In vitro site-directed mutagenesis: Generation and properties of an infectious extracistronic mutant of bacteriophage Qβ

Gene, 1 (1976) 3 - 2 5 © Elsevier/North-Holland Biomedical Press, Amsterdam- Printed in The Netherlands

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IN VITRO SITE-DIRECTED MUTAGENESIS: GENERATION AND PROPERTIES OF AN INFECTIOUS EXTRACISTRONIC MUTANT OF BACTE. RIOPHAGE QJ]* (N4-hydroxy-CMP; reversion rate; mutation rate; QO replicase; infectivity; competitive growth) E. DOMINGO**, R.A. FLAVELL** and C. WEISSM, .NN Institut IY~rM~ekularbiologie:I, Universit~t Zfiric~ 8t 93 Zfir/ch ($witzerZand) (Received August 11th, 1976) (Accepted August 17th, 1976)

SUMMARY An infectious extracistronic mutant of phage QO has been prepared by sitedirected mutagenesis. QJ] RNA minus strands containing the mutagenic base analog N4-hydroxy,CMP instead of UMP at position 39 from the 5' end were synthesized in vitro and used as template for Q~ replicase to synthesize one generation of plus strands. E. coli spheroplasts were infected with the newly synthesized plus strands and phage recovered from single plaques. RNA sequence analysis revealed that four out of the eighteen phage clones analyzed contained RNA with an A -~ G transition at position 40 from the 3'-end (which corresponds to position 39 of the minus strand**'). Thus, the viability of phage Q~ does n o t depend on a unique nucleotide sequence in the 3'extrac~tronic RNA segment. Upon in vivo propagation of mutant 40, spontaneous true revertants arose with high frequency and overgrew the parental clone within about 10 passages, indicating a selective disadvantage of the extracistronic mutant. Replication of mixtures of wild type and mutant RNA in vitro resulted in a decrease of the proportion of mutated RNA in the progeny plus strands. The fact that *This paper is dedicated to the memory of our eollequeDr. Donna Sabo, whose premature death early 1976 is a Meat lo~ to her friends and to the scientific community. **Present eddraum: (E.D.) Instituto "G. bf~sfion", Velazquez 144, Madrid 6 (Spain); (R.A.F.) Jan Swmnmerdam Institute, University of Amsterdam, Amsterdam (The Netherlands). ***Syuthesk of the minus strands of RNA phages starts at the penultimate nueleotide of the plus strand (Weith et al., 1968; Kamen, 1969; ]tensing and August, 1969; Weber and Weimmann, 1970); therefore nueleotide 39 from the 5'-end of the minus strand is complementary to nueleotide 40 from the 8'~nd of the plus strand. Abbreviations: n~o.i., multiplidty of infection; pfa, plaque-forming units; 8DS, sodium dodeeyl sulfate; TNB, 0.05 M Tris-HCI (pH 7), 0.1 M NaCI, 0.005 M EDTA; HOCp, N 4hydroxy~SP.

•

Q0 RNA containing an A -* G transition in nucleotide --40 of QO RNA is less efficiently replicated in vitro may explain the selective disadvantage of the mutant phage in vivo. The preparation of an infectious mutated RNA by site-directed mutagenesis shows that the method is suitable to produce specific nucleotide exchanges without impairing the biological competence of the RNA. INTRODUCTION The genomes of all RNA bacteriophages studied so far contain untranslated sequences at their termini and between cistrons (cf. review by Weissmann et al., 1973). The closely related RNA phages MS2 and R17 have identical nucleotide sequences at their 3'- and 5'-terminal extracistronic regions, while differing in about 3--4% of the nucleotides from the coding sequences (Adams and Cory, 1970; Nichols and Robertson, 1971; Min Jou et al., 1972; Robertson and Jeppesen, 1972; Min Jou and Fiers, 1976). This suggested that conservation of the precise extracistronic sequences, for which no functional or structural role has yet been found, may be essential for optimal multiplication of these Phages (Min Jou et al., 1972; Min Jou and Fiers, 1976). Recently, a procedure was described for the generation of purine transitions at specific positions of the 3'-terminus of Q0 RNA (Flavell et al., 1974). The method is based on the stepwise synthesis of Q0 minus strands (Bandle and Weissmann, 1972; Bandle, 1973), incorporation of the mutagenic base analog N4.hydroxy.CMP instead of CMP into a preselected position and use of the purified, substituted minus strands as template for QO replicase. The product consists of a mixture of RNAs containing either G or A in the position determined by the N4-hydroxy-CMP in the minus strand. Using this approach QO RNA with a G -, A transition in position 16 from the 3' end was synthesized. Despite the fact that this RNA was efficiently replicated by QO replicase in vitro (Flavell et al., 1974; 1975), infection of E. coli spheroplasts with the mixture of mutant and wild type RNA yielded no viable particles carrying the G -~ A mutation, suggesting that this nucleotide transition is lethal (Sabo et al., 1975). In order to test the hypothesis that the entire nucleotide sequence at the 3'-extracistronic region of QO RNA has to be strictly conserved for phage viability, it was necessary to generate other mutant RNAs with defined nucleotide changes within this region of the RNA. In the present paper we describe the preparation of QO RNA with an A -, G transition in position 40 from the 3' end. In contrast to mutant G-z6 -* A RNA, infection of spheroplasts with mutant A-40 -~ G RNA gave rise to infectious virus which retained the original mutation, showing that the 3'-extracistronic sequence does not have to be conserved for phage viability. When grown in competition with wild type phage, mutant 40 has a clear selective disadvantage and disappears from the mixed population after a few generations. In vitro, the mutant RNA is replicated less efficiently byQO replicase than wild type RNA. These results suggest that the decreased viability o f the

