volume 9 Number 91981
Nucleic Acids Research
The primary structure of oocyte and somatic SS rRNAs from the loach Misgumus fossilis
T.D.Mashkova, T.I.Serenkova, A.M.Mazo, T.A.Avdonina, M.Ya.Timofeyeva and L.L.Kisselev
Institute of Molecular Biology, USSR Academy of Sciences, Moscow 117984, USSR
Received 30 March 1981
ABSTRACT
Somatic and oocyte 5S rRNAs from the liver and unfertilized
eggs of the loach Misgurnus fossilis have been sequenced and
found to differ in six nucleotides. All the substitutions are
confined to the 5'-half of the molecules; 4 of them are pyrimidine-pyrimidine substitutions, and 2 are purine-pyrimidine
ones. Considerable differences, both in the position and the
character of substitutions, have been established when these
5S rRNAs were compared with somatic and oocyte 5S rRITAs from
Xenopus borealis and Xenopus laevis. Among the known primary
structures,somatic 5S rRNA of LI.fossilis is most similar to
trout 5S rRNA.
INTRODUCTION
5S rRNA genes in eukaryotic organisms are tandems of repeats and the number of their copies in the genome may be far
greater than that of ribosomal 18S and 28S genes . iith the
exception of certain lower organisms (fungi, yeasts), they are
located separately from 183 and 283 genes, and in different
chromosomes
. As was sho\/n for amphibians, tnere are several types of 53 genes differing in the primary structure ->~ :
genes that are transcribed mainly in oogenesis (oocyte genec),
and a minor type of genes transcribed in embryonic und coautic
cells (somatic genes). Moreover, trie co-called p^eudogenes
were found in Xenopus laevia, in the most of tue repeating
units carrying the gene of oocyte bS r.ii.n; thece pcjeudogene3
correspond to 5'-1-101 nucleotides of the 5d ?.i*~r. sequence
Q
-j Q
with a loss of the Ia3t 19 nucleotides from the 3'-terminus ' .
IIo data demonstrating expression of tnese goneo are available
at present.
© IRL Press Limited. 1 Falconberg Court, London W1V 5FG, U.K.
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Nucleic Acids Research
Certain teleost fishes are known to contain two types of 5S
1112
1"}
rRNA
'
, In our work
, for example, 5S rRNA from unferti-
lized eggs of the loach differs in its electrophoretic mobility from liver 5S rRNA when these are separated by electrophoresis in polyacrylamide gel with 7 M urea at 20°C though their
electrophoretic mobility is identical in the same gel at 50°C.
It appears therefore that the somatic and oocyte types of
5S
rRNA differ in the loach, just as in the amphibians, in primary structure though their length is identical as v/as found for
frog 5S rRNAs.
Our work was aimed at sequencing oocyte and somatic 5S rRNA
from the loach Misgurnus fossilis.
MATERIALS AND METHODS
RNAases T 1 , T« and Up were purchased from Sankyo, E.coli
alkaline phosphatase from Sigma, snake venom phosphodiesterase
from Worthington. RNAase A was a generous gift of Yu. Lebedev,
RNAase from Physarum polycephalum was kindly provided by A. Pusyriov, and T. RNA ligaae was a kind gift of V. Burd. Polynucleotide kinase was isolated from E,coli infected with T.
phage
. Carrier-free f P] orthophosphoric acid was obtained
from the Radiochemical Centre (Amersham), PEI cellulose from
Schleicher and Schull. t - P 2 P 7 A T P v/as synthesized as in ref. ,
5'-[32P]pCp as in ref. 16.
5S rRNA was isolated from loach liver and unfertilized eggs
by the phenol-detergent technique with the subsequent reprecipitation of total nucleic acid preparations by 1.5 M NaCl as
17
in ref.
. The isolated salt-soluble preparations contained
DNA, 5S rRlTA and tRUA as v/ell as an admixture of low molecular
weight nuclear RNAs.
The preparations of salt-soluble RIJAs were separated by electrophoresis in 12^5 polyacrylamide gel in the presence of 7 I.I
urea in 0.05 I.I tris-borate, pH 8.3, with 0.01 M EDTA at 180 V
for 12 h. Fractions corresponding to 5S rRNA v/ere eluted
from
the gel with 0.5 M NIL Ac, 0.01 1,1 Mg(Ac) o , 0.1% SDS, 0.1 mM
11 ft
ft
^
^
EDTA I O
After 5S rRITA had been treated with alkaline phosphatase,
it was labeled at the 5'-terminus using T. polynucleotide kinase
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Nucleic Acids Research
and y-P 2 PJATP (500-1500 Ci/mmole) as described earlier 1 8 .
