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volume 8 Number 231980 Nucleic Acids Research
The structure of the yeast ribosomal RNA genes. I. The complete nudeotide sequence of the18S ribosomal RNA gene from Saccharomyces cerevisiae+
P.M-Rubtsov, M.M.Musakhanov, V.M.Zakhaiyev, A.S.Krayev, K.G.Skryabin and A.A.Bayev
Institute of Molecular Biology, Academy of Sciences of the USSR, Vavilov str. 32, Moscow B-334,USSR
Received 7 October 1980
ABSTRACTThe cloned 18 S ribosomal RNA gene from Saocharomyoee
oerevieiae have been sequenced, using the Maxam - Gilbertprocedure. From this data the complete sequence of 1789nucleotides of the 18 S RNA was deduced. Extensive homologywith many eucaryotic as well as E.coli ribosomal small subu-nit rRNA (S-rRNA) has been observed in the 3'-end region ofthe rRNA molecule. Comparison of the yeast 18 S rRNA sequen-ces with partial sequence data, available for rRNAs of theother eucaryotes provides strong evidence that a substantialportion of the 18 S RNA sequence has been conserved in evo-lution.
INTRODUCTION
Understanding of biogenesis and the function of eucary-
otic ribosomes strongly depends upon our knowledge of the
primary structure of rRNA and ribosomal RNA precursors.
Recombinant DNA techniques and rapid gel sequencing
methods have greatly facilitated studies of eucaryotic
ribosomal genes. The efficiency of these approaches have
made it easier to study a gene sequence, instead of its
product RNA.
Genes of several eucaryotes that code for ribosomal
RNAs, have been cloned and are under extensive study (1).
Approximatly 140 tandemly repeated homogeneous-in-
length units code the ribosomal RNAs of baker's yeast
Saooharomycee aerevisiae. Each 9.5 kb repeat contains the
information for two transcription units. One unit conducts
the synthesis of the 5 S RNA and the other the 35 S precur-
sor of 18 S, 25 S and 5.8 S RNAs (2-6).
Our studies of the yeast ribosomal RNA genes are aimed at
several goals: first, the sequencing of rRNA structural genes
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and the identification of evolutionary divergence of different
parts of the moleculesj second, the mapping and determination
of the structures of the transcribed spacers which would be
likely to give information about the processing events; fina-
lly the elucidation of the nature of the start and stop signals
of the pre-rRNA transcription.
In this paper we present the complete sequence of the
S.aereviaiae 18 S RNA gene obtained from cloned DNA.
MATERIALS AND METHODS
The recombinant plasmlds. We have used EcoRI-fragments C
and D of the ribosomal DNA repeat isolated from recombinant
plasmid pY1rA3 (Fig. 1a) for this study (6). pY1rA3 contains
a substantial part of ribosomal DNA repeat from the yeast
S.cereviBiae + D4 inserted into the EcoR1-cleaved plasmid
pMB9 by the AT-connector method (7). An 1950 bp C fragment
contains the part of external transcribed spacer and the main
portion of the 18 S RNA gene from the 5'-end (8). An 670 bp D
fragment contains the 5'-end of 5.8 S rRNA gene, the left in-
ternal transcribed spacer and the 3'-end of 18 S rRNA gene
(9,10). These fragments were prepared by digestion of pY1rA3
with EcoRI followed by sucrose density gradient centrifugati-
on or electrophoresis on a preparative 5% acrylamide gel.
Later when the 5'- and 3'-ends of the 18 S rRNA gene
have been exactly located the 175O bp Hindlll-EcoRI-frag-
ment was subcloned in plasmid pBR322. For the construction
of derivative plasmids EcoRI-digested pY1rA3 DNA was centrifu-
ged in 10-30% sucrose gradient and fractions enriched in C
fragment were pooled, EtOH precipitated, cleaved with Hindlll
and ligated to EcoRI-Hlndlll-cleaved pBR322 DNA. E.ooli HB
101 was used for transformation and clones were selected
by colony hybridization with -P-labelled 18 S rRNA probe.
