by Fourier analysis, corresponded almost exactly to a value that was equal to twice the measured nucleosome repeat in all cases studied. Moreover, DNA regions that did not possess a single strong Fourier peak did not order nucleosomes into regular arrays in vitro. These observations suggested the hypothesis that nucleosome ordering by linker histones might be facilitated by a dinucleosome period signal consist- ing of regular period-10 VWG oscillations. It is not necessary for each nucleosome in an array to have its own positioning signal (8). Linker histone addition to nucleosomes can readily align one nucleosome with respect to another in the absence of signals in the DNA (15). We obtained further support for this hypothesis by making small alterations in the chicken ovalbumin gene sequence which affected nucleosome array formation in vitro in a computationally predictable way (16). We also showed that this oscillating signal appears to work because nucleosomes tend to avoid the DNA regions that have low counts of period-10 VWG; presumably they are less ﬂexi- ble than regions of DNA with high counts. Recently, we have suggested that it might be possible to extend our computational approach to the chromatin in animal tissues if the period-10 VWG oscillations are assessed over a 70–100 kb range (17). Here, we provide evidence for the ﬁrst time that it is possible to predict computationally, from the DNA sequence, loci that possess distinctive nucleosome arrays in mouse liver nuclei. MATERIALS AND METHODS Computational analysis Sequences were analyzed for long-range periodic oscillations in period-10 VWG content as described previously (14). Brieﬂy, the occurrences of the motif VWG/CWB (comple- ment) with a periodicity from 10.00 to 10.33 were counted in a sliding 102 bp window, ±51 bp from each VWG position. These histogram data were then averaged in a sliding 60 bp window (5 bp increments) to generate a continuous oscillat- ing curve of the average period-10 VWG count versus GenBank nucleotide number. The total number of VWG/ CWB occurrences in a sliding 600 bp window was also com- puted, and used to apply a small correction for the presence of VWG-poor or VWG-rich regions, as described previously. The extent of regularity and the period of the long-range period-10 VWG oscillations were assessed by Fourier analysis using a 100 kb window, unless otherwise stated. Approxi- mately 60 Mb of mouse genomic DNA (NCBI Build 35) from all chromosomes except Y was analyzed to obtain ten 100 kb regions predicted to have strong signals for form- ing a nucleosome array with a short repeat. These sequences are obtainable by ‘BLASTing’ the reported probe sequences (Supplementary Data), on which they are centered, against the mouse genome. This method, rather than reporting accession numbers, is independent of the NCBI build, which keeps changing over time. A short repeat is operationally deﬁned here as one having a value between 168 and 180 bp, at least 15 bp shorter than the bulk repeat in mouse liver (195 ± 5 bp). The very short yeast-like repeat value of 168 bp is theoretically possible, but may not exist in mouse liver chromatin, whereas the repeat value of 180 bp is closer to the ubiquitous bulk chromatin repeat value, but still readily distinguishable experimentally. The Fourier transform (FT) of the oscillating Nucleic Acids Research, 2006, Vol. 34, No. 7 1975 curve of period-10 VWG counts versus nucleotide number was decomposed into Gaussian peaks within and around the physiological dinucleosome region using the NORMDIST probability mass function of Excel. The mean, peak height and standard deviation of each peak was adjusted by trial and error until the sum of the peaks closely ﬁt the FT curve deﬁned by the computed points, rather than the Excel spline curve through the points. A strong signal is operationally deﬁned as one possessing a nearly symmetrical (Gaussian) Fourier Amplitude peak in the physiological dinucleosome period range (taken to be from 2 · 165 bp to 2 · 200 bp) with a standard deviation between 7.0 and 17 bp, a height (estimated ±5%) that is at least 800 VWG counts for the 100 kb window used, and a peak area that is at least 2.0 times the area of the next largest peak in the physiological dinucleosome region. For peaks possessing mean period values that are outside of the physiological dinucleosome region, only the area extending into the physiological region is considered. We also required that the signal was stable with respect to small variations in window size (±10 kb). The above-stated criteria for a strong signal are consistent with what we initially found for the mouse adenosine deaminase gene (17), and it eliminates pos- sible Fourier peaks that are sharp spikes, peaks that have low amplitudes or are very broad, and peaks that are unstable with respect to varying the window size. It does not imply that peaks that do not possess all of the characteristics deﬁned above cannot still be inﬂuential. We hypothesize, based on our previous study (17), that the presence of a strong signal in a particular DNA region should result in the formation of an extended nucleosome array in this region with a repeat value that is one-half the value of the dinucleosome period of the signal peak. In the much more common case where no appre- ciable signal in the DNA sequence exists, we hypothesize that the nucleosome arrays in these regions should possess the bulk chromatin repeat. Preparation of nuclei, micrococcal nuclease digestion and electrophoresis Nucleosome arrays in native chromatin were assessed by partial micrococcal nuclease (MNase) digestion of the DNA in the chromatin of mouse liver nuclei. Nuclei were prepared from mouse (strain C57BL/6J, the NCBI reference sequence) liver as described previously (17). Nuclei containing about 1 mg of DNA were gently pelleted and re-suspended in 1 ml of 0.1 M NaCl, 10 mM Tris–HCl (pH 8.0), 1 mM EDTA. After equilibration for 5 min at 37 C, 0.1 M CaCl2 was added to a ﬁnal concentration of 2 mM, then 30 U of MNase were added, and the sample was digested for 1.5 or 2 min. After deproteinization, the nucleic acid was treated with RNase A, and prepared for electrophoresis. The same DNA digests were repeatedly run on a 1.5% agarose bridge gel apparatus of dimensions 13.5 by 13.5 cm in TBE buffer for about 4 h, blotted, and the different DNA regions of interest were detected by Southern hybridization. Lambda DNA cut with AﬂIII (ascending band values in bp: 170, 277, 458, 493, 739, 956, 1268, 1399, 1520, 1712, 1913, 2360, 2691, 4091, 4920, 5733, 6236, 6631) and, in a separate lane, a 100 bp ladder (BioRad), each labeled with [a-32P]dATP, were used as size markers. Matthiola incana petals (two ﬂower stalks) were minced in a Virtis S 45 homogenizer in 50 ml chilled