Project description:One of the central goals of evolutionary biology is to explain and predict the molecular basis of adaptive evolution. We studied the evolution of genetic networks in Saccharomyces cerevisiae (budding yeast) populations propagated for more than 200 generations in different nitrogen-limiting conditions. We find that rapid adaptive evolution in nitrogen-poor environments is dominated by the de novo generation and selection of copy number variants (CNVs), a large fraction of which contain genes encoding specific nitrogen transporters including PUT4, DUR3 and DAL4. The large fitness increases associated with these alleles limits the genetic heterogeneity of adapting populations even in environments with multiple nitrogen sources. Complete identification of acquired point mutations, in individual lineages and entire populations, identified heterogeneity at the level of genetic loci but common themes at the level of functional modules, including genes controlling phosphatidylinositol-3-phosphate metabolism and vacuole biogenesis. Adaptive strategies shared with other nutrient-limited environments point to selection of genetic variation in the TORC1 and Ras/PKA signaling pathways as a general mechanism underlying improved growth in nutrient-limited environments. Within a single population we observed the repeated independent selection of a multi-locus genotype, comprised of the functionally related genes GAT1, MEP2 and LST4. By studying the fitness of individual alleles, and their combination, as well as the evolutionary history of the evolving population, we find that the order in which these mutations are acquired is constrained by epistasis. The identification of repeatedly selected variation at functionally related loci that interact epistatically suggests that gene network polymorphisms (GNPs) may be a frequent outcome of adaptive evolution. Our results provide insight into the mechanistic basis by which cells adapt to nutrient-limited environments and suggest that knowledge of the selective environment and the regulatory mechanisms important for growth and survival in that environment greatly increases the predictability of adaptive evolution. mRNA from each evolved clone or from the ancestral strain growing in the specificied nitrogen-limited condition was co-hybridized with mRNA from the ancestral strain grown in ammonium limited media

Project description:Cell growth rate is regulated in response to resource availability including the abundance, and molecular form, of essential nutrients. In the model eukaryotic cell, Saccharomyces cerevisiae (budding yeast), the molecular form of environmental nitrogen impacts both cell growth rate and mRNA expression. Disentangling causal relationships between nitrogen availability, cell growth rate and differential gene expression poses a considerable challenge. Using experimental control of cell growth rate using chemostats, we studied the effect of variation in environmental nitrogen on differential gene expression. We find that the primary determinant of nitrogen-regulated gene expression is nitrogen abundance whereas variation in nitrogen source affects the expression of only a small number of transcripts with highly specialized functions. To study the dynamics of nitrogen-responsive gene expression we perturbed steady-state nitrogen-limited chemostat cultures by addition of either proline or glutamine. Addition of either proline or glutamine to cells growing in nitrogen-limited chemostats results in repression of the nitrogen catabolite repression (NCR) regulon consistent with nitrogen abundance, and not nitrogen source, being the primary determinant of nitrogen-regulated gene expression. We find that a transition from nitrogen-limited to nitrogen-replete conditions is accompanied by rapid induction of transcripts required for protein translation. We identified a reciprocal relationship between specific regulons required for protein translation (RP and RiBi) and the NCR regulon. Using mathematical modeling we find evidence that cells adopt a metabolically inefficient growth mode during this transition. By means of high resolution time series analysis we find evidence that rapid, and potentially accelerated, mRNA degradation plays an important role in remodeling gene expression programs in response to change in environmental nitrogen. We propose that the evolutionarily conserved TORC1 signaling pathway orchestrates the balance between protein translation and assimilation of nitrogen sources at the transcriptional level to optimize rates of cell proliferation. A total of of 102 samples were analyzed in different nitrogen-limited conditions using chemostats in both steady-state and dynamic conditions. A common reference obtained from an ammonium-limited chemostat growing at a dilution rate of 0.12/hr was used for all two color hybridization experiments.

