A local electrostatic change is the cause of the large-scale protein conformation shift in bacteriorhodopsin

During light-driven proton transport bacteriorhodopsin shuttles between two protein conformations. A large-scale structural change similar to that in the photochemical cycle is produced in the D85N mutant upon raising the pH, even without illumination. We report here that (i) the pKa values for the change in crystallographic parameters and for deprotonation of the retinal Schiff base are the same, (ii) the retinal isomeric configuration is nearly unaffected by the protein conformation, and (iii) preventing rotation of the C13—C14 double bond by replacing the retinal with an all-trans locked analogue makes little difference to the Schiff base pKa. We conclude that the direct cause of the conformational shift is destabilization of the structure upon loss of interaction of the positively charged Schiff base with anionic residues that form its counter-ion.

Multiple-complete-digest restriction fragment mapping: Generating sequence-ready maps for large-scale DNA sequencing

Multiple-complete-digest mapping is a DNA mapping technique based on complete-restriction-digest fingerprints of a set of clones that provides highly redundant coverage of the mapping target. The maps assembled from these fingerprints order both the clones and the restriction fragments. Maps are coordinated across three enzymes in the examples presented. Starting with yeast artificial chromosome contigs from the 7q31.3 and 7p14 regions of the human genome, we have produced cosmid-based maps spanning more than one million base pairs. Each yeast artificial chromosome is first subcloned into cosmids at a redundancy of ×15–30. Complete-digest fragments are electrophoresed on agarose gels, poststained, and imaged on a fluorescent scanner. Aberrant clones that are not representative of the underlying genome are rejected in the map construction process. Almost every restriction fragment is ordered, allowing selection of minimal tiling paths with clone-to-clone overlaps of only a few thousand base pairs. These maps demonstrate the practicality of applying the experimental and software-based steps in multiple-complete-digest mapping to a target of significant size and complexity. We present evidence that the maps are sufficiently accurate to validate both the clones selected for sequencing and the sequence assemblies obtained once these clones have been sequenced by a “shotgun” method.

Large-scale protein structure modeling of the Saccharomyces cerevisiae genome

The function of a protein generally is determined by its three-dimensional (3D) structure. Thus, it would be useful to know the 3D structure of the thousands of protein sequences that are emerging from the many genome projects. To this end, fold assignment, comparative protein structure modeling, and model evaluation were automated completely. As an illustration, the method was applied to the proteins in the Saccharomyces cerevisiae (baker’s yeast) genome. It resulted in all-atom 3D models for substantial segments of 1,071 (17%) of the yeast proteins, only 40 of which have had their 3D structure determined experimentally. Of the 1,071 modeled yeast proteins, 236 were related clearly to a protein of known structure for the first time; 41 of these previously have not been characterized at all.

Linking genome and proteome by mass spectrometry: Large-scale identification of yeast proteins from two dimensional gels

The function of many of the uncharacterized open reading frames discovered by genomic sequencing can be determined at the level of expressed gene products, the proteome. However, identifying the cognate gene from minute amounts of protein has been one of the major problems in molecular biology. Using yeast as an example, we demonstrate here that mass spectrometric protein identification is a general solution to this problem given a completely sequenced genome. As a first screen, our strategy uses automated laser desorption ionization mass spectrometry of the peptide mixtures produced by in-gel tryptic digestion of a protein. Up to 90% of proteins are identified by searching sequence data bases by lists of peptide masses obtained with high accuracy. The remaining proteins are identified by partially sequencing several peptides of the unseparated mixture by nanoelectrospray tandem mass spectrometry followed by data base searching with multiple peptide sequence tags. In blind trials, the method led to unambiguous identification in all cases. In the largest individual protein identification project to date, a total of 150 gel spots—many of them at subpicomole amounts—were successfully analyzed, greatly enlarging a yeast two-dimensional gel data base. More than 32 proteins were novel and matched to previously uncharacterized open reading frames in the yeast genome. This study establishes that mass spectrometry provides the required throughput, the certainty of identification, and the general applicability to serve as the method of choice to connect genome and proteome.

Saccharomyces Genome Database provides tools to survey gene expression and functional analysis data

Upon the completion of the Saccharomyces cerevisiae genomic sequence in 1996 [Goffeau,A. et al. (1997) Nature, 387, 5], several creative and ambitious projects have been initiated to explore the functions of gene products or gene expression on a genome-wide scale. To help researchers take advantage of these projects, the Saccharomyces Genome Database (SGD) has created two new tools, Function Junction and Expression Connection. Together, the tools form a central resource for querying multiple large-scale analysis projects for data about individual genes. Function Junction provides information from diverse projects that shed light on the role a gene product plays in the cell, while Expression Connection delivers information produced by the ever-increasing number of microarray projects. WWW access to SGD is available at genome-www.stanford.edu/Saccharomyces/.

Real or virtual large-scale structure?

