ALBERT

Description: Background: Variation of gene expression can lead to phenotypic variation and have therefore been assumed to contribute the diversity of wine yeast (Saccharomyces cerevisiae) properties. However, the molecular bases of this variation of gene expression are unknown. We addressed these questions by carrying out an integrated genetical-genomic study in fermentation conditions. We report here quantitative trait loci (QTL) mapping based on expression profiling in a segregating population generated by a cross between a derivative of the popular wine strain EC1118 and the laboratory strain S288c. Results: Most of the fermentation traits studied appeared to be under multi-allelic control. We mapped five phenotypic QTLs and 1465 expression QTLs. Several expression QTLs overlapped in hotspots. Among the linkages unraveled here, several were associated with metabolic processes essential for wine fermentation such as glucose sensing or nitrogen and vitamin metabolism. Variations affecting the regulation of drug detoxification and export (TPO1, PDR12 or QDR2) were linked to variation in four genes encoding transcription factors (PDR8, WAR1, YRR1 and HAP1). We demonstrated that the allelic variation of WAR1 and TPO1 affected sorbic and octanoic acid resistance, respectively. Moreover, analysis of the transcription factors phylogeny suggests they evolved with a specific adaptation of the strains to wine fermentation conditions. Unexpectedly, we found that the variation of fermentation rates was associated with a partial disomy of chromosome 16. This disomy resulted from the well known 8--16 translocation. Conclusions: This large data set made it possible to decipher the effects of genetic variation on gene expression during fermentation and certain wine fermentation properties Our findings shed a new light on the adaptation mechanisms required by yeast to cope with the multiple stresses generated by wine fermentation. In this context, the detoxification and export systems appear to be of particular importance, probably due to nitrogen starvation. Furthermore, we show that the well characterized 8--16 translocation located in SSU1, which is associated with sulfite resistance, can lead to a partial chromosomic amplification in the progeny of strains that carry it, greatly improving fermentation kinetics. This amplification has been detected among other wine yeasts.

Description: Background: Trypanosoma cruzi, the causal agent of Chagas Disease, affects more than 16 million people in Latin America. The clinical outcome of the disease results from a complex interplay between environmental factors and the genetic background of both the human host and the parasite. However, knowledge of the genetic diversity of the parasite, is currently limited to a number of highly studied loci. The availability of a number of genomes from different evolutionary lineages of T. cruzi provides an unprecedented opportunity to look at the genetic diversity of the parasite at a genomic scale. Results: Using a bioinformatic strategy, we have clustered T. cruzi sequence data available in the public domain and obtained multiple sequence alignments in which one or two alleles from the reference CL-Brener were included. These data covers 4 major evolutionary lineages (DTUs): TcI, TcII, TcIII, and the hybrid TcVI. Using these set of alignments we have identified 288,957 high quality single nucleotide polymorphisms and 1,480 indels. In a reduced re-sequencing study we were able to validate ~ 97% of high-quality SNPs identified in 47 loci. Analysis of how these changes affect encoded protein products showed a 0.77 ratio of synonymous to non-synonymous changes in the T. cruzi genome. We observed 113 changes that introduce or remove a stop codon, some causing significant functional changes, and a number of tri-allelic and tetra-allelic SNPs that could be exploited in strain typing assays. Based on an analysis of the observed nucleotide diversity we show that the T. cruzi genome contains a core set of genes that are under apparent purifying selection. Interestingly, orthologs of known druggable targets show statistically significant lower nucleotide diversity values. Conclusions: This study provides the first look at the genetic diversity of T. cruzi at a genomic scale. The analysis covers an estimated ~ 60% of the genetic diversity present in the population, providing an essential resource for future studies on the development of new drugs and diagnostics, for Chagas Disease. These data is available through the TcSNP database (http://snps.tcruzi.org).

