Identification of an upstream regulatory sequence that mediates the transcription of mox genes in Methylobacterium extorquens AM1

Microbiology ◽  
2005 ◽  
Vol 151 (11) ◽  
pp. 3723-3728 ◽  
Author(s):  
Meng Zhang ◽  
Kelly A. FitzGerald ◽  
Mary E. Lidstrom
Keyword(s):  
Promoter Activity ◽  
Site Directed Mutagenesis ◽  
Regulatory Sequence ◽  
Methylobacterium Extorquens ◽  
Promoter Regions ◽  
Enhancer Element ◽  
Conserved Sequence ◽  
Normal Expression ◽  
Genetic Constructs ◽  
Transcriptional Fusions

A multiple A-tract sequence has been identified in the promoter regions for the mxaF, pqqA, mxaW, mxbD and mxcQ genes involved in methanol oxidation in Methylobacterium extorquens AM1, a facultative methylotroph. Site-directed mutagenesis was exploited to delete or change this conserved sequence. Promoter-xylE transcriptional fusions were used to assess promoter activity in these mutants. A fiftyfold drop in the XylE activity was observed for the mxaF and pqqA promoters without this sequence, and a five- to sixfold drop in the XylE activity was observed for the mxbD and mxcQ promoters without this sequence. Mutants were generated in the chromosomal copies in which this sequence was either deleted or altered, and these mutants were unable to grow on methanol. When one of these sequences was added to Plac of Escherichia coli, which is a weak constitutive promoter in M. extorquens AM1, the activity increased two- to threefold. These results suggest that this sequence is essential for normal expression of these genes in M. extorquens AM1, and may serve as a general enhancer element for genetic constructs in this bacterium.

Activation Control of pur Gene Expression inLactococcus lactis: Proposal for a Consensus Activator Binding Sequence Based on Deletion Analysis and Site-Directed Mutagenesis of purC and purD Promoter Regions

1998 ◽  
Vol 180 (15) ◽  
pp. 3900-3906 ◽  
Author(s):  
Mogens Kilstrup ◽  
Stine G. Jessing ◽  
Stephanie B. Wichmand-Jørgensen ◽  
Mette Madsen ◽  
Dan Nilsson
Keyword(s):  
Site Directed Mutagenesis ◽  
Deletion Analysis ◽  
Directed Mutagenesis ◽  
Promoter Regions ◽  
Conserved Sequence ◽  
Similar Region ◽  
Low Levels ◽  
High Level ◽  
Transcriptional Start Sites ◽  
Binding Sequence

ABSTRACT A comparison of the purC and purD upstream regions from Lactococcus lactis revealed the presence of a conserved ACCGAACAAT decanucleotide sequence located precisely between −79 and −70 nucleotides upstream from the transcriptional start sites. Both promoters have well-defined −10 regions but lack sequences resembling −35 regions for ς70 promoters. Fusion studies indicated the importance of the conserved sequence in purine-mediated regulation. Adjacent to the conserved sequence in purC is a second and similar region required for high-level expression of the gene. A consensus PurBox sequence (AWWWCCGAACWWT) could be proposed for the three regions. By site-directed mutagenesis we found that mutation of the central G in the PurBox sequence to C resulted in low levels of transcription and the loss of purine-mediated regulation at thepurC and purD promoters. Deletion analysis also showed that the nucleotides before the central CCGAAC core in the PurBox sequence are important. All results support the idea thatpurC and purD transcription is regulated by a transcriptional activator binding to the PurBox sequence.

scholarly journals Identification of the syr-syp Box in the Promoter Regions of Genes Dedicated to Syringomycin and Syringopeptin Production by Pseudomonas syringae pv. syringae B301D

2006 ◽  
Vol 188 (1) ◽  
pp. 160-168 ◽  
Author(s):  
Nian Wang ◽  
Shi-En Lu ◽  
Qingwu Yang ◽  
Sing-Hoi Sze ◽  
Dennis C. Gross
Keyword(s):  
Pseudomonas Syringae ◽  
Promoter Region ◽  
Genomic Island ◽  
Bioinformatic Analysis ◽  
Site Directed Mutagenesis ◽  
Signal Molecules ◽  
Promoter Regions ◽  
Promoter Sequences ◽  
Conserved Sequence ◽  
Transcriptional Start Sites

