Analysis of Promoters Recognized by HrpL, an Alternative σ-Factor Protein from Pantoea agglomerans pv. gypsophilae

HrpL, an alternative σ factor, activates the transcription of the Hrp regulon by its binding to a common “hrp box” promoter. Based on computational techniques, the hrp box previously was defined as a consensus bipartite cis element, 5′-GGAACC-N15–16-CCACNNA-3′. The present report combines a quantitative in vivo assay for measuring Hrp promoter activity with site-specific mutagenesis to analyze the effect of consensus and nonconsensus nucleotides on promoter activity. The analysis was carried out with Hop effectors of the tumorigenic bacterium Pantoea agglomerans pv. gypsophilae, in which HrpL is indispensable for gall formation. Mutational analysis indicates that the hrp box consensus can be divided into crucial and noncrucial nucleotides. The first 5 nucleotides (nt) of the -35 consensus motif (GGAAC) and the 3 nt of the -10 motif (ACNNA) are crucial, whereas other consensus and adjacent nonconsensus nucleotides exert a significant effect on the promoter's strength. With spacing of 13 or 17 nt between the two motifs, significant activity was still retained. Gel shift assays indicated that deletion of GG from the -35 consensus motif eliminated HrpL binding, whereas mutations in the -10 consensus motif or modification of the spacing, which eliminates promoter activity, did not elicit any effect. The degeneracy in Hrp promoters of four hrp and type III effector genes of P. agglomerans pv. gypsophilae indicated significant differences in promoter activity, whereas increasing the promoter strength of the Hop effector, HsvG, resulted in overexpression of gall formation.

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Dasatinib (SPRYCEL®) in Patients (pts) with Chronic Myelogenous Leukemia in Accelerated Phase (AP-CML) That Is Imatinib-Resistant (im-r) or -Intolerant (im-i): Updated Results of the CA180–005 ’START-A’ Phase II Study.

Blood ◽

10.1182/blood.v108.11.2160.2160 ◽

2006 ◽

Vol 108 (11) ◽

pp. 2160-2160 ◽

Cited By ~ 11

Author(s):

Jorge Cortes ◽

D.W. Kim ◽

F. Guilhot ◽

G. Rosti ◽

R.T. Silver ◽

...

Keyword(s):

Kinase Inhibitor ◽

Mutational Analysis ◽

Present Report ◽

Cytogenetic Response ◽

Myelogenous Leukemia ◽

Molecular Response ◽

Significant Activity ◽

Hematologic Response ◽

Grade 3

Abstract Dasatinib (SPRYCEL®, formerly BMS-354825) is a novel, oral, multi-targeted kinase inhibitor that induces complete hematologic and cytogenetic remissions in pts in all phases of im-r or im-i CML. START-A is an open-label study of dasatinib in pts with im-r or im-i AP-CML. Preliminary results with early follow-up suggested significant activity. The present report updates the results of this study with a minimum of 9 months of follow-up. Dasatinib was given orally at 70 mg twice daily (BID). Dose escalation to 100 mg BID was allowed for inadequate initial response and reduction to 50 or 40 mg BID for persistent toxicity. Evaluation included weekly blood counts and monthly bone marrow including cytogenetics. Molecular evaluation of BCR-ABL transcript levels by real-time qPCR was performed at baseline and upon documentation of complete cytogenetic response. A total of 174 pts (161 im-r and 13 im-i) were enrolled between December 2004 and July 2005 in 39 centers worldwide. There were 96 (55%) males; median age was 57 years (range 22–86); median time from original diagnosis of CML was 82 months. Prior therapy included im in all pts (>600 mg/day in 91 (52%), im for >3 years in 103 (59%) pts, interferon in 126 (72%) pts, stem cell transplantation in 23 (13%) pts. Major cytogenetic response (MCyR) to prior im had been seen in 57 (33%) pts. The average daily dose (median across all pts) was 130 mg/day (range 44–199). Major hematologic response (MaHR) was documented in 110 (63%) pts with complete hematologic response in 75 (43%) and no evidence of leukemia in 35 (20%). At 9 months, 85% of pts have maintained their MaHR. MCyR was documented in 65 (37%) pts, complete in 49 (28%), partial in 16 (9%). Median progression-free survival (PFS) has not been reached; estimated PFS at 9 months is 70%. In the 94 pts with BCR-ABL mutations at baseline the MaHR rate was 69%. Generally, response rates were similar in the im-i and im-r groups. Cytopenias were significant with grade 3–4 thrombocytopenia and neutropenia in 82% and 76% of pts, respectively. Non-hematologic toxicities were generally mild to moderate. The most frequent (% any grade, % grade 3–4) were diarrhea (51%, 8%), headache (29%, 1%), fatigue (26%, 4%), fever (25%, 3%) and pleural effusion (25%, 3%). Pleural effusions were optimally managed with dose interruption and diuretics and/or pulse steroids. Dasatinib is highly effective in pts with im-r or im-i AP-CML with high rates of durable MaHR and MCyR. An updated analysis with at least 12 months of follow-up, including molecular response data and mutational analysis at time of progression, will be presented.

