scholarly journals Repression of sigK Intervening (skin) Element Gene Expression by the CI-Like Protein SknR and Effect of SknR Depletion on Growth of Bacillus subtilis Cells

2010 ◽  
Vol 192 (23) ◽  
pp. 6209-6216 ◽  
Author(s):  
Tatsu Kimura ◽  
Yukie Amaya ◽  
Kazuo Kobayashi ◽  
Naotake Ogasawara ◽  
Tsutomu Sato
Keyword(s):  
Gene Expression ◽  
Bacillus Subtilis ◽  
Direct Interaction ◽  
Lethal Effect ◽  
Negative Regulator ◽  
Growth Defect ◽  
Mobility Shift ◽  
Gene Encoding ◽  
Initiation Of Replication ◽  
Mobility Shift Assay

ABSTRACT The Bacillus subtilis phage DNA-like sigK intervening (skin) element (48 kb) is excised from the chromosome by DNA rearrangement, and a composite gene, sigK (spoIIIC and spoIVCB), is created on the chromosome during sporulation. In this study, we first focused on the role of sknR (skin repressor), which has homology with the gene encoding the Xre repressor of defective phage PBSX. The depletion of SknR caused overexpression of the region between yqaF and yqaN (the yqaF-yqaN operon) and a growth defect in B. subtilis. Point mutation analysis and an electrophoretic mobility shift assay (EMSA) suggested that SknR functions as a negative regulator of gene expression in the yqaF-yqaN operon of the skin element through direct interaction with operators of 2-fold symmetry located in the intergenic region between sknR and yqaF. Deletion analysis revealed that the lethal effect of depletion of SknR was related to overexpression of yqaH and yqaM, whose products were previously reported to associate with DnaA and DnaC, respectively. Furthermore, overexpression of either yqaH or yqaM caused cell filamentation and abnormal chromosome segregation, which suggested that overproduction of these proteins inhibits DNA replication. Moreover, overexpression of yqaM inhibited the initiation of replication. Taken together, these data demonstrate that the B. subtilis skin element carries lethal genes, which are induced by the depletion of sknR.

scholarly journals FoxO1 Is a Negative Regulator of FSHβ Gene Expression in Basal and GnRH-Stimulated Conditions in Female

Endocrinology ◽  
2014 ◽  
Vol 155 (6) ◽  
pp. 2277-2286 ◽  
Author(s):  
Young-Suk Choi ◽  
Hyeon Jeong Lee ◽  
Cheol Ryong Ku ◽  
Yoon Hee Cho ◽  
Mi Ran Seo ◽  
...  
Keyword(s):  
Gene Expression ◽  
Negative Regulator ◽  
Pituitary Cells ◽  
Mobility Shift ◽  
Knockout Mouse Model ◽  
Forkhead Box ◽  
Direct Binding ◽  
Mobility Shift Assay ◽  
Phosphoinositide 3 Kinase ◽  
Kinase Pathway

The importance of forkhead box class O (FoxO) proteins in reproductive endocrinology has been confirmed by age-dependent infertility in females in a FoxO3a-knockout mouse model. In this study, FoxO1 was detected in gonadotropes in the anterior pituitary. Overexpression of FoxO1 in primary pituitary cells decreased FSHβ gene expression in both basal and GnRH-stimulated conditions, and this result was replicated by the human FSHβ promoter activity. Although direct binding of FoxO1 to FoxO-binding element (FBE) (at −124 to −119 bp of the human FSHβ promoter) was not detected in an electrophoretic mobility shift assay, a DNA pull-down assay and transfection study using the mutant FBE reporter vector revealed that FBE is necessary in FSHβ suppression by FoxO1, suggestive of other cofactor requirements. GnRH stimulated the phosphoinositide 3-kinase pathway, which induced posttranslational modification of FoxO1 and retained it in the cytoplasm. We also confirmed this result in primary cell cultures; most of the FoxO1 was detected in the cytoplasm when treated with GnRH but in the nucleus when the phosphoinositide 3-kinase pathway was inhibited. These findings suggest that FoxO1 is regulated by the GnRH signaling pathway and functions as a negative regulator of FSHβ gene expression.

