Transformation with Artificial Chromosomes in Oxytricha trifallax and Their Applications

Abstract

Abstract Oxytricha trifallax, like other ciliates, has separate germline and somatic nuclei. The diploid germline genome in the micronucleus is composed of long conventional chromosomes. The macronucleus contains a somatic genome which is naturally fragmented into thousands of kilobase-sized chromosomes. Here, we develop a method to stably incorporate artificial chromosomes into the macronucleus. We report two cases of successful transformation and demonstrate the use of somatic transformation to investigate gene regulation and gene function in Oxytricha. We show that the transformed artificial chromosomes are maintained through multiple asexual divisions. Furthermore, they support the transcriptional regulation of the native chromosome from which they were derived and are translated to produce functional proteins. To test if transformed chromosomes are amenable to practical applications, we generated a tagged version of a representative gene (AL1) and used it to co-precipitate associated proteins. This revealed an association with nucleic acid binding proteins, specifically RNA-binding proteins, and RNA immunoprecipitation of AL1 revealed its association with multiple RNAs. The use of artificial chromosomes in Oxytricha enables an array of genetic and molecular biological assays, as well as new avenues of inquiry into the epigenetic programming of macronuclear development and genome rearrangement.