Micronuclear DNA Fragmentation: A Model of Genome Rearrangement

Abstract

The ciliate Oxytricha trifallax engages in a genome-wide rearrangement process whereby its germline undergoes extensive removal of internal eliminated sequences (IESs) and unscrambling of macronuclear-destined sequences (MDSs). During conjugation, maternal RNA transcripts are transported to the developing macronucleus and perform sequence-dependent comparison between germline and somatic genomes (Nowacki et al. 2008). While templates are indispensable to genome assembly, little is known about the events surrounding the micronuclear DNA. The pointer-guided folding model proposes that pairs of repeats called pointers at IES-MDS junctions participate in recombination (Prescott et al. 2003). This study proposes an alternate model whereby long micronuclear DNA is fragmented first before being guided into the correct order on the templates. Our results demonstrate the existence of IES-containing fragments during rearrangement that are preferentially spliced near IES ends. After IES excision, internal telomeres are added to MDSs for further processing. In addition, a novel class of IES was identified and found to have internal telomeres as well. To experimentally verify the model, synthetic MIC fragments were injected into the developing macronucleus to see whether they are capable of assembling into nanochromosomes. The fragments failed to incorporate, although there is evidence that they can be stably maintained across several generations of vegetative growth. A terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay was also performed to directly detect the existence of MIC fragments but failed to produce a signal in the developing macronucleus. Overall, our results show that genome rearrangement displays features that are consistent with the fragmentation model, but further experiments are required to validate the model’s predictions.