Retrovirus-delivered siRNA

BACKGROUND: The ability of transfected synthetic small interfering (si) RNAs to suppress the expression of specific transcripts has proved a useful technique to probe gene function in mammalian cells. However, high production costs limit this technology's utility for many laboratories and experimental situations. Recently, several DNA-based plasmid vectors have been developed that direct transcription of small hairpin RNAs, which are processed into functional siRNAs by cellular enzymes. Although these vectors provide certain advantages over chemically synthesized siRNAs, numerous disadvantages remain including merely transient siRNA expression and low and variable transfection efficiency. RESULTS: To overcome several limitations of plasmid-based siRNA, a retroviral siRNA delivery system was developed based on commerically available vectors. As a pilot study, a vector was designed to target the human Nuclear Dbf2-Related (NDR) kinase. Cells infected with the anti-NDR siRNA virus dramatically downregulate NDR expression, whereas control viruses have no effect on total NDR levels. To confirm and extend these findings, an additional virus was constructed to target a second gene, transcriptional coactivator p75. CONCLUSION: The experiments presented here demonstrate that retroviruses are efficient vectors for delivery of siRNA into mammalian cells. Retrovirus-delivered siRNA provides significant advancement over previously available methods by providing efficient, uniform delivery and immediate selection of stable "knock-down" cells. This development should provide a method to rapidly assess gene function in established cell lines, primary cells, or animals

Induced sensitivity of Bacillus subtilis colony morphology to mechanical media compression

Bacteria from several taxa, including Kurthia zopfii, Myxococcus xanthus, and Bacillus mycoides, have been reported to align growth of their colonies to small features on the surface of solid media, including anisotropies created by compression. While the function of this phenomenon is unclear, it may help organisms navigate on solid phases, such as soil. The origin of this behavior is also unknown: it may be biological (that is, dependent on components that sense the environment and regulate growth accordingly) or merely physical. Here we show that B. subtilis, an organism that typically does not respond to media compression, can be induced to do so with two simple and synergistic perturbations: a mutation that maintains cells in the swarming (chained) state, and the addition of EDTA to the growth media, which further increases chain length. EDTA apparently increases chain length by inducing defects in cell separation, as the treatment has only marginal effects on the length of individual cells. These results lead us to three conclusions. First, the wealth of genetic tools available to B. subtilis will provide a new, tractable chassis for engineering compression sensitive organisms. Second, the sensitivity of colony morphology to media compression in Bacillus can be modulated by altering a simple physical property of rod-shaped cells. And third, colony morphology under compression holds promise as a rapid, simple, and low-cost way to screen for changes in the length of rod-shaped cells or chains thereof

Systems biology of gene regulation fulfills its promise

A report on the meeting 'Systems Biology: Global Regulation of Gene Expression' at Cold Spring Harbor, New York, USA, 23-26 March 2006

HIV-1 incorporates and proteolytically processes human NDR1 and NDR2 serine-threonine kinases

AbstractMammalian genomes encode two related serine-threonine kinases, nuclear Dbf2 related (NDR)1 and NDR2, which are homologous to the Saccharomyces cerevisiae Dbf2 kinase. Recently, a yeast genetic screen implicated the Dbf2 kinase in Ty1 retrotransposition. Since several virion-incorporated kinases regulate the infectivity of human immunodeficiency virus type 1 (HIV-1), we speculated that the human NDR1 and NDR2 kinases might play a role in the HIV-1 life cycle. Here we show that the NDR1 and NDR2 kinases were incorporated into HIV-1 particles. Furthermore, NDR1 and NDR2 were cleaved by the HIV-1 protease (PR), both within virions and within producer cells. Truncation at the PR cleavage site altered NDR2 subcellular localization and inhibited NDR1 and NDR2 enzymatic activity. These studies identify two new virion-associated host cell enzymes and suggest a novel mechanism by which HIV-1 alters the intracellular environment of human cells

Motion as a phenotype: the use of live-cell imaging and machine visual screening to characterize transcription-dependent chromosome dynamics

