Date of Award
Spring 2002
Document Type
Dissertation - Restricted
Degree Name
Doctor of Philosophy (PhD)
Department
Biological Sciences
First Advisor
Karrer, Kathleen
Second Advisor
Schlappi, Michael
Third Advisor
Waring, Gail
Abstract
The ciliated protozoan Tetrahymena is a unicellular eukaryote that contains two nuclei, a germline micronucleus and a somatic macronucleus. During conjugation, the sexual phase of the life cycle, the old macronucleus is degraded and the new macronucleus develops from the zygotic micronucleus. The formation of new macronucleus involves extensive DNA rearrangement and elimination. This process is a complex developmental program involving sequential regulation of dozens of genes. The technique of subtractive hybridization was applied to isolate genes that are specifically transcribed during postzygotic development. A library enriched for cDNA transcripts that are regulated during macronuclear development was constructed by subtraction of a cDNA library, complementary to RNA from conjugating cells at 12hr post-mixing of the two mating types, with mRNA from starved cells. A total of fourteen clones were obtained from the subtracted cDNA library. The insert size of the clones ranged from 150bp to 500bp. A cross-hybridization analysis showed each clone to be unique. Northern analysis of four ASI (a[barbelow]nlagen s[barbelow]tage i[barbelow]nduced) clones confirmed the isolation of genes that are transcribed during macronuclear development. The ASI1 gene was chosen for further analysis because it encodes a 2.8kb transcript, which is specifically induced during the period of macronuclear anlagen development. Southern analysis showed ASI1 is a single copy gene in both the micronucleus and the macronucleus. The ASI1 gene encodes a 95kd conceptual protein containing one intron and a leucine zipper near the amino terminus. The presence of a leucine zipper suggests the ASI1 protein may function as homodimer or heterodimer during the period of macronucleus development in Tetrahymena thermophila . The ASI2 gene encodes a 3.0kb transcript, which is induced during determination and differentiation of the new macronucleus. Southern analysis indicates it is a single copy gene in both the micronucleus and the macronucleus. The ASI3 gene encodes a transcript of 2kb induced during macronuclear anlagen development. BlastX analysis of ASI3 revealed significant homology to long-chain fatty-acid-CoA ligase. The ASI4 gene encodes a transcript of 0.85kb induced during the period of anlagen determination and differentiation. A low level expression is also observed in vegetatively growing cells.