Escherichia coli containing a mutation in recF are hypersensitive to UV. However, they exhibit normal levels of conjugational or transductional recombination unless the major pathway (recBC) is defective. This implies that the UV sensitivity of recF mutants is not due to a defect in recombination such as occurs during conjugation or transduction. Here, we show that when replication is disrupted, at least two genes in the recF pathway, recF and recR, are required for the resumption of replication at DNA replication forks, and that in their absence, localized degradation occurs at the replication forks. Our observations support a model in which recF and recR are required to reassemble a replication holoenzyme at the site of a DNA replication fork. These results, when taken together with previous literature, suggest that the UV hypersensitivity of recF cells is due to an inability to resume replication at disrupted replication forks rather than to a defect in recombination. Current biochemical and genetic data on the conditions under which recF-mediated recombination occurs suggest that the recombinational intermediate also may mimic the structure of a disrupted replication fork.
We have developed a novel workflow (sdDE-FACS, s̲ingle d̲roplet D̲ouble E̲mulsion FACS) that allows robust production, screening, and sorting of single double emulsion droplets with complete nucleic acid recovery.
Human T lymphocytes were stimulated using phorbol myristate acetate and ionomycin. Twenty-four hours post-activation the cells were harvested for DNA content and for measurements using a newly developed cell profiling system employing dielectrophoresis. This system provides individual cell size and dielectrophoresis data for statistically relevant numbers of control and activated cells. From this it was determined that the mean membrane specific capacitance decreased from 13.49 (+/- 4.72) mF/m(2) to 10.62 (+/- 5.13) mF/m(2). This can be related to a 21.3% reduction in the effective membrane surface area associated with membrane topography (e.g. reduction of membrane associated microvilli, blebs and folding), or to other changes of membrane architecture, following cell activation. From cytometric determinations of DNA content, it was concluded that these effects were related to a 3.0-fold decrease of cells in S-phase, and a 1.5-fold increase in G1 cells. This work demonstrates the powerful potential of using dielectrophoresis as a noninvasive tool to follow physiological changes that accompany transmembrane signaling events.
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