Biology and physiology of Hanseniaspora vineae: metabolic diversity and increase flavour complexity for food fermentation.

Apiculate yeasts belonging to the genus Hanseniaspora are predominant on grapes and other fruits. While some species, such as Hanseniaspora uvarum, are well known for their abundant presence in fruits, they are generally characterized by their detrimental effect on fermentation quality because the excessive production of acetic acid. However, the species Hanseniaspora vineae is adapted to fermentation and currently is considered as an enhancer of positive flavour and sensory complexity in foods. Since 2002, we have been isolating strains from this species and conducting winemaking processes with them. In parallel, we also characterized this species from genes to metabolites. In 2013, we sequenced the genomes of two H. vineae strains, being these the first apiculate yeast genomes determined. In the last 10 years, it has become possible to understand its biology, discovering very peculiar features compared to the conventional Saccharomyces yeasts, such as a natural and unique G2 cell cycle arrest or the elucidation of the mandelate pathway for benzenoids synthesis. All these characteristics contribute to phenotypes with proved interest from the biotechnological point of view for winemaking and the production of other foods.

Uncovering Pseudogenes and Intergenic Protein-coding Sequences in TriTryps' Genomes.

Trypanosomatids belong to a remarkable group of unicellular, parasitic organisms of the order Kinetoplastida, an early diverging branch of the phylogenetic tree of eukaryotes, exhibiting intriguing biological characteristics affecting gene expression (intronless polycistronic transcription, trans-splicing, and RNA editing), metabolism, surface molecules, and organelles (compartmentalization of glycolysis, variation of the surface molecules, and unique mitochondrial DNA), cell biology and life cycle (phagocytic vacuoles evasion and intricate patterns of cell morphogenesis). With numerous genomic-scale data of several trypanosomatids becoming available since 2005 (genomes, transcriptomes, and proteomes), the scientific community can further investigate the mechanisms underlying these unusual features and address other unexplored phenomena possibly revealing biological aspects of the early evolution of eukaryotes. One fundamental aspect comprises the processes and mechanisms involved in the acquisition and loss of genes throughout the evolutionary history of these primitive microorganisms. Here, we present a comprehensive in silico analysis of pseudogenes in three major representatives of this group: Leishmania major, Trypanosoma brucei, and Trypanosoma cruzi. Pseudogenes, DNA segments originating from altered genes that lost their original function, are genomic relics that can offer an essential record of the evolutionary history of functional genes, as well as clues about the dynamics and evolution of hosting genomes. Scanning these genomes with functional proteins as proxies to reveal intergenic regions with protein-coding features, relying on a customized threshold to distinguish statistically and biologically significant sequence similarities, and reassembling remnant sequences from their debris, we found thousands of pseudogenes and hundreds of open reading frames, with particular characteristics in each trypanosomatid: mutation profile, number, content, density, codon bias, average size, single- or multi-copy gene origin, number and type of mutations, putative primitive function, and transcriptional activity. These features suggest a common process of pseudogene formation, different patterns of pseudogene evolution and extant biological functions, and/or distinct genome organization undertaken by those parasites during evolution, as well as different evolutionary and/or selective pressures acting on distinct lineages.

Maxicircle architecture and evolutionary insights into Trypanosoma cruzi complex.

We sequenced maxicircles from T. cruzi strains representative of the species evolutionary diversity by using long-read sequencing, which allowed us to uncollapse their repetitive regions, finding that their real lengths range from 35 to 50 kb. T. cruzi maxicircles have a common architecture composed of four regions: coding region (CR), AT-rich region, short (SR) and long repeats (LR). Distribution of genes, both in order and in strand orientation are conserved, being the main differences the presence of deletions affecting genes coding for NADH dehydrogenase subunits, reinforcing biochemical findings that indicate that complex I is not functional in T. cruzi. Moreover, the presence of complete minicircles into maxicircles of some strains lead us to think about the origin of minicircles. Finally, a careful phylogenetic analysis was conducted using coding regions of maxicircles from up to 29 strains, and 1108 single copy nuclear genes from all of the DTUs, clearly establishing that taxonomically T. cruzi is a complex of species composed by group 1 that contains clades A (TcI), B (TcIII) and D (TcIV), and group 2 (1 and 2 do not coincide with groups I and II described decades ago) containing clade C (TcII), being all hybrid strains of the BC type. Three variants of maxicircles exist in T. cruzi: a, b and c, in correspondence with clades A, B, and C from mitochondrial phylogenies. While A and C carry maxicircles a and c respectively, both clades B and D carry b maxicircle variant; hybrid strains also carry the b- variant. We then propose a new nomenclature that is self-descriptive and makes use of both the phylogenetic relationships and the maxicircle variants present in T. cruzi.

