I propose to study the various developmental processes which alter the organization of an eukaryotic genome in the model system Tetrahymena. DNA elimination, rDNA amplification and chromosome breakage have all been shown to occur in this organism, and will be studied in detail. The rDNA exist as a single copy sequence in the chromosome of this organism. During development, chromosome breakage occurs near this gene and the rDNA is released from the chromosome for amplification. A protion of the flanking DNA is also eliminated during this process. We will isolate the rDNA and the flanking sequences from both the macro-and micronucleus by cloning and determine the nucleotide sequences in these junctions. The alteration process will also be studied during the development of the macronucleus by hybridization. This approach should reveal the nucleotide sequences that are involved in elimination, amplification and breakage, and the molecular mechanisms by which these processes take place. Elimination of DNA and breakage of chromosome also occur in other locations of the genome, and may be associated with a simple repeated hexanucleotide CCCCAA. The C4A2 repeats and the micronuclear specific sequences will be isolated together with their flanking sequences from the 2 nuclei by cloning. The flanking sequences will be used to determine the nature of elimination, and the role of the C4A2 repeats in these processes. This study should help us understand what specific nucleotide sequences are involved in the alteration, and how the entire genome may be reorganized, and perhaps also how these events may be related to the normal developmental process of an eukaryote.
