event would have occurred under our experimental conditions this would have led to a broadening of the accessible regions and would indicate that, in the absence of such hypothetical proteolytic attack, the accessible regions would be even narrower than those found. The possibility that an endogenous nuclease is already located close to some (the most redundant) cleavage sites cannot be ruled out, but is unlikely. While these points deserve further work, clearly apoptotic degradation opens up a new area of exploration of the most accessible open chromatin structures in the interphase nucleus. In any case the total number of sites, even taking into account those missed because of cloning problems, is in the range of a few hundreds, those of specifically targeted sites (as indicated by redundant clones) being at the lower end of the range. It is quite conceivable that the most accessible open chromatin regions which are repeatedly targeted by apoptotic degradation are different in different cell types, as suggested by preliminary experiments pointing to different rates of degradations in different tissues. In other words, the approach developed in this work should provide an overall view of the most accessible sites in interphase nuclei from different cell types. Methods Autolysis and DNA extraction Samples of tissues and blood were taken from adult chickens and immediately stored in sterile tubes for different times and at different temperatures. Autolysis was stopped by extracting DNA using the Genomix DNA extraction Kit (Talent). After different time lapses (30 min to 24 hours), at different temperatures (4uC, room temperature and 37uC), DNA was extracted, purified and analyzed by electrophoresis on 2% agarose gels, thus generating a nucleosome ladder. In the case of liver autolysis at 4uC, DNA digestion was extremely slow and there was no evidence of a nucleosome ladder even after 24 hours. At room temperature, the ladder started to form after 10 hours and was distinctly evident after 24 hours. At 37uC, the nucleosome ladder began to appear after 1 hour and DNA was hydrolyzed to oligonucleotides after 8 hours. Prelimi- nary results indicated that under the standard condition of 37uC, 1 hr, liver, kidney and lung were faster digested than brain, muscle, heart and pancreas. Erythrocytes were resistant to degradation, even after 24 hours of autolysis at 37uC. In this case a striking resistance to MNase was also evident. Nuclei preparation and MNase digestion Nuclei from the chicken tissues were prepared by a modified Hewish and Burgoyne [3] method as described by Kornberg et al [29]. Nuclei were then digested with MNase, 300 U/ml for 30 sec to 10 min at 37uC, the first time lapse being chosen as the standard procedure. Digestion was stopped by adding 0.5 M EDTA. DNA was extracted as mentioned above. Nucleosome ladder resolution by agarose gel electrophoresis The samples were run in 2% agarose gels and visualized by staining with ethidium bromide. High-performance liquid chromatography (HPLC) analysis LMW DNA fragments obtained by autolysis or MNase digestion were eluted from the gels using QIAquick Gel Extraction Kit and analyzed by HPLC as described by Varriale et al [30]. Apoptotic Attack on Chromatin Cloning and sequencing LMW DNA fragments DNA fragments, as released by either autolysis or MNase digestion, were filled-in by the Klenow fragment enzyme and ligated in PCR-II vector using Topo Cloning blunt-ends Kit (Invitrogen). The recombinant clones were detected by blue/white screening, and sequenced using T7 and M13 reverse universal primers in Applied Biosystems 37300 DNA analyzer. Data analysis The average GC level of the sequences was calculated by a computer program. The UCSC Genome Bionformatics Site contains the reference sequence and working draft assemblies for a large collection of genomes. In the case of the chicken genome, the May 2006 Gallus gallus draft assembly produced by the Genome Sequencing Center at the Washington University School of Medicine in St. Louis, MO, USA (WUSTL) was used. The DNA fragments cloned and their chromosome coordinates were identified by blast searching using URL: http://www.genome. ucsc.edu. The density of clones in the isochore families was calculated, on the basis of their locations and the coordinates of chicken isochores [14], by dividing the number of clones in each isochore family by the size (% of the genome size) of the family. The gene density in isochore families of a set of 12,663 chicken genes (retrieved from Ensembl) and the GC profiles of chicken chromosomes, are those reported by Costantini et al [14]. Supporting Information Figure S1 Clone density on chicken chromosomes. The chromosome location for all clones, for (A) autolysis and MNase digestion (B) respectively, was obtained by blast searching, using the URL: http://www.genome.ucsc.edu. The clone density was calculated as frequency of clones for chromosome / length of chromosome. A higher density of clones was found on micro- chromosomes. Found at: doi:10.1371/journal.pone.0005010.s001 (7.86 MB PDF) Figure S2 Mapping of apoptotic and MNase cuts on chicken chromosomes. The cut location, gene density and the GC level in the isochores (as in the Figure 3) are shown on all chicken choromosomes sequenced. Found at: doi:10.1371/journal.pone.0005010.s002 (0.14 MB PDF) Figure S3 Terminal dinucleotides analysis. Frequencies of the terminal dinucleotides of the autolytic sequences cloned after autolysis (A) and MNase digestion (C). The contribution of the clones (blue bars) and of redundant clones (white bars) in the dinucleotides were plotted together for each dinucleotide. All pairs of terminal dinucleotides after autolysis (B) and MNase digestion (D) are classified in the three possible groups: purine/purine (pur/ pur), pyrimidine/pyrimidine (pyr/pyr), and purine/pyrimidine or viceversa (pyr/pur). Found at: doi:10.1371/journal.pone.0005010.s003 (0.69 MB TIF) Table S1 Found at: doi:10.1371/journal.pone.0005010.s004 (0.36 DOC) Table S2 Found at: doi:10.1371/journal.pone.0005010.s005 (0.04 DOC) Table S3 Found at: doi:10.1371/journal.pone.0005010.s006 (0.04 DOC) MB MB MB PLoS ONE | www.plosone.org 6 April 2009 | Volume 4 | Issue 4 | e5010