Different kinetoplast degradation patterns in American Trypanosoma vivax strains: Multiple independent origins or fast evolution?

We analyzed the kinetoplast (mitochondrial genome) of Trypanosoma vivax strains from America and Africa to determine their precise architecture and to understand their adaptive response to mechanical transmission. The use of long-read based assemblies that retain individuality of tandem repeats, without erasing inter-copy variability, allowed us to investigate the evolutionary dynamics of repetitive kinetoplast-DNA. This analysis revealed that repeat elements located in edges of repeat clusters are less active in terms of renewal, whereas internal copies appear to undergo a permanent process of birth-and-death. Comparing different American strains with the African Y486 strain, we found that in the former, protein coding genes from the maxicircle contain several function disrupting mutations that with very few exceptions are present in one or the other American strain but not in both, suggesting the absence of common ancestry for most of the genomic changes that led to their loss of oxidative phosphorylation capacity. Analysis of another component of kinetoplast, the minicircles, revealed great loss of diversity, and loss of their encoded guideRNAs. Both groups of American strains retain minimal sets required to edit the still functional A6-APTase and RPS12 genes. The extensive maxi- and minicircle divergence suggests a history of multiple introduction events in America of strains that probably started to degrade their kinetoplast in Africa. The notion that kinetoplast degradation began after incursion in America would imply a pace of accumulation of genetic changes considerably faster than other trypanosomatids.

Expanding an expanded genome: long-read sequencing of Trypanosoma cruzi.

Although the genome of Trypanosoma cruzi, the causative agent of Chagas disease, was first made available in 2005, with additional strains reported later, the intrinsic genome complexity of this parasite (the abundance of repetitive sequences and genes organized in tandem) has traditionally hindered high-quality genome assembly and annotation. This also limits diverse types of analyses that require high degrees of precision. Long reads generated by third-generation sequencing technologies are particularly suitable to address the challenges associated with T. cruzi's genome since they permit direct determination of the full sequence of large clusters of repetitive sequences without collapsing them. This, in turn, not only allows accurate estimation of gene copy numbers but also circumvents assembly fragmentation. Here, we present the analysis of the genome sequences of two T. cruzi clones: the hybrid TCC (TcVI) and the non-hybrid Dm28c (TcI), determined by PacBio Single Molecular Real-Time (SMRT) technology. The improved assemblies herein obtained permitted us to accurately estimate gene copy numbers, abundance and distribution of repetitive sequences (including satellites and retroelements). We found that the genome of T. cruzi is composed of a 'core compartment' and a 'disruptive compartment' which exhibit opposite GC content and gene composition. Novel tandem and dispersed repetitive sequences were identified, including some located inside coding sequences. Additionally, homologous chromosomes were separately assembled, allowing us to retrieve haplotypes as separate contigs instead of a unique mosaic sequence. Finally, manual annotation of surface multigene families, mucins and trans-sialidases allows now a better overview of these complex groups of genes.

Transcriptomic analysis reveals metabolic switches and surface remodeling as key processes for stage transition in Trypanosoma cruzi.