scholarly journals CodY Is a Nutritional Repressor of Flagellar Gene Expression in Bacillus subtilis

2003 ◽  
Vol 185 (10) ◽  
pp. 3118-3126 ◽  
Author(s):  
F. Bergara ◽  
C. Ibarra ◽  
J. Iwamasa ◽  
J. C. Patarroyo ◽  
R. Aguilera ◽  
...  
Keyword(s):  
Gene Expression ◽  
Amino Acids ◽  
Bacillus Subtilis ◽  
Promoter Region ◽  
Stringent Response ◽  
Mobility Shift ◽  
Flagellin Gene ◽  
New Members ◽  
Environment Effects ◽  
Mobility Shift Assay

ABSTRACT Expression of the σD-dependent flagellin gene, hag, is repressed by the CodY protein in nutrient-rich environments. Analysis of a codY mutant bearing a hag-lacZ reporter suggests that the availability of amino acids in the environment is the specific signal that triggers this repression. Further, hag-lacZ expression appears to be sensitive to intracellular GTP levels, as demonstrated by increased expression upon addition of decoyinine. This result is consistent with the postulate that the availability of amino acids in the environment effects intracellular GTP levels through the stringent response. However, the levels of hag-lacZ measured upon the addition of subsets of amino acids suggest an additional mechanism(s). CodY is a DNA binding protein that could repress flagellin expression directly by binding to the hag promoter region, or indirectly by binding to the fla/che promoter region that governs expression of the σD transcriptional activator required for hag gene expression. Using an electrophoretic mobility shift assay, we have demonstrated that purified CodY protein binds specifically to both the hag and fla/che promoter fragments. Additionally, CodY acts as a nutritional repressor of transcription from the fla/che promoter region that contains two functional promoters. CodY binds to both the σD- and σA-dependent promoters in this region, as demonstrated by DNase I footprint analyses. Footprint analyses of the hag gene demonstrated that CodY binds downstream of its σD-dependent promoter. Taken together, these results identify new members of the CodY regulon that encode motility functions in Bacillus subtilis and are controlled by the σD alternate sigma factor.

scholarly journals A CpxR-Regulated zapD Gene Involved in Biofilm Formation of Uropathogenic Proteus mirabilis

2020 ◽  
Vol 88 (7) ◽  
Author(s):  
Hong-Han Chen ◽  
Chien-Che Chang ◽  
Yu-Han Yuan ◽  
Shwu-Jen Liaw
Keyword(s):  
Gene Expression ◽  
Proteus Mirabilis ◽  
Biofilm Formation ◽  
Extracellular Dna ◽  
Mobility Shift ◽  
Gene Encoding ◽  
Content Type ◽  
Mobility Shift Assay ◽  
Regulated Gene Expression

ABSTRACT Proteus mirabilis, a frequent uropathogen, forms extensive biofilms on catheters that are infamously difficult to treat. To explore the mechanisms of biofilm formation by P. mirabilis, we performed in vivo transposon mutagenesis. A mutant with impaired biofilm formation was isolated. The mutant was found to have Tn5 inserted in the zapD gene, encoding an outer membrane protein of the putative type 1 secretion system ZapBCD. zapBCD and its upstream zapA gene, encoding a protease, constitute an operon under the control of CpxR, a two-component regulator. The cpxR mutant and zapA mutant strains also had a biofilm-forming defect. CpxR positively regulates the promoter activities of zapABCD, cpxP, and cpxR. An electrophoretic mobility shift assay revealed that CpxR binds zapA promoter DNA. The loss of zapD reduced CpxR-regulated gene expression of cpxR, zapA, cpxP, and mrpA, the mannose-resistant Proteus-like (MR/P) fimbrial major subunit gene. The restoration of biofilm formation in the zapD mutant with a CpxR-expressing plasmid reinforces the idea that CpxR-mediated gene expression contributes to zapD-involved biofilm formation. In trans expression of zapBCD from a zapBCD-expressing plasmid also reestablished the biofilm formation ability of the cpxR mutant to a certain level. The zapD and cpxR mutants had significantly lower protease activity, adhesion, and autoaggregation ability and production of exopolysaccharides and extracellular DNA (eDNA) than did the wild type. Finally, we identified copper as a signal for CpxR to increase biofilm formation. The loss of cpxR or zapD abolished the copper-mediated biofilm upshift. CpxR was required for copper-induced expression of zapA and cpxR. Taken together, these data highlight the important role of CpxR-regulated zapD in biofilm formation and the underlying mechanisms in P. mirabilis.