55 rRNA was modified with a mixture of bisulfite and meth19 20
oxyamine according to refs
' . Once the reaction was over,
the RNA was purified using electrophoresis in 10% polyacryl'amide gel and eluted from the gel adding total yeast tRNA as a
carrier 1 8 . To convert G into U residues, 5 ' - [ 3 2 P ] 5 S rRNA
^ cpm, 1-5yug) was incubated in 0.15 ml of 1.5 M NagSgO,-,
10 mM EDTA and 7 M urea (pH 5.8) at 60°C for 3 h 2211. The material was desalted on a Sephadex G-50 (fine) column and treated
19 20
with a mixture of methoxyamine and bisulfite as described
' .
[
O p "*
labeled modified RNA (1 ug, lO^-IO3 cpm) was digested with
RNAases T1 and A in 5 p.1 of 0.1 M tris.HCl (pH 7.5), 10 mM
EDTA, and with RNAases U o , T o and Phy I in 5 ul of 50 ml NaAc
d.
C.
of) r/p
(pH 4.5), 2 mM EDTA as described in refs <:'J»". Non-modified
RNA was digested with RNAases T 1 , A, U 9 , Phy I and Phy M according to
• . 5 Ml of formamide containing 0.05% xylenecyanol
was added to the samples after hydrolyses. Partial non-specific
hydrolysis of RNA was conducted as in 20 , and the hydrolysates
were loaded on 8, 10 and 20% polyacrylamide gels (0.06x20x60 cm)
with 7 M urea
. After electrophoresis, the gels were exposed
with an X-ray RT-1 film at -70°C.
To determine 5'-terminal nucleotides, 5S rRNA (10 ,ug,
4
1O -1O5 cpm) in 0.15 ml of 0.01 M tris«HCl (pH 8.6), 0.01 M
Mg(Ac)p was digested with snake venom phosphodiesterase
(0.2 U/iug RNA), and the hydrolysate waa subjected to fractionation on PEI cellulose plates
. 5'-Nucleoside monophosphates
were used as markers. In order to determine 3'-terminal nucleotides, 5S rRNA (10yug, 1O4-1O5 cpm) was digested with T 2 RNAase
(0.02 U/ug RNA) at pH 4.5, and the hydrolysate was fractionated
24
as described in
. 3'-Nucleoside monophosphates were used as
markers.
RESULTS
The results of electrophoretic separation of salt-soluble
RNA preparations from loach liver and unfertilized eggs are
shown in Fig. 1. One can see that 5S rRNA from the unfertilized
eggs has a much greater electrophoretic mobility than 5S rRIIA
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Pig.
1. Electrophoretic separation
of salt-soluble KNA preparations
from loach liver (A) and
unfertilized eggs (B). The position of 5S rRNA is indicated with
arrov/s.
from the liver. 5S rRNA of a different type is present as a
minor component in the both preparations. To analyze the primary structure, RNA was isolated from the major electrophoretic fractions.
Analysis of terminal nucleotides in loach somatic and
oocyte 5S rRlTA has shown G to be located at the 5'-end of the
molecules. C and U were found in almost equal proportions at
the 3'-ends of both somatic and oocyte 5^ rltlJAs. At the same
tine, using the readout technique the
AAGC
sequence was
found to be adjacent to the 3'-ends of both type3 of 5S rRNAs.
Consequently, these iCJAs are a mixture of molecules ending
with
AAGC
and
iiAGCU
. Airthermore, the results of a
longer electrophoretic runs show that some molecules contain
also a 120th. nucleotide, possibly, 'J (shown in parentheses in
3),
just t.c in other animal 5S rrtMAs
determined only for 5'-[
. The sequence was
?] 53 rlUTA since the 3'-termini of
these lil'.k molecules are heterogeneous.
The sequencing was done using our procedure described else20
v/here
; the technique is based on selective modification of
•"32 "
C residues in 5'-[
xM labeled XIA •.vith a mixture of metnoxy-
amine and bisulfite, and on partial digestion of such R1JA(C*)
v/ith specific endonucleases. The principal advantage of this
technique is tnat it eliminates the effect of the secondary
structure in hydrolysis v/ith endonucleases as well as during
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Nucleic Acids Research
subsequent electrophoresis in polyacrylamide gel since the modification causes cleavage of G-C hydrogen bonds.
