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How selection affects phenotypic fluctuation.

Ito Y, Toyota H, Kaneko K, Yomo T - Mol. Syst. Biol. (2009)

Bottom Line: However, as fluctuation can increase phenotypic diversity, similar to mutation, it may contribute to the survival of individuals even under a single selective environment.To discuss whether the fluctuation increases over the course of evolution, cycles of mutation and selection for higher GFP fluorescence were carried out in Escherichia coli.In addition to the average phenotypic change by genetic mutation, the observed increase in phenotypic fluctuation acts as an evolutionary strategy to produce an extreme phenotype under severe selective environments.

View Article: PubMed Central - PubMed

Affiliation: Department of Bioinformatic Engineering, Graduate School of Information Science and Technology, Osaka University, Suita, Osaka, Japan.

ABSTRACT
The large degree of phenotypic fluctuation among isogenic cells highlighted by recent studies on stochastic gene expression confers fitness on some individuals through a 'bet-hedging' strategy, when faced with different selective environments. Under a single selective environment, the fluctuation may be suppressed through evolution, as it prevents maintenance of individuals around the fittest state and/or function. However, as fluctuation can increase phenotypic diversity, similar to mutation, it may contribute to the survival of individuals even under a single selective environment. To discuss whether the fluctuation increases over the course of evolution, cycles of mutation and selection for higher GFP fluorescence were carried out in Escherichia coli. Mutant genotypes possessing broad GFP fluorescence distributions with low average values emerged under strong selection pressure. These 'broad mutants' appeared independently on the phylogenetic tree and increased fluctuations in GFP fluorescence were attributable to the variance in mRNA abundance. In addition to the average phenotypic change by genetic mutation, the observed increase in phenotypic fluctuation acts as an evolutionary strategy to produce an extreme phenotype under severe selective environments.

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Related in: MedlinePlus

Evolutionary experiment. (A) GFP fluorescence intensity (FI) and forward scattering (FS) of individual cells measured by FCM (those of HLG10-6 are also shown). Cells in the R1 region (top 0.2% of the total in fluorescence density (FI/FS)) were sorted for the next selection cycle. (B) The selection threshold value, log(FI/FS) at the 99.8th percentile in the cell population, was measured. Open squares indicate values at each round of selection. Values of 12 clones picked at random from the last cycle of each generation are plotted (some clones had the same value). Blue and magenta bars indicate narrow and broad mutants, respectively. The selection threshold values in clone analysis (blue and magenta bars) were slightly lower than those in population analysis (open squares). This decrease was probably because of the differences in culture conditions. (C) Phylogenetic tree of the selected clones, 12 clones sampled from each generation (as in B) and 11 clones in our earlier experiment (Ito et al, 2004). Nodes of the 12 clones sampled from each of the first, second and third generations are indicated in orange, green and light blue, respectively. Broad mutants are encircled in red. Nodes of the clone selected at each generation, RP3-34H, first to ninth, and HLG10-6, of our earlier experiment, are indicated in red, grey and black, respectively. The evolutionary experiment was started from the clone of the 10th generation (black). Scale bar: five nucleotide substitutions.
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f1: Evolutionary experiment. (A) GFP fluorescence intensity (FI) and forward scattering (FS) of individual cells measured by FCM (those of HLG10-6 are also shown). Cells in the R1 region (top 0.2% of the total in fluorescence density (FI/FS)) were sorted for the next selection cycle. (B) The selection threshold value, log(FI/FS) at the 99.8th percentile in the cell population, was measured. Open squares indicate values at each round of selection. Values of 12 clones picked at random from the last cycle of each generation are plotted (some clones had the same value). Blue and magenta bars indicate narrow and broad mutants, respectively. The selection threshold values in clone analysis (blue and magenta bars) were slightly lower than those in population analysis (open squares). This decrease was probably because of the differences in culture conditions. (C) Phylogenetic tree of the selected clones, 12 clones sampled from each generation (as in B) and 11 clones in our earlier experiment (Ito et al, 2004). Nodes of the 12 clones sampled from each of the first, second and third generations are indicated in orange, green and light blue, respectively. Broad mutants are encircled in red. Nodes of the clone selected at each generation, RP3-34H, first to ninth, and HLG10-6, of our earlier experiment, are indicated in red, grey and black, respectively. The evolutionary experiment was started from the clone of the 10th generation (black). Scale bar: five nucleotide substitutions.

Mentions: Here, with mutagenesis of the selected clone at the 10th generation, HLG10-6, as the starting material, selection was done on the basis of individual cells, where the fluctuation could influence the clones that evolve. GFP, FI and forward scattering (FS) of the cells were measured by flow cytometry (FCM) for selection at the individual cell level (Figure 1A). The intensity of FS is generally regarded as proportional to the size of the cell (Bouvier et al, 2001). Thus, the GFP fluorescence density was estimated by FI/FS, which was used as an index of selection. Specifically, the selected genes were amplified from 20 000 cells (0.2% of total cells measured) in region R1 in Figure 1A, reinserted into fresh vector, and reintroduced into fresh E. coli cells to exclude unexpected mutations in other regions of the vector. After repeating the selection and amplification (FCM selection cycle) several times, the selected genes were mutated in the next generation. This evolutionary process was carried out for three generations. In this selection procedure, cells that happened to possess a higher value of FI/FS because of phenotypic fluctuations could be selected. Thus, the genes or clones causing larger fluctuations in FI/FS, as well as those with a higher average value, may evolve.


