ABSTRACT: We developed and validated a small-footprint array of miniature chemostats built from readily available parts for low cost. Physiological and experimental evolution results were similar to larger volume chemostats. The ministat array provides a compact, inexpensive, and accessible platform for traditional chemostat experiments, functional genomics, and chemical screening applications. Three experiments are gene expression comparisons between three ministat cultures and a single Sixfors sample. The four CGH arrays are individual clones evolved in four sulfate limitation ministats compared to a wt ancestor strain.
Project description:Aneuploidy is a hallmark of tumor cells and yet the precise relationship between aneuploidy and a cell’s proliferative ability, or cellular fitness, has remained elusive. In this study we have combined a detailed analysis of aneuploid clones isolated from laboratory-evolved populations of Saccharomyces cerevisiae with a systematic, genome-wide screen for the fitness effects of telomeric amplifications to address the relationship between aneuploidy and cellular fitness. We found that aneuploid clones rise to high population frequencies in nutrient-limited evolution experiments and show increased fitness relative to wild-type. Direct competition experiments confirmed that three out of four aneuploid events isolated from evolved populations were themselves sufficient to improve fitness. To expand the scope beyond this small number of exemplars, we created a genome-wide collection of >1,800 diploid yeast strains each containing a different telomeric amplicon (Tamp) ranging in size from 0.4 to 1,000kb. Using pooled competition experiments in nutrient-limited chemostats followed by high-throughput sequencing of strain-identifying barcodes, we determined the fitness effects of these >1,800 Tamps under three different conditions. Our data revealed that the fitness landscape explored by telomeric amplifications is much broader than that explored by single-gene amplifications. As also observed in the evolved clones, we found the fitness effects of most Tamps to be condition specific with a minority showing common effects in all three conditions. By integrating our data with previous work that examined the fitness effects of single-gene amplifications genome wide, we found that a small number of genes within each Tamp are centrally responsible for each Tamp’s fitness effects. Our genome-wide Tamp screen confirmed that telomeric amplifications identified in laboratory-evolved populations generally increased fitness. Our results show that Tamps are mutations that produce large, typically condition-dependent changes in fitness that are important drivers of increased fitness in asexually evolving populations. Each of these arrays is a Comparative Genomic Hybridization experiment to detect copy number differences between a reference strain and a strain of interest.
Project description:Aneuploidy, or an aberrant karyotype, results in developmental disabilities and has been implicated in tumorigenesis. However, the causes of aneuploidy-induced phenotypes and the consequences of aneuploidy on cell physiology remain poorly understood. We have performed a meta-analysis on gene expression data from aneuploid cells in diverse organisms, including yeast, plants, mice, and humans. We found highly-related gene expression patterns that are conserved between species: genes that were involved in the response to stress were consistently upregulated, while genes associated with the cell cycle and cell proliferation were downregulated in aneuploid cells. Within species, different aneuploidies induced similar changes in gene expression, independent of the specific chromosomal aberrations. Taken together, our results demonstrate that aneuploidies of different chromosomes and in different organisms impact similar cellular pathways and cause a stereotypical anti-proliferative response that must be overcome prior to transformation. These experiments are two-color hybridizations of RNA isolated from aneuploid samples vs matched wt cells, all grown to midlog phase.
