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In vivo DNase I sensitivity of the Streptomyces coelicolor chromosome correlates with gene expression: implications for bacterial chromosome structure.

McArthur M, Bibb M - Nucleic Acids Res. (2006)

Bottom Line: DNase I-sensitivity correlated positively with transcript levels, implying that it was predictive of gene expression, and indicating increased accessibility of transcribed DNA.The genome was fractionated based on the sensitivity to DNase I digestion, with the low molecular weight (frequently cut) fraction highly enriched for actively transcribed sequences when compared to the infrequently cut fraction, which was representative of the entire genome.This approach will allow comparison of nucleoid proteins, and any modifications thereof, associated with transcriptionally active and inactive regions of the bacterial genome.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Microbiology, John Innes Centre, Norwich, NR4 7UH, UK.

ABSTRACT
For a bacterium, Streptomyces coelicolor A3(2) contains a relatively large genome (8.7 Mb) with a complex and adaptive pattern of gene regulation. We discovered a correlation between the physical structure of the S.coelicolor genome and the transcriptional activity of the genes therein. Twelve genes were surveyed throughout 72 h of growth for both in vivo sensitivity to DNase I digestion and levels of transcription. DNase I-sensitivity correlated positively with transcript levels, implying that it was predictive of gene expression, and indicating increased accessibility of transcribed DNA. The genome was fractionated based on the sensitivity to DNase I digestion, with the low molecular weight (frequently cut) fraction highly enriched for actively transcribed sequences when compared to the infrequently cut fraction, which was representative of the entire genome. This approach will allow comparison of nucleoid proteins, and any modifications thereof, associated with transcriptionally active and inactive regions of the bacterial genome.

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Quantifying changes in DNase I-sensitivity with qrt-PCR. qrt-PCR was used to detected small changes in DNase I-sensitivity in two reciprocally expressed genes (redD and rlpA) at the earliest and latest time points. In vivo digestions were performed as described in Figure 1; qrt-PCR is described elsewhere (6). DNase I sensitivity was estimated by the percentage loss of copy number after the rate of digestion had reached a steady level (commonly after 4 U of enzyme had been used).
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fig3: Quantifying changes in DNase I-sensitivity with qrt-PCR. qrt-PCR was used to detected small changes in DNase I-sensitivity in two reciprocally expressed genes (redD and rlpA) at the earliest and latest time points. In vivo digestions were performed as described in Figure 1; qrt-PCR is described elsewhere (6). DNase I sensitivity was estimated by the percentage loss of copy number after the rate of digestion had reached a steady level (commonly after 4 U of enzyme had been used).

Mentions: The digestion profiles show a relatively rapid loss in copy number before the curves level out (Figure 3). This is consistent with results obtained with eukaryotic chromatin, where the final value represents accessibility of the chromatin, and allows an accurate comparison of the DNase I-sensitivity of each gene. Figure 3 shows the DNase I sensitivity of two of the genes analyzed by Southern blots in Figure 2A, redD and rlpA, at time points T1 and T3. Under limiting conditions of digestion, redD shows 8 and 58% relative copy number loss at T1 and T3, consistent with the late expression of the gene. The earlier expressed rlpA shows the opposite pattern, with 36 and 18% relative copy number loss at T1 and T3, respectively. These data correspond well with the Southern blots shown in Figure 2A.


In vivo DNase I sensitivity of the Streptomyces coelicolor chromosome correlates with gene expression: implications for bacterial chromosome structure.

McArthur M, Bibb M - Nucleic Acids Res. (2006)

Quantifying changes in DNase I-sensitivity with qrt-PCR. qrt-PCR was used to detected small changes in DNase I-sensitivity in two reciprocally expressed genes (redD and rlpA) at the earliest and latest time points. In vivo digestions were performed as described in Figure 1; qrt-PCR is described elsewhere (6). DNase I sensitivity was estimated by the percentage loss of copy number after the rate of digestion had reached a steady level (commonly after 4 U of enzyme had been used).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC1636467&req=5

fig3: Quantifying changes in DNase I-sensitivity with qrt-PCR. qrt-PCR was used to detected small changes in DNase I-sensitivity in two reciprocally expressed genes (redD and rlpA) at the earliest and latest time points. In vivo digestions were performed as described in Figure 1; qrt-PCR is described elsewhere (6). DNase I sensitivity was estimated by the percentage loss of copy number after the rate of digestion had reached a steady level (commonly after 4 U of enzyme had been used).
Mentions: The digestion profiles show a relatively rapid loss in copy number before the curves level out (Figure 3). This is consistent with results obtained with eukaryotic chromatin, where the final value represents accessibility of the chromatin, and allows an accurate comparison of the DNase I-sensitivity of each gene. Figure 3 shows the DNase I sensitivity of two of the genes analyzed by Southern blots in Figure 2A, redD and rlpA, at time points T1 and T3. Under limiting conditions of digestion, redD shows 8 and 58% relative copy number loss at T1 and T3, consistent with the late expression of the gene. The earlier expressed rlpA shows the opposite pattern, with 36 and 18% relative copy number loss at T1 and T3, respectively. These data correspond well with the Southern blots shown in Figure 2A.

Bottom Line: DNase I-sensitivity correlated positively with transcript levels, implying that it was predictive of gene expression, and indicating increased accessibility of transcribed DNA.The genome was fractionated based on the sensitivity to DNase I digestion, with the low molecular weight (frequently cut) fraction highly enriched for actively transcribed sequences when compared to the infrequently cut fraction, which was representative of the entire genome.This approach will allow comparison of nucleoid proteins, and any modifications thereof, associated with transcriptionally active and inactive regions of the bacterial genome.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Microbiology, John Innes Centre, Norwich, NR4 7UH, UK.

ABSTRACT
For a bacterium, Streptomyces coelicolor A3(2) contains a relatively large genome (8.7 Mb) with a complex and adaptive pattern of gene regulation. We discovered a correlation between the physical structure of the S.coelicolor genome and the transcriptional activity of the genes therein. Twelve genes were surveyed throughout 72 h of growth for both in vivo sensitivity to DNase I digestion and levels of transcription. DNase I-sensitivity correlated positively with transcript levels, implying that it was predictive of gene expression, and indicating increased accessibility of transcribed DNA. The genome was fractionated based on the sensitivity to DNase I digestion, with the low molecular weight (frequently cut) fraction highly enriched for actively transcribed sequences when compared to the infrequently cut fraction, which was representative of the entire genome. This approach will allow comparison of nucleoid proteins, and any modifications thereof, associated with transcriptionally active and inactive regions of the bacterial genome.

Show MeSH