Global expression mapping of mammalian genomes

he aim of genome projects is to decipher all the information contained within the DNA of an organism and to study the way this information is processed in physiological processes. It is believed that more than 95% of the information content of the mammalian genome is represented in the protein coding sequences that make up only approximately 2% of the DNA sequence. Consequently much effort is being invested in the study of coding sequences in the form of cDNA analysis. This thesis is concerned with the development of a new strategy for a highly parallel approach to analyse entire cDNA libraries. The strategy is based upon generating sufficient sequence information to identify uniquely more than 100,000 cDNA clones by hybridisation with short oligonucleotides, typically 7 - 10 mers. Each oligonucleotide is hybridised to all cDNA clones in parallel and under stringent conditions positively identifies a subset (3 - 10%) of clones. Oligonucleotides are designed in such a way that each will positively identify a different subset of clones and statistical simulations estimate that approximately 200 such hybridisation events are required to identify uniquely upto 100,000 cDNA sequences. Such a fingerprint can be generated from many cDNA libraries constructed from different tissue mRNAs and will not only lead to the identification of most sequecnes expressed from the genome but also indicate the level of expression by determining the number of times any given sequence is represented across different cDNA libraries. A human foetal brain cDNA library has been constructed and 100,000 clones arrayed into microtitre plates and on nylon membranes. All the required technological developments have been carried out successfully and are presented. In excess of 200 oligonucleotide hybridisations have been performed on a subset of 32,000 cDNA clones and 1,000 sequenced control clones. A detailed analysis of the data on the control clones is presented and the implications for cDNA fingerprinting discussed

The ORF, Regulated Synthesis, and Persistence-Specific Variation of Influenza C Viral NS1 Protein

AbstractThe open reading frame (ORF) and the regulated synthesis of the influenza C viral NS1 protein were analyzed in view of viruses possessing different biological activities. We provide evidence for a 246-amino-acid NS1-ORF, encoded by five viral strains and variants. Prokaryotic expression of the prototype NS1-ORF resulted in a product of 27 kDa, confirming the predicted molecular weight. Using an antiserum raised against recombinant NS1 protein, nonstructural proteins of wild-type virus were detected in infected cells for a limited course of time, whereas a persistent virus variant was characterized by a long-term nonstructural gene expression. As examined by infection experiments, the intracellular distribution of nonstructural protein was nuclear and cytoplasmic, whereas in NS1 gene-transfected cells, the cytoplasmic localization occurred in a fine-grained structure, suggesting an analogy to influenza A viral NS1 protein. Concerning persistent infection, NS1 protein species differing in sizes and posttranslational modifications were observed for a persistent virus variant, as particularly illustrated by a high degree of NS1 phosphorylation. Virus reassortant analyses proved the importance of the NS-coding genomic segment: the minimal viral properties required for the establishment of persistence were transferred with this segment to a monoreassortant virus. Thus the influenza C viral NS1 protein is a 246-amino-acid nuclear-cytoplasmic phosphoprotein that can be subject to specific variations being functionally linked to a persistent virus phenotype

The cell receptor level is reduced during persistent infection with influenza C virus

 Persistent influenza C virus infection of MDCK cells perpetuates the viral genome in a cell-associated form. Typically, virus production remains at a low level over extended periods, in the absence of lytic effects of replication. In this study, we demonstrate that persistently infected cells are very restricted in permissiveness for superinfection. By reconstitution experiments, using bovine brain gangliosides as artificial receptors, the degree of super-infection was markedly increased. Analysis of cellular receptor expression revealed reduced concentrations of sialoglycoproteins in general and a limited presentation of the major receptor gp40. Cocultures of persistently infected and uninfected cells (the latter carrying normal receptor levels) initiated a transient rise in virus titers. This kind of induction of virus synthesis appeared to be mainly receptor-linked, since a receptor-deprived subline, MDCK II, did not give rise to a similar effect. Susceptibility of MDCK II cocultures could be partly restored by ganglioside treatment. In accordance to related virus systems, these findings on influenza C virus suggest a role of cell receptor concentrations in the regulation of long-term persistence.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/42459/1/705-142-6-1155_71421155.pd

