Minichromosomal DNA replication in the macronucleus al the hypotrichous ciliate Stylonychia lemnae is independent off chromosome-internal sequences

The origins of DNA replication in prokaryotes and eukaryotes are typically defined by cis-acting sequences. However, in ciliates, evidence suggests th at the replication of short macronuclear minichromosomes may not require su ch determinants. In hypotrichous ciliates, macronuclei contain millions of gene-sized minichromosomes, which generally have a single protein-coding re gion, two short noncoding flanks and, on each end, a short telomere consist ing of a double-stranded repeat region and a single-stranded 3 ' overhang. Electron microscopic studies that showed that replication of minichromosome s initiates at or near telomeres and the discovery of a primase activity sy nthesizing RNA primers over the whole 3 ' telomeric overhang in vitro sugge sted that minichromosome replication starts directly at telomeres. Converse ly, many minichromosomes contain an AT-rich, semi-conserved, palindromic se quence motif in their subtelomeric regions and it has been proposed that th is motif is involved in regulating minichromosomal replication. To analyze what sequences or structures of the minichromosomes are essential for DNA r eplication, we stably transfected genetically modified al-tubulin-encoding minichromosomes into the hypotrichous ciliate Stylonychia lemnae. Cotransfe ction of mutated and control minichromosomes revealed that noncoding region s can be deleted or replaced with unrelated sequences without affecting min ichromosome replication efficiency in vegetatively growing cells. Similarly , replacement of the coding region resulted in a minichromosome that was st ably maintained in transfected cells at the same high copy number for many months. In contrast, alpha1-tubulin-encoding minichromosomes without telome res were rapidly lost after transfection. Hence, DNA replication of the al- tubulin-encoding minichromosome does not depend on chromosome-internal sequ ences but may depend on telomeres.