Published online 9 December 2005
Methods Online |
New approaches to the analysis of palindromic sequences from the human genome: evolution and polymorphism of an intronic site at the NF1 locus
1Program in Genetics and Genomic Biology, Hospital for Sick Children Research Institute Toronto, ON, Canada 2Graduate Department of Molecular and Medical Genetics, University of Toronto Canada 3Gene Regulation and Chromosome Biology Laboratory, NCI Frederick, MD, USA
*To whom correspondence should be addressed at Program in Genetics and Genomic Biology, Hospital for Sick Children Research Institute, TMDT Building, East Tower rm 15-308, 101 College Street, Toronto, ON, Canada M5G 1L7. Tel: +1 416 813 8980; Fax: +1 416 813 4931; Email: susanna{at}sickkids.ca
Received October 24, 2005. Revised November 23, 2005. Accepted November 23, 2005.
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. We describe and validate a practical alternative to the analysis of naturally-occurring palindromes based upon cloning and propagation in Saccharomyces cerevisiae. 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 find that the site is highly polymorphic, exhibiting different degrees of palindromy in different individuals. A side-by-side comparison of the same plasmids in E.coli versus. S.cerevisiae demonstrated that the more palindromic alleles were inevitably corrupted upon cloning in E.coli, but could be propagated intact in yeast. 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.