HAMAP: a database of completely sequenced microbial proteome sets and manually curated microbial protein families in UniProtKB/Swiss-Prot

The growth in the number of completely sequenced microbial genomes (bacterial and archaeal) has generated a need for a procedure that provides UniProtKB/Swiss-Prot-quality annotation to as many protein sequences as possible. We have devised a semi-automated system, HAMAP (High-quality Automated and Manual Annotation of microbial Proteomes), that uses manually built annotation templates for protein families to propagate annotation to all members of manually defined protein families, using very strict criteria. The HAMAP system is composed of two databases, the proteome database and the family database, and of an automatic annotation pipeline. The proteome database comprises biological and sequence information for each completely sequenced microbial proteome, and it offers several tools for CDS searches, BLAST options and retrieval of specific sets of proteins. The family database currently comprises more than 1500 manually curated protein families and their annotation templates that are used to annotate proteins that belong to one of the HAMAP families. On the HAMAP website, individual sequences as well as whole genomes can be scanned against all HAMAP families. The system provides warnings for the absence of conserved amino acid residues, unusual sequence length, etc. Thanks to the implementation of HAMAP, more than 200 000 microbial proteins have been fully annotated in UniProtKB/Swiss-Prot (HAMAP website: http://www.expasy.org/sprot/hamap)

Evolutionary History of the HAP2/GCS1 Gene and Sexual Reproduction in Metazoans

The HAP2/GCS1 gene first appeared in the common ancestor of plants, animals, and protists, and is required in the male gamete for fusion to the female gamete in the unicellular organisms Chlamydomonas and Plasmodium. We have identified a HAP2/GCS1 gene in the genome sequence of the sponge Amphimedon queenslandica. This finding provides a continuous evolutionary history of HAP2/GCS1 from unicellular organisms into the metazoan lineage. Divergent versions of the HAP2/GCS1 gene are also present in the genomes of some but not all arthropods. By examining the expression of the HAP2/GCS1 gene in the cnidarian Hydra, we have found the first evidence supporting the hypothesis that HAP2/GCS1 was used for male gamete fusion in the ancestor of extant metazoans and that it retains that function in modern cnidarians

Improved accuracy of multiple ncRNA alignment by incorporating structural information into a MAFFT-based framework

<p>Abstract</p> <p>Background</p> <p>Structural alignment of RNAs is becoming important, since the discovery of functional non-coding RNAs (ncRNAs). Recent studies, mainly based on various approximations of the Sankoff algorithm, have resulted in considerable improvement in the accuracy of pairwise structural alignment. In contrast, for the cases with more than two sequences, the practical merit of structural alignment remains unclear as compared to traditional sequence-based methods, although the importance of multiple structural alignment is widely recognized.</p> <p>Results</p> <p>We took a different approach from a straightforward extension of the Sankoff algorithm to the multiple alignments from the viewpoints of accuracy and time complexity. As a new option of the MAFFT alignment program, we developed a multiple RNA alignment framework, X-INS-i, which builds a multiple alignment with an iterative method incorporating structural information through two components: (1) pairwise structural alignments by an external pairwise alignment method such as SCARNA or LaRA and (2) a new objective function, Four-way Consistency, derived from the base-pairing probability of every sub-aligned group at every multiple alignment stage.</p> <p>Conclusion</p> <p>The BRAliBASE benchmark showed that X-INS-i outperforms other methods currently available in the sum-of-pairs score (SPS) criterion. As a basis for predicting common secondary structure, the accuracy of the present method is comparable to or rather higher than those of the current leading methods such as RNA Sampler. The X-INS-i framework can be used for building a multiple RNA alignment from any combination of algorithms for pairwise RNA alignment and base-pairing probability. The source code is available at the webpage found in the Availability and requirements section.</p

An efficient genetic algorithm for structural RNA pairwise alignment and its application to non-coding RNA discovery in yeast

