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Date of Award

Spring 2011

Author's School

College of Arts & Sciences

Author's Department/Program

Biology

Abstract

Tetrahymena thermophila is a ciliate, a group of single-celled eukaryotes that possess nuclear dimorphism. Tetrahymena has both a silent, germ-line micronucleus, and an actively expressed macronucleus that develops from a copy of the micronucleus through an intricate mechanism where thousands of repetitive sequences are physically eliminated. To recognize these sequences to be deleted, Tetrahymena relies on a mechanism related to RNA interference where small RNAs homologous to the internal eliminated sequences (IES) search the developing macronucleus, and mark IES loci for methylation. One of these internal eliminated sequences called the M Element rearranges very efficiently among predictable borders. Interestingly, a class of sequences that are up to 95 percent homologous to ~200bp of the M Element (dubbed M-like sequences, or MLSs) undergo inefficient rearrangement in their native genomic contexts, despite their homology to an efficiently eliminated IES. When one MLS was cloned out of its native environment into a high-copy number artificial chromosome, it rearranged with significantly higher efficiency. This conforms to the hypothesis that MLSs in their native chromosomal loci have chromatin modifications or other epigenetic factors that hide these IES-like sequences from the rearrangement machinery. Furthermore, when the M Element homologous region is absent in the plasmid-based constructs that become artificial chromosomes, the M like sequences are still rearranged, but with even less efficiency. Although the specific epigenetic modifications are still not known, preliminary rtPCR evidence suggests that these M-like sequences are still transcribed. These results may suggest that MLSs have the potential to behave like IESs, but their native genomic contexts suppress their IES behavior. The project has the potential to improve the understanding of how Tetrahymena determines which sequences should be rearranged, which can improve knowledge of molecular genetics and potentially have future applications in gene therapy and DNA-targeting drugs.

Language

English (en)

Advisor/Committee Chair

Douglas Chalker