The primary structure of the 5 S rRNA isolated from the cryptobiotic cysts of the brine shrimp Artemia salina is pACCAACGGCCAUACCACGUUGAAAGUACCCAGUCUCGUCAGAUCCUGGAAGUCACACAACGUCGGGCCCGGUCAGUACUUGGAUGGGUGACCGCCUGGGAACACCGGGUGCUGUUGGCAU (OH)

De Baere, Raymond

De Wachter, Rupert

Diels, Ludo

Vandenberghe, Antoon

Nucleic Acids Research

PubMed

Diels, L.

De Baere, R.

Vandenberghe, A.

De Wachter, R.

Open Marine Archive

volume 9 Number 19 1981 Nucle ic Acids Research
The sequence of the 5S ribosomal RNA of the crustacean Artemia salina
Ludo Diels, Raymond De Baere, Antoon Vandenberghe and Rupert De Wachter
Departement Celbiologie, Universiteit Antwerpen, Universiteitsplein 1, B-2610 Wilrijk, Belgium
Received 30 July 1981
ABSTRACT
The primary s t ruc ture of the 5 S rRNA i so la ted from the c ryp tob io t i c
cysts of the br ine shrimp Artemia sa l ina i s pACCAACGGCCAUACCACGUUGAAAGU
ACCCAGUCUCGUCAGAUCCUGGAAGUCACACAACGUCGGGCCCGGUCAGUACUUGGAUGGGUG
ACCGCCUGGGAACACCGGGUGCUGUUGGCAUOLJ .
Un
INTRODUCTION
The availability of gel sequencing techniques applicable to small RNA
molecules is resulting in a rapid growth of the collection of known
5 S rRNA primary structures . These data can be used in phylogenetic stud-
1 R
ies ' , and in attempts do derive general secondary structure models for
7,9~125 S RNA ' by a search for common sequence complementarities. We report
the primary structure of 5 S rRNA isolated from the cysts of the brine shrimp
Artemia salina (Phylum arthropoda, class Crustacea).
MATERIALS AND METHODS
Dry cysts of Artemia salina were obtained from San Francisco Bay Brand
(NewarK, California). The ribosomes were isolated according to Zasloff and
Ochoa and ribosomal RNA was obtained from these by phenol extraction
5 S RNA was isolated by gel filtration on SephadexG100 as described by
15
Monier , except that the column was not recycled. It was further purified
by electrophoresis on slab gels containing 10% polyacrylamide, 7 M urea,
0.05 M Tris-borate buffer pH 8.3 and 0.001 M EDTA.
Three different gel-sequencing methods were used to derive the primary
structure : the methods of Diels and De Wachter and of TanaKa, Dyer and
4
Brownlee , both of which are two-dimensional adaptations of Stanley and
Vassilenko's procedure , and Peattie's chemical degradation method. RNA
labeled at the 3'-terminus by [32P] pCp ligation was also used for a confir-
mation of the terminal sequence by a moving spot analysis . The 5'-terminal
©IRL Press Limited, 1 Falconberg Court, London W1V5FG, U.K. 5141
Nucleic Acids Research
basa was determined by alkaline hydrolysis of 20 vg unlabeled RNA, followed
by HPLC identification of the resulting nucleoside bisphosphate. The hydro-
lysate was fractionated on a 4.6 x 250 mm Partisil SAX (Reeve Angel, Clifton,
N.J.) column eluted with 120 ml of a concentration gradient of H P0 adjusted
to pH 2.2 with NH , rising from 0.01 M to 1 M in 100 min. Nucleotides are
eluted in the order Cp, Ap, Up, Gp, pCp, pAp, pUp, pGp.
RESULTS
Different gel sequencing methods were used to establish the complete
sequence, as summarized in Table 1. Each part of the sequence was confirmed
by at least three succesfull experiments. In the area of positions 62 to 70,
which is rich in G's andC' s probably involved in base pairing, the chemical
degradation method was preferable to the methods based on aspecific hydro-
4 5lysis ' which gave poor resolution. The 3'-terminal sequence was confirmed
by a moving spot analysis of a partial hydrolysate of 5 S RNA labeled at the
3'-end with [32P ] pCp. We have found it difficult to dephosphorylate the
