UNIPROT:P51532 - FACTA Search

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Query: UNIPROT:P51532 (transcriptional activator)
6,546 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

An inducible middle promoter from the lactococcal bacteriophage phi31 was isolated previously by shotgun cloning an 888-bp fragment (P15A10) upstream of the beta-galactosidase (beta-Gal) gene (lacZ.st) from Streptococcus thermophilus (D. J. O'Sullivan, S. A. Walker, S. G. West, and T. R. Klaenhammer, Bio/Technology 14:82-87, 1996). The promoter showed low levels of constitutive beta-Gal activity which could be induced two- to threefold over baseline levels after phage infection. During this study, the fragment was subcloned and characterized to identify a smaller, tightly regulated promoter fragment which allowed no beta-Gal activity until after phage infection. This fragment, defined within nucleotides 566 to 888 (P(566-888); also called fragment 566-888), contained tandem, phage-inducible transcription start sites at nucleotides 703 and 744 (703/744 start sites). Consensus -10 regions were present upstream of both start sites, but no consensus -35 regions were identified for either start site. A transcriptional activator, encoded by an open reading frame (ORF2) upstream of the 703/744 start sites, was identified for P(566-888). ORF2 activated P(566-888) when provided in trans in Escherichia coli. In addition, when combined with pTRK391 (P15A10::lacZ.st) in Lactococcus lactis NCK203, an antisense ORF2 construct was able to retard induction of the phage-inducible promoter as measured by beta-Gal activity levels. Finally, gel shift assays showed that ORF2 was able to bind to promoter fragment 566-888. Deletion analysis of the region upstream from the tandem promoters identified a possible binding site for transcriptional activation of the phage promoters. The DNA-binding ability of ORF2 was eliminated upon deletion of part of this region, which lies centered approximately 35 bp upstream of start site 703. Deletion analysis and mutagenesis studies also elucidated a critical region downstream of the 703/744 start sites, where mutagenesis resulted in a two- to threefold increase in beta-Gal activity. With these improvements, the level of expression achieved by an explosive-expression strategy was elevated from 3,000 to 11,000 beta-Gal units within 120 min after induction.
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PMID:Molecular characterization of a phage-inducible middle promoter and its transcriptional activator from the lactococcal bacteriophage phi31. 947 48

The roles of the nitrogen fixation regulatory proteins NifA, FixK1, and FixK2 in the symbiotic regulation of hydrogenase structural gene expression in Bradyrhizobium japonicum have been investigated. Bacteroids from FixJ and FixK2 mutants have little or no hydrogenase activity, and extracts from these mutant bacteroids contain no hydrogenase protein. Bacteroids from a FixK1 mutant exhibit wild-type levels of hydrogenase activity. In beta-galactosidase transcriptional assays with NifA and FixK2 expression plasmids, the FixK2 protein induces transcription from the hup promoter to levels similar to those induced by HoxA, the transcriptional activator of free-living hydrogenase expression. The NifA protein does not activate transcription at the hydrogenase promoter. Therefore, FixK2 is involved in the transcriptional activation of symbiotic hydrogenase expression. By using beta-galactosidase transcriptional fusion constructs containing successive truncations of the hup promoter, the region of the hup promoter required for regulation by FixK2 was determined to be between 29 and 44 bp upstream of the transcription start site.
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PMID:The FixK2 protein is involved in regulation of symbiotic hydrogenase expression in Bradyrhizobium japonicum. 962 Sep 82