Identification of a killer cell-specific regulatory element of the mouse perforin gene: an Ets-binding site-homologous motif that interacts with Ets-related proteins

1993 ◽  
Vol 13 (11) ◽  
pp. 6690-6701
Author(s):  
H Koizumi ◽  
M F Horta ◽  
B S Youn ◽  
K C Fu ◽  
B S Kwon ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dna Binding ◽  
Binding Site ◽  
Protein Interactions ◽  
Regulatory Element ◽  
Killer Cell ◽  
Mobility Shift ◽  
Specific Expression ◽  
Native Molecular Mass ◽  
Mobility Shift Assay

The gene encoding the cytolytic protein perforin is selectively expressed by activated killer lymphocytes. To understand the mechanisms underlying the cell-type-specific expression of this gene, we have characterized the regulatory functions and the DNA-protein interactions of the 5'-flanking region of the mouse perforin gene (Pfp). A region extending from residues +62 through -141, which possesses the essential promoter activity, and regions further upstream, which are able to either enhance or suppress gene expression, were identified. The region between residues -411 and -566 was chosen for further characterization, since it contains an enhancer-like activity. We have identified a 32-mer sequence (residues -491 to -522) which appeared to be capable of enhancing gene expression in a killer cell-specific manner. Within this segment, a 9-mer motif (5'-ACAGGAAGT-3', residues -505 to -497; designated NF-P motif), which is highly homologous to the Ets proto-oncoprotein-binding site, was found to interact with two proteins, NF-P1 and NF-P2. NF-P2 appears to be induced by reagents known to up-regulate the perforin message level and is present exclusively in killer cells. Electrophoretic mobility shift assay and UV cross-linking experiments revealed that NF-P1 and NF-P2 may possess common DNA-binding subunits. However, the larger native molecular mass of NF-P1 suggests that NF-P1 contains an additional non-DNA-binding subunit(s). In view of the homology between the NF-P motif and other Ets proto-oncoprotein-binding sites, it is postulated that NF-P1 and NF-P2 belong to the Ets protein family. Results obtained from the binding competition assay, nevertheless, suggest that NF-P1 and NF-P2 are related to but distinct from Ets proteins, e.g., Ets-1, Ets-2, and NF-AT/Elf-1, known to be expressed in T cells.

scholarly journals PgMYB2, a MeJA-Responsive Transcription Factor, Positively Regulates the Dammarenediol Synthase Gene Expression in Panax Ginseng

2019 ◽  
Vol 20 (9) ◽  
pp. 2219 ◽  
Author(s):  
Tuo Liu ◽  
Tiao Luo ◽  
Xiangqian Guo ◽  
Xian Zou ◽  
Donghua Zhou ◽  
...  
Keyword(s):  
Gene Expression ◽  
Transcription Factor ◽  
Panax Ginseng ◽  
Growth Regulation ◽  
Ginseng Root ◽  
Myb Transcription Factor ◽  
Mobility Shift ◽  
Binding Interaction ◽  
Mobility Shift Assay ◽  
Dammarenediol Synthase

