Intramolecular recombination in bacteria Escherichia coli and Agrobacterium tumefaciens

Abstract

Uzastopno ponovljeni sljedovi nalaze se u genomima mnogih organizama i podložni su delecijama i amplifikacijama, te su velik uzrok genomske nestabilnosti. Genetička analiza rearanžmana ponovljenih sljedova u bakteriji Escherichia coli je pokazala da je u njihov nastanak uključeno nekoliko mehanizama. Mogu se ugrubo podijeliti na RecA-ovisne i RecA-neovisne mehanizme. Ovim radom željeli smo ispitati efikasnost intramolekularne rekombinacije u bakteriji Agrobacterium tumefaciens, te je usporediti s onom u bakterije Escherichia coli. U tu svrhu napravili smo plazmid koji sadrži direktno ponovljene nepotpune gene za otpornost na spektinomicin razmaknute genom za otpornost na ampicilin. Otpornost na spektinomicin može nastati ili RecA-ovisnom homolognom rekombinacijom, pri čemu dolazi do delecije gena za otpornost na ampicilin ili RecA-neovisno pri čemu se zadržava gen za otpornost na ampicilin i amplificiraju geni za otpornost na spektinomicin. U ovom radu smo pokazali da je intramolekularna rekombinacija u A. tumefaciens uglavnom RecA-neovisna.Tandemly repeated DNA sequences are common in the genomes of many organisma and are susceptive to deletions and amplifications. That way they are a major cause of genome instability. Genetic analysis of repeated sequence rearrangements in Escherichia coli has revealed that multiple mechanisms participate in the process. They can be roughly divided into RecA-dependant and RecA-independent mechanisms. In this graduation thesis we wanted to test intramolecular recombination efficiency of bacterium Agrobacterium tumefaciens and compare it to that in Escherichia coli. For that purpose we have designed a plasmid which has tandem repeats of incomplete spectinomycin genes separated with ampicillin resistance gene. Spectinomycin resistance can be achieved either by RecA-dependant homologous recombination between direct repeats, while simultaneously deleting ampicillin resistance gene, or by RecAindependent mechanisms, which yields both ampicillin resistance gene and spectinomycin resistance genes amplification as a product. In this graduation thesis we have shown that intramolecular recombination in A. tumefaciens is mostly RecAindependent