DNA Research 11, 83–91 (2004)
The Minimal Eukaryotic Ribosomal DNA Units in the Primitive
Red Alga Cyanidioschyzon merolae
Shinichiro Maruyama,1,∗,† Osami Misumi,2 Yasuyuki Ishii,3 Shuichi Asakawa,3 Atsushi Shimizu,3
Takashi Sasaki,3 Motomichi Matsuzaki,4 Tadasu Shin-i,5 Hisayoshi Nozaki,1 Yuji Kohara,5
Nobuyoshi Shimizu,3 and Tsuneyoshi Kuroiwa2
Department of Biological Sciences, Graduate School of Science, University of Tokyo, 7-3-1 Hongo,
Bunkyo, Tokyo 113-0033, Japan,1 Department of Life Science, College of Science, Rikkyo (St. Paul’s)
University, 3-34-1 Nishiikebukuro, Toshima, Tokyo 171-8501, Japan,2 Department of Molecular Biology,
Keio University School of Medicine, 35 Shinanomachi, Shinjyuku, Tokyo 160-8582, Japan,3 Department
of Biomedical Chemistry, Graduate School of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo, Tokyo
113-0033, Japan,4 and Center for Genetic Resource Information, National Institute of Genetics, 1111 Yata,
Mishima, Shizuoka 411-8540, Japan5
(Received 5 January 2004; revised 22 January 2004)
Abstract
Cyanidioschyzon merolae is a small unicellular red alga that is considered to belong to one of the most
deeply branched taxa in the plant kingdom. Its genome size is estimated to be 16.5 Mbp, one of the
smallest among free-living eukaryotes. In the nucleus containing this small genome, one nucleolus is clearly
observed, but the molecular basis for the intranuclear structure including ribosomal DNA organization is
still unclear. We constructed a bacterial artificial chromosome library for C. merolae 10D composed of
two subsets with different insert size distributions. The two subsets have average insert sizes of 97 and
48 kb, representing 10.0- and 6.9-fold genome-equivalent coverage of the haploid genome, respectively. For
application to whole-genome shotgun sequencing, the termini of each clone were sequenced as sequencetagged connectors and mapped on the contigs assigned to chromosomes. Screening for rRNA genes by
conventional colony hybridization with high-density filter blots and subsequent sequencing revealed that
the C. merolae genome contained the smallest number of ribosomal DNA units among all the eukaryotes
examined to date. They consist of only 3 single units of rRNA genes distributed on separate chromosomal
loci, representing an implication for concerted evolution. Based on these results, the origin and evolution
of the nucleolus are discussed.
Key words: BAC library; Genome organization; Nucleolus
1.
Introduction
The red algae are considered to be the most basal
taxa in the plant kingdom but, unfortunately, they have
not received as much attention in the field of genomics
as other taxa. Cyanidioschyzon merolae has a nuclear
genome of 16.5 Mb, a distinctly small genome relative to known photosynthetic eukaryotes.1 Phylogenetic
∗
†
‡
Communicated by Masahiro Sugiura
To whom correspondence should be addressed. Tel. +81-35841-7826, Fax. +81-3-5841-8476, E-mail: [email protected].
u-tokyo.ac.jp
Present Address: Institute of Molecular and Cellular Biosciences, University of Tokyo, 1-1-1 Yayoi, Bunkyo, Tokyo, 1130032, Japan
Data deposition: Database submissions of BAC end-sequences,
AG266951 to AG275742 and AB158482; accessions for the
clone H1D17, H1L17 (partial) H1A11 (partial); AB158483,
AB158484 and AB158485, respectively.
analyses of several nuclear-encoded genes and organellar genome sequences suggest that C. merolae belongs to
one of the most deeply branched lineages in the plant
kingdom.2,3 Furthermore, these results are consistent
with the ancestral features of factors involved in organellar division4−6 and nucleoid organization.7 The characteristics of the C. merolae cell are small and simple: one
nucleus, one mitochondrion with a 32-kb genome8 and
one chloroplast with a 150-kb genome3 in a small cell
(2 µm in diameter). This small nucleus contains a clearly
observed nucleolus (Fig. 1), but the molecular basis of the
intranuclear organization is unclear.
