Evolution and assembly of an extremely scrambled gene

Abstract

The process of gene unscrambling in hypotrichous ciliates represents one of nature's ingenious solutions to the problem of gene assembly. With some essential genes scrambled in as many as 51 pieces, these ciliates rely on sequence and structural cues to rebuild their fragmented genes and genomes. Here we report the complex pattern of scrambling in the DNA polymerase alpha gene of Stylonychia lemnae. The germline (micronuclear) copy of this gene is broken into 48 pieces with 47 dispersed over two loci, with no asymmetry in the placement of coding segments on either strand. Direct repeats present at the boundaries between coding and noncoding sequences provide pointers to help guide assembly of the functional (macronuclear) gene. We investigate the evolution of this complex gene in three hypotrichous species.

Figure 1

(a) A schematic map of the macronuclear DNA-polymerase α gene of S. lemnae. The 48 MDS are shown as colored boxes. Green and blue MDSs are both present on the major locus, but in opposite orientation. Red MDSs are derived from the minor locus. The yellow MDS is missing. Pointer sequences are indicated by black vertical bars, telomeres by hatched bars. PCR primers indicated by small arrows; degenerate primers, in magenta, were based on an alignment of this gene in 10 hypotrich species (33). The macronuclear copy is derived from the micronuclear copy (large arrow). (b) A schematic alignment of the micronuclear genes encoding the large catalytic subunit of DNA-polymerase α in S. lemnae, O. trifallax (7), and O. nova (6), with MDSs aligned based on predicted amino acid sequences; open boxes within MDSs indicate gaps in alignment. MDSs are drawn to scale with the length (excluding pointer sequences when available) underlined in italics and parentheses. Green and blue MDSs are encoded on opposite strands of the major locus, indicated by the inversion. Red MDSs are derived from the minor locus. Black vertical bars indicate pointers. IESs are indicated by thin lines, not to scale, but IES length is given in italics and brackets. IES-specific primers are circled. The yellow highlighted region indicates 193/199 bp overlap between the IES that marks the 5′ end of the major locus and MDS 30 in the minor locus of S. lemnae.

Figure 2

Proposed steps in the evolution of the scrambled DNA polymerase α gene through a series of recombinations between MDSs and IESs. MDSs are shown as boxes (black numbers and color stripes reference cognate regions in the extant S. lemnae gene; Fig. 1); IESs or noncoding DNA are drawn as thin lines; and pointers are shown as black bars. The first general step takes place in a nonscrambled ancestral version of the gene [shown here with one IES between MDS 1–2 because this IES is conserved in all three species and IESs flanked by TA repeats are the most common in other ciliate species (1)]. Reciprocal exchange at the ×s between an MDS and upstream noncoding DNA creates a scrambled hypothetical MDS order (3-1–2-4; new MDSs in purple) and an inversion (up arrow) between 3 and 1; these MDSs are numbered from the 5′ end (… ) and italicized if on the reverse strand. The juxtaposition of MDSs and IESs now promotes homologous recombination at chance matches, perhaps even at favored sites (Table 1), creating the new pattern 5–3-1–2-4–6. Continued exchange at the ×s, in any order (not just the reasonable one shown here), propagates the odd/even MDS splitting: 17–15-13–11-9–7-5–3-1–2-4–6-8–10-12–14-16–18 is shown as one evolutionary intermediate, finally reaching a dense set of 43 contiguous MDSs very similar to O. nova. At any stage in an ancestor of all three species there could be insertion of an IES in the last MDS and finally translocation of a cluster of MDSs at the 5′ end (probably maintained as a polymorphism initially, allowing substantial overlap in the orange region) by reciprocal exchange with a distant DNA fragment. The precise ordering of the previous steps does not matter, but the most recent step is splitting of MDSs 5–7 (by exchange with an unknown fragment) and 11–13 in an ancestor of S. lemnae and O. trifallax, leading to the pattern in S. lemnae.

Figure 3

Comparison of MDS versus IES length (nt) in S. lemnae for exchanged regions flanked by the same set of pointers (such as x_ij and x_jk). [Pointer length is not included in the comparison; hence, this graph compares the length of each α and opposing ɛ (see text).] The data are listed in Table 2. Mean MDS length is 49 nt; mean IES length is 23 nt.