Evidence that Two Types of 18s rDNA Coexist in the Genome of

Evidence that Two Types of 18s rDNA Coexist in the Genome of Dugesia
(Schmidtea) mediterranea (Platyhelminthes,
Turbellaria, Tricladida)
Salvador
Carranza,”
*Departament
de Genetica
Gonzalo Giribet,?
and tDepartament
Caries Ribera,? Jaume Baguii&* and Marta Riutort”
de Biologia Animal, Facultat de Biologia,
Universitat
de Barcelona
Sequences of 18s ribosomal DNA (rDNA) are increasingly being used to infer phylogenetic relationships among
living taxa. Although the 18s rDNA belongs to a multigene family, all its copies are kept homogeneous by concerted
evolution (Dover 1982; Hillis and Dixon 1991). To date, there is only one well-characterized
exception to this rule,
the protozoan Plasmodium (Gunderson et al. 1987; Waters, Syin, and McCutchan 1989; Qari et al. 1994). Here we
report the 1st case of 18s rDNA polymorphism
within a metazoan species. Two types (I and II) of 18s rDNA have
been found and sequenced in the platyhelminth Dug&a (Schmidtea) mediterrunea (Turbellaria, Seriata, Tricladida).
Southern blot analysis suggested that both types of rDNA are present in the genome of this flatworm. This was
confirmed through sequence comparisons and phylogenetic analysis using the neighbor-joining
method and bootstrap
test. Although secondary structure analysis suggests that both types are functional, only type I seems to be transcribed to RNA, as demonstrated by Northern blot analysis. The finding of different types of 18s rDNAs in a single
genome stressess the need for analyzing a large number of clones whenever 18s sequences obtained by PCR
amplification and cloning are being used in phylogenetic reconstruction.
Introduction
Ribosomal
DNA (rDNA)
serves a pivotal role in
the protein
synthesis
machinery
of all prokaryotic
and
eukaryotic
cells. The eukaryotic
rDNA array typically
consists of several hundred tandemly repeated copies of
the transcription
unit separated by nontranscribed
spacers (NTS). The transcription
unit codes for the 18S,
5.8S, and 28s genes and for external and internal transcribed spacers (ETS and ITS) (for a review see Long
and Dawid 1980). The multiple copies of this cluster
appear to be nearly identical within a given organism
(Hillis and Dixon 1991; Fox, Pechman,
and Woese
1977; Lane et al. 1985). This conservation
of sequence
presumably
reflects functional constraints on the molecules that are required for optimal translational efficiency. The process postulated to keep the different copies
of the repeat so similar is called concerted evolution
(Dover 1982; Hillis and Dixon 1991), and two molecular mechanisms
are assumed to account for it, gene
conversion (Nagylaki 1984a, 1984b; Lassner and Dvorak 1986; Hillis et al. 1991) and unequal crossing over
(Petes 1980; Szostak and Wu 1980; Arnheim
1983;
Coen and Dover 1983).
Among the genes within each transcription unit, the
18s ribosomal DNA (18s rDNA) is increasingly
being
used to infer phylogenetic
relationships
for reasons that
have been repeatedly
reviewed
(Woese 1987; Sogin
1991; Adoutte and Philippe 1993). The main ones are
that it is sufficiently long to provide a statistically valid
amount of information,
it is among the slowest evolving
sequences found throughout living organisms, and different regions of the molecule evolve at different rates
which allows the inference
of phylogenetic
history
Key
words:
18s
rDNA,
polymorphism,
Dug&a
variability,
phylogenetic
family.
mrditerranea, Platyhelminthes,
tion, metazoan,
multigene
(Schmidfea)
reconstruc-
Address for correspondence
and reprints: Salvador Carranza, Departament de Ge&tica, Facultat de Biologia, Universitat de Barcelona,
Av. Diagonal,
645, 08071
Barcelona,
Spain. E-mail:
carranza@porthos.bio.ub.es.
ML Biol. Evol. 13(6):824-832.
1996
0 1996 by the Society for Molecular Biology and Evolution.
824
ISSN: 0737.4038
across a very broad taxonomic
range. Moreover, the
presence of many copies of 18s rDNA per genome and
its homogeneization
through concerted evolution greatly
reduces intraspecific variation compared to what one can
expect based on data of interspecific variation. The low
(or negligible)
levels of intraspecific
variation of 18s
rDNA genes avoids the extensive sampling required for
most single-copy genes. Therefore, small sample sizes,
even from single individuals,
have been used in most
phylogenetic
studies of rDNA (Hillis and Davis 1988;
Baverstock and Moritz 1990).
However, it is important to point out that although
gene homogenization
appears to be the rule, intragenomic variation is known. The most important cases seem
to be growth-stage-specific
rRNAs. The first cases were
described in the amphibian
Xenopus laevis (Wegnez,
Monier, and Denis 1972; Peterson, Doering, and Brown
1980) and in the mammal Misgurnus fossilis (Mashkova
et al. 1981) where two classes of 5s rRNA transcripts
that are specific to either somatic or oocyte ribosomes
have been reported. More recently, it has been shown in
some species of the protist Plasmodium that two different types of 18s rDNA exist, whose expression is linked
to different stages of the parasitic life cycle of these
organisms
(Gunderson
et al. 1987; Waters, Syin, and
McCutchan 1989; Qari et al. 1994). Although such cases
can be taken as exceptions to a general rule, small sampling sizes may hide a greater variability in 18SrDNA
genes than expected. If this turns out to be the case, it
may have implications
for the extent and mechanisms
of concerted evolution and will introduce some degree
of caution in using rDNA for phylogenetic
analysis.