Project description:One of the central goals of evolutionary biology is to explain and predict the molecular basis of adaptive evolution. We studied the evolution of genetic networks in Saccharomyces cerevisiae (budding yeast) populations propagated for more than 200 generations in different nitrogen-limiting conditions. We find that rapid adaptive evolution in nitrogen-poor environments is dominated by the de novo generation and selection of copy number variants (CNVs), a large fraction of which contain genes encoding specific nitrogen transporters including PUT4, DUR3 and DAL4. The large fitness increases associated with these alleles limits the genetic heterogeneity of adapting populations even in environments with multiple nitrogen sources. Complete identification of acquired point mutations, in individual lineages and entire populations, identified heterogeneity at the level of genetic loci but common themes at the level of functional modules, including genes controlling phosphatidylinositol-3-phosphate metabolism and vacuole biogenesis. Adaptive strategies shared with other nutrient-limited environments point to selection of genetic variation in the TORC1 and Ras/PKA signaling pathways as a general mechanism underlying improved growth in nutrient-limited environments. Within a single population we observed the repeated independent selection of a multi-locus genotype, comprised of the functionally related genes GAT1, MEP2 and LST4. By studying the fitness of individual alleles, and their combination, as well as the evolutionary history of the evolving population, we find that the order in which these mutations are acquired is constrained by epistasis. The identification of repeatedly selected variation at functionally related loci that interact epistatically suggests that gene network polymorphisms (GNPs) may be a frequent outcome of adaptive evolution. Our results provide insight into the mechanistic basis by which cells adapt to nutrient-limited environments and suggest that knowledge of the selective environment and the regulatory mechanisms important for growth and survival in that environment greatly increases the predictability of adaptive evolution. DNA from each evolved clone or population is hybridized vs DNA from the ancestral strain

Project description:The counts of total white blood cells (WBCs) and WBC subsets are well-established diagnostic factors for various diseases. It has been shown that variations in WBC counts are significantly controlled by individuals’ genetics in swine. However, despite detection of quantitative trait loci (QTLs) for these phenotypes, little is known on the molecular basis underlying their variations. Our aim was to study gene profiling variations according to variations in WBC counts and to connect results with available QTL mapping. Whole blood transcriptome of animals contrasted for levels of WBC counts were compared. A pig generic microarray enriched in immunity-related genes was used. 378 probes representing 334 genes were found significantly differentially expressed between high- and low-count WBC groups. 65 genes were associated with hematological system development and function. 336 probes could be mapped on all autosomes and the X chromosome, and 59 transcripts fell within 28 QTLs reported to affect the counts of WBC and WBC subsets. By combining probe mapping results and biological functions, 6 genes (CDKN2A, TCIRG1, SIPA1, RGL2, FLT1 and CFLAR) were found as putative relevant positional candidate genes for the WBC traits. Genetic linkage experiments are warranted to validate these candidate genes, and further investigating a possible pleiotropic effect of these genes could contribute to elucidate molecular mechanisms involved in WBC development. differentially expressed genes or transccripts between high- and low-count white blood cells groups The dual channel microarray experiments were carried out using a common reference hybridization design. In this study the reference RNA sample was prepared by pooling of total RNAs from different porcine tissues. The test samples were RNA samples isolated from total porcine blood. According to the total white blood cells count, 18 animals were selected from the edges of the distribution, named high-count group (HC, 9 animals) and low-count group (LC, 9 animals).

Project description:The engulfment of live cells by other live cells, also known as cannibalism, can result in the death of engulfed cells. Recently, we show that autophagy favors the accumulation of E-cadherins on the surface of cannibal cells, thereby enhancing their capacity to engulf and eliminate live cells. Importantly, cannibal cells exhibited genomic instability and activated a senescence program that we termed “entescence”. In breast cancer patients, neoadjuvant chemotherapy response was associated with the induction of entescence. Conversely, the suppression of entescence by dominant negative mutants of p53 favored the proliferation of aneuploid cannibal cells and predicts cancer progression. Our data indicate that entescence is a non-cell-autonomous program of senescence that compromises cancer development. In this context, we analyzed using CGH array the genomic features of cells that escape from entescence. The first goal of this experiment is to confirm the aneuploidy of cannibal cells and the second aim is to identify genomic alterations associated to cellular cannibalism. To isolate and purify cellular clones obtained from cannibal cells that escaped entescence, homotypic cultures of HCT116 p53R248W/- cells that have been half labeled with 5-chloromethylfluorescein diacetate (CMFDA) and half labeled with 5-(and-6)-(((4-chloromethyl)benzoyl)amino) tetramethylrhodamine (CMTMR) were irradiated (or not) with 4 Gy, cultured for 24 hours and purified using the BD Influx™cell sorter (Becton Dickinson). Single cells and cannibal cells were seeded in microtiter 96-well plates (with one single structure per well). Sorted populations (single cell and cannibal cell) were then validated by fluorescence microscopy. Thus, 3 control clones (samples 1-3), 16 clones that ‘escaped’ to spontaneous entescence (samples 4-19), 5 irradiated clones (samples 20-24) and 16 clones that ‘escaped’ to IR-mediated entescence (samples 25-40) were generated and analyzed using CGH array. Entescence is a novel senescence program initiated by cellular cannibalism that prevents genomic instability and tumor progression.

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Molecular Specificity, Convergence and Constraint Shape Adaptive Evolution in Nutrient-Poor Environments

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