Nucleic Acids Research, 2006, Vol. 34, No. 10 3059 Figure 1. Sequences of DNA used in the AFM experiments. (a) Schematic representation of the PPC (2885) and sequence of the region analyzed (953 bp) that contains the hyperperiodic fragment W4 from C.elegans (923 bp). In the PPC the hyperperiodic region is located at both ends of the molecule, hence tracing of the molecule is independent of the particular end chosen as a starting point. Capital case letters denote the hyperperiodic fragment W4 from C.elegans. (b) Sequence of SQ100 fragment (185 bp, non-periodic) that contains the fragment W7 and (c) sequence of SQ322 (182 bp, hyperperiodic) that contains the fragment W6 from C.elegans. Two or more consecutive As or Ts were colored in green and red, respectively. The sequences were plotted in columns of 10 bases to highlight 10 bp periodicities. Capital letters in (a) denote the hyperperiodic fragment W4 from C.elegans and in (b and c) denote the W7 and W6 fragments, respectively. in air at room temperature and humidity using tapping mode. We used an AFM from Nanotec (Nanotec Electro´ nica, Mad- rid, Spain) and also a Nanoscope IIIa (Digital Instruments, Santa Barbara, CA). We utilized tapping mode SuperSharp- Silicon tips, type SSS-NCH-8 (Nanosensors, Neuchaˆtel, Swit- zerland). Image processing, including plane subtraction and ﬂattening, was done using WSxM freeware (www.nanotec. es). Traces were obtained using the tracing routine described in (P.A. Wiggins, T. van der Heijden, F. Moreno-Herrero, A. Spakowitz, R. Phillips, J. Widom, C. Dekker and P.C. Nelson, manuscript submitted). Calculation of periodicity value and predictions of curvature Calculations of predicted radius of curvature for DNA seg- ments and of overall periodicity used several available algorithms (5,7,19) (see also Supplementary Data). Source code for local analysis and display of these parameters is available on request. RESULTS Periodic DNA segments from C.elegans We chose a number of hyperperiodic DNA fragments from C.elegans for analysis based on the relevance of the proteins that they encode and on the periodicity value provided by the PATC algorithm described in Ref. (19). This algorithm is by no means unique and we note that many different algorithms identify a strongly periodic character to C.elegans DNA (data not shown) and could have been used to select speciﬁc fragments. For reference, the PATC algorithm returns a numerical score with greater numbers indicating more highly periodic character. A score of >95 is reached at <10 6 fre- quency in a random sequence with base composition equival- ent to C.elegans. There is no segment in the entire unique genomes of Drosophila melanogaster (euchromatin) or Schizosaccharomyces pombe (two genomes with comparable AT content to C.elegans) with a periodicity score >200. Of the natural bent DNAs that have been analyzed previously on a biophysical level, only the mitochondrial DNA of Crithidia shows a substantial signal in the PATC assay (a score of 235 maintained over a distance of 175 bp). Supplementary Figure S2 shows the periodicity values as a function of sequence for the DNAs used in this work. Fragment W4 is a 932 bp segment comprising the fourth intron of C.elegans gene F54C4.1 on chromosome III. The F54C4.1 gene is essential, encodes mitochondrial ribosomal protein L40, and is likely expressed in both somatic and germline tissue of the animal. The intron 4 fragment W4 was chosen from among the many hyperperiodic segments in the C.elegans genome based on its localization in a well- deﬁned essential gene. Informatic analysis of fragment W4 using the PATC algorithm (19) or standard bending algorithms (5,7) indicated a degree of periodicity that is char- acteristic of a subset of germline-expressed C.elegans genes but which has not been seen outside of nematode DNA. Using the PATC algorithm, an average score of 433 for the W4 fragment was observed. This magnitude of score has