1976 Nucleic Acids Research, 2006, Vol. 34, No. 7 400 mM sucrose, 10 mM MgCl2, 50 mM Tris–HCl (pH 8.0), 10 mM NaCl isolation buffer (IB) to a ﬁne pulp, ﬁltered through four layers of cheesecloth, two layers of miracloth. Triton X-100 to 0.1% ﬁnal concentration was added drop-wise while stirring on ice. The ﬁltrate was centrifuged in a Sorvall SS34 rotor at 3000 r.p.m. for 5 min at 4 C. The pellet was re-suspend and washed twice with 40 ml chilled IB. Finally, the cream-colored nuclear pellet was re-suspended in 2 ml of chilled IB. MNase digestion was as above. Southern blots and hybridizations Probes with sizes ranging from 336 to 820 bp located in the center of each 100 kb region were PCR-ampliﬁed from mouse liver genomic DNA. Each probe used is described by the chromosome number, followed by the probe size in base pairs (see Supplementary Data for the DNA sequence of each probe). To purify the PCR-ampliﬁed DNA fragments to be used as probes, the DNA was run on an agarose gel, the gel was stained with ethidium bromide to visualize the band of interest, and the DNA fragment was excised from the gel. The DNA was puriﬁed from the gel slice using the Qiaex II gel puriﬁcation kit (Qiagen). The pure denatured DNA probe fragment (25–50 ng) was labeled with [a-32P]dATP as described previously (17). After stopping the reaction, the labeled DNA was supplemented with about 2 mg of salmon sperm DNA, denatured and chilled on ice. The pre-hybridization and the hybridization solution contained 100 mg/ml denatured salmon sperm DNA. Final probe concentrations in the hybridization buffer were 5 ng/ ml. All pre-hybridizations, hybridizations and washes were performed in a Hyb-Aid oven at 65 C using the membrane manufacturer’s recommendations, except for the high strin- gency washes. The high stringency washes were generally done in 0.1· SSC, 0.1% SDS at 63 C, with a room temperature wash in 0.1· SSC. The blots were exposed to Biomax MR ﬁlm (Kodak) from 1–3 days at 80 C using a Kodak Biomax MS intensifying screen. To assess the speciﬁcity of hybrid- ization, puriﬁed mouse genomic DNA digested with appropri- ate restriction enzymes (usually PstI plus HindIII) were included in lanes labeled D. Nearly half of the probes initially prepared failed to hybridize speciﬁcally and could not be used; some of these could be rescued by cleaving with a restriction enzyme and re-purifying a sub-fragment of the initial PCR product (see Supplementary Data for restriction sites). Nucleosome repeat analysis The midpoint of each of the nucleosome oligomer bands from the 2 min digest was sized based upon the 100 bp ladder, always present in an adjacent lane. The slope of the best-ﬁt straight line in the plot of nucleosome oligomer size versus nucleosome oligomer number gives the nucleosome repeat length (18). Y-intercepts and the standard deviation of the ﬁt, calculated as the square root of the variance of the residu- als, are reported for each ﬁt (Supplementary Data, summarized in Table 1). Negative y-intercepts are expected for most genomic DNA regions at the extent of digestion used because of the exonuclease activity associated with MNase, whereas regions that are resistant to digestion and regions with shorter repeats should have y-intercepts that are near zero. A large positive y-intercept would not be consistent with a regular nucleosome array that contained multiples of a unit repeat. RESULTS Specific loci can possess short nucleosome repeats in mouse liver nuclei The bulk chromatin nucleosome repeat in mouse liver nuclei is 195 ± 5 bp, as assessed by total DNA staining using ethidium bromide (17,19). Early on it was shown that in mouse liver chromatin transcribed regions, rDNA regions and satellite DNA all have the same nucleosome repeat as the bulk chro- matin (19). Thus, the 195 ± 5 bp repeat is representative of most of the chromatin. However, there could be some regions of the genome, possibly having distinctive chromatin struc- tures, which possess nucleosome repeats that differ from the bulk chromatin. A few such regions have been observed in other animal tissues (20–22). In mouse liver nuclei it was recently shown that the ubiquitously expressed adenosine deaminase gene possesses an unusually regular nucleosome array with a repeat length that is about 12 bp shorter than the bulk chromatin value, consistent with the idea that some genomic DNA regions differ from the bulk chromatin (17). To assess whether there are additional genomic DNA regions with perhaps even shorter nucleosome repeats, we attempted to use our computational methods to search 60 Mb of DNA to identify 100 kb regions predicted to have short nucleo- some repeats. After identifying each candidate region com- putationally, we then prepared a hybridization probe (400– 800 bp) by PCR from the center of the 100 kb window, and performed an experiment to test our computational prediction. One such result is shown in Figure 1. Figure 1A (left) shows the FT of the period-10 VWG oscil- lations in a 90 kb window from a region of chromosome 9. For this DNA region, there is no predominant Fourier peak in the physiological dinucleosome region, and therefore no signal for the formation of a nucleosome array. The prediction in this case is that this region of chromatin should possess the bulk chromatin repeat. In contrast, Figure 1A (right) shows the FT (black points/ black spline curve) of the period-10 VWG oscillations in a 100 kb window from a region of chro- mosome 4, which has a large peak in the physiological dinu- cleosome region. The curve was decomposed into Gaussian peaks (blue curves) in and around the physiological dinucleo- some region (from 330 to 400 bp) such that the black points of the Fourier were well-represented by the curve consisting of the sum of the Gaussian peaks (orange circles). The broad apparent Fourier peak at around 385 bp, possessing a shoulder at 370 bp, required two Gaussian peaks to adequately ﬁt it. The predominant Fourier peak at 337 bp has a height of 1174 VWG counts, a standard deviation of 9.0 bp, and is slightly more than twice as large (by area) as the next largest peak area in the physiological dinucleosome region (Table 1). This peak meets all of the criteria that we set (Materials and Methods) for a strong signal for this 100 kb region of DNA, and predicts that the chromatin in this region of the genome should tend to form a nucleosome array with an extremely short repeat (in mouse liver nuclei) of 337 bp/2 ¼ 169 bp. These predictions were tested experimentally. Hybridiza- tion probes were prepared from the center of each 100 kb