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Mutational Analysis of the Myxococcus xanthus Ω4499 Promoter Region Reveals Shared and Unique Properties in Comparison with Other C-Signal-Dependent Promoters

Journal of Bacteriology ◽

10.1128/jb.186.12.3766-3776.2004 ◽

2004 ◽

Vol 186 (12) ◽

pp. 3766-3776 ◽

Cited By ~ 14

Author(s):

Deborah R. Yoder ◽

Lee Kroos

Keyword(s):

Base Pair ◽

Promoter Region ◽

Promoter Activity ◽

Mutational Analysis ◽

Myxococcus Xanthus ◽

Promoter Regions ◽

Multicellular Development ◽

Σ Factor ◽

Signal Dependence ◽

Promoter Mutations

ABSTRACT The bacterium Myxococcus xanthus undergoes multicellular development during times of nutritional stress and uses extracellular signals to coordinate cell behavior. C-signal affects gene expression late in development, including that of Ω4499, an operon identified by insertion of Tn5 lac into the M. xanthus chromosome. The Ω4499 promoter region has several sequences in common with those found previously to be important for expression of other C-signal-dependent promoters. To determine if these sequences are important for Ω4499 promoter activity, the effects of mutations on expression of a downstream reporter gene were tested in M. xanthus. Although the promoter resembles those recognized by Escherichia coli σ54, mutational analysis implied that a σ70-type σ factor likely recognizes the promoter. A 7-bp sequence known as a C box and a 5-bp element located 6 bp upstream of the C box have been shown to be important for expression of other C-signal-dependent promoters. The Ω4499 promoter region has C boxes centered at −33 and −55 bp, with 5-bp elements located 7 and 8 bp upstream, respectively. A multiple-base-pair mutation in any of these sequences reduced Ω4499 promoter activity more than twofold. Single base-pair mutations in the C box centered at −33 bp yielded a different pattern of effects on expression than similar mutations in other C boxes, indicating that each functions somewhat differently. An element from about −81 to −77 bp exerted a twofold positive effect on expression but did not appear to be responsible for the C-signal dependence of the Ω4499 promoter. Mutations in sigD and sigE, which are genes that encode σ factors, reduced expression from the Ω4499 promoter. The results provide further insight into the regulation of C-signal-dependent genes, demonstrating both shared and unique properties among the promoter regions so far examined.

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Analysis of the Ferric Citrate Transport Gene Promoter of Escherichia coli

Journal of Bacteriology ◽

10.1128/jb.185.7.2387-2391.2003 ◽

2003 ◽

Vol 185 (7) ◽

pp. 2387-2391 ◽

Cited By ~ 14

Author(s):

Sabine Enz ◽

Susanne Mahren ◽

Claudia Menzel ◽

Volkmar Braun

Keyword(s):

Transcription Initiation ◽

Promoter Activity ◽

Mutational Analysis ◽

Regulatory Element ◽

Gene Promoter ◽

Ferric Citrate ◽

Nucleotide Substitutions ◽

Citrate Transport ◽

Σ Factor ◽

Subsequent Step

ABSTRACT FecI, an extracytoplasmic-function σ factor, is required for initiation of transcription of the ferric citrate transport genes. A mutational analysis of the fecA promoter revealed that the nonconserved −10 region and a downstream regulatory element are important for fecA promoter activity. However, nucleotide substitutions in the well-conserved −35 region also have an effect on the fecA promoter activity. Titration of FecI suggests that the FecI-RNA polymerase holoenzyme does not bind strongly to the downstream regulatory element, which is therefore probably involved in a subsequent step of transcription initiation.