<p>Abstract</p> <p>Background</p> <p>Aligning RNA sequences with low sequence identity has been a challenging problem since such a computation essentially needs an algorithm with high complexities for taking structural conservation into account. Although many sophisticated algorithms for the purpose have been proposed to date, further improvement in efficiency is necessary to accelerate its large-scale applications including non-coding RNA (ncRNA) discovery.</p> <p>Results</p> <p>We developed a new genetic algorithm, Cofolga2, for simultaneously computing pairwise RNA sequence alignment and consensus folding, and benchmarked it using BRAliBase 2.1. The benchmark results showed that our new algorithm is accurate and efficient in both time and memory usage. Then, combining with the originally trained SVM, we applied the new algorithm to novel ncRNA discovery where we compared <it>S. cerevisiae </it>genome with six related genomes in a pairwise manner. By focusing our search to the relatively short regions (50 bp to 2,000 bp) sandwiched by conserved sequences, we successfully predict 714 intergenic and 1,311 sense or antisense ncRNA candidates, which were found in the pairwise alignments with stable consensus secondary structure and low sequence identity (≤ 50%). By comparing with the previous predictions, we found that > 92% of the candidates is novel candidates. The estimated rate of false positives in the predicted candidates is 51%. Twenty-five percent of the intergenic candidates has supports for expression in cell, i.e. their genomic positions overlap those of the experimentally determined transcripts in literature. By manual inspection of the results, moreover, we obtained four multiple alignments with low sequence identity which reveal consensus structures shared by three species/sequences.</p> <p>Conclusion</p> <p>The present method gives an efficient tool complementary to sequence-alignment-based ncRNA finders.</p

The Dawn of Open Access to Phylogenetic Data

The scientific enterprise depends critically on the preservation of and open access to published data. This basic tenet applies acutely to phylogenies (estimates of evolutionary relationships among species). Increasingly, phylogenies are estimated from increasingly large, genome-scale datasets using increasingly complex statistical methods that require increasing levels of expertise and computational investment. Moreover, the resulting phylogenetic data provide an explicit historical perspective that critically informs research in a vast and growing number of scientific disciplines. One such use is the study of changes in rates of lineage diversification (speciation - extinction) through time. As part of a meta-analysis in this area, we sought to collect phylogenetic data (comprising nucleotide sequence alignment and tree files) from 217 studies published in 46 journals over a 13-year period. We document our attempts to procure those data (from online archives and by direct request to corresponding authors), and report results of analyses (using Bayesian logistic regression) to assess the impact of various factors on the success of our efforts. Overall, complete phylogenetic data for ~60% of these studies are effectively lost to science. Our study indicates that phylogenetic data are more likely to be deposited in online archives and/or shared upon request when: (1) the publishing journal has a strong data-sharing policy; (2) the publishing journal has a higher impact factor, and; (3) the data are requested from faculty rather than students. Although the situation appears dire, our analyses suggest that it is far from hopeless: recent initiatives by the scientific community -- including policy changes by journals and funding agencies -- are improving the state of affairs

Photo-antagonism of the GABAA receptor

Neurotransmitter receptor trafficking is fundamentally important for synaptic transmission and neural network activity. GABAA receptors and inhibitory synapses are vital components of brain function, yet much of our knowledge regarding receptor mobility and function at inhibitory synapses is derived indirectly from using recombinant receptors, antibody-tagged native receptors and pharmacological treatments. Here we describe the use of a set of research tools that can irreversibly bind to and affect the function of recombinant and neuronal GABAA receptors following ultraviolet photoactivation. These compounds are based on the competitive antagonist gabazine and incorporate a variety of photoactive groups. By using site-directed mutagenesis and ligand-docking studies, they reveal new areas of the GABA binding site at the interface between receptor β and α subunits. These compounds enable the selected inactivation of native GABAA receptor populations providing new insight into the function of inhibitory synapses and extrasynaptic receptors in controlling neuronal excitation

Large introns in relation to alternative splicing and gene evolution: a case study of Drosophila bruno-3