Here we report the first case of 18s rDNA polymorphism within a metazoan species, Dugesia (Schmidtea) mediterranea,
a free-living
platyhelminth.
D. (S.)
mediterranea
has sexually and asexually reproducing
populations
or races (Benazzi et al. 1975). Sexual populations are diploids (2n = 8) or parthenogenetic
triploids (3n = 12), whereas asexual populations, also diploid (2n = 8) or triploid (3n = 12), bear a chromosomal
translocation
from the 1st to the 3rd pair of chromo-
Two Types of 18s rDNA in the Genome
somes (Ribas 1990) and reproduce exclusively
by fission. There is some evidence for multiple origins of
these asexual lineages from sexual forms (unpublished
data). The results shown here refer to diploid sexual and
asexual populations.
Materials
Organisms
and Methods
Specimens
of the asexual populations
of D. (S.)
mediterrunea were collected from a pond in the city of
Barcelona (Spain). Individuals
from sexual populations
came from Rio di Montes in Sardinia (Italy). Prior to
DNA extraction, organisms were fasted for a week.
Obtention
of the Sequences
High-molecular-weight
DNA was purified according to a modification
(Garcia-Fernandez,
Bagufia, and
Sal6 1993) of the guanidine isothiocyanate
method initially described for RNA (Chirgwing et al. 1979). The
entire length of the 18s rDNA was amplified by the
polymerase chain reaction (Saiki et al. 1985) using the
primers lF, TACCTGGTTG
ATCCTGCCAG
TAG and
9R, GATCCTTCCG
CAGGTTCACC
TAC. The reaction conditions were: 1.6 ng/pl template DNA; first denaturing step 5’ 94°C; 30” 94°C; 30” 50°C; 45” 72°C 35
cycles. The amplified DNA was electrophoresed
on an
agarose gel containing
ethidium bromide to check the
product band size, and then it was purified by DEAEcellulose transference.
DNA samples were redissolved
in 400 l.~l of elution buffer (T.E. pH 7.5 and NaCl 1.5
M) for 2 h at 65°C. The DEAE-cellulose
paper was discarded and the solution containing the DNA was washed
once with 1 volume of phenol-chloroform-isoamilalcoho1 (25:24:1) and precipitated with 1 volume of isopropanol. The pellet was dissolved in 15 ~1 of sterile distilled water. The purified PCR products were ligated into
pUC 18 Sma I dephosphorilated
vector using the Sure
Clone ligation kit (Pharmacia P-L Biochemicals).
The
constructs were transformed
into E. coZi JM105 competent cells. The alkaline lysis method from the smallscale preparation
of plasmidic
DNA protocol (Sambrook, Frischt, and Maniatis 1989) was used. In addition
to the 2 primers described above, 12 internal primers,
3F GTTCGATTCC
GGAGAGGGA,
4F
CCAGCCAGCC
GCGGTAATTC,
5F GCGAAAGCAT
TTGCCAAGAA,
6F AAACTTAAAG
GAAT, 7F
GCAATAACAG
GTCTGTGATG
CCC, and SF GTACACACCG CCCGT and its antisense analogues, called
3R, 4R, 5R, 6R, 7R, and 8R, were used to completely
sequence both chains using the dideoxy chain termination method (Sanger, Nicklen, and Coulsen 1977) (sequencing kit [Pharmacia]).
Direct sequencing
of PCR
products was also performed. Single-strand
DNA was
produced by asymmetric PCR and precipitated (McCabe
1990); the sequences were obtained by the same methods described above for cloned DNA.
Southern
Blot Analyses
Total genomic DNAs of asexual (6 pg) and of sexual (2 kg) D. (S.) mediterranea were independently
single-digested
with either EcoRI or Hind111 and double-
of a Metazoan
825
digested with EcoRIIHindIII.
DNAs were separated in
0.9% agarose gel and transferred to Hybond-N membranes (Amersham)
by capillary blotting. Two oligonucleotides specifically complementary
to type I or type
II 18s rDNA (see fig. 2) were used as probes. The hybridization conditions were optimized in order to avoid
cross-hybridization
between the two types. The resulting
conditions were as follows: filter hybridization
was carried out at 62°C (type I probe) and 45°C (type II probe)
for 18 h in: 6~ SSC, 5X Denhardt’s,
0.05% Na-pyrophosphate, 1% SDS, 100 pg/ml yeast RNA and 15X
lo6 cpm of 32P-labeled probe. Filters were washed twice
in 6X SSC, 0.1% Na-pyrophosphate:
2 X 10’ at room
temperature for type I probe; and 10’ at room temperature, 10 min 50°C for type II probe.
