiang ◽  
S.Y. Lu ◽  
M. Musso ◽  
G. Karsenty ◽  
W.H. Klein
Keyword(s):  
Binding Site ◽  
Regulatory Element ◽  
Regulatory Elements ◽  
Gene Promoters ◽  
Mobility Shift ◽  
Specific Expression ◽  
Lytechinus Pictus ◽  
Nuclear Extracts ◽  
Nuclear Factors ◽  
Electrophoretic Mobility Shift Assays

The LpS1 alpha and beta genes of Lytechinus pictus are activated at the late cleavage stage of embryogenesis, with LpS1 mRNAs accumulating only in lineages contributing to aboral ectoderm. We had shown previously that 762 bp of 5' flanking DNA from the LpS1 beta gene was sufficient for proper temporal and aboral ectoderm specific expression. In the present study, we identified a strong positive cis-regulatory element at −70 bp to −75 bp in the LpS1 beta promoter with the sequence (G)6 and a similar, more distal cis-element at −721 bp to −726 bp. The proximal ‘G-string’ element interacted with two nuclear factors, one specific to ectoderm and one to endoderm/mesoderm nuclear extracts, whereas the distal G-string element interacted only with the ectoderm factor. The ectoderm and endoderm/mesoderm G-string factors were distinct based on their migratory behavior in electrophoretic mobility shift assays, binding site specificities, salt optima and EDTA sensitivity. The proximal G-string element shared homology with a binding site for the mammalian transcription factor IF1, a protein that binds to negative cis-regulatory elements in the mouse alpha 1(I) and alpha 2(I) collagen gene promoters. Competition experiments using wild-type and mutant oligonucleotides indicated that the ectoderm G-string factor and IF1 have similar recognition sites. Partially purified IF1 specifically bound to an oligonucleotide containing the proximal G-string of LpS1 beta. From our results, we suggest that the ectoderm G-string factor, a member of the G-rich DNA-binding protein family, activates the LpS1 gene in aboral ectoderm cells by binding to the LpS1 promoter at the proximal G-string site.

The lung-specific CC10 gene is regulated by transcription factors from the AP-1, octamer, and hepatocyte nuclear factor 3 families

1993 ◽  
Vol 13 (7) ◽  
pp. 3860-3871
Author(s):  
P L Sawaya ◽  
B R Stripp ◽  
J A Whitsett ◽  
D S Luse
Keyword(s):  
Transcription Factors ◽  
Rat Liver ◽  
Transcription Initiation ◽  
Clara Cell ◽  
Clara Cells ◽  
Mobility Shift ◽  
Specific Expression ◽  
Fetal Sheep ◽  
Region I

We have shown that a large fragment (-2339 to +57) from the rat CC10 gene directed lung-specific expression of a reporter construct in transgenic animals. Upon transfection, a smaller fragment (-165 to +57) supported reporter gene expression exclusively in the Clara cell-like NCI-H441 cell line, suggesting that a Clara cell-specific transcriptional element resided on this fragment (B. R. Stripp, P. L. Sawaya, D. S. Luse, K. A. Wikenheiser, S. E. Wert, J. A. Huffman, D. L. Lattier, G. Singh, S. L. Katyal, and J. A. Whitsett, J. Biol. Chem. 267:14703-14712, 1992). The interactions of nuclear proteins with a particular segment of the CC10 promoter which extends from 79 to 128 bp upstream of the CC10 transcription initiation site (CC10 region I) have now been studied. This sequence can stimulate both in vitro transcription in H441 nuclear extract and transient expression of reporter constructs in H441 cells. Electrophoretic mobility shift assays using extracts from H441, HeLa, rat liver, and fetal sheep lung cells were used to demonstrate that members of the AP-1, octamer, and HNF-3 families bind to CC10 region I. Transcription factors from H441 cells which are capable of binding to CC10 region I are either absent in HeLa, rat liver, and fetal sheep lung extracts or enriched in H441 extracts relative to extracts from non-Clara cells.

scholarly journals Characterization of the promoter elements required for hepatic and intestinal transcription of the human apoB gene: definition of the DNA-binding site of a tissue-specific transcriptional factor.