Project description:Aneuploidy causes a proliferative disadvantage in all cells analyzed to date, yet this condition is associated with a disease characterized by unabated proliferative potential, cancer. The mechanisms that allow cancer cells to tolerate the adverse effects of aneuploidy are not known. To probe this question, we identified aneuploid yeast strains with high proliferative abilities and characterized their genetic alterations. We found both strain-specific genetic alterations and mutations shared between different aneuploid strains. One such mutation, a loss of function mutation in the gene encoding the deubiquitinating enzyme UBP6, improves growth rates in four different aneuploid yeast strains. Our data further suggest that deletion of UBP6 attenuates the effects of aneuploidy on cellular protein composition. Our results demonstrate the existence of aneuploidy-tolerating mutations that improve the fitness of multiple different aneuploidies and highlight the importance of ubiquitin-proteasomal degradation in suppressing the adverse effects of aneuploidy. This dataset contains both expression analysis and CGH of yeast strains bearing extra chromosomes. In all cases, the wt euploid strain grown under the same conditions was used as the reference sample. Reference nucleic acid was generally labeled with Cy3, though some were labeled with Cy5 as indicated in the associated annotations for each array. No replicate arrays are included. Expression Samples: GSM513249-GSM513277 CGH Samples: GSM513278-GSM513369
Project description:The experimental evolution of laboratory populations of microbes provides an opportunity to observe the evolutionary dynamics of adaptation in real time.Until very recently, however, such studies have been limited by our inability to systematically find mutations in evolved organisms.We overcome this limitation by using a variety of DNA microarray-based techniques to characterize genetic changes, including point mutations, structural changes, and insertion variation, that resulted from the experimental adaptation of 24 haploid and diploid cultures of Saccharomyces cerevisiae to growth in glucose-, sulfate, or phosphate-limited chemostats for ~ 200 generations.We identified frequent genomic amplifications and rearrangements as well as novel retrotransposition events associated with adaptation.Global mutation detection in 10 clonal isolates identified 32 point mutations. On the basis of mutation frequencies, we infer that these mutations and the subsequent dynamics of adaptation are determined by the batch phase of growth prior to initiation of continuous phase in the chemostat.We relate these genotypic changes to phenotypic outcomes, namely global patterns of gene expression, and to increases in fitness by 5-50%. We found that the spectrum of available mutations in glucose or phosphate-limited environments combined with the batch phase population dynamics early in our experiments to allow several distinct genotypic and phenotypic evolutionary pathways in response to these nutrient limitations. By contrast, sulfate-limited populations were much more constrained in both genotypic and phenotypic outcomes.Thus, the reproducibility of evolution varies with specific selective pressures reflecting the constraints inherent in the system-level organization of metabolic processes in the cell.We were able to relate some of the observed adaptive mutations (e.g. transporter gene amplifications) to known features of the relevant metabolic pathways, but many of the mutations pointed to genes not previously associated with the relevant physiology. Thus, in addition to answering basic mechanistic questions about evolutionary mechanisms our work suggests that experimental evolution can also shed light on the function and regulation of individual metabolic pathways. Keywords: gene expression, CGH, and TSE analysis consult individual records for details of analysis This Dataset consists of several experiments: Gene expression experiments found in Figure 1a of paper: Design: RNA from each evolved clone or population grown in chemostat culture is compared to RNA from matching ancestor strains grown in the same conditions. Samples: GSM339025-GSM339088 CGH experiments found in Figure 2A, Figure 3, and Table S2: Experiment design: DNA from each evolved clone or population is hybridized vs DNA from the matched ancestor strain Samples: GSM339089-GSM339146 CGH experiments found in Table S3: Experiment design: DNA from each segregant derived from an evolved strain is hybridized vs DNA from the matched ancestor strain Samples: GSM339147-GSM339158 Gel band CGH experiments found in Figure S1: Experiment design: Chromosomes from evolved strains were run on a gel and excised. DNA from the bands is hybridized vs matched ancestor genomic DNA Samples: GSM339159-GSM339184 TSE CGH experiments found in Table S4: Experiment design: DNA linked to Ty1/Ty2 sequences was extracted from evolved strains and ancestor strains, and compared to ancestral extracted or genomic DNA as indicated. Samples: GSM339185-GSM339212 CGH experiments for experiments in Figure S7: Experiment design: DNA from 2 strains transformed with a SUL1 plasmid hybridized vs DNA from the matched ancestor strain Samples: GSM339213 and GSM339214
Project description:Aneuploidy and aging are correlated; however, a causal link between these two phenomena has remained elusive. Here we show that yeast disomic for a single native yeast chromosome generally have a decreased replicative lifespan. In addition, the extent of this lifespan deficit correlates with the size of the extra chromosome. We identified a mutation in BUL1 that rescues both the lifespan deficit and a protein trafficking defect in yeast disomic for chromosome 5. Bul1 is an E4 ubiquitin ligase adaptor involved in a protein quality-control pathway that targets membrane proteins for endocytosis and destruction in the lysosomal vacuole thereby maintaining protein homeostasis. Concurrent suppression of the aging and trafficking phenotypes suggests that disrupted membrane protein homeostasis in aneuploid yeast may contribute to their accelerated aging. The data reported here demonstrate that aneuploidy can impair protein homeostasis, shorten lifespan, and may contribute to age-associated phenotypes. These are all CGH arrays comparing DNA content between the indicated strain of interest and a wt control.
