INTRON MOBILITY IN THE PHAGE T4/E COLI SYSTEM

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The group I introns of phage T4 self-splice at the RNA level. During the past funding period we discovered that the td and sunY introns of phage T4 are capable of DNA-based "mobility" reactions as well. In accordance with their dual properties, the introns are bipartite elements. They comprise sequences that fold into a characteristic RNA structure, the ribozyme core, out of which is looped an open reading frame (ORF), whose product is required for mobility. This mobility process, also termed "homing", involves transfer of the intron into an intronless allele via a non-reciprocal gene conversion event. We have shown that the intron ORF encodes a site-specific DNA endonuclease which cleaves the recipient allele to initiate gene conversion. Recombination is hypothesized to occur via the double-strand-break repair (DSBR) pathway. The mobile phage T4 introns provide the opportunity to study the gene conversion process in a well-defined and manipulable genetic background, with well-characterized mutants in DNA recombination and repair functions. This application is directed toward using the phage/bacterial model system to dissect the intron mobility process. This will involve defining the recombination pathway in detail. We shall also exploit the gene conversion system to facilitate gene manipulation in Escherichia coli, and to test the role of yeast recombination functions in DSBR. Furthermore, we wish to explore the possibility of intron transposition to new sites via either the DSBR pathway or by an RNA-mediated pathway. Finally, we shall further characterize the self-splicing introns which we recently identified in purple bacteria (close relatives of E. coli) and study the distribution of similar introns in the eubacterial kingdom. Thus we hope to enhance our fundamental understanding of the intron mobility process, to utilize the process for practical purposes, and to address evolutionary hypotheses for intron mobility and intron distribution.