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At least 505 records · Page 28Linked to original sources
PubMed1997

The binding of two dimers of IciA protein to the dnaA promoter 1P element enhances the binding of RNA polymerase to the dnaA promoter 1P.

Transcription of the dnaA gene from the promoter 1P has been shown to be activated in vitro and in vivo by the binding of IciA protein to two sites on the dnaA promoter region [Lee, Y. S., Kim, H., and Hwang, D. S. (1996) Mol. Microbiol . 19, 389-396; Lee, Y. S., and Hwang, D. S. (1997) J. Biol. Chem. 272, 83-88]. In vitro transcription assays using DNA fragments carrying variable combinations of two IciA binding sites revealed that IciA binding site I (IciA I site), which is located upstream of the promoter 1P, is responsible for the transcriptional activation. Binding of one dimeric IciA protein to the IciA I site is followed by binding of the second dimer. Two dimers of IciA protein, rather than one dimer, on the IciA I site appeared to enhance the binding of RNA polymerase to the promoter 1P, resulting in the activation of transcription from the promoter 1P.

Bacterial Proteins↗
PubMed1998

Nucleosomes are not necessary for promoter-proximal pausing in vitro on the Drosophila hsp70 promoter.

RNA polymerase II has been found to pause stably on several metazoan genes in a promoter-proximal region located 20-40 nt downstream from the start site of transcription. Escape of polymerase from this paused state has been proposed to be a rate limiting step in transcription of some genes. A study of the human hsp70 promoter showed that a nucleosome positioned downstream from the transcription start was a key component in establishing a stably paused polymerase in one cell-free system. We tested whether these results could be extended to the Drosophila hsp70 promoter in a Drosophila cell-free system and found that polymerase paused stably on the promoter even when the length of DNA downstream from the transcription start was not sufficient for assembly of a nucleosome. Our results indicate that a downstream nucleosome is not a universal requirement for stably pausing RNA polymerase in the promoter-proximal region.

Animals↗
PubMed2002

Contribution of downstream promoter elements to transcriptional regulation of the rice tungro bacilliform virus promoter.

Downstream sequences influence activity of the rice tungro bacilliform virus (RTBV) promoter in protoplasts derived from cultured rice cells. We previously identified a DNA element located between positions +50 and +90 relative to the transcription start site to which rice nuclear proteins bind. In this study, using DNA UV crosslinking assays, we show that two rice nuclear proteins bind specifically to this DNA element. We demonstrate that the DNA element enhances RTBV promoter activity in a copy number-dependent manner when transferred to a position upstream of the promoter. In addition, using electrophoretic mobility shift assays, we show that at least two novel nuclear proteins from rice cell suspension cultures bind to a subregion (from +50 to +59) of the DNA element and that a protein from rice root, but not shoot, nuclear extracts interacts with a perfect palindromic sequence motif located within the sequence +45 to +59. Furthermore, a position-dependent GAGA motif, present in three copies within downstream promoter sequences from +1 to +50, is involved in the regulation of RTBV promoter activity.

Badnavirus↗
PubMed1993

Photochemical cross-linking of influenza A polymerase to its virion RNA promoter defines a polymerase binding site at residues 9 to 12 of the promoter.

A previous study of the 12 nucleotide-long influenza A virion RNA promoter has shown that three nucleotides, residues 9 to 11, were crucial for transcription in vitro, although other nucleotides play a significant but less important role. A model for polymerase-promoter recognition was proposed, according to which there were two sites: a binding site at residues 9 to 11 and a regulatory site at or near the site of initiation at residue 1. By studying the effect of point mutations in the promoter on the binding efficiency of the polymerase using a photochemical cross-linking assay, we now show that residues 9 to 12 are crucial for binding. In addition residues 4 to 8, though not as important, are involved in binding, possibly by stabilizing the polymerase-promoter complex. Both PB1 and PB2 apparently play an important role during virion RNA promoter recognition and binding.

