Architectural specificity in chromatin structure at the TATA box in vivo: Nucleosome displacement upon β-phaseolin gene activation

Extensive studies of the β-phaseolin (phas) gene in transgenic tobacco have shown that it is highly active during seed embryogenesis but is completely silent in leaf and other vegetative tissues. In vivo footprinting revealed that the lack of even basal transcriptional activity in vegetative tissues is associated with the presence of a nucleosome that is rotationally positioned with base pair precision over three phased TATA boxes present in the phas promoter. Positioning is sequence-dependent because an identical rotational setting is obtained upon nucleosome reconstitution in vitro. A comparison of DNase I and dimethyl sulfate footprints in vivo and in vitro strongly suggests that this repressive chromatin architecture is remodeled concomitant with gene activation in the developing seed. This leads to the disruption of histone-mediated DNA wrapping and the assembly of the TATA boxes into a transcriptionally competent nucleoprotein complex.

Upstream A-tracts increase bacterial promoter activity through interactions with the RNA polymerase α subunit

Upstream A-tracts stimulate transcription from a variety of bacterial promoters, and this has been widely attributed to direct effects of the intrinsic curvature of A-tract-containing DNA. In this work we report experiments that suggest a different mechanism for the effects of upstream A-tracts on transcription. The similarity of A-tract-containing sequences to the adenine- and thymine-rich upstream recognition elements (UP elements) found in some bacterial promoters suggested that A-tracts might increase promoter activity by interacting with the α subunit of RNA polymerase (RNAP). We found that an A-tract-containing sequence placed upstream of the Escherichia coli lac or rrnB P1 promoters stimulated transcription both in vivo and in vitro, and that this stimulation required the C-terminal (DNA-binding) domain of the RNAP α subunit. The A-tract sequence was protected by wild-type RNAP but not by α-mutant RNAPs in footprints. The effect of the A-tracts on transcription was not as great as that of the most active UP elements, consistent with the degree of similarity of the A-tract sequence to the UP element consensus. A-tracts functioned best when positioned close to the −35 hexamer rather than one helical turn farther upstream, similar to the positioning optimal for UP element function. We conclude that A-tracts function as UP elements, stimulating transcription by providing binding site(s) for the RNAP αCTD, and we suggest that these interactions could contribute to the previously described wrapping of promoter DNA around RNAP.

Conversion of DNA gyrase into a conventional type II topoisomerase

DNA gyrase is unique among topoisomerases in its ability to introduce negative supercoils into closed-circular DNA. We have demonstrated that deletion of the C-terminal DNA-binding domain of the A subunit of gyrase gives rise to an enzyme that cannot supercoil DNA but relaxes DNA in an ATP-dependent manner. Novobiocin, a competitive inhibitor of ATP binding by gyrase, inhibits this reaction. The truncated enzyme, unlike gyrase, does not introduce a right-handed wrap when bound to DNA and stabilizes DNA crossovers; characteristics reminiscent of conventional type II topoisomerases. This new enzyme form can decatenate DNA circles with increased efficiency compared with intact gyrase and, as a result, can complement the temperature-sensitive phenotype of a parCts mutant. Thus these results suggest that the unique properties of DNA gyrase are attributable to the wrapping of DNA around the C-terminal DNA-binding domains of the A subunits and provide an insight into the mechanism of type II topoisomerases.

The [(G/C)3NN]n motif: a common DNA repeat that excludes nucleosomes.

Nucleosomes, the basic structural elements of chromosomes, consist of 146 bp of DNA coiled around an octamer of histone proteins, and their presence can strongly influence gene expression. Considerations of the anisotropic flexibility of nucleotide triplets containing 3 cytosines or guanines suggested that a [5'(G/C)3 NN3']n motif might resist wrapping around a histone octamer. To test this, DNAs were constructed containing a 5'-CCGNN-3' pentanucleotide repeat with the Ns varied. Using in vitro nucleosome reconstitution and electron microscopy, a plasmid with 48 contiguous CCGNN repeats strongly excluded nucleosomes in the repeat region. Competitive reconstitution gel retardation experiments using DNA fragments containing 12, 24, or 48 CCGNN repeats showed that the propensity to exclude nucleosomes increased with the length of the repeat. Analysis showed that a 268-bp DNA containing a (CCGNN)48 block is 4.9 +/- 0.6-fold less efficient in nucleosome assembly than a similar length pUC19 fragment and approximately 78-fold less efficient than a similar length (CTG)n sequence, based on results from previous studies. Computer searches against the GenBank database for matches with a [(G/C)3NN]48 sequence revealed numerous examples that frequently were present in the control regions of "TATA-less" genes, including the human ETS-2 and human dihydrofolate reductase genes. In both cases the (G/C)3NN repeat, present in the promoter region, co-maps with loci previously shown to be nuclease hypersensitive sites.

