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.

Project Start
1986-12-01
Project End
1988-11-30
Budget Start
1987-12-01
Budget End
1988-11-30
Support Year
5
Fiscal Year
1988
Total Cost
Indirect Cost
Name
Fred Hutchinson Cancer Research Center
Department
Type
DUNS #
075524595
City
Seattle
State
WA
Country
United States
Zip Code
98109
Yao, M C; Yao, C H; Monks, B (1990) The controlling sequence for site-specific chromosome breakage in Tetrahymena. Cell 63:763-72
Yao, M C; Yao, C H (1989) Accurate processing and amplification of cloned germ line copies of ribosomal DNA injected into developing nuclei of Tetrahymena thermophila. Mol Cell Biol 9:1092-9
Sweeney, R; Yao, M C (1989) Identifying functional regions of rRNA by insertion mutagenesis and complete gene replacement in Tetrahymena thermophila. EMBO J 8:933-8
Austerberry, C F; Snyder, R O; Yao, M C (1989) Sequence microheterogeneity is generated at junctions of programmed DNA deletions in Tetrahymena thermophila. Nucleic Acids Res 17:7263-72
Karrer, K M; Yao, M C (1988) Transformation of Tetrahymena with hypermethylated ribosomal RNA genes. Gene 74:105-7
Austerberry, C F; Yao, M C (1988) Sequence structures of two developmentally regulated, alternative DNA deletion junctions in Tetrahymena thermophila. Mol Cell Biol 8:3947-50
Karrer, K M; Yao, M C (1988) Transformation of Tetrahymena thermophila with hypermethylated rRNA genes. Mol Cell Biol 8:1664-9
Austerberry, C F; Yao, M C (1987) Nucleotide sequence structure and consistency of a developmentally regulated DNA deletion in Tetrahymena thermophila. Mol Cell Biol 7:435-43
Altschuler, M I; Yao, M C (1985) Macronuclear DNA of Tetrahymena thermophila exists as defined subchromosomal-sized molecules. Nucleic Acids Res 13:5817-31
Yao, M C; Zhu, S G; Yao, C H (1985) Gene amplification in Tetrahymena thermophila: formation of extrachromosomal palindromic genes coding for rRNA. Mol Cell Biol 5:1260-7