As is generally known, rRNAs are essential components for protein synthesis. In most eukaryotic genomes,
18S, 5.8S and 28S rRNA genes are clustered as a single ribosomal DNA (rDNA) unit, or an operon, and
transcribed into a polycistronic transcript by RNA poly-
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The minimal eukaryotic rDNA units in C. merolae
[Vol. 11,
Figure 1. Nucleoli in C. merolae. Acridine Orange staining shows that an RNA signal (orange), distinct from the DNA signal (yellow-green), is located in the nucleus (A). It corresponds to a portion of the nucleus poorly stained by DAPI (B) and an electron-dense
intranuclear structure observed by electron microscopy (C). N, nucleus; No, nucleolus; M, mitochondrion; C, chloroplast. Bars =
1 µm.
merase I. More distinctively, the rDNA units usually represent a higher order organization of long tandemly repeated arrays, which consist of several hundred copies of
the individual unit.
There are no rules without exceptions, and several
eukaryotes are known for their characteristic rDNA
organization and composition. In the nucleomorph
genomes of the cryptomonad Guillardia theta and the
chlorarachniophyte Bigellowiella natans, which are modified and highly reduced in size, rRNA gene units are
distributed at both ends of three chromosomes adjacent to the telomeric regions.9,10 The malarial parasites
Plasmodium berghei and P. falciparum possess four and
seven unlinked and divergent rDNA units per haploid
genome, respectively, which are distributed among the
genome as singletons.11,12 These recent studies indicate
that rDNA composition in lower eukaryotic genomes is
still an open question and that a deep-coverage genomic
library is required for genome-wide analysis of rDNA
structure.
In recent years, the bacterial artificial chromosome
(BAC) cloning system has been developed to clone large
DNA fragments for a variety of applications for wholegenome analysis of many organisms. Although inferior
in cloning capacity, the BAC has the important ad-
vantages of high cloning efficiency, high stability and a
low degree of chimerism compared to the yeast artificial
chromosomes (YACs). The combination of BAC library
construction and shotgun sequencing has now become
a practical and general strategy for obtaining accurate
and abundant information about genome structures, especially those of organisms lacking genetic maps or sufficient genetic markers, as is the case for the primitive
red alga C. merolae. Since the C. merolae whole-genome
shotgun (WGS) sequencing project has been completed,1
the availability of authentic genome resources, that is, a
deep-coverage genomic library, is a crucial advantage for
facilitating further analysis of the genome structure including centromeres and telomeres.
Here, we report the construction and characterization
of a 16.9 genome-equivalent BAC library for the primitive
red alga C. merolae 10D and analysis of the rDNA distribution in the genome. Furthermore, the origin of the
nucleolus in eukaryotic evolution is discussed. We hope
that this study will provide the foundation for investigating the structure and evolutionary pathway of the plant
genome.
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S. Maruyama et al.
Materials and Methods
2.1. Fluorescence and electron microscopy
Cells were cultured in Allen’s medium13 at pH 2.5
under continuous light at 30◦ C. For fluorescence microscopy, cells were stained with a mixture of 1 µg/ml
4 ,6-diamidino-2-phenylindole (DAPI), 60 µg/ml Acridine Orange and 1% glutaraldehyde, and were examined
using an Olympus BH 2 microscope. Images were captured with a HAMAMATSU 3CCD digital camera C7780
and processed using Adobe Photoshop software. Electron
microscopy was performed as described previously.14
2.2. Preparation of high-molecular-weight DNA
High molecular weight DNA was prepared from C.
merolae strain 10D cells cultured as described above.
Cells were embedded in 1% low-melting-point agarose
(SeaPlaque Agarose, BMA) and applied to plug molds.