mutated phage in vivo may be due to a diminished rate of the replication of its RNA. MATERIALS AND METHODS

Q~ replicase, host factor, Q~ RNA and unlabeled ribonucleoside triphosphates were prepared as described by Flavell et al. (1974).

(a) Pretmmtion of minus strands with N4-hydroxy-CMP in position 39 The methods of stepwise synthesis of Q~ RNA minus strands and mutagenesis by incorporation of N4-hydroxy-CMP into minus strands are based on the procedures of Bandle (1973) and Flavell et al. (1974). For the synthesis of minus strands with N4-hydroxy-CMP in position 39 (cf. Fig. 1) 500 units of Q/I replicase, 600 #g of Q~ RNA (including 1 #g of [32P]Q/3 RNA (4300 cpm/#g, as marker for the chromatography steps) and 500 units of host factor I were incubated for 5 min at 37°C with 0.4 mM GTP, 0.1 mM ATP, /

(1) (10) (20) ,], (30) ppPG-G-G-A-G-G-A-G-A-G-A-6-G-G-C-A-A-A-G-C-A-G-A-U-C-C-C-C-C-U-C-U-C-

[1

(,)

(6o)

Fig. 1. Nucleotide sequence at the §'-end of Q# minus strand RNA (Goodman et al., 197~; Bandle, 1973). The lint nucleotide incorporated at each step of the minus strand synthesi~ dNcribed in MATERIALS AND METHODS is indicated by an arrow. First step: incubation with GTP, ATP and CTP. Second step: incubation with ATP, CTP and UTP. Third step: incubation with GTP. Fourth step: incubation with ATP and UTP (or ATP and N 4 -hydroxy-CTP, for generation of substituted minus strands).

0.1 mM CTP, 80 mM Tris--HCl (pH 7.5), 12 mM MgCI2 and I mM EDTA (final volume, 1.25 ml). The replicating complex was freed from unincorporated substrates by chromatography through a 0.7 X 35 cm column of Sephadex G-100 equilibrated with 80 mM Tris--HCl (pH 7.5) at 4°C, at a flow rate of 0.1 ml/min; 0.4--0.5 ml fractions were collected. The excluded fractions were located by the Cerenkov radiation of the RNA template. The recovery of enzymatic activity was 98% of the initial, as determined by the chain elongation assay (Flavell et al., 1974). The pooled fractions (1.5 ml) were then adjusted to final concentrations of 10/~M each of CTP, UTP and ATP, 12 mM MgCI2, I mM EDTA, 50/zg/ml polyethylene sulfonate and incubated 5 rain at 37°C. The replicating complex was purified as in the previous step (Sephadex G-100 column, 0.7 X 47 era). The yield of enzymatic activity was 65% of the initial value. The excluded fractions (1.8 ml) were adjusted to final concentrations of 10/zM GTP, 12 mM MgCI~, I mM EDTA and incubated § m|n at 87°C. After removal of the unincorporated GTP on a Sephadex G-100 column (0.7 X 55 cm) as in the previous steps, 49% of the initial replicare activity were recovered in the replicating complex. The ex-

