Origin Usage during Euplotes Ribosomal DNA Amplification

Abstract

The macronuclear genome of the ciliate Euplotes is comprised of millions of small linear DNA molecules that have telomeres on each end. These molecules are generated during the sexual stage of the life cycle, when the new macronucleus is formed by a series of DNA processing events and multiple rounds of DNA amplification. We have used two-dimensional gels to compare the location of the replication origins used during vegetative growth and the two periods during macronuclear development when DNA amplification takes place. When we examined the pattern of ribosomal DNA (rDNA) replication intermediates, we observed almost identical Y arcs regardless of when in the Euplotes life cycle the DNA was isolated. No bubble or bubble-to-Y arcs could be detected. This indicates that replication of the macronuclear rDNA initiates at or near the telomere even when these molecules are being differentially amplified. Since replication rarely initiated from both ends of the rDNA, we examined the direction of replication fork movement to determine which end of the rDNA served as the origin. Fork movement gels indicated that replication initiated at the 5 end. As transcription also starts near the telomere at the 5 end, our findings suggest that the telomere and the promoter region cooperate to recruit Euplotes replication initiation complexes.