Apoptotic Attack on Chromatin Figure 4. Map distances (as % of total distances) among clones from autolysis and MNase. Histogram are presented in Mb bins (top yellow segments refer to redundant clones; inserts refer to distances lower than 500 kb). doi:10.1371/journal.pone.0005010.g004 In the case of MNase digestion a sequence specificity was found for TG/CA dinucleotides, namely for purine/pyrimidine dinucle- otides (Figure S3B and S3D). The analysis of the next nucleotides also indicated a specificity for the palindromic trinucleotides Table 2. Number of clones, redundancies, sites, and minimal distances between the location of the clones derived from apoptotic or MNase degradation and between the two kinds of cuts. Autolysis MNase Autolysis/Mnase Site redundant clones 117 (36.9%) 28 (14.5%) Number of Clones Sites Distances, Mb ,4 Mb Relative amounts (%) ,4 Mb ,0.01 Mb 317 200 4.6 1.8 65.4 1.7 doi:10.1371/journal.pone.0005010.t002 193 165 5.7 1.5 47.6 2.8 2.9 1.5 77.6 6.5 TGC/GCA (see Table S2). These results confirm and extend the previous findings that MNase cleaves DNA preferentially in (A– T)n regions (n$1) flanked by a 59C or G [17–21]. Interestingly, in both apoptotic and MNase degradation the doublets TG and CA were the most represented in terminal positions. In the apoptotic case other dinucleotides were, however, more abundant than in the MNase degradation. Since the larger distances could be, in the case of MNase, due to the fact that the number of non-redundant clones was lower (165 vs 200; see Table 2), 165 randomly chosen apoptotic clones were investigated and distances were found to be comparable to those of MNase clones (5.4 Mb). Discussion Apoptosis via autolysis Apoptosis is usually induced in cell cultures by chemical or physical agents, such as X-rays, UV-light, bleomycin, ectoposide, dimethylsulphoxide (DMSO). In our work, apoptosis was simply induced by leaving tissues in sterile tubes, an entirely new way of inducing apoptosis. In this case, the most likely trigger that activates programmed cell death is deprivation of oxygen. Indeed, the time after which the nucleosomal ladder is already visible (309) is too short for other causes (e.g. lack of nutrients or of growth factors) to be responsible for it. Since this induction of apoptosis is PLoS ONE | www.plosone.org 4 April 2009 | Volume 4 | Issue 4 | e5010