A recessive mutation, aarG1, has been identified that resulted in an 18-fold increase in the expression of beta-galactosidase from an aac(2')-lacZ fusion. Transcriptional fusions and Northern blot analysis demonstrated that the aarG1 allele also resulted in a large increase in the expression of aarP, a gene encoding a transcriptional activator of aac(2')-Ia. The effects of aarG1 on aac(2')-Ia expression were mediated by aarP-dependent and -independent mechanisms. The aarG1 allele also resulted in a multiple antibiotic resistance (Mar) phenotype, which included increased chloramphenicol, tetracycline and fluoroquinolone resistance. This Mar phenotype also resulted from aarP-dependent and -independent mechanisms. Sequence analysis of the aarG locus revealed the presence of two open reading frames, designated aarR and aarG, organized in tandem. The putative AarR protein displayed 75% amino acid identity to the response regulator PhoP, and the AarG protein displayed 57% amino acid identity to the sensor kinase PhoQ. The aarG1 mutation, a C to T substitution, resulted in a threonine to isoleucine substitution at position 279 (T279I) in the putative sensor kinase. The AarG product was functionally similar to PhoQ, as it was able to restore wild-type levels of maganin resistance to a Salmonella typhimurium phoQ mutant. However, expression of the aarP and aac(2')-Ia genes was not significantly affected by the levels of Mg2+ or Ca2+, suggesting that aarG senses a signal other than divalent cations.
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PMID:A regulatory cascade involving AarG, a putative sensor kinase, controls the expression of the 2'-N-acetyltransferase and an intrinsic multiple antibiotic resistance (Mar) response in Providencia stuartii. 968 Feb 22

An equine herpesvirus 1 (EHV-1) mutant was constructed by inserting a lacZ expression cassette into the intergenic region upstream of gene 62 (glycoprotein L; gL) and downstream of gene 63 (a homologue of the herpes simplex virus transcriptional activator ICP0). The recombinant lacZ62/63-EHV-1 had similar growth kinetics in cell culture to those of the parental wild type (wt) virus, with indistinguishable cytopathic effects and plaque morphology. Reverse transcriptase PCR confirmed that the lacZ insertion did not interfere with transcription of gL and immunoblot analysis indicated there was no modification to late gene expression as monitored by synthesis of EHV-1 glycoproteins C and D. The parental EHV-1 isolate HVS25A used here had almost identical nucleotide sequence to that published for isolate Ab4, in a 1200 bp region surrounding the insert, but lacked a HindIII site corresponding to Ab4 position 109,048. The lacZ62/63-EHV-1 caused respiratory disease in BALB/c mice with clinical signs, histopathology and virus titres in lungs throughout days 1-5 post infection similar to those induced by wt EHV-1. X-gal staining for beta-galactosidase expression in murine lungs clearly demonstrated EHV-1 infection in cells of the bronchiolar epithelium and pulmonary parenchyma, with a peak of infection evident at day 2 post infection, when up to 50% of bronchioles demonstrated blue-staining and thus virus-infected epithelial cells. The construction of this replication competent virus carrying a reporter gene identifies a site for insertion of foreign genes and will facilitate studies on the pathogenesis of EHV-1.
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PMID:An equine herpesvirus 1 mutant with a lacZ insertion between open reading frames 62 and 63 is replication competent and causes disease in the murine respiratory model. 985 3

We describe a method for the genetic analysis of the DNA-binding properties of Xenopus transcription factor IIIA (TFIIIA). In this approach, a transcriptional activator with the DNA-binding specificity of Xenopus TFIIIA is expressed in yeast cells, where it specifically activates expression of a beta-galactosidase reporter gene containing one or more Xenopus 5 S rRNA genes that function as upstream activator sequences. This transcription-promoting activity was used as the basis for a genetic assay of Xenopus TFIIIA's DNA-binding function in yeast, an assay that we show can be calibrated quantitatively to allow the affinity of the Xenopus TFIIIA-5 S rRNA gene interaction to be deduced from measurements of beta-galactosidase activity. We have combined this genetic assay with a simple and efficient method of mutagenesis that makes use of error-prone PCR and homologous recombination to generate and screen large numbers of TFIIIA mutants for those with altered 5 S rRNA gene-binding affinity. Over 30 such mutants have been identified and partially characterized. The mutants we have obtained provide strong support for the application to intact TFIIIA of recent structural models of the N-terminal zinc fingers of the protein bound to fragments of the 5 S rRNA gene. Other mutants permit identification of important residues in more C-terminal zinc fingers of TFIIIA for which high-resolution structural information is not currently available. Finally, our results have interesting implications with respect to the mechanism of activation of transcription by RNA polymerase II in yeast.
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PMID:Genetic analysis of Xenopus transcription factor IIIA. 987 52