How selection affects phenotypic fluctuation.

Ito Y, Toyota H, Kaneko K, Yomo T - Mol. Syst. Biol. (2009)

Evolutionary experiment. (A) GFP fluorescence intensity (FI) and forward scattering (FS) of individual cells measured by FCM (those of HLG10-6 are also shown). Cells in the R1 region (top 0.2% of the total in fluorescence density (FI/FS)) were sorted for the next selection cycle. (B) The selection threshold value, log(FI/FS) at the 99.8th percentile in the cell population, was measured. Open squares indicate values at each round of selection. Values of 12 clones picked at random from the last cycle of each generation are plotted (some clones had the same value). Blue and magenta bars indicate narrow and broad mutants, respectively. The selection threshold values in clone analysis (blue and magenta bars) were slightly lower than those in population analysis (open squares). This decrease was probably because of the differences in culture conditions. (C) Phylogenetic tree of the selected clones, 12 clones sampled from each generation (as in B) and 11 clones in our earlier experiment (Ito et al, 2004). Nodes of the 12 clones sampled from each of the first, second and third generations are indicated in orange, green and light blue, respectively. Broad mutants are encircled in red. Nodes of the clone selected at each generation, RP3-34H, first to ninth, and HLG10-6, of our earlier experiment, are indicated in red, grey and black, respectively. The evolutionary experiment was started from the clone of the 10th generation (black). Scale bar: five nucleotide substitutions.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2683726&req=5

f1: Evolutionary experiment. (A) GFP fluorescence intensity (FI) and forward scattering (FS) of individual cells measured by FCM (those of HLG10-6 are also shown). Cells in the R1 region (top 0.2% of the total in fluorescence density (FI/FS)) were sorted for the next selection cycle. (B) The selection threshold value, log(FI/FS) at the 99.8th percentile in the cell population, was measured. Open squares indicate values at each round of selection. Values of 12 clones picked at random from the last cycle of each generation are plotted (some clones had the same value). Blue and magenta bars indicate narrow and broad mutants, respectively. The selection threshold values in clone analysis (blue and magenta bars) were slightly lower than those in population analysis (open squares). This decrease was probably because of the differences in culture conditions. (C) Phylogenetic tree of the selected clones, 12 clones sampled from each generation (as in B) and 11 clones in our earlier experiment (Ito et al, 2004). Nodes of the 12 clones sampled from each of the first, second and third generations are indicated in orange, green and light blue, respectively. Broad mutants are encircled in red. Nodes of the clone selected at each generation, RP3-34H, first to ninth, and HLG10-6, of our earlier experiment, are indicated in red, grey and black, respectively. The evolutionary experiment was started from the clone of the 10th generation (black). Scale bar: five nucleotide substitutions.
Mentions: Here, with mutagenesis of the selected clone at the 10th generation, HLG10-6, as the starting material, selection was done on the basis of individual cells, where the fluctuation could influence the clones that evolve. GFP, FI and forward scattering (FS) of the cells were measured by flow cytometry (FCM) for selection at the individual cell level (Figure 1A). The intensity of FS is generally regarded as proportional to the size of the cell (Bouvier et al, 2001). Thus, the GFP fluorescence density was estimated by FI/FS, which was used as an index of selection. Specifically, the selected genes were amplified from 20 000 cells (0.2% of total cells measured) in region R1 in Figure 1A, reinserted into fresh vector, and reintroduced into fresh E. coli cells to exclude unexpected mutations in other regions of the vector. After repeating the selection and amplification (FCM selection cycle) several times, the selected genes were mutated in the next generation. This evolutionary process was carried out for three generations. In this selection procedure, cells that happened to possess a higher value of FI/FS because of phenotypic fluctuations could be selected. Thus, the genes or clones causing larger fluctuations in FI/FS, as well as those with a higher average value, may evolve.

Bottom Line: However, as fluctuation can increase phenotypic diversity, similar to mutation, it may contribute to the survival of individuals even under a single selective environment.To discuss whether the fluctuation increases over the course of evolution, cycles of mutation and selection for higher GFP fluorescence were carried out in Escherichia coli.In addition to the average phenotypic change by genetic mutation, the observed increase in phenotypic fluctuation acts as an evolutionary strategy to produce an extreme phenotype under severe selective environments.

View Article: PubMed Central - PubMed

Affiliation: Department of Bioinformatic Engineering, Graduate School of Information Science and Technology, Osaka University, Suita, Osaka, Japan.

ABSTRACT
The large degree of phenotypic fluctuation among isogenic cells highlighted by recent studies on stochastic gene expression confers fitness on some individuals through a 'bet-hedging' strategy, when faced with different selective environments. Under a single selective environment, the fluctuation may be suppressed through evolution, as it prevents maintenance of individuals around the fittest state and/or function. However, as fluctuation can increase phenotypic diversity, similar to mutation, it may contribute to the survival of individuals even under a single selective environment. To discuss whether the fluctuation increases over the course of evolution, cycles of mutation and selection for higher GFP fluorescence were carried out in Escherichia coli. Mutant genotypes possessing broad GFP fluorescence distributions with low average values emerged under strong selection pressure. These 'broad mutants' appeared independently on the phylogenetic tree and increased fluctuations in GFP fluorescence were attributable to the variance in mRNA abundance. In addition to the average phenotypic change by genetic mutation, the observed increase in phenotypic fluctuation acts as an evolutionary strategy to produce an extreme phenotype under severe selective environments.

Show MeSH
Related in: MedlinePlus