REGULATORY SEQUENCES FOR THE AMPLIFICATION AND REPLICATION OF THE RIBOSOMAL DNA MINICHROMOSOME IN TETRAHYMENA-THERMOPHILA

We have analyzed the cis-acting sequences that regulate rRNA gene (rDN A) replication in Tetrahymena thermophila. The macronucleus of this ci liated protozoan contains 9,000 copies of a 21-kbp minichromosome in t he form of a palindrome comprising two copies of the rDNA, These are d erived from a single chromosomally integrated copy during conjugation through selective amplification and are maintained by replicating once per cell cycle during vegetative growth. We have developed a transfor mation vector and carried out a deletion analysis to determine the min imal sequences required for replication, amplification, and/or stable maintenance of the rDNA molecule. Using constructs containing progress ively longer deletions, we show that only a small portion (similar to 900 bp) of the rDNA is needed for extrachromosomal replication and sta ble maintenance of this molecule. This core region is very near but do es not include the rRNA transcription initiation site or its putative promoter, indicating that replication is not dependent on normal rRNA transcription, It includes two nearly identical nuclease-sensitive dom ains (D1 and D2), one of which (D1) corresponds to the physical origin of replication determined previously, Deletion of both domains abolis hes replication, whereas deletion of either domain allows the molecule s to replicate, indicating that only one domain is required, In additi on to this core region, we have found several DNA segments, including a tandem array of a 21-nucleotide repeat (type II repeats) and sequenc es within the rRNA coding region, that play distinctive and important roles in maintaining the rDNA at a high copy number.