5 S RNA without partially degrading it, hence we could not label the 5'-
terminus satisfactorily. However, the 5'-terminal residue was identified un-
equivocally as pAp by complete alkaline hydrolysis and HPLC-analysis of un-
labeled material. In pig. 1, the sequence of Artemia salina 5 S RNA is
compared with that of the two other arthropods thus far investigated,
1 fl 1 Q
Drosophila melanogaster and Bombyx mori
DISCUSSION
The primary structure of Drosophila melanogaster 5 S rRNA was originally
reported20 as GGGCGCm , but later corrected to GCGGGC69. The recent
determination of the 5 S RNA gene sequence and the comparison with other
arthropods (Fig. 1) now make it evident that the first version was the
correct one. The primary structure of Artemia 5 S rRNA differs with that of
Table 1. Methods used to establish the Artemia 5 S rRNA sequence
Method
Gel sequencing :
Other :
Diels 8 De Wachter5
4
Tanaka et a l .
Peattie3
moving spot
alkaline hydrolysis, hPLC
Positions ident i f ied
4-25,27-65,70-117
2-61, 70-111
4-120
102-120
1
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Nucleic Acids Research
A . s .
B . m .
D . m .
60
CAACGU:GG
10 20 30 40 50
AJCCAACGGCCAUACCACGUUGAAAGUACCCAGUCUCGUCAGAUCCUGGLAAGUCACA
[GCCAACGUCCAUACCAUGUUGAAUACACCGGUUCUCGLICCGAUCACCGAAGIICAAG
GCCAACGACCAUACCACGCUCAAUACAUCGGUUCUCGUCCGAUCACCGAAAUUAAG
A B C C '
70 80 90 100 110 1 20
CAACAU
CAGCGU
iCCCGGUCAGUA
;CGUGGUCAGUA
;CGCGGUUAGUA
CUUGGAUGG
CUUGGAUGG
CUUAGAUGG
;UGA
;UGA
;GGA
CCG
CCG
CCG
CUGGGAACACCGGGU
CUGGGAACACCACGU
UUGGGAACACCGCGU
GCUGUUGGEAU
GAUGUUGGdUU
iUUGUUGGqCU
B' D '
Fig. 1. Conservation of 5 S RNA primary and secondary structure within the
phylum arthropods (A.s., Artemia salina; B.m., Bombyx mori; D.m., Drosophila
melanogaster). The five double-stranded areas of our secondary structure
model are indicated by the boxes A-A1, B-B' etc.. Nested boxes delimit
bulges and small interior loops within these areas.
Drosophila in 28 positions and with that of the silkworm in 21 positions. The
two insect sequences show 1B differences. Among the vertebrates, the mean
divergence between two organisms belonging to different classes tfish,
amphibia, reptiles, birds, and mammals have been investigated) is limited to
9 positions.
A common base pairing scheme can be applied to the three arthropod
sequences, as indicated by the boxes in Fig. 1. The resulting secondary
structure model is illustrated with the Artemia 5 S RNA in Fig. 2. There now
seems to be a consensus among the majority of investigators that there is a
B
A C
C C A A C G G C
A U
G G U U G U C G
C
A
u n
U G
G -C
G C
G C
C G
C G
A - U C
CC AC GUUG
GGUGC AAC
C '
u c u c
A C C C A G uu
U G G G U C c
C A c A-A C _A
G-C A
G U
GU
UG
CG
C A
GU
CG
CG
A G
G U
A
G
U
Fig. 2. Secondary
structure model for
Artemia 5 S RNA.
5143
Nucleic Acids Research
basic difference between the secondary structures of bacterial and eukaryotic
5 S RNAs, the former having four helical regions, the latter five9"1^. We do
not share this opinion and we present evidence elsewhere (De Wachter, Chen
and Vandenberghe, manuscript submitted) that the model of Fig. 2 applies,
with minor variations, to all 5 S RNAs hitherto sequenced.
ACKNOWLEDGMENTS
We are grateful to Drs. G.G. Brownlee and P. Arcari for instructing
4
one of us (A.V.) in the gel sequencing method of Tanaka et al. . This work
was supported by the belgian foundation for basic research F K F O .
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The sequence of the 5S ribosomal RNA of the crustacean Artemia salina

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