Comparison of the nucleotide sequences at the 3′-terminal region of RNAs from RNA coliphages

I. Mol. Riol. (1982) 158, 71 l-730

Comparison of the Nucleotide Sequences at the 3’-Terminal Region of RNAs from RNA Coliphages

Department of Molecular Biology, School of Medicine Keio liniversity, Shinjuku-ku, Tokyo 160, Japan (Received

25 November

1981, and in revised form

29 March

1982)

In order to study the genealogical relationships among four groups (I to IV) of RNA coliphages, we sequenced 200 to 260 nucleotides from the 3’ termini of 14 phage RNAs according to the method of Sanger et al. (1977), and compared the results. It, was found that the sequences of phage RNAs in the same group were extremely homologous (about 90%). On the other hand, when the sequences were compared with those from other groups, they were seen to be only about 50 to 60% homologous between group I and group II, and about 50% homologous between group III and group IV. In other combinations. such as groups I (or II) and III, and groups I (or II) and IV, however, the extent of homology was small. Furthermore. the sequences up to 30 residues from the 3’ end were found to be

about 90% homologous These

results

proximity (Inokuchi

between

confirm

our

groups I and II, and between groups III and IV.

previous

findings.

of the 3’ end of phage RNA et al.,

and between

1979),

and

that

close

that

the

sequences

in the same group

relationships

exist

located

in the

were well-conserved

between

groups

I and

II.

groups III and IV (Furuse et al., 1979).

1. Introduction RNA phages of Escherichia coli (RNA coliphages) can be classified into four groups (I to IV) on the basis of the serological and certain physicochemical properties of the phage particles (Watanabe et al., 1967; Miyake et al., 1969; Furuse et al., 1979). Among these groups, it has been shown that the RNA genomesfrom group I and group

II phages

comprise

three

genetically

defined

cistrons

(maturation,

coat and

replicase subunit proteins: Valentine et al., 1964; Aoi, 1973), whereas t,hose from groups III and IV phages contain an additional protein, designated read-through protein (or Al protein: Horiuchi et a1.,1971; Ando et al., 1976; Aoi & Kaesberg, 1976). Recently, a cistron for lysis prot,ein was found in the genome of group I phages, such as MS2 and f’2 (Atkins et al., 1979), but little is known about this in other phages. It has also been shown that long untranslated regions exist at the 5’, and 3’ termini of phage MS2, R17, f2, and Qfi RNA (Weissmann et al., 1973). Analysis of the complete nucleotide sequence of MS2 RNA by Fiers et al. (1976) and partial analysis of phage RNAs by other workers (Cory et al., 1970,1972; Goodman et al.. 1970; Billeter et al., 1969) allows comparison of the nucleotide 0022~2836/82/2007

I I-20

803.001’0

711

IQ 1982 Academic

Press Inc. (London)

Ltd.

il2

Y. ISOKl’CHI.

A.

HIKASHIMA

ASI)

I. \VATANAHE

sequences in various bacteriophages (Gory et al.. 1970: Min Jou & Fiers, 1976). It was found that the primary structure of the coat protein gene was very similar. that, that of the 3’.terminal region was st,rictly conserved wibhin the group I phages. and t,hat the 5’-terminal sequences of group I phages and group III phage Q/3 were similar. Nevertheless, sequence analysis of other phage RNAs among the fout groups has long been delayed. Taking advantage of our many stocks of RNA coliphages representing groups 1 through IV. we have attempted to correlate the structure and function of various phage RNAs with the classification of RNA coliphages. Miyake ef al. (1971) and Yonesaki & Aoyama (1981) compared the template specificity of various RNA replicases, and Furuse et al. (1979) compared the size of various phage RNAs and the size and kind of various virion proteins. These studies demonstrated the existence of a close relationship between groups I and II, and between groups III and IV. In t’his work, in relation to studies on the classification of RSA coliphages. web determined the sequences up to 200 to 260 nucleotides from the 3’ termini of 14 phage RNAs, and compared the results with each other. From the comparison of these RNA sequences, we conclude that the nucleotide sequences in each group are well-conserved and that groups I and II phages, and groups III and IV phages. have close relationships in terms of sequence homology in the 3’-t,erminal region.

