This proposal focuses upon the control of the replication, amplification and segregation of one specific macronuclear replicon, the rRNA gene(rDNA) of T. thermophila. T. thermophila has been studied extensively(l), because of the ease of biochemical, molecular and genetic studies with this species, and it is a model for other ciliates. The overall goal is to continue to exploit the unique advantages afforded by the Tetrahymena system and its rDNA for molecular genetic studies on the control of DNA replication. We plan to: 1) Analyze the replication, maintenance and segregation properties of rDNA -origin plasmids in Tetrahymena macronuclei, and determine the cis-acting sequences important for these in vivo properties; 2) obtain and characterize additional macronuclear rDNA maturation and/or maintenance mutants (rmm mutants), using a genetic strategy we established previously for obtaining rmm mutants. The emphasis will be on new mutations affecting amplification or the early steps in the rDNA maturation process. We will relate the findings on these mutants to previous findings on both mutations affecting vegetative cell rDNA replication and transformation with rDNA origin-based vectors; 3) Modify the genetic strategy to obtain rmm mutants by using an anisomycin-resistant C3 rDNA allele, to increase the detection sensitivity of mutations occurring at low frequencies. A long term aim is to use the genetic strategy to obtain suppressors of rmm mutations; 4) Investigate one step in rDNA maturation, de novo telomere addition to the rDNA, using transformation of parental macronuclei with a mutated telomerase RNA gene, and treatment of cells with AZT to slow rDNA maturation; 5) Determine the role in amplification of a transient form of the rDNA, the 11 kb rDNA. Arising directly out of the work we have done on the control of rDNA replication, we have developed transformation vectors for introducing other genes into the Tetrahymena macronucleus. Building on this work, to extend the utility of Tetrahymena as an experimental organism, further aims are to : 6) continue development of rDNA-origin based overexpression vectors for transforming Tetrahymena macronuclei with genes transcribed by RNA polymerases II and III; 7) construct a non-rDNA dominant selectable marker, with the aim of developing an integrative vector for gene replacement or disruption in Tetrahymena. A long term goal will be to use these vectors for complementation in vivo.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM032565-11
Application #
3281530
Study Section
Molecular Cytology Study Section (CTY)
Project Start
1983-08-01
Project End
1995-07-31
Budget Start
1993-08-01
Budget End
1994-07-31
Support Year
11
Fiscal Year
1993
Total Cost
Indirect Cost
Name
University of California San Francisco
Department
Type
Schools of Medicine
DUNS #
073133571
City
San Francisco
State
CA
Country
United States
Zip Code
94143
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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