cluded fractions (2.3 ml) were adjusted to a final concentration of 10 pM N 4hydroxy-CTP and 3 #M [a-32P]ATP (specific activity 1.5.106 cpm/nmole), 12 mM MgCI2, 1 mM EDTA and incubated 10 rain at 37°C. The replicating complex, into which 0.08 nmole of [a-32P]AMP had been incorporated, was purified by Sephadex chromatography as detailed for the previous step. The minus strands were then elongated by adjusting the pooled excluded fractions (3.3 ml) to final concentrations of 0.8 mM each of GTP, ATP, CTP and UTP, 12 mM MgCI2, I mM EDTA, 50 pg/ml polyethylene sulfonate and incubating for 15 rain at 37°C. The reaction mixture was made 0.1%/n SDS and extracted twice with I vol. of phenol equilibrated with 0.05 M Tris--HCI (pH 7.5), 0.1 M NaCI, 0.005 M EDTA (TNE). The RNA was precipitated from the aqueous phase with 2 vol. of ethanol and left at--20°C overnight. The precipitate was collected by eentrifugation (15 rain at 10 000 r~m, 0°C) in the HB-4 Sorvall rotor, resuspended in 0.4 ml of 0.5 mM EDT A and adjusted to the composition of TNE. Following phenol extraction the n~wly synthesized minus strand hybridizes to the template RNA (Feix et c~/., 1967). This double-stranded RNA was separated from ribonucleoside triphosphates and the excess of Q# plus strand template by Chromatography on a 0.7 X 30 em column of Sepharose 2B layered on 2 cm of Chelex-100 (Na÷); the flow rate was about 0.6 ml/h; 0.2 ml fractions were collected. The excluded fractions contained a total of 67 pmoles of [a-32P]AMP. Assuming that 2 residues of AMP had been incorporated into positions 40 and 41 respectively (compare Fig. I), and that the minus strands were full-length, this would correspond to 50 ~g of product. The minus strands were separated from the plus strand template by the method of Poller et al. (1967) as described by Flavell et al. (1974). The yield of intact purified minus strand was 12 pg. Since the final preparation contained about 5% of infectious plus strands it was heated at an RNA concentration of 0.1 pg/pl for 15 min at 55°C in 0.5 X SSC to convert the residual plus strands into a double-stranded form, which is known to be non-infectious and to lack ternplate activity (Pollet et al., 1967; Feix et al., 1968). Wild type minus strands were synthesized in vitro in a parallel reaction (Flavell et al., 1974) and purified as above. (b) RNA infectivity assay A suspension of £. coli sp:heroplasts (0.4 ml) prepared from £. coli K12 W6 according to Strauss and Sinsheimer (1967) was mixed with 2 ~1 of a solution of protamine sulfate (5 pg/Izl) and immediately mixed with 9.--4 ng of the RNA sample to be tested, in 0.9. ml of 0.5 mM EDTA at 37°C. The addition of protamine sulfate to the spheropt~st suspension is essential if the RNA sample contains polyethylene sulfo~ate. It should not be added to the RNA sample directly (Bandle, unpublished results) since it complexes with RNA and strongly inhibits its infectivity. The mixture was kept at 37°C for 40 sec and then plated on E. ¢oU Q13 (Billeter and Weissmarm, 1966). The specific infectivity of QO RNA was 20--40 pfu/nB,

(¢) Single.round synthesis of plus rtrand RNA using minus strands with N 4. hydroxy.CMP in position 39 as template A mixtuze of 0.9 #g of substituted minus stnmcb, 2 units of Q~ replicase, 0.8 mM each of GTP and ATP, 80 mM Tris--HCl (pH 7.5), 12 mM MgCI~, I mM EITI'A (final volume, 20 #1) was incubated for 5 min at 37°C. Then 20 #1 of a mixture containing 0.8 mM each of GTP and ATP, 1.6 mM UTP, 0.3 mM [ a - 3 ~ P ] ~ (3.3-10 ~ cpm/nmole), 80 mM Tzis-HCl (pH 7.5), 12 mM MgCI2, 1 mM EDTA and 100 #g/ml polyethylene sulfonate were added and the incubation was continued for 15 min at 37°C to allow the synthesis of one round of plus strands. The reaction mixture was diluted with 0.1 m] of TNE and an aliquot removed to determine the acid-insoluble radioactivity. A total of 5.4.106 cpm was incorporated. The reaction mixture was then extracted with phenol and chromatographed through a column of Sephadex G-100 (0.5 × 6 cm) layezed on 0,5 cm of Chelex-100 (Na*), equilibrated with 20 mM Tris-HCI (pH 7.5), 2 mM EDTA. The excluded fractions (0.3 ml) were heated in a boiling water bath for 60 sec. The product was then purified by zonal centzifugation through a 5% to 23% sucrose gradient (5 ml) in 50 mM Tz~--HCI (pH 7.5), for 150 min at 60 000 rpm and 5°C in a SW65 rotor. Prior to analysis the plus strands were purified on a column of cellulose CF-11 (Franklin, 1966) as described by Flavell et al. (1974).

(d) Preparation of 32P-labeled RNA from phage isolated from single plaques Plaques formed after plating infected spheroplasts on a bacterial lawn were punched out, resuspended in I ml of t ~ p t o n e medium (Billeter and Weissmann, 1966), incubated for 1 h at 37°C and stored at 4°C over chloroform. When requizvd, cloned phage were recloned by diluting and plating to obtain a single plaque on a plate (end-point dilution). 10.ml cultures of K coli Q13, grown to a density of 2- 10 s cells/ml in twptone medium depleted of inorganic phosphate to about 0.02 mM (C. Escazmis et all. in preparation), were infected at a multiplicity of about 4 with phage from the single clone isolates. Each infected culture was labeled by the addition of 0.3--0.6 mCi of [32P]phosphate 15 min after infection. After 2 h at 37°C chloroform was added. About 0.4 mg of unlabeled Q~ phage was added as carrier to the crude lysates, and the phage was purified and the RNA extracted as described earlier (Weissmann et al., 1968). From 2 to 10-10 ~ cpm of each 32P-labeled phage RNA were obtained.

