Removal of 8-oxo-GTP by MutT hydrolase is not a major contributor to transcriptional fidelity.
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.
Affiliation: Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX 77030, USA.Show MeSH
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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).
Affiliation: Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX 77030, USA.