Alternative processing of scrambled genes generates protein diversity in the ciliate Chilodonella uncinata

Abstract

In ciliates, chromosomal rearrangements occur during the development of the somatic macronuclear genome from the germline micronuclear genome. These rearrangements are extensive in three ciliate classes -Armophorea, Spirotrichea and Phyllopharyngea -generating a macronucleus with up to 20,000,000 gene-sized chromosomes. Previously, we have shown that these three classes also share elevated rates of protein evolution relative to other ciliates. To assess the evolution of germline-limited sequences in the class Phyllopharyngea, we used a combination of traditional and walking PCR to analyze micronuclear copies of multiple genes from two lines of the morphospecies Chilodonella uncinata for which we had previously characterized macronuclear sequences. Analyses of the resulting data yield three main results: 1) conserved macronuclear (somatic) regions are found within rapidly evolving micronuclear (germline) regions; 2) gene scrambling exists within this ciliate lineage; and 3) alternative processing of micronuclear regions yields diverse macronuclear β-tubulin paralogs. To our knowledge, this is the first study to demonstrate gene scrambling outside of the non-sister class Spirotrichea, and to show that alternative processing of scrambled genes generates diversity in gene families. Intriguingly, the Spirotrichea and Phyllopharyngea are also united in having transient 'giant' polytene chromosomes, gene-sized somatic chromosomes, and elevated rates of protein evolution. We hypothesize that this suite of characters enables these ciliates to enjoy the benefits of asexuality while still maintaining the ability to go through sexual cycles. The data presented here add to the growing evidence of the dynamic nature of eukaryotic genomes within diverse lineages across the tree of life.

Evolution of Germline-Limited Sequences in Two Populations of the Ciliate Chilodonella uncinata

“…The structure of these loci is similar to the micronuclear structure of other C. uncinata loci examined, however no gene scrambling was found in any of the loci described here ( Fig. 1; Zufall and Katz 2007;Katz and Kovner 2010). For two additional genes, we were unable to recover micronuclear sequence (data not shown); it is possible that this failure was due to gene scrambling.…”

“…One of the genes, PK, contains three very short (19-21 bp) introns. Short IES and rare introns now appear to be a general feature of the C. uncinata genome (Katz et al 2003;Zufall and Katz 2007;Katz and Kovner 2010), similar to other ciliates with White triangles in PK MDS3 indicate the location, and approximate size, of introns. Beneath EIF2G, PP, and PK are shown the polymorphic sites in the US alleles and the structure of clones that were recombinants between alleles and/or contained some, but not all IES (''partial'' excision).…”

“…Previous studies of variation in IES suggest a pattern of rapid divergence in IES sequence (Seegmiller et al 1996;DuBois and Prescott 1997;Ardell et al 2003;Huvos 2007;Mollenbeck et al 2006;Katz and Kovner 2010). Because these sequences are non-coding, the evolutionary forces that would result in this rapid diversification are unclear.…”

“…Because these sequences are non-coding, the evolutionary forces that would result in this rapid diversification are unclear. The presence of conserved motifs among IES in some species, including C. uncinata, suggests a possible role of primary sequence in regulating IES excision Katz and Kovner 2010), however in other species, IES excision is dependent on RNA template-guided comparison of somatic and germline genomes (Mochizuki and Gorovsky 2004;Nowacki et al 2008;Duharcourt et al 2009). …”

“…In some ciliates, including Chilodonella uncinata, the subject of this study, this process results in macronuclei with thousands of genesized chromosomes (Prescott 1994;Riley and Katz 2001). Such extensive genome fragmentation in ciliates is associated with the presence of highly polytenized chromosomes (Ammermann 1987), gene scrambling (Nowacki and Landweber 2009;Katz and Kovner 2010), and elevated rates of protein evolution .…”