(e) Analysis of 3~P-labeled RNA Two-dimensional polyacrylamide gel electrophoresis for the preparative fractionation of large T~ oligonucleotides was carried out essentially according to de Wachter and Fiers (1972). For the complete digestion of QO ttNA by T1 RNAase 12 units of the enzyme were mixed with 100 #g of RNA in 10#1 of 20 mM Tris--HCl (pH 7.6), 2 mM EDTA and incubated 30 min at

37°C. Under the conditions used previously (60 units of T~ ttNAase and 100 pg of RNA (Flavell et al., 1974)) the digestion of Q~ RNA resulted in the

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formation of about 0.1-0.2 moles of a large oligonucleofide desit~mted 1' (1! in Plate H of Flavell et al., 1974). Oligon~=leotide 1'- ~ t e s close to oligonucleotide 1", the counterpart of oligonucleotide I ~ m the extracistronic mutant--40 ( c o m p m Plate 2for the locations of I and 1"). Oligonucleotide 1' is an overdigest/on p r o d u c t ~ t i n g fromcleav ,~e Tz

RN~

between residues A (~40)antiC (--39), l ~ n ~

~ e 3!.end of

1 (M. Billet~,~~, unpublished observation;see Fig. 2 for the relevant sequences). Under the milder digestion conditions currently ~ theradioactivity located in the position of 1' was 1 t o 3% of that found for oligonucleotide 1 . , liilJ) (-6O)

(-50)

(-~0)

-q;...

T1

T1

A-A-U-A-A-A-U-U-A-U-C-A-C-A-A-U-U-A-C-U-C-U-U°A-£-6 (OL| GONUCLEOTIDE1) ,

C i PANCREAT; RNAAsE

1 A-A-A-U, 2 A-A-U

1 C-U-C-U-U-A

1 A-U, 3 A-C

2 U-U-A. 1 U-C-A

5U, 2C, 1 6

1 U-A, 1 C-A, 1 C-6, 5 A

A

P,UTANT-qO (-60) (-50) (-q0) ... G-U-G,~A-A-U-A-A-A-U-U-A-U-C-A-C-A-A-d-U-A-C-U-C-U-U-G-C-G-A-G...

"t

T1

T1 RN~sE

11

A-A-U-A-A-A-U-U-A-U-C-A-C-A-A-U-U-A-C-UoC-U-U-6 (OLIGONUCLEOTIDE1*) I

I PANCREATIC

RNAAsE 1 A-A-A-U, 2 AoA-U,

1 C-U-C-U-U-G

1 A-U. 2 A-C

2 U-U-A. 1 U-C-A

5U. 2 £ . 1 G

z U-A, Z C-A, S A

B

Fig. 2. Nucleotide sequence from residue --36 to --66 at the :}'-end of wild type Qp RNA (Goodman et al., 1970; Weissmann et 8.1., 1973) and of mutant A-40 -* G RNA. Comparison of the expected products of hydrolysis of oligonucleotides I and 1" by pancreatic and Uz RNAueL

RESULTS

(a) Synthesis of Q# ItNA minus Strands'with N4.hydroxy.CMP in ~osition B9 nu

(Flavell et aL, 1974) but also for UTP (data not shown), when incorporation was memsured at a single, defined position. In order to insert N4-hydroxy-CMP into poI/tlo~ 89, ~/S minus strands were initiated and elongated to the appropr/ate posit/on ~ the stepwise synthesis procedure described earlier (Bandle, 1973; Flavell et ~., 1974). QiS replicase was incubated with Q/I RNA as template and GRIT, ATP and U o n l y substrates. This resulted in the synthesis of a minus strand segmerit extending t o position 23 from the 5' end, as shown in the scheme of Fig. 1 (Bandle, 1973). After removal of the substrates by Sephadex chromatography the replicating complex was incubated with ATP, CTP and UTP in order to elongate the minus strand up to nucleotide 37. The complex was again purified by Sephadex chromatography and incubated with GTP to TABLE I ANALYSIS OF OLIGONUCLEOTIDESDERIVED FROM MINUS STRANDS SYNTHESIZED WITHN'-HYDROXY-CTP AND [~-32P]ATP IN THE FOURTH STEP (POSITIONS 39 TO 41) Aliquots of the minus strand preparation were digested either with T, or pancreatic RNAue, the digestion products were separated by two-dimensionalpaper electrophoresis as detailed in the legend to Plate land characterized by further digestion. Nueleotide imalyseswere performed as described in Flavellet al. (1974). (1) Products of digestion with T, RNAmm Oligonueleotide