Description: Background: Vomeronasal receptors (VRs), expressed in sensory neurons of the vomeronasal organ, are thought to bind pheromones and mediate innate behaviours. The mouse reference genome has over 360 functional VRs arranged in highly homologous clusters, but the vast majority are of unknown function. Differences in these receptors within and between closely related species of mice are likely to underpin a range of behavioural responses. To investigate these differences, we interrogated the VR gene repertoire from 17 inbred strains of mice using massively parallel sequencing. Results: Approximately half of the 6222 VR genes that we investigated could be successfully resolved, and those that were unambiguously mapped resulted in an extremely accurate dataset. Collectively VRs have over twice the coding sequence variation of the genome average; but we identify striking non-random distribution of these variants within and between genes, clusters, clades and functional classes of VRs. We show that functional VR gene repertoires differ considerably between different Mus subspecies and species, suggesting these receptors may play a role in mediating behavioural adaptations. Finally, we provide evidence that widely-used, highly inbred laboratory-derived strains have a greatly reduced, but not entirely redundant capacity for differential pheromone-mediated behaviours. Conclusions: Together our results suggest that the unusually variable VR repertoires of mice have a significant role in encoding differences in olfactory-mediated responses and behaviours. Our dataset has expanded over nine fold the known number of mouse VR alleles, and will enable mechanistic analyses into the genetics of innate behavioural differences in mice.

Description: Background: Redox homeostasis is essential to sustain metabolism and growth. We recently reported that yeast cells meet a gradual increase in imposed NADPH demand by progressively increasing flux through the pentose phosphate (PP) and acetate pathways and by exchanging NADH for NADPH in the cytosol, via a transhydrogenase-like cycle. Here, we studied the mechanisms underlying this metabolic response, through a combination of gene expression profiling and analyses of extracellular and intracellular metabolites and 13C-flux analysis. Results: NADPH oxidation was increased by reducing acetoin to 2,3-butanediol in a strain overexpressing an engineered NADPH-dependent butanediol dehydrogenase cultured in the presence of acetoin. An increase in NADPH demand to 22 times the anabolic requirement for NADPH was accompanied by the intracellular accumulation of PP pathway metabolites consistent with an increase in flux through this pathway. Increases in NADPH demand were accompanied by the successive induction of several genes of the PP pathway. NADPH-consuming pathways, such as amino-acid biosynthesis, were upregulated as an indirect effect of the decrease in NADPH availability. Metabolomic analysis showed that the most extreme modification of NADPH demand resulted in an energetic problem. Our results also highlight the influence of redox status on aroma production. Conclusions: Combined 13C-flux, intracellular metabolite levels and microarrays analyses revealed that NADPH homeostasis, in response to a progressive increase in NADPH demand, was achieved by the regulation, at several levels, of the PP pathway. This pathway is principally under metabolic control, but regulation of the transcription of PP pathway genes can exert a stronger effect, by redirecting larger amounts of carbon to this pathway to satisfy the demand for NADPH. No coordinated response of genes involved in NADPH metabolism was observed, suggesting that yeast has no system for sensing NADPH/NADP+ ratio. Instead, the induction of NADPH-consuming amino-acid pathways in conditions of NADPH limitation may indirectly trigger the transcription of a set of PP pathway genes.

Description: Background: Studies conducted with gilthead sea bream (Sparus aurata L.) have determined the maximum dietary replacement of fish meal and oil without compromising growth or product quality. The present study aimed to analyze the effect of the nutritional background on fish health and fish fed plant protein-based diets with fish oil (FO diet) or a blend of vegetable oils (66VO diet) were exposed for 102 days to the intestinal myxosporean parasite Enteromyxum leei, and the intestine transcriptome was analyzed with a customized oligo-microarray of 7,500 annotated genes. Results: Infection prevalence was high and similar in the two diet groups, but the outcome of the disease was more pronounced in fish fed the 66VO diet. No differences were found in the transcriptome of both diet control groups, whereas the number of differentially expressed genes in infected groups was considerable. K-means clustering of these differentially expressed genes identified four expression patterns that reflected the progression of the disease with the magnitude of the fold-change being higher in infected 66VO fish. A positive correlation was found between the time of infection and the magnitude of the transcriptional change within the 66VO group, being higher in early infected animals. Within this diet group, a strong up-regulation of many components of the immune specific response was evidenced, whereas other genes related to complement response and xenobiotic metabolism were down-regulated. Conclusions: The high replacement of fish oil by vegetable oils in practical fish feeds did not modify the intestine transcriptome of gilthead sea bream, but important changes were apparent when fish were exposed to the myxosporean E. leei. The detected changes were mostly a consequence rather than a cause of the different disease progression in the two diet groups. Hence, the developed microarray constitutes an excellent diagnostic tool to address changes associated with the action of intestinal pathogens, but lacks a prognostic value to predict in advance the different susceptibility of growing fish to the current pathogen.