The MYB transcription factor family members have been reported to play different roles in plant growth regulation, defense response, and secondary metabolism. However, MYB gene expression has not been reported in Panax ginseng. In this study, we isolated a gene from ginseng adventitious root, PgMYB2, which encodes an R2R3-MYB protein. Subcellular localization revealed that PgMYB2 protein was exclusively detected in the nucleus of Allium cepa epidermis. The highest expression level of PgMYB2 was found in ginseng root and it was significantly induced by plant hormones methyl jasmonate (MeJA). Furthermore, the binding interaction between PgMYB2 protein and the promoter of dammarenediol synthase (DDS) was found in the yeast strain Y1H Gold. Moreover, the electrophoretic mobility shift assay (EMSA) identified the binding site of the interaction and the results of transiently overexpressing PgMYB2 in plants also illustrated that it may positively regulate the expression of PgDDS. Based on the key role of PgDDS gene in ginsenoside synthesis, it is reasonable to believe that this report will be helpful for the future studies on the MYB family in P. ginseng and ultimately improving the ginsenoside production through genetic and metabolic engineering.

scholarly journals Contrasting Effects of σE on Compartmentalization of σF Activity during Sporulation of Bacillus subtilis

2004 ◽  
Vol 186 (7) ◽  
pp. 1983-1990 ◽  
Author(s):  
David W. Hilbert ◽  
Vasant K. Chary ◽  
Patrick J. Piggot
Keyword(s):  
Gene Expression ◽  
Transcription Factor ◽  
Bacillus Subtilis ◽  
Mother Cell ◽  
Cell Fates ◽  
Gene Encoding ◽  
Primitive Form ◽  
Small Proteins ◽  
Dna Translocase

ABSTRACT Spore formation by Bacillus subtilis is a primitive form of development. In response to nutrient starvation and high cell density, B. subtilis divides asymmetrically, resulting in two cells with different sizes and cell fates. Immediately after division, the transcription factor σF becomes active in the smaller prespore, which is followed by the activation of σE in the larger mother cell. In this report, we examine the role of the mother cell-specific transcription factor σE in maintaining the compartmentalization of gene expression during development. We have studied a strain with a deletion of the spoIIIE gene, encoding a DNA translocase, that exhibits uncompartmentalized σF activity. We have determined that the deletion of spoIIIE alone does not substantially impact compartmentalization, but in the spoIIIE mutant, the expression of putative peptidoglycan hydrolases under the control of σE in the mother cell destroys the integrity of the septum. As a consequence, small proteins can cross the septum, thereby abolishing compartmentalization. In addition, we have found that in a mutant with partially impaired control of σF, the activation of σE in the mother cell is important to prevent the activation of σF in this compartment. Therefore, the activity of σE can either maintain or abolish the compartmentalization of σF, depending upon the genetic makeup of the strain. We conclude that σE activity must be carefully regulated in order to maintain compartmentalization of gene expression during development.

scholarly journals The retinoblastoma gene product RB stimulates Sp1-mediated transcription by liberating Sp1 from a negative regulator.

1994 ◽  
Vol 14 (7) ◽  
pp. 4380-4389 ◽  
Author(s):  
L I Chen ◽  
T Nishinaka ◽  
K Kwan ◽  
I Kitabayashi ◽  
K Yokoyama ◽  
...  
Keyword(s):  
Dna Binding ◽  
Gene Product ◽  
Binding Activity ◽  
Negative Regulator ◽  
Control Element ◽  
Dependent Manner ◽  
Mobility Shift ◽  
Dna Binding Activity ◽  
Cell Extracts ◽  
Mobility Shift Assay