2. Materials and Methods High specific activity grade [ r-32P]dC:TP. [y-32t’]ATP and ]~?‘-~~l’]pCp were purchased from the Radiochemical Centre (Amersham). and (3H]dCTP and [ 14C]ATP from New England Nuclear Co. Four kinds of deoxynucleoside triphosphate (dNTP) were obtained from Sigma, and 2’3.dideoxynucleoside triphosphate (ddNTP). p(dT),-d($. cytidine X,5’bisphosphate (pCp) and phage T4 polynucleotide kinase from PI, Biochemicals. Bacterial alkaline phosphatase was purchased from Worthington Biochemical Co. Oligonucleotide p(dT)s-dG, obtained from PL Biochemicals, was further purified by co electrophoresis on polyacrylamide gels with [5’-32P]p(dT)s-d( 4 as reported by Donis-Keller rt al. (1977) and used as a primer for reverse transcription of various phage RNAs. Phage T4 RNA ligase was purified from T4 phage-infected E. co/i according to the method of Sugiura et al. (1979). with additional purification by column chromatography on Affigel Blue as reported by McCoy et al. (1979).

(b)

Preparation

of RXA

All the RNA phages used were grown medium supplemented with 025;i, yeast phages and extraction of their RNAs were

(c) I’olyadenylation

of phage

RSAcy

phages

atLd phage

R.VAs

separately in E. coli A/h in a Peptone-glucose extract and 10 mM-CaCl,. Purification of the carried out as described b?- Pace et al. (1968).

and

isolutiorr

of polyudetLylated

R:VAs

Poly(A) was joined to the 3’ end of the phage RNA by ATl’ : RNA adenyltransferase purified by Sippel’s method (1973). The reaction mixture for polyadenylation of the phage RNA was prepared according to Devos et al. (1976) as follows (in 100 ~1) : 50 miv-Tris .HCl (pH 8.0), 200 mM-NaCl, I2 mM-MgCl,. 2 mM-MnCl,. 1 mM-dithiothreitol. 64 mM-EDTA. 0.2 mM-]i4C]ATB (diluted to 5 Ci/mol). 50 pg of phage RNA and 5 ~1 of enzyme fraction.

3’.TERMINAL

SEQUENCES

OF

PHAGE

RNAs

713

After 20 min incubation at 37”C, the reaction was stopped by the addition of 0~59~ (v/v) 1auroy.l sarcosine (Sigma), 20m~-EDTA and 2 vol. ethanol. The radioactive, alcoholpreclpltable materials were suspended in Tris/Mg buffer (10 mM-Tris. HCI (pH 7.5), 5 mMMgCl,), loaded on a 5% to 20% linear sucrose density gradient containing 50 mM-Tris . HCl (pH 8.0). @l M-NaCl and @2% lauroyl sarcosine, and centrifuged at 45,000 revs/min for 200 to 210 min at 4°C in a Beckman SW50.1 rotor. The phage RNA containing poly(A) that sedimented to the position of the intact size of the RNA was collected, and further purified by poly(U)-Sepharose 4B (Pharmacia Fine Chemicals) column chromatography according to Lindberg & Persson (1972). The resultant poly(A)- containing RNA was designated phage RNA-poly(A). such as GA RNA-poly(A) etc.. and used for reverse transcription as a template. (d) Reverse