American trypanosomiasis is a chronic and endemic disease which affects millions of people. Trypanosoma cruzi, its causative agent, has a life cycle that involves complex morphological and functional transitions, as well as a variety of environmental conditions. This requires a tight regulation of gene expression, which is achieved mainly by post-transcriptional regulation. In this work we conducted an RNAseq analysis of the three major life cycle stages of T. cruzi: amastigotes, epimastigotes and trypomastigotes. This analysis allowed us to delineate specific transcriptomic profiling for each stage, and also to identify those biological processes of major relevance in each state. Stage specific expression profiling evidenced the plasticity of T. cruzi to adapt quickly to different conditions, with particular focus on membrane remodeling and metabolic shifts along the life cycle. Epimastigotes, which replicate in the gut of insect vectors, showed higher expression of genes related to energy metabolism, mainly Krebs cycle, respiratory chain and oxidative phosphorylation related genes, and anabolism related genes associated to nucleotide and steroid biosynthesis; also, a general down-regulation of surface glycoprotein coding genes was seen at this stage. Trypomastigotes, living extracellularly in the bloodstream of mammals, express a plethora of surface proteins and signaling genes involved in invasion and evasion of immune response. Amastigotes mostly express membrane transporters and genes involved in regulation of cell cycle, and also express a specific subset of surface glycoprotein coding genes. In addition, these results allowed us to improve the annotation of the Dm28c genome, identifying new ORFs and set the stage for construction of networks of co-expression, which can give clues about coded proteins of unknown functions.

Gene Expression Profiling in the Injured Spinal Cord of Trachemys scripta elegans: An Amniote with Self-Repair Capabilities.

Slider turtles are the only known amniotes with self-repair mechanisms of the spinal cord that lead to substantial functional recovery. Their strategic phylogenetic position makes them a relevant model to investigate the peculiar genetic programs that allow anatomical reconnection in some vertebrate groups but are absent in others. Here, we analyze the gene expression profile of the response to spinal cord injury (SCI) in the turtle Trachemys scripta elegans. We found that this response comprises more than 1000 genes affecting diverse functions: reaction to ischemic insult, extracellular matrix re-organization, cell proliferation and death, immune response, and inflammation. Genes related to synapses and cholesterol biosynthesis are down-regulated. The analysis of the evolutionary distribution of these genes shows that almost all are present in most vertebrates. Additionally, we failed to find genes that were exclusive of regenerating taxa. The comparison of expression patterns among species shows that the response to SCI in the turtle is more similar to that of mice and non-regenerative Xenopus than to Xenopus during its regenerative stage. This observation, along with the lack of conserved "regeneration genes" and the current accepted phylogenetic placement of turtles (sister group of crocodilians and birds), indicates that the ability of spinal cord self-repair of turtles does not represent the retention of an ancestral vertebrate character. Instead, our results suggest that turtles developed this capability from a non-regenerative ancestor (i.e., a lineage specific innovation) that was achieved by re-organizing gene expression patterns on an essentially non-regenerative genetic background. Among the genes activated by SCI exclusively in turtles, those related to anoxia tolerance, extracellular matrix remodeling, and axonal regrowth are good candidates to underlie functional recovery.

The Anolis Lizard Genome: An Amniote Genome without Isochores?

Two articles published 5 years ago concluded that the genome of the lizard Anolis carolinensis is an amniote genome without isochores. This claim was apparently contradicting previous results on the general presence of an isochore organization in all vertebrate genomes tested (including Anolis). In this investigation, we demonstrate that the Anolis genome is indeed heterogeneous in base composition, since its macrochromosomes comprise isochores mainly from the L2 and H1 families (a moderately GC-poor and a moderately GC-rich family, respectively), and since the majority of the sequenced microchromosomes consists of H1 isochores. These families are associated with different features of genome structure, including gene density and compositional correlations (e.g., GC3 vs flanking sequence GC and intron GC), as in the case of mammalian and avian genomes. Moreover, the assembled Anolis chromosomes have an enormous number of gaps, which could be due to sequencing problems in GC-rich regions of the genome. In conclusion, the Anolis genome is no exception to the general rule of an isochore organization in the genomes of vertebrates (and other eukaryotes).

A strategy to recover a high-quality, complete plastid sequence from low-coverage whole-genome sequencing.

PREMISE OF THE STUDY: We developed a bioinformatic strategy to recover and assemble a chloroplast genome using data derived from low-coverage 454 GS FLX/Roche whole-genome sequencing. METHODS: A comparative genomics approach was applied to obtain the complete chloroplast genome from a weedy biotype of rice from Uruguay. We also applied appropriate filters to discriminate reads representing novel DNA transfer events between the chloroplast and nuclear genomes. RESULTS: From a set of 295,159 reads (96 Mb data), we assembled the chloroplast genome into two contigs. This weedy rice was classified based on 23 polymorphic regions identified by comparison with reference chloroplast genomes. We detected recent and past events of genetic material transfer between the chloroplast and nuclear genomes and estimated their occurrence frequency. DISCUSSION: We obtained a high-quality complete chloroplast genome sequence from low-coverage sequencing data. Intergenome DNA transfer appears to be more frequent than previously thought.