1990 ◽  
Vol 10 (6) ◽  
pp. 2653-2659 ◽  
Author(s):  
D Kardassis ◽  
M Hadzopoulou-Cladaras ◽  
D P Ramji ◽  
R Cortese ◽  
V I Zannis ◽  
...  
Keyword(s):  
Binding Site ◽  
Regulatory Elements ◽  
Apob Gene ◽  
Mobility Shift ◽  
Promoter Elements ◽  
Nuclear Extracts ◽  
Dna Binding Site ◽  
Gene Definition ◽  
Definition Of ◽  
Electrophoretic Mobility Shift Assays

The promoter elements important for intestinal and hepatic transcription of the human apoB gene have been localized downstream of nucleotide -150. Footprinting analysis using hepatic nuclear extracts identified four protected regions, -124 to -100, -97 to -93, -86 to -33, and +33 to +52. Gel electrophoretic mobility shift assays showed that multiple factors interact with the apoB sequence -86 to -33, while the region -88 to -61 binds a single nuclear factor. Methylation interference analysis and nucleotide substitution mutagenesis identified the binding site of the factor between residues -78 and -68. Binding competition experiments indicate that this factor recognizes the regulatory elements of other liver-specific genes.

Constitutive activation of LIL-Stat in adult T-cell leukemia cells

Blood ◽  
2000 ◽  
Vol 95 (8) ◽  
pp. 2715-2718 ◽  
Author(s):  
Junichi Tsukada ◽  
Yoko Toda ◽  
Masahiro Misago ◽  
Yoshiya Tanaka ◽  
Philip E. Auron ◽  
...  
Keyword(s):  
T Cell ◽  
Dna Binding ◽  
Leukemic Cells ◽  
T Cell Leukemia ◽  
Cell Leukemia ◽  
Mobility Shift ◽  
Adult T Cell Leukemia ◽  
Nuclear Extracts ◽  
Responsive Element ◽  
Electrophoretic Mobility Shift Assays

Abstract The activation status of a recently identified STAT (signal transducers and activators of transcription) factor, LIL-Stat (lipopolysaccharide [LPS]/IL-1–inducible Stat) in adult T-cell leukemia (ATL) cells was investigated by electrophoretic mobility shift assays using nuclear extracts of leukemic cells from 7 patients with ATL and a GAS (gamma interferon activation site)-like element termed LILRE (LPS/IL-1–responsive element), which is found in the human prointerleukin 1β (IL1B) gene. Spontaneous DNA binding of LIL-Stat was observed in all ATL cells examined. However, in normal human peripheral lymphocytes, DNA binding of LIL-Stat was detected only after stimulation with IL-1. These results demonstrated that LIL-Stat is constitutively activated in ATL cells. Furthermore, our transient transfection studies using LILRE chloramphenicol acetyltransferase (CAT) reporters argue that LIL-Stat in ATL cells functions as a transcriptional activator through binding to the LILRE in theIL1B gene.

Brief hypoxic stress suppresses postbacteremic NF-κB activation and TNF-α bioactivity in perfused liver

2000 ◽  
Vol 279 (1) ◽  
pp. R99-R108 ◽  
Author(s):  
Laura L. Loftis ◽  
Cheryl A. Johanns ◽  
Andrew J. Lechner ◽  
George M. Matuschak
Keyword(s):  
Gene Expression ◽  
Nuclear Translocation ◽  
Nuclear Factor Κb ◽  
Mobility Shift ◽  
Gram Negative ◽  
Nuclear Extracts ◽  
Tnf Α ◽  
Cellular Hypoxia ◽  
Electrophoretic Mobility Shift Assays ◽  
Rat Livers