Northern
Blot Analyses
Total cellular RNA was isolated from sexual and
asexual populations
by the guanidinium
thiocyanate
method (Chirgwin et al. 1979) and separated in 1.2%
agarose/formaldehyde
gels. After electrophoresis,
the
RNA was transferred to a Hybond-N membrane (Amersham) and hybridized with the same probes and in similar conditions as for Southern blot experiments. As control, rat RNA was electrophoresed
and probed.
Sequence
Data Comparison
and Phylogenetic
Trees
18s rDNA sequences from D. (S.) mediterrunea
were compared to sequences of other platyhelminthes
and to those of related and distant groups, and a phylogenetic tree was obtained. Briefly, sequences of D. (S.)
were aligned and compared to sequences
mediterranea
from 11 species (5 platyhelminthes
and 6 belonging to
other phyla). The platyhelminths
were four freshwater
triclads belonging to the three families of the group (Dugesiidae: D. (G.) tigrina and D. (D.) iberica; Planariidae: Crenobia alpina; and Dendrocoelidae:
Dendrocoelineata.
lum lacteum) and one proseriate, Monocelis
Representatives
from other phyla were: an annelid (Ianice conchilega, X79873), an arthropod (Eutypelma californica, X 13457), an echinoderm
(Asterias amurensis,
Dl4358),
a cnidarian
(Anemonia sulcatu, X53498), a
ciliate (Colpidium campylum, X56532), and a sporozoan
(Theiluria parva, L02366). Triclad sequences were almost complete (1,630 nucleotides)
sequences obtained
using direct sequencing from rRNA by reverse transcriptase (Riutort et al. 1992). Sequence from the proseriate
(U45961) is a new complete sequence obtained by PCR.
The sequences were aligned by hand using a computer
editor with the help of the secondary structure. Only
conserved regions that could unambiguously
be aligned
were used (797 positions, of which 353 were informative). Kimura’s two-parameter
distances were calculated
and a tree was obtained by the neighbor-joining
method
(Saitou and Nei 1987) and a bootstrap test was performed (1,000 replications)
using the MEGA program
(Kumar, Tamura, and Nei 1993).
Results
High-molecular-weight
DNA was independently
isolated from a pool of intact adult individuals of each
826
Camnza
et al
of the two populations of D. (S.) mediterranea (see Materials and Methods).
18s rDNA was independently
PCR-amplified
from total DNA using specific primers.
Amplified bands were cloned. Full length (1,795 nucleotides) sequencing of the clones revealed the existence
of two different types of 18s rDNA in both populations
(fig. 1). One type was identical in sequence to the 18s
rRNA partial sequences already published (Riutort et al.
1992) (which we will refer to as type I; GenBank accession number U31084). The other (type II; GenBank
accesion number U31085) had an overall 8% difference
(130 sites) compared to type I. Positions that vary between the two sequences are not randomly dispersed
throughout the length of the molecule. Six regions (nucleotides 69-81, 220-241, 645-743, 1046-1072,
13351375, 1465-1567; shaded background in fig. 1) showed
considerable
variation, and secondary structure analysis
shows that most changes are conservative
(when a
change occurs in a stem a compensatory
change in the
complementary
sequence restores the pairing of bases
and the secondary structure is conserved) or situated in
loops. The two types of 18s rDNA were also found
when DNA was isolated, amplified, and cloned from a
single organism in both populations. This shows that the
two 18s types coexist within an individual genome.
Previous hints that different (at least two) types of
ge18s rDNAs coexisted within D. (S.) mediterrunea
nome came from double bands seen in particular positions after direct sequencing
from PCR (fig. 2). These
bands were at first thought to be artifactual; however, as
they appear in constant positions giving a uniform pattern (see double bands in fig. 2 matching the four variable positions within the fragment 1713-1732 in fig. l),
a more likely explanation,
it appears, is the presence of
different 18s rDNA types. Further evidence came from
Southern blots of genomic DNA from both races, singleand double-digested
with EcoRI and Hind111 and probed
using two oligonucleotides
matching specifically type I
or II sequences (shaded boxes within the variable region
645-743, fig. 1). As expected, blots showed both types
of rDNAs to be present in the genome of both races (fig.
3). The band obtained for type II when DNA was cut
with HindIIUEcoRI
(lanes 3) was around 1.3 kb, which
matches the expected size for this fragment as deduced
from restriction enzyme sites for type II 18s rDNA sequence (fig. 1, nucleotides 339-343 HindIII, 1576-l 58 1
EcoRI).
On the other hand, double-digested
DNA
probed with type I gave a band of the same size as the
EcoRI fragment, which means that Hind111 does not cut
inside this cluster; this could also be deduced from the
large band obtained (more than 21 kb, lane 2, type I)
when DNA was only cut with HindIII. For type II,
Hind111 cuts in at least two places, one inside the 18s
sequence (nucleotides
339-343, fig. 1) and another that
should be located in the ITS-l region or within the 5.8s
gene (at aproximately 400 bp from the 3’ end of the 18s
sequence). This will explain the 1.8-kb band obtained
(type II, lane 2 in fig 3). Restriction patterns between
both types were thus substantially
different, whereas
conservation
of restriction sites within each type (only
one band per enzyme is observed; fig. 3) indicates a lack
of internal polymorphism.
