Understanding Complex Gene Editing Systems in Protists

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DESCRIPTION (provided by applicant): The goal of this research is to study unusual forms of biological information processing in protists, with emphasis on gene unscrambling in stichotrichous ciliates. The proposed research combines different approaches to this problem to address questions and test the hypotheses at the genomic, evolutionary, and developmental levels and time scales. The global aim is to use this model system to explore the mechanisms underlying complex rewriting systems in microbial eukaryotic genomes. Specific aims include: a) Probing the global distrbution of scrambled genes in Oxytricha trifallax, our model system for studies on a genomic scale, and the presence of scrambled orthologs in earlier diverging spirotrichous species for comparisons on an evolutionary time scale. b) Probing the populations of intermediates during the process of gene unscrambling on a developmental time scale. c) Functional experiments introducing foreign DNA or RNA into the developing macronucleus. d) Testing the role of small RNAs in macronuclear development in Oxytricha. e) Identification and testing of candidate genes involved in macronuclear development in Oxytricha. Computational analysis of both molecular evolutionary and direct experiment data-such as the time course of scrambled gene rearrangements-will provide a window into the complexity and rules of such biological computations, such as the sequence and molecular requirements for correct gene processing. The combination of molecular biology, functional genomics, computational biology, and evolutionary experiments in this proposal offers a unique opportunity to build a strong understanding of gene unscrambling in ciliates-a phenomenon that still mystifies the research community.