The Role of Lia1 in the DNA Deletion Process of Tetrahymena thermophila

Student: Rajat Jain Mentor: Douglas Chalker, Ph.D. Biology Department, Washington University

Tetrahymena thermophila belongs to the ciliates, a successful lineage of aquatic unicellular eukaryotes. As is common for ciliates, Tetrahymena thermophila has two structurally and functionally distinct nuclei. The transcriptionally inactive micronucleus (mic) acts as a repository of genetic information that is passed on to sexual progeny.. The macronucleus (mac) is actively expressed during vegetative multiplication. The diploid mic contains five pairs of chromosomes. The mac’s DNA consists of smaller chromosomes derived from the mic’s genome by chromosome breakage and site-specific deletion.

Although cell reproduction occurs exclusively by binary fission, Tetrahymena thermophila can introduce sexual variation through conjugation. In a mating pair, following meiosis, conjugation entails reciprocal fertilization of the micronucleus which then divides twice by mitosis to form four nuclei. Two of these differentiate into macronuclei. In the exconjugants the old macronucleus and one of the new micronuclei are destroyed. Pair separation followed by cell division, results four karyonides; this completes the sexual cycle and vegetative growth is resumed. DNA deletion occurs in the developmental macronuclei, and is completed before the first cell division. Approximately 30 Megabases or 15% of the mic DNA is deleted.

It has been shown that the protein Pdd1 (Programmed DNA Degradation) is involved in the deletion of DNA in the developmental macronuclei and co-localizes with sections of DNA known to be deleted. A screen was previously performed to find proteins that localize similarly to Pdd1 and thus may also be involved in DNA deletion. mRNA from developmental cells was randomly inserted into a vector containing Green Fluorescence Protein (GFP). Tetrahymena thermophila were then transformed with this library. A test for protein localization was performed on the transformants. Ten novel proteins were identified from 22,700 transformants. One of these, coded the Lia1 by gene, was then modified with myc and flag tags to aid in characterization. These tagged versions were inserted into a vector and used to transform cells. It was hoped that the tagged version of Lia1 would be co-expressed with the endogenous gene. However, the endogenous protein was far more prevalent.

My experiments sought to replace the endogenous gene completely with an HA tagged version within the genome rather than co-expressing the protein. A sequential PCR strategy was used to modify the Lia1 gene with an upstream HA tag and to clone two flanking sections where potential recombination can occur with the endogenous gene. At this time, the construct has been completed. Further steps include Tetrahymena thermophila transformation using biolistics. The goal is to replace the endogenous gene through homologous recombination. A neomycin selectable marker within the vector will allow drug selection of transformants. Transformants will be mated and Lia1 expression will be monitored throughout the developmental phase by using immunohistochemistry and Western blot analysis to determine whether Lia1 associates with Pdd1 and with segments that undergo developmentally programmed deletion.