This strongly suggests that
each type of rDNA is organized in a different array of
repeat units which are independently
kept homogeneous
by concerted evolution.
To further prove the internal homogeneity
of type
I and type II genes, we sequenced, from the pool of
clones obtained from a sexual single organism, 5 clones
of type I and 13 clones of type II, using the 4F primer.
The sequence length analyzed was 209 bp for type I
clones and 190 bp for type II clones. The mean percentage of differences
between
type I clones was
0.188%, and that between type II clones was 0.45% (results not shown). In other words, if all the 18s molecule
were as variable as it is in the zone analyzed, we would
expect a mean of 3 changes between two 18s type I
molecules and a mean of 8 changes between two 18s
type II molecules randomly choosen from their respective rDNA clusters. Given that the 4F primer sequence
comprises the most variable zone of the molecule, we
can assume that the level of variability between copies
within each cluster is very low.
To test whether type II sequence is of planarian
origin and not from any contaminant
or potential planarian parasite (e.g., protozoans), both type I and type
II sequences were compared to 18s sequences from other platyhelminthes,
two protostomians,
a deuterostomian, a cnidarian and two protozoans, and a neighbor-joining tree was obtained (fig. 4). Type I sequence forms a
group with the other two species belonging to the Dugesiidae family, while type II branches off after families
Planariidae and Dendrocoelidae
diverged from the main
stem but before family Dugesiidae did. The bootstrap
test clearly shows that the new sequence (type II) belongs to Tricladida (100%) and that is more related to
Dugesiidae than to the other two families (80%). This
result clearly indicates that type II sequence originated
within the planarian genome.
From our primary and secondary structure analyses
we infer that neither type I nor type II genes are pseudogenes. Indeed, differences between them are nonrandom and restricted to regions that are not evolutionary
conserved, whereas a perfect match exists between those
regions required for optimal functionality.
Moreover, restriction patterns suggest that type I and type II genes
are in different clusters and kept internally
homogeneous. This led us to think of both genes as functional
genes. To test this hypothesis, we compared the expression of both genes by Northern blots using the same
probes as used for Southern blots. RNA was prepared
from intact adult individuals of both sexual and asexual
populations of D. (S.) mediterranea, with rat RNA as a
negative control. Unexpectedly,
the results show that although both types of rDNA are present in the genome
of each population of D. (S.) mediterranea only type I
is transcribed in adult intact animals (fig. 5).
Discussion
One of the main consequences
of concerted evolution is that sequence identity between multiple copies
of repeated DNA within a species is higher than that
Two Types of 18s rDNA in the Genome
I
II
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TATAACAGCTATGATTTGAGAGATTTTATCATTTTGCCAC~GAT~CTGTGGT~TTCTAGAGCT~TATTTTT~G~TGCCGTGACTCACG~GCGG
. . . . . . . . . . . . . . . . . . . . . . ..A.........-.......................................AT........................
100
I
II
170
140
260
180
220
130
150
201
210
CGGATTTATTAGATCAAAAT~~~~~G~G~~TATTTTGATGACTCTGGATAATTTTACTGATCGTACGACCTTTGTGTTGACGACATATCTCT
. . . . . . . . . . . . . . . . . ..CT . ..FT.TC-CT..A.A..TA..................C...................C............GA......
300
I
II
I
II
310
330
301
310
TGAAGTGGCTGACCTATCAACTTTCGATGGTAAGAT
. . ..A...............................
340
290
360
370
380
350
390
ACCATGGTTGTAACGGGTAACGGGG~TCAGTGTTCGATTCCGGAGAGGGAGCCTG
400
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
I
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470
440
450
460
480
490
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430
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401
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AGAAACGGCTACCACATCCACGG~GGCAGCAGGCGCGT~TTACCCMTACCGGTTCGGTGAGGTAGTGAC~
TAACAATATGGGCCCTAGTGG
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..A............G.........C.............T..............A.........
I
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570
540
550
560
580
590
510
530
501
510
TTTCATAATTGCAATGAGAACATTTTAAATACTTTACTTTATC~GTATC~TTGGAGGGC~GTCTGGTGCCAGCAGCCGCGT~TTCCAGCTCC~TAGCGTA
. . . . . . . . . . . . . . . . . ..TT..............T.........................................................A......
I
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670
640
650
660
680
690
601
610
630
700
610
TA~'1'AAA‘.'.":':;~',:;:'A:.:~~AAAA:.:;L"T~.'!.;'IA:':i'T::AAA:'?'~
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A”3A.T’P~ACA
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770
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870
840
850
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880
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810
830
801
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TGGAATAATAAAATAGGACTTCGGTTTTATTTTGTTGGTTTTCGAAACTGAAGTAATGATTAAAAGAGAGACTGCCGGGGGCATATGTATGCTGGTGCTAGA
. . . . . . . . . . . . . . . . . . . ..T.....................TT....A.....................~...........................
900
I
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970
940
950
960
980
990
901
910
930
910
GGTGAAATTCTTAGATCATCAGCAGACAGTCTACTGCG~AGCATTTGCC~G~TGTTTTCATT~TC~GNNCG~GTCAGAGGATCG~GACGATC
. . . . . . . . . . . . . ..c............AA.......................................................................