Description: Background: Polyploidization is an important mechanism in plant evolution. By analyzing the leaf transcriptomes taken from the allotetraploid Nicotiana tabacum (tobacco) and parental genome donors, N. Sylvesteris (S-Genome) and N. tomentosiformis (T-Genome), a phylogenomic approach was taken to map the fate of homeologous gene pairs in this plant. Results: A comparison between the genes present in the leaf transcriptomes of N. tabacum and modern day representatives of its progenitor species demonstrated that only 33 % of assembled transcripts could be distinguished based on their sequences. A large majority of the genes (83.6 % of the non parent distinguishable and 87.2 % of the phylogenetic topology analyzed clusters) expressed above background level (more than 5 reads) showed similar overall expression levels. Homeologous sequences could be identified for 968 gene clusters, and 90 % (6 % of all genes) of the set maintained expression of only one of the tobacco homeologs. When both homeologs were expressed, only 15 % (0.5 % of the total) showed evidence of differential expression, providing limited evidence of subfunctionalization. Comparing the rate of synonymous nucleotide substitution (Ks) and non-synonymous nucleotide substitution (Kn) provided limited evidence for positive selection during the evolution of tobacco since the polyploidization event took place. Conclusions: Polyploidization is a powerful mechanism for plant speciation that can occur during one generation; however millions of generations may be necessary for duplicate genes to acquire a new function. Analysis of the tobacco leaf transcriptome reveals that polyploidization, even in a young tetraploid such as tobacco, can lead to complex changes in gene expression. Gene loss and gene silencing, or subfunctionalization may explain why both homeologs are not expressed by the associated genes. With Whole Genome Duplication (WGD) events, polyploid genomes usually maintain a high percentage of gene duplicates. The data provided little evidence of preferential maintenance of gene expression from either the T- or S-genome. Additionally there was little evidence of neofunctionalization in Nicotiana tabacum suggesting it occurs at a low frequency in young polyploidy.

Description: Background: In fleshy fruit, abscission of fully ripe fruit is a process intimately linked to the ripening process. In many fruit-tree species, such as olive (Olea europaea L. cv. Picual), there is a coupling of the full ripening and the activation of the abscission-zone (AZ). Although fully ripe fruit have marked physiological differences with respect to their AZs, dissimilarities in gene expression have not been thoroughly investigated. The present study examines the transcriptome of olive fruit and their AZ tissues at the last stage of ripening, monitored using mRNA-Seq. Results: Roche-454 massive parallel pyrosequencing enabled us to generate 397,457 high-quality EST sequences, among which 199,075 were from ripe-fruit pericarp and 198,382 from AZ tissues. We assembled these sequences into 19,062 contigs, grouped as 17,048 isotigs. Using the read amounts for each annotated isotig (from a total of 15,671), we identified 7,756 transcripts. A comparative analysis of the transcription profiles conducted in ripe-fruit pericarp and AZ evidenced that 4,391 genes were differentially expressed genes (DEGs) in fruit and AZ. Functional categorization of the DEGs revealed that AZ tissue has an apparently higher response to external stimuli than does that of ripe fruit, revealing a higher expression of auxin-signaling genes, as well as lignin catabolic and biosynthetic pathway, aromatic amino acid biosynthetic pathway, isoprenoid biosynthetic pathway, protein amino acid dephosphorylation, amino acid transport, and photosynthesis. By contrast, fruit-enriched transcripts are involved in ATP synthesis coupled proton transport, glycolysis, and cell-wall organization. Furthermore, over 150 transcripts encoding putative transcription-factors (TFs) were identified (37 fruit TFs and 113 AZ TFs), of which we randomly selected eight genes and we confirmed their expression patterns using quantitative RT-PCR. Conclusion: We generated a set of EST sequences from olive fruit at full ripening, and DEGs between two different olive tissues, ripe fruit and their AZ, were also identified. Regarding the cross-talk between fruit and AZ, using qRT-PCR, we confirmed a set of TF genes that were differentially expressed, revealing profiles of expression that have not previously been reported, this offering a promising beginning for studies on the different transcription regulation in such tissues.

