Isolation and Characterization of the Tlr Family of Germ Line-Limited Mobile Genetic Elements in Tetrahymena Thermophila
Date of Award
Dissertation - Restricted
Doctor of Philosophy (PhD)
In the ciliated protozoan Tetrahymena thermophila , extensive DNA elimination is associated with differentiation of the somatic macronucleus from the germ line micronucleus. Tlr elements are a family of approximately 30 closely related DNA sequences in the micronuclear genome. All copies of these elements are deleted from the macronucleus during development. The primary goals of this research were to characterize the complete structure of Tlr elements and identify the cis -acting internal DNA signals that control their programmed elimination. The composite structure of a typical Tlr element was assembled from a series of overlapping DNA clones isolated by successively screening a plasmid library of micronuclear genomic DNA. Analysis of the resulting sequences revealed that Tlr elements consist of a ∼22 kb internal region flanked by long, complex terminal inverted repeats. The Tlr internal region is 90-97% conserved at the nucleotide level among family members and contains 15 major open reading frames. The conceptual translation products from several of the open reading frames resemble proteins encoded by transposable elements and viruses. Taken together, these results suggest that Tlr elements comprise a novel family of mobile genetic elements that are confined to the germ line genome in Tetrahymena . In order to examine the mechanism by which Tlr elements are recognized for deletion from the macronuclear genome, an rDNA-based in vivo rearrangement assay was utilized to identify the internal sequences required in cis for faithful elimination of the most thoroughly characterized Tlr family member, Tlr1. These analyses revealed that multiple, non-overlapping regions of the Tlr1 element are independently sufficient to stimulate developmentally regulated DNA deletion within the context of normal flanking sequences. Since complete removal of Tlr1 DNA abolishes construct rearrangement activity, as does replacement of element sequences with macronuclear DNA, the data suggest that unique features of Tlr sequences provide cis -acting regulatory signals for programmed deletion from the differentiating macronuclear genome. Furthermore, replacement of Tlr1 DNA with the micronucleus-limited region of a non-Tlr internally eliminated sequence, the M element, resulted in accurate rearrangement activity. Thus, Tlr1 and M appear to contain functionally similar internal signals that promote programmed DNA elimination in Tetrahymen.