Maintenance of genome integrity requires that chromosomes be accurately and
faithfully replicated. We are using Tetrahymena thermophila as a model system for
studying the initiation and regulation of eukaryotic DNA replication. This organism
contains a diploid micronucleus and polyploid macronucleus. During macronuclear
development, the five diploid chromosomes of the micronucleus are fragmented into 280
macronuclear minichromosomes that are subsequently replicated to ~45 copies. In stark
contrast, the 21 kb ribosomal DNA minichromosome (rDNA) is amplified from 2 to
10,000 copies in the same nucleus. Previous characterization of the rDNA replicon has
led to the localization of its origin and the cis-acting regulatory determinants to the 1.9
kb 5'non-transcribed spacer region.
The objective of this study was to identify and characterize non-rDNA origins of
replication in Tetrahymena. This will help determine the underlying basis for differential
regulation of rDNA and non-rDNA origins during development, as well as provide a
better understanding of the organization of eukaryotic replicons. To this effect, I developed a DNA transformation assay that I used to isolate new Tetrahymena
replication origins. A 6.7 kb non-rDNA fragment, designated TtARS1, was shown to
support stable autonomous replication of circular plasmids in Tetrahymena. Genetic
dissection revealed that TtARS1 contains two independent replicons, TtARS1-A and
TtARS1-B. Full TtARS1-A function requires a minimal sequence of 700 bp, and two
small regions in this fragment have been shown to be essential for origin function.
TtARS1-B replicon function was localized to a 1.2 kb intergenic segment that contains
little sequence similarity to TtARS1-A. Both non-rDNA replicons lack sequence
similarity to the rDNA 5' NTS, suggesting that each replicon interact with a different set
of regulatory proteins. This study indicates that the rDNA and the non-rDNA replicons
have a modular organization, containing discrete, cis-acting replication determinants.
| Identifer | oai:union.ndltd.org:tamu.edu/oai:repository.tamu.edu:1969.1/4666 |
| Date | 25 April 2007 |
| Creators | Datta, Shibani |
| Contributors | Kapler, Geoffrey M. |
| Publisher | Texas A&M University |
| Source Sets | Texas A and M University |
| Language | en_US |
| Detected Language | English |
| Type | Book, Thesis, Electronic Thesis, text |
| Format | 1177316 bytes, electronic, application/pdf, born digital |
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