Yield

32P-Labeled products after complete hydrolysis

Conclusiona

A~, ~ / 5 A,p, U/5 AI3, ~

HOCI~AISApGp UiSAiSApGp HOC~A~b

(epm) (%) 1 2 3

2042 429 393

71 15

14

(2) Products of digestion with pancreatic RNAase

Oligonueleotide

Yield

32P-Labeled products after hydrolysis by T, RNAase

Conclusiona

A~ApGp AiSApGp H'--OC~!(00%), Ui~(10%)

A~ApGpApGpUp! A/SApGpApGpUp GpH--0-C~!, GpU~ GpH-'0"C~ GpUis,

(epm) (%) 1 2 3

1300 2240 2560

19 33 38

4

550

8

~J'--OCI3~

5

140

2

uiS,

aBss~ on the nearut neighbor labeling pattern, the mobilities of the fragments (Sanger andBrownlee, 1967) and the known sequence in this region of the minus strand (compare

Fit. 1).

bThis product may have arisen by overdigestionof product I by T, RNAase; it was not d e ~ d in fingerprints from similar minUSstrand preparations.

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insert a GMP residue in position 38. The substrates were removed and the replicating complex was incubated with N 4 .hydroxy·CTP and [a_ 32P]ATP, to introduce the sequence ... pHOCpApAOH' which could be later detected by appropriate nucleotide analysis. The complex was again separated from the substrates and elongated to completion with the four unlabeled, standard nucleoside triphosphates. The mBlor part of the preparation was purified free of plus strands by the method of Pollet et al. (1967). A sample of the product was digested with T 1 RNAase and the resulting oligonucleotides were separated by two·dimensional paper electrophoresis. As seen from the fingerprint in Plate l(a), only one major labeled oligonucleotide was recovered (7~% of total radioactivity). This oligonucleotide, upon digestion with Tl RNAase, yielded HOCp and Ap as the only labeled nucleotides (Table J) and was thus assigned the structure HOCpApApGp. A second minor oligonucleotide (15% of the radioactivity), identified as UpApApGp by its nearest neighbor analysis and mobility (Sanger and Brownlee, 1967) was derived from the wild type sequence, which arose when UMP was incorporated into position 39 instead of HOCMP. Traces of UTP may contaminate the incubation mixture and be incorporated preferentially by the replicase (cf. Flavell et al., 1974). The product of digestion of minus strands with pancreatic RNAase shown in Plate l(b) are also consistent with th~ proposed sequence around position 39 of the QI3 minus strand RNA (compare Fig. 1 and Table I). The analysis thus shows that about 85% of the minus strands were substituted with N 4-hydroxy·CMP in position 39; there was no evidence that substitution had occurred elsewhere. (b) One-round synthesis of QI3 plus strands using N 4 -hydroxy-CMP-substituted minus strands as template We expected that a certain proportion of plus strands synthesized on a N 4 _ hydroxy-eMP-substituted minus strand would carry a base substitution in the position corresponding to the nucleotide analog (cf. Flavell et al., 1974). We first examined the plus strands resulting from a one-fold transcription of the minus strand, since we did not know whether mutant plus strands, once generated, would be replicated as efficiently in vitro as their wild type counterparts. Purified minus strands (0.6 pmoles) substituted in position 39 with ~­ hydroxy-eMP were incubated with Q(j replicase and GTP and ATP as only substrates to allow initiation and limited elongation of the plus strands. Polyethylene sulfonate was added to inhibit subsequent initiation (Kondo and Weissmann, 1972) and UTP and [a-32 P1CTP were then added to allow completion of the plus strand. The product, 0.15 pmoles of plus strands, was purified, digested with T 1 RNAase and analyzed by two-dimensional polyacrylamide gel electrophoresis. The autoradiogram of the fingerprint showed a new large Tl oligonucleotide (named 1*) which was not present in fingerprints from T1-digested wild type RNA (compare Plate 2). The radioactivity found in 1* was about 26% of that in 1. Fig. 2 shows the sequence of oligonucleotide 1, which extends from positions -63 to -38 at the 3' end of wild type Q~ RNA (Goodman et aI., 1970; Weissmann et al., 1973) as well as

11 (a'}

o

o

collulooO aeotato, pH3.5 i

(b')

.............__.._...._.~.......... cellulose acetate,~[0H 3.5 . i

)

Hate I. Fingerprints of minus st=ands synthesized with N'-bydroxy-CTP and (a-s:P]ATP in the fourth step (position 39 to 41). The minus strands were synthesized and purified as described ~ MATERIALS AND METHODS. An aliquot of the preparation (7.3 • 103 cpm, total RNA 1 ~¢) i dilasted with 2 units of T, RNAase for 30 min at 37°C. A second aUquot (2.8-104 epm) m mixed with 20 #g of carrier yeast RNA and digested with 1 ~g of panereatie RNAsse for 80rain at 87°C. The digested samples were analyzed by two~onal paper eleetrophoresis (Banger and Brownlee, !967). (a) Autoradiogram of the T, R N ~ diffestion productL (b) Autoradiogram of the pancreatic RNAase digestion products. (a') and (b'), tracinp of a and b, respectively. For identification of the spots, cf. Table I.