In the present study we describe the set-up of a new one-hybrid reporter gene assay in Saccharomyces cerevisiae composed of the human progesterone receptor fused to the DNA-binding domain of the yeast transcriptional activator Gal4. This assay allows the convenient estimation of receptor mediated progestogenic as well as antiprogestogenic actions of compounds. The induction of the beta-galactosidase reporter gene expression correlated well with the progesterone receptor affinity and the concentration of the progestins tested. The results corresponded to those obtained from a reporter gene assay in the cancer cell line CV-1 and in vitro binding experiments using rabbit uterus cytosol. In both the yeast and CV-1 cells the activity of antiprogestins was detectable by inhibition of the progestin-induced reporter gene expression. Secondary reporter genes under the transcriptional control of receptor unrelated promoters have been introduced into yeast and mammalian test strains to distinguish between specific receptor mediated antihormone actions and nonspecific effects on cellular metabolism.
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PMID:Dual reporter systems in yeast and mammalian cells for assessing progesterone receptor modulators. 1008 31

A gene encoding a third alpha-galactosidase (AglB) from Aspergillus niger has been cloned and sequenced. The gene consists of an open reading frame of 1,750 bp containing six introns. The gene encodes a protein of 443 amino acids which contains a eukaryotic signal sequence of 16 amino acids and seven putative N-glycosylation sites. The mature protein has a calculated molecular mass of 48,835 Da and a predicted pI of 4.6. An alignment of the AglB amino acid sequence with those of other alpha-galactosidases revealed that it belongs to a subfamily of alpha-galactosidases that also includes A. niger AglA. A. niger AglC belongs to a different subfamily that consists mainly of prokaryotic alpha-galactosidases. The expression of aglA, aglB, aglC, and lacA, the latter of which encodes an A. niger beta-galactosidase, has been studied by using a number of monomeric, oligomeric, and polymeric compounds as growth substrates. Expression of aglA is only detected on galactose and galactose-containing oligomers and polymers. The aglB gene is expressed on all of the carbon sources tested, including glucose. Elevated expression was observed on xylan, which could be assigned to regulation via XlnR, the xylanolytic transcriptional activator. Expression of aglC was only observed on glucose, fructose, and combinations of glucose with xylose and galactose. High expression of lacA was detected on arabinose, xylose, xylan, and pectin. Similar to aglB, the expression on xylose and xylan can be assigned to regulation via XlnR. All four genes have distinct expression patterns which seem to mirror the natural substrates of the encoded proteins.
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PMID:Differential expression of three alpha-galactosidase genes and a single beta-galactosidase gene from Aspergillus niger. 1034 26

During murine spermatogenesis, beginning in late pachytene spermatocytes, the beta4-galactosyltransferase-I (beta4GalT-I) gene is transcribed from a male germ cell-specific start site. We had shown previously that a 796-bp genomic fragment that flanks the germ cell start site and contains two putative CRE (cyclic AMP-responsive element)-like motifs directs correct male germ cell expression of the beta-galactosidase reporter gene in late pachytene spermatocytes and round spermatids of transgenic mice (N. L. Shaper, A. Harduin-Lepers, and J. H. Shaper, J. Biol. Chem. 269:25165-25171, 1994). We now report that in vivo expression of beta4GalT-I in developing male germ cells requires an essential and previously undescribed 14-bp regulatory element (5'-GCCGGTTTCCTAGA-3') that is distinct from the two CRE-like sequences. This cis element is located 16 bp upstream of the germ cell-specific start site and binds a male germ cell protein that we have termed TASS-1 (transcriptional activator in late pachytene spermatocytes and round spermatids 1). The presence of the Ets signature binding motif 5'-GGAA-3' on the bottom strand of the TASS-1 sequence (underlined sequence) suggests that TASS-1 is a novel member of the Ets family of transcription factors. Additional transgenic analyses established that an 87-bp genomic fragment containing the TASS-1 regulatory element was sufficient for correct germ cell-specific expression of the beta-galactosidase reporter gene. Furthermore, when the TASS-1 motif was mutated by transversion, within the context of the original 796-bp fragment, transgene expression was reduced 12- to 35-fold in vivo.
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PMID:A novel 14-base-pair regulatory element is essential for in vivo expression of murine beta4-galactosyltransferase-I in late pachytene spermatocytes and round spermatids. 1040 68