Plugs were placed in TEM buffer (0.45 M EDTA, 25 mM
Tris-HCl pH 7.5, 1 M 2-mercaptoethanol), followed by
incubation at 37◦ C for 24 hr. After washing four times
with 0.1 M EDTA, plugs were transferred into lysis buffer
(0.1 M EDTA, 50 mM Tris-HCl pH 7.5, 1% SDS, 1 mg/ml
proteinase K [Merck]), before incubation at 50◦ C for
24 hr. Plugs were washed with 0.1 M EDTA four times
and then stored in an excess of 0.1 M EDTA at 4◦ C.
2.3.
Construction and characterization of the BAC library
High-molecular-weight DNA in agarose gel was partially digested with either Hind III or Mbo I (Fermentus),
and subjected to pulsed-field gel electrophoresis (PFGE)
(CHEF DR III, Bio-Rad) to obtain fragments ranging
in size from 97 to 145 kb (designated the Hind III library) or from 48 to 97 kb (designated the Mbo I library), respectively.15 These fragments were then ligated into the pBAC-Lac vector, and transformed into
DH10B Escherichia coli by electroporation using Gene
Pulser II (Bio-Rad) with the following settings: 25 µF,
200 ohms and 2.0 kV. Individual white colonies selected
by the well-established lacZ color selection method were
automatically transferred using Q-Pix (Genetix Ltd.) to
the wells of 384-well plates containing LB medium with
6.25 µg/ml chloramphenicol and 7.5% glycerol, and the
plates were incubated overnight at 37◦ C, followed by storage at −80◦ C. DNAs prepared from randomly selected
clones from both libraries were digested with Not I (Fermentus) to release the inserts from the BAC vectors and
were subjected to PFGE with the molecular size markers
of a λ phage DNA ladder to estimate the insert size in
each clone.
2.4. High density filter production and hybridization
The entire Hind III and Mbo I libraries, respectively containing 1920 and 3072 clones, were gridded
85
onto one piece each of nylon membrane (Biodyne B,
Pall) using a Bio Grid (Bio Robotics). Individual
clones were doubly spotted. A total of 1920 BAC
clones in the Hind III-digested library were subjected
to colony hybridization screening with C. merolae putative 18S and 28S rDNA partial sequences as probes,
using a DIG-High Prime DNA Labeling and Detection
Starter Kit (Roche Diagnostics) according to the manufacturer’s instructions. The following primers were
used to amplify the probes from C. merolae genomic
DNA: 5 -AACCTGGTTGATCCTGCCAGT-3 and 5 GCTGGCACCAGACTTGCCCTC-3
for 18S; and 5 -CGAGTTAGGAGGTGTAGCAT-3 and
5 -CGACAACGACGCATCAGTAG-3 for 28S. The sequences for the putative 18S and 28S rDNA of C. merolae
were defined by BLAST searches16 against the GenBank
database.
2.5. Identification of rDNA sequences
For Southern blotting analysis of C. merolae genomic
and BAC DNAs, each DNA sample was separately digested with BamH I, Pst I or Sac I (Takara Bio Inc.)
and electrophoresed in a 1% agarose gel. Blotting onto
Biodyne B nylon membranes (Pall) and detection using the AlkPhos Direct Labelling and Detection System
(Amersham Biosciences) were performed according to
the recommended protocols of the manufacturers. Clone
H1D17 was subjected to shotgun sequencing and data
assembly using the Phred/Phrap/Consed System.17−19
The sequences of clones H1L17 and H1A11, each including one rRNA unit, were determined by the PCR direct sequencing strategy based on the sequencing results
of clone H1D17. Sequences were established using ABI
3100/3700/3730 sequencers (Applied Biosystems).
3.
Results and Discussion
3.1. Deep coverage C. merolae BAC library
We constructed a C. merolae BAC library containing two subsets derived from genomic DNAs digested by
two different endonucleases with different size selectivity
in order to reduce locus-dependent cloning bias. Highmolecular-weight DNA embedded in agarose gel plugs
was subjected to partial digestion and single size selection using PFGE. For one subset with a larger average
insert length, Hind III-digested DNA fragments in the
region of 97 to 145 kb in the agarose gel were used for
ligation (the Hind III library, 1920 clones), while Mbo
I-digested DNA fragments in the region of 48 to 97 kb
were used for the other subset with a smaller average
insert length (the Mbo I library, 3072 clones).