ABSTRACT The phytotoxins syringopeptin and syringomycin are synthesized by nonribosomal peptide synthetases which are encoded by the syringomycin (syr) and syringopeptin (syp) genomic island of Pseudomonas syringae pv. syringae. Previous studies demonstrated that expression of the syr-syp genes was controlled by the salA-syrF regulatory pathway, which in turn was induced by plant signal molecules. In this study, the 132-kb syr-syp genomic island was found to be organized into five polycistronic operons along with eight individual genes based on reverse transcriptional PCR and bioinformatic analysis. The transcriptional start sites of the salA gene and operons III and IV were located 63, 75, and 104 bp upstream of the start codons of salA, syrP, and syrB1, respectively, using primer extension analysis. The predicted −10/−35 promoter region of operon IV was confirmed based on deletion and site-directed mutagenesis analyses of the syrB1::uidA reporter with β-glucuronidase assays. A 20-bp conserved sequence (TGtCccgN6cggGaCA, termed the syr-syp box) with dyad symmetry around the −35 region was identified via computer analysis for the syr-syp genes/operons responsible for biosynthesis and secretion of syringomycin and syringopeptin. Expression of the syrB1::uidA fusion was decreased 59% when 6 bp was deleted from the 5′ end of the syr-syp box in the promoter region of operon IV. These results demonstrate that the conserved promoter sequences of the syr-syp genes contribute to the coregulation of syringomycin and syringopeptin production.

scholarly journals Mechanism of Interferon Action: Identification of Essential Positions within the Novel 15-Base-Pair KCS Element Required for Transcriptional Activation of the RNA-Dependent Protein Kinasepkr Gene

1998 ◽  
Vol 72 (12) ◽  
pp. 9934-9939 ◽  
Author(s):  
Kelli L. Kuhen ◽  
Jill W. Vessey ◽  
Charles E. Samuel
Keyword(s):  
Transcriptional Activation ◽  
Base Pair ◽  
Promoter Activity ◽  
Mutational Analysis ◽  
Consensus Sequence ◽  
Transient Transfection ◽  
Site Directed Mutagenesis ◽  
Type I ◽  
Conserved Sequence ◽  
Dependent Protein

ABSTRACT RNA-dependent protein kinase PKR is an important regulator of gene expression in interferon (IFN)-treated and virus-infected cells. The 50-kb gene encoding human PKR kinase (pkr) is inducible by IFN. Transfection analyses, using chloramphenicol acetyltransferase (CAT) as the reporter in constructs possessing various 5′-flanking fragments of the human pkr gene, led to the identification of a functional TATA-less promoter that directed IFN-inducible transcription. Sequence determination and mutational analysis of thepkr promoter region revealed, in addition to a functional copy of the IFN-stimulated response element (ISRE) responsible for inducibility by type I IFN, a novel 15-bp element required for optimal promoter activity mediated by the ISRE. This element (5′ GGGAAGGCGGAGTCC 3′), designated KCS for kinase-conserved sequence, is exactly conserved between the human and mousepkr promoters in sequence and position relative to the ISRE. We have now carried out an extensive mutational analysis of the 15-bp KCS element. Site-directed mutagenesis was performed, whereby every base pair position within the KCS element was replaced by each of the other three alternatives. Forty-five substitution mutants were analyzed for promoter activity by transient transfection analysis of untreated and IFN-treated human cells. The results establish 5′ NNRRRGG(C,A,T)GGRGYYN 3′, where R stands for purine and Y stands for pyrimidine, as the consensus sequence for the KCS element, both for basal and for IFN-inducible promoter activity. KCS-binding proteins were detected by electrophoretic mobility shift analysis (EMSA). Competition EMSA established that constitutively expressed nuclear proteins bound the KCS element selectively; KCS protein binding activity correlated with promoter activity in the transient transfection reporter assay.

scholarly journals Identification and Analysis of a Conserved Tcfap2a Intronic Enhancer Element Required for Expression in Facial and Limb Bud Mesenchyme

