CIRCULAR RDNA REPLICONS PERSIST IN TETRAHYMENA-THERMOPHILA TRANSFORMANTS SYNTHESIZING GGGGTC TELOMERIC REPEATS

Site-directed mutagenesis of the telomerase RNA from Tetrahymena therm ophila was used previously to demonstrate the templating function of a sequence within this RNA; this sequence specifies the sequence of tel omeric DNA in vivo. The possible functional importance of a phylogenet ically conserved nucleotide outside the telomerase RNA template region was investigated by a similar experimental approach. The telomerase R NA gene was altered by site-directed mutagenesis, cloned in a circular selectable transformation vector consisting of an rRNA gene carrying a selectable drug resistance marker, and introduced into macronuclei o f vegetatively dividing Tetrahymena thermophila by microinjection. Cha nging an invariant A to U at position 16 of the telomerase RNA (A16U) had no effect detectable by phenotype on telomerase function in vivo. However these experiments showed that a telomerase template alteration that dictates the synthesis of the mutant telomeric DNA sequence GGGG TC leads to a profound change in the population of rDNA replicons. The addition of GGGGTC mutant repeats leads to selective pressure for the loss of high copy linear rDNA, and the rRNA genes are maintained in t he form of the circular rDNA replicons introduced during transformatio n.