transcription

a,nd polyacrylamide gel vlectrophorrsia sequencing phage R.VA s

for

The reaction mixtures for reverse transcription of the phage RNA were prepared according to Hamlyn et al. (1978), with slight modifications. For example, a reaction mixture designed to produce complementary DNA terminated in all possible “adenine” positions contained the following (in 12.5 ~1) : 50 mM-Tris. HCl (pH 8.3). 50 mM-NaCl, 6 mM-MgC1,. 5 mw-dithiothreitol, @7 to 1 pg of polyadenylated phage RNA as template, 0+)025 .4260 unit,s of p(dT),-dG as primer, 1 unit of avian myeloblastosis virus (AMV) reverse transcriptase, 1 mx-dCTP and dTTP, 5 PM-dATP. 5 pM-[R-32P]dGTP (40 Ci/mmol) and an appropriate concentration of ddATP as terminator. The ratio of ddATP to dATP was set at 4, 1.0.4 and @I, respectively. After the reaction mixture had been incubated for 30 to 60 min at 37°C. and chased by the addition of dGTP to 170 PM for 15 min. the complementary DNA synthesized was precipitated with 1 ml of ethanol. dissolved in 5 ~1 of dye solution (003”/, (w/v) xylene cyan01 FF, OOSq/b (w/v) bromphenol blue, and 925% (v/v) d elonized . formamide), boiled for 3 min, and loaded onto polyacrylamide gel containing 127; (or So/b, w/v) acrylamide, 0674, (w/v) methylene bisacrylamide, 8 M-urea, 90 mw-Tris-borate (pH %3), 2.5 mM-EDTA, 007O, (w/v) ammonium persulfate. Gel electrophoresis was performed as described by Maxam & (iilhert (3977) and Sanger & Coulson (1978). (e) Post-lrrheling

of phagp

Rn’As direct

with [5’-321’]pCp RN.4 sequencin,g

u&g

T4 R,V.4

liyasr

nnd

The 3’ end of the phage RNA was post-labeled with [5’-32P]pCp using T4 RXA ligase according to England et al. (1980). The reaction mixture (150 ~1) contained 50 mi\l-Tris HCI (pH 8.3), 10 mM-MgCl,, 3 rnx-dithiothreitol, 5 PM-ATP, 10 pg bovine serum albumin/ml, 20yo (v/v) dimethylsulfoxide. 2 ~~“-[fi’-~~P]pCp (10 Ci/mmol ; diluted with non-labeled pCp), 3.5 mg phage RNA/ml, and 250 units T4 RNA ligase/ml. After 18 h incubation at 4”C, the reaction was stopped by the addition of 0.5% lauroyl sarcosine. 20 mM-EDTA and 2 vol. ethanol. The phage RNA so labeled was first precipitated by low-speed centrifugation. and loaded on a 5% to 20% linear sucrose density gradient to obtain the intact size of the phage RNA. After centrifugation at 45,000 revs/min for 200 to 210 min at 4°C in a Beckman SW50.1 rotor, the first main radioactive peak from the bottom of the tube was collected by precipitation with ethanol as the intact size of the phage RNA. and segmented with E. co/i RNAase IV. which was partially purified according to the method of Gesteland & Spahr (1969). The radioactive segment (about 7 S in size) was then further purified by polyacrylamide gel electrophoresis (Maxam & Gilbert, 1977), and used for direct RNA sequencing as reported by Peattie (1979). (f) Determination of the residue at the 3’ end of phage RIVA4 The residue at the 3’ end of the phage RNA was determined as follows. The 3’ end of the phage R?;A was post,-labeled with [5’-32P\pCp as described above, and the labeled RNA was

71-l

Y. INOKI’CHI.

A. HIHASHIMA

ASI)

I. WATASAHE

purified by sucrose density-gradient centrifugation. The 3’ end-labeled RXA was then completely digested with 2.5 units of RNAase TJ25 ~1 containing 50 mwsodium acetate (pH 43). 2 mM-EDTA at 37°C overnight. The Wabeled nucleoside monophosphatr

produced 1)~t.his digestion wasseparatedby 2-dimensionalthin-layer chromatography HS described

by Harada

rl nl. (1971).

