MBE Advance Access originally published online on September 14, 2005
Molecular Biology and Evolution 2006 23(1):4-6; doi:10.1093/molbev/msj017
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Letter |
Complex Germline Architecture: Two Genes Intertwined on Two Loci
Department of Ecology and Evolutionary Biology, Princeton University
E-mail: lfl{at}princeton.edu.
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Abstract |
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The germline micronuclear genome of some ciliated protists can be scrambled, with coding segments disordered relative to the expressed macronuclear genome. Here, we report a surprisingly complex pair of genes that assemble from interwoven segments on two germline loci in the ciliate Uroleptus. This baroque organization requires two scrambled genes to be disentangled from each other from two clusters in the genome, one containing segments 1-2-4-5-6-8-11-13-15-16 and the other 7-9-3-10-12-14, with pieces 1–5 comprising the first gene and 6–16 the second gene. Both genes remain linked in the somatic genome on a 1.5-kb "nanochromosome." This study is the first to reveal that two genes can become scrambled during evolution with their coding segments intertwined. These twin scrambled genes underscore the beauty and exceptions of protist genome architecture, pointing to the critical need for evolutionary biologists to survey protist genomes broadly.
Key Words: micronucleus • scrambled gene • Uroleptus • hypotrich • spirotrich • ciliate
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Introduction |
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Ciliates possess two types of nuclei, a germline micronucleus and a somatic macronucleus that develop from a copy of the germline after cell mating. In spirotrichous ciliates, massive deletion and rearrangement of the 1- to 2.5-Gb germline genome constructs an
50-Mb set of approximately 2-kb tiny chromosomes (Prescott 1994
), sometimes called nanochromosomes (Doak et al. 2003) because of their size and because they typically contain just one gene each. These together comprise the gene-dense somatic genome. The process of deletion of up to 98% of the germline DNA removes internal eliminated segments (IES) that interrupt genes, as well as transposons and intergenic DNA. The remaining macronuclear-destined segments (MDS), containing mostly coding DNA with limited regulatory sequences and introns, join together to form the nanochromosomes. In some cases, the germline order of coding segments is permuted, requiring their decryption (unscrambling) to create functional genes in the macronucleus (Prescott 2000). Scrambled segments can be present on either strand within a locus or even dispersed over unlinked loci in the germline (Ardell, Lozupone, and Landweber 2003). While three scrambled genes have been extensively studied to date (
-telomere–binding protein [Mitcham, Lynn, and Prescott 1992]; DNA polymerase [Hoffman and Prescott 1996; Landweber, Kuo, and Curtis 2000]; and actin I [Greslin et al. 1989; Hogan et al. 2001; Dalby and Prescott 2004]), no new scrambled genes have been reported for almost a decade.
To search for new scrambled genes, we constructed a small macronuclear library from the spirotrichous ciliate Uroleptus, known to have scrambled genes (e.g., Dalby and Prescott 2004; Chang et al. 2005), and randomly selected 11 clones whose sequences were used to search for their counterparts in the germline micronuclear DNA by polymerase chain reaction (PCR). One of the clones contained a 1,554-bp macronuclear chromosome, including 36-bp telomeric repeats (C4A4) at each end, with two predicted open reading frames that we confirmed by 5'- and 3'-rapid amplification of cDNA ends (RACE) (fig. 1D). Because few two-gene chromosomes have been described (e.g., Seegmiller, Williams, and Herrick 1997), we examined this molecule further. The upstream mRNA putatively encodes a 77-aa peptide of unknown function (positions 146–379), which shares no significant database matches at the protein or nucleotide level. The downstream mRNA encodes a protein of 198 aa with high similarity to eukaryotic 60S ribosomal protein L13 (58% identical/75% similar to ribosomal protein L13a in Homo sapiens).
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We determined the germline organization of these genes by a variation of walking PCR (Myrick and Gelbart 2002
) that is successful on ciliate micronuclear DNA. The genetic map splits this gene into two regions (fig. 1C and table 1). The overall germline architecture is quite surprising: while both genes are located in tandem on a single 1.5-kb macronuclear nanochromosome, their 16 segments are scrambled and comingled on two clusters. One locus contains 10 segments in the order 1-2-4-5-6-8-11-13-15-16 and the other contains 6 segments in the order 7-9-3-10-12-14, with pieces 1–5 comprising the first gene and 6–16 the second gene. Pointer sequence repeats of 3–14 bp are always present at the end of segment n and the beginning of segment n + 1, with one copy of the repeat retained in the macronuclear sequence (table 1) and longer pointers between scrambled segments that have to be reordered to make a functional copy. These pointer sequence repeats are thought to participate in homologous DNA recombination during macronuclear development (Prescott 2000). Curiously, we also found a duplicated region of 1859 bp (91% identity) between these two loci (highlighted in fig. 1C) that probably indicates where these two loci broke apart during evolution (fig. 1A and B) because this region contains paralogous copies of four segments for the first gene. Three of these segments contain frameshifts and are too divergent to contribute to a functional gene (table 1). So far, these two genes have not been found to be linked in the macronucleus in three other spirotrichous ciliates that contain scrambled genes (A. J. Li, W.-J. Chang, and L. Landweber, unpublished data), suggesting that this unusual architecture may be specific to the Uroleptus lineage.
