Date of Award
Fall 1997
Document Type
Dissertation - Restricted
Degree Name
Doctor of Philosophy (PhD)
Department
Biological Sciences
First Advisor
Karrer, Kathleen M.
Second Advisor
Fredricks, Walter
Third Advisor
Kumaran, A. K.
Abstract
In the macronuclear DNA of Tetrahymena, about 0.8% of adenine residues are modified to N6-methyladenine. 3% ofmethyladenine occurs in the sequence 5'-GmethATC-3'. Methylation is site-specific, and the pattern of methylation is consistent between cell clones. Some sites are methylated on >90% of the macronuclear DNA molecules, while others are methylated on a lower but constant percentage of the molecules. Experiments described herein were designed to investigate the relationship between DNA methylation and chromatin structure at several genomic loci in Tetrahymena. Transformation of eucaryotic cells can be used to test potential signals for DNA methylation. Cloned fragments of Tetrahymena macronuclear DNA containing a 5' - GATC-3' site that is fully methylated on the chromosome were cloned into the rDNA vector p947H8 and microinjected into mating cells. The methylation state of the DNA in transformant lines was determined by digestion with methyl-sensitive restriction endonucleases. There is a position effect for DNA methylation in Tetrahymena: The 5'GATC- 3' site that is methylated on the genomic copy of the sequence was unmethylated on the transformed copy in analyzed cell lines regardless of orientation relative to the origin of replication on the rDNA vector and of methylation state of the construct prior to transformation of cells. This suggests that localized chromatin structure affects DNA methylation in Tetrahymena. Chromatin structure at specific genomic loci was assessed by Southern hybridization and indirect endlabelling of DNA from isolated nuclei treated with micrococcal nuclease. The localization of nucleosomes was assessed relative to both DNA sequence and methylated and unmethylated 5'-GATC-3' sites. The results demonstrated that nucleosomes are phased at the three macronuclear loci analyzed, and phasing occurs such that six methylated 5'-GATC-3' sites are present within internucleosomal linker DNA regions. Three unmethylated 5'-GATC-3' sites are located in intranucleosomal core DNA sequences. The pattern of nucleosome phasing is maintained in a strain (L'i.Hl) from which the gene encoding macronuclear HI is deleted. Thus, Hl is not required for positioning of nucleosomal core proteins relative to DNA sequence. Site-specific methylation patterns in Tetrahymena were compared between the wild type strain CU428 and the L'i.Hl strain lacking HI. Methylation at seven sites in the macronuclear genome is the same in the L'i.Hl knock-out strain, indicating that HI is not required for the establishment of methyladenine patterns in Tetrahymena.