3. Results (a) Sequen.ce analysis

of phage HXAs

In advance of sequencing RNA using the dideoxynuoleotide method, we det,ermined a dinucleotide from the 3’ terminus of various phage RNAs. The 3’. terminal residue of these RNAs was determined as adenine by two-dimensional thin-layer chromatography as described above. The penultimate base was determined as cytosine by the slippage reaction of AMV rrverse transcriptase on the poly(A) tail of phage RNA-poly(A) as reported by Devos et al. (1976). (Data not shown.) In this experiment, we used the oligodeoxythymidilate p(dT),o as a primer. In the presence of [3H]dTTP, the reaction wit’h dTTP plus dGTP resulted in a decreaseof dTMP incorporation, while t,he reactions with dTTP plus dATP or dTTP plus dCTP did not. Therefore, we decided to utilize p(dT),-dG as the primer for further eDNAt synthesis during t)he course of sequencing by the dideoxynucleotide method. Figure 1 gives examples of the cDNA sequencing up to 240 residues from the 3’ end of VK RNA (Fig, l(a), (c). (d), (e) and (f)). 5 i‘ince the 3rd. 6th and 12th basefrom the 3’ end were hidden by heavy shadow in the autoradiogram (Fig. l(a)): we determined them by Peattie’s method (1979: Fig. l(b)). The 124th and 162nd base (Fig l(e)). and 169th base (Fig. l(f)) were determined as G, A and C, respectively, by other sequencing gels (data not shown). The 88th and 145t,h base, (i-s8 and c- 145? respectively, were not detected clearly due to strong band compressionsat the sites (Fig. l(c) to (e), arrows). Similar band compressionsat the sites of C’or (: clusters were observed by our method for group I phage RNAs (seeFig. 2 for cDNA sequencing of MS2 RNA, filled arrows) and other group III phage RNAs. Our sequencing results for MS2 and Q/3 RNA were slightly different from those reported by Vandenberghe et al. (1975) for MS2 RNA and by Weissmann et al. (1973) for Q/l RNA. In our sequence, as shown in Figure 3. six positions of the sequencereported by Vandenberghe et al., and five positions of that reported b) Weissmann et ab.were not detected, probably due t’o band compression of the C or (: cluster. On the other hand, for the MS2 RNA sequence. the fact that a (’ residue was found between G - I, 9 and U - , 2. of the sequenceremains puzzling (Fig. 2, open arrow). However, since the gel patterns for sequencing were very reproducible, and since our major objective was to compare a large number of isolates, we believe that it is relevant to use our sequencing data for further comparison. (b) Homology

in the sequence wt the 3’-frrminal phage RiVA4s from the .samr group

region

of

Figure 3 shows t,he nucleotide sequencesup t’o 200 t,o 260 residuesfrom the 3’ end of 14 phage RXAs, and up t,o 100 residues from the 3’ end of two phage RNAs. In f Abbreviation

used.

cI)NA.

complementary

I)NA.

3’-TERMINAL

SEQIIENCES

ddC ddA abababab

OF

ddG

PHAGE

RNAs

ddT

(a) Frc:. 1. Autoradiograph of polyacrylamide gels used to sequence cDNA of VK RNA and to sequeno the 3’.labeled VK RNA. (a) and (c) to (f) Autoradiographs of gels of cDNA of VK RNA sequenced by th’ chain-termination method (Hamlyn et nl.. 1978). The ratio of dideoxynucleotide to deoxynucleotid (ddNTP/dNTP) was 4 (lane b in (a)). 1 (lane a in (a). and (c)) and 0.4 ((d), (e) and (f)). BPB Number Bromophenol blue, XC: Xylene cyanol FF. Filled arrows show the sites of band compression. start from the 3’ end of VK RNA.

Y. INOKIT(‘HI.

A. HIRASHIMA

lb) IQc:. l(1))

ASI)

I. LVATANA

3'.TERMINAI,SEQITENCESOFPHAC:E

PI<:. I(c)

RNAs

71X

Y. INOfil~CHI.

A. HIKASHTMA

(d) FIG;. I(d)

ANI)

I. M'A'I'ANAKE

3’.TERMINAL

SEQUENCES

iel mc:. l(e)

OF YHAGE

RNAs

719

if) PlCi.I (t’)

3’.TERMINAL

SEQUENCES

OF

PHAGE

RNAs

of polyacrylamide gel used to sequence cDNA of MS2 RNA. The ratio of Fit i. 2 Autoradiograph didec EqT lucleotide to deoxynucleotide (ddNTP/dNTP) was 0.4. Filled arrows show the sites of band COnll n%SI lion. The open arrow shows the extra G residue observed in our sequencing gel. Numb ers start from the 3’ end of MS2 RNA.