Toxoplasma invasion: the parasitophorous vacuole is formed from host cell plasma membrane and pinches off via a fission pore.

Most intracellular pathogens avoid lysing their host cells during invasion by wrapping themselves in a vacuolar membrane. This parasitophorous vacuole membrane (PVM) is often retained, serving as a critical transport interface between the parasite and the host cell cytoplasm. To test whether the PVM formed by the parasite Toxoplasma gondii is derived from host cell membrane or from lipids secreted by the parasite, we used time-resolved capacitance measurements and video microscopy to assay host cell surface area during invasion. We observed no significant change in host cell surface area during PVM formation, demonstrating that the PVM consists primarily of invaginated host cell membrane. Pinching off of the PVM from the host cell membrane occurred after an unexpected delay (34-305 sec) and was seen as a 0.219 +/- 0.006 pF drop in capacitance, which corresponds well to the predicted surface area of the entire PVM (30-33 microns2). The formation and closure of a fission pore connecting the extracellular medium and the vacuolar space was detected as the PVM pinched off. This final stage of parasite entry was accomplished without any breach in cell membrane integrity.

Bacteriophage T7 helicase/primase proteins form rings around single-stranded DNA that suggest a general structure for hexameric helicases.

Most helicases studied to date have been characterized as oligomeric, but the relation between their structure and function has not been understood. The bacteriophage T7 gene 4 helicase/primase proteins act in T7 DNA replication. We have used electron microscopy, three-dimensional reconstruction, and protein crosslinking to demonstrate that both proteins form hexameric rings around single-stranded DNA. Each subunit has two lobes, so the hexamer appears to be two-tiered, with a small ring stacked on a large ring. The single-stranded DNA passes through the central hole of the hexamer, and the data exclude substantial wrapping of the DNA about or within the protein ring. Further, the hexamer binds DNA with a defined polarity as the smaller ring of the hexamer points toward the 5' end of the DNA. The similarity in three-dimensional structure of the T7 gene 4 proteins to that of the Escherichia coli RuvB helicase suggests that polar rings assembled around DNA may be a general feature of numerous hexameric helicases involved in DNA replication, transcription, recombination, and repair.

Biochemical characterization and DNA binding properties of the MocR family member GabR, a PLP-dependent transcription factor

