Genomic Characterization of Adaptive Mutations that Effect Minimal Fitness Trade-offs in Evolved Clones of Saccharomyces cerevisiae
ABSTRACT: In some of the earliest uses of genome-wide gene-expression microarrays and array-based Comparative Genomic Hybridization (aCGH), a set of diploid yeasts that had undergone experimental evolution under aerobic glucose limitation was used to explore how gene expression and genome structure had responded to this selection pressure. To more deeply understand how adaptation to one environment might constrain or enhance performance in another we have now identified the adaptive mutations in this set of clones using whole-genome sequencing, and have assessed whether the evolved clones had become generalists or specialists by assaying their fitness under three contrasting growth environments: aerobic and anaerobic glucose limitation and aerobic acetate limitation. Additionally, evolved clones and their common ancestor were assayed for gene expression, biomass estimates and residual substrate levels under the alternative growth conditions. Relative fitnesses were evaluated by competing each clone against a common reference strain in each environment. Unexpectedly, we found that the evolved clones also outperformed their ancestor under strictly fermentative and strictly oxidative growth conditions. We conclude that yeasts evolving under aerobic glucose limitation become generalists for carbon limitation, as the mutations selected for in one environment are advantageous in others. High-throughput sequencing of the evolved clones uncovered mutations in genes involved in glucose sensing, signaling, and transport that in part explain these physiological phenotypes, with different sets of mutations found in independently-evolved clones. Earlier gene expression data from aerobic glucose-limited cultures had revealed a shift from fermentation towards respiration in all evolved clones explaining increased fitness in that condition. However, because the evolved clones also show higher fitness under strictly anaerobic conditions and under conditions requiring strictly respirative growth, this switch cannot be the sole source of adaptive benefit. Furthermore, because independently evolved clones are genetically distinct we conclude that there are multiple mutational paths leading to the generalist phenotype. Strain Name: Parental strain (CP1AB) or evolved clones (E1 - E5) Media: aerobic / anaerobic 36 hybridizations
Project description:Hybrid progeny can enjoy increased fitness and stress tolerance relative to their ancestral species, a phenomenon known as hybrid vigor. Though this phenomenon has been documented throughout the Eukarya, evolution of hybrid populations has yet to be explored experimentally in the lab. To fill this knowledge gap we created a pool of Saccharomyces cerevisiae and S. bayanus homoploid and aneuploid hybrids, and then investigated how selection in the form of incrementally increased temperature or ethanol impacted hybrid genome structure and adaptation. During 500 generations of continuous ammonia-limited, glucose-sufficient culture, temperature was raised from 25C to 46??C. This selection invariably resulted in nearly-complete loss of the S. bayanus genome, although the dynamics of genome loss differed among independent replicates. Temperature-evolved isolates were significantly more thermal tolerant and exhibited greater phenotypic plasticity than parental species and founding hybrids. By contrast, when the same hybrid pool was subjected to increases in exogenous ethanol from 0% to 14%, selection favored euploid S. cerevisiae x S. bayanus hybrids. Ethanol-evolved isolates exhibited significantly greater ethanol tolerance relative only to S. bayanus and one of the founding hybrids tested. Adaptation to thermal and ethanol stress manifested as heritable changes in cell wall structure demonstrated by resistance to zymolyase or micafungin treatment. This is the first study to show experimentally that the fate of interspecific hybrids critically depends on the type of selection they encounter during the course of evolution. Array-CGH was performed on the S. cerevisiae parent strain CEN.PK (GSY2160), the S. bayanus parent strain CBS7001 (GSY2161) and on the F1 interspecific hybrid resulting from mating the 2 parents (GSY2168). Additionally, three rare viable spores obtained after sporulation of the F1 were assayed by array-CGH (F2a, F2b, F2c). A large pool of F2 spores (and probably some number of F1 hybrid cells) were subjected to gradually increasing temperatures, in three independent vessels, with populations sampled at various generation times. Likewise, the same pool was used to found populations in an additional three independent vessels, which were then subjected to gradually increasing ethanol concentrations (at constant temperature). Array-CGH was performed on three different clones from each of the three temperature vessels at the final 500 generation time point (T500 clones). Biological replicates of the T500 clones were performed (T500-new). Two self-self array-CGH hybridization controls were also performed (self-control). Array-CGH was performed on one clone from each of the three ethanol vessels taken at the 400 generation timepoint (EtOH400gen clones).
