Date of Award

Summer 2007

Degree Type

Thesis - Restricted

Degree Name

Master of Science (MS)

Department

Biology

Abstract

DNA methylation in Tetrahymena thermophila involves modification of adenine to N6- methyladenine (m6A). About 0.8% of adenines in the macronuclear (Mac) DNA are methylated to N6-methyladenine, but no methylation was detected in the micronucleus (Mic) DNA. Adenine methylation occurs at the sequence 5'-NAT-3' site in vivo, but not all adenines at NAT sites are methylated. De novo DNA methylation occurs in the developing Mac at 13.5-15.5 hours of conjugation. The biological function of DNA adenine methylation in Tetrahymena is unknown. In order to investigate the possible roles of DNA methylation in Tetrahymena, a potential DNA adenine methy1transferase I (MTase) gene was investigated. A putative MTase gene was found by a BLAST search of · the predicted genes in the Tetrahymena genome data base. The open reading frame (ORF) of this putativ~ gene contains conserved amino acid motifs I, II and IV of both DNA MTases and the yeast tRNA MTase TRMll. Sequencing of cDNAs showed that there was a single intron of 341 bp in the putative MTase gene of 21 08bp. The size of its mRNA was estimated by Northern blot analysis as about 2800bp. The relative RNA abundance of the putative MTase gene was determined in vegetatively growing cells, starved cells, and conjugating cells at 6 hours, 8 hours, 10 hours, 12 hours, 14 hours and 16 hours of mating. Real-time PCR showed that the relative abundance of RNA from the putative MTase gene was 2-5 fold higher in vegetatively growing cells than in starved or mating cells.

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