3058 Nucleic Acids Research, 2006, Vol. 34, No. 10 several hundred nucleotides (18,19). In the Caenorhabditis elegans genome, a remarkable periodicity of 10 bp AA/TT dinucleotides has been described elsewhere (20–24). One proposed role for such sequences has been to limit the capability of nucleosomes to slide into the kind of ordered arrays needed for heterochromatin formation (19). Although such long DNAs can be readily modeled using the extensive data available for shorter bent segments, it is by no means clear that such models continue to provide an accurate approximation as the length of the Periodic An/Tn Cluster (PATC) increases. At extremes of possibility, it could be imagined that such DNAs would form complex coils and knots in solution or, alternatively, might behave as relat- ively straight segments perhaps with unusual microscopic ﬂexibility properties. In order to understand the genesis and role for extended PATCs in eukaryotic genomes, we ﬁrst need to deﬁne their physical properties. Genomes for C.elegans and other nemat- ode species have been shown to exhibit an unusual high fre- quency of extended PATCs (20,23,24), particularly in regions of the genome that are transcribed in germline tissues. The C.elegans genome has the additional advantage that it is among the most extensively characterized both in structure and function, since the sequence is known and individual genetic studies, genome expression and phenotype analysis have been done. In this work, we have used gel electrophoresis and AFM to carry out an extensive analysis of several extended PATCs from the genome of the nematode C.elegans. We ﬁnd that the hyperperiodic DNA shows a signiﬁcant and readily discernible degree of bending compared with non-periodic DNAs, adopting correlated angles over long distances, and that increased ﬂexibility alone cannot explain the mechanical properties of this DNA. MATERIALS AND METHODS AFM constructs For AFM imaging, we fabricated a pseudo-palindrome construct of 2885 bp using a method similar to that described in Ref. (14). This construct contained two segments of 923 bp of hyperperiodic W4 fragments from C.elegans in an inverse orientation each located at the ends of the construct (for description of the fragments of C.elegans used, see Results). We refer to this construct as the periodic palindrome con- struct (PPC). Our AFM analysis focused on the ﬁrst 953 bp at both ends, which included the hyperperiodic sequences. We analyzed a fragment of DNA slightly longer than the pure hyperperiodic fragment because the very ends were not considered in the analysis. The extra non-periodic 979 bp, located in the middle part of the construct, was chosen to be non-palindromic, although it was also part of the C.elegans genome. In doing so, we avoided the synthesis of a real palindromic construct, which could generate two inde- pendent double-stranded DNA molecules of length equal to half of the original one. The scheme of the PPC, including the sequence of one of the hyperperiodic DNA fragments (capital letters), is shown in Figure 1a. PPC fabrication and long-term stability was veriﬁed using gel electrophoresis. We also analyzed two short DNAs, which contained frag- ments W6 and W7 (for description of these fragments see Res- ults). Fragments W7 and W6 were ampliﬁed by cloning into the pCR4Blunt-TOPO vector (Promega), yielding two short DNAs (SQ100 and SQ322) that were analyzed with gel elec- trophoresis and AFM. SQ100 (185 bp) contained the non-periodic fragment W7 (Figure 1b) and SQ322 (182 bp) contained the hyperperiodic fragment W6 (Figure 1c). Capital letters in Figure 1b and c denote the W7 and W6 sequence components. Sequences are speciﬁed excluding the single- stranded overhangs. All sequences in Figure 1 are displayed in a 10 bp arrange- ment to facilitate visualization of same-residue coincidence every helical turn. To assist this, two or more consecutive Ts are colored in red and two or more consecutive As in green. The sequences in Figure 1a and c, corresponding to the hyperperiodic fragment in the PPC and the SQ322 con- struct fragment, respectively, demonstrate a propensity for an AA/TT to be present every 10 bp. In contrast, the degree of periodicity in SQ100 (Figure 1b) is negligible. Gel electrophoresis Both 1D and 2D electrophoresis techniques were used to identify anomalous migration behavior of fragments of DNA from C.elegans. 2D gels were cast in 4% NuSieve agar- ose gels using 1· TAE Buffer in the absence of ethidium bromide. For DNA samples run with an internal standard, it was important to avoid overloading of DNA and consequent smearing of the ﬁrst dimension during electrophoresis; this was achieved by constructing single-tooth combs that were relatively wide (2.5–5 mm) rather than the narrower combs used in other 2D gel systems. The ﬁrst ‘vertical’ direction in all 2D gels was run at 53 V for 3 h in a cold room at 4 C. At this voltage, heating by the electrophoretic current was observed to be minimal. Each gel was then soaked for 2 h in 1· TAE Buffer with ethidium bromide (0.3 mg/ml) at room temperature. Next, the second dimension (horizontal direction in the 2D gels) was run with the gel box at room temperature at 80 V for 2 h. Temperatures are somewhat raised by the higher voltage of the second dimension electro- phoresis, and were estimated to be 37 C. Similar analysis using 1D electrophoresis was carried out by running two separate gels on each sample. One gel was cast with 0.3 mg/ml ethidium bromide present and run at 60 V for 6 h at room temperature, while the other was cast with no intercalator present and run at 30 V for 15 h in a cold room at 4 C and then stained with ethidium bromide (2.0 mg/ml) in 1· TAE for 1 h. Although the two gel systems are comparable in resolution, the paired 1D gels offer the ability to analyze greater numbers of samples, while the 2D gels allow more sensitive detection of modest electrophoretic anomalies. Sample preparation and AFM imaging DNA samples for AFM imaging were prepared by depositing 5 ng of DNA, diluted in 6 ml of 10 mM Tris–HCl (pH 8.0) and then supplemented with 15 mM MgCl2, onto freshly cleaved mica. After 60 s, the mica was washed with MilliQ- ﬁltered water and blown dry in a gentle stream of nitrogen gas. Samples were imaged at 1.95 nm per pixel resolution