One of the positive clones that gave very strong signal was
shown to contain two copies of a 175O bp Hindlll-EcoRI-frag-
ment as well as one copy of G fragment and one copy of an
194 bp EcoRI-Hindlll-fragment (fig. 1). This derivative plas-
mid prYC was used for further experiments. Plasmid DNA was iso-
lated according to Tanaka and Weisblum (11).
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pMB9 I 1pY1rA3
DMB9
I * 18 S
b
pBR352
prYC
J IpBR322
t * 18SrRNAgene100bp
18 S rRNA gene
J-EcoRI j-Hindlll X-po\y(4m<fT)
Figure 1. The restriction maps of two recombinant plasmidsused in this work, a - pY1rA3, b - prYC.~A-G - EcoRI-fragments of the S.aerevieiae rDNArepeat unit. In the plasmid pY1rA3 only portionsof A and B fragments are presented.
Sequencing procedures. DNA sequencing was done according
Maxam and Gilbert (12), using a revised protocol with seve-
ral modifications. DNA fragments were labelled either at 5'-
end with T4-polynucleotide kinase or at the 3'-end with T4-
DNA polymerase. Excess of unreacted NTP was removed by pas-
sing a reaction mixture through a small Sephadex G-50 co-
lumn. Labelled fragments were either subjected to secondary
restriction enzyme cleavage, or strand-separated according
to Szalay et al. (13).
At the first steps of sequencing work, when restriction
map was not known, we deliberately sequenced all fragments
and used almost exclusively strand-separation. Later on,
doubledigestion was also useful.
Chemical cleavage reactions used were G as described
in (12), A + G as described in (14) and T + C, C as desc-
ribed in (12), but with additional step, that is described
below- Hydrazine cleavage reactions are very frequently
associated with problems, that appear as non-specific
"ladder", additional bands, doublebands, shifted bands
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etc. An efficient stoppage of the hydrazinolysis can be
obtained by quick addition of 250 ul benzaldehyde to the
mixture, to which "stop-solution" has already been added.
After thorough vortexing, the mixture is put on ice for
10-15 rain (it should become yellowish, if it does not, it
should be put to 20° bath until it develops bright yellow
colouration), then extracted with ether to remove yellow
water-insoluble residue. The subsequent steps are as desc-
ribed (12). The inclusion of this step, though it does not
simplify the procedure, greatly enhances its reproducibili-
ty, provided, of course, that all reagents used are fresh
(these include hydrazine, benzaldehyde and piperidine).
Initially we used 1.5 mm thick 10% and 20% gels for frac-
tionation of chemical cleavage products. Later on, thin
gels according to (15) were used exclusively (8% and 2O% acry-
lamide, 1 : 20, 7 M urea, 1OO mM TBE, pH 8.3). For thin gels
with small slots (5 mm) carrier DNA was omitted from chemical
cleavage reactions. Gels were exposed at -70°, in many cases
with intensifying screens and preflashed medical X-ray films
(16).
Hybridization of the 18 S rRNA with the 42 bp DNA frag-
ment Sau 3A'2-Sau 3A»3. 5 fig of the yeast 18 S rRNA was hyb-
ridizied with about O.O5-O.1 pg of the 5'-ends labelled 42 bp
Sau 3A»2-Sau 3A-3 fragment isolated from preparative 6% po-
lyacrylamide gel. The hybridization was done in 5XSSC and
50% formamlde in a total volume of 20 ul. The incubation
mixture was heated for 5 min at 75°C followed by cooling to
20 C. After annealing the mixture was electxophoresed on
10% non-denaturing polyacrylamide gel (60:1). Partially
and entirely denatured Sau 3A.2-Sau 3A.3 fragments were
used as controls.
Restriction endonucleases EcoRI, TagI, Sau3A, T4-poly-
nucleotide-kinase, T4 DNA ligase and T4 DNA polymerase were
prepared in our laboratory by standard procedures. Restric-
tion endonucleases HindiII and Alul were a gift by Dr. A.Yanu-
laitis, Hinfl by Dr. G.S.Monastyrskaya, Bspl by Dr. A.Botcha-32
rov and Mspl by Dr. V.G.Korobko. Carrier free P-orthophos-
phate was obtained from the Radiochemical Centre, Amersham,
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U.K.Gamma- P-ATP (about 1OOO Ci/mmol) was prepared by the
method of Glynn and Chappel (17). The 18 S rRNA from the
S.oereviaiae was a generous gift by Prof. A.A.Hadjiolov.