7%P

\‘.

MSZa MSZb JP501 FRl BOl

A.

HIRASHIMA

AND

I. WATANABE

-200 * * * * * UUCCCUCAGGAGUGUGGGCCAGCGAGCUCUCCUCGGUAGCUGACCGAGGGAC

MS2a MSZb JP501 FRl B01

MS2a MSZb JP501 FRl B01

INOKU(‘HI.

*

A u u

U U *

* * *d GUGGGUGUGCUCGAAAGAGCACGGGU-GCGAAAGCGGU C C C C

-100 * * CC GGCUCCACCGAAAGGUGGGC (CC) (CC) v (CC) (CC)

-150 * CCC CGUAAACGGG (CCC) (CCC) (CCCjc (CCC) * G GGCUUCGGCC (G) (G) (G) (G)

-50 * * * * * * * CAGGGACCUCCCCCUAAAGAGAGGACCCGGGAUUCUCCCGAUUUGGUllACUAGCUGCUUGGCUAGUUACCACCCA U G

U (a)

GA JP34 THl KU1 BZ13

GA JP34 THl KU1 B7.13

GA JP34 THl KU1 BZ13

-200 * * ?r * * * * UGAGACCCUAUCCUCCGCCAGGUUUAGGUGCAAACCUAACUCGGAAUGGAGAACCCAGAUCCCUCUAUUUC U U U

CAUA A

UG

U UA

-100 * * * * x * CUCAGGAAUUAGAGGCCUGCGUUCUCUCCUGAUAGUAUCAGGACCUCCCCGGAUGGGGUGGGUGUGACCGAAAGG C G G

-15( *

*

u G G

-50 * * * * * * * CCACUAUGGAGGUGA-ACCCUCCCGCACCAAAAGGCGGUUCUCGGUGACUAGUUUGCUUGGCUAGUCACCACCCA A -au A -u ACGC uuc U cuuu uu - UAA uuc uu - C GUG - UAA (b)

FIG;. 3. Nucleotide sequences at the 3’.terminal region of RNAs from RN.4 coliphages. (a) Group I phage RNAs. M82” sequence of Vandenberghe et nl. (1975). M%Zb sequence determined in this study. ’ Parentheses show bases of uncertain identity due to band compression. d The bar indicates that a base is missing from the site. (b) Group II phage RNAs. a The bar shows that a base is missing from the site. (c) Group III phage RNAs. Q,Y RNA sequence determined in this study. Qflb RNA sequence from Weissmann et crl. (1973). ’ Left bracket shows that furt,her sequencing data have not been published. d Parentheses show bases of uncertain identity due to band compression. ’ The bar shows that a base is missing from the site. (d) Group IV phage RNAs. a The bar shows that a base is missing from the sit?. h Left bracket shows that further nucleotide sequencing has not been done. Hyphens haw been omitted from the sequences for claritv.

3’-TERMlNAI,

SEQUENCES

OF

PHAGE

* VK ST QP

VK

VK ST

QB; QB

VK ST

i23

RNAx

*

*

CUAUCGAUCAGCUUAUUUGUAAGAGUAAUCCUA C C G c C G -200 -150 * * * * * * * CAAAAAUAAGCAGAUCUACUGGUAAGUUCGAUAUACAGUAUAUCGCCUGCAGUAGCCGUG~CUGGCACC(C)UA G C G G C CA C G G C CA r= -100 * * * * * CGGGGUUUUCCAGGGCACGAAGGUUGCGUCUCUACACGAGGCGUAACCU C C C

