NOVEL GENETIC MANIPULATION APPROACHES TO INVESTIGATE GENOMIC REARRANGEMENT IN OXYTRICHA TRIFALLAX

Abstract

The dual nuclear structure of ciliates, and the derivation of the somatic nucleus from the germline nucleus makes ciliates great model system for studying genomic rearrangement. Oxytricha trifallax is a ciliate with a complex genomic rearrangement process, converting megabase sized conventional chromosomes into 16,000 kilobase sized unique chromosomes. While Oxytricha trifallax is an excellent model organism for studying complex programmed genomic rearrangement, the unusual nature of its dual nuclear structure makes conventional genetic manipulation approaches challenging or inaccessible in Oxytricha. In this thesis, I describe various approaches for genetic manipulation I have developed and their implications to Oxytricha biology.

In my thesis, I show that injecting synthetic DNA molecules homologous to native somatic chromosomes into Oxytricha at different stages in its life cycle result in divergent outcomes. Injections of synthetic chromosomes during its genomic rearrangement programs the deletion of those somatic chromosomes from the next generation. This phenomenon is robust across a variety of different somatic chromosomes. In addition, by backcrossing the programmed chromosome deletion lines to parental wild type, the programmed deletion can be inherited, implying an unidentified mechanism of elimination from soma through epigenetic inheritance in Oxytricha.

While injections of artificial chromosomes during genomic rearrangement cause deletion, injections during regular growth incorporate the construct into the somatic nucleus. These introduced artificial chromosomes are stably maintained, transcribed, and successfully translated. In addition, they can be used for various applications including characterizing the functional role of Oxytricha genes with an example of such applications shown here. In essence, these phenomena provide biological insights and are exploitable as means to investigate in previously unavailable ways.