Reductions in hepatic O2 delivery are common early after gram-negative bacteremic sepsis owing to cardiopulmonary dysfunction and derangements in sinusoidal perfusion. Although gram-negative endotoxin and cellular hypoxia independently enhance activation of nuclear factor-κB (NF-κB) via generation of reactive O2 species (ROS), the combination of these stimuli downregulates hepatic TNF-α gene expression. Here we tested the hypothesis that hypoxic suppression of postbacteremic TNF-α gene expression is transcriptionally mediated by reduced activation of NF-κB. Buffer-perfused rat livers ( n = 52) were studied over 180 min after intraportal infection at t = 0 with 109 live Escherichia coli (EC), serotype O55:B5, or 0.9% NaCl controls under normoxic conditions, compared with 0.5 h of constant-flow hypoxia (Po 2 ∼41 ± 7 Torr) beginning at t = 30 min, followed by 120 min of reoxygenation. In parallel studies, tissue was obtained at peak hypoxia ( t = 60 min). To determine the role of xanthine oxidase (XO)-induced ROS in modulating NF-κB activity after hypoxia/reoxygenation (H/R), livers were pretreated with the XO inhibitor allopurinol, with results confirmed in organs of tungstate-fed animals. Electrophoretic mobility shift assays were performed on nuclear extracts of whole liver lysates using32P-labeled oligonucleotides specific for NF-κB. Compared with normoxic EC controls, hypoxia reduced postbacteremic NF-κB nuclear translocation and TNF-α bioactivity, independent of reoxygenation, tissue levels of reduced glutathione, or posthypoxic O2 consumption. XO inhibition reversed the hypoxic suppression of NF-κB nuclear translocation and ameliorated decreases in cell-associated TNF-α. Thus decreases in hepatic O2delivery reduce postbacteremic nuclear translocation of NF-κB and hepatic TNF-α biosynthesis by signaling mechanisms involving low-level generation of XO-mediated ROS.

scholarly journals A novel cis-acting element controlling the rat CYP2D5 gene and requiring cooperativity between C/EBP beta and an Sp1 factor.

1994 ◽  
Vol 14 (2) ◽  
pp. 1383-1394 ◽  
Author(s):  
Y H Lee ◽  
M Yano ◽  
S Y Liu ◽  
E Matsunaga ◽  
P F Johnson ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Ternary Complex ◽  
Sequence Similarity ◽  
Consensus Sequence ◽  
Regulatory Region ◽  
Mrna Levels ◽  
Binding Studies ◽  
Mobility Shift ◽  
Specific Expression ◽  
Nuclear Extracts

The rat CYP2D5 gene encodes a cytochrome P450 and is expressed in liver cells. Its expression commences a few days after birth, and maximal mRNA levels are achieved when animals reach puberty. Transfection and DNA binding studies were performed to investigate the mechanism controlling developmentally programmed, liver-specific expression of CYP2D5. Transfection studies using a series of CYP2D5 upstream DNA chloramphenicol acetyltransferase gene fusion constructs identified a segment of DNA between nucleotides -55 and -156 that conferred transcriptional activity in HepG2 cells. Activity was markedly increased by cotransfection with a vector expressing C/EBP beta but was unaffected by vectors producing other liver-enriched transcription factors (C/EBP alpha, HNF-1 alpha, and DBP). DNase I footprinting revealed a region protected by both HepG2 and liver cell nuclear extracts between nucleotides -83 and -112. This region displayed some sequence similarity to the Sp1 consensus sequence and was able to bind the Sp1 protein, as assessed by a gel mobility shift assay. The role of Sp1 in CYP2D5 transcription was confirmed by trans activation of the 2D5-CAT construct in Drosophila melanogaster cells by using an Sp1 expression vector. C/EBP beta alone was unable to directly bind the -83 to -112 region of the promoter but was able to produce a ternary complex when combined with HepG2 nuclear extracts or recombinant human Sp1. C/EBP alpha was unable to substitute for C/EBP beta in forming this ternary complex. A poor C/EBP binding site is present adjacent to the Sp1 site, and mutagenesis of this site abolished formation of the ternary complex with the CYP2D5 regulatory region. These result establish that two transcription factors can work in conjunction, possibly by protein-protein interaction, to activate the CYP2D5 gene.

scholarly journals Genomic organization and transcriptional analysis of the human l-glutaminase gene

