volume 10 Number 71982
Nucleic A c i d s Research
The nucleotide seqaence of chJoroplast 4.5S rRNA from a fern, Dryopteiis acuminata
Fumio Takaiwa, Mie Kusuda and Masahiro Sugiura
National Institute of Genetics, Mishima, Shizuoka-ken 411, Japan
Received 11 February 1982; Accepted 8 March 1982
ABSTRACT
The 4.5S rRNA was isolated from the chloroplast ribosomes
from Dryopteris acuminata. The complete nucleotide sequence was
determined to be: 0HUAAGGUCACGGCAAGACGAGCCGUUUAUCACCACGAUAGGUGCU
AAGUGGAGGUGCAGUAAUGUAUGCAGCUGAGGCAUCCUAAUAGACCGAGAGGUUUGAACQH-
The 4.5S rRNA is composed of 103 nucleotides and shows strong
homology with those from flowering plants.
INTRODUCTION
A 4.5S rRNA is an unique component of chloroplast ribosomes
from higher plants, which is associated with the large subunit
(1-9). The nucleotide sequences of the 4.5S rRNA species from
tobacco (6), wheat (8) and maize (9) chloroplasts have recently
been determined. The sequences of these 4.5S rRNA are found to
be homologous with that of the 3'-terminal region of E. coli 23S
rRNA (9-11). Therefore, it was proposed that chloroplast 4.5S
rRMAs are functionally equivalent to the 3'-terminal region of
prokaryotic 23S rRNA. The appearance of 4.5S rRNA during evolution is quite interesting. He, therefore, surveyed the RNA
species in lower plants and found it in the fern chloroolast
ribosomes and determined its nucleotide sequence.
HATERIALS AND METHODS
Chloroplasts were prepared from freshly harvested leaves of
a fern (Dryopteris acuminata) (12). Total RNA was extracted
twice with an equal volume
of 80% phenol from the chloroplasts
after lysis with 2% Triton X-100 in 10 mM Tris-HCl (pH 8.0), 30
mM KC1, 20 mM MgCl2 and 0.1% dietylpyrocarbonate, and the 4.5S
© IRL Press Limited, 1 Falconbara. Court. London W1V 5FG, U.K.
0306-1048^2/1007-2267S 2.00/0
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Nucleic Acids Research
Fig. 1 Polyacrylamide gel
electrophoresis of lowmolecular-weight RNA from D.
acuminata chloroplasts.
~
-5SL
-5SM'5S S
-4.5S
4S
rRNA was purified by electrophoresis in a 10% polyacrylamide gel
as described (5) . The 5 1 and 31 terminal labeling and its
sequencing were carried out as described (6, 13). The T4 polynucleotide kinase and RNA ligase were prepared as described (14,
15).
[ Y 3 2 P ] A T P and [5'32P]pCp were obtained from Radiochemical
Centre.
RESULTS AND DISCUSSION
Chloroplast 4.5S rRNA has been found so far in the flowering plants (3). When -the D. acuminata chloroplast RNA was fractionated by electrophoresis in a 10% polyacrylaraide gel, an
additional low-molecular-weight RNA component was found between
txie 5S rRNA and 4S RNA (Fig. 1) . V7e then isolated and sequenced
this RNA species.
The sequence of at least 93 nucleotides from the 3' end was
unambigously determined by the chemical method using the [31 P]
RNA (16). The sequence from the 5' end to position 40 was determined by the enzymatic method using the [5 |32 P]RNA (17, 18).
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Nucleic Acids Research
20
II
ill
SI
D. acuminata
5' UAAGGU-CACGGCAAGACGAGCCGUUUAUCACCACGAUAGGUGCUAAGUGG
Tobacco
5' G
Wheat
5'
GAG
G
A
UC
Maize
5'
-AG
G
A
UC
G
to
D. acuminata
UU
70
UC
10
90
100
AGGUGCAGUAAUGUAUGCAGCUGAGGCAUCCUAAUAGACCGAGAGGUUUGAAC 3'
A
Tobacco
3'
G
C
-
AC A
3'
-
AC A
3'
Wheat
A
G
CGA
Maize
A
G -
CGA
GU
AC
Fig. 2 Alignment of 4.5S rRNA species from chloroplasts of D.
acuminata, tobacco (5), wheat (8) and maize (9). Nucleotides
identical to those found in D. acuminata chloroplast 4.5S
sequence were not typed. Bars denote gaps introduced to maximize
sequence homology.
The 5' and 31 terminal residues were further confirmed by PEI
thin-layer chromatography after complete digestion of the [5'32P]
RNA with nuclease Pa and the [3'32P]RNA with RNase T2.
The 5'
end was identified not to be phosphorylated because it could be
labeled with polynucleotide kinase and [ Y ' 2 P ] A T P without prior
dephosphorylation.
The total nucleotides sequence is shown in
ABSTRACT and in Fig. 2.
It is 103 nucleotides long and its
sequence is very similar to those of 4.5S rRNA species reported
so far, thus indicating that this fern RNA species is classified
as,
so called, 4.5S rRNA.
It is the first case that the
presence of chloroplast 4.5S rRNA was established in non-flowering plants.
A comparison of the nucleotide sequence of chloroplast 4.5S
rRNA from £. acuminata, tobacco (6), wheat (8) and maize (9) is
presented in Fig. 2.
As an unique feature, the sequence corres-
ponding to positions 28 to 34 of tobacco 4.5S rRNA, which is
missing from wheat and maize 4.5S rRNAs, is found in the D.
acuminata 4.5S rRNA.
The D. acuminata 4.5S rRNA shows 87%, 78%
and 78% homology with the tobacco, wheat and maize 4.5S species,
respectively.
The degree of homology between tobacco and D.
acuminata 4.5S rRNAs is very similar to that of these 5S rRNAs
(87%) (in preparation).
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Nucleic Acids Research
comparable to chloroplast 4.5S rRNA was not detected in
blue green algae, Anabaena sp. (3) and Anacystis nidulans (19),
Euglena gracilis (20) , Chlamydomonas reinhardii (21) , liverwort
(3), moss (3) and a fern, Adiantum sp. (3). Present work showed
unarabiguosly tiie existence of 4.5S rRNA in a fern, D. acuminata.
It is therefore suggested that Pteridophyta is a borderline
class as to the existence of 4.5S rRNA.
ACKNOWLEDGEMENTS
We thank Dr. N. Naruhashi (Toyama Univ.) for identification
of the fern. This work was supported in part by Grants-In-Aid
from the Ministry of Education.
*To whom correspondence should be addressed.
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