Project description:DNA replication errors are a major driver of evolution—from single nucleotide polymorphisms to large-scale copy number variations (CNVs). Here we test a specific replication-based model to explain the generation of interstitial, inverted triplications. While no genetic information is lost, the novel inversion junctions and increased copy number of the included sequences create the potential for adaptive phenotypes. The model—Origin-Dependent Inverted-Repeat Amplification (ODIRA)—proposes that a replication error at pre-existing short, interrupted, inverted repeats in genomic sequences generates an extrachromosomal, inverted dimeric, autonomously replicating intermediate; subsequent genomic integration of the dimer yields this class of CNV without loss of distal chromosomal sequences. We used a combination of in vitro and in vivo approaches to test the feasibility of the proposed replication error and its downstream consequences on chromosome structure in the yeast Saccharomyces cerevisiae. We show that the proposed replication error—the ligation of leading and lagging nascent strands to create a "closed" fork—can occur in vitro at short, interrupted inverted repeats. The removal of molecules with closed forks results in a hairpin-capped linear duplex that we show replicates in vivo to create an inverted, dimeric plasmid that subsequently integrates into the genome by homologous recombination, creating an inverted triplication. While other models have been proposed to explain inverted triplications and their derivatives, our model can also explain the generation of human, de novo, inverted amplicons that have a 2:1 mixture of sequences from both homologues of a single parent—a feature readily explained by a plasmid intermediate that arises from one homologue and integrates into the other homolog prior to meiosis. Our tests of key features of ODIRA lend support to this mechanism and suggest further avenues of enquiry to unravel the origins of interstitial, inverted CNVs pivotal in human health and evolution These are all CGH arrays comparing DNA copy number between evolved yeast strains and a euploid wt strain.
Project description:Reproductive cessation is perhaps the earliest aging phenotypes humans experience. Similarly, C. elegans' reproduction ceases in mid-adulthood. Although somatic aging has been studied in both worms and humans, mechanisms regulating reproductive aging are not yet understood. Here we show that TGF-beta Sma/Mab activity regulates reproductive aging transcriptionally separable from its regulation of body size growth. This SuperSeries is composed of the following subset Series: GSE23446: Reproductive aging: sma-2;fem-1 day 8 oocyte vs fem-1 day 8 oocyte GSE23447: Reproductive aging: fem-1 day 3 oocyte vs fem-1 day 8 oocyte GSE23448: Body size regulation and TGF-beta Sma/Mab pathway: sma L4 vs N2 L4 Refer to individual Series
Project description:To find genes in C. elegans oocytes associated with reproductive aging. Five replicates comparing RNA from oocyte samples collected from day 3 fem-1(hc17) animals with RNA from oocyte samples collected from day 8 fem-1(hc17) animals. Three out of five are dye-flipped.
Project description:To find genes downstream of the TGF-beta Sma/Mab pathway associated with body size regulation in C. elegans. Three replicates comparing RNA from sma-2(e502) L4 whole animal with RNA from wild-type L4 whole animal, in which one is dye flipped. Plus one array comparing RNA from sma-4(e729) L4 whole animal with RNA from wild-type L4 whole animal.
Project description:To find genes downstream of the TGF-beta Sma/Mab pathway in C. elegans oocytes associated with reproductive aging. Eight replicates comparing RNA from oocyte samples collected from day 8 sma-2(e502);fem-1(hc17) animals with RNA from oocyte samples collected from day 8 fem-1(hc17) animals. Five out of eight are dye-flipped.