* -% GGGAG(G)GC c(G) (GG) (GG) C(G) GG G

(0, CC) C

* G CUAUUAUGG WGC A (G)Gc A CA

-50 * * * * * * * CGCCUGGUUGUGAAUAAAUUAUCACAACUACUACUCUUACGAGUGAGAGAGGGAUCUGCU~GCCCUCUCUCCUCCCA AA U G AA U G AA U G (cl

TW28 SP

-250 * * * * * CGCGUGUCGCAGAUGGGACUGGGUUUGAUGAAGCGCCGCUAGCUAC A A uu

TW28 SP

-200 -150 * * * * * * * UAGCCUUCGUCGCAAGACAGGUCGGUACAGUGGCCUGGAUUCAGGACAGUGCCUUCAUCCGGCCCCCGUAUUU -a

Tw28 SP Tw19 FI

-100 * * * * * * UAUUACGGGACUUCCCGAGGUGMGCUUGCAAGCUAGACAC-UAGCUUGUGAUGGGAAGGG--GUCCAUUGA-A G C UC GU ; C [ [

TW28 SP TW19 FI

-50 * * * * * GCCCCGGAAG-GAGAAAGAAAGA-CU----CCUCCUGCGAGGGUGGGCUCUGCUUUGCCCACUCUCCUCCCA -AG c cccc - ---c U - --c AG A - uu A GU Cd) Flc:. 3(c),

(d)

* C C C

*

* CC G--A

I-. INOKI:CHI.

i24

A. HlltASHlMA

AND I. WATANABE

general, the nucleotide sequences at the 3’.terminal region of phage RNAs were found to be well-conserved in each group. In group I, we determined more than 200 residues of four phage RNAs (Jl’501, MS2, FRl and BOl: Fig. 3(a)). Wh en our MS2 sequence was used as a master sequence for comparison; the overall frequency of variability in this region was 3.3Ob. In group II. the sequences of five phage RXAs are shown in Figure 3(b). The overall frequency of variability in the region of 220 residues from the 3’ end was 14.4%. In addition, most of the base changes were found in the region of positions - 33 to -59, where a hairpin loop structure is formed, as shown in the Discussion (see Fig. 6). In group III. the sequences of ST, VK, and Q/3 RNA were det,ermined (Fig. 3(c)). The overall frequency of variability in this region among the three phage RNAs was 8.40/6. if our Q/l sequence was used for comparison. An adenine-rich sequence, which is known to represent an Sl and another host factor for the Q/3 replicasrbinding site in Q/3 RNA (Senear bz Steitz, 1976), was also seen in ST and VK RNA at the same position as in Qp RNA (-38 to -63). In group IV, we tried to determine four phage RNAs (Fig. 3(d)). However, in the case of phages FI and TW19 we failed t’o sequence more than 100 residues from the 3’ end, because of non-specific t,erminations by the inhibitors in each reaction. The overall frequency of variability up to residue -73 among four phages was very high (25.9(),,). The frequency between TW28 and SY was again 20.2°+J in the region up to residue - 80 from the 3’ end, whereas it was 4.49;) in t’he region from positions - 81 to - 260. This suggests that, most of the changes occurred in the region from T.\BlX !l’he frequency

(:roup

Group

MS2 JP501 HOI FRl

(houp

ii

J1’501

KC)1

FRI

09 -

4.3 I3

I .4 2.3 I .4

ST

Qp

8.4

84 0.4

III

(iA .JP34 THI RZ13 KVl

+JJ’34

THl

HZ13

Kl‘l

09

4.5 45

8.5 8.1 8.5

I 1.3 IO.4 135 63 -

iv TWZX

TWS TW19 SP Fl

reglow

II (:A

(;roup vii

VK

of sequence variability in the X-terminal among phage RiVAs in each group

I MS2

1

-

TWIO .

ISl’

FI

4.9

160 I64

135 II.1 18.3 -

Group I phage RNAs. our MS2 sequence was used. Group III phage RNAs. our Qp sequence was used. In the case of group IV phage RNAs, the region up to 73 residues from the 3’ end was compared. The values in the Table are percentage homology. The data are taken from Fig. :I.