1000
I
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600
1070
1040
1060
1080
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1010
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1170
1140
1150
1160
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1110
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. . .._........................................................A
. . . . . . . . . . . ..NNNN.....................
1140
1160
1280
1190
1101
1110
1110
1130
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1300
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AATCTCACCCGGTCCGGACACTGTGAGGATTGACAGATTGATAGCTCTTTCTTGATTCGGTGGGTGGTGGTGCATGGCCGTTCTTAGTTGGTGGAGCGAT
II
. . . .. . . . . . .. . . . . . . . . . . . . . . . . . .. . . . . . . . . . . .. . . . . . .. . . . . .. . . . . . . . . . . . . . . . . . . . .. . . .. . . . . . . . . . . . . . . . . .. .
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TAOAPqTOOCC~TAACCTGCT~TCACTGTf~TGTCAGGIIATACTGG~'~TTbTTCCAC;TrGAAcGAc
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1601
1640
1660
1610
1610
1630
1650
1670
1680
1690
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AGCTTGCGCTGATTACGTCCCTGCCCTTTGTACACACCGCCCGTCGCTACTACCGATTGAATGGTTTAGTGAGATCGTTGGATTTAAOTGG
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. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..T.............C....A.....
1701
1710
1740
1750
1760
1710
1730
1770
1780
I
NCAACATTTCTATAATTGGGAGAAGACAATCAAACTTGATGGAT
II
. . . . . . . . . . . ..G.A.....A......C....................................................................
1790
FIG. I.--Sequences
of Dugesia
(S.) meditrrrunm
type I and type II 18s rDNA genes. Synthetic oligonucleotides
complementary
to
evolutionary
conserved regions of the 18s rDNA were used as primers in dideoxynucleotide
chain termination sequencing reactions. The type
I gene sequence (GenBank U3 1084) is presented, and nucleotide positions that are different in the type II gene (GenBank U3 1085) are indicated.
The six highly variable regions (nucleotides 69-8 1,220-24 I, 645743,
1046-I 072, 1335-l 375, 1465-l 567) are indicated in shaded gray. Boxes
within the variable region 645-743 show the location of the synthetic oligonucleotides
which were used in Southern and Northern blot analyses.
Open boxes indicate restriction sites for Hind111 (nucleotides 339-343) and EcoRI (nucleotides 1576-I 581). Note that Hind111 only cuts type
II. N: uncertain nucleotide.
830
Carranza et al
minthes. Second, a protozoan type II gene would be
functional and, unless the number of parasites was extremely low, detectable in Northern blots, which is not
the case (fig. 5). On the other hand, a low number of
parasites undetectable
in Northern blots cannot explain
the strong bands of hybridization
for type II seen in
Southern blots (fig. 3).
The overall difference between type I and type II
genes is around 8%. This is higher than values found in
interspecific comparison between 18s rDNA sequences
of different species and genera of freshwater planarians
(2.5%-3.9%;
table 5 in Riutort et al. 1992) and close to
the values found for interfamilial
comparisons
of freshwater planarians
(6.6%-8.9%;
table 5 in Riutort et al.
1992) and to those separating
classes of Vertebrata
(5.0%-7.0%
in table 5, Riutort et al. 1992). This means
that the divergence
between types I and II should be
ancient. Indeed, the phylogenetic
tree obtained (fig. 4)
shows that the type II gene branches off before the divergence of the species of the Dugesiidae family, but
after families Planariidae and Dencrocoelidae
branched
off from the main stem. This indicates that divergence
betwen type I and type II occurred between these two
events and anticipates that species of the family Dugesiidae should have both types of sequences
whereas
those from families
Planariidae
and Dendrocoelidae
should not (unpublished
data).
Heterogeneity
in 18s rDNA coding sequences has
only been reported for the A and C genes of several
species of the protozoan Plasmodium. These genes code
for 18s rRNAs that differ in 4.5% of their sequences
(Gunderson
et al. 1987). In metazoans, most variation
reported results from length heterogeneity.
In the parasitic platyhelminth
Schistosoma mansoni, length heterogeneity revealed by extrabands
of hybridization
are
thought to result from insertions of DNA sequences in
more than one place within the 18s rDNA sequence
(Simpson et al. 1984). The 28s rRNA of Drosophila
melanogaster
exhibit length heterogeneity
due to the
presence, in some copies, of a large intron (Barnett and
Rae 1979). However, single bands of hybridization
in
Southern blots (fig. 3) for type I and type II in D. (S.)
mediterranea and the extensive differences in sequence
between them clearly show that nucleotide changes, and
not length heterogeneity,
are at the base of such polymorphism. Thus, type I and type II genes of D. (S.)
mediterranea
bear a striking parallelism
to A and C
genes of Plasmodium species.
The only platyhelminth
studied so far, Schistosoma
mansoni, bears 100 copies per haploid genome of a standard repeat unit 10 kb long (Simpson et al. 1984; Van
Keulen et al. 1985). In situ hybridization
studies show
its rDNA to be located in the secondary constriction
region of chromosome
2 (Tanaka et al. 1995). In contrast, the rDNA organization
of the protozoan Plasmodium is unusual and is thought to be required for the
maintenance
of the two types of ribosomal RNA transcripts encoded by the A and C genes (Gunderson et al.