Project description:Array data from HeLa cells that remained Uninfected or were infected with wildtype S. flexneri serotype 2a strain 2457T. These groupings are further divided such that Uninfected or Infected cells remained untreated or were treated with the apopotosis inducer staurosporine (STS). The design of the experiment was a time course and samples were harvested at the indicated time points (hr). All hybridizations were conducted against a common reference sample. time_series_design
Project description:The number of relevant and well-characterized cell lines and xenograft models for studying human breast cancer are few, and may represent a limitation for this field of research. With the aim of developing new breast cancer model systems for in vivo studies of hormone dependent and independent tumor growth, progression and invasion, and for in vivo experimental therapy studies, we collected primary mammary tumor specimens from patients, and implanted them in immunodeficient mice. Primary tumor tissue from 29 patients with breast cancer was implanted subcutaneously with matrigel in SCID mice, in the presence of continuous release of estradiol. The tumors were transferred into new animals when reaching a diameter of 15mm and engrafted tumors were harvested for morphological and molecular characterization from passage six. Further, gene expression profiling was performed using Agilent Human Whole Genome Oligo Microarrays, as well as DNA copy number analysis using Agilent Human Genome CGH 244K Microarrays. Of the 30 primary tumors implanted into mice (including two implants from the same patient), two gave rise to viable tumors beyond passage ten. One showed high expression levels of estrogen receptor-alpha protein (ER) while the other was negative. Histopathological evaluation of xenograft tumors was carried out at passage 10-12; both xenografts maintained the morphological characteristics of the original tumors (classified as invasive grade III ductal carcinomas). The genomic profile of the ER-positive xenograft tumor resembled the profile of the primary tumor, while the profile obtained from the ER-negative parental tumor was different from the xenograft. However, the ER-negative parental tumor and xenograft clustered on the same branch using unsupervised hierarchical clustering analysis on RNA microarray expression data of "intrinsic genes". A significant variation was observed in the expression of extracellular matrix (ECM)-related genes, which were found downregulated in the engrafted tumors compared to the primary tumor. By IHC and qRT-PCR we found that the downregulation of stroma-related genes was compensated by the overexpression of such molecules by the mouse host tissue. The two established breast cancer xenograft models showed different histopathological characteristics and profound diversity in gene expression patterns that in part can be associated to their ER status and here described as basal-like and luminal-like phenotype, respectively. These two new breast cancer xenografts represent useful preclinical tools for developing and testing of new therapies and improving our knowledge on breast cancer biology. Set of arrays organized by shared biological context, such as organism, tumors types, processes, etc.