Animals↗
PubMed2001

Negative regulatory regions of the PAT1 promoter of Hz-1 virus contain GATA elements which associate with cellular factors and regulate promoter activity.

The persistence-associated transcript 1 (PAT1) is actively expressed during persistent infection with Hz-1 virus, while transcription of the rest of the viral genes is shut down. Previously, results of a series deletion of the PAT1 promoter suggested that the regions from nucleotides -312 to -212 and nucleotides -158 to -90 negatively regulate the promoter activity. Here, the negative regulatory effect of the -312/-90 fragment was confirmed using a heterologous IE0 promoter of Autographa californica multiple nucleopolyhedrovirus. Further, the negative regulation of the -312 to -212 region was orientation-independent. The results of electrophoresis mobility shift assays showed that cellular protein(s) bind specifically to DNA fragments -312/-212 and -158/-90. In each of these fragments, a GATA element was identified by computer-assisted analysis. Mutating both GATA elements in the -312/-90 fragment completely eliminated its negative effect on IE0 promoter activity, while mutating only one of these elements had little or no effect. Together, these results suggest that the GATA element has a negative regulatory role on the IE0 and PAT1 promoters.

Animals↗
PubMed2001

Enhancer-promoter specificity mediated by DPE or TATA core promoter motifs.

To investigate the basis for enhancer-promoter specificity, we compared the ability of enhancers to activate transcription in vivo from core promoters containing either downstream promoter element (DPE) or TATA box motifs. To eliminate position effects, we generated and analyzed pairs of sister Drosophila lines that contain a DPE- or TATA-dependent reporter gene at precisely the same genomic position relative to each enhancer. These studies revealed transcriptional enhancers that are specific for promoters that contain either DPE or TATA box elements. Thus, the core promoter not only mediates the initiation of transcription, but also functions as a regulatory element.

Animals↗
PubMed2006

Characterization of a novel plant promoter specifically induced by heavy metal and identification of the promoter regions conferring heavy metal responsiveness.

The bean (Phaseolus vulgaris) stress-related gene number 2 (PvSR2) gene responds to heavy metals but not to other forms of environmental stresses. To elucidate its heavy metal-regulatory mechanism at the transcriptional level, we isolated and characterized the promoter region (-1623/+48) of PvSR2. Deletions from the 5' end revealed that a sequence between -222 and -147 relative to the transcriptional start site was sufficient for heavy metal-specific induction of the promoter region of PvSR2. Detailed analysis of this 76-bp fragment indicated that heavy metal-responsive elements were localized in two regions (-222/-188 and -187/-147), each of which could separately confer heavy metal-responsive expression on the beta-glucuronidase gene in the context of a minimal cauliflower mosaic virus 35S promoter. Region I (-222/-188) contains a motif (metal-regulatory element-like sequence) similar to the consensus metal-regulatory element of the animal metallothionein gene, and mutation of this motif eliminated the heavy metal-inducible function of region I. Region II (-187/-147) had no similarity to previously identified cis-acting elements involved in heavy metal induction, suggesting the presence of a novel heavy metal-responsive element. Transformed tobacco (Nicotiana tabacum) seedlings expressing beta-glucuronidase under control of the PvSR2 promoter region (-687/+48) showed heavy metal-specific responsive activity that depended on the type and concentration of the heavy metal and the type of organ. These findings further our understanding of the regulation of PvSR2 expression and provide a new heavy-metal-inducible promoter system in transgenic plants.

Base Sequence↗
PubMed2004

Stimulus-dependent, promoter-specific binding of transcription factor WRKY1 to Its native promoter and the defense-related gene PcPR1-1 in Parsley.

