Transformation with Artificial Chromosomes in Oxytricha trifallax and Their Applications

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.

Keywords: Ciliates; Epigenetics; Genome Rearrangement.

Figure 1

Detection and quantification of transformed nanochromosome in cell lines. Schematic maps of the transformed artificial chromosome AL1 with a C-terminal histidine-tag with the location of primers used to amplify the construct (A) and the artificial chromosome containing GFP in place of the Hsp70 gene (C). PCR amplification of genomic DNA collected from cell lines shows the successful transformation of the AL1 with a C-terminal histidine-tag (B) and the GFP constructs (D) using the detection primers. The AL1 construct was injected into the F1 progeny of JRB310 (labeled 310 in the figure) cells mated to JRB510 (510). These cells also contained a programed deletion of the entire AL1 chromosome. The GFP construct, on the other hand, was introduced into WT JRB310 cells. PCR of the native TEBP-β locus provided a loading control. Quantitative PCR shows the abundance of the transformed chromosomes of histidine-tagged (His tag) AL1 (E) and GFP (F). TEBP-α and the wild type HSP70 chromosome were quantified as controls. Error bars for the quantitative PCR results represent standard deviation.

Figure 2

Quantitation of expression patterns of transformant and relevant native genes. A) Quantitative PCR on cDNA collected from vegetative JRB310 or the GFP transformed cells (JRB310 cells transformed with the GFP in the Hsp70 nanochromosome construct) shows the levels of GFP, HSP70, and TEBP-α expression after exposure to either heat shock for 10 min (10’ HS) or control, room temperature for 10 min (10’ RT). B) Schematic map of the AL1+His-tag construct and the location of the qPCR primer used with the AL1 primer in green and the His-tag specific qPCR primers in pink. C) Quantitative PCR on cDNA collected from JRB310 x JRB510 mated cells or from the AL1+His-tag transformant mated to JRB510 at 0, 10, and 20 hr post mixing. Error bars represent the standard deviation of the three biological replicates.

Figure 3

Detection of translated protein from the transformant constructs. Fluorescence microscopy of live cells immobilized under mineral oil, for the GFP transformant cell line and JRB310. Images were taken 2 hr after incubation either at room temperature or at 37° C for 20 min (heat shock), as labeled.

Figure 4

Application of the transformed AL1 tagged protein for immunoprecipitation and identification of associated proteins. Spectrum counts of protein domains from the domains identified in the top co-immunoprecipitated proteins (exclusive to the tagged sample, greater than 25 spectra counts across the three replicates, along with the presence of only one of any set of indistinguishable proteins) from AL1 pulldown by mass spectrometry. The number in parentheses represents the number of proteins in this set that contain the domain.