3'-TERMINALSEQLTENCESOFPHAGE

RNAs

i25

positions -43 t’o -80, where mutations may occur, as in the case ofgroup II phage RNAs. In an attempt to show intragroup relationships among the phages, we examined the frequency of sequence variability in each group. As shown in Table 1, it was noticed that phages in each group, especially in groups II and IV, can be further classified into a few subgroups. For example, in group II, phages GA, JP34 and THl are classified as subgroup i, in which phage THl is somewhat different from the others, and phages BZ13 and KU1 are classified as subgroup ii. In the same way, in group IV’, phage SP is classified as subgroup i, phages TW19 and TW28 as subgroup ii, though the frequency of variability between them is somewhat high, and phage FI as subgroup iii. This subgrouping coincides with that reported according to sequence homology by RNA-cDNA hybridization (Inokuchi et al., 1979) and that on the basis of the serological property of phage particles and t.he molecular weight of phage virion proteins (Miyake et al., 1969; Furuse et al., 1979), except that phage JP34 is serologically distinguished from GA (Furuse et al., 1973). Of the base substitutions observed among phage RNAs in each group, the percentage of transitions was 44% in group I, 47% in group II, 80% in group III, and 52% in group IV, which are relatively low when compared with that (86Oi,) among MS2, R17 and f2 RNA (Min Jou & Fiers, 1976).

(c) Homology phage

in the sequence at the 3’-terminal region RNAs between the different groups

of

To compare the sequencesof phage RNAs among the four groups, we picked sequencesof our MS2 RNA (group I), GA RNA (group II), VK RNA (group III) and TW28 RNA (group IV) at random from each group. Since the sequencesof the phage RNAs in each group are fairly homologous, as mentioned above, the results obtained with other phage RNAs from each group were essentially the same as those described below. Table 2 shows the numbers of common sequences (more than pentanucleotide) in the 200 residuesfrom the 3’ end in each combination of the four phage RNAs. Two sequencesof identical nona- and dodecanucleotide were found in the combination of MS2 and GA, and one each of identical undeca- and

rlnalysis No. of nurleotides 6 I 8 9 10 11 12

of nucleotide

sequences identical No.

of identical

between two phage RNAs sequences

MSZ/BA

MS2/VK

GA/VK

VK/TW28

GA/TW28

38 23 16 11 I 4 2

12 4 I 0 0 0 0

I 2 1 0 0 0 0

17 12 9 7 5 3 1

19 6 1 0 0 0 0

MS2,TW2X 26 I 2 0 0 0 0

7%

Y. INOKl’(:HI.

A. HIRASHIMA

n

VK

nn#jn

\w TW20

ANI)

1. LVA’I’ANAHK

rjjqjn \

\\

mnnjjj,f#+H \\

‘\

\\\\

hl

\;\

\\

/

/ ,!