GabR è un fattore di trascrizione chimerico appartenente alla famiglia dei MocR/GabR, costituito da un dominio N-terminale elica-giro-elica di legame al DNA e un dominio effettore e/o di oligomerizzazione al C-terminale. I due domini sono connessi da un linker flessibile di 29 aminoacidi. Il dominio C-terminale è strutturalmente omologo agli enzimi aminotransferasici fold-type I, i quali, utilizzando il piridossal-5’-fosfato (PLP) come cofattore, sono direttamente coinvolti nel metabolismo degli aminoacidi. L’interazione contemporanea di PLP e acido γ-aminobutirrico (GABA) a GabR fa sì che questa promuova la trascrizione di due geni, gabT e gabD, implicati nel metabolismo del GABA. GabR cristallizza come un omodimero con una configurazione testa-coda. Il legame con la regione promotrice gabTD avviene attraverso il riconoscimento specifico di due sequenze dirette e ripetute (ATACCA), separate da uno spacer di 34 bp. In questo studio sono state indagate le proprietà biochimiche, strutturali e di legame al DNA della proteina GabR di Bacillus subtilis. L’analisi spettroscopica dimostra che GabR interagisce con il PLP formando l’aldimina interna, mentre in presenza di GABA si ottiene l’aldimina esterna. L’interazione fra il promotore gabTD e le forme holo e apo di GabR è stata monitorata mediante Microscopia a Forza atomica (AFM). In queste due condizioni di legame è stata stimata una Kd di circa 40 ηM. La presenza di GABA invece, determinava un incremento di circa due volte della Kd, variazioni strutturali nei complessi GabR-DNA e una riduzione del compattamento del DNA alla proteina, indipendentemente dalla sequenza del promotore in esame. Al fine di valutare il ruolo delle caratteristiche topologiche del promotore, sono state inserite cinque e dieci bp all’interno della regione spacer che separa le due sequenze ripetute dirette riconosciute da GabR. I significativi cambiamenti topologici riscontrati nel frammento aggiunto di cinque bp si riflettono anche sulla forte riduzione dell’affinità di legame verso la proteina. Al contrario, l’inserzione di 10 bp provoca solamente l’allontanamento delle sequenze ripetute dirette. L’assenza quindi di cambiamenti significativi nella topologia di questo promotore fa sì che l’affinità di legame per GabR rimanga pressoché inalterata rispetto al promotore non mutato. L’analisi del potenziale elettrostatico superficiale di GabR mostra la presenza di una fascia carica positivamente che si estende lungo un’intera faccia della proteina. Per verificare l’importanza di questa caratteristica di GabR nel meccanismo di interazione al DNA, sono stati preparati ed indagati i mutanti R129Q e K362-366Q, in cui la carica positiva superficiale risultava indebolita. L’affinità di legame dei mutanti di GabR per il DNA era inferiore rispetto alla proteina non mutata, in particolar modo nel mutante K362-366Q. Le evidenze acquisite suggeriscono che la curvatura intrinseca del promotore ed il corretto orientamento delle sequenze sulla doppia elica, più della distanza che le separa, siano critici per sostenere l’interazione con GabR. Oltre a questo, la superficie positiva di GabR è richiesta per accomodare la curvatura del DNA sul corpo della proteina. Alla luce di questo, l’interazione GabR-gabTD è un esempio di come il riconoscimento specifico di sequenze, la topologia del DNA e le caratteristiche strutturali della proteina siano contemporaneamente necessarie per sostenere un’interazione proteina-DNA stabile.

The Broadway

Plymouth at Broadway 761-4280, party goods - candles - books - candy - gifts. Gift wrapping - wrap and mail service - Autotyping - bulk mailing - Bigby ice-skate exchange.

Charges for Ginning Cotton

Title Varies: Rates for Ginning and Wrapping American Cotton, and Related Data, Seasons 1928-29 To 1935-36

Clinically active Crohn's disease in the presence of a low C-reactive protein

Objective. Clinical interest in C-reactive protein (CRP) - a component of the innate immune system - has focused mainly on its worth as an indicator of disease activity. There has been a resurgence of interest in CRP in the Crohn's disease ( CD) literature because several trials of new treatments for active CD have been characterized by both a large proportion of patients with low CRP ( < 10 mg/l) at entry to the trials and by a negative therapeutic outcome. It is therefore of interest to study the clinical characteristics of patients who are thought to have at the same time both active CD and a low CRP. Material and methods. Patients were prospectively recruited as part of the Brisbane IBD clinical and research programme. Subjects were included in the low CRP group only if there were complete datasets for CRP on all occasions of active CD, and CRP was < 10 mg/l. Active disease was defined as CD activity index (CDAI) > 200. The low CRP group was compared with patients in the raised CRP group for a range of clinical variables as well as the major NOD2 variants. Results. There were data sets for 223 CD patients, with a mean disease duration of 12 years. Of these, 22 patients fulfilled the criteria for low CRP. The low CRP group ( group 1) showed significant differences for disease site (p < 0.01) and for BMI (p = 0.006) compared to the raised CRP group ( group 2). Specifically, group 1 had a predominance of pure ileal disease (95% versus 53%) and lack of pure colonic disease (0% versus 24%) compared to group 2, and their BMI was significantly lower (20.3 kg/m(2) versus 25.0 kg/m(2)). Groups 1 and 2 did not differ with respect to Vienna behaviour at diagnosis, smoking, appendicectomy, extra-intestinal manifestations of CD, or NOD2 SNP variants. There was a trend for low CRP patients with previous ileal resection to evolve to a stricturing phenotype. Fat wrapping was noted in 11/13 (85%) of low CRP patients undergoing ileal resections. Conclusions. Patients with CD and a persistently low CRP in the face of active disease were characterized by an almost exclusive ileal disease distribution and a low BMI, compared to those with a raised CRP. These patients had a similar frequency and distribution of NOD2/CARD15 variants. Stricturing ( v inflammatory or penetrating) behaviour may explain some low CRP. Despite the abnormally low BMI, fat wrapping was noted in the majority of low CRP patients undergoing ileal resection.

