Chromosomes in the cells of higher organisms consist of DNA-protein complexes, referred to as chromatin. A mass of histone proteins approximately equal to the mass of DNA is largely responsible for the high degree of compaction of the very long DNA molecule in a chromosome. About 150 bp of DNA is tightly wrapped around an octamer consisting of two copies each of the core histones H2A, H2B, H3, and H4 forming the nucleosome, the fundamental building block of chromatin. Nucleosomes are separated from each other by approximately 45 bp of linker DNA, which is associated with the linker histone H1. This "string of beads" or nucleosome array is further condensed into an irregular approx 30-nm fiber to form interphase chromatin, in which the overall level of DNA compaction is about 40-fold (1). The irregularities and the variability among chromatin fibers viewed by cryoelectron microscopy (2,3) or scanning force microscopy (4,5) are considerable, with some fibers appearing significantly more condensed than others. Moreover, some fibers

From: Methods in Molecular Biology, vol. 338: Gene Mapping, Discovery, and Expression: Methods and Protocols Edited by: M. Bina © Humana Press Inc., Totowa, NJ

exhibit abrupt bends, and others possess loops that bring distal regions of the DNA molecule into close proximity. The possibility exists that such variations might not simply arise from the random coiling of the samples prepared for microscopy, rather, different regions of DNA might tend to form different chromatin higher order structures, and these structural differences might have functional significance (6,7).

Variations also exist among the nucleosome arrangements in different regions of DNA (6,8). Some regions of DNA have more ordered nucleosome arrangements than others, and the value of the nucleosome repeat length often differs from that of the bulk chromatin, sometimes over large DNA regions (9). In the very plausible models of chromatin higher order structure in which the linker DNA is straight (10-12), small changes in linker DNA length can generate large differences in the appearance of the chromatin fiber (2). This sensitivity is a consequence of the structural fact that the histone-DNA contacts in the nucleosome occur across the minor groove of DNA (13), which rotates 360 degrees every 10 bp for straight DNA. Thus, for each base pair change in the length of linker DNA between nucleosomes, the plane of one nucleosome should be expected to rotate by an angle of 36 degrees with respect to the other. Because all the nucleosomes in an array are connected, this effect will propagate, unless constrained, throughout the chain of nucleosomes, with each linker DNA variation contributing, and could thus generate many different higher order structures. Therefore, it is of interest to be able to specifically probe different regions of DNA to assess the nucleosome arrangement in a particular region. Recent work strongly suggests that computationally recognizable longrange periodic sequence motifs in genomic DNA are responsible for the variations in nucleosome arrangements that have been observed (9).

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