RESULTS AND DISCUSSION
The Location of the 18 S rRNA Gene Boundaries
Cloning, RNA-DNA hybridization and EM data have allowed
the construction of a physical map for the yeast rDNA repea-
ting unit (2-6, 18). The position of the 18 S rRNA gene in
these works was estimated to be in EcoRI-fragments C and D.
Moreover, some 20 residues have been sequenced at the 3' end
of the 18 S rRNA of the yeast Saaoharomyaee carlabergenaia
(19). Using these data as a guide, we have first sequenced
the ends of fragment C and the whole of the fragment D
( ' the complete sequence of this fragment will be discussed
elsewhere) (Fig. 2). In the EcoRI-D sequence, 225 bases down-
stream from its boundary with EcoRI-C, we have found a se-
quence, which closely resembles a 3'-end of S.oarlabergenaia
18 S RNA, as well as some other eucaryotic S-rRNA.A prelimi-
nary location of the 3'-end was assigned to be 225 bp down-
stream from the EcoRI site between fragments C and D. To
locate the 5'-end, the method of Donis-Keller et al. (20)
on 5'-end labelled yeast 18 S RNA we used (Bayev et al. in
preparations). The comparison of purine sequence of the 18 S
rRNA with the DNA sequence near the EcoRI site between frag-
ments G and C revealed the existence of the corresponding
region, which begins 182 bases downstream the Hindlll site
of the C fragment or 376 bp downstream the EcoRI site.
The Sequencing the 18 S rRNA Gene
For the determination the complete sequence of the 18 S
rRNA gene the 1750 bp Hindlll-EcoRI fragment isolated from
plasmid prYC was used. This fragment consists of 182 nucleo-
tides of internal transcribed spacer and 1564 nucleotides
of the 18 S rRNA gene.
The restriction endonucleases and the positions of their
cleavage sites which were used for generation of DNA subfrag-
ments via sequencing are presented in fig. 2.
The sequencing strategy presented in this figure shows
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tOObp
Rl H'xdOl
IBS rRNA |«n«
1750 bp Hindi-EcoRI- fragment
Xbil
Bipl
Alu I
Mipl
T iq l
Hind
Sau3A
1
]
1
J
Iff
1 j
1
M
IffJJ
J
f
7
•4
a 9
7
Figure 2. Schematic map of the 18 S rRNA gene: restrictionendonuclease cleavage sites and the DNA fragmentsactually used in the sequence determination.
a. The part of the S.oerevieiae rDNA repeat unit.The positions of the 18 S rRNA gene and the 1750 bpHlndlll-EcoRI-fragment subcloned in the plasmidpBR322 are shown.
b. The map of the 18S rRNA gene for restriction endo-nucleases which were used for generation of DNAfragments.
c. The DNA fragments used tn the sequence determi-nation.
that the most of the DNA sequence was determined by sequen-
cing both strands.
The examples of the typical sequencing gels are presen-
ted in fig. 3. All restriction enzymes cleavage sites were
overlapped by sequencing DNA fragments prepared with other
restriction endonucleases, except for the EcoRI site that
separates C and D fragments and two Sau 3A cleavage sites -
Sau3A.2 and Sau3A.3.
For the orientation of the 42 bp Sau3A.2-Sau3A3 frag-
ment the hybrydization of its strands with 18 S rRNA was
carried out. This experiment revealed that the "slow"
strand of this fragment is hybridized with 18 S rRNA
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1510
380
15601480
Figure 3. The examples of the sequencing gels.a - MspI.1-BspI.2-fragment; b - EcoRI-HinfI.7-fragment; c - HinfI.7-EcoRI-fragment.
(fig. 4). Therefor the RNA sequence was derived from the
sequence of the "fast" strand of this fragment.
The complete nucleotide seguence of the 18 S RNA as deri-
ved from the gene sequence is presented in fig. 5. The total
length of the chain is 1789 nucleotides, in close agreement
with earlier estimates based on physical methods (21).