12 {a9 0 ¸

'~

•

•

~i I

N|seryllnldo tlOl, uroo,.pH I.I)

o 0

~o~ o o"

U

Wtpolyser~OS!4o sol, woo, pH S.~ Plate 2. Two-dimensional polyacrylamide gel eleetrophoresis of the T, oligonucleotidu of plus strand RNA synthesized from wild type mlnus stnmds and from minus strands substituted with N'-hydroxy-CMP in position 39. Plus strands were prepared from minus strands in a ~ l e round of synthesis, purified, digested with Tt RNANo and eleetrophoresed as detailed in MATERIALS AND METHODS, except that in the tint dimension the bromphenolblue marker was run to 30 cm from the origin to provide better qmmtitation of the oligonucleotides, the corresponding gel pieces were excised and their Cerenkov radiation was determined. (a) Autoradiogram of a T~ digest of wild type plus strands; (a') trancingof (a); (b) autoradiof~unof a Ts digest of plus strands synthmized using s u i t e d minus strands as template; (b) tracing of (b). Oligonucleotide 1" is the mutant em~nterpart of I. The spots marked x and y are partial digestion products of Qp RNA which oecur in varying yields. the J

with with. Tabl~ label,

18

.o o ~ . ~ ~,~' |, •

' ~ +"++++I i+m

''

-~,< .,.; imZ~

•.~~.~~ "~.

:

+

+

+

+

ii ++

,

.

++_+ i,m.

.,,+~

~o

8< ..,. ~ ,...,

|

1

r~

o

+i+

I ~ ¢ 0 +-I 0

I,,,-I o~II v-I 1,-I

0 0 0 0

0 1 - 1 ~1 m

14

1. In addition, 1" yielded only two moles of A ~ p whereas I yielded three. The migration of oligonucleotide 1" in the t w ~ e u s i o n a l gelelectrophoresis is compatible with the length and nucleotide composition ascribed to it in Table II (of. de Wachter and Fiefs, 1972). These results suggest ~ a t oligonucleotide I * is derived from posit/ons - 6 3 t o --40 at ~ e : S ? ~ d of a modified Q/I RNA, and that it aro--because nucleotide 40 (which ~!eomplementaw ~ the i¢4-hydroxy-CMP present in position 39 ~ m t h e § ' end of the minus s t r a n d ) * w a s mutated from an A to a G. A more complete ~alysis of oligonucleotide I* is given below.

(¢) Synthesis of infectious Q[J RNA using the N4-hydroxy-CMP-substituted minus strands as template The analysis described above strongly suggested that a mixture of about 75% wild type and 25% plus strands c ~ g an A -* G substitution in position 40 from the 3' end (A-40 -~ G mutation) had.been synthesized in a singleround of in vitro synthesis using as template minus strands containing ~r4. hydroxy-CMP in position 39*. In order to determine whether the mutant RNA could give rise to viable mutant phage, spheroplasts were infected with the mixture of wild type and mutant RNA and phage clones obtained from individual spheroplasts were analyzed in regard to their nucleotide sequence in the 3' terminal region, around position--40. Feix et al. (1968) showed that Q~ minus strands, which are inherently noninfectious, can serve as template for the synthesis of infectious plus strands in vitro Minus strands substituted with N4-hydroxy-CMP in position 39 were incubated with Q~ replicase and GTP and ATP as the only substrates, then polyethylene sulfonate was added and the chains were elongated by the addition of CTP and [a-32P]UTP. As shown in Table IH, no infectivity was detectable prior to the addition of the missing triphosphates, showing that no biologically active plus strands were present initially. After the reaction, a total of 9 ng of labeled plus strands and 300 pfu of infectious RNA had been formed per 120 ng of minus strands used as template; the specific infectivity of the product synthesized on the substituted minus strand (33 pfu/ng) was thus similar to that of plus strands synthesized on wild type minus strands (25 pfu/ng) or plus strands extracted from virions (27 pfu/ng). (d) ~adiochemical ana/ys/8 of RNA prepared from cloned phage Phage was recovered from each of 18 plaques formed in thespheroplast infectivity assay of RNA from a single-round synthesis of plus strands in which N4-hydroxy-CMP-substituted minus strands h ~ been used as template (Table III). 3ZP-Labeledphages were prepared from each clone, their RNAs purified, digested with TI RNAase and analyzed by two~limensional gel eleetrophoresis. As set forth above, the mutated site on the plus strand is located within the region corresvonding to T, oli~onucleotide I in the wild type (Fig. 2). Thus, oligonucleo carrying an A -, G transition in *See footnote ~

on p. 3.