In addition to the above approach, we used also hydrolyses
of RNAs in which almost all cytosine residues were converted
21
into uridine ones by bisulfite treatment
, and then treated
with a mixture of methoxyamine and bisulfite to modify unreacted C residues. The product of such double treatment is designated as RNA (C-"-U, C * ) . Since N-U bonds are usually split with
endonucleases faster than N-C* bonds
20
» 2 ° j this strategy makes
it possible to establish not only the positions of C converted
into U, but also the nature of residues adjacent to them. Comparison between hydrolysates of modified RNA(C*) and RNA(C-*-U,
C*) enabled us to determine the complete nucleotide sequence of
5S rRNA, with the exception of four pyrimidines in positions
18, 29, 35 and 45 in somatic 5S rRNA and three pyrimidines in
positions 29, 35 and 45 in oocyte 5S rRNA. This confusion was
overcome by hydrolyzing unmodified RNAs with endonucleases
Phy I
22
and Phy M
23
(data not shown). It should be noted that
hydrolyses of native (unmodified) RNAs do not allow to determine
unambiguously a considerable part of the sequence due to the
difficulties caused by the stable secondary structure.
As an
illustration, analysis of the nucleotide sequence of
fragments from two 5S rRIIAs is shown in Pig. 2. Intensification
of bands in the U 2 track for 5S rRNA(C-»-U, C*) comparing to
5S rRNA(C*) confirms that C is the 3'-neighbour of these A residues. In the region presented in Pig. 2, these bands correspond to Ar, A.,, k*r and Ap,- in somatic RNA and to A,- and A..-,
in oocyte RNA. Appearance of new, as compared to 5S rRNA(C*j,
bands in the A track for 5S rRNA(C-«-U, C*) indicates that the
bands correspond to C residues. The presence of bands for C 1 0 ,
C 1 2 and C 1 4 in the U 2 and T 2 tracks for 5S oocyte rRHA(C*)
should be attributed to a great lability of CJ Q -A, C*?-A and
Cf.-A bonds. Hydrolysis of oocyte 5S rRNA(C-»-U, C*) makes these
bands disappear in the Up track but intensify in the A track;
therefore, these bands correspond to C nucleotide3.
The RNA region depicted in Pig. 2 shows four out of six differences found between the sequences of somatic and oocyte
5S rKNAs. A longer electrophoretic separation (not shown) makes
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4.IT*
Pig. 2. Autoradiograms of partial digestion products of 5'-L' 32
PJ'
labeled loach somatic (A) and oocyte (B) 5S rRIIAs treated with
a mixture of methoxyamine and bisulfite (left side of gel3),
and 5S rRIJAs treated with bisulfite and then with a mixture of
methoxyamine and bisulfite (right side of gels, narked with a
plus sign), ./ithout endonucleases (-1)); the ladder (L) prepared
p
as
RIIAases ?i (1O~4-1O"5 U/>ig
as described in
in '9;
9; incubation
incubation with RIIAase
R1IA), U2 (10-3-10-4 U/>ig
(10-4-10-^ig/ug HIT^.),
/>g RIJA), A (104-10^ig/ug
HIT^.), T2
T2 '
(10-4-10-5 U/^ug RITA)
RITA) and
and Fny
Fny I (C.5-0.05
( C U / U//ig &:k)
) as described
in Llaterials and Llethods. The samples v/ere loaded on 20^J polyacrylamide gel (40 x 20 x 0.06 cm) and electrophorezed at 1 . k
for 3 h. Xylenecyanol migrated by 10 en.
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Nucleic Acids Research
it possible to establish the complete sequences of these molecules which are presented in Pig. 3.
DISCUSSION
The sequence of oocyte 5S rRNA from the loach Ilisgurnus
fossilis (UFO) differs from that of somatic 5S rRNA (MPS) in
six positions: 12, 17, 26, 40, 53 and 63. Pig. 3 shows differences between the sequences of 5S rRNAs from somatic cells and
?fic ?Q
oocytes of two frogs, Xenopus borealis cPV
'»«>»
-7 an(j Xenopus
?T tJ30 31
laevis '
' . The former species has six differences between
the sequences of somatic (XBS) and oocyte (XBO) 5S rRNAs, and
the latter one has seven differences between the sequences of
somatic (XLS) and oocyte (XLO) 5S rRNAs.
The sequence of loach somatic 5S rRNA differs from those of
somatic 5S rRNAs of X.borealis and X.laevis in 10 and 11 nucleotides, respectively; the sequence of loach oocyte 5S rRNA
differs from those of 5S rRNAs of X.borealis and X.laevia in
15 and 17 nucleotides, respectively. Comparison of somatic and
oocyte 5S rRNAs from M.fossilis. X.borealis and X.laevis reveals also the following differences and common features
(Pig. 3).