The Extensive Homology of the 3'End of the Yeast 18 S
rRNA with the Other RNA's of the Same Class
As stated in the previous section the preliminary loca-
tion of the 18S rRNA gene 3'-end was based on its structu-
ral similarity to S.aaplabergensie 18 S rRNA 3'-end. Sequen-
ce data for the other 18 S species that appeard later pro-
vided further evidence that this initial assumption was
correct. Sequences, that for E.coli 16 S rRNA, and several
ones of eucaryotic origin, showed extensive homology at
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c d
Figure 4. Autoradiograph of 10% non-denaturing acrylamidegel (6O:1) of 42 bp Sau3A.2-Sau3A.3-fragment.1 - ds-DNA fragment; 2 - "slow" strand; 3 - "fast"strand.
a. appr. 0.05 pg the 5'end labelled DNA fragmentwas denaturated in 5XSSC and 5O% formamide intotal volume 20 ul for 5 rain, at 75 C followedby slow annealing to 20 C. 5 pi 1 mM EDTA, 0.1%(w/v) xylene cyanol, 0.1% (w/v) bromphenol bluewas added and the sample was loaded on the gel.
b. The same as a., but 5 pg of the yeast 18S rRNAwas added before denaturation.
c. appr. 0.05 pg DNA fragment was heated at 95 for5 rain, in 20 pi of 50% (v/v) dimethylsulfoxide,10 mM tris-borate, pH 8.3, 1 mH EDTA, O.o5%(w/v) xylene cyanol, 0.05% (w/v) bromphenol blueand immediately transferred into ethanol-dry icebath. The sample was loaded on the gel.
their 3'-ends with the yeast sequence. In particular, the
homologous sequence totals over 50 bases between yeast and
E.coli 16 S (22, 23) human mitochondrial (24) and Zea mays
chloroplast (25) S-rRNA. All published 2OO bases of Bombyx
mori 18 S rRNA (26) strongly resemble the corresponding
nucleotides in yeast 18 S rRNA (Fig. 6). Unfortunately,
there are no other 18 S rRNAs that have been sequenced
completely, but short sequences available agree that the
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3'-end of eucaryotic 18 S RNAs is a strikingly conserved
region.
The 3'-end of the 16 S rRKA molecule: as pointed out
by Shine-Dalgarno model, is involved in a complex with an
mRNA that is indispensable for effective translation, the
interaction do occurring via the sequence CCUCC (27) . All
eucaryotic 18 S rRNAs, sequenced to date, lack this parti-
cular sequence (28). So does the S.cereviaiae rRNA, though
it shows striking homology with adjacent region of 16 S
procaryotic RNA. The conservation of the large stretches of
3'-ends of the S-rRNA molecules both in procaryotic and
in different eucaryotic organisms as well as in organelle
ribosomes makes the involvement of these sequences very
likely in some essential interactions.
The Evolutionary Stability of the Other Regions of
the 18 S rRNA's
The 18 S rRNA primary structure derived from the gene
sequence presented here - to our knowledge - is the first
complete sequence of the eucaryotic cytoplasmic riboso-
mal S-rRNA.
Partial sequence data available from the literature
allows some comparison with other eucaryotic species SrrRNAs.
The homology between these species is apparently not rest-
ricted to the 3'-end region. This conclusion comes from the
attempts to locate some large Tl-oligonucleotides homolo-
gous in chicken and in rat 18 S rRNA (29) within the yeast
sequence. We have found, that most of these oligonucleotides
completely or at least closely correspond to portions of the
yeast sequence. A summary of homologous parts between yeast
and rat 18 S rRNA oligonucleotides is given in Table 1. As
it is shown in the table 13 (a-h) from 18 large conservati-
ve oligonucleotides of the rat and chicken 18 S rRNA are
homologous with the nucleotide sequence of the yeast 18 S
rRNA. Four Tl-oligonucleotides (a, c, i, k) are absolutely
identical in the 18 S RNA's of the three species; other
oligonucleotides have some differences.