Evolutionary volatile Cysteines and protein disorder in the fast evolving tunicate Oikopleura dioica.

Cysteine (Cys) is regarded as the most conservative amino acid in nature, something that does not occur in the tunicate Oikopleura dioica, where this amino acid is one of the fastest evolving. In this work we analyze some of the causes of this intriguing absence of conservation. Considering the well-known stabilizing role of Cys, it was first investigated whether the lack of conservation was accompanied by an increase in intrinsic protein disorder. In contrast to expectations, it was found that O. dioica is the chordate that has the lowest levels of intrinsic disorder, while vertebrates (represented by Bos taurus) contain the most disordered proteins. Oikopleura proteins are shorter than their homologs in other Chordates (Ciona and B. taurus proteins are respectively 11% and 18% longer). This process of protein shortening was more intense in intrinsic disordered regions. As a result proteins became not only shorter but also more compact. It is also reported here that the conservation/divergence behavior of Cys depends on whether they are located in ordered or disordered regions. In the four species analyzed, disordered Cys are majorly (> 75%) not conserved at all. Ordered Cys instead, are much more free to diverge in Oikopleura than in the other chordates. We hypothesize that the preferential deletion of disordered regions resulted in a decreased protein disorder and a direct elimination (by deletion) of many ancestral Cys. Besides, the alterations (shortening or complete elimination) of some disordered regions (loops/random coils) probably promoted further Cys evolutionary volatility, because some ancestral Cys (and other amino acids which play a role in stability like Trp) located outside deleted regions became redundant due to the loss of their stabilizing partners.

A stochastic microscopic model for the dynamics of antigenic variation.

We present a novel model that describes the within-host evolutionary dynamics of parasites undergoing antigenic variation. The approach uses a multi-type branching process with two types of entities defined according to their relationship with the immune system: clans of resistant parasitic cells (i.e. groups of cells sharing the same antigen not yet recognized by the immune system) that may become sensitive, and individual sensitive cells that can acquire a new resistance thus giving rise to the emergence of a new clan. The simplicity of the model allows analytical treatment to determine the subcritical and supercritical regimes in the space of parameters. By incorporating a density-dependent mechanism the model is able to capture additional relevant features observed in experimental data, such as the characteristic parasitemia waves. In summary our approach provides a new general framework to address the dynamics of antigenic variation which can be easily adapted to cope with broader and more complex situations.

Conservation of CFTR codon frequency through primates suggests synonymous mutations could have a functional effect.

Cystic fibrosis is an inherited chronic disease that affects the lungs and digestive system, with a prevalence of about 1:3000 people. Cystic fibrosis is caused by mutations in CFTR gene, which lead to a defective function of the chloride channel, the cystic fibrosis transmembrane conductance regulator (CFTR). Up-to-date, more than 1900 mutations have been reported in CFTR. However for an important proportion of them, their functional effects and the relation to disease are still not understood. Many of these mutations are silent (or synonymous), namely they do not alter the encoded amino acid. These synonymous mutations have been considered as neutral to protein function. However, more recent evidence in bacterial and human proteins has put this concept under revision. With the aim of understanding possible functional effects of synonymous mutations in CFTR, we analyzed human and primates CFTR codon usage and divergence patterns. We report the presence of regions enriched in rare and frequent codons. This spatial pattern of codon preferences is conserved in primates, but this cannot be explained by sequence conservation alone. In sum, the results presented herein suggest a functional implication of these regions of the gene that may be maintained by purifying selection acting to preserve a particular codon usage pattern along the sequence. Overall these results support the idea that several synonymous mutations in CFTR may have functional importance, and could be involved in the disease.

Kinetoplast adaptations in American strains from Trypanosoma vivax.