WRKY transcription factors form a large family that plays a role in plant responses to biotic stress and during senescence. Defining in vivo relevant WRKY/promoter relationships has been hampered by the factors' indiscriminate binding to known W box DNA elements and their possible genetic redundance. Employing chromatin immunoprecipitations (ChIP) of cultured cells, we show that parsley (Petroselinum crispum) WRKY1 protein binds to the W boxes of its native promoter as well as to that of PcWRKY3 and the defense-related PR10-class marker gene Pathogenesis-Related1-1 (PcPR1-1). Although present at low concentrations in resting cells, WRKY1 does not appear to play a role in the immediate early gene response upon elicitation because it does not bind to the promoter at this time. Paradoxically, in vivo binding at the PcWRKY1 promoter correlates more with downregulation of gene expression, whereas previous overexpression studies suggested an activating function of WRKY1 on PcWRKY1 expression. By contrast, PcPR1-1 expression remains strong when its promoter is occupied in vivo by WRKY1. Unexpectedly, ChIP revealed that W boxes at promoter sites are constitutively occupied by other WRKY transcription factors, indicating that site recruitment does not seem to play a major role in their regulation. Rather, WRKY proteins very likely act in a network of mutually competing participants with temporal displacement occurring at defined preoccupied sites by other family members in a stimulus-dependent manner.

Base Sequence↗
PubMed2002

Deletion of dinucleotide repeat (Delta 14 allele) in the methylthioadenosine phosphorylase (MTAP) promoter and the allelotype of MTAP promoter in the Japanese population.

5'-deoxy-5'-methylthioadenosine phosphorylase (MTAP) is an enzyme involved in purine and polyamine metabolism and is ubiquitously expressed in normal human tissues and cells. However, this enzyme has been found to be deficient in a variety of human cancers. Although the enzyme deficiency is known to be caused by MTAP gene deletion, human diffuse histiocytic lymphoma cell line DHL-9 without any detectable MTAP activity has been found to possess the intact MTAP gene. These lines of evidence suggested that promoter abnormality might cause the MTAP deficiency in DHL-9. Therefore, we analyzed the MTAP promoter region of DHL-9 and found the deletion of 14 bases in its sequence. We designated the allele lacking (GT)(6)GC as dinucleotide repeat deletion (Delta 14 allele) and determined the effect of the Delta 14 allele on the MTAP promoter activity by a luciferase reporter assay. We have also analyzed the distribution of the Delta 14 allele and wild-type (WT) allele in the Japanese population by PCR assay. A reporter plasmid harboring the Delta 14 allele exhibited luciferase activity comparable to that of a plasmid containing the WT allele. Forty-six (22%) out of 210 people were homozygous for WT allele in the MTAP promoter, whereas 43 (20.5%) were homozygous for Delta 14 allele. The remaining 121 people (57.5%) possessed Delta 14/WT alleles in the MTAP promoter region. These results indicated that the Delta 14 allele has nothing to do with MTAP deficiency in DHL-9. The Delta 14 allele is distributed among the general population irrespective of gender.

Alleles↗
PubMed1991

Organ-dependent regulation of a plant promoter isolated from rice by 'promoter-trapping' in tobacco.

A vector containing a transcriptionally inactive neomycin phosphotransferase II gene was used to select promoter sequences from a pool of random genomic DNA fragments. This paper describes how one such sequence (P4.7) isolated from Oryza sativa acts as a hormonally regulated promoter in Nicotiana tabacum. Relative expression ratios in leaf, root, midrib, callus, and stem tissue of tobacco plants are 1:5:4:10:17. Histochemical assays show that P4.7 activates the uidA reporter gene throughout the phloem and cortex of tobacco stems. Transcription from the P4.7 fragment is inducible in leaf tissue by low levels of alpha-naphthalene acetic acid or 6-benzyl-aminopurine, even when cell proliferation is inhibited by colchicine or hydroxyurea. Conversely, 1% DMSO was found to inhibit activation of P4.7 without interfering with callus formation. The fragment contains TATA and CAAT sequences normally found at the 5' end of many plant genes, and an additional region homologous to sequences located in similar positions in a variety of similarly regulated promoters. Promoter deletion and fusion experiments have indicated the location of a stem enhancer element in P4.7. The promoter trap system we have described may potentially be used to characterize transcriptional factors common to monocot and dicot species.

