Federico Abascal

TranslatorX: multiple alignment of nucleotide sequences guided by amino acid translations

We present TranslatorX, a web server designed to align protein-coding nucleotide sequences based on their corresponding amino acid translations. Many comparisons between biological sequences (nucleic acids and proteins) involve the construction of multiple alignments. Alignments represent a statement regarding the homology between individual nucleotides or amino acids within homologous genes. As protein-coding DNA sequences evolve as triplets of nucleotides (codons) and it is known that sequence similarity degrades more rapidly at the DNA than at the amino acid level, alignments are generally more accurate when based on amino acids than on their corresponding nucleotides. TranslatorX novelties include: (i) use of all documented genetic codes and the possibility of assigning different genetic codes for each sequence; (ii) a battery of different multiple alignment programs; (iii) translation of ambiguous codons when possible; (iv) an innovative criterion to clean nucleotide alignments with GBlocks based on protein information; and (v) a rich output, including Jalview-powered graphical visualization of the alignments, codon-based alignments coloured according to the corresponding amino acids, measures of compositional bias and first, second and third codon position specific alignments. The TranslatorX server is freely available at http://translatorx.co.uk.

Reductive genome evolution in Buchnera aphidicola

We have sequenced the genome of the intracellular symbiont Buchnera aphidicola from the aphid Baizongia pistacea. This strain diverged 80–150 million years ago from the common ancestor of two previously sequenced Buchnera strains. Here, a field-collected, nonclonal sample of insects was used as source material for laboratory procedures. As a consequence, the genome assembly unveiled intrapopulational variation, consisting of ≈1,200 polymorphic sites. Comparison of the 618-kb (kbp) genome with the two other Buchnera genomes revealed a nearly perfect gene-order conservation, indicating that the onset of genomic stasis coincided closely with establishment of the symbiosis with aphids, ≈200 million years ago. Extensive genome reduction also predates the synchronous diversification of Buchnera and its host; but, at a slower rate, gene loss continues among the extant lineages. A computational study of protein folding predicts that proteins in Buchnera, as well as proteins of other intracellular bacteria, are generally characterized by smaller folding efficiency compared with proteins of free living bacteria. These and other degenerative genomic features are discussed in light of compensatory processes and theoretical predictions on the long-term evolutionary fate of symbionts like Buchnera.

Diversity and evolution of membrane intrinsic proteins

BACKGROUND Membrane intrinsic proteins (MIPs) are the proteins in charge of regulating water transport into cells. Because of this essential function, the MIP family is ancient, widespread, and highly diverse. SCOPE OF REVIEW The rapidly accumulating genomic and transcriptomic data from previously poorly known groups such as unicellular eukaryotes, fungi, green algae, mosses, and non-vertebrate animals are contributing to expand our view of MIP evolution throughout the diversity of life. Here, by analyzing more than 1700 sequences, we provide an updated and comprehensive phylogeny of MIPs MAJOR CONCLUSIONS The reconstructed phylogeny supports (i) deep orthology of X intrinsic proteins (XIPs; present from unicellular eukaryotes to plants); (ii) that the origin of small intrinsic proteins (SIPs) traces back to the common ancestor of all plants; and (iii) the expansion of aquaglyceroporins (GLPs) in Oomycetes, as well as their loss in vascular plants and in the ancestor of endopterygote insects. Additionally, conserved positions in the protein, and residues involved in glycerol selectivity are reviewed within a phylogenetic framework. Furthermore, functional diversification of human and Arabidopsis paralogs are analyzed in an evolutionary genomic context. GENERAL SIGNIFICANCE Our results show that while bacteria and archaea generally function with one copy of each a water channel (aquaporin or AQP) and a GLP, recurrent independent expansions have greatly diversified the structures and functions of the different members of both MIP paralog subfamilies throughout eukaryote evolution (and not only in flowering plants and vertebrates, as previously thought). This article is part of a Special Issue entitled Aquaporins.

LRRC8 proteins share a common ancestor with pannexins, and may form hexameric channels involved in cell-cell communication.

Leucine‐rich repeat‐containing 8 (LRRC8) proteins are composed of four transmembrane helices and 17 leucine‐rich repeats (LRR). Although LRRC8 proteins have been associated with important processes, like maturation of B cells or adipocyte differentiation, their biology and molecular function are largely unknown. We found that LRRC8 proteins originated from the combination of a pannexin and an LRR domain (most likely related to the SHOC2, LAP, RSU1 and LRRIQ4 protein families) before the diversification of chordates. We propose that, like pannexins, LRRC8 proteins form hexameric channels, which participate in cell‐cell communication processes. According to the inferred topological model, and contrary to what was previously assumed, the six LRR domains are located in the cytoplasm, and could participate in the organisation of signalling cascades. By compiling available proteomics and gene expression data, and on the basis of the LRRC8 proposed hexameric channel structure, we present clues to the function of this family.

