Download Complex Germline Architecture: Two Genes

Complex Germline Architecture: Two Genes Intertwined on Two Loci
Shiuhyang Kuo, Wei-Jen Chang, and Laura F. Landweber
Department of Ecology and Evolutionary Biology, Princeton University
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.
Introduction
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 (a-telomere–binding protein [Mitcham,
Lynn, and Prescott 1992]; DNA polymerase a [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 1,554bp 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 twoKey words: micronucleus, scrambled gene, Uroleptus, hypotrich,
spirotrich, ciliate.
E-mail: [email protected].
Mol. Biol. Evol. 23(1):4–6. 2006
doi:10.1093/molbev/msj017
Advance Access publication September 14, 2005
Ó The Author 2005. Published by Oxford University Press on behalf of
the Society for Molecular Biology and Evolution. All rights reserved.
For permissions, please e-mail: [email protected]
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).
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 1-2-4-5-6-8-11-13-1516 and the other contains 6 segments in the order 7-9-310-12-14, 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 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,
Two Scrambled Genes Intertwined on Two Loci 5
FIG. 1.—Schematic maps of the somatic and corresponding germline genes. Segments (macronuclear-destined segments [MDS]) 1–16 are marked in
each case and numbered according to (D). (A) and (B) present an evolutionary model for the origin of the architecture in (C), based on these data, with (A) the
inferred ancestral state, after the region became scrambled but before translocation of segment 3. (B) The inferred ancestral state containing both genes on the
same locus. (C) Germline map. Segments derived from the longer germline locus are white boxes, and the shorter loci are dark gray boxes. Internal eliminated segments and flanking DNA are not drawn to scale but as solid black lines with sizes (bp) in parentheses. The flanking DNA from the end of MDS 14
to another gene downstream (white arrow in opposite orientation) is subdivided into three sections with lengths indicated in brackets. Gray highlight indicates
that the middle section (1859 bp) overlaps both loci, containing paralogous copies of segments 1, 2, 4, and 5. The geometric representation reflects the
presence of pointer sequence repeats at the end of each segment that recombine with their copy at the beginning of the next segment (table 1). (D) Somatic
map. Hatched ends represent telomeres. Arrowed lines illustrate mRNA transcripts, with dots representing mRNA cap sites (positions 69 and 721) and
arrows representing poly-adenylation sites (437, 577, and 1420). Small vertical arrows mark inferred start and stop codons for both genes.
Oxytricha trifallax (Sterkiella histriomuscorum), currently
underway (Doak et al. 2003), we anticipate that more
surprises are yet to come.
Methods
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
6
Kuo et al.
Table 1
Features of DNA Segments for Both Scrambled Genes
5# Pointer Sequencea
5# Telomere addition site
GTT
AATTCTTATCATaT
CAGGATAATtAAA
ATAAATTTT
ATA
GTATGTTCgAAAAG
CACTTGCTC
AGAAACAAGCT
AAGTTCCACGAATT
CAACCCAA
TCAGTT
GTATTTTCT
TCAGAG
TTGGCtGCATGAA
TCTCGTcGAA
Segment (MDS)
number n
3# Pointer Sequencea
MDS Length
(bp)b
Percent Similarity
to MDS n#c
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
GTT
AATTCTTATCATtT
CAGGATAATaAAA
ATAAATTTT
ATA
GTATGTTCaAAAAG
CACTTGCTC
AGAAACAAGCT
AAGTTCCACGAATT
CAACCCAA
TCAGTT
GTATTTTTCT
TCAGAG
TTGGCcGCATGAA
TCTCGTtGAA
3# Telomere addition site
3
321
65
64
46
167
24
96
1
21
7
20
9
39
144
313
100
91.6
92.3
80.8
NOTE.—MDS: macronuclear-destined segments.
a
Lowercase nucleotides indicate pointer mismatches, with the retained somatic sequence underlined.
b
MDS length excludes pointer sequence repeats.
c
See figure 1C.
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
Primer sequences and additional detail of methods are
available at Molecular Biology and Evolution online (http://
www.mbe.oxfordjournals.org/).
Acknowledgments
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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Jennifer Wernegreen, Associate Editor
Accepted August 30, 2005