Role of selfish restriction-modification gene complexes in genome evolution : comparative genomics and experimental evolution

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1.Conditions for post-segregational host killing (genetic addiction)We found that EcoRI modification enzyme is as stable as EcoRI restriction enzyme and bulk cellular proteins. We analyzed conditions for evolution of addiction genes using evolutionary ecology approach. Importance of space structure was demonstrated.2.Restriction enzyme and modification enzyme obtained through comparative genomics with hyperthermophilic archaeaThrough comparison of the genomes of Pyrococcus abyssi and Pyrococcus horikoshii, we identified a putative restriction-modification gene complex inserted into the former genome. We identified restriction enzyme PabI through expression screening in wheat germ-derived cell-free protein synthesis system. It generates a novel terminus TA3' at 5'GTAC.3.Reconstruction of formation of large genome polymorphisms through comparison of closely-related genomesComparing 7 genomes of Staphylococcus aureus, we found evolution of S (specificity) subunit of Type I restriction enz … More yme through combination of two target recognition domains. From analysis of three tandem paralogue gene clusters, we concluded that some genome rearrangments have resulted from homologous recombination between sequence within each unit conserved for its functional importance. Comparing 4 genomes of Neisseria, we identified filamentous phages likely integrated by its transposase and occasionally rearranged by it.4.DNA breakage and genome rearrangements triggered by restriction breaks and othersWe found that Type III restriction breakage after single infection of bacteriophage lambda is repaired by homologous recombination function of prophage. Homology-driven illegitimate recombination was found to depend on RecA and Type I restriction sites. We developed a sensitive assay of linearization of circular bacterial chromosome and analyzed various recombination-defective mutants of E.coli. We demonstrated high level of DNA double-strand break repair in BJ5183 strain of E.coli. We modeled asymmetric branch migration of Holliday structure and analyzed data in vitro.5.Multiplication of a restriction-modification gene complexWe showed that tandem amplification of BamHI restriction-modification gene complex on Bacillus subtilis chromosome is dependent on function of the restriction enzyme. Less