Base Sequence↗
PubMed1992

Characterization of the promoter region of the porcine opn (osteopontin, secreted phosphoprotein 1) gene. Identification of positive and negative regulatory elements and a 'silent' second promoter.

Osteopontin (secreted phosphoprotein-1, Opn) is a phosphorylated glycoprotein expressed by transformed cells, macrophages, activated T-lymphocytes, specialized epithelial cells and bone cells that is characteristically enriched in milk and in the mineralized matrix of bone. The synthesis of Opn by bone cells is regulated by glucocorticoids and growth factors, which promote bone formation, and by the osteotropic hormone calcitriol (1,25-dihydroxycholecalciferol) and retinoic acid, which mediate bone resorption, indicating a bifunctional role for this protein in bone remodelling. To study the transcriptional regulation of the opn gene, two genomic clones (10 and 15 kb) encoding the opn gene were isolated from a porcine liver genomic library cloned into lambda phage. From the 15-kb clone a 4-kb EcoRI fragment containing the first two exons and 2.6 kb of the 5' flanking region of the opn gene was sequenced, and the transcriptional start site determined by primer extension analysis and S1 nuclease mapping. To identify the opn promoter, chimeric chloramphenicol acetyltransferase constructs were prepared using fragments from the first intron and the 5' flanking region of the opn gene. Transient transfection of porcine bone cells with these constructs showed strong promoter activity located within 74 bp upstream from the transcription initiation site. Within this region a TATA sequence, TTTAAA, was identified at positions -26 to -31. However, the highest transcription rate was observed in a construct extending 180 bp upstream that included a CCGCCC Sp1 binding sequence (-63 to -68), and an AP1 site (-74 to -80). Further upstream in the 5' flanking region and within the first intron of the opn, a number of consensus sequences could be identified. Chimeric constructs containing a GGGTCAtatGGTTCA direct repeat consensus sequence for a vitamin D3 response element located at nucleotides -2245 to -2259 responded to the addition of 0.1 microM calcitriol by a 2.5-fold stimulation of transcription, although a greater than 2-fold increase was also observed in shorter constructs -180 to -905 lacking such a consensus sequence. Promoter activity was also exhibited by a region containing a TTTAAA sequence in the first intron that corresponded to the putative promoter site reported for mouse opn in macrophages (Miyazaki, Y., Setoguchi, M., Yoshida, S., Higuchi, Y., Akizuki, S. & Yamamoto, S. (1990) J. Biol. Chem. 265, 14432-14438).(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗
PubMed1994

Promoting prenatal care: what do community leaders know and believe about it? (community leader beliefs about promotion).

Community leaders directly affect prenatal services in their local communities. Twenty-nine leaders in an urban community and 47 in a rural community in Wisconsin were interviewed to determine their knowledge and beliefs about and intent to promote prenatal care for low-income women in their communities. The findings showed that leaders generally assigned high importance to prenatal care for low-income women and intended to promote it during the next six months; and they demonstrated good general knowledge about prenatal care and perceived broad-based community support for their decision to promote it. Disturbingly, many community leaders did not know what percentage of local pregnant women entered prenatal care late or how many medical care providers were available to their constituents on Medicaid, cost was cited as a disadvantage to promoting prenatal care for low-income women, and several leaders in both communities believed that promoting prenatal care would result in low-income women wanting to have more babies or increasing their dependence on free programs. Public health nurses can use the findings of this study in their work with community leaders to ensure prenatal services for low-income populations.

Community Participation↗
PubMed1992

Transcription factor loading on the MMTV promoter: a bimodal mechanism for promoter activation.

