It is highly desirable to learn where perfect, or near-perfect palindromes exist naturally in the human genome, but this is difficult to determine because such sequences become corrupted or eliminated as soon as they are introduced into E.coli. A recent observation of note is that yeast strains compromised for telomere maintenance can escape senescence concurrent with the generation of long palindrome-like structures near chromosome ends ( 17). The origin of these structures may be mechanistically related to long palindromes or near-palindromes that can be experimentally generated in yeast ( 14– 16). Long inverted repeats that may be palindromic arise de novo at tumor-specific sites in the genome during oncogenesis ( 13). Natural palindromes or near-palindromes of about 200–800 bp exist at sites of some inherited chromosomal rearrangements in humans ( 10– 12). Suspected palindromes in higher eukaryotes have surfaced in diverse pathological situations. In both cases, the ultra-large palindromes were well above a size that might be propagated in E.coli, and both required special approaches in order to obtain DNA sequence information. More recently a synthetic, pure palindrome with 900 bp arms was demonstrated to be stably maintained in Saccharomyces cerevisiae lacking SAE2 gene function ( 9).

This huge palindrome, despite moderate instability, was inherited in a Mendelian ratio, and could be maintained in tissue culture cells without any apparent lethal effects. In mice, a 15.7 kb perfect palindrome, transgenically introduced, could be transmitted in the germline ( 7, 8). The suggestion that organisms other than E.coli tolerate long stretches of perfect palindromy to different extents ( 2, 6) is supported by a handful of examples where introduced perfect palindromes (no central spacer) have been documented by DNA sequence analysis. The tendency to adopt a cruciform structure underlies at least some of the observed inviability and instability of palindromes in bacteria ( 2, 6). Palindromes and near-palindromes have overlapping properties, but a pure palindrome is significantly more susceptible to effects that drive cruciform extrusion than are inverted repeats separated by a central spacer or quasipalindromes with incompletely matched arms ( 5). The word ‘palindrome’ is applied non-specifically to different types of inverted repeat in the literature, but for consistency we use the term only in its narrowest sense here. For these reasons, pure palindromes longer than roughly 200 bp become impossible to clone in Escherichia coli, and no mutant has been described that fully overcomes the cloning block ( 4). Palindromic sequences are able to self-pair, forming intrastrand hairpin structures and extensive stretches of palindromy can interfere with key aspects of DNA metabolism whether located chromosomally or on extrachromosomal replicons ( 1– 3). True DNA palindromes, where a sequence is immediately followed by its exact inverse complement (with no central asymmetry or spacer), can be extremely difficult to analyze by standard molecular genetic approaches. The high quality sequence obtained from the yeast-based approach provides insight into the various mechanisms that destabilize a palindrome in E.coli, yeast and humans, into the diversification of a highly polymorphic site within the NF1 locus during primate evolution, and into the association between palindromy and chromosomal translocation. S.cerevisiae demonstrated that the more palindromic alleles were inevitably corrupted upon cloning in E.coli, but could be propagated intact in yeast. A side-by-side comparison of the same plasmids in E.coli versus. We find that the site is highly polymorphic, exhibiting different degrees of palindromy in different individuals. With this approach we have investigated an intronic sequence in the human Neurofibromatosis 1 (NF1) locus that is represented by multiple conflicting versions in GenBank. We describe and validate a practical alternative to the analysis of naturally-occurring palindromes based upon cloning and propagation in Saccharomyces cerevisiae. The nature of any long palindrome that might exist in the human genome is obscured by the instability of such sequences once cloned in Escherichia coli.

Example of palindromic sequence mcat