Characterization of cis-acting replication control elements in ribosomal RNA genes of Tetrahymena thermophila

This dissertation describes the identification and characterization of specific cis-acting replication control elements in the replication origin of Tetrahymena rDNA. Earlier studies of a mutant rDNA allele, C3-rmm1, identified a cis-acting sequence (the Type I repeat) within the 5[superscript]\u27 non-transcribed spacer of rDNA that affects rDNA replication (Larson et al. 1986). Characterization of two additional, independently-isolated mutants (C3-rmm3 and C3-rmm4) and a revertant of C3-rmm4 strongly argue that the Type I repeat is indeed a cis-acting replication control element. Mutations causing rDNA replication defects were identified in copies of the Type I repeat that are separated by \u3e600 bp. Therefore, the rDNA replication origin may be much larger than typical origins defined in prokaryotes. In addition, the mutation in C3-rmm3 is located within the rRNA gene transcriptional promoter region, suggesting a possible dual role for the Type I repeat in transcription and replication. Together these results provide genetic evidence for a novel sequence-specific element that affects eukaryotic DNA replication;Chromosomal origins of replication in higher eukaryotes appear to share common modular sequence elements (Benbow et al. 1992). Several of these modular sequence elements were found clustered in multiple copies within the origin region of Tetrahymena rDNA. Furthermore, an intrinsically bent DNA structure and nuclear matrix-associated regions were directly demonstrated in the origin region. Replicative intermediates of rDNA that contain bubble structures within the 5[superscript]\u27NTS were detected;A high resolution method for mapping sites of replication initiation was developed. Individual DNA molecules were tagged with streptavidin-gold and visualized using atomic force microscopy. This work provides the basis for a novel technique to examine the first nucleotides incorporated into replicating rDNA