A genomic approach to gene fusion technology

Gene expression profiling provides powerful analyses of transcriptional responses to cellular perturbation. In contrast to DNA array-based methods, reporter gene technology has been underused for this application. Here we describe a genomewide, genome-registered collection of Escherichia coli bioluminescent reporter gene fusions. DNA sequences from plasmid-borne, random fusions of E. coli chromosomal DNA to a Photorhabdus luminescens luxCDABE reporter allowed precise mapping of each fusion. The utility of this collection covering about 30% of the transcriptional units was tested by analyzing individual fusions representative of heat shock, SOS, OxyR, SoxRS, and cya/crp stress-responsive regulons. Each fusion strain responded as anticipated to environmental conditions known to activate the corresponding regulatory circuit. Thus, the collection mirrors E. coli's transcriptional wiring diagram. This genomewide collection of gene fusions provides an independent test of results from other gene expression analyses. Accordingly, a DNA microarray-based analysis of mitomycin C-treated E. coli indicated elevated expression of expected and unanticipated genes. Selected luxCDABE fusions corresponding to these up-regulated genes were used to confirm or contradict the DNA microarray results. The power of partnering gene fusion and DNA microarray technology to discover promoters and define operons was demonstrated when data from both suggested that a cluster of 20 genes encoding production of type I extracellular polysaccharide in E. coli form a single operon.

Exploring plant genomes by RNA-induced gene silencing

The nucleotide sequences of several animal, plant and bacterial genomes are now known, but the functions of many of the proteins that they are predicted to encode remain unclear. RNA interference is a gene-silencing technology that is being used successfully to investigate gene function in several organisms - for example, Caenorhabditis elegans. We discuss here that RNA-induced gene silencing approaches are also likely to be effective for investigating plant gene function in a high-throughput, genome-wide manner.

A new plasmid display technology for the in vitro selection of functional phenotype-genotype linked proteins

Background Display technologies which allow peptides or proteins to be physically associated with the encoding DNA are central to procedures which involve screening of protein libraries in vitro for new or altered function. Here we describe a new system designed specifically for the display of libraries of diverse, functional proteins which utilises the DNA binding protein nuclear factor κB (NF-κB) p50 to establish a phenotype-genotype link between the displayed protein and the encoding gene. Results A range of model fusion proteins to either the amino- or carboxy-terminus of NF-κB p50 have been constructed and shown to retain the picomolar affinity and DNA specificity of wild-type NF-κB p50. Through use of an optimal combination of binding buffer and DNA target sequence, the half-life of p50-DNA complexes could be increased to over 47 h, enabling the competitive selection of a variety of protein-plasmid complexes with enrichment factors of up to 6000-fold per round. The p50-based plasmid display system was used to enrich a maltose binding protein complex to homogeneity in only three rounds from a binary mixture with a starting ratio of 1:108 and to enrich to near homogeneity a single functional protein from a phenotype-genotype linked Escherichia coli genomic library using in vitro functional selections. Conclusions A new display technology is described which addresses the challenge of functional protein display. The results demonstrate that plasmid display is sufficiently sensitive to select a functional protein from large libraries and that it therefore represents a useful addition to the repertoire of display technologies.

Genetic studies in osteoporosis: The end of the beginning

Osteoporosis and disorders of bone fragility are highly heritable, but despite much effort the identities of few of the genes involved has been established. Recent developments in genetics such as genome-wide association studies are revolutionizing research in this field, and it is likely that further contributions will be made through application of next-generation sequencing technologies, analysis of copy number variation polymorphisms, and high-throughput mouse mutagenesis programs. This article outlines what we know about osteoporosis genetics to date and the probable future directions of research in this field.

Mapping genes for osteoporosis-Old dogs and new tricks

In stark contrast to its horticultural origins, modern genetics is an extremely technology-driven field. Almost all the major advances in the field over the past 20 years have followed technological developments that have permitted change in study designs. The development of PCR in the 1980s led to RFLP mapping of monogenic diseases. The development of fluorescent-tagged genotyping methods led to linkage mapping approaches for common diseases that dominated the 1990s. The development of microarray SNP genotyping has led to the genome-wide association study era of the new millennium. And now the development of next-generation sequencing technologies is about to open up a new era of gene-mapping, enabling many potential new study designs. This review aims to present the strengths and weaknesses of the current approaches, and present some new ideas about gene-mapping approaches that are likely to advance our knowledge of the genes involved in heritable bone traits such as bone mineral density (BMD) and fracture.

