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

Abstract

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

α-Tubulin Minichromosome Promoters in the Stichotrichous Ciliate Stylonychia lemnae

“…These pTZ18U vectors contained, inserted into their SmaI restriction sites, the complete double-stranded S. lemnae ␣1 tubulin minichromosome sequence fused to an ApaI restriction site on either end. Besides several inserted restriction sites (33), the main manipulation of the minichromosomal sequence was the insertion of the 19-nucleotide tag sequence 5Ј-TTCCATGGTATGGCGCCAG-3Ј (includes an NcoI restriction site) at position ϩ103 relative to the transcription initiation site (TIS). pTuba1h-st is a derivative of pTuba1g-st and contains an additional SmaI restriction site immediately after the 5Ј telomeric repeats.…”

“…Linear Tuba1-lt (long-tag) and Tuba1-st minichromosomes were obtained by restriction digestion of the corresponding plasmids and Klenow treatment as described previously (33). They possessed the correct double-stranded telomeric repeats on either end but no single-stranded overhangs.…”

“…They possessed the correct double-stranded telomeric repeats on either end but no single-stranded overhangs. lt and st minichromosomes were mixed in equimolar amounts and microinjected directly into the macronuclei of immobilized S. lemnae cells as described elsewhere (32,33). For each mutation, a minimum of three clonal cell lines were raised from individual transfected cells.…”

Origin Usage during Euplotes Ribosomal DNA Amplification

“…This could explain why in both mated and vegetatively growing cells, replication does in some instances initiate at the end of the molecule where transcription terminates (seen in the molecules undergoing random termination). It could also explain why no conserved origin sequence has been found in any of the hypotrichous ciliates and why in Stylonychia the sequence of the 5Ј UTR, 3Ј UTR, and coding region can be completely replaced with an unrelated sequence (35). In the absence of a promoter complex, the telomere would still recruit the replication initiation factors and the replication complex would still form, but more slowly.…”

“…However, sequencing of Euplotes and Stylonychia macronuclear DNA molecules has not revealed a conserved sequence that might function as an origin (2,17,23). Moreover, Stylonychia molecules that have had the 5Ј untranslated region (UTR), 3Ј UTR, or coding region replaced by an unrelated sequence can be maintained at a normal copy number (35). These findings have raised the possibility that the telomere itself may serve as the origin during vegetative growth.…”