BACKGROUND: Gene transcriptional activity is well correlated with intra-nuclear position, especially relative to the nuclear periphery, which is a region classically associated with gene silencing. Recently however, actively transcribed genes have also been found localized to the nuclear periphery in the yeast Saccharomyces cerevisiae. When genes are activated, they become associated with the nuclear pore complex (NPC) at the nuclear envelope. Furthermore, chromosomes are not static structures, but exhibit constrained diffusion in real-time, live-cell studies of particular loci. The relationship of chromosome motion with transcriptional activation and active-gene recruitment to the nuclear periphery has not yet been investigated. RESULTS: We have generated a yeast strain that enables us to observe the motion of the galactose-inducible GAL gene locus relative to the nuclear periphery in real-time under transcriptionally active and repressed conditions. Using segmented geometric particle tracking, we show that the repressed GAL locus undergoes constrained diffusive movement, and that transcriptional induction with galactose is associated with an enrichment in cells with GAL loci that are both associated with the nuclear periphery and much more constrained in their movement. Furthermore, we report that the mRNA export factor Sac3 is involved in this galactose-induced enrichment of GAL loci at the nuclear periphery. In parallel, using a novel machine visual screening technique, we find that the motion of constrained GAL loci correlates with the motion of the cognate nuclei in galactose-induced cells. CONCLUSION: Transcriptional activation of the GAL genes is associated with their tethering and motion constraint at the nuclear periphery. We describe a model of gene recruitment to the nuclear periphery involving gene diffusion and the mRNA export factor Sac3 that can be used as a framework for further experimentation. In addition, we applied to the analysis of chromosome motion a machine visual screening approach that used unbiased visual data rather than segmented geometric data. This novel analytical approach will allow for high-throughput study of processes that can be monitored via alterations in chromosome motion and connectivity with the nuclear periphery

An objective function exploiting suboptimal solutions in metabolic networks

Background: Flux Balance Analysis is a theoretically elegant, computationally efficient, genome-scale approach to predicting biochemical reaction fluxes. Yet FBA models exhibit persistent mathematical degeneracy that generally limits their predictive power. Results: We propose a novel objective function for cellular metabolism that accounts for and exploits degeneracy in the metabolic network to improve flux predictions. In our model, regulation drives metabolism toward a region of flux space that allows nearly optimal growth. Metabolic mutants deviate minimally from this region, a function represented mathematically as a convex cone. Near-optimal flux configurations within this region are considered equally plausible and not subject to further optimizing regulation. Consistent with relaxed regulation near optimality, we find that the size of the near-optimal region predicts flux variability under experimental perturbation. Conclusion: Accounting for suboptimal solutions can improve the predictive power of metabolic FBA models. Because fluctuations of enzyme and metabolite levels are inevitable, tolerance for suboptimality may support a functionally robust metabolic network

Spatial and Temporal Organization of Chromosome Duplication and Segregation in the Cyanobacterium Synechococcus elongatus PCC 7942

The spatial and temporal control of chromosome duplication and segregation is crucial for proper cell division. While this process is well studied in eukaryotic and some prokaryotic organisms, relatively little is known about it in prokaryotic polyploids such as Synechococcus elongatus PCC 7942, which is known to possess one to eight copies of its single chromosome. Using a fluorescent repressor-operator system, S. elongatus chromosomes and chromosome replication forks were tagged and visualized. We found that chromosomal duplication is asynchronous and that the total number of chromosomes is correlated with cell length. Thus, replication is independent of cell cycle and coupled to cell growth. Replication events occur in a spatially random fashion. However, once assembled, replisomes move in a constrained manner. On the other hand, we found that segregation displays a striking spatial organization in some cells. Chromosomes transiently align along the major axis of the cell and timing of alignment was correlated to cell division. This mechanism likely contributes to the non-random segregation of chromosome copies to daughter cells

Enabling community-based metrology for wood-degrading fungi

Background: Lignocellulosic biomass could support a greatly-expanded bioeconomy. Current strategies for using biomass typically rely on single-cell organisms and extensive ancillary equipment to produce precursors for downstream manufacturing processes. Alternative forms of bioproduction based on solid-state fermentation and wood-degrading fungi could enable more direct means of manufacture. However, basic methods for cultivating wood-degrading fungi are often ad hoc and not readily reproducible. Here, we developed standard reference strains, substrates, measurements, and methods sufficient to begin to enable reliable reuse of mycological materials and products in simple laboratory settings. Results: We show that a widely-available and globally-regularized consumer product (Pringles™) can support the growth of wood-degrading fungi, and that growth on Pringles™-broth can be correlated with growth on media made from a fully-traceable and compositionally characterized substrate (National Institute of Standards and Technology Reference Material 8492 Eastern Cottonwood Whole Biomass Feedstock). We also establish a Relative Extension Unit (REU) framework that is designed to reduce variation in quantification of radial growth measurements. So enabled, we demonstrate that five laboratories were able to compare measurements of wood-fungus performance via a simple radial extension growth rate assay, and that our REU-based approach reduced variation in reported measurements by up to ~ 75%. Conclusions: Reliable reuse of materials, measures, and methods is necessary to enable distributed bioproduction processes that can be adopted at all scales, from local to industrial. Our community-based measurement methods incentivize practitioners to coordinate the reuse of standard materials, methods, strains, and to share information supporting work with wood-degrading fungi