2003 ◽  
Vol 370 (3) ◽  
pp. 771-784 ◽  
Author(s):  
Cristina PÉREZ-GÓMEZ ◽  
José M. MATÉS ◽  
Pedro M. GÓMEZ-FABRE ◽  
Antonio del CASTILLO-OLIVARES ◽  
Francisco J. ALONSO ◽  
...  
Keyword(s):  
Transcription Initiation ◽  
Genomic Organization ◽  
Core Promoter ◽  
Regulatory Elements ◽  
Initiation Site ◽  
Genome Project ◽  
Transcriptional Analysis ◽  
Cdna Clones ◽  
Mobility Shift ◽  
Specific Expression

In mammals, glutaminase (GA) is expressed in most tissues, but the regulation of organ-specific expression is largely unknown. Therefore, as an essential step towards studying the regulation of GA expression, the human liver-type GA (hLGA) gene has been characterized. LGA genomic sequences were isolated using the genome walking technique. Analysis and comparison of these sequences with two LGA cDNA clones and the Human Genome Project database, allowed the determination of the genomic organization of the LGA gene. The gene has 18 exons and is approx. 18kb long. All exon/intron junction sequences conform to the GT/AG rule. Progressive deletion analysis of LGA promoter—luciferase constructs indicated that the core promoter is located between nt −141 and +410, with several potential regulatory elements: CAAT, GC, TATA-like, Ras-responsive element binding protein and specificity protein 1 (Sp1) sites. The minimal promoter was mapped within +107 and +410, where only an Sp1 binding site is present. Mutation experiments suggested that two CAAT recognition elements near the transcription-initiation site (-138 and −87), play a crucial role for optimal promoter activity. Electrophoretic mobility-shift assays confirmed the importance of CAAT- and TATA-like boxes to enhance basal transcription, and demonstrated that HNF-1 motif is a significant distal element for transcriptional regulation of the hLGA gene.

scholarly journals Gata4 Regulates Testis Expression of Dmrt1

2004 ◽  
Vol 24 (1) ◽  
pp. 377-388 ◽  
Author(s):  
Ning Lei ◽  
Leslie L. Heckert
Keyword(s):  
Transient Transfection ◽  
Mobility Shift ◽  
Specific Expression ◽  
Dnase I Footprinting ◽  
Unknown Factor ◽  
Related Transcription Factor ◽  
Important Regulator ◽  
Functional Relevance ◽  
Electrophoretic Mobility Shift Assays

ABSTRACT The doublesex and mab-3 related transcription factor 1 (Dmrt1) is a putative transcriptional regulator that is expressed exclusively in the gonads and is required for postnatal testis differentiation. Here we describe the transcriptional mechanisms regulating testis-specific expression of the Dmrt1 gene. Transient-transfection analysis identified a region of the promoter between kb −3.2 and −2.8 that is important for Sertoli cell-specific expression. DNase I footprinting revealed four sites of DNA-protein interaction within this region, three of which were prominent in primary Sertoli cells. Analysis of these sites, using electrophoretic mobility shift assays, revealed that Gata4 and another unknown factor bound within these regions. Further transient-transfection assays of various mutant promoters established the functional relevance of the Gata4-response and unknown factor-response elements, while studies of Dmrt1 expression in 13.5 days postcoitum Fog2 null gonads supported the in vivo importance of Gata4's regulation. As a whole, these studies identify Gata4 as an important regulator in the Dmrt1 transcriptional machinery that is responsible for robust expression of Dmrt1 in the testis.

3,5-Diiodo-L-thyronine (T2) has selective thyromimetic effects in vivo and in vitro

1997 ◽  
Vol 19 (2) ◽  
pp. 137-147 ◽  
Author(s):  
SG Ball ◽  
J Sokolov ◽  
WW Chin
Keyword(s):  
Gh3 Cells ◽  
Mrna Levels ◽  
Thyroid Status ◽  
Hormone Activity ◽  
Mobility Shift ◽  
Nuclear Extracts ◽  
Electrophoretic Mobility Shift Assays ◽  
Gh3 Cell

