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Removal of 8-oxo-GTP by MutT hydrolase is not a major contributor to transcriptional fidelity.

Gordon AJ, Satory D, Wang M, Halliday JA, Golding I, Herman C - Nucleic Acids Res. (2014)

Bottom Line: We do not observe any increase in epigenetic switching in mutT cells.Moreover, we observe a fluctuation type of distribution of β-galactosidase appearance in a growing culture, consistent with Lac+ DNA revertant events.We conclude that the absence of MutT produces a DNA mutator but does not equally create an RNA mutator.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX 77030, USA.

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Related in: MedlinePlus

Stochastic switching in the lac bistable gene network. (A) Under maintenance conditions, the lac operon is OFF when the lac repressor is bound to the lac operator (indicated by the solid red line) and the inducer thio-methylgalactoside (TMG) remains extracellular; stochastic events that lead to a transient derepression of the lac operon will result in a burst of lac operon functions and the appearance of permease will initiate an autocatalytic positive-feedback response (indicated by solid blue lines), which will heritably maintain the ON state (TMG induces an allosteric transition in lac repressor, indicated by the dashed red line, so that it no longer binds to the lac operator), and the cell will exhibit green fluorescence. (B) Wild-type (CH458) and ΔmutT (CH505) cells that were originally ON or OFF were sub-cultured and grown in media containing various concentrations of TMG. Each value is the average ±SD from four independent cultures. The shaded area highlights the maintenance concentration of 6-μM TMG for these strains. (C) OFF wild-type cells (red histograms) and OFF ΔmutT cells (blue histograms) were diluted and grown in media containing 6-μM TMG. After 42 h growth, flow cytometry was performed to determine the frequency of epigenetically ON cells in 49 independent cultures of each strain; the ΔmutT histograms are superimposed over the wild-type histograms (104 cells interrogated for each histogram). (D) The ΔmutT epigenetic-switch frequency is not significantly increased over the wild-type value (Mann–Whitney Rank Sum Test, P = 0.13).
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Figure 2: Stochastic switching in the lac bistable gene network. (A) Under maintenance conditions, the lac operon is OFF when the lac repressor is bound to the lac operator (indicated by the solid red line) and the inducer thio-methylgalactoside (TMG) remains extracellular; stochastic events that lead to a transient derepression of the lac operon will result in a burst of lac operon functions and the appearance of permease will initiate an autocatalytic positive-feedback response (indicated by solid blue lines), which will heritably maintain the ON state (TMG induces an allosteric transition in lac repressor, indicated by the dashed red line, so that it no longer binds to the lac operator), and the cell will exhibit green fluorescence. (B) Wild-type (CH458) and ΔmutT (CH505) cells that were originally ON or OFF were sub-cultured and grown in media containing various concentrations of TMG. Each value is the average ±SD from four independent cultures. The shaded area highlights the maintenance concentration of 6-μM TMG for these strains. (C) OFF wild-type cells (red histograms) and OFF ΔmutT cells (blue histograms) were diluted and grown in media containing 6-μM TMG. After 42 h growth, flow cytometry was performed to determine the frequency of epigenetically ON cells in 49 independent cultures of each strain; the ΔmutT histograms are superimposed over the wild-type histograms (104 cells interrogated for each histogram). (D) The ΔmutT epigenetic-switch frequency is not significantly increased over the wild-type value (Mann–Whitney Rank Sum Test, P = 0.13).

Mentions: To determine the proportion of cells that switch to the ON state for lac operon expression, we used a lacZYA::gfp construct expressing β-galactosidase, galactoside permease and green fluorescent protein (5). During growth of OFF cells in a maintenance concentration of TMG (6-μM TMG; see Figure 2A and B), if a cell suffers a stochastic event leading to derepression of the lac operon, this transient derepression will trigger permease synthesis and activation of the autocatalytic positive-feedback loop, resulting in green fluorescent cells (5,6). As a result, the OFF state will transition to the ON state and be heritably maintained in the following generations, mimicking lacI mutation in this system (Figure 2A). To determine the epigenetic switch frequency, we measured the number of green cells within the resulting cultures by flow cytometry (Figure 2C). We calculate the epigenetic switch frequency as number of ON cells over the total number of cells interrogated, following the convention used in determining lacI− mutation frequencies in a population (21). The observed ON switch frequency is therefore dependent on both the number of switch events that have occurred and the number of generations after a discrete switch event has occurred, as in a classical fluctuation test (see ‘Materials and Methods’ section).


