Apoptotic Attack on Chromatin Figure 1. Nucleosome ladder and compositional distribution of low molecular weight (LMW) DNA fragments. (A) Chicken liver was cut into small pieces and immediately put in sterile tubes for 1 hr at 37uC (AU). Chicken liver nuclei were digested with 300 U of MNase for 30 sec. at 37uC (MN). DNA was purified from both preparations and run on 2% agarose gel producing a ‘‘nucleosome ladder’’. The marker Smart Ladder Eurogentec is also shown (M). Compositional distribution of chicken DNA and of low molecular weight (LMW) DNA fragments after autolysis (B) or MNase digestion (C). The GC level of the LMW DNA fragments (average length ,200 bp, red line) was superimposed on the GC profile of the chicken genome (blue line), as assessed at a window size of 200 bp. The frequency of the segments (61026) was indicated on the left and right ordinates for the chicken genome and the LMW DNA fragments respectively. doi:10.1371/journal.pone.0005010.g001 cloned fragments in 1% GC bins was superimposed on the GC profile of total chicken genome obtained using a window size of 256 base pairs (Figure 1B), the clones were shown to be enriched in GC-rich sequences. In fact, the clones covered a range of 30% to 75% GC, indicating that a small number of very GC-rich fragments could still be cloned in spite of the bias mentioned above. A similar result, with a slightly less pronounced shift, was found with 245 cloned MNase fragments in the 200 bp range (Figure 1C). Localization of the clones in chicken isochores We localized all the sequences cloned from both apoptosis and MNase degradation on chicken chromosomes. This localization obviously could not be done for the clones (representing about 20% of all clones) that corresponded to unassembled scaffolds, unsequenced microchromosomes and repeats. This reduced the number of localized clones to 317 and 193 for apoptotic and micrococcal degradation, respectively. Taking into consideration that a number of clones from each degradation coincided in chromosome location, the numbers of ‘‘non-redundant’’ clones were 200 and 165 for the two degradations. The coordinates of these clones on chromosomes are presented in Table S1. The full maps of both series of cuts on chicken chromosomes are displayed on 24 panels (Figure S1) along with the GC profiles of chicken chromosomes and the corresponding isochore maps. The clone density in the isochore families as defined by Costantini et al [14] was found to show a similar trend in both degradations, in that it increased with increasing GC level, to decrease in the very GC-rich family H3 (Figure 2A and 2B). The Figure 2. Density of apoptotic and MNase LMW cloned DNA fragments in isochore families. The bottom panel shows the amount of DNA in each isochore family of the chicken genome. doi:10.1371/journal.pone.0005010.g002 latter result is, in all likelihood, due to the loss of some DNA fragments from the isochore family H3, because of the cloning bias against GC-rich fragments and of the unavailability of sequences from some microchromosomes (and also from macrochromosome W). As for the first reason, it should be pointed out that, in spite of the cloning bias, clones higher than 60% GC were obtained from both degradations, as already mentioned (see Figure 1B and 1C). As far as the second reason is concerned, it should be stressed that microchromosomes are GC-richer than macrochromosomes [15] and that cloned fragments have a higher density in the former compared to the latter (see Figure S2). Identification of the sequences The maps of apoptotic and MNase cuts provided several interesting results: (i) In both degradations a preference was observed for genes, as well as narrow (,50 kb) and wide (50– 100 kb) chromosomal surroundings of genes; this preference concerned about 60% of all cleavage sites (see Table 1); another Table 1. Sequences cleaved by autolysis and MNase. APOPTOSIS MNASE GENES+SURROUNDINGS Genes ,50 kb from genes .50 kb ,100 kb from genes INTERGENIC (.100 kb FROM GENES) REPEATS CR1 GGXHOI family satellite GGL ERV LTR-repeats Mariner1b_GG family other repeats doi:10.1371/journal.pone.0005010.t001 % 60.3 39.2 16.5 4.6 25.7 14.3 8.6 1.1 0.5 1.3 2.8 % 61.6 32.6 18 11 18.8 19.5 4.9 8.2 2.5 1.6 2.3 PLoS ONE | www.plosone.org 2 April 2009 | Volume 4 | Issue 4 | e5010