39. In an experiment, intact chromatin was isolated and digested with micrococcal nuclease in independent tubes for 30 min, 1 h, 2h, and 4h.
Further, the DNA was purified from each tube, separated on agarose gel and Southern hybridization was performed with rRNA gene probe and a centromeric DNA probe. Which one of the following patterns of signal intensity from both of the probes is likely to be obtained following Southern hybridization?
(1) With increasing time, compared to centromeric probe, a rapid increase in signal intensity of rRNA gene probe was observed.
(2) With increasing time, compared to centromeric probe, a rapid decrease in signal intensity of rRNA gene probe was observed.
(3) Irrespective of incubation period, both probes produced identical band intensities.
(4) Treatment with micrococcal nuclease would instantly degrade the DNA, hence, no hybridization signal would be obtained, in any of the samples.
Introduction
Micrococcal nuclease (MNase) digestion is a powerful technique used to probe chromatin structure by preferentially cutting accessible DNA regions. When intact chromatin is digested with MNase over increasing time intervals, differences in chromatin accessibility between genomic regions become evident. This article explains the expected Southern hybridization patterns of rRNA gene and centromeric DNA probes following MNase digestion, highlighting how chromatin compaction influences nuclease sensitivity.
MNase Digestion and Chromatin Accessibility
MNase preferentially cleaves DNA in the linker regions between nucleosomes and more accessible chromatin. The degree of digestion depends on chromatin compaction:
-
Euchromatin (e.g., rRNA gene regions): Generally less compact, more transcriptionally active, and more accessible to MNase. These regions are digested faster and more extensively.
-
Heterochromatin (e.g., centromeric DNA): Highly compact and transcriptionally silent, making DNA less accessible to MNase. These regions are more resistant to digestion and degrade more slowly.
Expected Southern Hybridization Patterns
In the described experiment, chromatin is digested with MNase for varying durations (30 min, 1 h, 2 h, 4 h), DNA is purified, and Southern hybridization is performed using probes for rRNA genes and centromeric DNA.
Key Predictions:
-
rRNA Gene Probe:
-
rRNA genes are located in relatively open chromatin regions due to active transcription.
-
With increasing digestion time, MNase will progressively digest these regions, leading to a rapid decrease in signal intensity as DNA fragments become too small or degraded to hybridize effectively.
-
-
Centromeric DNA Probe:
-
Centromeric DNA is embedded in heterochromatin, which is more resistant to MNase digestion.
-
The signal intensity from centromeric probes will decrease more slowly compared to rRNA genes, reflecting the compact and protected nature of centromeric chromatin.
-
Why Other Outcomes Are Less Likely
-
A rapid increase in rRNA gene signal intensity with digestion time is unlikely because MNase digestion reduces intact DNA available for hybridization.
-
Identical band intensities for both probes across time points contradict the known differences in chromatin accessibility.
-
Instantaneous DNA degradation by MNase is unrealistic; digestion is progressive and time-dependent.
Conclusion
The expected pattern following MNase digestion and Southern hybridization is that the rRNA gene probe signal decreases rapidly with increasing digestion time, reflecting its presence in accessible euchromatin, whereas the centromeric DNA probe signal decreases more slowly, consistent with its localization in compact heterochromatin. This differential sensitivity to MNase digestion highlights the distinct chromatin environments of these genomic regions.
Answer:
The correct pattern is:
(2) With increasing time, compared to centromeric probe, a rapid decrease in signal intensity of rRNA gene probe was observed.
