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Project description:The NS1 protein of influenza A virus (IAV) is a multifunctional virulence factor. Mouse adaptive mutations in the NS1 protein of the human isolate A/Hong Kong/1/1968(H3N2) (HK) have been previously reported to increase virulence, viral fitness, and interferon antagonism, but differ in binding to post-transcriptional processing factor CPSF30. Because nuclear trafficking is a major genetic determinant of influenza virus host adaptation, we assessed subcellular localization and host gene expression of NS1 adaptive mutations. Recombinant HK viruses with adaptive mutations in the NS1 gene were assessed for NS1 protein subcellular localization in mouse and human cells using confocal microscopy and cellular fractionation. HK-wt virus NS1 partitioned equivalently between the cytoplasm and nucleus in human cells but was defective in cytoplasmic localization in mouse cells. The adaptive mutations either increased the proportion or abundance of NS1 in the cytoplasm, and/or the nucleus. NS1 mutations that increased cytoplasmic distribution identified a putative second nuclear export signal (NES) spanning aa positions 98-106 LSEDWFMLM, (mutation sites in bold); with the strongest effect seen for mutation M106I. The putative NES in the NS3 protein was associated with cytoplasmic localization. The host gene expression profile of the adaptive mutants was determined by microarray analysis of infected mouse cells to show either high or low gene regulation (HGR or LGR) phenotypes that mapped to the amino-terminal and the carboxy-terminal regions respectively. The HGR and LGR mutations were predominantly down regulating versus up regulating respectively. The greatest effect on host gene expression in the HGR group correlated with the ability of the NS1 protein to bind CPSF30. To our knowledge this is the first report of roles of adaptive NS1 mutations that affect intracellular localization and regulation of host gene expression. biological triplicates of mock infected mouse M1 cells; cells infected with A/HK/1/1968(H3N2) wt and NS1 mtuatn with mutaions, D2N, V23A, L98S, L98S + M106I, F103L, M106V, M106V + M124I, D125G, V180A, V226I, M106I, and R227K. Cells were infected at a multiplicty of infection of 2 and cels were incubated for 8 hr at 37 C for 8 hrs before RNA extraction and analysis relative to mock PBS infected cells.
2013-06-22 | E-GEOD-48217 | biostudies-arrayexpress
Project description:High-throughput sequencing has enabled genetic screens that can rapidly identify mutations that occur during experimental evolution. The presence of a mutation in an evolved lineage does not, however, constitute proof that the mutation is adaptive, given the well-known and widespread phenomenon of genetic hitchhiking, in which a non-adaptive or even detrimental mutation can co-occur in a genome with a beneficial mutation and the combined genotype is carried to high frequency by selection. We approximated the spectrum of possible beneficial mutations in Saccharomyces cerevisiae using sets of single-gene deletions and amplifications of almost all the genes in the S. cerevisiae genome. We determined the fitness effects of each mutation in three different nutrient-limited conditions using pooled competitions followed by barcode sequencing. Although most of the mutations were neutral or deleterious, ~500 of them increased fitness. We then compared those results to the mutations that actually occurred during experimental evolution in the same three nutrient-limited conditions. On average, ~35% of the mutations that occurred during experimental evolution were predicted by the systematic screen to be beneficial. We found that the distribution of fitness effects depended on the selective conditions. In the phosphate-limited and glucose-limited conditions, a large number of beneficial mutations of nearly equivalent, small effects drove the fitness increases. In the sulfate-limited condition, one type of mutation, the amplification of the high-affinity sulfate transporter, dominated. In the absence of that mutation, evolution in the sulfate-limited condition involved mutations in other genes that were not observed previously—but were predicted by the systematic screen. Thus, gross functional screens have the potential to predict and identify adaptive mutations that occur during experimental evolution. Previously version available on bioRXiv.
2015-01-01 | GSE58497 | GEO
Project description:The NS1 protein of influenza A virus (IAV) is a multifunctional virulence factor. Mouse adaptive mutations in the NS1 protein of the human isolate A/Hong Kong/1/1968(H3N2) (HK) have been previously reported to increase virulence, viral fitness, and interferon antagonism, but differ in binding to post-transcriptional processing factor CPSF30. Because nuclear trafficking is a major genetic determinant of influenza virus host adaptation, we assessed subcellular localization and host gene expression of NS1 adaptive mutations. Recombinant HK viruses with adaptive mutations in the NS1 gene were assessed for NS1 protein subcellular localization in mouse and human cells using confocal microscopy and cellular fractionation. HK-wt virus NS1 partitioned equivalently between the cytoplasm and nucleus in human cells but was defective in cytoplasmic localization in mouse cells. The adaptive mutations either increased the proportion or abundance of NS1 in the cytoplasm, and/or the nucleus. NS1 mutations that increased cytoplasmic distribution identified a putative second nuclear export signal (NES) spanning aa positions 98-106 LSEDWFMLM, (mutation sites in bold); with the strongest effect seen for mutation M106I. The putative NES in the NS3 protein was associated with cytoplasmic localization. The host gene expression profile of the adaptive mutants was determined by microarray analysis of infected mouse cells to show either high or low gene regulation (HGR or LGR) phenotypes that mapped to the amino-terminal and the carboxy-terminal regions respectively. The HGR and LGR mutations were predominantly down regulating versus up regulating respectively. The greatest effect on host gene expression in the HGR group correlated with the ability of the NS1 protein to bind CPSF30. To our knowledge this is the first report of roles of adaptive NS1 mutations that affect intracellular localization and regulation of host gene expression.
2013-06-22 | GSE48217 | GEO
Project description:Adaptive mutations associated with oxacillin treatment failure in Staphylococcus aureus infections
| PRJEB40246 | ENA
Project description:High-throughput identification of adaptive mutations in experimentally evolved yeast populations
| PRJNA252936 | ENA
Project description:Studies of the RNA polymerase-binding molecule ppGpp in bacteria and plants have shown that changes to the kinetics of the RNA polymerase can have dramatic biological effects in the short-term as a stress response. Here we describe the reprogramming of the kinetic parameters of the RNAP through mutations arising during laboratory adaptive evolution of Escherichia coli in minimal media. The mutations cause a 10- to 30-fold decrease in open complex stability at a ribosomal promoter and approximately a 10-fold decrease in transcriptional pausing in the his operon. The kinetic changes coincide with large scale transcriptional changes, including strong downregulation of motility, acid-resistance, fimbria, and curlin genes which are observed in site-directed mutants containing the RNA polymerase mutations as well as the evolved strains harboring the mutations. Site-directed mutants also grow 60% faster than the parent strain and convert the carbon-source 15% to 35% more efficiently to biomass. The results show that long-term adjustment of the kinetic parameters of RNA polymerase through mutation can be important for adaptation to a condition. Mutations in the RNA polymerase beta prime subunit (rpoC) were discovered in E. coli following adaptation to continual logarithmic growth (OD <= 0.3) in glycerol M9 minimal media at 30 C. We used site-directed mutagenesis to make strains of E. coli isogenic to wild-type except for single adaptive rpoC mutations. We found they increase growth rate in this condition by 60%. In order to understand how the mutations affect gene expression in this condition, we extracted total mRNA from the strains, which had been growing in the adaptive evolution condition, at OD=0.3. There were three RNAP mutants and the wild-type (E. coli K-12 MG1655). Each strain had three flasks from which RNA was extracted (three biological replicates). There were no technical replicates. The mRNA was synthesized into cDNA, labeled, and hybridized to an Affymetrix E. coli 2.0 GeneChip.
2010-04-12 | E-GEOD-15500 | biostudies-arrayexpress