2007 ◽  
Vol 28 (1) ◽  
pp. 315-325 ◽  
Author(s):  
Weiguo Feng ◽  
Jian Huang ◽  
Jian Zhang ◽  
Trevor Williams
Keyword(s):  
Specific Activity ◽  
Comparative Sequence Analysis ◽  
Limb Bud ◽  
Regulatory Sequence ◽  
Enhancer Element ◽  
Conserved Sequence ◽  
Intronic Enhancer ◽  
The Face ◽  
Multiple Structures

ABSTRACT Tcfap2a, the gene encoding the mouse AP-2α transcription factor, is required for normal development of multiple structures during embryogenesis, including the face and limbs. Using comparative sequence analysis and transgenic-mouse experiments we have identified an intronic enhancer within this gene that directs expression to the face and limb mesenchyme. There are two conserved sequence blocks within this intron, and the larger of these directs tissue-specific activity and is found in all vertebrate Tcfap2a genes analyzed. To assess the role of the enhancer in regulating endogenous mouse Tcfap2a expression, we have deleted this cis-regulatory sequence from the genome. Loss of this element severely impairs Tcfap2a expression in the limb bud mesenchyme but generates only a modest reduction in the facial mesenchyme. The reduction in Tcfap2a transcription is accompanied by altered patterning of the forelimb, resulting in postaxial polydactyly. These results indicate that the major role for this enhancer resides within the limb bud, and it serves to maintain a level of Tcfap2a expression that limits the size of the hand plate and the associated number of digit primordia. The potential role of this cis-acting sequence in modeling the size and shape of the face and limbs during evolution is discussed.

scholarly journals Characterization of the Autographa californica Nucleopolyhedrovirus Ubiquitin Gene Promoter

2008 ◽  
Vol 63 (3-4) ◽  
pp. 277-283 ◽  
Author(s):  
Xu Ai Lin ◽  
Yin Chen ◽  
Wei Hua Xu ◽  
Yong Zhu Yi ◽  
Zhi Fang Zhang
Keyword(s):  
Promoter Region ◽  
Promoter Activity ◽  
Gene Promoter ◽  
Active Regions ◽  
Autographa Californica ◽  
Negative Regulator ◽  
Site Directed Mutagenesis ◽  
Promoter Regions ◽  
Transient Expression Assay ◽  
Native Promoter

Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) encodes an ubiquitin protein, which may be involved in virus infection. Functional analysis of the AcMNPV ubiquitin promoter was performed by progressive deletion of sequence or mutation of putative cis-activating motifs in the promoter region. In the presence of viral factors, a transient expression assay demonstrated that the active regions responsive to promoter transcription are mainly located within the range of -595 to -382 bp upstream of ATG. A 196-bp fragment (-383 to -187 bp), consisting of the distal TAAG, CAAT motif and TATA box, could also drive the expression of a reporter gene. Site-directed mutagenesis analyses indicated that mutations of TATA boxes and TAAG motifs reduce the promoter activity remarkably, while CAAT mutations enhance the promoter activity by about 3- or 4-fold as compared to the native promoter. All the results suggested that two continuous promoter regions are involved in the transcription of the ubiquitin gene and the cis-activating motifs corresponding to viral factors are mainly present within the 5′ region of the promoter. In addition, CAAT motifs in the promoter region function as negative regulator(s) binding sites

Optimization of the Expression of 1,3-1,4-β-glucanase Gene from Rhizomucor miehei in the Komagataella kurtzmanii yeast

2019 ◽  
Vol 35 (5) ◽  
pp. 3-11 ◽  
Author(s):  
I.I. Gubaidullin ◽  
A.S. Fedorov ◽  
D.G. Kozlov
Keyword(s):  
Target Gene ◽  
Rhizomucor Miehei ◽  
Leader Peptide ◽  
Promoter Regions ◽  
Functional Elements ◽  
Resource Center ◽  
The Status ◽  
Pichia Pastoris Yeast ◽  
The Difference ◽  
Genetic Constructs

