the structure of the yeast ribosomal rna genes. i. the complete

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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Nucleic Acids Research

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).

5780



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

5781



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,

5782



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

6783



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

5784



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

6785



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

5786



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

5787


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


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.



(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

5790



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

5791



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

5792



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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the structure of the yeast ribosomal rna genes. i. the complete

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