Studies have demonstrated that the retinoblastoma susceptibility gene product, RB, can either positively or negatively regulate expression of several genes through cis-acting elements in a cell-type-dependent manner. The nucleotide sequence of the retinoblastoma control element (RCE) motif, GCCACC or CCACCC, and the Sp1 consensus binding sequence, CCGCCC, can confer equal responsiveness to RB. Here, we report that RB activates transcription of the c-jun gene through the Sp1-binding site within the c-jun promoter. Preincubation of crude nuclear extracts with monoclonal antibodies to RB results in reduction of Sp1 complexes in a mobility shift assay, while addition of recombinant RB in mobility shift assay mixtures with CCL64 cell extracts leads to an enhancement of DNA-binding activity of SP1. These results suggest that RB is directly or indirectly involved in Sp1-DNA binding activity. A mechanism by which RB regulates transactivation is indicated by our detection of a heat-labile and protease-sensitive Sp1 negative regulator(s) (Sp1-I) that specifically inhibits Sp1 binding to a c-jun Sp1 site. This inhibition is reversed by addition of recombinant RB proteins, suggesting that RB stimulates Sp1-mediated transactivation by liberating Sp1 from Sp1-I. Additional evidence for Sp1-I involvement in Sp1-mediated transactivation was demonstrated by cotransfection of RB, GAL4-Sp1, and a GAL4-responsive template into CV-1 cells. Finally, we have identified Sp1-I, a approximately 20-kDa protein(s) that inhibits the Sp1 complexes from binding to DNA and that is also an RB-associated protein. These findings provide evidence for a functional link between two distinct classes of oncoproteins, RB and c-Jun, that are involved in the control of cell growth, and also define a novel mechanism for the regulation of c-jun expression.

scholarly journals Pituitary homeobox 1 (Pitx1) stimulates rat LHβ gene expression via two functional DNA-regulatory regions

2005 ◽  
Vol 35 (1) ◽  
pp. 145-158 ◽  
Author(s):  
Qiaorong Jiang ◽  
Kyeong-Hoon Jeong ◽  
Cheryl D Horton ◽  
Lisa M Halvorson
Keyword(s):  
Gene Expression ◽  
Molecular Mechanisms ◽  
Gene Promoter ◽  
Orphan Nuclear Receptor ◽  
Mobility Shift ◽  
Regulatory Regions ◽  
Steroidogenic Factor 1 ◽  
Reproductive Axis ◽  
Functional Dna ◽  
Mobility Shift Assay

Luteinizing hormone (LH) plays a central role in the reproductive axis, stimulating both gonadal steroid biosynthesis and the development of mature gametes. Over the past decade, significant progress has been made in characterizing the transcription factors and associated DNA-regulatory sites which mediate expression of the LH β-subunit gene (LHβ). One of these factors, pituitary homeobox 1 (Pitx1), has been shown to stimulate LHβ gene promoter activity, both alone and in synergy with the orphan nuclear receptor, steroidogenic factor-1 (SF-1), and the early growth response gene 1 (Egr-1). Prior reports have attributed the Pitx1 response to a cis-element located at position -101 in the rat LHβ gene promoter. While investigating the role of Pitx1 in regulating rat LHβ gene expression, we observed a small, but significant, residual Pitx1 response despite mutation or deletion of this site. In the studies presented here, we identify the presence of a second functional Pitx1 region spanning positions −73 to −52 in the rat LHβ gene promoter. Based on electrophoretic mobility shift assay, Pitx1 binds to both the initially described 5′Pitx1 site as well as this putative 3′Pitx1 region. In transient transfection analysis, mutation of the LHβ-3′Pitx1 site significantly blunted Pitx1 responsiveness, with elimination of the Pitx1 response in a construct containing mutations in both Pitx1 cis-elements. We also analyzed the importance of each of these Pitx1 sites for providing functional synergy with SF-1 and with Egr-1. We observed a markedly decreased synergistic response with mutation of the 5′Pitx1 site with further loss following mutation of the 3′Pitx1 site. In contrast, functional interaction between Pitx1 and Egr-1 persisted with mutation of both Pitx1 regions. We conclude that Pitx1 stimulates the rat LHβ gene promoter via two Pitx1 DNA-regulatory regions. These results further our understanding of the molecular mechanisms that regulate expression of this critical reproductive gene promoter.