Project description:We created a multi-species microarray platform, containing probes to the whole genomes of seven different Saccharomyces species, with very dense coverage (one probe every ~500 bp) of the S. cerevisiae genome, including non-S288c regions, mitochondrial and 2 micron circle genomes, plus probes at fairly dense coverage (one probe every ~2,100 bp) for each of the genomes of six other Saccharomyces species: S. paradoxus, S. mikatae, S. kudriavzevii, S. bayanus, S. kluyveri and S. castellii. We performed array-Comparative Genomic Hybridization (aCGH) using this platform, examining 83 different Saccharomyces strains collected across a wide range of habitats; of these, 69 were widely used commercial S. cerevisiae wine strains, while the remaining 14 were from a wide range of other industrial and natural habitats. Thus, we were able to sample much of the pan-genome space of the Saccharomyces genus. We observed interspecific hybridization events, introgression events, and pervasive copy number variation (CNV) in all but a few of the strains. These CNVs were distributed throughout the strains such that they did not produce any clear phylogeny, suggesting extensive mating in both industrial and wild strains. To validate our results and to determine whether apparently similar introgressions and CNVs were identical by descent or recurrent, we also performed whole genome sequencing on nine of these strains. These data may help pinpoint genomic regions involved in adaptation to different industrial milieus, as well as shed light on the course of domestication of S. cerevisiae. Control arrays of the following types are included in the dataset: (1) "Self-self" hybridizations (called "Self cntrl" in the dataset), where the reference DNA mix was labeled with either Cy3 or Cy5 (in separate reactions) and then mixed and hybridized. (2) Reference sub-pool hybridizations: the mix of 6 non-cerevisiae strains' DNA was labeled with Cy5, mixed with the final Reference pool (labeled in Cy3) and hybridized (called "OtherSppCompletePool" control) or the mix of 42 S. cerevisiae strains' DNA was labeled with Cy5, mixed with the final Reference pool (labeled in Cy3) and hybridized (called "Scer only ref pool") (3) Single Saccharomyces species hybridizations, where each of the 6 non-cerevisiae species, and also the S288c S. cerevisiae lab strain, was labeled with Cy5, mixed with the final Reference pool (labeled in Cy3) and hybridized (these arrays are called by the strain number followed by the species nickname). Four separate replicates of the self-self hybridizations were performed, and duplicate arrays (done on different days using different DNA preparations) were performed for three of the wine strains: GSY2175, GSY2176 and GSY2196.
Project description:BACKGROUND & AIMS: Gastric cancer results from a combination of Helicobacter pylori (H pylori) infection, exposure to dietary carcinogens, and predisposing genetic make-up. Because the role of these factors in gastric carcinogenesis cannot be determined readily in human beings, the present study examined the role of an oral carcinogen and H pylori infection in rhesus monkeys. METHODS: Gastroscopies were performed in 23 monkeys assigned to 4 groups: controls; nitrosating carcinogen ethyl-nitro-nitrosoguanidine administration alone; and inoculation of a virulent H pylori strain alone (H), or in combination with ethyl-nitro-nitrosoguanidine (EH). Follow-up gastroscopies and biopsies were performed at 3-month intervals for 5 years for pathologic and molecular studies. RESULTS: Postinoculation, H and EH groups showed persistent infection and antral gastritis. Starting at 2 and 5 years, respectively, gastric intestinal metaplasia and intraepithelial neoplasia developed in 3 EH monkeys but in no other groups. Transcriptional analysis of biopsies at 5 years revealed group-specific expression profiles, with striking changes in EH monkeys, plus a neoplasia-specific expression profile characterized by changes in multiple cancer-associated genes. Importantly, this neoplastic profile was evident in nonneoplastic mucosa, suggesting that the identified genes may represent markers preceding cancer. CONCLUSIONS: Gastric intraglandular neoplasia is induced in primates when H pylori infection is associated with consumption of a carcinogen similar to the nitrosaminesfound in pickled vegetables, suggesting that H pylori and the carcinogen synergistically induce gastric neoplasia in primates. Monkey arrays used for analysis of the affect of Helicobacter pylori and diet on development of gastric cancer. Each RNA was hybridized against a common reference. Biopsy numbers represent the following in terms of descriptions used in publication of the paper... Each set of numbers/descriptions is organized in the following order: Biopsy#; Monkey#; Group Code on Fig 5 Biopsy #4674; AB24; E; E1 Biopsy #4684; 16G; E; E2 Biopsy #4716; 89G; H; H1 Biopsy #4724; 54H; H; H2 Biopsy #4720; 