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.
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