scholarly journals Glucocorticoid Stimulation of Corticotropin-Releasing Hormone Gene Expression Requires a Cyclic Adenosine 3′,5′-Monophosphate Regulatory Element in Human Primary Placental Cytotrophoblast Cells*

2000 ◽  
Vol 85 (5) ◽  
pp. 1937-1945
Author(s):  
You-Hong Cheng ◽  
Richard C. Nicholson ◽  
Bruce King ◽  
Eng-Cheng Chan ◽  
John T. Fitter ◽  
...  
Keyword(s):  
Gene Expression ◽  
Regulatory Element ◽  
Mobility Shift ◽  
Messenger Ribonucleic Acid ◽  
Specific Effects ◽  
Placental Cells ◽  
Mobility Shift Assay ◽  
Human Placental Cells ◽  
Human Parturition ◽  
Stimulation Of

Abstract Production of placental CRH, which is identical to the peptide synthesized and secreted in the hypothalamus, has been linked to human parturition. Glucocorticoids stimulate placental CRH secretion and messenger ribonucleic acid expression, in contrast to their inhibition of CRH synthesis in the hypothalamus. A positive feedforward loop involving glucocorticoid-CRH-ACTH-glucocorticoid is thought to drive the exponential increase in placental CRH leading to delivery. Tissue-specific effects of glucocorticoids on CRH expression are therefore of interest. Using human primary placental cells, we investigated the mechanism by which glucocorticoids stimulate placental CRH gene expression. Nuclear run-on transcription shows that in human placental cells glucocorticoids up-regulate transcription of human CRH (hCRH). Using transient transfection assays we demonstrate that dexamethasone up-regulates both basal and cAMP-stimulated hCRH promoter activity, correlating well with the increase in endogenous CRH peptide levels. Through mutagenesis and deletion analyses we show that dexamethasone stimulation of hCRH gene transcription requires a functional cAMP regulatory element (CRE); this CRE is adequate to confer dexamethasone stimulation upon a heterologous promoter, and electrophoretic mobility shift assay studies show that a placental nuclear protein specifically binds to the hCRH CRE.

scholarly journals DegU-P Represses Expression of the Motility fla-che Operon in Bacillus subtilis

2004 ◽  
Vol 186 (18) ◽  
pp. 6003-6014 ◽  
Author(s):  
Giuseppe Amati ◽  
Paola Bisicchia ◽  
Alessandro Galizzi
Keyword(s):  
Bacillus Subtilis ◽  
Adaptive Strategies ◽  
Promoter Sequence ◽  
Two Component System ◽  
Mobility Shift ◽  
Flagellin Gene ◽  
Transcriptional Termination ◽  
Phosphorylated Form ◽  
Mobility Shift Assay

ABSTRACT Bacillus subtilis implements several adaptive strategies to cope with nutrient limitation experienced at the end of exponential growth. The DegS-DegU two-component system is part of the network involved in the regulation of postexponential responses, such as competence development, the production of exoenzymes, and motility. The degU32(Hy) mutation extends the half-life of the phosphorylated form of DegU (DegU-P); this in turn increases the production of alkaline protease, levan-sucrase, and other exoenzymes and inhibits motility and the production of flagella. The expression of the flagellum-specific sigma factor SigD, of the flagellin gene hag, and of the fla-che operon is strongly reduced in a degU32(Hy) genetic background. To investigate the mechanism of action of DegU-P on motility, we isolated mutants of degU32(Hy) that completely suppressed the motility deficiency. The mutations were genetically mapped and characterized by PCR and sequencing. Most of the mutations were found to delete a transcriptional termination signal upstream of the main flagellar operon, fla-che, thus allowing transcriptional readthrough from the cod operon. Two additional mutations improved the σA-dependent promoter sequence of the fla-che operon. Using an electrophoretic mobility shift assay, we have demonstrated that purified DegU binds specifically to the PA promoter region of the fla-che operon. The data suggest that DegU represses transcription of the fla-che operon, and they indicate a central role of the operon in regulating the synthesis and assembly of flagella.

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