These twin scrambled genes underscore the complexities of ciliate genome architecture, with segments for more than one gene woven together during germline evolution. With the genome project of a spirotrichous ciliate, Oxytricha trifallax (Sterkiella histriomuscorum), currently underway (Doak et al. 2003), we anticipate that more surprises are yet to come.
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Methods |
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Uroleptus sp. (similar to Uroleptus gallina) was isolated from soil in a meadow close to Plainsboro township in Princeton, N.J., and grown as previously described (Chang et al. 2004
). Monoclonal culture of Uroleptus sp. was achieved by isolating one single cell and growing it into a population. Macronuclei and micronuclei were isolated from vegetative Uroleptus cells as previously described (Chang et al. 2004). DNA was purified by phenol/chloroform extractions and treated with RNase A to remove RNA. Micronuclear DNA was further purified from residual macronuclear DNA by low melting point agarose gel electrophoresis. Uroleptus macronuclear DNA was treated with exonuclease I (New England BioLabs, Ipswich, Mass.) to trim telomeric overhangs and with shrimp alkaline phosphatase (Promega, Madison, Wis.) to remove 5'-phosphates. Blunt-ended DNA was then cloned into a pCR4Blunt-TOPO vector (Invitrogen, Carlsbad, Calif.), and 11 clones were randomly selected for sequencing. The sequence in figure 1D (GenBank AY875978) was verified by several independent PCR clones, as in Chang et al. (2004). Southern hybridization confirmed that there were no alternatively processed versions of this macronuclear chromosome (data not shown). Micronuclear organizations of these two genes were determined by using traditional PCR against 20 ng micronuclear DNA. Sequences flanking these micronuclear PCR fragments were obtained by Universal Fast Walking (UFW) PCR (Myrick and Gelbart 2002). Sequences of the major (accession number AY875979) and minor (accession number AY875980) micronuclear loci including flanking sequences are deposited in GenBank. RNA isolation and 5'- and 3'-RACE were as described (Chang et al. 2004). All primer sequences are available in Supplementary Material online. |
Supplementary Material |
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Primer sequences and additional detail of methods are available at Molecular Biology and Evolution online (http://www.mbe.oxfordjournals.org/).
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Acknowledgements |
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We thank Jingmei Wang for ciliate culture, Mark Daley and Ian McQuillan for suggesting the representation in fig. 1C, and Glenn Herrick for suggesting and Kyl Myrick for discussing UFW PCR. This study was supported by National Institute of General Medical Sciences grant GM59708 and National Science Foundation grant 0121422. S.K. thanks H. Nick for comments.
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Footnotes |
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Jennifer Wernegreen, Associate Editor
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References |
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Ardell, D. H., C. A. Lozupone, and L. F. Landweber. 2003. Polymorphism, recombination and alternative unscrambling in the DNA polymerase alpha gene of the ciliate Stylonychia lemnae (Alveolata; class Spirotrichea). Genetics 165:1761–1777.
Chang, W. J., P. D. Bryson, H. Liang, M. K. Shin, and L. F. Landweber. 2005. The evolutionary origin of a complex scrambled gene. Proc. Natl. Acad. Sci. USA (in press).
Chang, W. J., N. A. Stover, V. M. Addis, and L. F. Landweber. 2004. A micronuclear locus containing three protein-coding genes remains linked during macronuclear development in the spirotrichous ciliate Holosticha. Protist 155:245–255.[Medline]
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Hogan, D. J., E. A. Hewitt, K. E. Orr, D. M. Prescott, and K. M. Muller. 2001. Evolution of IESs and scrambling in the actin I gene in hypotrichous ciliates. Proc. Natl. Acad. Sci. USA 98:15101–15106.
Landweber, L. F., T. C. Kuo, and E. A. Curtis. 2000. Evolution and assembly of an extremely scrambled gene. Proc. Natl. Acad. Sci. USA 97:3298–3303.
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