1987). Thus, the number of gene copies of each type is
two and they are unlinked in the genome as opposed to
being tandemly repeated. In the case of D. (S.) mediter-
ranea the differences in the restriction pattern between
both types and, conversely, conservation
of restriction
sites (lack of minor extrabands
of hybridization
in
Southern blots; fig. 3) and of the overall sequence within
each type suggest, though do not prove, that type I and
type II could be organized in separate, but internally
homogeneous,
arrays of repeat units. The location of the
two different arrays of clusters in different positions of
the same chromosome or even in different chromosomes
could help to prevent the homogeneization
between
types by concerted evolution. Attempts to localize type
I and type II into planarian chromosomes
by in situ hybridization, a technique still not developed in planarians,
has so far not worked well in our hands (unpublished
data).
Gene Expression
Identity between the sequence of type I gene described here and the partial sequence obtained by reverse transcriptase from total RNA (Riutort et al. 1992)
indicated that the type I gene is functional. Besides, positions that vary between type I and type II genes are
not randomly
dispersed throughout
the length of the
molecule, but are concentrated in six regions, which typically display considerable
variation among eukaryotic
18s rDNAs. Altogether, these features suggested that the
type II gene was not a pseudogene but a functional gene.
Northern blot analyses (fig. 5) shows this not to be the
case. Type I shows a very clear transcription
band in
both sexual and asexual races, whereas the type II gene
does not. In Plasmodium species, both A and C genes
are functional. A gene transcripts predominate in bloodstream and hepatic-stage parasites in the warm-blooded
host while C gene transcripts predominate
in parasites
developing
in the mosquito stages (Gunderson
et al.
1987). The switch from C to A transcripts has been related to its parasitic way of life.
In contrast to the parasitic Plasmodium species, D.
is a free-living platyhelminth
with a
(S.) mediterranea
rather simple life cycle (Bagufia et al. 1990). Since they
are hermaphrodite
animals, differences between sexual
and asexual populations
lie in the presence of gonads
(two ovaries and many testes), germ cells (eggs and
spermatozoa),
and the copulatory apparatus in the former. In the amphibian Xenopcls laevis and in the mammal Misgurnusfossilis,
two types of 5s rDNA genes are
differentially
expressed in somatic and germ cells. Since
sexual and asexual populations of D. (S.) mediterranea
do not express type II 18S, we can not assign, as in
Xenopus or Misgurnus, any type either to somatic (expected in both populations)
or germinal (only to be expected in sexual populations)
cell lineages. However,
minor tissue-specific
or even germ-cell-specific
expression of type II 18s cannot be ruled out. In situ hybridization in sections using probes specific to type I or type
II or isolation of germ cells will be necessary to detect
such minor transcriptional
activities. Moreover, stagespecific expression of both type I and type II during
embryonic development
could also be worthy of study.
Two Types of 1% rDNA
Phylogenetic
Implications
The findings described here may have important
methodological
implications
for phylogenetic
reconstruction using 18s rDNA sequences. A basic advantage
of this molecule is that it occurs in multiple copies that
are kept very similar through concerted
evolution.
Hence, small sample sizes, often single individuals,
are
currently used in most phylogenetic
studies. The finding
of divergent
18s rDNA variants in the genome of D.
(S.) mediterrunea
(extensive to other platyhelminth
species of different orders; unpublished
data) introduces a
degree of caution when using this molecule in phylogenetic studies. If 18s rDNA is polymorphic,
and if different types of 1% rDNA coexist and evolve independently within a species or group, we take a risk in comparing nonhomologous
sequences.
This is especially
true when DNA samples come from a single individual
or fragments
of it. The existence
of these polymorphisms, however, is not a drawback for the use of 18s
rDNA as a phylogenetic
tool. It only stressess the need
for analysis of a large number of clones when PCR amplification and cloning are used to obtain 18s rDNA
sequences. Moreover, direct sequencing from PCR could
be used as a first strategy (see fig. 2) to detect the existence of hidden polymorphisms.
Acknowledgments
We would like to thank Phillip Newmark (Departament de Genktica, Universitat
de Barcelona) for his
helpful comments and English correction. Rat RNA was
supplied by Rebeca Valero. We also thank Jose Ram6n
Bayascas and Teresa Luque for technical
support. This
research was supported by CICYT PB90-0477
(Ministerio de Educacidn y Ciencia) and by 2192-PGC 94A
(Generalitat de Catalunya).
LITERATURE CITED
ADOUTTE, A., and H. PHILIPPE. 1993. The major lines of metazoan evolution: summary of traditional evidence and lessons from ribosomal RNA sequence analysis. Pp. I-30 in
Y. PICHON, ed. Comparative molecular neurobiology. Birkhguser Verlag, Base], Switzerland.
ARNHEIM, N. 1983. Concerted evolution of multi-gene families. Pp. 38-61 in M. NEI and R. K. KOEHN, eds. Evolution
of genes and proteins. Sinauer Associates,
Sunderland,
Mass.