Modeling the development of structure in the universe on galactic and larger scales is the challenge that drives the field of computational cosmology. Here, photorealism is used as a simple, yet expert, means of assessing the degree to which virtual worlds succeed in replicating our own.

Galaxies and large scale structure at high redshifts

It is now straightforward to assemble large samples of very high redshift (z ∼ 3) field galaxies selected by their pronounced spectral discontinuity at the rest frame Lyman limit of hydrogen (at 912 Å). This makes possible both statistical analyses of the properties of the galaxies and the first direct glimpse of the progression of the growth of their large-scale distribution at such an early epoch. Here I present a summary of the progress made in these areas to date and some preliminary results of and future plans for a targeted redshift survey at z = 2.7–3.4. Also discussed is how the same discovery method may be used to obtain a “census” of star formation in the high redshift Universe, and the current implications for the history of galaxy formation as a function of cosmic epoch.

Large-scale isolation of candidate virulence genes of Pseudomonas aeruginosa by in vivo selection.

Pseudomonas aeruginosa, an opportunistic human pathogen, is a major causative agent of mortality and morbidity in immunocompromised patients and those with cystic fibrosis genetic disease. To identify new virulence genes of P. aeruginosa, a selection system was developed based on the in vivo expression technology (IVET) that was first reported in Salmonella system. An adenine-requiring auxotrophic mutant strain of P. aeruginosa was isolated and found avirulent on neutropenic mice. A DNA fragment that can complement the mutant strain, containing purEK operon that is required for de novo biosynthesis of purine, was sequenced and used in the IVET vector construction. By applying the IVET selection system to a neutropenic mouse infection model, genetic loci that are specifically induced in vivo were identified. Twenty-two such loci were partially sequenced and analyzed. One of them was a well-studied virulence factor, pyochelin receptor (FptA), that is involved in iron acquisition. Fifteen showed significant homology to reported sequences in GenBank, while the remaining six did not. One locus, designated np20, encodes an open reading frame that shares amino acid sequence homology to transcriptional regulators, especially to the ferric uptake regulator (Fur) proteins of other bacteria. An insertional np20 null mutant strain of P. aeruginosa did not show a growth defect on laboratory media; however, its virulence on neutropenic mice was significantly reduced compared with that of a wild-type parent strain, demonstrating the importance of the np20 locus in the bacterial virulence. The successful isolation of genetic loci that affect bacterial virulence demonstrates the utility of the IVET system in identification of new virulence genes of P. aeruginosa.

Unification and large-scale structure.

The hypothesis of relativistic flow on parsec scales, coupled with the symmetrical (and therefore subrelativistic) outer structure of extended radio sources, requires that jets decelerate on scales observable with the Very Large Array. The consequences of this idea for the appearances of FRI and FRII radio sources are explored.

The effect of the geomagnetic field on cosmic ray energy estimates and large scale anisotropy searches on data from the Pierre Auger Observatory

We present a comprehensive study of the influence of the geomagnetic field on the energy estimation of extensive air showers with a zenith angle smaller than 60 degrees, detected at the Pierre Auger Observatory. the geomagnetic field induces an azimuthal modulation of the estimated energy of cosmic rays up to the similar to 2% level at large zenith angles. We present a method to account for this modulation of the reconstructed energy. We analyse the effect of the modulation on large scale anisotropy searches in the arrival direction distributions of cosmic rays. At a given energy, the geomagnetic effect is shown to induce a pseudo-dipolar pattern at the percent level in the declination distribution that needs to be accounted for.

Large scale SLAM with visual features

Comunicación presentada en el X Workshop of Physical Agents, Cáceres, 10-11 septiembre 2009.

Probing large-scale wind structures in Vela X–1 using off-states with INTEGRAL

Vela X–1 is the prototype of the class of wind-fed accreting pulsars in high-mass X-ray binaries hosting a supergiant donor. We have analysed in a systematic way 10 years of INTEGRAL data of Vela X–1 (22–50 keV) and we found that when outside the X-ray eclipse, the source undergoes several luminosity drops where the hard X-rays luminosity goes below ∼3 × 1035 erg s−1, becoming undetected by INTEGRAL. These drops in the X-ray flux are usually referred to as ‘off-states’ in the literature. We have investigated the distribution of these off-states along the Vela X–1 ∼ 8.9 d orbit, finding that their orbital occurrence displays an asymmetric distribution, with a higher probability to observe an off-state near the pre-eclipse than during the post-eclipse. This asymmetry can be explained by scattering of hard X-rays in a region of ionized wind, able to reduce the source hard X-ray brightness preferentially near eclipse ingress. We associate this ionized large-scale wind structure with the photoionization wake produced by the interaction of the supergiant wind with the X-ray emission from the neutron star. We emphasize that this observational result could be obtained thanks to the accumulation of a decade of INTEGRAL data, with observations covering the whole orbit several times, allowing us to detect an asymmetric pattern in the orbital distribution of off-states in Vela X–1.