DNA Rearrangements and Macronuclear Development in Cilated Protozoa

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Genome remodeling occurs throughout the eukaryotic kingdom and includes well known examples such as mating type switching in yeast and recombination of immunoglobulin genes in mammals. The ciliated protozoa present an extreme example in which large amounts of DNA are eliminated and rearranged to form the transcriptionally active macronucleus as a normal part of sexual reproduction. In Paramecium tetraurelia, an estimated 50,000 short DNA elements called internal eliminated elements (IESs) are removed from the genome via site specific recombination. Previous research led to the hypothesis that Paramecium IESs are related to transposable elements. This project will define the regions of a 28 bp Paramecium IES that are required for accurate DNA splicing and compare these regions with those critical for excision of transposable elements. One series of experiments will involve the construction of specific mutations in the cloned IES. Each mutant IESs will be tested for accurate splicing by injection into cells that are undergoing macronuclear development. A second approach will involve sequencing several examples of evolutionarily related IESs from different Paramecium strains. Comparison of these sequences will identify conserved nucleotides that are presumed to be important due to their evolutionary conservation. This assumption will be tested by constructing appropriate mutations in the IES. Investigations in the related ciliate Tetrahymena thermophila will focus on a recently identified developmentally regulated translation elongation factor-2. Gene disruption and replacement experiments will determine whether this gene and its corresponding protein are involved in a translational control mechanism that is required for the genome remodeling process. The research will contribute to a broader understanding of the relationship between ciliate DNA elimination and transposable element insertion-excision, and perhaps reveal novel biochemical features of DNA splicing in this organism. The study of a developmentally regulated elongation factor 2 may reveal a novel mechanism for the control of protein synthesis in eukaryotes.