It is interesting to point out that half of the conser-
ved oligonucleotides have the modified nucleotides in rat
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10 20 30 49 50 £0 70 80 90 180UAUCUGGUUG PlUCCUGCCAu IMGUCfl'JflUG CUUGUCUCM AGAUUAAGCC AUGCAUGUCU FlftGUfiUfifiGC AAUUUAUACA GUGfiflflCUGC GAAUGGCUCA
113 1.M 130 1-13 158 160 170 180 190 200UUAAAUCrrjJ UAUCGU'XI.I't L"Jrif.'-'.":••"."" CU'J"aC'J 1~ 1 UGGUAUAqCC GUGGUAAUUC UAGAGCUAAU ACAUGCUUAA flflUCUCGRCC CUUUGGAAGA
a210 2?0 ?3T 241 256 268 278 288 296 300
GftUGUAUL'Ufl UUAGAUA:>WJTAJJCJWJ£UC UUCSGflCUCU UUGAUGqu'JC AUAAUfiftCUU UUCGAflUCGC flUGGCCUUGU GCUGGCGflUG GUUCAUUCAA
b310 320 333 340 359 360 373 388 390 408
AUUUCUGCCC UAUCAACUUH CGflUI''J C-G flUflGUGICCU QCCAUGGUUU CAACGGGUAA CGGGGAALIflA GGGUUCGAUU CCGGAGAGGG AGCCUGAGAA
c d410 420 430 440 458 460 470 488 490 508
ACGGCUACCfl CAUCCAAGGA AGGCPGCAGG CGCGCAflflUU ACCCflftUCCU AAUUCAGGGA GGUAGUGACA AUftAAUflftCG AUACftGGGCC CAUUCGGGUC
e f g510 nzri JTO ^ ? 550 568 570 580 593 6P0
UUGUflAUUbG fiAUGAuUPC.1 AUG'JA'q ac Cl'UAACPA'IG MCPAUUGGO G3GCAAGUCU GGUGCCAGCA GCCGCGGUAA UUCCAGCUCC AAUAGCGUAU
h610 C20 333 S-13 658 668 678 680 690 780
AUUAfiAGL'JG UUGCAGL".'.*!) flAAr.r''CJ'JA GUUGA r.'J'J'J GGGCCCGGUU GGCCGGUCCG AUUUUUUCGU GUACUGGAUU UCCAftCGGGG CCUUUCCUUC
7 lfi 7:(5 :33 7 4? 758 768 778 780 798 800UGGCUAACC'J UGAGUCC'J'i IJGGC'JC'JuUG CGflACC.jG^ CUUUl'Qruuu GflflAflAAUUA GAGUGUUCAA AGCAGGCC-UA UUGCUCGAAU AUAUUAGCAU
i810 """) 330 "543 850 860 870 880 890 900
GGAAUAAL'A'"i AAUAGGAC'J UUGG'JLCL'ftU UUUG'JUGr-'JU UCUAGGACCA UCGUAAUGAU UAAUAGGGAC GGUCGGGGGC AUCGGUAUUC AAUUGUCGAG
k313 ?:r T30 943 958 960 970 980 990 1800
GUGAflAUUCU UGGAU'JUTJ'J GAAG'iCU^flC USCUGCMAA GCGUUUGCCfl fiGGACGUUUU CGUUAAUCAA GAACGAAAGU UGAGGGAUCU GAUACCGUCG
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1010 1020 1030 1940 1050 1060 1070 1060 1090 1100UAGUCUUAAC CAUAAACUAU GCCGACUAGA UCGGGUGGUG UUUUUUUflAU GACCCACUCG.GUACCUUACG AGAAAUCAAA GUCUUUGGGU UCUGGGGGGA
1119 1120 1130 1140 1150 1160 1170 1180 1190 1200GUAUGGUCGC AAAGGCUG3A ACUUAAAGGA BUUGflCGGSfi GGGCACCACU AGGAGUGGAG CCUGCGGCUA AUUUGACUCA ACACGGGGAA ACUCACCAGG
O
