Site-Specific DNA Deletion in Tetrahymena thermophila

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9600715 Cox Cilliates possess two nuclei, a vegetative macronucleus and a germline micronucleus. The macronucleus is produced from the micronucleus by a programmed series of fragmentation events of the germline chromosomes, deletion of some sequences and amplification of the remaining DNA. There is only limited information about the mechanisms involved in this process as well as the biological function of the deletion reactions. An in vitro system for two of these developmentally programmed DNA deletion reactions will be developed, the M and R region deletions from the ciliate, Tetrahymena thermophila. Previous work from this laboratory has suggested a likely deletion pathway and a protocol is envisioned in which new DNA substrates are first tested in vivo and then assays are carried out to detect individual steps of the projected recombination process in vitro. To facilitate the purification of any relevant enzymes detected, methods to produce large-scale cultures of synchronously mating cells will be developed. The ultimate goal is a complete molecular description of the deletion pathway derived from studies of an in vitro system reconstituted from purified enzymes and substrates. %%% Programmed DNA rearrangements are not only a normal part of mammalian genome mechanisms (i.e. immunoglobulin genes) but also of aberrant genome phenomena (i.e. tumorogensis). An understanding of the biochemistry of genome restructuring in simple one-celled organisms such as Tetrahymena can give insight into the mechanisms utilized by more advanced organisms such as humans. ***