On the functional ordering of biomembranes : Implications for drug binding

Cells are packed with membrane structures, defining the inside and outside, and the different subcellular compartments. These membranes consisting mainly of phospholipids have a variety of functions in addition to providing a permeability barrier for various compounds. These functions involve cellular signaling, where lipids can act as second messengers, or direct regulation of membrane associating proteins. The first part of this study focuses on relating some of the physicochemical properties of membrane lipids to the association of drug compounds to membranes. A fluorescence based method is described allowing for determination of the membrane association of drugs. This method was subsequently applied to a novel drug, siramesine, previously shown to have anti-cancer activity. Siramesine was found to associate with anionic lipids. Especially interesting is its strong affinity for a second messenger lipid phosphatidic acid. This is the first example of a small molecule drug compound specifically interacting with a cellular lipid. Phosphatidic acid in cells is required for the activation of many signaling pathways mediating growth and proliferation. This provides an intriguing possibility for a simple molecular mechanism of the observed anti-cancer activity of siramesine. In the second part the thermal behavior and self assembly of charged and uncharged membrane assemblies was studied. Strong inter-lamellar co-operativity was observed for multilamellar DPPC vesicles using fluorescence techniques together with calorimetry. The commonly used membrane models, large unilamellar vesicles (LUV) and multilamellar vesicles (MLV) were found to possess different biophysical properties as interlamellar interactions of MLVs drive segregation of a pyrene labeled lipid analogue into clusters. The effect of a counter-ion lattice on the self assembly of a cationic gemini surfactant was studied. The presence of NaCl strongly influenced the thermal phase behavior of M-1 vesicles, causing formation of giant vesicles upon exceeding a phase transition temperature, followed by a subsequent transition into a more homogenous dispersion. Understanding the underlying biophysical aspects of cellular membranes is of fundamental importance as the complex picture of the structure and function of cells is evolving. Many of the cellular reactions take place on membranes and membranes are known to regulate the activity of many peripheral and intergral membrane associating proteins. From the point of view of drug design and gene technology, membranes can provide an interesting target for future development of drugs, but also a vehicle sensitive for environmental changes allowing for encapsulating drugs and targeting them to the desired site of action.

Use of a lux-marked rhizobacterium as a biosensor to assess changes in rhizosphere C flow due to pollutant stress

The flow of carbon from plant roots into soil supports a range of microbial processes and is therefore critical to ecosystem function and health. Pollution-induced stress, which influences rhizosphere C flow is of considerable potential importance, and therefore needs to be evaluated. This paper reports on a method, based on reporter gene technology, for quantifying pollutant effects on rhizosphere C flow. The method uses the lux-marked rhizobacterium Pseudomonas fluorescens, where bioluminescence output of this biosensor is directly correlated with the metabolic activity and reports on C flow in root exudate. Plantago lanceolata was treated with paraquat (representing a model pollutant stress) in a simple microcosm system. The lux-biosensor response correlated closely with C concentrations in the exudate and demonstrated that the pollutant stress increased the C flow from the plantago roots, 24 h after application of the herbicide. The lux-reporter system therefore potentially offers a technique for use in assessing the impact of pollutant stress on rhizosphere C flow through the soil microbial biomass.

Impacto de las normas de ecoetiquetado de Japón y La Unión Europea, y la posición de Estados Unidos en este aspecto, en las exportaciones de organismos genéticamente modificados canadienses, en los últimos 10 años

La hipótesis de este trabajo de grado es el desarrollo de definiciones estándares de Estados Unidos, Japón y la Unión Europea en materia de ecoetiquetado, han dificultado el crecimiento de las exportaciones de Organismos Genéticamente Modificados canadienses y la aplicación de su estrategia

A study of seed viability following consumption by birds

This project aimed to quantify the potential for wild birds to spread genetically modified seeds from their area of planting to adjacent areas, through ingestion and transport in the gut (endozoochorous transmission). We addressed this by assessing whether seed viability was affected by passage through the avian gut. We sought to address this question by testing the effects of ingestion of a number of key agricultural crops by three common wild bird pest species.