The mitochondrion role changes during the digenetic life cycle of African trypanosomes. Owing to the low abundance of glucose in the insect vector (tsetse flies) the parasites are dependent upon a fully functional mitochondrion, capable of performing oxidative phosphorylation. Nevertheless, inside the mammalian host (bloodstream forms), which is rich in nutrients, parasite proliferation relies on glycolysis, and the mitochondrion is partially redundant. In this work we perform a comparative study of the mitochondrial genome (kinetoplast) in different strains of Trypanosoma vivax. The comparison was conducted between a West African strain that goes through a complete life cycle and two American strains that are mechanically transmitted (by different vectors) and remain as bloodstream forms only. It was found that while the African strain has a complete and apparently fully functional kinetoplast, the American T. vivax strains have undergone a drastic process of mitochondrial genome degradation, in spite of the recent introduction of these parasites in America. Many of their genes exhibit different types of mutations that are disruptive of function such as major deletions, frameshift causing indels and missense mutations. Moreover, all but three genes (A6-ATPase, RPS12 and MURF2) are not edited in the American strains, whereas editing takes place normally in all (editable) genes from the African strain. Two of these genes, A6-ATPase and RPS12, are known to play an essential function during bloodstream stage. Analysis of the minicircle population shows that its diversity has been greatly reduced, remaining mostly those minicircles that carry guide RNAs necessary for the editing of A6-ATPase and RPS12. The fact that these two genes remain functioning normally, as opposed to that reported in Trypanosoma brucei-like trypanosomes that restrict their life cycle to the bloodstream forms, along with other differences, is indicative that the American T. vivax strains are following a novel evolutionary pathway.

Evolutionary genomics of fast evolving tunicates.

Tunicates have been extensively studied because of their crucial phylogenetic location (the closest living relatives of vertebrates) and particular developmental plan. Recent genome efforts have disclosed that tunicates are also remarkable in their genome organization and molecular evolutionary patterns. Here, we review these latter aspects, comparing the similarities and specificities of two model species of the group: Oikopleura dioica and Ciona intestinalis. These species exhibit great genome plasticity and Oikopleura in particular has undergone a process of extreme genome reduction and compaction that can be explained in part by gene loss, but is mostly due to other mechanisms such as shortening of intergenic distances and introns, and scarcity of mobile elements. In Ciona, genome reorganization was less severe being more similar to the other chordates in several aspects. Rates and patterns of molecular evolution are also peculiar in tunicates, being Ciona about 50% faster than vertebrates and Oikopleura three times faster. In fact, the latter species is considered as the fastest evolving metazoan recorded so far. Two processes of increase in evolutionary rates have taken place in tunicates. One of them is more extreme, and basically restricted to genes encoding regulatory proteins (transcription regulators, chromatin remodeling proteins, and metabolic regulators), and the other one is less pronounced but affects the whole genome. Very likely adaptive evolution has played a very significant role in the first, whereas the functional and/or evolutionary causes of the second are less clear and the evidence is not conclusive. The evidences supporting the incidence of increased mutation and less efficient negative selection are presented and discussed.

The Purine Bias of Coding Sequences is Determined by Physicochemical Constraints on Proteins.

For this report, we analyzed protein secondary structures in relation to the statistics of three nucleotide codon positions. The purpose of this investigation was to find which properties of the ribosome, tRNA or protein level, could explain the purine bias (Rrr) as it is observed in coding DNA. We found that the Rrr pattern is the consequence of a regularity (the codon structure) resulting from physicochemical constraints on proteins and thermodynamic constraints on ribosomal machinery. The physicochemical constraints on proteins mainly come from the hydropathy and molecular weight (MW) of secondary structures as well as the energy cost of amino acid synthesis. These constraints appear through a network of statistical correlations, such as (i) the cost of amino acid synthesis, which is in favor of a higher level of guanine in the first codon position, (ii) the constructive contribution of hydropathy alternation in proteins, (iii) the spatial organization of secondary structure in proteins according to solvent accessibility, (iv) the spatial organization of secondary structure according to amino acid hydropathy, (v) the statistical correlation of MW with protein secondary structures and their overall hydropathy, (vi) the statistical correlation of thymine in the second codon position with hydropathy and the energy cost of amino acid synthesis, and (vii) the statistical correlation of adenine in the second codon position with amino acid complexity and the MW of secondary protein structures. Amino acid physicochemical properties and functional constraints on proteins constitute a code that is translated into a purine bias within the coding DNA via tRNAs. In that sense, the Rrr pattern within coding DNA is the effect of information transfer on nucleotide composition from protein to DNA by selection according to the codon positions. Thus, coding DNA structure and ribosomal machinery co-evolved to minimize the energy cost of protein coding given the functional constraints on proteins.

