1980 Nucleic Acids Research, 2006, Vol. 34, No. 7 Figure 4. Mixing experiment providing an estimation of the percentage of mouse liver chromatin that possesses a short repeat. (A) Nuclei from mouse liver (ML) or Matthiola petals (MP) were digested with MNase, and the purified DNA fragments were run on an agarose gel which was stained with ethidium bromide to visualize the nucleosome ladders from total genomic DNA for each sample (lanes ML and MP). The shorter nucleosome repeat (183 ± 5 bp) of the MP chromatin compared to the ML chromatin (195 ± 5 bp) is evident. The DNA from the two chromatin samples was mixed together in the proportions indicated (%MP) and analyzed on the same gel. Nucleosome oli- gomer bands for the MP chromatin are indicated. The brackets denote the upper region of the gel containing the oligomer DNA fragments greater than 5mers. (B) The 20% MP lane is shown adjacent to the ML lane, and the photograph was expanded for comparing the upper region of the gel. The lanes were precisely aligned using the 100 bp ladder markers immediately flanking the gel. (C) Densitometer scan of the ML and 20% MP lanes shown in (B). estimates based upon analysis of 60 Mb of DNA sequence. We observed strong unambiguous signals (deﬁned in Materi- als and Methods), as reported in Table 1, in fewer than 2% of the loci that we analyzed. However, it is likely that signals less strong than those selected here can still be inﬂuential. Our computational analysis further suggests that nucleosome repeats with values as short as 175 bp occur very infrequently in mouse liver; signals for such short repeats were observed in less than 1% of the loci analyzed. In addition to predicting short nucleosome repeats from the DNA sequence, we attempted to deﬁne the upper and lower limits for nucleosome repeats that exist in mouse liver chro- matin. We have never observed a repeat value longer than the bulk chromatin repeat of 195 ± 5 bp. This was true even when the computational prediction was 210 bp (probe 19–395, Table 1). Thus, nucleosome repeat values longer than the bulk value may not exist in mouse liver chromatin. It is inter- esting that probe 19–395 detected an 187 ± 5 bp repeat, a value that is slightly shorter than the bulk repeat. For the 100 kb window centered on this probe, a Fourier peak is present at 370 bp ¼ 2 · 185 bp that meets our stated criterion for a strong signal, despite the presence of the larger peak at 420 bp ¼ 2 · 210 bp. Thus, this 370 bp peak could explain the experi- mentally observed 187 ± 5 bp repeat. The shortest nucleosome repeat that we observed was 172 ± 5 bp (Table 1). This occurred when the computational prediction was 169 bp (Table 1). The observed value of 172 bp in this case was within our experimental uncertainty of ±5 bp of the 169 bp prediction. However, repeats shorter than about 172 bp may not exist in mouse liver chromatin. From our computational analysis of only 60 Mb of mouse DNA we do not yet know whether there is anything in common among the loci possessing short nucleosome repeats. The num- ber of annotated genes in the ten short-repeat loci identiﬁed varied between zero and ﬁve for the 100 kb windows. This variation is not signiﬁcantly different from what would be expected from ten randomly selected 100 kb windows. It would be feasible to analyze the whole mouse genome, after the still signiﬁcant numbers of gaps present are elimi- nated, and to compare the results with those obtained from the human genome analyzed in the same way. It is interesting that the range over which the signal for the formation of a particu- lar nucleosome repeat extends is close to 100 kb in most cases. It is clear from Figure 2 and Table 1 that DNA windows for analysis with sizes less than about 70 kb would not have good predictive power. Moreover, we have found only one example thus far of a phased period-10 VWG signal that was not evi- dent until a window larger than 100 kb was examined. The DNA length of 100 kb could conceivably correspond to some element of large-scale chromatin structure, such as a loop emanating from a scaffold (23–25). It is plausible that 100 kb regions of DNA with distinctive nucleosome repeats also possess distinctive chromatin struc- tures. Recent work has supported non-solenoid-like models for chromatin structure containing straight internucleosomal (lin- ker) DNA segments (10). Computer modeling studies (9) have suggested that such structures are highly sensitive to linker DNA lengths and their degree of uniformity. Therefore, even if chromatin higher-order structures are dynamic in vivo, differ- ent nucleosome arrangements could cause the structures to bend and ﬂex in different ways. Moreover, a genome-wide study of (human) chromatin structure suggested that there is not a simple structural division between heterochromatin and euchromatin, and that there is not a simple correlation between gene expression and chromatin compaction (26). These obser- vations are consistent with the existence of a variety of dif- ferent higher-order chromatin structures, rather than just open or closed chromatin. The DNA sequence could play a role in the formation of these structures. SUPPLEMENTARY DATA Supplementary Data are available at NAR Online. ACKNOWLEDGEMENTS The authors would like to thank Chad Pitschka for assistance with Visual Basic programming/Excel Macros and Andrew Bentz for help in selecting some of the mouse GenBank files suitable for this analysis and Dr Jody Banks for assistance with the plant nuclei preparation. This work was supported by NIH grant GM62857, NIGMS to A.S. The Open Access