The mouse mammary tumor virus (MMTV) promoter attains a phased array of six nucleosomes when introduced into rodent cells. This architecture excludes nuclear factor 1/CCAAT transcription factor (NF1/CTF) from the promoter before glucocorticoid treatment and hormone-dependent access of nucleolytic agents to promoter DNA. In contrast, when the promoter was transiently introduced into cells, NF1/CTF was bound constitutively and nucleolytic attack was hormone-independent. Thus, induction at this promoter was a bimodal process involving receptor-dependent remodeling of chromatin that allows NF1/CTF loading and direct receptor-mediated recruitment of additional transcription factors.

Animals↗
PubMed2000

Broad-host-range shuttle vectors for screening of regulated promoter activity in viridans group streptococci: isolation of a pH-regulated promoter.

Viridans group streptococci are major constituents of the normal human oral flora and are also identified as the predominant pathogenic bacteria in native valve infective endocarditis. Little information is available regarding the regulation of gene expression in viridans group streptococci, either in response to changes in the oral environment or during development of endocarditis. We therefore constructed a set of broad-host-range vectors for the isolation of promoters from viridans group streptococci that are activated by specific environmental stimuli in vitro or in vivo. A genomic library of Streptococcus gordonii strain CH1 was constructed in one of the new vectors, and this library was introduced into a homologous bacterium by using an optimized electroporation protocol for viridans group streptococci. Because viridans group streptococci entering the bloodstream from the oral cavity encounter an increase in pH, we selected promoters upregulated by this specific stimulus. One of the selected promoter sequences showed homology to the promoter region of the hydA gene from Clostridium acetobutylicum, the expression of which is known to be regulated by the environmental pH. The isolation of this pH-regulated promoter shows that S. gordonii can sense an increase in the environmental pH, which serves as a signal for bacterial gene activation. Furthermore, this demonstrates the usefulness of these new selection vectors in research on adaptive gene expression of viridans group streptococci and possibly also of other gram-positive bacteria.

Base Sequence↗
PubMed1997

Bacillus subtilis PhoP binds to the phoB tandem promoter exclusively within the phosphate starvation-inducible promoter.

Several gene products, including three two-component systems, make up a signal transduction network that controls the phosphate starvation response in Bacillus subtilis. Epistasis experiments indicate that PhoP, a response regulator, is furthest downstream of the known regulators in the signaling pathway that regulates Pho regulon genes. We report the overexpression, purification, and use of PhoP in investigating its role in Pho regulon gene activation. PhoP was a substrate for both the kinase and phosphatase activities of its cognate sensor kinase, PhoR. It was not phosphorylated by acetyl phosphate. Purified phosphorylated PhoP (PhoPP) had a half-life of approximately 2.5 h, which was reduced to about 15 min by addition of the same molar amount of *PhoR (the cytoplasmic region of PhoR). ATP significantly increased phosphatase activity of *PhoR on PhoPP. In gel filtration and cross-linking studies, both PhoP and PhoPP were shown to be dimers. The dimerization domain was located within the 135 amino acids at the N terminus of PhoP. Phosphorylated or unphosphorylated PhoP bound to one of the alkaline phosphatase gene promoters, the phoB promoter. Furthermore, PhoP bound exclusively to the -18 to -73 region (relative to the transcriptional start site +1) of the phosphate starvation-inducible promoter (Pv) but not to the adjacent developmentally regulated promoter (Ps). These data corroborate the genetic data for phoB regulation and suggest that activation of phoB is via direct interaction between PhoP and the phoB promoter. Studies of the phosphorylation, oligomerization, and DNA binding activity of the PhoP protein demonstrate that its N-terminal phosphorylation and dimerization domain and its C-terminal DNA binding domain function independently of one another, distinguishing PhoP from other response regulators, such as PhoB (Escherichia coli) and NtrC.

Bacillus subtilis↗
PubMed1998

Demonstration that the TyrR protein and RNA polymerase complex formed at the divergent P3 promoter inhibits binding of RNA polymerase to the major promoter, P1, of the aroP gene of Escherichia coli.