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Mutational Analysis of the ompA Promoter from Flavobacterium johnsoniae

Journal of Bacteriology ◽

10.1128/jb.00401-07 ◽

2007 ◽

Vol 189 (14) ◽

pp. 5108-5118 ◽

Cited By ~ 39

Author(s):

Shicheng Chen ◽

Michael Bagdasarian ◽

Michael G. Kaufman ◽

Adam K. Bates ◽

Edward D. Walker

Keyword(s):

Promoter Activity ◽

Mutational Analysis ◽

Core Promoter ◽

Gc Content ◽

Pronounced Effect ◽

Promoter Elements ◽

Spacer Length ◽

Promoter Sequences ◽

E Coli ◽

Flavobacterium Johnsoniae

ABSTRACT Sequences that mediate the initiation of transcription in Flavobacterium species are not well known. The majority of identified Flavobacterium promoter elements show homology to those of other members of the phylum Bacteroidetes, but not of proteobacteria, and they function poorly in Escherichia coli. In order to analyze the Flavobacterium promoter structure systematically, we investigated the −33 consensus element, −7 consensus element, and spacer length of the Flavobacterium ompA promoter by measuring the effects of site-directed mutations on promoter activity. The nonconserved sequences in the spacer region and in regions close to the consensus motifs were randomized in order to determine their importance for promoter activity. Most of the base substitutions in these regions caused large decreases in promoter activity. The optimal −33/−7 motifs (TTTG/TANNTTTG) were identical to Bacteroides fragilis σABfr consensus −33/−7 promoter elements but lacked similarity to the E. coli σ70 promoter elements. The length of the spacer separating the −33 and −7 motifs of the ompA promoter also had a pronounced effect on promoter activity, with 19 bp being optimal. In addition to the consensus promoter elements and spacer length, the GC content of the core promoter sequences had a pronounced effect on Flavobacterium promoter activity. This information was used to conduct a scan of the Flavobacterium johnsoniae and B. fragilis genomes for putative promoters, resulting in 188 hits in B. fragilis and 109 hits in F. johnsoniae.

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Spo0A-Dependent Activation of an Extended −10 Region Promoter in Bacillus subtilis

Journal of Bacteriology ◽

10.1128/jb.188.4.1411-1418.2006 ◽

2006 ◽

Vol 188 (4) ◽

pp. 1411-1418 ◽

Cited By ~ 13

Author(s):

Guangnan Chen ◽

Amrita Kumar ◽

Travis H. Wyman ◽

Charles P. Moran

Keyword(s):

Bacillus Subtilis ◽

Base Pair ◽

Promoter Activity ◽

Mutational Analysis ◽

Dna Binding Protein ◽

Base Pairs ◽

Dnase I Footprinting ◽

Level Of Activity ◽

High Level ◽

Activity Dependent

ABSTRACT At the onset of endospore formation in Bacillus subtilis the DNA-binding protein Spo0A directly activates transcription from promoters of about 40 genes. One of these promoters, Pskf, controls expression of an operon encoding a killing factor that acts on sibling cells. AbrB-mediated repression of Pskf provides one level of security ensuring that this promoter is not activated prematurely. However, Spo0A also appears to activate the promoter directly, since Spo0A is required for Pskf activity in a ΔabrB strain. Here we investigate the mechanism of Pskf activation. DNase I footprinting was used to determine the locations at which Spo0A bound to the promoter, and mutations in these sites were found to significantly reduce promoter activity. The sequence near the −10 region of the promoter was found to be similar to those of extended −10 region promoters, which contain a TRTGn motif. Mutational analysis showed that this extended −10 region, as well as other base pairs in the −10 region, is required for Spo0A-dependent activation of the promoter. We found that a substitution of the consensus base pair for the nonconsensus base pair at position −9 of Pskf produced a promoter that was active constitutively in both ΔabrB and Δspo0A ΔabrB strains. Therefore, the base pair at position −9 of Pskf makes its activity dependent on Spo0A binding, and the extended −10 region motif of the promoter contributes to its high level of activity.