Polykation-DNA-Komplexe : Eigenschaften und Anwendung in der Gentransfektion

Polykationen bilden mit DNA spontan Komplexe. Triebkraft ist der Entropiegewinn durch Freisetzung der Gegenionen auf den Polyelektrolyten. Solche Komplexe können in der Gentechnik verwendet werden, um fremde DNA in eine Zelle einzuschleusen. Dies bezeichnet man als Gentransfektion. In dieser Arbeit werden erstmals bürstenförmige Polykationen mit wurmförmiger Topologie zur Gentransfektion verwendet. Dazu wurde die Komplexierung von DNA mit Bürstenpolymeren mit Poly-L-Lysin- und Polyvinylpyridinium-Seitenketten und linearen Polykationen untersucht. Die Komplexbildung verläuft in allen Fällen kinetisch kontrolliert, alle Polykationen bilden sphärische Komplexe, die Topologie hat keinen Einfluss auf die Komplexgröße. Komplexe aus Bürstenpolymeren transfizieren mehr als 25% der gesamten Zellpopulation bei Schweinehirnendothelzellen. Gegenüber dem kommerziellen Transfektionsmittel Lipofektamin konnte eine deutliche Steigerung um bis zu 400% erreicht werden. Komplexe, die mit linearen Analoga gebildet wurden, zeigten bei gleicher Komplexgröße Transfektionsraten unter 5%. Freisetzungsversuche zeigen, dass die Komplexe, die gut transfizieren, recht labil sind, also die DNA unter Kompetitoreinfluss freisetzen können. Stabile Komplexe haben geringe Transfektionseffizienzen. Ebenso wichtig ist der Schutz der DNA vor Abbau durch DNase. Die PVP-Bürste bietet als einziges der untersuchten Polykationen diesen Schutz und zeigt auch die besten Transfektionsraten. Zusätzlich zu der medizinischen Anwendung wurde die Kinetik der Komplexbildung untersucht. Dazu wurde ein spezieller Aufbau entwickelt, der es ermöglicht die Streuintensität der Komplexlösung bei kleinen Streuwinkeln zeitaufgelöst im Millisekundenbereich zu detektieren. Die Komplexbildung verläuft diffusionskontrolliert, im Bereich von Ladungsverhältnissen (positive zu negativen Ladungen) von 1.8 bis 4.0 schließt sich ein fraktales Wachstum an.

The use of artificial MicroRNA technology to control gene expression in Arabidopsis thaliana

In plants, double-stranded RNA (dsRNA) is an effective trigger of RNA silencing, and several classes of endogenous small RNA (sRNA), processed from dsRNA substrates by DICER-like (DCL) endonucleases, are essential in controlling gene expression. One such sRNA class, the microRNAs (miRNAs) control the expression of closely related genes to regulate all aspects of plant development, including the determination of leaf shape, leaf polarity, flowering time, and floral identity. A single miRNA sRNA silencing signal is processed from a long precursor transcript of nonprotein-coding RNA, termed the primary miRNA (pri-miRNA). A region of the pri-miRNA is partially self-complementary allowing the transcript to fold back onto itself to form a stem-loop structure of imperfectly dsRNA. Artificial miRNA (amiRNA) technology uses endogenous pri-miRNAs, in which the miRNA and miRNA*(passenger strand of the miRNA duplex) sequences have been replaced with corresponding amiRNA/ amiRNA*sequences that direct highly efficient RNA silencing of the targeted gene. Here, we describe the rules for amiRNA design, as well as outline the PCR and bacterial cloning procedures involved in the construction of an amiRNA plant expression vector to control target gene expression in Arabidopsis thaliana. © 2014 Springer Science+Business Media New York.