Systematic identification and analysis of frequent gene fusion events in metabolic pathways.

BACKGROUND: Gene fusions are the most powerful type of in silico-derived functional associations. However, many fusion compilations were made when <100 genomes were available, and algorithms for identifying fusions need updating to handle the current avalanche of sequenced genomes. The availability of a large fusion dataset would help probe functional associations and enable systematic analysis of where and why fusion events occur. RESULTS: Here we present a systematic analysis of fusions in prokaryotes. We manually generated two training sets: (i) 121 fusions in the model organism Escherichia coli; (ii) 131 fusions found in B vitamin metabolism. These sets were used to develop a fusion prediction algorithm that captured the training set fusions with only 7 % false negatives and 50 % false positives, a substantial improvement over existing approaches. This algorithm was then applied to identify 3.8 million potential fusions across 11,473 genomes. The results of the analysis are available in a searchable database at http://modelseed.org/projects/fusions/ . A functional analysis identified 3,000 reactions associated with frequent fusion events and revealed areas of metabolism where fusions are particularly prevalent. CONCLUSIONS: Customary definitions of fusions were shown to be ambiguous, and a stricter one was proposed. Exploring the genes participating in fusion events showed that they most commonly encode transporters, regulators, and metabolic enzymes. The major rationales for fusions between metabolic genes appear to be overcoming pathway bottlenecks, avoiding toxicity, controlling competing pathways, and facilitating expression and assembly of protein complexes. Finally, our fusion dataset provides powerful clues to decipher the biological activities of domains of unknown function.

High throughput screening using acoustic droplet ejection to combine protein crystals and chemical libraries on crystallization plates at high density.

We describe a high throughput method for screening up to 1728 distinct chemicals with protein crystals on a single microplate. Acoustic droplet ejection (ADE) was used to co-position 2.5nL of protein, precipitant, and chemicals on a MiTeGen in situ-1 crystallization plate™ for screening by co-crystallization or soaking. ADE-transferred droplets follow a precise trajectory which allows all components to be transferred through small apertures in the microplate lid. The apertures were large enough for 2.5nL droplets to pass through them, but small enough so that they did not disrupt the internal environment created by the mother liquor. Using this system, thermolysin and trypsin crystals were efficiently screened for binding to a heavy-metal mini-library. Fluorescence and X-ray diffraction were used to confirm that each chemical in the heavy-metal library was correctly paired with the intended protein crystal. A fragment mini-library was screened to observe two known lysozyme ligands using both co-crystallization and soaking. A similar approach was used to identify multiple, novel thaumatin binding sites for ascorbic acid. This technology pushes towards a faster, automated, and more flexible strategy for high throughput screening of chemical libraries (such as fragment libraries) using as little as 2.5nL of each component.

Solvent minimization induces preferential orientation and crystal clustering in serial micro-crystallography on micro-meshes, in situ plates and on a movable crystal conveyor belt.

X-ray diffraction data were obtained at the National Synchrotron Light Source from insulin and lysozyme crystals that were densely deposited on three types of surfaces suitable for serial micro-crystallography: MiTeGen MicroMeshes™, Greiner Bio-One Ltd in situ micro-plates, and a moving kapton crystal conveyor belt that is used to deliver crystals directly into the X-ray beam. 6° wedges of data were taken from ∼100 crystals mounted on each material, and these individual data sets were merged to form nine complete data sets (six from insulin crystals and three from lysozyme crystals). Insulin crystals have a parallelepiped habit with an extended flat face that preferentially aligned with the mounting surfaces, impacting the data collection strategy and the design of the serial crystallography apparatus. Lysozyme crystals had a cuboidal habit and showed no preferential orientation. Preferential orientation occluded regions of reciprocal space when the X-ray beam was incident normal to the data-collection medium surface, requiring a second pass of data collection with the apparatus inclined away from the orthogonal. In addition, crystals measuring less than 20 µm were observed to clump together into clusters of crystals. Clustering required that the X-ray beam be adjusted to match the crystal size to prevent overlapping diffraction patterns. No additional problems were encountered with the serial crystallography strategy of combining small randomly oriented wedges of data from a large number of specimens. High-quality data able to support a realistic molecular replacement solution were readily obtained from both crystal types using all three serial crystallography strategies.

A linear relationship between crystal size and fragment binding time observed crystallographically: implications for fragment library screening using acoustic droplet ejection.

High throughput screening technologies such as acoustic droplet ejection (ADE) greatly increase the rate at which X-ray diffraction data can be acquired from crystals. One promising high throughput screening application of ADE is to rapidly combine protein crystals with fragment libraries. In this approach, each fragment soaks into a protein crystal either directly on data collection media or on a moving conveyor belt which then delivers the crystals to the X-ray beam. By simultaneously handling multiple crystals combined with fragment specimens, these techniques relax the automounter duty-cycle bottleneck that currently prevents optimal exploitation of third generation synchrotrons. Two factors limit the speed and scope of projects that are suitable for fragment screening using techniques such as ADE. Firstly, in applications where the high throughput screening apparatus is located inside the X-ray station (such as the conveyor belt system described above), the speed of data acquisition is limited by the time required for each fragment to soak into its protein crystal. Secondly, in applications where crystals are combined with fragments directly on data acquisition media (including both of the ADE methods described above), the maximum time that fragments have to soak into crystals is limited by evaporative dehydration of the protein crystals during the fragment soak. Here we demonstrate that both of these problems can be minimized by using small crystals, because the soak time required for a fragment hit to attain high occupancy depends approximately linearly on crystal size.

Green roof adoption in atlanta, georgia: the effects of building characteristics and subsidies on net private, public, and social benefits.

This research draws on and expands previous studies that have quantified the costs and benefits associated with conventional roofs versus green roofs. Using parameters from those studies to define alternative scenarios, we estimate from a private, public, and social perspective the costs and benefits of installing and maintaining an extensive green roof in Atlanta, GA. Results indicate net private benefits are a decreasing function of roof size and vary considerably across scenarios. In contrast, net public benefits are highly stable across scenarios, ranging from $32.49 to $32.90 m(-2). In addition, we evaluate two alternative subsidy regimes: (i) a general subsidy provided to every building that adopts a green roof and (ii) a targeted subsidy provided only to buildings for which net private benefits are negative but net public benefits are positive. In 6 of the 12 general subsidy scenarios the optimal public policy is not to offer a subsidy; in 5 scenarios the optimal subsidy rate is between $20 and $27 m(-2); and in 1 scenario the optimal rate is $5 m(-2). The optimal rate with a targeted subsidy is between $20 and $27 m(-2) in 11 scenarios and no subsidy is optimal in the twelfth. In most scenarios, a significant portion of net public benefits are generated by buildings for which net private benefits are positive. This suggests a policy focused on information dissemination and technical assistance may be more cost-effective than direct subsidy payments.

Farm-level irrigation and the marginal cost of water use: evidence from Georgia.

We create a proxy for the cost of irrigation water in Georgia from a sample of Georgia irrigators by investigating the marginal cost of pumping groundwater. We then combine this proxy with agronomic and climatic variables to estimate the response of agricultural water use to differences in the marginal cost of irrigation. The results show that pumping costs are a significant determinant of water use, and imply that agricultural water use would be moderately affected by institutional changes that would explicitly price water.