15

16

Plate 3. Two-dimensional polyacrylamide gel electrophorasis of the T,~lipnucleotidas of uniformly labeled wild type RNA and mutant A-40 ~ G RNA prepared from clonmi phqe. 3'P-Labeled phage was prepared from wild type and mutant clones obtained in the sl~her~ plast infectivity assay of RNA synthesized with substituted minus Strand as template (Tab|e HI). The RNAs were purified, digested with T, RNAsse and electrophoresed as indicated in MATERIALS AND METHODS. (a) wild type RNA (b) mutant A-4o *-~G RNA (c) mixture of wild type and mutant A-4o -* G RNAs.

17 oligonucleotide 1". Four out of the eighteen RNA preparations analyzed indeed showed the oligonucleotide pattern diagnostic for the mutant (Plate 3). In two case.s oligonucleotide 1 was not detectable (~ 1% of oligonucleotide I*) and in t w o of them the radioactivity of oligonucleotide 1 was about 5% of t h a t of 1", probably due to some reversion (see below). Fourteen RNAs contained oligonucleotide I and no detectable 1". The uniformly labeled oligonucleotides I and I* were digestedwith pancreaticRNAase A and RNAase U2 and the digestion products analyzed. As shown in Table IV, the analytical data are in full agreement with the structures shown for I and 1" in Fig.2. In part:cular it may be noted that I yields an RNAase U2 digestion product (C2U3)A which in the case of 1" is replaced by (C2U3)G, a further, more direct confirmation of the conclusion that A in position --40 of the wild type was replaced by G in the ml~tant RNA. Two of the phage clones (which were wild type in regard to position 40) showed unique changes in their Tl fingerprints. In clone EDS8 oligonucleotide 6 (located in the A2 cistron) was replaced by 6" which differed from its wild type counterpart by a U -* C transition, and in EDS45 a new, large T oligonucleotide appeared, presumably as consequence of a G -* A transition. The occurrence of sequence variants at similar frequencies in natural populations of Q~ and in phage generated in spheroplasts by natural Q~ RNA has been reported elsewhere (Domingo et al., 1976). We may note however that among 113 clones examined (not including the ones described above), 16 were variants as judged by their Tz fingerprints but none showed the 1 -~ 1" transition. (e) Competitive growth in vivo between mutant A-4o -~ G and wild type phage; reversion o f the mutant to wild type phage To test if wild type phage has a selective advantage over mutant 40 in vivo, liquid cultures of E. ¢oli Q13 were infected with both cloned mutant and wild type phage at an m.o.i, of about 10 each; a high m.o.i, was chosen to ensure intracellular competition. The resulting lysate was then used to infect a fresh bacterial culture at an m.o.i, of about 20. A total of 10 such cycles of infection was carried out. Cultures infected with wild type phage or mutant 40 alone were propagated in parallel. 32P-Labeled phage was prepared from several of the intermediate lysates, and the RNA analyzed as described above in order to determine the ratio of wild type and mutant. The result of such a competition experiment is shown in Fig. 3. The proportion of the mutant phage decreased with the number of in vivo passages. After four cycles of infection the proportion of the mutant was only about 2% and no detectable mutant was found after ten cycles. The cloned wild type phage propagated in parallel showed no change in its oligonucleotide fingerprint. Propagation of the mutant A-40 -~ G in the absence of added wild type resulted in the appearance of an increasing proportion of wild type phage, as judged by the [pradual increase of TI oligonucleotide 1 in the fingerprints

18 _+..

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19

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NUMBER OF TRANSFERS

Fig. 3. Competitive growth of mutant A.,o -* G and wild type Q# in vivo. Wild type and mutan~ A-+o -~ O phage were prepared by resuspending single plaques obtained in the exper. iment of Table IH in I ml of tryptone medium (Billeter and Weksmann, 1966) and incubating I h at 37°C. £. ¢off QI3 (I0 ml) was grown in tryptone medium to a cell density of about 2 • 10' cells/rnl. Then either mutant A-40 -~ G or a 1 : 1 mixture of mutant and wild type phage were added to give an m.o.i, of 16--25. The lysate resulting after 2 h of incubation was used to infect a fresh culture of £. ¢01i QI3 at an m.o.i, of 15--25. This procedure was repeated for a total of I 0 transfers. A sample of the bacterial culture was taken immediately prior to infection, in order to ascertain that it was free from phage. 32P-Labeled lysates were prepared from the initial phage mixture and from several intermediate lysates. The preparation of "P-labeled p h a p is counted as a transfer; i.e. transfer I corresponds to the ["P]lysate prepared from the initial phage mixture. The [3zP]RNAs were purified, digested with T, RNAsse and the large T,-oligonucleotides separated and quantitated as d~eribed in Plate 2. Experiment I, o o, infection with'mutant A.40 -~ G (clone I); Experiment 2, a , , infection with mutant A . , -~ G (phage repurified by end-point dilution of clone I ); Experiment 3, ~ 4, infection with mutant A.40 -~ G (end-point repurified clone 2); Experiment 4, • e, infection with a mixture of a wild type clone (m.o.i. ~- 10) end mutant A-+o -* G (clone 1) (m.o.i. = 10).