In previous studies, we have identified three promoters (P1, P2, and P3) in the regulatory region of the Escherichia coli aroP gene (P. Wang, J. Yang, and A. J. Pittard, J. Bacteriol. 179:4206-4212, 1997). Both P1 and P2 can direct mRNA synthesis for aroP expression, whereas P3 is a divergent promoter which overlaps with P1. The repression of transcription from the major promoter, P1, has been postulated to involve the activation of the divergent promoter, P3, by the TyrR protein (P. Wang, J. Yang, B. Lawley, and A. J. Pittard, J. Bacteriol. 179:4213-4218, 1997). In the present study, we confirmed the proposed mechanism of P3-mediated repression of P1 transcription by studying the binding of RNA polymerase to the promoters P1 and P3 in vitro in the presence and absence of TyrR protein and its cofactors. Our results show that (i) only one RNA polymerase molecule can bind to the DNA fragment carrying the aroP regulatory region, (ii) RNA polymerase has a higher affinity for P1 than for either P2 or P3 and binds to P1 in the absence of TyrR protein, (iii) in the presence of TyrR protein and its cofactor, phenylalanine or tyrosine, RNA polymerase preferentially binds to P3, and (iv) RNA polymerase does not respond to the activation-defective mutant TyrR protein TyrR-RQ10 and remains bound to P1 in the presence of TyrR-RQ10 and either of the cofactors.

Amino Acid Transport Systems↗
PubMed1991

Adenovirus E4-dependent activation of the early E2 promoter is insufficient to promote the early-to-late-phase transition.

The adenovirus E4 ORF6/7 protein has been shown to activate the cellular transcription factor E2F. E2F activation leads to activation of the adenovirus early E2 promoter which controls the production of viral DNA replication proteins. In the present study an adenovirus type 5 cDNA mutant, H5ilE4L, was constructed. This mutant is capable of making the ORF6/7 polypeptide but lacks the coding sequences for all other E4 products. H5ilE4L trans activates the early E2 promoter to wild-type levels, but still it is defective for viral DNA replication. A mutant expressing ORF6 in addition to ORF6/7, H5ilE4I, is normal for viral DNA replication. This indicates that activation of the early E2 promoter is insufficient to promote efficient viral DNA replication and that another E4-encoded function is necessary. The ORF6 protein seems to provide this function. We suggest that ORF6/7-induced activation of E2F is not necessary for adenovirus growth in HeLa cells. Rather, this activation might be of importance in the normal, growth-arrested host cell, since E2F has been shown to bind to the promoter regions of a number of immediate-early genes involved in regulation of cell proliferation (M. Mudryj, S. W. Hiebert, and J. R. Nevins, EMBO J. 9:2179-2184, 1990).

Adenovirus Early Proteins↗
PubMed1992

Analysis of the gB promoter of herpes simplex virus type 1: high-level expression requires both an 89-base-pair promoter fragment and a nontranslated leader sequence.

To investigate the cis-acting sequences involved in regulation of a herpes simplex virus gamma 1 gene, deletion analyses of the glycoprotein B (gB) gene promoter were performed. In transfection assays with gB-chloramphenicol acetyltransferase plasmids, high-level constitutive expression from the gB promoter was found with an 89-bp sequence (-69 to +20). Additional sequences in the 5'-transcribed noncoding leader region (+20 to +136) were required for full stimulation by herpes simplex virus infection. Plasmids with progressive deletions of the gB leader sequence demonstrated that chloramphenicol acetyltransferase expression in infected cells was proportional to the length of the leader region retained. In recombinant viruses containing a gB-gC gene fusion, a similar 83-bp (-60 to +23) region of the gB gene was found to promote accurately initiated gC mRNA from the viral genome with the same kinetics as the wild-type gB gene. Although the kinetics of expression remained the same, RNA abundance was greater with a 298-bp (-260 to +38) promoter than with the 83-bp promoter.

Animals↗