1. In the loach, all differences in the nucleotide sequence
are found in the 5'-half of a 5S rRNA molecule; in the frogs,
these appear also in the 3'-half of a 5S rRNA molecule.
2. Loach oocyte and somatic 5S rRNAs differ one from the
other in positions (17, 26, 40, 63) other than those in which
somatic 5S rRNA differs from oocyte 5S rRNA in X.borealis (55,
62, 79, 90) and X.laevis (30, 47, 55, 56, 79, 90). The only
position at which a nucleotide varies in all of the three species is 53.
3. In M.fossilis. four out of six substitutions in 5S rRNA
are of a pyrimidine-pyrimidine type; in X.borealis. three substitutions in 5S rRIIA are pyrimidine-pyrimidine, two are purine-pyrimidine, and one is a purine-purine substitution; in
X.laevis 5S rRIIA, there are tv/o pyrimidine-pyrimidine, two purine-pyrimidine and three purine-purine substitutions.
Therefore, fishes and amphibians have more differences than
similarities in the position and character of substitutions in
2147
SCE
G G
HKB
UC
U UA
THO
MPS
G
A AAG A GUU
C
U
"A U
UAA A
C
ACG
0 GUAG
CAUAC C
ACU
C
AG
C
C
AU
C
C
C
UC
G
1
V
ZP
3P
40
5P
60
'JO
8.0
90
WO
110
120
GCUUACGGCCAUACCACCCUOAGCACGCCCGAUCUCGUCCGAUCTCGGAAGOTAAGCAGGGUCGGGCCUGGUUAGUACUUGGAUGGGAGACUGCCUGGGAAUACCAGGUGUUGUAAOCU(U)
CCUO
HFO
C
G
XBS
C
U
C
U
G
C AG
UC
U
A C
C
C
C
C
AAGU
XBO
AAGU
C
U
G
G UO
CC
C
G C
C
G
XLS
C
C
C
AAGU
C
U
G
C AG
UC
U
A C
C
G
XLO
C
C
C
AAGU
U
U
A
G UA
UC
c
G C
C
G
y 3. Comparison of the nucleotide sequence of loach somatic (MPS) and oocyte (MPO) 5S
rKTAu with the sequences of 5S r*iIIAs from Xenopus borealis (somatic, XBS; oocyte, XBO),
Xenopua Iaevi3 (somatic, XLS; oocyte, XL0) # Saccharomyces cerevisiae. SCE, human KB cells,
HKJ3, trout, "SdO.
Nucleic Acids Research
5S rRNA. Consequently, it would be expedient to compare
the
two types of 5S genes in a greater number of species in order
to find certain features in common, if any.
The sequence of loach somatic 5S rRNA is most similar to
that of trout 5S rRNA (5differences) -32 which differs from
those of 5S rRNAs in X.borealis and X.laevis in 10 and 11 nucleotides, respectively. Loach somatic 5S rRNA differs from human KB 5S RNA
' in 9 nucleotides, and from Saccharomyces cere-
visiae 5S KNA ^ in 47 nucleotides.
2S
Hori and Ozawa J hold that all the eukaryotic 5S rRNAs
known at present contain sequences constituting four palindromes.
In Pig. 3, these sequences are indicated with arrows num-
bered after the corresponding complementary regions. In loach
oocyte and somatic 5S rRNAs, five out of six substitutions lie
outside these complementary regions; an A for C substitution
is found only in position 17 in the third palindrome, causing
a decreased extent of complementarity in this region.
In contrast to M.fossilis. X.borealis has substitutions in
the second and third palindrome regions, and X.laevis in the
second and fourth regions. However, complementarity is decreased only if a substitution occurs in the third region (U for C
in position 62); otherv/ise, it either increases (C for U in position 79) or a new complementary pair appears v/hen substitutions occur simultaneously in two
positions of one pa-
lindrome (U for C in position 30, and A for C in position 47
in X.laevis). It is noteworthy that there are no differences
between oocyte and somatic 5S rRNAs in neither the loach or
Xenopus. in the first palindromic sequence comprising nine nucleotides from the 5'- and 3'-termini. II. fossilis and the two
Xenopus species have differences v/ithin these sequences in positions 3, 112 and 116, but without destabilisation of the secondary structure in these regions. Apparently, the first, second and fourth palindrome regions are actually conservative
ones whereas, in the third palindrome, only four out of six
nucleotides are conservative.
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