Ciliate Biology Background

Ciliated protists (Phylum Ciliophora) are microbial eukaryotes, characterized by the presence of cilia used for locomotion, and they all share two types of nuclei: a somatic nucleus - macronucleus (MAC) - which provides templates for the transcription of all genes required for vegetative growth, and a genetic nucleus - micronucleus (MIC) - used for the exchange of meiotic products during sexual reproduction.

Figure 1: In the macronucleus, gene-sized nanochromosomes assemble from their scrambled building blocks; telomere repeats (dark blue) mark and protect the surviving ends.

In the micronucleus, the precursor DNA segments (Macronuclear-Destined Sequences, MDS) for all somatic chromosomes are dispersed over the long chromosome, and interrupted by Internally-Eliminated Sequences (IES).

During conjugation (sexual reproduction), haploid gametic nuclei exchange between pairs of mating cells form a diploid zygotic nucleus, a copy of which develops into a new MIC and MAC. DNA in the MIC remains organized in large chromosomes. In contrast, the much smaller chromosomes in the MAC genome form via extensive fragmentation, elimination, and even rearrangement of germline DNA, coupled to DNA amplification and telomere addition (Prescott 1994). This process produces a set of over 16,000 small acentric MAC chromosomes in Oxytricha (Swart et al. 2013) and 181 in Tetrahymena (Coyne et al. 2012).

The extent of MAC genome reorganization varies greatly among ciliate species. In ciliates belonging to the class Spirotrichea (which includes Oxytricha trifallax), the level of DNA processing in the formation of a new MAC is extraordinary: the original zygotic chromosomes are fragmented into over 225,000 DNA pieces (MDSs), and approximately 90% of the DNA complexity is lost (Chen et al. 2014). The resulting MAC chromosomes – sometimes referred to as "nanochromosomes" – are amplified to thousands of copies each (Prescott 1994). In spirotrichs, approximately 90% of MAC chromosomes encode a single gene, flanked at the 5' and 3' ends by very short (average 50 bp) untranslated regions plus telomeres (Swart et al. 2013). The size of these molecules ranges from approximately 0.31 kb to 66 kb (Swart et al. 2013).

In ciliates such as Oxytricha and Paramecium, genes in the MIC are interrupted by the non-coding, A–T rich IES segments that must be removed. IESs in Tetrahymena mostly fall between genes, with few exceptions (Fass et al. 2011). Approximately 20% of Oxytricha's macronuclear contigs contain MDSs that are present in a permuted order or orientation, relative to the precursor MDSs in the micronucleus (Chen et al. 2014). These MDSs rearrange during MAC development according to a long noncoding RNA templates as guides (Nowacki et al. 2008). This added layer allows Oxytricha to rebuild its functional somatic chromosomes from a scrambled genome (see Figure 1).

The last few nucleotides of a MDS are repeated in the beginning of the next consecutive MDS; these sequences are called the pointers, or junction sequences. DNA recombination between these 2-20 bp direct repeats in Oxytricha (2bp TA repeats in Paramecium) leaves precisely one copy in the macronucleus. Except for the longest pointers, however, these short sequences are usually present in multiple locations in the precursor MIC gene loci (Landweber et al. 2000). Hence, this underscores the need for an RNA-guided and error-correcting mechanism that accurately establishes and maintains wild-type versions of somatic genes across generations (Nowacki et al. 2008).

For a more thorough review of our current knowledge of the mechanism of RNA guided DNA rearrangement and DNA descrambling in the ciliate Oxytricha, see (Yerlici and Landweber 2014).


References:

  1. Yi Feng, Rafik Neme, Leslie Y. Beh, Xiao Chen, Jasper Braun, Michael Lu, Laura F. Landweber (2022). Comparative genomics reveals insight into the evolutionary origin of massively scrambled genomes. bioRxiv, 2022.05.09.490778. [article]
  2. Chen, X., Jung, S., Beh, L. Y., Eddy, S. R., & Landweber, L. F. (2015). Combinatorial DNA Rearrangement Facilitates the Origin of New Genes in Ciliates. Genome biology and evolution, 7 (10), 2859–2870. [pubmed] [article]
  3. Chen X., Bracht J.R., Goldman A.D., Dolzhenko E., Clay D.M., Swart E.C., Perlman D.H., Doak T.G., Stuart A., Amemiya C.T., Sebra R.P., Landweber L.F. (2014) The Architecture of a Scrambled Genome Reveals Massive Levels of Genomic Rearrangement during Development. Cell 158 (5): 1187–98.
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  4. Coyne R.S., Stover N.A., and Miao W. (2012) Whole genome studies of Tetrahymena, In Collins K. (ed.) Methods in Cell Biology, 109: Tetrahymena Thermophila, Academic Press, pp. 53–81.
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  5. Fass J.N., Joshi N.A., Couvillion M.T., Bowen J., et al. (2011) Genome-Scale Analysis of Programmed DNA Elimination Sites in Tetrahymena thermophila. G3 1: 515–522.
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  6. Landweber L.F., Kuo T.C., Curtis E.A. (2000) Evolution and assembly of an extremely scrambled gene. PNAS 97: 3298–3303.
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  7. Nowacki M., Vijayan V., Zhou Y., Schotanus K., Doak T.G., Landweber L.F. (2008) RNA-mediated epigenetic programming of a genome-rearrangement pathway. Nature 451: 153–158.
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  8. Prescott D.M. (1994) The DNA of Ciliated Protozoa. Microbiol. Rev. 58 (2): 233–267.
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  9. Swart E.C., Bracht J.R., Magrini V., Minx P., Chen X., Zhou Y., Khurana J.S., Goldman, M. Nowacki A.D., Schotanus K., et al. (2013) The Oxytricha trifallax macronuclear genome: a complex eukaryotic genome with 16,000 tiny chromosomes. PLoS Biol. 11 (1): e1001473
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  10. Yerlici T.V., Landweber L.F. (2014) Programmed Genome Rearrangements in the Ciliate Oxytricha. Microbiol. Spectr. 2 (6): MDNA3-0025-2014
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