The structure, amplification and replication properties of rRNA genes in the ciliated protozoan Tetrahymena thermophila will be further analyzed.
The aims are: 1) to follow the molecular pathway of rDNA amplification, which results in the conversion of a single rRNA gene in the germline nucleus to 104 extrachromosomal, linear, palindromic rDNA molecules in the somatic macronucleus; 2) to analyze thee regulation of macronuclear DNA replication, to test a copy number control model applied to rDNA. Mutants altered in their rDNA amplification and replication properties will be analyzed. The behavior of these mutant genes during the early steps of the amplification process, and in their subsequent replication in the macronucleus, will be examined. Two such mutants with cis-acting rDNA mutations which alter their amplification and macronuclear replication respectively have already been isolated and partly characterized, and we will continue our molecular studies of them. Further rDNA mutants, obtained by genetic selection similar to that already used to obtain the first two, will be analyzed by restriction digestion to determine any gross structural alterations of the rDNA, time course analysis during macronuclear development and subsequent vegetative growth, and DNA sequence comparisons of the rDNAs of mutant and wild-type strains. The molecular details of the DAN rearrangements involved in rDNA amplification will be investigated by a primer-extension assay. Treatment of cells by temperature changes or other conditions which affect or inhibit new macronuclear development will be used to selectively block steps in amplification. A genetic selection for cells resistant to the normally lethal 40 degree during macronuclear development will be done to investigate the basis of this lethality. The replication properties of the rDNA of one of the mutants already partially characterized will be exploited in DNA mediated transformation of Tetrahymena macronuclei. A mutant rDNA which we have shown confers resistance to t he drugparamamycin will be used in transformation experiments to analyze functionally the rDNA replication origins of both wild type and mutant rDNAs. Finally, as part of a long term goal of micronuclear transformation, a transposon-like family of micronuclear genomic elements will be further studies.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM032565-07
Application #
3281528
Study Section
Genetics Study Section (GEN)
Project Start
1983-08-01
Project End
1990-07-31
Budget Start
1989-08-01
Budget End
1990-07-31
Support Year
7
Fiscal Year
1989
Total Cost
Indirect Cost
Name
University of California Berkeley
Department
Type
Schools of Arts and Sciences
DUNS #
094878337
City
Berkeley
State
CA
Country
United States
Zip Code
94704
Blackburn, E; Gilley, D; Ware, T et al. (2000) Studying the telomerase RNA in Tetrahymena. Methods Cell Biol 62:417-32
Gallagher, R C; Blackburn, E H (1998) A promoter region mutation affecting replication of the Tetrahymena ribosomal DNA minichromosome. Mol Cell Biol 18:3021-33
Pan, W J; Blackburn, E H (1995) Tandem repeats of the 5' non-transcribed spacer of Tetrahymena rDNA function as high copy number autonomous replicons in the macronucleus but do not prevent rRNA gene dosage regulation. Nucleic Acids Res 23:1561-9
Romero, D P; Blackburn, E H (1995) Circular rDNA replicons persist in Tetrahymena thermophila transformants synthesizing GGGGTC telomeric repeats. J Eukaryot Microbiol 42:32-43
Kirk, K E; Blackburn, E H (1995) An unusual sequence arrangement in the telomeres of the germ-line micronucleus in Tetrahymena thermophila. Genes Dev 9:59-71
Kapler, G M; Orias, E; Blackburn, E H (1994) Tetrahymena thermophila mutants defective in the developmentally programmed maturation and maintenance of the rDNA minichromosome. Genetics 137:455-66
Kapler, G M; Blackburn, E H (1994) A weak germ-line excision mutation blocks developmentally controlled amplification of the rDNA minichromosome of Tetrahymena thermophila. Genes Dev 8:84-95
Lundblad, V; Blackburn, E H (1993) An alternative pathway for yeast telomere maintenance rescues est1- senescence. Cell 73:347-60
Lee, M S; Gallagher, R C; Bradley, J et al. (1993) In vivo and in vitro studies of telomeres and telomerase. Cold Spring Harb Symp Quant Biol 58:707-18
Blackburn, E H (1992) Telomerases. Annu Rev Biochem 61:113-29

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