mnnnn J

~~~~~~~~~~~~~~ -250

I -200

-150

IllI

IIll

III

-50

-100

-1

FIG:. 4. Possible homologies between MS2 and G-4 RNA. and Iwtwrn \‘K and TW28 RNA. connected by unbroken lines indicate identical nuclcotide squrnces longer than a trinucleotidr

Hoxrs

dodecanucleotides were found in the combination of VK and TW28. In contrast, only sequences smaller than octamers were common in the other combinations. When the sequence data were slightly rearranged for further comparison of RNAs, several homologous sites in the sequence up to 860 residues from the 3’ end were detected between MS2 and GA, and between VK and TW28 (Fig. 4). In the combination of MS2 and GA. 64% of the GA RN,4 was homologous with MS2 RNA. and 48”/;, of the MS2 RNA was homologous with GA RNA. In addition, one interesting region was present at positions - 101 to - 136 of MS2 RNA, since this region was missing from GA RNA. Similarly, in the combination of VK and TW28. 540/b of TW28 RNA was homologous with VK RNA. and 46O/,, of VK RNA was homologous with TW28 RNA. As shown in Figure 5, it should be emphasized that sequences up to 30 residues

Group

I

MS 2 JP501 FRl B01

Group

II

GA JP34 THl KU1 BZ13

Group

IE

VK Gil

Grow

ISL

FIG:. 5. Sequence homologies nucleotide sequences. Hyphens

TW28 SP TW19 FI near the 5’.terminal have been omitted

UCUCCUCCCA

end among for clarity.

phagr

RNAs.

Boxes

indicate

identical

Y’-TERMINBL

SEQUENCES

OF

PHAGE

RNAs

727

from the 3’ end of various phage RNAs were found to be : (1) almost completely conserved among the phage RNAs in each group ; (2) approximately 90% homologous between groups I and II, and between groups III and IV; (3) most of the variations observed among the above combinations were base transitions ; and (4) one pentanucleotide (U-G-C-U-U), w h’ic h is common to every phage RNA, was found at around the same position in all phage RNA tested,

4. Discussion In the present study, we determined the nucleotide sequences up to 200 to 260 residues from the 3’ terminus of 14 phage RNAs from four groups of RNA coliphages. To study the homology in the sequences of this region among four groups, we searched for randomly common sequences (more than pentamer) among MS2 RNA (group I), GA RNA (group II), VK RNA (group III) and TW28 RN,4 (group IV). In this survey, we found only one common heptamer, C-U-C-U-C-C-U, and five pentamers. However, as shown in Table 2, when the homology search was focused on the sequences of two RNAs, high levels of homology were observed in each combination. In particular, between MS2 and GA, and between VK and TW28, the number and length of common sequences were high. These relationships were further strengthened when the sequence data were duly arranged for comparison of the RNAs (Fig. 4). These results support our previous proposal (Furuse et al., 1979), that close genealogical relationships exist between groups I and II, and between groups III and IV’. Figure 6 shows a possible secondary structure at the 3’-terminal region of GA, VK and TW28 RNA constructed according to Tinoco et al. (1973). For MS2 RNA, data are cit’ed from Fiers et al. (1976). The features of these secondary structures generally resemble each other and also those reported by Fiers et al. (1976), Flavell et al. (1975) and Senear & Steitz (1976). Comparing MS2 with GA, it can be seen that the region of positions - 101 to - 136 in MS2 RN-4 is missing from GA RNA (Fig. 6). If, besidesthe above region, in MS2 RNA the regions of positions -32 to - 38, -48 to -56 and - 76 to - 90. which are missing or short in the corresponding regions of GA RNA, are omitted we can picture the secondary structure of MS2 RNA as very similar to t’hat of GA RNA (Fig. 6). In the processes underlying the evolution of each RNA phage, base substitution, insertion and deletion of one or a few nucleotides, as well as sequence reduplication and block deletion must be considered. In this respect, the above finding is significant, since it strongly suggests a recent speciation between groups I and II phages. The sequence up to about residue -30 presents two interesting features. One is that this region is very homologous bet’ween the RNA of groups I and II, and of groups III and IV. Since most of the base substitutions observed in this region in both combinations were base transitions, the prototype of RNA in this region must be the same. The other point is that the sequencesof this region were similar among the four groups, and this region can form a common shape of stem and loop structure, which contains a common pentamer (U-G-C-U-U) in the hairpin loop. These findings support the idea that RNA coliphages are derived from a common ancestor, as suggested by Cory et al. (1970) and McLachlan (1971), who

3’.TEK1IIlNAL

SEQCEXCES

OF

PHA(:E

RN;\s

it29

demonstrated that the nucleotide sequences near the 5’ ends of R17 RNA (group I) and Q/3 RNA (group III) were very similar to each other. In relation to the function of the 3’.terminal region of RNA molecules, it can be said that this region may contain part of the recognition site for RNA replicate and its cofactors such as the host factor for Q/3 replicase (Renear & Steitz, 1976). In this respect, the very high degree of homology of the nucleotide sequences in this region between phage RNAs of groups I and II, and of groups III and IV, facilitates the explanation of the results presented by Miyake et al. (1971) and Yonesaki B Aoyama (1981), which show ambiguous template specificity of RNA replication between groups III and IV. and between groups I and II in vitro. We thank Dr K. Furuse computer programming in grateful to Dr Y. Okada transcriptase. This work was Science. and Culture of Japan, Fund. from the Keio rniversity

for supplying the RNA phages and Dr M. Katsuki for his the search for identical nucleotide sequences. We are also (Tokyo University) for his generous gift of ,4hIV reverse supported in part by grants from the Ministry of Education. from the Keio r’mversitp School of Medicine Research-Aid Research-Aid Fund and the Takrda Science Foundation.

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