To estimate the genome coverage of the C. merolae
BAC library, randomly selected clones were subjected
to Not I digestion and sizing by PFGE in 1% agarose
gels. However, the length of each insert was not easily
86
The minimal eukaryotic rDNA units in C. merolae
[Vol. 11,
Figure 2. Insert size distribution in the Hind III and Mbo I libraries of C. merolae 10D. (A) Ethidium bromide-stained gel after PFGE.
Randomly selected BAC DNAs were digested with Not I and separated in a 1% agarose gel. (B) Size distribution of the inserts of
the end-sequenced clones calculated by contig-based digital mapping using the C. merolae WGS sequence. The average insert sizes
of the Hind III and Mbo I libraries are 97 and 48 kb, resulting in 10.0- and 6.9-fold genome equivalents, respectively. Left, Hind III
library; right, Mbo I library.
recognized because C. merolae genomic DNA contains
Not I restriction sites at high frequency (Fig. 2A). On
the other hand, all the clones were subjected to endsequencing for computational mapping of BAC end sequences onto the C. merolae WGS sequence, what we call
“contig-based digital mapping.” We selected the clones
in which both ends were sequenced and mapped onto the
same contig assembled by WGS sequencing. The average insert sizes of the Hind III and Mbo I libraries calculated from 275 and 895 clones were 97 and 48 kb, respectively (Fig. 2B). These results appear to reflect the precise sizing by PFGE. The end-sequence information also
resulted in the development of STCs (sequence-tagged
connectors). The BAC library with the STC dataset provided a reliable foundation for the contig scaffolding.
We determined the insert size distribution and genome
coverage of the C. merolae BAC library mainly by contigbased digital mapping, rather than conventional restriction analysis and colony hybridization. The C. merolae
genomic DNA showed a high frequency of Not I cutting
sites, implying the existence of scattered small G+C-rich
regions, and the insert fragments were often recognized as
multiple bands on PFGE gels. Restriction digestion with
Swa I, which recognizes ATTTAAAT and cleaved the
pBAC-Lac vector at one site, led to the result that most
clones were recognized as a single band (data not shown).
Contig-based digital mapping enabled us to comprehensibly visualize the insert size distribution and coverage.
The contribution of the organelle DNA to the libraries was estimated by BLAST searches16 using the
end-sequence data as query sequences. The mitochondrial DNA was not detected at all, whereas the clones
harboring chloroplast DNA accounted for 12% and 23%
of the Hind III and Mbo I libraries, respectively. This
result was not surprising because whole cells, and not
isolated nuclei, were embedded in the agarose gel plugs
for the high-molecular-weight DNA preparation and partial digestion. The actual coverages of the Hind III and
Mbo I libraries were equivalent to 10.0- and 6.9-fold, respectively. The deep coverage of the library itself compensates for chloroplast DNA contamination, although
its percentage is relatively high compared to other plant
BAC libraries.20 The properties of the library that excludes the mitochondrial DNA (32 kb genome) and includes the chloroplast DNA (150 kb genome) reinforced
the accuracy of the size selection.
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S. Maruyama et al.
87
Figure 3. Chromosomal mapping of the clones containing rDNA. Two regions in chromosome 17 and one region in chromosome 18 are
common to the positive clones. Only the clones in which both ends were successfully sequenced are shown.
3.2.
Only three separate rDNA units are present in the
C. merolae genome
We screened the entire Hind III library using the partial sequences of C. merolae 18S and 28S rRNA genes as
probes. Conventional colony hybridization against high
density replica filters showed 29 identical positive clones
for both probes. Individual end-sequences of the positive clones were searched against the C. merolae genome
sequence using BLASTN to map these clones on the chromosomes. As a result, although 7 clones (H1B11, H2N04,
H3H21, H3H01, H4B04, H4J21 and H4N07) were only
sequenced successfully at one end, all the positive clones
were mapped on two distinct regions on chromosome 17
and one region on chromosome 18 (Fig. 3). Strangely, we
could not find the entire sequence of rDNA units in the
WGS sequence. By analyzing the BAC mapping data
onto the WGS sequence, we found that portions of the
rDNA units were completely hidden within the gaps of
the contigs.