Habitual short sleep duration and circulating endothelial progenitor cells

Chronic short sleep duration has been linked to endothelial dysfunction and increased risk of cardiovascular disease. Circulating endothelial progenitor cells (EPCs) are vital to endogenous vascular repair processes and cardiovascular health. We tested the hypothesis that habitual short sleep duration is associated with impairment in EPC number and function. Cells with phenotypic EPC characteristics were isolated from 37 healthy, sedentary adults: 20 with normal sleep duration (13M/7F; age: 59±1 years; sleep duration: 7.7±0.1 h/night) and 17 with short sleep duration (9M/8F; 56±2 years; 6.0±0.2 h/night). EPC number was assessed by flow cytometric analysis of the percentage of peripheral blood mononuclear cells negative for CD45 and positive for CD34, VEGFR-2, and CD133 antigens. EPC colony-forming capacity was determined by colony-forming unit (CFU) assay; migration by Boyden chamber; and intracellular caspase-3 concentrations by immunoassay. There were no significant differences between groups in EPC number (0.001±0.0004 vs. 0.001±0.0003 %), colony-forming capacity (6.1±1.5 vs. 5.4±1.7 CFUs), or migration to VEGF (1410.1±151.2 vs. 1334.3±111.1 AU). Furthermore, there were no group differences in basal and staurosporine-stimulated intracellular concentrations of active caspase-3 (0.3±0.03 vs. 0.5±0.1 ng/mL; and 2.9±0.4 vs. 2.7±0.3 ng/mL), a marker of apoptotic susceptibility. Taken together, these data indicate that short sleep duration is not associated with EPC dysfunction in healthy adults. Numerical and functional impairment in circulating EPCs may not contribute to the increased cardiovascular risk with habitual short sleep duration

Package mountaineer tourists holidaying in the French Alps: An evaluation of key influences encouraging their participation

This study investigates the key influences that encourage mountaineer tourists, classified as a type of adventure tourist, to participate in package mountaineering holidays. There is limited understanding of why tourists take package adventure holidays, yet the demand for such holidays has grown dramatically in recent years. The author conducted in-depth interviews with mountaineer tourists either during or at the end of their package mountaineering holiday in the Chamonix region of the French Alps. Interview findings provide an insight into package mountaineer tourists. Firstly, mountaineering was an important part of respondents’ lifestyles. Secondly, contrary to previous research on experienced mountaineers, respondents did not consider risk as an important motive and they did not view themselves as risk takers. Thirdly, skills development and experience were key motives encouraging package mountaineering holiday participation. Fourthly, a major concern for respondents was to have a safe mountaineering experience in which the mountaineering organisation and the guide played a key role

Microwave stray radiation losses in vacuum windows

Vacuum windows are required in magnetically confined fusion experiments to provide possibilities to observe the plasma in a wide range of electromagnetic wavelengths. The window disk consists of a dielectric, e.g. Fused Silica (SiO$_2$), Sapphire or Chemically Vapourised Diamond (CVD). As electromagnetic waves pass through the disk, a fraction of the beam power is dissipated resulting in a temperature increase of the disk. In Electron Cyclotron Waves (ECW) heated plasmas the dissipation in the window disk can be very high. The computation of dielectric losses for a collimated beam with known incidence angle, polarisation and loss tangent (measure for the intrinsic dielectric loss) is well established. However, the dielectric losses in diagnostic windows mostly result from microwave stray radiation, which results from a modest, but inevitable, fraction of non-absorbed ECW. This fraction diffuses in the vessel by many reflections into rays with random k-vector and with random polarisation. In this work the thermal load on the window disk by microwave stray radiation is assessed. The load by a collimated beam is studied as a function of incidence angle and polarisation allowing to average over a distribution of incident rays. An experiment was commissioned measuring the loss tangent of a number of commercially available SiO$_2$ disks at low power in an open resonator, and subsequently measuring the dielectric heating of these disks at high power stray radiation using the facility ’MISTRAL’ at Wendelstein-7X. The experimental results are compared to modelling and it is demonstrated that, in the parameter range considered, single-pass fractional absorption may be applied while taking a safety margin that arises from the minima and maxima due to multiple reflections

A Three-Hybrid Approach to Scanning the Proteome for Targets of Small Molecule Kinase Inhibitors

AbstractIn this study, we explored the application of a yeast three-hybrid (Y3H)-based compound/protein display system to scanning the proteome for targets of kinase inhibitors. Various known cyclin-dependent kinase (CDK) inhibitors, including purine and indenopyrazole analogs, were displayed in the form of methotrexate-based hybrid ligands and deployed in cDNA library or yeast cell array-based screening formats. For all inhibitors, known cell cycle CDKs as well as novel candidate CDK-like and/or CDK-unrelated kinase targets could be identified, many of which were independently confirmed using secondary enzyme assays and affinity chromatography. The Y3H system described here may prove generally useful in the discovery of candidate drug targets