Bacterial chromosomes are condensed about 1,000-fold, in part by DNA gyrase which introduces negative supercoils, so that they fit inside the cell. Supercoiled DNA is arranged into domains and stabilized by proteins and possibly RNAs, so that one strand break doesn't unwind the whole chromosome, which would kill the cell.
By inhibiting DNA gyrase, more than 300 supercoiling-sensitive genes (SSGs) were identified in E. coli. Postow et al. realized that monitoring the expression of the SSG gene-set might allow chromosome domain size to be accurately delineated. They used an inducible restriction enzyme to randomly cut the chromosome, then monitored levels of SSG transcripts using microarrays. The distance from random cut sites to the domain boundary will affect the extent of local unwinding (relaxation) of the supercoils, so that the level of an SSG transcript will only change if the cut site is in the same domain as the SSG. Differences in SSG transcript abundance were detected well before the chromosome was completely degraded—proving that the method works. Expression of individual SSGs was quantitated using RNase protection. The results were compared with Monte Carlo simulations of different models of domain organization and closely matched a model in which the chromosome is packed into randomly placed domains with an average size of 9—11 kb. Gentle chromosome preparation and analysis with electron microscopy confirmed these experimental observations—domains are much smaller than was previously thought.
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