Background: Alternative splicing (AS) of maturing mRNA can generate structurally and functionally distinct transcripts from the same gene. Recent bioinformatic analyses of available genome databases inferred a positive correlation between intron length and AS. To study the interplay between intron length and AS empirically and in more detail, we analyzed the diversity of alternatively spliced transcripts (ASTs) in the Drosophila RNA-binding Bruno-3 (Bru-3) gene. This gene was known to encode thirteen exons separated by introns of diverse sizes, ranging from 71 to 41,973 nucleotides in D. melanogaster. Although Bru-3's structure is expected to be conducive to AS, only two ASTs of this gene were previously described. Results: Cloning of RT-PCR products of the entire ORF from four species representing three diverged Drosophila lineages provided an evolutionary perspective, high sensitivity, and long-range contiguity of splice choices currently unattainable by high-throughput methods. Consequently, we identified three new exons, a new exon fragment and thirty-three previously unknown ASTs of Bru-3. All exon-skipping events in the gene were mapped to the exons surrounded by introns of at least 800 nucleotides, whereas exons split by introns of less than 250 nucleotides were always spliced contiguously in mRNA. Cases of exon loss and creation during Bru-3 evolution in Drosophila were also localized within large introns. Notably, we identified a true de novo exon gain: exon 8 was created along the lineage of the obscura group from intronic sequence between cryptic splice sites conserved among all Drosophila species surveyed. Exon 8 was included in mature mRNA by the species representing all the major branches of the obscura group. To our knowledge, the origin of exon 8 is the first documented case of exonization of intronic sequence outside vertebrates. Conclusion: We found that large introns can promote AS via exon-skipping and exon turnover during evolution likely due to frequent errors in their removal from maturing mRNA. Large introns could be a reservoir of genetic diversity, because they have a greater number of mutable sites than short introns. Taken together, gene structure can constrain and/or promote gene evolution

Molecular characterization of beta-tubulin from Phakopsora pachyrhizi, the causal agent of Asian soybean rust

β-tubulins are structural components of microtubules and the targets of benzimidazole fungicides used to control many diseases of agricultural importance. Intron polymorphisms in the intron-rich genes of these proteins have been used in phylogeographic investigations of phytopathogenic fungi. In this work, we sequenced 2764 nucleotides of the β-tubulin gene (Pp tubB) in samples of Phakopsora pachyrhizi collected from seven soybean fields in Brazil. Pp tubB contained an open reading frame of 1341 nucleotides, including nine exons and eight introns. Exon length varied from 14 to 880 nucleotides, whereas intron length varied from 76 to 102 nucleotides. The presence of only four polymorphic sites limited the usefulness of Pp tubB for phylogeographic studies in P. pachyrhizi. The gene structures of Pp tubB and orthologous β-tubulin genes of Melampsora lini and Uromyces viciae-fabae were highly conserved. The amino acid substitutions in β-tubulin proteins associated with the onset of benzimidazole resistance in model organisms, especially at His 6 , Glu 198 and Phe 200 , were absent from the predicted sequence of the P. pachyrhizi β-tubulin protein

Specificity of the E. coli LysR-Type Transcriptional Regulators

Families of paralogous oligomeric proteins are common in biology. How the specificity of assembly evolves is a fundamental question of biology. The LysR-Type Transcriptional Regulators (LTTR) form perhaps the largest family of transcriptional regulators in bacteria. Because genomes often encode many LTTR family members, it is assumed that many distinct homooligomers are formed simultaneously in the same cell without interfering with each other's activities, suggesting specificity in the interactions. However, this assumption has not been systematically tested.A negative-dominant assay with λcI repressor fusions was used to evaluate the assembly of the LTTRs in E. coli K-12. Thioredoxin (Trx)-LTTR fusions were used to challenge the homooligomeric interactions of λcI-LTTR fusions. Eight cI-LTTR fusions were challenged with twenty-eight Trx fusions. LTTRs could be divided into three classes based on their interactions with other LTTRs.Multimerization of LTTRs in E. coli K-12 is mostly specific. However, under the conditions of the assay, many LTTRs interact with more than one noncognate partner. The physiological significance and physical basis for these interactions are not known