Exchanging uncertainty: interoperable geostatistics?

Traditionally, geostatistical algorithms are contained within specialist GIS and spatial statistics software. Such packages are often expensive, with relatively complex user interfaces and steep learning curves, and cannot be easily integrated into more complex process chains. In contrast, Service Oriented Architectures (SOAs) promote interoperability and loose coupling within distributed systems, typically using XML (eXtensible Markup Language) and Web services. Web services provide a mechanism for a user to discover and consume a particular process, often as part of a larger process chain, with minimal knowledge of how it works. Wrapping current geostatistical algorithms with a Web service layer would thus increase their accessibility, but raises several complex issues. This paper discusses a solution to providing interoperable, automatic geostatistical processing through the use of Web services, developed in the INTAMAP project (INTeroperability and Automated MAPping). The project builds upon Open Geospatial Consortium standards for describing observations, typically used within sensor webs, and employs Geography Markup Language (GML) to describe the spatial aspect of the problem domain. Thus the interpolation service is extremely flexible, being able to support a range of observation types, and can cope with issues such as change of support and differing error characteristics of sensors (by utilising descriptions of the observation process provided by SensorML). XML is accepted as the de facto standard for describing Web services, due to its expressive capabilities which allow automatic discovery and consumption by ‘naive’ users. Any XML schema employed must therefore be capable of describing every aspect of a service and its processes. However, no schema currently exists that can define the complex uncertainties and modelling choices that are often present within geostatistical analysis. We show a solution to this problem, developing a family of XML schemata to enable the description of a full range of uncertainty types. These types will range from simple statistics, such as the kriging mean and variances, through to a range of probability distributions and non-parametric models, such as realisations from a conditional simulation. By employing these schemata within a Web Processing Service (WPS) we show a prototype moving towards a truly interoperable geostatistical software architecture.

Volume strain sensor based on spectra analysis of in-fiber modal interferometer

An optical in-fiber modal interferometer-based volume strain sensor for earthquake prediction is proposed and experimentally demonstrated. The sensing element is formed by wrapping a multimode-singlemode-multimode fiber structure onto a polyurethane hollow column. Due to the modal interference between the excited guided modes in the fiber, strong interference pattern could be observed in the transmission spectrum. Theoretical analysis verifies that the resonant wavelength shifts as a result of the volume strain variation caused by the column deformation with square root relationship. Sensitivity > 3.93 pm/με within the volume strain ranging from 0 to 1300 με is also experimentally demonstrated. By taking the response of bidirectional change of volume strain and the sluggish character of the employed sensing material into consideration, the sensing system presents good repeatability and stability. © 2001-2012 IEEE.

Normal ECG Recognition for Express-Diagnostics Based on Scale-Space Representation and Dynamic Matching

A novel approach of normal ECG recognition based on scale-space signal representation is proposed. The approach utilizes curvature scale-space signal representation used to match visual objects shapes previously and dynamic programming algorithm for matching CSS representations of ECG signals. Extraction and matching processes are fast and experimental results show that the approach is quite robust for preliminary normal ECG recognition.

Leveraging Existing Plasma Simulation Codes

This paper describes the process of wrapping existing scientific codes in the domain of plasma physics simulations through the use of the Sun’s Java Native Interface. We have created a Java front-end for a particular functionality, offered by legacy native libraries, in order to achieve reusability and interoperability without having to rewrite these libraries. The technique, introduced in this paper, includes two approaches – the one-to-one mapping for wrapping a number of native functions, and using peer classes for wrapping native data structures.

Automatic ECG Analysis for Preliminary and Detailed Diagnostics Based on Scale-space Representation

A novel approach of automatic ECG analysis based on scale-scale signal representation is proposed. The approach uses curvature scale-space representation to locate main ECG waveform limits and peaks and may be used to correct results of other ECG analysis techniques or independently. Moreover dynamic matching of ECG CSS representations provides robust preliminary recognition of ECG abnormalities which has been proven by experimental results.