Description: Background: Bulk segregant analysis (BSA) coupled to high throughput sequencing is a powerful method to map genomic regions related with phenotypes of interest. It relies on crossing two parents, one inferior and one superior for a trait of interest. Segregants displaying the trait of the superior parent are pooled, the DNA extracted and sequenced. Genomic regions linked to the trait of interest are identified by searching the pool for overrepresented alleles that normally originate from the superior parent. BSA data analysis is non-trivial due to sequencing, alignment and screening errors. Results: To increase the power of the BSA technology and obtain a better distinction between spuriously and truly linked regions, we developed EXPLoRA (EXtraction of over-rePresented aLleles in BSA), an algorithm for BSA data analysis that explicitly models the dependency between neighboring marker sites by exploiting the properties of linkage disequilibrium through a Hidden Markov Model (HMM).Reanalyzing a BSA dataset for high ethanol tolerance in yeast allowed reliably identifying QTLs linked to this phenotype that could not be identified with statistical significance in the original study. Experimental validation of one of the least pronounced linked regions, by identifying its causative gene VPS70, confirmed the potential of our method. Conclusions: EXPLoRA has a performance at least as good as the state-of-the-art and it is robust even at low signal to noise ratio's i.e. when the true linkage signal is diluted by sampling, screening errors or when few segregants are available.

Description: Background: Human Mesenchymal Stromal/Stem Cells (MSCs) are adult multipotent cells that behave in a highly plastic manner, inhabiting the stroma of several tissues. The potential utility of MSCs is nowadays strongly investigated in the field of regenerative medicine and cell therapy, although many questions about their molecular identity remain uncertain. Results: MSC primary cultures from human bone marrow (BM) and placenta (PL) were derived and verified by their immunophenotype standard pattern and trilineage differentiation potential. Then, a broad characterization of the transcriptome of these MSCs was performed using RNA deep sequencing (RNA-Seq). Quantitative analysis of these data rendered an extensive expression footprint that includes 5,271 protein-coding genes. Flow cytometry assays of canonical MSC CD-markers were congruent with their expression levels detected by the RNA-Seq. Expression of other recently proposed MSC markers (CD146, Nestin and CD271) was tested in the placenta samples, finding only CD146 and Nestin. Functional analysis revealed enrichment in stem cell related genes and mesenchymal regulatory transcription factors (TFs). Analysis of TF binding sites (TFBSs) identified 11 meta-regulators, including factors KLF4 and MYC among them. Epigenetically, hypomethylated promoter patterns supported the active expression of the MSC TFs found. An interaction network of these TFs was built to show up their links and relations. Assessment of dissimilarities between cell origins (BM versus PL) disclosed two hundred differentially expressed genes enrolled in microenvironment processes related to the cellular niche, as regulation of bone formation and blood vessel morphogenesis for the case of BM-MSCs. By contrast genes overexpressed in PL-MSCs showed functional enrichment on mitosis, negative regulation of cell-death and embryonic morphogenesis that supported the higher growth rates observed in the cultures of these fetal cells and their closer links with development processes. Conclusions: The results present a transcriptomic portrait of the human MSCs isolated from bone marrow and placenta. The data are released as a cell-specific resource, providing a comprehensive expression footprint of the MSCs useful to better understand their cellular and molecular biology and for further investigations on the isolation and biomedical use of these multipotent cells.

Description: Background: The genetic determinism of blood lipid concentrations, the main risk factor for atherosclerosis, is practically unknown in species other than human and mouse. Even in model organisms, little is known about how the genetic determinants of lipid traits are modulated by age-specific factors. To gain new insights into this issue, we have carried out a genome-wide association study (GWAS) for cholesterol (CHOL), triglyceride (TRIG) and low (LDL) and high (HDL) density lipoprotein concentrations measured in Duroc pigs at two time points (45 and 190 days). Results: Analysis of data with mixed-model methods (EMMAX, GEMMA, GenABEL) and PLINK showed a low positional concordance between trait-associated regions (TARs) for serum lipids at 45 and 190 days. Besides, the proportion of phenotypic variance explained by SNPs at these two time points was also substantially different. The four analyses consistently detected two regions on SSC3 (124 Mb, CHOL and LDL at 190 days) and SSC6 (135 Mb, CHOL and TRIG at 190 days) with highly significant effects on the porcine blood lipid profile. Moreover, we have found that SNP variation within SSC3, SSC6, SSC10, SSC13 and SSC16 TARs is associated with the expression of several genes mapping to other chromosomes and related to lipid metabolism. Conclusions: Our data demonstrate that the effects of genomic determinants influencing lipid concentrations in pigs, as well as the amount of phenotypic variance they explain, are influenced by age-related factors.