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Identification of functional TTF-1 and Sp1/Sp3 sites in the upstream promoter region of rabbit SP-B gene

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.2000.278.3.l477 ◽

2000 ◽

Vol 278 (3) ◽

pp. L477-L484 ◽

Cited By ~ 14

Author(s):

Ramgopal Margana ◽

Kiflu Berhane ◽

M. Nurul Alam ◽

Vijayakumar Boggaram

Keyword(s):

Promoter Region ◽

Promoter Activity ◽

Mutational Analysis ◽

Surfactant Protein ◽

Alveolar Type ◽

Deletion Mapping ◽

Clara Cells ◽

Mobility Shift ◽

Upstream Promoter ◽

Upstream Promoter Region

Surfactant protein B (SP-B) is essential for the maintenance of biophysical properties and physiological function of pulmonary surfactant. SP-B mRNA is expressed in a cell type-restricted manner in alveolar type II and bronchiolar (Clara) epithelial cells of the lung and is developmentally induced. In NCI-H441 cells, a lung cell line with characteristics of Clara cells, a minimal promoter region comprising −236 to +39 nucleotides supports high-level expression of chloramphenicol acetyltransferase reporter activity. In the present investigation, we characterized the upstream promoter region, −236 to −140 nucleotides, that is essential for promoter activity. Deletion mapping identified two segments, −236 to −170 and −170 to −140 nucleotides, that are important for promoter activity. Mutational analysis and gel mobility shift experiments identified thyroid transcription factor-1, Sp1, and Sp3 as important trans-acting factors that bind to sequences in the upstream promoter region. Our data suggest that SP-B promoter activity is dependent on interactions between factors bound to upstream and downstream regions of the promoter.

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A new MEFV gene mutation in an Iranian patient with familial Mediterranean fever

Reumatismo ◽

10.4081/reumatismo.2019.1141 ◽

2019 ◽

Vol 71 (2) ◽

pp. 85-87

Author(s):

S. Farjadian ◽

F. Bonatti ◽

A. Soriano ◽

M. Reina ◽

A. Adorni ◽

...

Keyword(s):

Familial Mediterranean Fever ◽

Mutational Analysis ◽

Novel Mutation ◽

Present Report ◽

Case Reports ◽

Mefv Gene ◽

Recurrent Fever ◽

Iranian Patient ◽

Mediterranean Fever ◽

Exon 10

Familial mediterranean fever (FMF) is an inherited autoinflammatory disorder characterized by recurrent episodes of fever and painful inflammation involving the intra-abdominal organs, the lungs and the joints, which is highly prevalent in specific ethnic groups including the Iranians. We report a 12-year-old boy from Iran, with a clinical history of recurrent fever. Based on the suggestive clinical data, mutational analysis revealed the presence of the novel c.1945C>T heterozygous variant in exon 10, which leads to a leucine to phenylalanine change at position 649 of the protein. The mutation was inherited from the mother. This novel mutation lies in exon 10 of the MEFV gene, which encodes for a domain called B30.2-SPRY, located in the C-terminal region of the pyrin protein and contains the most frequent mutations associated with FMF. The present report expands the spectrum of MEFV gene mutations associated with FMF. The uniqueness of this study, compared with other published case reports, consists in the new mutation found in the MEFV gene. In fact, new mutations in this gene are of high interest, in order to better understand the role of this gene in autoinflammation.

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Influence of cis Element Arrangement on Promoter Strength in Trichoderma reesei

Applied and Environmental Microbiology ◽

10.1128/aem.01742-17 ◽

2017 ◽

Vol 84 (1) ◽

Cited By ~ 12

Author(s):

Daniel P. Kiesenhofer ◽

Robert L. Mach ◽

Astrid R. Mach-Aigner

Keyword(s):

Trichoderma Reesei ◽

Transcription Start Site ◽

Binding Sites ◽

Inverted Repeat ◽

Promoter Activity ◽

Promoter Strength ◽

Start Site ◽

Transcription Start ◽

Content Type ◽

Sheer Number

ABSTRACTTrichoderma reeseican produce up to 100 g/liter of extracellular proteins. The major and industrially relevant products are cellobiohydrolase I (CBHI) and the hemicellulase XYNI. The genes encoding both enzymes are transcriptionally activated by the regulatory protein Xyr1. The first 850 nucleotides of thecbh1promoter contain 14 Xyr1-binding sites (XBS), and 8 XBS are present in thexyn1promoter. Some of these XBS are arranged in tandem and others as inverted repeats. One suchciselement, an inverted repeat, plays a crucial role in the inducibility of thexyn1promoter. We investigated the impact of the properties of suchciselements by shuffling them by insertion, exchange, deletion, and rearrangement ofciselements in both thecbh1andxyn1promoter. A promoter-reporter assay using theAspergillus nigergoxAgene allowed us to measure changes in the promoter strength and inducibility. Most strikingly, we found that an inverted repeat of XBS causes an important increase incbh1promoter strength and allows induction by xylan or wheat straw. Furthermore, evidence is provided that the distances ofciselements to the transcription start site have important influence on promoter activity. Our results suggest that the arrangement and distances ofciselements have large impacts on the strength of thecbh1promoter, whereas the sheer number of XBS has only secondary importance. Ultimately, the biotechnologically importantcbh1promoter can be improved byciselement rearrangement.IMPORTANCEIn the present study, we demonstrate that the arrangement ofciselements has a major impact on promoter strength and inducibility. We discovered an influence on promoter activity by the distances ofciselements to the transcription start site. Furthermore, we found that the configuration ofciselements has a greater effect on promoter strength than does the sheer number of transactivator binding sites present in the promoter. Altogether, the arrangement ofciselements is an important factor that should not be overlooked when enhancement of gene expression is desired.