Key functional elements of the vector (promoter, leader and terminator regions) that provide the expression of a target l,3-l,4-(3-glucanase gene from Rhizomucor miehei in the Komagataella kurtzmanii yeast have been optimized. It was shown that the promoter regions of the gene AOX1 from the Pichia pastoris yeast currently reclassified as Komagataella phaffti and from К. kurtzmanii yeast as parts of a vector provided equal levels of expression of the target gene in the cells of the recipient strain К. kurtzmanii Y727his4, i.e. they were completely interchangeable. This means that genetic constructs that were previously developed for the biosynthesis of recombinant proteins in К. phajfii are able to provide an effective expression in the К kurtzmanii yeast. The leader peptide MF4I (used as a variant of mif4I containing one amino acid substitution) and the leader peptide maxHH (containing the double proregion of the Hspl50 protein from Saccharomyces cerevisiae) confirmed the status of the most powerful elements among the five leader sequences analyzed. Their efficiency was 1.7 times higher than that of the standard leader from the yeast alpha-factor, and by 20% higher than the characteristics of the second group of artificial leaders. At the same time, it was found that, the choice of the terminator region had the strongest influence on the expression of the target gene among all of the vector functional elements. The best terminator elements were variants derived from the transcription termination region of the AOX1 gene, and the difference in the expression level of the target gene using different terminators was approximately 4.5 times. Based on the analysis of the obtained data, the optimal composition of the key functional elements of the expression vector was determined ; it included the promoter and terminator regions of the AOX1 yeast gene and one of the artificial leaders, mif4I or maxHH. β-glucanase, Komagataella kurtzmanii, yeast, secretion, strain producer The work was financially supported by the Ministry of Science and Higher education of the Russian Federation (Unique Project Identifier RFMEFI60717X0179) using the Unique Scientific Facility of the National Bio-Resource Center «All-Russian Collection of Industrial Microorganisms», NRC «Kurchatov Institute» - GOSNIIGENETIKA

scholarly journals DNA Methylation of Human Choline Kinase Alpha Promoter-Associated CpG Islands in MCF-7 Cells

Genes ◽  
2021 ◽  
Vol 12 (6) ◽  
pp. 853
Author(s):  
Siti Aisyah Faten Mohamed Sa’dom ◽  
Sweta Raikundalia ◽  
Shaharum Shamsuddin ◽  
Wei Cun See Too ◽  
Ling Ling Few
Keyword(s):  
Dna Methylation ◽  
Transcription Factor ◽  
Binding Site ◽  
Promoter Activity ◽  
Cytosine Methylation ◽  
Cpg Island ◽  
Cpg Islands ◽  
Site Directed Mutagenesis ◽  
Choline Kinase ◽  
Mcf 7

Choline kinase (CK) is the enzyme catalyzing the first reaction in CDP-choline pathway for the biosynthesis of phosphatidylcholine. Higher expression of the α isozyme of CK has been implicated in carcinogenesis, and inhibition or downregulation of CKα (CHKA) is a promising anticancer approach. This study aimed to investigate the regulation of CKα expression by DNA methylation of the CpG islands found on the promoter of this gene in MCF-7 cells. Four CpG islands have been predicted in the 2000 bp promoter region of ckα (chka) gene. Six CpG island deletion mutants were constructed using PCR site-directed mutagenesis method and cloned into pGL4.10 vectors for promoter activity assays. Deletion of CpG4C region located between –225 and –56 significantly increased the promoter activity by 4-fold, indicating the presence of important repressive transcription factor binding site. The promoter activity of methylated full-length promoter was significantly lower than the methylated CpG4C deletion mutant by 16-fold. The results show that DNA methylation of CpG4C promotes the binding of the transcription factor that suppresses the promoter activity. Electrophoretic mobility shift assay analysis showed that cytosine methylation at MZF1 binding site in CpG4C increased the binding of putative MZF1 in nuclear extract. In conclusion, the results suggest that DNA methylation decreased the promoter activity by promoting the binding of putative MZF1 transcription factor at CpG4C region of the ckα gene promoter.

scholarly journals Reverse Genetic Analysis of the Transcription Regulatory Sequence of the Coronavirus Transmissible Gastroenteritis Virus

2004 ◽  
Vol 78 (11) ◽  
pp. 6061-6066 ◽  
Author(s):  
Kristopher M. Curtis ◽  
Boyd Yount ◽  
Amy C. Sims ◽  
Ralph S. Baric
Keyword(s):  
Genetic Analysis ◽  
Full Length ◽  
Transmissible Gastroenteritis Virus ◽  
Regulatory Sequence ◽  
Reverse Genetic ◽  
Conserved Sequence ◽  
Gastroenteritis Virus ◽  
Flanking Sequences ◽  
Transmissible Gastroenteritis ◽  
Discontinuous Transcription

ABSTRACT Coronavirus discontinuous transcription uses a highly conserved sequence (CS) in the joining of leader and body RNAs. Using a full-length infectious construct of transmissable gastroenteritis virus, the present study demonstrates that subgenomic transcription is heavily influenced by upstream flanking sequences and supports a mechanism of transcription attenuation that is regulated in part by a larger domain composed of primarily upstream flanking sequences which select appropriately positioned CS elements for synthesis of subgenomic RNAs.