Evolutionary analyses of gap junction protein families

Gap junctions are intercellular channels that link the cytoplasm of neighboring cells in animals, enabling straight passage of ions and small molecules. Two different protein families, pannexins and connexins, form these channels. Pannexins are present in all eumetazoans but echinoderms (and are termed innexins in non-chordates) whereas connexins are exclusive of chordates. Despite little sequence similarity, both types of proteins assemble into a common secondary structure with four hydrophobic transmembrane domains linked by one cytoplasmic and two extracellular loops. Although all pannexins and connexins are packed into hexamers forming single channels, only non-chordate pannexins (innexins) and connexins form gap junctions. Here, we revisit and review evolutionary features of pannexin and connexin protein families. For that, we retrieved members of both families from several complete genome projects, and searched for conserved positions in the independent alignments of pannexin and connexin protein families. In addition, the degree of evolutionary conservation was mapped onto the 3D structure of a connexon (i.e. the assembly of six connexins). Finally, we reconstructed independent phylogenies of pannexins and connexins using probabilistic methods of inference. Non-chordate (Drosophila and Caenorhabditis) pannexins (i.e. innexins) were recovered as sister group of chordate pannexins, which included Ciona paralogs and vertebrate pannexins (pannexin-1 and pannexin-3 were recovered as sister groups to the exclusion of pannexin-2). In the reconstructed phylogeny of connexins, subfamilies α and β were recovered as sister groups to the exclusion of subfamily γ, whereas δ and (the newly identified) ζ subfamilies were recovered at the base of the tree. A sixth highly divergent subfamily (ε) was not included in the phylogenetic analyses. Several groups of paralogy were identified within each subfamily. This article is part of a Special Issue entitled: The Communicating junctions, roles and dysfunctions.

Alternative Splicing May Not Be the Key to Proteome Complexity.

Alternative splicing is commonly believed to be a major source of cellular protein diversity. However, although many thousands of alternatively spliced transcripts are routinely detected in RNA-seq studies, reliable large-scale mass spectrometry-based proteomics analyses identify only a small fraction of annotated alternative isoforms. The clearest finding from proteomics experiments is that most human genes have a single main protein isoform, while those alternative isoforms that are identified tend to be the most biologically plausible: those with the most cross-species conservation and those that do not compromise functional domains. Indeed, most alternative exons do not seem to be under selective pressure, suggesting that a large majority of predicted alternative transcripts may not even be translated into proteins.

Clustering of proximal sequence space for the identification of protein families

MOTIVATION The study of sequence space, and the deciphering of the structure of protein families and subfamilies, has up to now been required for work in comparative genomics and for the prediction of protein function. With the emergence of structural proteomics projects, it is becoming increasingly important to be able to select protein targets for structural studies that will appropriately cover the space of protein sequences, functions and genomic distribution. These problems are the motivation for the development of methods for clustering protein sequences and building families of potentially orthologous sequences, such as those proposed here. RESULTS First we developed a clustering strategy (Ncut algorithm) capable of forming groups of related sequences by assessing their pairwise relationships. The results presented for the ras super-family of proteins are similar to those produced by other clustering methods, but without the need for clustering the full sequence space. The Ncut clusters are then used as the input to a process of reconstruction of groups with equilibrated genomic composition formed by closely-related sequences. The results of applying this technique to the data set used in the construction of the COG database are very similar to those derived by the human experts responsible for this database. AVAILABILITY The analysis of different systems, including the COG equivalent 21 genomes are available at http://www.pdg.cnb.uam.es/GenoClustering.html.

Automatic annotation of protein function based on family identification.