Recent data have suggested that the iodothyronine, 3,5-diiodo-l-thyronine (T2), has selective thyromimetic activity. In vivo, T2 has been shown to suppress TSH levels at doses that do not produce significant peripheral manifestations of thyroid hormone activity. Furthermore, T2 has been shown to produce smaller increments in peripheral indices of thyroid status than does T3, when doses resulting in equivalent suppression of circulating TSH are compared. We have assessed the selective thyromimetic activity of T2 in vivo and in vitro, and performed in vitro studies to assess the potential molecular basis for these selective properties. T2 was 100-fold less potent than T3 in stimulating GH mRNA levels in GH3 cells. In contrast, the iodothyronines were almost equivalent in their ability to downregulate TRbeta2 mRNA levels in this cell line. Both 3,3'-diiodo-L-thyronine and thyronine exhibited no significant thyromimetic effects on either process. In vivo, doses of T2 and T3 that were equivalent in their induction of hepatic malic enzyme (ME) mRNA did not produce equivalent suppression of circulating TSH, with T2 being only 27% as effective as T3. T2 was up to 500-fold less potent than T3 in displacing [125I]-T3 from in vitro translated specific nuclear receptors (TRs) and GH3 cell nuclear extracts. Electrophoretic mobility shift assays, assessing the ability of T2 to produce dissociation of TRbeta1 homodimers from inverted palindrome T3 response elements, indicated that T2 was also 1000-fold less potent than T3 in this respect. These data confirm that T2 has significant thyromimetic activity, and that this activity is selective both in vivo and in vitro. However, there are no data to support a selective central effect, T2 being relatively more potent in stimulating hepatic ME mRNA than in suppression of TSH in vivo. The basis for this differential thyromimetic activity is not selective affinity of the different TR isoforms for T2, or divergent properties of T2 in competitive binding and functional assays in vitro.

Regulation of the Bacillus subtilis mannitol utilization genes: promoter structure and transcriptional activation by the wild-type regulator (MtlR) and its mutants

Microbiology ◽  
2014 ◽  
Vol 160 (1) ◽  
pp. 91-101 ◽  
Author(s):  
Kambiz Morabbi Heravi ◽  
Josef Altenbuchner
Keyword(s):  
Bacillus Subtilis ◽  
Transcriptional Activation ◽  
Transcription Initiation ◽  
Phosphotransferase System ◽  
Mobility Shift ◽  
Dnase I Footprinting ◽  
Proposed Model ◽  
Rna Polymerase Holoenzyme ◽  
Electrophoretic Mobility Shift Assays

Expression of mannitol utilization genes in Bacillus subtilis is directed by P mtlA , the promoter of the mtlAFD operon, and P mtlR , the promoter of the MtlR activator. MtlR contains phosphoenolpyruvate-dependent phosphotransferase system (PTS) regulation domains, called PRDs. The activity of PRD-containing MtlR is mainly regulated by the phosphorylation/dephosphorylation of its PRDII and EIIBGat-like domains. Replacing histidine 342 and cysteine 419 residues, which are the targets of phosphorylation in these two domains, by aspartate and alanine provided MtlR-H342D C419A, which permanently activates P mtlA in vivo. In the mtlR-H342D C419A mutant, P mtlA was active, even when the mtlAFD operon was deleted from the genome. The mtlR-H342D C419A allele was expressed in an Escherichia coli strain lacking enzyme I of the PTS. Electrophoretic mobility shift assays using purified MtlR-H342D C419A showed an interaction between the MtlR double-mutant and the Cy5-labelled P mtlA and P mtlR DNA fragments. These investigations indicate that the activated MtlR functions regardless of the presence of the mannitol-specific transporter (MtlA). This is in contrast to the proposed model in which the sequestration of MtlR by the MtlA transporter is necessary for the activity of MtlR. Additionally, DNase I footprinting, construction of P mtlA -P licB hybrid promoters, as well as increasing the distance between the MtlR operator and the −35 box of P mtlA revealed that the activated MtlR molecules and RNA polymerase holoenzyme likely form a class II type activation complex at P mtlA and P mtlR during transcription initiation.

Sign in / Sign up

Export Citation Format

Share Document