Date of Award
Summer 1998
Document Type
Dissertation - Restricted
Degree Name
Doctor of Philosophy (PhD)
Department
Biological Sciences
First Advisor
Karrer, Kathleen
Second Advisor
Fredericks, Walter
Third Advisor
Kumaran, Krishna
Abstract
Programmed DNA rearrangements occur in several biological systems, including mammalian immune systems. Tetrahymena is a model system to study developmentally regulated DNA rearrangements. One such DNA rearrangement in Tetrahymena is Tlrl (Tetrahymena long repeat). Tlrl deletes over 13 kb of DNA and has an 825 bp inverted repeat near the junctions. Within the inverted repeat are tandem repeats of two distinct 19 bp sequences. The Tlrl major rearrangement product shows junctional microheterogeneity over 6-7 bp. Sequence analysis of the junctions suggested Tlrl rearrangement may occur via a different mechanism than the current transesterification model for Tetrahymena DNA rearrangements. To determine the cis-acting sequences required for Tlrl rearrangements, a series of constructs were made and assayed in vivo. The first construct consisting of the entire inverted repeat and about one kilobase of flanking DNA on each side, rearranged accurately and showed microheterogeneity similar to the endogenous Tlrl. Therefore, the inverted repeat plus a kilobase of flanking sequences is sufficient to direct precise Tlrl rearrangement and the 11 kb or more of internal DNA is not required. The second construct tested the relevance of the 19 bp tandem repeats that bind developmentally regulated proteins in vitro. The construct lacking the 19mer repeats also rearranged precisely, showing the 19mer repeats are not required on both halves of the inverted repeat. In a supplementary experiment, excess 19mer sequences were introduced into the Tetrahymena cell. If the introduced sequences competed with endogenous sequences for binding rearrangement proteins, Tlrl rearrangement would be suppressed. However, Tlrl rearrangement was not inhibited, suggesting sequences other than the 19mers may also be involved in recruiting Tlrl rearrangement factors. The importance of flanking sequence for the Tlrl rearrangement was demonstrated by aberrant rearrangement of the third construct with only 51 bp of the DNA flanking the right junction. Thus suggesting that 51-851 bp of the right flanking DNA contains an important signal for determination of the Tlrl junction sequences. Therefore, despite its unique structure, Tlrl is similar to other DNA rearrangements in Tetrahymena in showing junctional microheterogeneity and possessing cis-acting sequences outside the deleted DNA.