DNA loop domain organization as revealed by single-cell gel electrophoresis

Afanasieva, K. S.; Chopei, Marianna; Zazhytska, Marianna; Vikhreva, M. A.; Sivolob, Andrei

doi:10.1016/j.bbamcr.2013.09.021

Cited by 21 publications

(69 citation statements)

References 43 publications

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“…As we have shown earlier , for lysed cells the kinetic plot of the average relative amount of DNA in the comet tails has a two‐step shape (Fig. C).…”

Section: Resultssupporting

confidence: 68%

“…This reasoning was supported in our previous works focused on physical mechanisms of the comet formation . Applying a kinetic approach we have shown that several factors influence the rate of the exit of DNA loops into the tail at neutral pH (only the neutral variant of the comet assay will be considered here; the peculiarities of the DNA exit in alkaline conditions were reported in ).…”

Section: Introductionsupporting

confidence: 62%

“…In any case, an attachment of the loop ends to nucleoid proteins is a prerequisite for the loops to be able to exit: since the tail is formed by the loops with the contour length that is not higher than ∼200 kb (larger loops apparently cannot move in the agarose gel), the loop detachment should decrease the number of loops that can migrate into the tail. A perturbation of the attaching interactions by high intercalator concentrations or proteins denaturation (in presence of 8 M urea or 0.3% SDS) essentially decreased the saturation amount of DNA in the tails that can be reached after long time of electrophoresis . This observation shows that an additional cell treatment during lysis procedure may induce a redistribution of the loop sizes and hence influence the results of the comet assay.…”

Section: Introductionmentioning

confidence: 80%

“…All the procedures of slide preparation, electrophoresis, and microscopic analysis were described earlier . Briefly, cells or nuclei were embedded in the 0.67% agarose gel (low‐melting point agarose; Sigma) on the surface of a microscope slide.…”

Section: Methodsmentioning

confidence: 99%

“…Kinetic plots (the relative amount of DNA in the tail f vs. electrophoresis time t ) obtained for the nuclei‐derived nucleoids were fitted with the standard equation of monomolecular kinetics:

f = A (1 - e x p (k t)),

where A is the maximum relative amount of DNA that can exit, k is the rate constant. The two‐step plots obtained for cell‐derived nucleoids were fitted as described with the equation:

f = A_{1} (1 - e x p (k_{1} t)) + A_{2} / ([], 1 + e x p ((), k_{2} ((), t_{0} - t))),

where A 1 and A 2 are the maximum amplitudes of two components, k 1 and k 2 are the rate constants, and t 0 is the transition half‐time. The first term in Eq.…”

Section: Methodsmentioning

confidence: 99%

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Single nucleus versus single‐cell gel electrophoresis: Kinetics of DNA track formation

2015

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Single-cell gel electrophoresis, or the comet assay, is usually performed with nucleoids prepared after a lysis of either whole cells (more often) or isolated cell nuclei (rarely). Electrophoretic properties of the second type of nucleoids have never been investigated carefully. We measured the kinetics of the DNA exit from nuclei-derived nucleoids in comparison with cell-derived nucleoids. The results show that general organization of the nuclei-derived nucleoids is not changed very much in comparison with nucleoids commonly obtained from whole cells. At the same time, in contrast to the cell-derived nucleoids, for which the exit is stepwise and cooperative, the DNA exit from the nuclei-derived nucleoids can be described by a simple monomolecular kinetics. This difference is probably due to agarose penetration into nuclei (but not into cells) before polymerization of the agarose gel. We suggest that single-nucleus gel electrophoresis may be a way for the comet assay standardization.

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“…As we have shown earlier , for lysed cells the kinetic plot of the average relative amount of DNA in the comet tails has a two‐step shape (Fig. C).…”

Section: Resultssupporting

confidence: 68%

Section: Introductionsupporting

confidence: 62%

Section: Introductionmentioning

confidence: 80%

Section: Methodsmentioning

confidence: 99%

f = A (1 - e x p (k t)),

where A is the maximum relative amount of DNA that can exit, k is the rate constant. The two‐step plots obtained for cell‐derived nucleoids were fitted as described with the equation:

f = A_{1} (1 - e x p (k_{1} t)) + A_{2} / ([], 1 + e x p ((), k_{2} ((), t_{0} - t))),

where A 1 and A 2 are the maximum amplitudes of two components, k 1 and k 2 are the rate constants, and t 0 is the transition half‐time. The first term in Eq.…”