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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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Single-molecule FISH (smFISH) to characterize lacI mRNA copy number statistics. Typical images of smFISH-labeled cells in different strain backgrounds (ΔlacI, CH5201; wild-type lacI, CH458; lacIq, CH1143). An overlay of the phase contrast (grayscale) and smFISH probes targeting the lacI gene (red) is shown. n, number of cells monitored. Mean and standard error values for absolute lacI mRNA numbers per cell are shown for each strain. The histogram shows the distribution of lacI mRNA molecules per cell observed within each strain population monitored.
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Figure 1: Single-molecule FISH (smFISH) to characterize lacI mRNA copy number statistics. Typical images of smFISH-labeled cells in different strain backgrounds (ΔlacI, CH5201; wild-type lacI, CH458; lacIq, CH1143). An overlay of the phase contrast (grayscale) and smFISH probes targeting the lacI gene (red) is shown. n, number of cells monitored. Mean and standard error values for absolute lacI mRNA numbers per cell are shown for each strain. The histogram shows the distribution of lacI mRNA molecules per cell observed within each strain population monitored.

Mentions: Although it is clear that incorporation of 8-oxo-dGTP into DNA can have mutagenic consequences for the cell, with heritable phenotypic consequences, it remains unknown if incorporation of 8-oxo-GTP into RNA can also have heritable phenotypic consequences for the cell (26). To study the effect of mutT on heritable phenotypic change, we used the bistable lac switch assay in E. coli. This bistable switch assay is sensitive to transcription errors: we previously observed that a 5-fold decrease in transcription fidelity due to an RNA polymerase mutation [measured both in vitro and in vivo (27)] leads to a 4- to 6-fold increase in epigenetic switching frequency in our bistable lac assay (5). Moreover, the number of lacI mRNA per cell is very low making this system susceptible to transcription errors. Based on equilibrium dialysis against radioactive isopropyl-thio-galactoside it was estimated that there are about five lac repressor tetramers per cell (10). It was therefore suggested that the lacI gene has an inefficient promoter and that only one or two mRNA molecules are synthesized per lacI gene per generation (28). We directly measured the number of lacI mRNA molecules in single cells using single-molecule mRNA fluorescent in situ hybridization (smFISH) (24,25) to quantify mRNA statistics in three E. coli strains: an entire deletion of the lacI gene (CH5201), the wild-type lacI promoter (CH458) and the lacI up-promoter Iq (CH1143; Figure 1). Fully 86% of wild-type lacI cells do not exhibit any lacI mRNA, 11% of cells have one lacI mRNA and 3% have two or more lacI mRNA per cell at any given time (2081 cells monitored); see Figure 1. No lacI mRNA molecules were observed in cells that have the entire lacI gene deleted. A mutant that makes about 10× more lac repressor, lacIq, has been previously isolated (28) which carries an up-mutation at the −35 position of the lacI promoter (29). smFISH analysis showed that the lacIq strain exhibited an average of 3.05 lacI mRNA per cell (2638 cells monitored; Figure 1). This is the first demonstration of wild-type lacI mRNA statistics and is in excellent agreement with the low lac repressor numbers indirectly determined during the initial isolation of the lac repressor almost 50 years ago (10). Thus, these results suggest that wild-type lac repressor production is subject to large fluctuations in protein number, due to rare stochastic transcription events (30), making the bistable switch system sensitive to transcription errors during lacI mRNA production (31).


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)

Single-molecule FISH (smFISH) to characterize lacI mRNA copy number statistics. Typical images of smFISH-labeled cells in different strain backgrounds (ΔlacI, CH5201; wild-type lacI, CH458; lacIq, CH1143). An overlay of the phase contrast (grayscale) and smFISH probes targeting the lacI gene (red) is shown. n, number of cells monitored. Mean and standard error values for absolute lacI mRNA numbers per cell are shown for each strain. The histogram shows the distribution of lacI mRNA molecules per cell observed within each strain population monitored.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4231768&req=5

Figure 1: Single-molecule FISH (smFISH) to characterize lacI mRNA copy number statistics. Typical images of smFISH-labeled cells in different strain backgrounds (ΔlacI, CH5201; wild-type lacI, CH458; lacIq, CH1143). An overlay of the phase contrast (grayscale) and smFISH probes targeting the lacI gene (red) is shown. n, number of cells monitored. Mean and standard error values for absolute lacI mRNA numbers per cell are shown for each strain. The histogram shows the distribution of lacI mRNA molecules per cell observed within each strain population monitored.
Mentions: Although it is clear that incorporation of 8-oxo-dGTP into DNA can have mutagenic consequences for the cell, with heritable phenotypic consequences, it remains unknown if incorporation of 8-oxo-GTP into RNA can also have heritable phenotypic consequences for the cell (26). To study the effect of mutT on heritable phenotypic change, we used the bistable lac switch assay in E. coli. This bistable switch assay is sensitive to transcription errors: we previously observed that a 5-fold decrease in transcription fidelity due to an RNA polymerase mutation [measured both in vitro and in vivo (27)] leads to a 4- to 6-fold increase in epigenetic switching frequency in our bistable lac assay (5). Moreover, the number of lacI mRNA per cell is very low making this system susceptible to transcription errors. Based on equilibrium dialysis against radioactive isopropyl-thio-galactoside it was estimated that there are about five lac repressor tetramers per cell (10). It was therefore suggested that the lacI gene has an inefficient promoter and that only one or two mRNA molecules are synthesized per lacI gene per generation (28). We directly measured the number of lacI mRNA molecules in single cells using single-molecule mRNA fluorescent in situ hybridization (smFISH) (24,25) to quantify mRNA statistics in three E. coli strains: an entire deletion of the lacI gene (CH5201), the wild-type lacI promoter (CH458) and the lacI up-promoter Iq (CH1143; Figure 1). Fully 86% of wild-type lacI cells do not exhibit any lacI mRNA, 11% of cells have one lacI mRNA and 3% have two or more lacI mRNA per cell at any given time (2081 cells monitored); see Figure 1. No lacI mRNA molecules were observed in cells that have the entire lacI gene deleted. A mutant that makes about 10× more lac repressor, lacIq, has been previously isolated (28) which carries an up-mutation at the −35 position of the lacI promoter (29). smFISH analysis showed that the lacIq strain exhibited an average of 3.05 lacI mRNA per cell (2638 cells monitored; Figure 1). This is the first demonstration of wild-type lacI mRNA statistics and is in excellent agreement with the low lac repressor numbers indirectly determined during the initial isolation of the lac repressor almost 50 years ago (10). Thus, these results suggest that wild-type lac repressor production is subject to large fluctuations in protein number, due to rare stochastic transcription events (30), making the bistable switch system sensitive to transcription errors during lacI mRNA production (31).

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