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.
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