Removal of 8-oxo-GTP by MutT hydrolase is not a major contributor to transcriptional fidelity.

Gordon AJ, Satory D, Wang M, Halliday JA, Golding I, Herman C - Nucleic Acids Res. (2014)

Stochastic switching in the lac bistable gene network. (A) Under maintenance conditions, the lac operon is OFF when the lac repressor is bound to the lac operator (indicated by the solid red line) and the inducer thio-methylgalactoside (TMG) remains extracellular; stochastic events that lead to a transient derepression of the lac operon will result in a burst of lac operon functions and the appearance of permease will initiate an autocatalytic positive-feedback response (indicated by solid blue lines), which will heritably maintain the ON state (TMG induces an allosteric transition in lac repressor, indicated by the dashed red line, so that it no longer binds to the lac operator), and the cell will exhibit green fluorescence. (B) Wild-type (CH458) and ΔmutT (CH505) cells that were originally ON or OFF were sub-cultured and grown in media containing various concentrations of TMG. Each value is the average ±SD from four independent cultures. The shaded area highlights the maintenance concentration of 6-μM TMG for these strains. (C) OFF wild-type cells (red histograms) and OFF ΔmutT cells (blue histograms) were diluted and grown in media containing 6-μM TMG. After 42 h growth, flow cytometry was performed to determine the frequency of epigenetically ON cells in 49 independent cultures of each strain; the ΔmutT histograms are superimposed over the wild-type histograms (104 cells interrogated for each histogram). (D) The ΔmutT epigenetic-switch frequency is not significantly increased over the wild-type value (Mann–Whitney Rank Sum Test, P = 0.13).
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Figure 2: Stochastic switching in the lac bistable gene network. (A) Under maintenance conditions, the lac operon is OFF when the lac repressor is bound to the lac operator (indicated by the solid red line) and the inducer thio-methylgalactoside (TMG) remains extracellular; stochastic events that lead to a transient derepression of the lac operon will result in a burst of lac operon functions and the appearance of permease will initiate an autocatalytic positive-feedback response (indicated by solid blue lines), which will heritably maintain the ON state (TMG induces an allosteric transition in lac repressor, indicated by the dashed red line, so that it no longer binds to the lac operator), and the cell will exhibit green fluorescence. (B) Wild-type (CH458) and ΔmutT (CH505) cells that were originally ON or OFF were sub-cultured and grown in media containing various concentrations of TMG. Each value is the average ±SD from four independent cultures. The shaded area highlights the maintenance concentration of 6-μM TMG for these strains. (C) OFF wild-type cells (red histograms) and OFF ΔmutT cells (blue histograms) were diluted and grown in media containing 6-μM TMG. After 42 h growth, flow cytometry was performed to determine the frequency of epigenetically ON cells in 49 independent cultures of each strain; the ΔmutT histograms are superimposed over the wild-type histograms (104 cells interrogated for each histogram). (D) The ΔmutT epigenetic-switch frequency is not significantly increased over the wild-type value (Mann–Whitney Rank Sum Test, P = 0.13).
Mentions: To determine the proportion of cells that switch to the ON state for lac operon expression, we used a lacZYA::gfp construct expressing β-galactosidase, galactoside permease and green fluorescent protein (5). During growth of OFF cells in a maintenance concentration of TMG (6-μM TMG; see Figure 2A and B), if a cell suffers a stochastic event leading to derepression of the lac operon, this transient derepression will trigger permease synthesis and activation of the autocatalytic positive-feedback loop, resulting in green fluorescent cells (5,6). As a result, the OFF state will transition to the ON state and be heritably maintained in the following generations, mimicking lacI mutation in this system (Figure 2A). To determine the epigenetic switch frequency, we measured the number of green cells within the resulting cultures by flow cytometry (Figure 2C). We calculate the epigenetic switch frequency as number of ON cells over the total number of cells interrogated, following the convention used in determining lacI− mutation frequencies in a population (21). The observed ON switch frequency is therefore dependent on both the number of switch events that have occurred and the number of generations after a discrete switch event has occurred, as in a classical fluctuation test (see ‘Materials and Methods’ section).

Bottom Line: We do not observe any increase in epigenetic switching in mutT cells.Moreover, we observe a fluctuation type of distribution of β-galactosidase appearance in a growing culture, consistent with Lac+ DNA revertant events.We conclude that the absence of MutT produces a DNA mutator but does not equally create an RNA mutator.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX 77030, USA.

Show MeSH
Related in: MedlinePlus