The focus of this proposal is on understanding the process of genome reorganization found in the ciliated protozoan, Tetrahymena thermophila, that occurs as part of the normal developmental process. To coordinate and carry out this genome rearrangement, a large number of trans-acting factors must be required, but thus far, only two, named Pdd1p and Pdd2p have been identified. Pdd1p, the more well characterized of the two, belongs to the chromo family of proteins, which are usually associated with condensed chromatin. It is found in close association with that fraction of genomic DNA that is eliminated, at the time of its processing, suggesting a direct role in the DNA elimination mechanism. Disruption of the parental, somatic PDD1 gene causes a defect in DNA elimination and chromosome breakage and a failure to complete successful conjugation. Exconjugants are unable to divide and eventually die. The activities of this protein will be studied by disrupting the gene encoding it in both the germline and somatic nuclei of Tetrahymena, as well as by generating site-directed mutations in the gene. These mutations will be reintroduced into the Tetrahymena genome by gene replacement technology and analyzed for their effects on specific DNA rearrangements, the progression of nuclear differentiation events, and proper nuclear targeting of Pdd1p.
Stability of the genome is normally a hallmark of development and inheritance. Some well-known exceptions include the rearrangement of vertebrate immunoglobulin genes and the yeast mating type loci. Intriguingly, however, many organisms undergo massive genome reorganization as part of their normal developmental program. The experiments proposed will help define the mechanisms involved in such DNA rearrangements. They will also clarify the connection between chromatin structure formation and genome rearrangement. The experiments are particularly suited to the training of undergraduate students in the rigor and excitement of experimental biological research.