1210 1220 1230 1249 1250 1260 1279 1289 1290 1388UCCAGACACA AUAAGGAUUG ACAGflUUGAG AGCUCUUUCU UGAUUUUGUG GGUGGUGGUG CAUGGCCGUU UCUCAGUUGG UGGAGUGAUU UGUCUGCUUA
1310 1320 1330 1340 1350 1360 1370 1380 1390 1400
ftUUGCGAUAA CGAACGAGHC CUUAACCUAC UAftAUAGUGG UGCUAGCAUU UGCUGGUUAU CCACUUCUUA GAGGGACUAU CGGUUUCAAG CCGAUGGflAG
I m1410 1420 1430 1440 1450 1460 1470 1480 1490 1500
UUUGAGGCAA UAACAGGUCU GUGAUGCCCU UAGAACGU'JC UGGGCCGCAC GCGCGCUACA CUGACGGAGC CAGCGAGUCU AACCUUGGCC GAGAGGUCUU
1510 1520 1530 1540 1550 1560 1570 1580 1590 1600GGUAAUCUUG UGAAACUCCG UCGUGCUGGG GAUAGAGCAU UGUAAUUAUU GCUCUUCAPlC GAGGAAUUCC UAGUPAGCGC AAGUCAUCAG CUUGCGUUGA
n p1610 1620 1630 1S40 1650 1660 1670 1689 1690 1700
UUACGUCCCU GCCCUUUGUA CACACCGCCC GUCGCUAGUA CCGAUUGAAU GGCUUAGUGA GGCCUCAGGA UCUGCUUAGA GAAGGGGGCA ACUCCAUCUC
1710 1720 1730 1743 1750 1769 1770 1780 1790 1800AGAGCGGAGP AUUUGGACAA ACUUGGUCAU UUGGAGGAAC UAAAAGUCGU AACAAGGUUU CCGUAGGUGA ACCUGCGGAA GGAUCAUUA
Figure 5. The complete nucleotide sequence of the 18S rRNA deduced from the gene sequence.
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(1337) 200
IAAUUI AAGCGCGAGUCAUAACCOCCCGUUGUJUACG
IACACCGCCCCUC
iC lACACCGCCCGUCGCUACUACC
^CACCGCCCGUCGCO/fljACC
(ISM) 200 150
iAUUGMUCkUUUAGUGAGGU
JAUUGAAUGtnJUAGUi .CUCCAUCUCAG,
Figure 6. The homologous sequences at the 3'-ends ofS.oerevieiae, Bombyx mori (26) and E.coli (22,23) S-rRNA. The sequences are aligned for maximumhomology and numbered from the 3'-terminus (thenumbers from the 5'-terminus are shown in bracketsfor the E.coli and S.aereviaiae S-rRNA).
18 S RNA.
In contrast with 40 sites of 2'-0-methylation and 38 si-
tes of pseudouridylate formation there were only five major
sites of base methylation in rat 18 S RNA (30).
(a) oligonucleotide U-A-m A-C-A-A-Gp with 6-methyl ade-
nylate has the homologous sequence in yeast 18 S RNA (nucle-
Otide N 1750- 1756) J
(b) oligonucleotide mjA-m^A-C-C-U-Gp with 6-dimethyl
adenylates also conserved in positions 1770-1775;
(c) T1 oligonucleotide with 7-methyl guanylate in rat
18 S RNA was deduced to be m7G-A-A-U-(V,C3)-A-Gp.
The homologous structure in yeast 18 S RNA could be G-A-
A-U-U-C-C-U-A-G ( 1564 - 1573) .
The similar sequence GAAUUCCAG was found in the corres-
ponding position of S-rRNA in Bombyx mori (26) .