Compositional patterns in the genomes of unicellular eukaryotes.

BACKGROUND: The genomes of multicellular eukaryotes are compartmentalized in mosaics of isochores, large and fairly homogeneous stretches of DNA that belong to a small number of families characterized by different average GC levels, by different gene concentration (that increase with GC), different chromatin structures, different replication timing in the cell cycle, and other different properties. A question raised by these basic results concerns how far back in evolution the compartmentalized organization of the eukaryotic genomes arose. RESULTS: In the present work we approached this problem by studying the compositional organization of the genomes from the unicellular eukaryotes for which full sequences are available, the sample used being representative. The average GC levels of the genomes from unicellular eukaryotes cover an extremely wide range (19%-60% GC) and the compositional patterns of individual genomes are extremely different but all genomes tested show a compositional compartmentalization. CONCLUSIONS: The average GC range of the genomes of unicellular eukaryotes is very broad (as broad as that of prokaryotes) and individual compositional patterns cover a very broad range from very narrow to very complex. Both features are not surprising for organisms that are very far from each other both in terms of phylogenetic distances and of environmental life conditions. Most importantly, all genomes tested, a representative sample of all supergroups of unicellular eukaryotes, are compositionally compartmentalized, a major difference with prokaryotes.

Transcriptome analysis of the bloodstream stage from the parasite Trypanosoma vivax.

BACKGROUND: Trypanosoma vivax is the earliest branching African trypanosome. This crucial phylogenetic position makes T. vivax a fascinating model to tackle fundamental questions concerning the origin and evolution of several features that characterize African trypanosomes, such as the Variant Surface Glycoproteins (VSGs) upon which antibody clearing and antigenic variation are based. Other features like gene content and trans-splicing patterns are worth analyzing in this species for comparative purposes. RESULTS: We present a RNA-seq analysis of the bloodstream stage of T. vivax from data obtained using two complementary sequencing technologies (454 Titanium and Illumina). Assembly of 454 reads yielded 13385 contigs corresponding to proteins coding genes (7800 of which were identified). These sequences, their annotation and other features are available through an online database presented herein. Among these sequences, about 1000 were found to be species specific and 50 exclusive of the T. vivax strain analyzed here. Expression patterns and levels were determined for VSGs and the remaining genes. Interestingly, VSG expression level, although being high, is considerably lower than in Trypanosoma brucei. Indeed, the comparison of surface protein composition between both African trypanosomes (as inferred from RNA-seq data), shows that they are substantially different, being VSG absolutely predominant in T. brucei, while in T. vivax it represents only about 55%. This raises the question concerning the protective role of VSGs in T. vivax, hence their ancestral role in immune evasion.It was also found that around 600 genes have their unique (or main) trans-splice site very close (sometimes immediately before) the start codon. Gene Ontology analysis shows that this group is enriched in proteins related to the translation machinery (e.g. ribosomal proteins, elongation factors). CONCLUSIONS: This is the first RNA-seq data study in trypanosomes outside the model species T. brucei, hence it provides the possibility to conduct comparisons that allow drawing evolutionary and functional inferences. This analysis also provides several insights on the expression patterns and levels of protein coding sequences (such as VSG gene expression), trans-splicing, codon patterns and regulatory mechanisms. An online T. vivax RNA-seq database described herein could be a useful tool for parasitologists working with trypanosomes.

Different mutation profiles associated to P53 accumulation in colorectal cancer.

