Regulatory Sequences for the Amplification and Replication of the Ribosomal DNA Minichromosome in Tetrahymena thermophila

Abstract

We have analyzed the cis-acting sequences that regulate rRNA gene (rDNA) replication in Tetrahymena thermophila. The macronucleus of this ciliated protozoan contains 9,000 copies of a 21-kbp minichromosome in the form of a palindrome comprising two copies of the rDNA. These are derived 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 transformation vector and carried out a deletion analysis to determine the minimal sequences required for replication, amplification, and/or stable maintenance of the rDNA molecule. Using constructs containing progressively longer deletions, we show that only a small portion (ϳ900 bp) of the rDNA is needed for extrachromosomal replication and stable maintenance of this molecule. This core region is very near but does 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 domains (D1 and D2), one of which (D1) corresponds to the physical origin of replication determined previously. Deletion of both domains abolishes replication, whereas deletion of either domain allows the molecules to replicate, indicating that only one domain is required. In addition to this core region, we have found several DNA segments, including a tandem array of a 21-nucleotide repeat (type II repeats) and sequences within the rRNA coding region, that play distinctive and important roles in maintaining the rDNA at a high copy number.

TIF1 Represses rDNA Replication Initiation, but Promotes Normal S Phase Progression and Chromosome Transmission inTetrahymena

“…The 1.9-kb 5Ј nontranscribed spacer (5Ј NTS) is necessary and sufficient for developmentally programmed amplification and cell cycle-regulated replica-tion of the macronuclear rDNA minichromosome (reviewed in Kapler et al, 1996). Origin-proximal and distal type I elements act in concert to control replication that initiates within two nucleosome-free regions ( Figure 1A; Larson et al, 1986;Blomberg et al, 1997;Zhang et al, 1997;Gallagher and Blackburn, 1998;Reischmann et al, 1999). In addition to their role in replication initiation, type I elements induce the pausing of replication forks at adjacent pause site elements (PSEs; MacAlpine et al, 1997).…”

Characterization of a Novel Origin Recognition Complex-Like Complex: Implications for DNA Recognition, Cell Cycle Control, and Locus-Specific Gene Amplification

“…By comparison, non-rDNA chromosomes achieve a copy number of Ϸ45. The 1.9-kb 5Ј nontranscribed spacer is both necessary and sufficient for replication of artificial rDNA minichromosomes (6,41,43). Dispersed type I and pause site elements are required for rDNA replication (16,25,47).…”

Tetrahymena-specific biomass evaluation with a DNA-based method

“…2) for our further biomass determination. As the 18S rRNA gene copies of Tetrahymena are relatively stable 8,25 , here we applied 18S rDNA as a marker to explore the Tetrahymena-specific biomass evaluation. Although relative lower of Tetrahymena DNA (0.14 ng) could be detected in Q-PCR analysis, we did not use that lower level of DNA in order to get a relative higher Ct value for sample quantification.…”

“…With rapid development of molecular techniques and dramatically increased collection of nucleotide sequences, it is easy to design primers targeting different taxonomic levels to amplify particular genes. Some genes present stable copies per cell (e.g., rRNA gene in unicellular Tetrahymena) 8 , and some genes even can be used as DNA barcoding for taxonomic classification (e.g., cytochrome c oxidase I gene for animals) 9 . Therefore, taxa-specific biomass perhaps can be realized with a bridge of specifically amplified gene.…”