EXPRESSION OF AN INTERRUPTED PROKARYOTIC GENE

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The long-term objective of this research is to use the split td gene of phage T4 as the molecular paradigm for understanding the RNA and DNA transactions of a self-splicing, mobile intron. During the first 5-year funding period (supported by NSF grant DMB8502961) we established that the td intervening sequence is a group I self-splicing intron. We used this knowledge to detect two other introns in T4 and discovered that one of these as well as the td intron are mobile, transferring efficiently to intronless alleles of their respective genes. As for their eukaryotic group I counterparts, intron mobility is dependent upon an endonuclease encoded by the intron itself. The proposed research is intended to further our fundamental understanding of aspects of both the RNA-based (splicing) and DNA-based (mobility) processes, using the td gene as model system. To both ends we shall continue to exploit the powerful positive and negative selections provided by this phage genetic system and by the td gene in particular. For the splicing studies we shall continue to use molecular genetics to analyze the function of a prokaryotic ribozyme. We shall conduct parallel studies to investigate the possible role of Escherichia coli functions as accessories to the self-splicing process. With respect to DNA mobility, we shall examine the function of the td endonuclease, which mediates the process, and its interaction with its DNA target, the intron homing site. We shall also ask some more general questions about the intron-phage relationship and use the td gene as a model for exploring the possibility of intron loss in a prokaryotic system. Finally, to rationalize the existence of introns , in streamlined phage genomes, we wish to address the hypothesis that introns provide a selective advantage by enhancing phage recombinogenicity. Together, the proposed experiments represent a continuation of ongoing work that exploits facile prokaryotic genetic techniques to shed light on the multifaceted reactions of group I introns, which are dynamic genetic elements in common to the pro- and eukaryotic kingdoms.