20G; H; H3 Biopsy #4710; 92F; C; C1 Biopsy #4714; 26G; C; C2 Biopsy #4712; 92G; C; C3 Biopsy #4726; 08G; H; H4 Biopsy #4722; 57G; H; H5 Biopsy #4718; 85G; H; H6 Biopsy #4694; 48H; HE NON; HE1 Biopsy #4686; 59H; HE NON; HE2 Biopsy #4690; 81G; HE NEO; HE3 Biopsy #4696; 36G; HE NEO; HE4 Biopsy #4692; 63G; HE NEO; HE5 Biopsy #4680; 76G; E; E3 Biopsy #4682; 52G; E; E4 Biopsy #4678; 15G; E; E5 Biopsy #4676; 93G; E; E6 Biopsy #4688; 47H; HE NON; HE6 A pathogenicity experiment design type is where an infective agent such as a bacterium, virus, protozoan, fungus etc. infects a host organism(s) and the infective agent is assayed. Infection: Rhesus monkeys were infected with H. pylori (+/-) Compound Based Treatment: Rhesus monkeys were treated with ENNG (N-ethyl-N-nitrosoguanidine) (+/-) pathogenicity_design
Project description:Saccharomyces pastorianus is the yeast used to make lager beer; it is known to be an interspecific hybrid formed by the fusion between S. cerevisiae and S. bayanus genomes. This data set queries 17 S. pastorianus strains, collected at various times over the last 125 years from various breweries located in different geographical locations, which were obtained from CBS and DBVPG culture collections. The data in this set represent array-CGH experiments performed with these strains, using "2-species" custom Agilent arrays (the "2-species" arrays contain probes spaced every ~2 kb across the whole genomes of both S. cerevisiae and S. bayanus; the probes are unique and specific for each genome). The data set also contains 3 self-self hybridizations (S. cerevisiae + S. bayanus DNA mixed together in equimolar amounts, then labeled green or red in separate reactions, then hybridized to the "2-species" arrays) used for normalization in CGH-Miner analysis. A strain or line experiment design type assays differences between multiple strains, cultivars, serovars, isolates, lines from organisms of a single species. Keywords: strain_or_line_design
Project description:We have identified a novel xylose-utilization phenotype controlled by a single gene, and we sought to characterize how both the presence and absence of xylose in the presence or absence of the trait affected the S. cerevisiae transcriptional program over time. To do so, we measured global steady state mRNA levels on three pairs of sister spores from a Simi White strain that was backcrossed twice to S288c. Each pair of spores was from an independent tetrad, and contained one xylose utilizing ("positive", GSY2465, 2466, 2469) and one xylose non-utilizing spore ("negative", GSY2464, 2467, 2470). We grew each of the six spores in the presence or absence of xylose for 72 hours with samples taken every 8 hours. We then assayed relative RNA abundance versus a pooled reference, containing equimolar amounts of each sample, using Agilent yeast catalog arrays. time_series_design
Project description:Study of host response in mice to uropathogenic E. coli (UPEC) infection and the contribution of deletion of the Cnf and HlyA1 toxins to the host response. Mice were infected with UPEC in the bladder for 24 hrs and RNA was isolated from whole bladder to analyze on Agilent whole mouse 2-channel arrays against normal untreated mouse bladder RNA. Biological replicates were analyzed for each condition. Infection: genotype of E. coli CP9 strain strain_or_line_design
Project description:Study of host response in mice to uropathogenic E. coli (UPEC) infection and the contribution of deletion of the Cnf and HlyA1 toxins to the host response. Mice were infected with UPEC in the bladder for 24 hrs and RNA was isolated from whole bladder to analyze on Agilent whole mouse 2-channel arrays against normal untreated mouse bladder RNA. Biological replicates were analyzed for each condition. Infection: genotype of E. coli CP9 (UPEC) strain strain_or_line_design
Project description:Question Addressed: What is the host response (specifically apoptosis pathways) to Shigella infection and what is the contribution of MxiE to the host response? The arrays are apoptosis Exon hit arrays, thus splice variation can be determined. Array data from HeLa cells that remained Uninfected or were infected with wildtype S. flexneri serotype 2a strain 2457T or an isogenic mixE mutant. These groupings are further divided such that Uninfected or Infected cells remained untreated or were treated with the apopotosis inducer staurosporine (STS). The design of the experiment was a time course and samples were harvested at the indicated time points (hr). All hybridizations were conducted against a common reference sample that was made from pooled samples of normal uninfected healthy HeLa cells. Infection: HeLa cells were infected with wt Shigella (wt), mxiE mutant (mixE) or were left uninfected (uninfected) Compound Based Treatment: Cultures were treated with Staurosporin (STS) or were left untreated (untreated) Infection time: Cells were harvested at the indicated time after treatment 20 Samples