3060 Nucleic Acids Research, 2006, Vol. 34, No. 10 never been observed in random DNA or in the genomes of non-nematode species. Of sequences in the W4 fragment 89.7% have PATC scores above 95 (the cutoff for 10 6 occurrence in random DNA). Several other fragments have also been analyzed in this work. Fragment W6 is a strongly periodic 147 bp segment comprising the third intron of C.elegans gene ZK792.7 on chromosome IV. The gene is predicted to encode a protein with metallophosphoesterase activity. The periodicity value of W6 is 122 with 76.9% of the sequences with PATC scores above 95. Fragment W6 was originally isolated among a set Table 1. Relevant DNAs used in this study DNA (size) Fragment W4 (932 bp) Fragment W6 (147 bp) Fragment W7 (150 bp) Control Pgem-3Z (2743 bp) Gene in C.elegans F54C4.1 ZK792.7 Y38F1A.3 0.93, (0%) — 3.21, (0%) Periodicity value Ave, AFM sample (Frac>95 cutoff) (size) 433, (89.7%) 122, (76.9%) PPC (2885 bp) SQ322 (182 bp) SQ100 (185 bp) Control (2743 bp) Periodicity values were calculated using the algorithm described in Ref. (19). of random clones of Microccocal nuclease treated C.elegans lysates, consistent with the possibility that this fragment rep- resents a single nucleosome core (25). We stress, however, that no further in vivo analysis has been carried out on this segment of the chromosome. As controls, we have examined the behavior of several DNAs which had no unusual periodic character. These include lambda DNA, pUC plasmid backbones and W7, a dis- tinct C.elegans genome fragment. Fragment W7 was 150 bp and was also derived from Micrococcal nuclease digestion of C.elegans lysates, but did not show distinct periodicity. W7 is an exonic fragment from gene Y38F1A.3. Table 1 summarizes the DNAs employed in this study including the origin of the DNA, the periodicity value and the derived AFM construct. From the periodicities above one would expect unusually bent DNA structures for the fragments W4 and W6, but not for W7. We tested this prediction by gel electrophoresis, which exactly conﬁrmed the expectations (Figure 2). Each of the hyperperiodic fragments (W4 and W6) was retarded compared to size markers, with retardation relieved at higher temperatures and with the addition of an intercalating agent (ethidium bromide) during electrophoresis. In contrast, the non-periodic fragment W7 did not show any retardation. Interestingly, our observations were made using agarose gel electrophoresis, which has not been seen to distinguish Figure 2. Migration of hyperperiodic and control DNA in agarose gels. (a) Two-dimensional 4% NuSieve agarose gel electrophoresis of lambda DNA fragments (cut with NciI and PvuII) and (b) lambda fragments plus W4 fragments cut with the same restriction enzymes. Gels were run first at 4 C in the absence of intercalator (Ethidium Bromide, vertical) and then at 37 C in the presence of intercalator in the perpendicular direction (horizontal). The hyperperiodic W4 fragments (indicated by arrows) migrated more slowly than one would expect on the basis of its length. Similar anomalies were observed when using 2% agarose gels (data not shown). Gel electrophoresis on fragments SQ100 (185 bp, non-periodic, lane 3) and SQ322 (182 bp, hyperperiodic, lane 4) run at 37 C with EtBr (c) and at 4 C without EtBr (d). Lanes 1, 2, 5 and 6 correspond to 100 and 25 bp DNA ladders. At low temperature, the hyperperiodic fragment migrates more slowly than expected.