To analyze the rRNA gene structure in more detail,
three representative clones corresponding to each region
were digested with three different restriction enzymes
(BamH I, Pst I and Sac I) and subjected to Southern
hybridization with the same probes used for the colony
hybridization (Fig. 4). The results indicated that each
clone contained one set of 18S and 28S rRNA genes and
that at least three rDNA units were encoded in the C.
merolae genome.
Furthermore, full-length sequencing of clone H1D17,
mapped on chromosome 17, revealed that this clone encoded a single rDNA unit comprised of putative 18S, 5.8S
and 28S rRNA genes without redundancy. This result
was consistent with the Southern hybridization analysis.
We employed the PCR direct sequencing method to determine the sequences of the other two clones (H1L17 and
H1A11). Consequently, we found that the three rDNA
units were nearly identical and homogenous except for
the putative internal transcribed spacer (ITS) regions,
where the sequences were slightly diversified (Fig. 5 and
Table 1). The ITS sequences were significantly long compared to those of related species, although the biological
importance of this finding is unknown (Table 2). We
conclude that concerted evolution has occurred among
the three rDNA units but accelerated rates of sequence
diversification in the ITS regions have overcome it.
The C. merolae genome encodes only three single
rDNA units, in contrast to the long tandemly repeated
arrays of rDNA units in most eukaryotic genomes. This
is striking, and suggests that the free-living unicellular
alga C. merolae possesses the smallest number of rDNA
units among all the eukaryotes tested to date, including parasites and nucleomorphs. The sequence similarity
between the individual rDNA units in C. merolae implies the presence of a sequence homogenization mechanism. Sequence homogenization is believed to be driven
by unequal crossing over and gene conversion, causing reciprocal and nonreciprocal sequence replacement,
respectively.21,22 Although the relative contributions of
these processes in the homogenization between the three
separate rDNA units is not known, it is likely that these
rDNA units are arranged spatially adjacent to, and interact with, each other, and that this homogenization
88
The minimal eukaryotic rDNA units in C. merolae
[Vol. 11,
Figure 4. Southern blotting analysis of the genomic DNA and representative positive clones digested by BamH I, Pst I and Sac I.
Upper, analysis with the partial 18S rRNA gene as a probe; lower, analysis with the partial 28S gene as a probe. G, genomic DNA;
A, clone H1A11 (chromosome 18); D, H1D17 (chromosome 17, distal); L, H1L17 (chromosome 17, proximal).
Figure 5. Schematic representation of rDNA organization in the C. merolae genome. Putative rRNA genes are homogenous except
putative ITS regions and distributed among the separate chromosomal loci. Chromosome 17 distal and proximal rDNAs represent
head-to-head orientation on the chromosome. Asterisks indicate fragments used as probes in Southern blotting analysis (Fig. 4). B,
P and S indicate restriction sites of BamH I, Pst I and Sac I, respectively.
is probably mediated by recombination machinery in the
nucleolus. The sequence diversity between units found in
Plasmodium species,11,12 which is thought to be a consequence of adaptive evolution to a particular life cycle,
was not detected in the C. merolae rDNA units. Mean-
while, the telomere-associated organization of rDNA seen
in the nucleomorph genomes,9,10 which might be acquired
through extensive genome reduction, was also not found.
The overall organization of rDNA in C. merolae is similar to that of prokaryotes,22 and it remains to be seen
No. 2]
S. Maruyama et al.
89
Table 1. Sequence diversity between the three rDNA units.
region
pairwise diversity (%)
(min. - max.)
5' boundary (2 kb)
1.2-1.5
18S
0.17-0.33
ITS1
1.3-1.8
5.8S
0
ITS2
1.4-4.7
28S
0.19-1.2
3' boundary (0.5 kb)
0.20-1.0
5 and 3 boundary region represent upstream of the
putative 18S gene and downstream of the putative
28S gene, respectively.