BAGU&& J., R. ROMERO, E. SALO, J. COLLET, C. AULADELL,
M. RIBAS, M. RIUTORT, J. GARCIA-FERNANDEZ, F. BURCAYA, and D. BUENO. 1990. Growth, degrowth and regeneration as developmental
phenomena
in adult freshwater
planarians. Pp. 129-162 in H. J. MARTHY, ed. Experimental
embriology in aquatic plants and animals. Plenum Press,
New York.
BALL, I. R., and T. B. REYNOLDSON. 1981. British planarians.
Cambridge University Press.
BARNETT, T., and I? M. M. RAE. 1979. A 9.6 kb intervening
sequence in Drosophila viridis rDNA, and sequence homology in rDNA interruptions of diverse species of Drosophila and other Diptera. Cell 16:763-775.
BAVERSTOCK, I? R., and C. MORITZ. 1990. Sampling design.
Pp. 13-24 in D. M. HILLIS, and C. MORITZ, eds. Molecular
systematics. Sinauer Associates, Sunderland, Mass.
in the Genome
of a Metazoan
831
BENAZZI, M., J. BAGuRIA, R. BALLESTER, I. PUCCINELLI, and
R. DEL PAPA. 1975. Further contribution to the taxonomy
group” with descripof the “Dugesia luguhris-polychroa
tion of Dugesia mediterranea n. sp. (Tricladida, Paludicola).
Boll. Zool. 42:8l-89.
CHIRGWIN, J. M., A. E. PRZYBYLA, R. J. MACDONALD, and W.
RUTTER. 1979. Isolation of biologically active ribonucleic
acid from sources enriched in ribonuclease. Biochemistry
l&5294-5299.
COEN, E. S., and G. A. DOVER. 1983. Unequal exchanges and
the coevolution of X and Y rDNA arrays in Drosophila
melanogaster. Cell 33:849-855.
DEVRIES, E. J., J. BAGuN;IA,and I. R. BALL. 1984. Chromosomal polymorphism
in planarians (Turbellaria, Tricladida) and
the plate tectonics of the western Mediterranean. Genetica
62:187-191.
DOVER, G. A. 1982. Molecular drive: a cohesive model of species evolution. Nature 299: 1 I 1-l 17.
-.
1986. Molecular drive in multigene families: how biological novelties arise, spread and are assimilated. TIG 2:
159-165.
Fox, G. E., K. R. PECHMAN, and C. R. WOESE. 1977. Comparative cataloging of 16s ribosomal ribonucleic acid. Molecular approach to procaryotic systematics.
Int. J. Syst.
Bacterial. 27:44-57.
GARCIA-FERNANDEZ, J., J. BAGuRA, and E. SALK. 1993. Genomic organization and expression of the planarian homeobox genes Dth-1 and Dth-2. Development 118:241-253.
GUNDERSON, J. H., M. L. SOGIN, G. WOLLETT, M. HOLLINGDALE, V. E DE LA CRUZ, A. I? WATERS, and T. E McCUTCHAN. 1987. Structuraly distinct, stage-specific
ribosomes occur in Plasmodium. Science 238:933-937.
HILLIS, D. M., and S. K. DAVIS. 1988. Ribosomal DNA: intraspecific polymorphism,
concerted evolution, and phylogeny
reconstruction.
Syst. Zool. 37:63-66.
HILLIS, D. M., and M. T. DIXON. 199 1. Ribosomal DNA: molecular evolution and phylogenetic inference. Q. Rev. Biol.
66:41 l-453.
HILLIS, D. M., C. MORITZ, C. A. PORTER, and R. J. BAKER.
1991. Evidence for biased gene conversion in concerted
evolution of ribosomal DNA. Science 251:308-310.
KUMAR, S., K. TAMURA, and M. NEI. 1993. MEGA: molecular
evolutionary genetics analysis, version 1.O1. The Pennsylvania State University, University Park, Pa.
LANE, D., B. PACE, G. J. OLSEN, D. A. STAHL, M. L. SOGIN,
and N. R. PACE. 1985. Rapid determination
of 16s ribosomal RNA sequences for phylogenetic
analyses. Proc.
Natl. Acad. Sci. USA 82:6955-6959.
LASCOMBE, C. 1973. L’affinitC de deux protozoaires Cpibiontes
pour les deux espttces jumelles de planaries d’eau deuce
Polycelis nigra et Polycelis tenuis. Ann. Limnol. 9125-44.
LASSNER, M., and J. DVORAK. 1986. Preferential homogenization between adjacent and alternate subrepeats in wheat
rDNA. Nucleic Acids Res. 14:5499-55 12.
LONG, E. O., and I. B. DAWID. 1980. Repeated genes in eukaryotes. Annu. Rev. Biochem. 49:727-764.
MASHKOVA, T. D., T. I. SERENKOVA, A. M. MAZO, T. A.
AVDONINA, Y. TIMOFEYEVA,and L. L. KISSELEV. 198 I. The
primary structure of oocyte and somatic 5S rRNAs from
the loach Misgurnus fossilis. Nucleic Acids Res. 9:21412151.