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Transcriptional Activation of the Decidual/Trophoblast Prolactin-Related Protein Gene1

Endocrinology ◽

10.1210/endo.140.9.6954 ◽

1999 ◽

Vol 140 (9) ◽

pp. 4032-4039 ◽

Cited By ~ 9

Author(s):

Kyle E. Orwig ◽

Michael J. Soares

Keyword(s):

Transcriptional Activation ◽

Promoter Activity ◽

Mutational Analysis ◽

Regulatory Element ◽

Gene Activation ◽

Cell Formation ◽

Regulatory Elements ◽

Related Protein ◽

Trophoblast Cells ◽

Decidual Cells

Abstract The decidual/trophoblast PRL-related protein (d/tPRP) is dually expressed by decidual and trophoblast cells during pregnancy. We have characterized the proximal d/tPRP promoter responsible for directing d/tPRP expression in decidual and trophoblast cells. We have demonstrated that the proximal 93 bp of d/tPRP 5′-flanking DNA are sufficient to direct luciferase gene expression in primary decidual and Rcho-1 trophoblast cells, but not in fibroblast, undifferentiated uterine stromal cells or trophoblast cells of a labyrinthine lineage. The 93-bp d/tPRP promoter was also sufficient to direct differentiation-dependent expression in trophoblast giant cells. Mutational analysis demonstrated the differential importance of activating protein-1 and Ets regulatory elements (located within the proximal 93 bp of d/tPRP 5′-flanking DNA) for activation of the d/tPRP promoter in decidual vs. trophoblast cells. Disruption of the activating protein-1 regulatory element inhibited d/tPRP promoter activity by more than 95% in decidual cells, and approximately 80% trophoblast cells. Disruption of the Ets regulatory element reduced d/tPRP promoter activity by approximately 50% in decidual cells, while inactivating the d/tPRP promoter in trophoblast cells. Protein interactions with the trophoblast Ets regulatory element were shown to be cell type specific and to change during trophoblast giant cell formation. In conclusion, a 93-bp region of the d/tPRP promoter is shown to contain regulatory elements sufficient for gene activation in decidual and trophoblast cells.

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Transcriptional Regulation of BK Virus by Nuclear Factor of Activated T Cells

Journal of Virology ◽

10.1128/jvi.01918-09 ◽

2009 ◽

Vol 84 (4) ◽

pp. 1722-1730 ◽

Cited By ~ 18

Author(s):

Joslynn A. Jordan ◽

Kate Manley ◽

Aisling S. Dugan ◽

Bethany A. O'Hara ◽

Walter J. Atwood

Keyword(s):

T Cells ◽

Promoter Activity ◽

Mutational Analysis ◽

Adult Population ◽

Bk Virus ◽

Human Polyomavirus ◽

Luciferase Reporter ◽

Nuclear Factor ◽

Activated T Cells ◽

Transplant Patients

ABSTRACT The human polyomavirus BK virus (BKV) is a common virus for which 80 to 90% of the adult population is seropositive. BKV reactivation in immunosuppressed patients or renal transplant patients is the primary cause of polyomavirus-associated nephropathy (PVN). Using the Dunlop strain of BKV, we found that nuclear factor of activated T cells (NFAT) plays an important regulatory role in BKV infection. Luciferase reporter assays and chromatin immunoprecipitation assays demonstrated that NFAT4 bound to the viral promoter and regulated viral transcription and infection. The mutational analysis of the NFAT binding sites demonstrated complex functional interactions between NFAT, c-fos, c-jun, and the p65 subunit of NF-κB that together influence promoter activity and viral growth. These data indicate that NFAT is required for BKV infection and is involved in a complex regulatory network that both positively and negatively influences promoter activity and viral infection.

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