volume 11 Number 20 1983
Nucleic Acids Research
The nodeotide sequence* of SS rRNAs from two ribbon worms: Emplectonema gracile contains two
SS rRNA species differing considerably in their sequences
Tsutomu Kumazaki1, Hiroshi Hori and Syozo Osawa
Laboratory of Molecular Genetics, Department of Biology, Faculty of Science, Nagoya University,
Chikusa, Nagoya 464, Japan
Received 19 August 1983; Accepted 30 September 1983
ABSTRACT
The nucleotide sequences of 5S rRNAs from two nemerteans (ribbon worms),
LineuB geniculatus and Empleotonema gracile have been determined.
Emplectonema has two 5S rRNA species that are composed of 119 and 120
nucleotides, respectively. The sequences of these two 5S rRNAs differ at 22
positions. On the other hand, only a single 5S rRNA species was found in
LineuB. The sequence similarity percents are 88Z (Lineue I Empleotonema longer
5S rRNA), 82Z (.Empleotonema longer I Emplectonema shorter) and 80Z
{LineusI'Emplectonema shorter). The comparisons of these sequences with those
of other organisms suggest that the phylum Nemertinea is most related to the
Mollusca (91Z) and the Rotifera (89Z), but not to fresh-water planariaa
(72Z).
INTRODUCTION
The nemerteans have often been treated on the anatomical basis as an
offshoot from the free-living flatworms (planarias) (1), but there has been
no approach to settle their phylogenic position on the molecular basis.
In
this study, we have determined the 5S rRNA sequences from a Japanese
nemertean Lineus geniculatua, and a Naples nemertean Empleotonema gracile,
and compared them with those from flatworms (marine and fresh-water
planarias) and other eukaryotes as described before (2).
MATERIALS AND METHODS
Several individuals of Linens genioulatus were collected at an area of
the Ise-Bay near the Sugaehima Marine Biological Station, Nagoya University.
Specimens of Empleotonema graoile were collected at the seashore near the
Stazlone Zoologica di Napoli, Italy.
The 5S rRNA of Lineus was directly isolated and purified from whole
organisms by phenol method followed by gel electrophoresis as previously
described (3). The 5S rRNAs of Empleotonema were also treated as above, but
the crude RNAs were subjected to chromatography on a DE52 column before gel
electrophoresis to remove polysaccharide-like substances.
The sequences were determined by the chemical method of Peattie (4) or
the enzymatic method of Donis-Keller (5) using [31- or 5'- P] RNA. Certain
parts of the sequence were confirmed by the electrophoresis on a hot plate at
70°C (6). The 3'- and 5'-terminal bases were determined by chromatography on
a cellulose TLC plate (7).
© IR L Press Limited, Oxford, England.
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Nucleic Acids Research
Mussel
Lineua
Emplecto.
Emplecto.
Marine P.
2
1
123456789 012345 678901 2345678
GUCUACGAC CAUAJUC ACGUUG AAAACAC
GUCUACGAC CAUAjUC ACGUUG AAAACAC
GUCUACAAC CAUACC ACGUUG AACACAC
GGCAACGAC1 CAUAJCC AUGCUG AAAAUAC
(fijAGCCflC CAUA£C ACACUG AAAACAC
aLb
bLc
B
3
9012
CGGU
CGGU
CGGU
CAGU
CGGU
345678901234
UCUCGUCCGAUC
UCUCGUCCGAUC
UCUCGUCCGAUC
UCCCGUCCGAUC
UCUCGUCCGAUC
cLc1
5678
ACCG
ACCG
ACCG
ACUG
ACCG
C
90123456
AAGUUAAG
AAGUUAAG
AAGUCAAG
AAGUCAAG
CAGUUAAG
c'Lb1
1
1
1
0
1
2
6
8
789012 345 6789012 34567 890123456 7890 123456789 012 3456789 012345678 90
CAACGU
GCCCGGU UAGUA
GUGA CCG"CUGGG AAU ACCGGGU GUUGUAGAC
GCGUGGU UAGUA
GUGA
AAU ACCACGU GUUGTJAGGC
CAACGU
GUGA CCG C UUGGG AAU ACCGAGU GUUGUAGAC
CAACGU 'AlGGl GCUCGGU UAGUA
GUGA CCG C SUGGG AAC ACCGGGU GUCGUUi
CAGCAU |CJGG| GCCCGGU UAGUA
GUGA CCCCCUGGG AAU ACUGGGU GQCGCUAQC
CAGUGU < Q G G | G C C C A G U UAGUA
a*Ld'
eLd
Fig. 1
AAC
2 A
A 3
cue
C 0 U
0 A
CGGU
1 UAUCACGUUG
****
0A ,',' ******
C ,',' UGCAAC
GCCA
!.'C 6 G
GUCUACGAC
*********
UAG4
k
A
CGGAUGUUG
0
A G5
1U*G
UU 0
U
1G*C
OC*G
A*U
C*G70
C*G
A*U
U
Fig. 1. Sequence alignment of 5S rRNAs of
U
Lineua genioulatua, Empleatonema gradle,
A
a mussel (8) and a marine planaria (9). The
G
squared-off sequences correspond to the
U
100A
base-paired regions in secondary structures.