Another indication on conservation of this sequence came
from rDNA restriction mapping of various organisms. The se-
quence GAATTC (EcoRI recognition site) maped in the region
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Table 1. Comparison the large Tl-ollgonucleotides sequencesof the rat 18 S rRNA with the yeast 18 S rRNAsequence
Identical sequences are underlined
Rat 18S rRNA T l - o l i g o - T h e sequence homology of ratnucleotides numbers 18 S rRNA (upper) and yeast
18 S rRNA (lower)ref.(30)
13 93 CUCAmUUAAAVCAGpCUCA UUAAAUCAG
c
d
e
f
g
h
i
k
1
m
n
1 0
9
14
4
2
1
19
18
5
3 ( 6 )
7
109
16 101 AAACCAAAGpAAAUCAAUG
108 CCCUAUCAACUUUCGpCCCUAUCAACUUUCG
228
322
354109 CDACCAC—ADCmCAAGp
CUACCAUGGUUU CAAC111 AAAC—CUCACCCGp
AAACGGCU-ACCAC 4 1 1
1 1 5 CAmAAUUACCCACUCCCGpCA AAUUACCCAAUCCUA 4 5 1
1 1 4 AAAAAUAACAAUACAGpAUAAAUAACGAUACAG .„,— r 486
112 UCCACUUUAAAmU-CCUUUAACGpUACAAUGUAAA CACCUU-AACG 5 3 7
34 UU ACUUUGpUUUACPUUG 7 5 1
33 UUCUAUUUUGpUUCUAUUUUG 8 3 4
1 1 6 AUCAAAACCAACCCGpGCGAUAACGAACGAG
1 13 UCCCCCAACUUmCUUAGmAGpUUAUCC-ACUU CUUAG AG
106 CAAUUAUUCCCCAUGpUAAUUAUUGCUCUUC
10 2 AACXCA-CACGpACUCAACACG TTQ,
86 GmAAU(VC3)AGpG AAUUCCU AG I5?3
78 UAAmCAAGpUAA CAAG I?56
42 AmAmCCUGpA A C C 0 G 1775
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approximately 250 nucleotides left to the end of the 18 S
RNA gene of X.leavie (31),D.melanogaeter (32J;
(d) The T1 oligonucleotide possessing the unique 18 S
RNA hypennodified nucleotide m cap V was found both in
yeast (33) and rat (30). In these works the sequence of
oligonucleotide was proposed to be A-A-C-m cap y-C-A-C-A-
C-Gp.
We could not find such oligonucleotide within S.eerevi-
eiae 18 S RNA sequence. The only appropriate T1 oligonucleo-
tide which could be deduced was A-C-U-C-A-A-C-A-C-Gp.
It is necessary to mention that this structure does
not contradict the results of the RNAse A and U, digestions
of the oligonucleotide which was obtained by Maden et al.
(33) and Choi and Busch (30).
The positions of the homologous sequences in the 18 S
rRNA of yeast and rat are marked on the yeast 18 S rRNA
sequence (fig. 5). It is clear from this data that homolo-
gous regions are dispersed over the entire 18 S rRNA mole-
cule. To the same conclusion came Gourse and Gerbi who used
the alternative approach (34). They employed heterologous
DNA-DNA hybridization techniques to localize conserved re-
gions of restriction fragments of Xenopus laevia rDNA.
Some new data about the conserved regions of 18 S rRNA
was provided recently by T.Moss et al. (35) . They used our
sequence data (8) to locate the X&nopue laevie 18 S rRNA ge-
ne 5'-end and found a substantional homology between 5'-end
portions of these genes.
Further information about 18 S sequences, which is
expected to emerge in the future, would add to our know-
ledge of how similar different 18 S rRNAs are, but at the
present time this information is limited to the examples
listed above.
The availability of complete primary structure now per-
mits direct probing of secondary structure of the yeast 18 S
RNA molecule by several existent methods.
Acknowledgements
Authors are grateful to A.Bocharov, A.Yanulaitis, G.Mo-
nastyrskaya and V.Korobko for restriction enzymes and to
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Nucleic Acids Research
G.Lysov for computer handling of the sequence data. We whish
to thankN.Batchikova and I.Bespalova for expert technikal
assistance. Authors are also grateful to Dr. T.Petes for
providing recombinant clones with yeast rDNA fragments, and
to Dr. A.M.Maxam for providing sequencing protocols prior
to publication and for helpful disctBsions and to Prof.
A.A.Hadjiolov for providing 18 S rRNA from the S.cersviaiae.
*)
The summary of the substantial portion of this sequence
has been published (36), and presented by K.G.S. at the
1979 Meeting on the Molecular Biology of Yeast (Cold
Spring Harbor Laboratory).
"•Tor Part II of this series see Nucleic Acids Research (1980) 8, 4919^*926.
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