The SpoC1-C1C gene is centrally located within the A. nidulans conidium-specific SpoC1 gene cluster. With one exception, the 14 genes within the cluster are coordinately regulated. C1C transcript is first detected late in conidiation, coincidental with the appearance of mature conidia, and accumulates approximately 1000-fold in conidia. We show that C1C expression is restricted to conidia, with mRNA abundance decreasing immediately after induction of germination. C1C transcription and translation are not temporally separated and, similar to C1C RNA abundance, a C1C::beta-galactosidase fusion protein is first detected with the appearance of mature conidia and decreases after induction of germination. Cell-specific C1C expression requires both a position-dependent mechanism of regulation, responsible for repression in hyphae, and a position-independent mechanism of regulation, responsible for developmental expression. We show by functional analysis of upstream DNA sequences that a 10-bp sequence and two adjacent 6-bp direct repeats are necessary for position-independent, condium-specific expression of both the intact C1C gene and the reporter gene. At least one repeat (CAACAT) is required for normal levels of expression. We find that the C1C gene is not a direct target of the BrlAp and AbaAp developmental regulators, but of a yet unidentified conidium-specific transcriptional activator.
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PMID:Functional analysis of DNA sequences required for conidium-specific expression of the SpoC1-C1C gene of Aspergillus nidulans. 1044 49

Conjugal transfer of the Ti plasmid pTiC58 is regulated by a quorum-sensing system involving the transcriptional activator TraR and the acyl homoserine lactone autoinducer N-(3-oxo-octanoyl)-L-homoserine lactone (AAI). Activation of tra gene expression by TraR and AAI is inhibited by TraM, an 11 kDa protein also coded for by the Ti plasmid. Previous studies suggested that TraM interferes with TraR activity by directly interacting with the activator protein. Using the yeast two-hybrid system, constructs of Saccharomyces cerevisiae containing a fusion of traR to the B42 domain of the prey plasmid pJG4.5 and a fusion of traM to the lexA gene of the bait plasmid pEG202 produced beta-galactosidase and grew on medium lacking leucine, both phenotypes indicative of an interaction between the two proteins. Early termination mutants and substitution mutants mapping to the C-terminus of TraM were isolated by screening for alleles unable to interfere with TraR activity in Agrobacterium tumefaciens. These mutants all failed to interact with the TraR fusion in the two-hybrid system. An N-terminal deletion mutant of TraM lacking the first 27 residues weakly interacted with TraR in the two-hybrid system whereas deletions of 48 amino acids or more abolished the interaction. As assessed by Western blot analysis, the mutant fusion proteins were produced at levels indistinguishable from that of the wild-type TraM in the yeast tester strain. Mutants of TraR that were not inhibited by TraM in A. tumefaciens were isolated and fell into two classes. In the first, the mutation resulted in increased expression of wild-type TraR. In the second, a proline residue at position 176 was changed to serine (P176 --> S) or to leucine (P176 --> L). The P176 --> S mutant interacted with wild-type TraM, but at a detectably lower level, in the two-hybrid assay. Mutants of TraR with N-terminal deletions as large as 105 amino acids interfered with the ability of TraM to inhibit wild-type TraR in A. tumefaciens. Two-hybrid assays indicated that these mutants, as well as a C-terminal 49 residue fragment of TraR, can interact with TraM. We conclude that TraM and TraR interact in vivo and that this interaction is responsible for inhibition of TraR-mediated activation. We also conclude that the two proteins interact with each other through domains located at their respective C-termini.
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PMID:Modulating quorum sensing by antiactivation: TraM interacts with TraR to inhibit activation of Ti plasmid conjugal transfer genes. 1056 72

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