Although genomes are being sequenced at an impressive rate, the information generated tells us little about protein function, which is slow to characterize by traditional methods. Automatic protein function annotation based on computational methods has alleviated this imbalance. The most powerful current approach for inferring the function of new proteins is by studying the annotations of their homologues, since their common origin is assumed to be reflected in their structure and function. Unfortunately, as proteins evolve they acquire new functions, so annotation based on homology must be carried out in the context of orthologues or subfamilies. Evolution adds new complications through domain shuffling: homology (or orthology) frequently corresponds to domains rather than complete proteins. Moreover, the function of a protein may be seen as the result of combining the functions of its domains. Additionally, automatic annotation has to deal with problems related to the annotations in the databases: errors (which are likely to be propagated), inconsistencies, or different degrees of function specification. We describe a method that addresses these difficulties for the annotation of protein function. Sequence relationships are detected and measured to obtain a map of the sequence space, which is searched for differentiated groups of proteins (similar to islands on the map), which are expected to have a common function and correspond to groups of orthologues or subfamilies. This mapmaking is done by applying a clustering algorithm based on Normalized cuts in graphs. The domain problem is addressed in a simple way: pairwise local alignments are analyzed to determine the extent to which they cover the entire sequence lengths of the two proteins. This analysis determines both what homologues are preferred for functional inheritance and the level of confidence of the annotation. To alleviate the problems associated with database annotations, the information on all the homologues that are grouped together with the query protein are taken into account to select the most representative functional descriptors. This method has been applied for the annotation of the genome of Buchnera aphidicola (specific host Baizongia pistaciae). Human inspection of the annotations allowed an estimation of accuracy of 94%; the different kinds of error that may appear when using this approach are described. Results can be accessed at http://www.pdg.cnb.uam.es/funcut.html. The programs are available upon request, although installation in other systems may be complicated. Proteins 2003;53:000–000.

The origin of modern frogs (Neobatrachia) was accompanied by acceleration in mitochondrial and nuclear substitution rates

BackgroundUnderstanding the causes underlying heterogeneity of molecular evolutionary rates among lineages is a long-standing and central question in evolutionary biology. Although several earlier studies showed that modern frogs (Neobatrachia) experienced an acceleration of mitochondrial gene substitution rates compared to non-neobatrachian relatives, no further characterization of this phenomenon was attempted. To gain new insights on this topic, we sequenced the complete mitochondrial genomes and nine nuclear loci of one pelobatoid (Pelodytes punctatus) and five neobatrachians, Heleophryne regis (Heleophrynidae), Lechriodus melanopyga (Limnodynastidae), Calyptocephalella gayi (Calyptocephalellidae), Telmatobius bolivianus (Ceratophryidae), and Sooglossus thomasseti (Sooglossidae). These represent major clades not included in previous mitogenomic analyses, and most of them are remarkably species-poor compared to other neobatrachians.ResultsWe reconstructed a fully resolved and robust phylogeny of extant frogs based on the new mitochondrial and nuclear sequence data, and dated major cladogenetic events. The reconstructed tree recovered Heleophryne as sister group to all other neobatrachians, the Australasian Lechriodus and the South American Calyptocephalella formed a clade that was the sister group to Nobleobatrachia, and the Seychellois Sooglossus was recovered as the sister group of Ranoides. We used relative-rate tests and direct comparison of branch lengths from mitochondrial and nuclear-based trees to demonstrate that both mitochondrial and nuclear evolutionary rates are significantly higher in all neobatrachians compared to their non-neobatrachian relatives, and that such rate acceleration started at the origin of Neobatrachia.ConclusionsThrough the analysis of the selection coefficient (ω) in different branches of the tree, we found compelling evidence of relaxation of purifying selection in neobatrachians, which could (at least in part) explain the observed higher mitochondrial and nuclear substitution rates in this clade. Our analyses allowed us to discard that changes in substitution rates could be correlated with increased mitochondrial genome rearrangement or diversification rates observed in different lineages of neobatrachians.

GenDecoder: genetic code prediction for metazoan mitochondria

Although the majority of the organisms use the same genetic code to translate DNA, several variants have been described in a wide range of organisms, both in nuclear and organellar systems, many of them corresponding to metazoan mitochondria. These variants are usually found by comparative sequence analyses, either conducted manually or with the computer. Basically, when a particular codon in a query-species is linked to positions for which a specific amino acid is consistently found in other species, then that particular codon is expected to translate as that specific amino acid. Importantly, and despite the simplicity of this approach, there are no available tools to help predicting the genetic code of an organism. We present here GenDecoder, a web server for the characterization and prediction of mitochondrial genetic codes in animals. The analysis of automatic predictions for 681 metazoans aimed us to study some properties of the comparative method, in particular, the relationship among sequence conservation, taxonomic sampling and reliability of assignments. Overall, the method is highly precise (99%), although highly divergent organisms such as platyhelminths are more problematic. The GenDecoder web server is freely available from .