MECHANISM OF SEQUENCE SPECIFIC RETROTRANSPOSITION OF R2

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Eukarytic genomes are filled with sequences derived from the reverse flow of information from RNA to DNA. The retrotransposable elements responsible for these reverse transcripts have undoubtedly played a significant role in the general shaping of genomes, can account for many current insertional mutations and chromosomal rearrangements, and are believed to be the origin of numerous viruses, eg. retroviruses. The most abundant, yet poorly understood, class of these retrotransposable elements are the non-long terminal repeat (non-LTR) retrotransposable elements. The best characterized non-LTR elements are the R2 elements of insects. The initial steps in the R2 retrotransposition mechanism have been defined because R2 specifically inserts into a unique sequence in the 28S ribosomal genes of its host. Reverse transcription of the R2 RNA template is primed by a nick at the DNA target site, thus retrotransposition is by a mechanism completely different from that of the well-described retroviral mechanism. This target primed reverse transcription mechanism is believed to used by most other non-LTR elements, the group II introns, an is likely the origin of SINEs, processed pseudogenes and maybe even of introns. The specific aim of this proposal is to fully characterize athe R2 retrotransposition mechanism as a model for all non-LTR elements. Direct enzymatic studies of the mechanism will be conducted using purified R2 protein and nucleic acid components. These in vitro studies will be complemented with in vivo studies of the integration reaction in Drosophila melanogaster. Purified R2 protein and R2 RNA templates have been shown to integrate R2 sequences into the 28S genes of somatic and germline cells of D. melanogaster embryos. Further development of this in vivo integration system will enable studies of the transcription and translation of the R2 element within the context of the rDNA units. These studies will address general questions of the mechanism of non-LTR retrotransposition, as well as questions concerning the transcription and recombinational mechanism of the rRNA genes. Finally, these studies will serve as the basis for the development of a transformation system that offers the unique advantage of inserting foreign sequences into a specific sequence of the genome, the rDNA units. Because R2 protein and RNA templates from a moth will be used to integrate sequences in a fruit fly this system may serve as a general transformation system for insects and possibly other organisms.