Replication cell-free conditions

The cyclin-dependent kinase inhibitor p21 is another downstream effector of p53 [161-163], There is evidence for p53-independent induction of p21 [162], and under these conditions the protein may be responsible for cisplatin-induced apoptosis [210][211], The p21 protein usually plays a protective role in response to cisplatin [212] [213], however, an effect which correlated with enhanced repair of a damaged reporter plasmid [212-214], These observations are consistent with the hypothesis that DNA-damage induced p53 activates a G, cell cycle arrest through p21, affording the cell time to repair the lesions and precluding the genetic instability produced by replication of damaged DNA. In accord with this model, the addition of p21 to cell free extracts blocked DNA replication but not excision repair... 

The replication complex can be isolated with the particulate fraction of the cytoplasm of picornavirus-infected cells (71) Under proper conditions the ECs are able to continue vitro the synthesis of ENA, providing a unique tool to study the mechanism of ENA synthesis with all the advantages (and all the limitations, too) of an m vitro cell-free system. 

Oxidative damage that leads to replication fork arrest is common during bacterial growth and probably occurs at least once during each cell division even under the most favorable conditions (48, 51). Most lesions are not dealt with by mutageiuc polymerases, but instead they are repaired by recombination, which is an error-free process. Recombinational repair can take many different paths and employs several different proteins, but ultimately, it involves high-fidelity DNA polymerases such as Pol I, Pol II, and/or Pol III (6, 49, 56). For additional information, we refer the reader to more thorough reviews of the various recombination repair pathways (44, 48-50,70). 

Cell growth during the time of the assay might influence the results, in the sense that the filling of the artificial wound could be contributed from both cell replication and movement. In order to avoid this problem, the assay should be conducted in serum-free or at least low-serum conditions, to limit cell growth. Alternatively, inhibitors of DNA synthesis (mitomycin-C or aphidicolin at 0.5 /Lig/ml), can be included in the culture medium, since it has been shown that active migration may depend on protein, but not DNA synthesis (Geimer and Bade, 1991 Chen et al., 1994). 

In any case, working with complete virus particles requires some caution with respect to an evaluation of results. The observable amplification of many viruses appears in the form of single bursts defining infectious cycles. In each burst, however, many copies of virus particles are set free, of which only a minor fraction turns out to be infectious. Within a replication cycle inside the host cell the replication machinery is available to both viable and nonviable (or less efficiently) replicating virions, while after the burst only those particles will survive that have encoded the correct machinery for further infection. Moreover, one may start under conditions where single infections (i.e., one... 

A wide variety of environmental, chemical and physiological conditions affect the structure of DNA and its repair mechanisms. A potent environmental agent to affect the DNA, its replication and repair in the eye is UV radiation. It has also been described that UV radiation provokes peroxidation of membrane lipids which themselves can provoke DNA damage as welF . To differentiate between DNA damage directly induced by UV-B and damage caused by UV-B induced oxgen free radicals it appeared necessary to quantify first the overall effects of UV-B on DNA. However, so far nobody had document the extent of overall UV-B induced DNA damage in relation to the effects on lens epithelial cell proliferation. 

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