A
Dotted lines represent the potential base-pairs
G*C
according to Peattie et al. (13). Symbols,
G G
A,A', B,B' etc. in the lowest line are for
C U80
base-paired regions. Symbols, aLb, bLc, etc.
are for loop regions (see ref. 2 ) .
" C*G ••
C A
G*U
Fig. 2. Secondary structure model of 5S rRNA from
C*G
Lineua genioulatua. Dotted lines indicate
C*G
potential base-pairs according to Peattie
90A G
et al. (13).
G U
Fig. 2
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Nucleic Acids Research
RESULTS
After the labelling of 3'- or 5'-terminus of the Emplectonema 5S rRNA
with
P, the labelled material was subjected to electrophoresis on a 6.5Z
polyacrylamlde gel (800 x 200 x 0.5 mm).
The autoradiogram showed that two
5S rRNA species, a longer (L) and a shorter (S), exsisted
radioactivity ratio of 5 : 4.
with the
On the other hand, the labelled product of the
Linens 5S rRNA gave a clear single band on the gel.
The Lineua and the Empleotonerm L 5S rRNAs were both 120 nucleotides
long, while that of the Emplectonema S 5S rRNA was 119 nucleotides long.
Their sequences are shown in Fig. 1 with the sequences of a mussel (8) and a
marine planaria (9) for comparison.
differed at 22 positions.
The Empteotonema L and S sequences
Furthermore, the position 117 of the Emplectonema
L 5S rRNA was heterogeneous (A or G).
No heterogeneity was found in the
Lineua 5S rRNA sequence.
DISCUSSION
The secondary structure of the 5S rRNAs from the nemerteans is
essentially the same as that previously reported for the multicellular animal
5S rRNAs (3), which have five base-paired regions and an A/C mismatch at the
D-D' base-paired region (Fig. 2 ) . The Lineus 5S rRNA has, in addition to the
above A/C mismatch, two more mismatches at the D-D' region (Fig. 1).
The microheterogenelty of the 5S rRNA sequences in one organism has been
reported in many cases throughout prokaryotes and eukaryotes.
Beside such a
microheterogeneity, the presence in one organism of two major 5S rRNA
populations differing considerably in their sequences has been known in
certain eukaryotic species such as in a toad Xenopua laevis (differing in 7
positions) (10,11), a nematode Rhabditie tokai (in 13 positions) (12), etc.
The Emplectonema L and S sequences are extreme in the sense that they differ
at 22 positions.
In contrast to this, only one species of 5S rRNA can be
detected in a related nemertean, LineuB.
The sequence similarities among the nemertean 5S rRNAs are 88Z
{Lineua IEmplectonema L ) , 82Z (Empl. L/Empl. S) and 80* (Lin./Empl. S ) ,
respectively.
These values suggest that the ancestor for these nemerteans
possessed at least two kinds of 5S rRNA genes, L and S, and these two
evolved independently.
After the separation of Lineua and Emplectonema, the
S-type gene might have disappeared or have been repressed in Lineua, while
both L- and S-types have been functioning in Emplectcmema.
The similarity matrix of the 5S rRNA sequences (not shown) indicates
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Nucleic Acids Research
that the nemerteans are most related to molluscs (91Z identity) and a rotifer
(89Z), considerably to vertebrates (79Z), arthropods (84Z), a marine planaria
(79Z), coelenterates (83Z) and sponges (85Z), and less to fresh-water
planarias (72Z), protozoa (68Z), plants (65Z), the Basidiomycetes (66Z) and
the Ascoinycetes (64%).
For the above calculation, the average of the LineiiB
and Empleatonema L sequences, but excluding the Empleotonema S sequence, has
been used, since the meaning of the Empleatonema S sequence is not clear at
present.
Thus, in contrast to the anatomical evidence, the nemerteans are
not particularly connected to flatworms when Judged from their 5S rRNA
sequences.
Especially notable is that the 5S rRNA of the nemerteans is less
similar to that of fresh-water planarias than to those of many other animal
groups.
ACKNOWLEDGEMENTS
We thank Professor Motoki Hoshi of this department who kindly collected
Empleotonema for us during his stay at the Statione Zoologica di Napoli,
Italy. Thanks are also due to Professor Fumio Iwata of Hokkaido University
for the identification of Empleatonema. This work was supported by grants
56480377, 56570178 and 57121003 (Special Project Research) from the Ministry
of Education of Japan.
Present address: Department of Biochemistry and Biophysics, Research
Institute for Nuclear Medicine and Biology, Hiroshima University, Hiroshima
734, Japan.
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