Table 2. Length of the ITS sequences in red algal rRNA genes.
species
C. merolae (chr.17 distal)
(chr.17 proximal)
(chr.18)
Porphyra yezoensis
Porphyra umbilicalis
Palmaria palmata
Laurencia viridis
Halymenia floridana
Gracilaria vermiculophylla
Guillardia theta nucleomorph
ITS1 (bp) ITS2 (bp)
868
1744
AB158483
866
1747
AB158484
852
1724
AB158485
335
561
AY368576
333
483
AJ318959
373
452
AJ318958
162
221
AF082345
173
292
AF412019
524
740
511
558
whether this intermediate organization between eukaryotes and prokaryotes might be a remnant of the ancestral
rDNA units or the result of reductive evolution.
3.3.
Accession
Hypothesis for the origin and evolution of the nucleolus
The nucleolus is often referred to as a ribosome
factory, but recent studies have revealed that it
has additional functions not directly linked to ribosome biogenesis.23 The components involved in prerRNA processing, which form large ribonucleoprotein
(RNP) complexes in the nucleolus in the budding yeast
Saccharomyces cerevisiae,24 are universally conserved between archaea and eukaryotes.25 Clues for understanding
the molecular bases of nucleolus maintenance were found
through genetic and mutational studies of RNA polymerase I (Pol I), which were intensively conducted in S.
cerevisiae.26,27 These studies showed that the transcription by Pol I, tandemly repeated arrays of rDNA, and
the nucleolus are not absolutely required for normal ribosome functions. Therefore, the origin and evolution of
nucleoli remain to be discussed. In this study, we have
shown that C. merolae intrinsically possesses single sepa-
AY465829
AF165818, AJ010592, AF083031
rated rDNA units, distributed between different chromosomal loci, while the nucleolus is obviously observed in
the nucleus (Fig. 1). This indicates that the long tandem
repeats of rDNA units, which have been believed to coalesce in or around the nucleolus in most eukaryotes, are
not required to maintain the nucleolar structure and the
innate ribosome functions in C. merolae. This dispersed
organization may allow the rDNA units with a low copy
number to undergo flexible spatial arrangement, leading
to the formation of a nucleolus.
We propose the hypothesis that at least two directions
of driving forces facilitated the evolution of the nucleolus. First, as eukaryotes emerged, the ancestral RNA
polymerase is thought to have diverged into three classes
of functionally distinct enzymes: RNA polymerase I, II
and III. Among these, RNA polymerase I is specialized
for rRNA transcription and has the rRNA genes under
its control, away from the complex transcriptional network of the other genes. This designated polymerase
accomplishes a huge workload of transcribing essential
rRNAs to form a specialized intranuclear structure. Second, the nuclear envelope (NE) could aid the maintenance of higher-order intranuclear structures. We assume
90
The minimal eukaryotic rDNA units in C. merolae
that the NE plays important roles not only in compartmentalization of the cytoplasm, to allow efficient material flow and distribution, but also in intranuclear spatial
information management. By means of projecting the
enormous amount of three-dimensional spatial information of the separate eukaryotic linear chromosomes into
the two-dimensional field of the NE, more elaborate gene
expression systems and higher-order intranuclear structures, such as the nucleolus, might have been developed.
In conclusion, our results lead to the basic concept that
functional (qualitative) conservation of the genome underlies structural (quantitative) diversification, accompanied by size expansion and reduction, during a long period of evolution. Genome analysis of this primitive photosynthetic eukaryote may open the way to explore the
evolution of the eukaryotic genome.
Acknowledgements: We thank our colleagues at
Keio University School of Medicine and the Center for
Genetic Resource Information for their support, and K.
Tanaka at the University of Tokyo for helpful discussions
on the manuscript. This work was supported by Grantsin-Aid for JSPS Fellows (No. 11911 to S. M.) and for
Scientific Research on Priority Areas (C) “Genome Biology” from the Ministry of Education, Culture, Sports,
Science, and Technology of Japan, and from the Promotion of Basic Research Activities for Innovative Biosciences (ProBRAIN).
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