MCCABE, I? C. 1990. Production of single-stranded
DNA by
asymmetric PCR. Pp. 76-83 in M. A. INNIS, D. H. GELFAND, J. J. SNINSKY, and T. J. WHITE, eds. PCR protocols,
a guide to methods and applications. Academic Press, San
Diego, Calif.
832
Camanza et al.
T. 1984~. The evolution of multigene families under intrachromosomal
gene conversion. Genetics 106:529548.
1984b. The evolution of multigene families under in-.
terchromosomal
gene conversion.
Proc. Natl. Acad. Sci.
USA 81:3796-3800.
PETERSON, J. L., J. L. DOERING, and D. D. BROWN. 1980. Characterization of two Xenopus somatic 5S DNAs and one minor oocyte-specific
5S DNA. Cell 20:131-141.
PETES, T. D. 1980. Unequal meiotic recombination within tandem arrays of yeast ribosomal DNA genes. Cell 19:765774.
QARI, S. H., I. E GOLDMAN, N. J. PIENIAZEK, W. E. COLLINS,
and A. A. LAL. 1994. Blood and sporozoite stage-specific
small subunit ribosomal RNA-encoding
genes of the human
malaria parasite Plusmodium vivux. Gene 1.50:43-49.
RIBAS, M. 1990. Cariologia, sistematica i biogeografia de les
planaries d’aigues dolces als Pai’sos Catalans. Ph.D. thesis,
Universitat de Barcelona.
RIUTORT, M. 1991. Filogenia molecular i taxonomia de planaries d’aigties dolces (Platihelmints,
Turbellaris, Tricladides). Ph.D. thesis, Universitat de Barcelona.
RIUTORT, M., K. G. FIELD, R. A. RAFF, and J. BAGU~A. 1993.
18s rRNA sequences and phylogeny of Platyhelminthes.
Biochem. Syst. Ecol. 21:71-77.
R~UTORT,M., K. G. FIELD, J. M. TURBEVILLE,R. A. RAFF, and
J. BAGU&%. 1992. Enzyme electrophoresis,
18s rRNA sequences, and levels of phylogenetic resolution among severa1 species of freshwater planarians (Platyhelminthes,
Tricladida, Paludicola). Can. J. Zool. 70: 1425-1439.
SAFFO, M. B. 1992. Invertebrates in endosymbiontic
associations. Am. Zool. 32:557-565.
SAIKI, R. K., S. J. SCHARF, E FALOONA, K. B. MULLIS, G. T.
HORN, H. A. ERLICH, and N. ARNHEIM. 1985. Enzymatic
amplification of B-globin genomic sequences and restriction
site analysis for diagnosis of sickle cell anaemia. Science
233: 1076-1078.
NAGYLAKI,
SAITOU, N., and M. NEI. 1987. The neighbor-joining
method:
a new method for reconstructing phylogenetic trees. Mol.
Biol. Evol. 4:406-425.
SAMBROOK, J., E. E FRISCHT, and T. MANIATIS. 1989. Molecular cloning. A laboratory manual. Cold Spring Harbor University Press, New York.
SANGER, E, S. NICKLEN, and A. R. COULSEN. 1977. DNA sequencing with chain termination
inhibitors. Proc. Natl.
Acad. Sci. USA 74:5463-5467.
SIMPSON, A. J. G., J. B. DAME, E A. LEWIS, and E McCUTCHAN. 1984. The arrangement of ribosomal RN.4 genes
in Schistusoma mansoni identificati:ln ot’ poiy
mo;_ph~c xtructural variants. Eur. J. Biochem. 1;9,3!-15.
SLUYS, R. 1989. A monograph of the marine trtclads. A. A.
Balkema, Rotterdam.
SOGIN, M. L. 1991. Early evolution and the origin of eukaryotes. Cut-r. Opin. Genet. Dev. 1:457-463.
SZOSTAK, J. W., and R. Wu. 1980. Unequal crossing over in
the ribosomal DNA of Sucharum~ces
cerevisiue. Nature
284:426430.
TANAKA, M., H. HIRAI, I? T. LOVERDE, S. NAGAFUCHI, G. R.
FRANCO, A. J. G. SIMPSON, and S. D. J. PENA. 1995. Yeast
artificial chromosome
(YAC)-based
genome mapping of
Schistosoma mansoni. Mol. Biochem. Parasitol. 69:41-51.
VAN KEULEN, H., P T. LOVERDE, L. A. BOBEK, and D. M.
REKOSH. 1985. Organization of the ribosomal RNA genes
in Schistosomu mansoni. Mol. Biochem. Parasitol. 15:215230.
WATERS, A. P., C. SYIN, and T. E MCCUTCHAN. 1989. Developmental regulation of stage-specific ribosome populations
in Plasmodium. Nature 342:438-440.
WEGNEZ, M., R. MONIER, and H. DENIS. 1972. Sequence heterogeneity of 5S rRNA in Xenopus luevis. FEBS Lett. 25:
13-20.
WOESE, C. R. 1987. Bacterial evolution. Microbial. Rev. 51:
221-271.
MANOLO GOUY, reviewing
Accepted
February
editor
26, 1996