The tumor suppressor TP53 gene is one of the most frequently mutated in different types of human cancer. Particularly in colorectal cancer (CRC), it is believed that TP53 mutations play a role in the adenoma-carcinoma transition of tumors during pathological process. In order to analyze TP53 expressed alleles in CRC, we examined TP53 mRNA in tumor samples from 101 patients with sporadic CRC. Samples were divided in two groups defined according to whether they exhibit positive or negative P53 protein expression as detected by immunohistochemistry (IHC). The presence of TP53 mutation was a common event in tumors with an overall frequency of 54.5%. By direct sequencing, we report 42 different TP53 sequence changes in 55 CRC patients, being two of them validated polymorphisms. TP53 mutations were more frequent in positive than in negative P53 detection group (p<0.0001), being the precise figures 79.6% and 30.8%, respectively. In addition, the mutation profiles were also different between the two groups of samples; while most of the mutations detected in P53 positive group were missense (38 out of 39), changes in P53 negative detection group include 7 insertions/deletions, 6 missense, 2 nonsense and 1 silent mutation. As previously observed, most mutations were concentrated in regions encoding P53 DNA binding domain (DBD). Codons 175, 248 and 273 together account for 36.7% of point mutations, in agreement with previous observations provided that these codons are considered mutation hotspots. Interestingly, we detected two new deletions and two new insertions. In addition, in three samples we detected two deletions and one insertion that could be explained as putative splicing variants or splicing errors.

Peculiar patterns of amino acid substitution and conservation in the fast evolving tunicate Oikopleura dioica.

We analyze the patterns and rates of amino acid evolution in tunicates with special interest on the extremely fast evolving Oikopleura dioica. We show that this species, on average, is twice as fast as the already fast evolving Ciona intestinalis. The acceleration in both species seems to be affected by similar evolutionary forces yet to different extent, since a substantial proportion of the most and less accelerated genes are orthologous between the two species. Among the possible causes that underlie the genome wide acceleration in Oikopleura, relaxation of functional constraints appears to be an important one, since all amino acids exhibit surprisingly homogenous levels of divergence. Such homogeneity, however, is not observed in Ciona. Apart from the genome wide acceleration, detailed analysis of functional groups of genes revealed that genes associated with regulatory functions (transcription regulators, chromatin remodeling proteins and metabolic regulators), have been subjected to an even more extreme process of acceleration, suggesting that adaptive evolution is the most probable cause of their unusual exacerbated rates. Another remarkable observation is that cysteine is among the less conserved amino acids, contrary to what is commonly observed in other species. The possible causes of this particular behavior are discussed.

Survey of transcripts expressed by the invasive juvenile stage of the liver fluke Fasciola hepatica.

BACKGROUND: The common liver fluke Fasciola hepatica is the agent of a zoonosis with significant economic consequences in livestock production worldwide, and increasing relevance to human health in developing countries. Although flukicidal drugs are available, re-infection and emerging resistance are demanding new efficient and inexpensive control strategies. Understanding the molecular mechanisms underlying the host-parasite interaction provide relevant clues in this search, while enlightening the physiological adaptations to parasitism. Genomics and transcriptomics are still in their infancy in F. hepatica, with very scarce information available from the invasive newly excysted juveniles (NEJ). Here we provide an initial glimpse to the transcriptomics of the NEJ, the first stage to interact with the mammalian host. RESULTS: We catalogued more than 500 clusters generated from the analysis of F. hepatica juvenile expressed sequence tags (EST), several of them not detected in the adult stage. A set of putative F. hepatica specific transcripts, and a group of sequences conserved exclusively in flatworms were identified. These novel sequences along with a set of parasite transcripts absent in the host genomes are putative new targets for future anti-parasitic drugs or vaccine development. Comparisons of the F. hepatica sequences with other metazoans genomes or EST databases were consistent with the basal positioning of flatworms in the bilaterian phylogeny. Notably, GC content, codon usage and amino acid frequencies are remarkably different in Schistosomes to F. hepatica and other trematodes. Functional annotation of predicted proteins showed a general representation of diverse biological functions. Besides proteases and antioxidant enzymes expected to participate in the early interaction with the host, various proteins involved in gene expression, protein synthesis, cell signaling and mitochondrial enzymes were identified. Differential expression of secreted protease gene family members between juvenile and adult stages may respond to different needs during host colonization. CONCLUSION: The knowledge of the genes expressed by the invasive stage of Fasciola hepatica is a starting point to unravel key aspects of this parasite's biology. The integration of the emerging transcriptomics, and proteomics data and the advent of functional genomics tools in this organism are positioning F. hepatica as an interesting model for trematode biology.