An amphibian cytoskeletal-type actin gene is expressed exclusively in muscle tissue

Development ◽  
1987 ◽  
Vol 101 (2) ◽  
pp. 393-402 ◽  
Author(s):  
T.J. Mohun ◽  
N. Garrett
Keyword(s):  
Nucleotide Sequence ◽  
Protein Isoforms ◽  
Actin Gene ◽  
Promoter Regions ◽  
Conserved Sequence ◽  
Beta Actin ◽  
Genes Encoding ◽  
Actin Mrna ◽  
Ccaat Box ◽  
Equivalent Region

The complete nucleotide sequence of two Xenopus actin genes encoding cytoskeletal protein isoforms has been determined. Transcripts from these genes are remarkably similar in nucleotide sequence throughout their length and code for type-5 and type-8 cytoskeletal actins. Both share some sequence homology with human gamma-actin mRNA within the 3′ untranslated region but none with the equivalent region of any vertebrate beta-actin transcript. The promoter regions of the two Xenopus genes are virtually identical from the cap site to the CCAAT box and show extensive homology further upstream. Despite such similarity, the two genes are divergently expressed during embryonic development. The type-5 actin gene is expressed in all regions of the developing embryo whilst the type-8 gene is coregulated with the muscle-specific skeletal actin gene. In common with mammalian and avian cytoskeletal actin counterparts, the Xenopus genes possess a conserved sequence within their promoter that has previously been identified as a transcription-factor-binding site.

scholarly journals Transcriptional Regulation of the Copper Transporter Mfc1 in Meiotic Cells

2013 ◽  
Vol 12 (4) ◽  
pp. 575-590 ◽  
Author(s):  
Jude Beaudoin ◽  
Raphaël Ioannoni ◽  
Stéphane Mailloux ◽  
Samuel Plante ◽  
Simon Labbé
Keyword(s):  
Transcriptional Activation ◽  
Regulatory Element ◽  
Specific Protein ◽  
Copper Transport ◽  
Site Directed Mutagenesis ◽  
Regulatory Sequence ◽  
Transcriptional Level ◽  
Binding Studies ◽  
Copper Transporter ◽  
Content Type

ABSTRACT Mfc1 is a meiosis-specific protein that mediates copper transport during the meiotic program in Schizosaccharomyces pombe . Although the mfc1 + gene is induced at the transcriptional level in response to copper deprivation, the molecular determinants that are required for its copper starvation-dependent induction are unknown. Promoter deletion and site-directed mutagenesis have allowed identification of a new cis -regulatory element in the promoter region of the mfc1 + gene. This cis -acting regulatory sequence containing the sequence TCGGCG is responsible for transcriptional activation of mfc1 + under low-copper conditions. The TCGGCG sequence contains a CGG triplet known to serve as a binding site for members of the Zn (2) Cys (6) binuclear cluster transcriptional regulator family. In agreement with this fact, one member of this group of regulators, denoted Mca1, was found to be required for maximum induction of mfc1 + gene expression. Analysis of Mca1 cellular distribution during meiosis revealed that it colocalizes with both chromosomes and sister chromatids during early, middle, and late phases of the meiotic program. Cells lacking Mca1 exhibited a meiotic arrest at metaphase I under low-copper conditions. Binding studies revealed that the N-terminal 150-residue segment of Mca1 expressed as a fusion protein in Escherichia coli specifically interacts with the TCGGCG sequence of the mfc1 + promoter. Taken together, these results identify the cis -regulatory TCGGCG sequence and the transcription factor Mca1 as critical components for activation of the meiotic copper transport mfc1 + gene in response to copper starvation.

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