(Fig. 3). To ensure that this effect was due to spontaneous reversion and not to contamination of the original isolates with wild type phage, two mutant clones were picked from plates containing single plaques. Again, the transfers resulted in the appearance of wild type phage after one or a few generations. The results show that r e v e r t s accumulated rapidly during the propagation of mutant A-a0 + G on E. ¢off Q13. In addition, the revertants overgrew the parental mutant, again suggesting that the wild type had a selective advantage over mutant phage. We did not carry out a serial transfer experiment under noncompetitive conditions, i.e., keeping the m.o.i, low at all times. We did note, however, that the average burst size of wild type and mutant phage was very similar when+the p h q e s were grown singly: cultures of £. eoU ( 2 - 1 0 s cells/m/were infected with either wild type or mutant A-40 + G phage at m.o.i. -- 10 and the yield of progeny particles per infected cell was determined. In five exper4ments the average value for the wild type phage was 1300 ± 396 (S.D.) and for the mutant A-4o + G 1410 ± 535 (S.D.).

20

(f) Competitive replication in vitro o f wild type and mutant A_4o -~ G RNA The selective disadvantage shown by the mutant phage vis-a.vis its wild type counterpart could be due to a less effective replication of its RNA. It was therefore of interest to determine whether a difference could be detected in the relative rate of in vitro replication by Q~ replicase of mutant A-do -~ G RNA and of wild type RNA. A mixture of wild ~ e QORNA and :mutant (A-4o -~ G) Q~I RNA was used as template for Q~ replicase in a 20-min reaction which resulted in the synthesis of an amount of product about equivalent to that of the original template. Half of the reaction mixture was used as template for a second 20-min reaction. This serial reaction was continued for a total of 18 transfers. The results shown in Table V indicate t h a t while the content of oligonucleotide 1" was 52% in plus strand RNA synthesized in the first incubation, this proportion decreased gradually to a value of 3 0 ~ after the 18th transfer. Similar results were obtained in tl~ree additional experiments conducted essentially under the same conditions. These results indicated that mutant A-d0 -" G RNA is less efficiently synthesized by QO replicase than wild type RNA. DISCUSSION

It has previously been shown that QO replicase can incorporate the nucleotide analog Nd-hydroxy-CMP in place of CMP into a preselected position (the 15th from the 5' end) of Q~ minus strands. Once incorporated, the analog directed the insertion of AMP as well as of GMP into the corresponding position of the plus strand (Flavell et al., 1974). This behavior of the nucleotide analog is ascribed to its occurrence in either of two tautomeric forms, of which one resembles UMP and the other CMP in regard to its biochemical behavior (Budowsky, 1976). The experiments reported in this paper show that, in keeping with this hypothesis, N4-hydroxy-CMP is also efficiently incorporated in place of UMP. The analog, when present in position 15, directed the incorporation of GMP somewhat more efficiently than that of AMI~ (about I : 0.7) while the opposite was true for position 39 (about I : 3.3). It h~s been suggested that the immediate environment of the analog may preferentially stabilize one of the tautomeric states (Bdnks et al., 1971). Since the nearest neighbors to positions 15 ~nd 39 are the same, namely G to the 5' and A to the 3' side, a more subtle effect.may be responsible for this discrimination. The specific infectivity of the mixture of mutant and wild type RNA generated on minus strands with a ~ M P substitution in position 39 was the same as that of RNA synthesized using wild type minus strands as template,

suggestingthat the mutated R N A was as infectiousM..the~wildtype, Since the specificinfectivity~of the mutated RNA.could n o t ~ determined directly, spheroplasts were infected with the RNA ~ and Ph e clones w e ~ molated ~from individual plaques, Four o u t o f I 8 c l o n e s had RNA with an

21

A~

~o~

I

~ -o

° .

22

A-* G substitution in position --40 (A-40 -~ G) reflecting rather accurately the proportion of mutant RNA present in the preparation used for transfecfion. No mutant A-4o -* G was found among 120 phage clones derived from infection of spheroplasts with RNA synthesized from minus stlands substihated with/V%hydroxy-CMP in position 15 (Sabo et al., 197§). We may thus conclude that the mutant RNA A-40 -~ G was about as effective as wild type RNA in initiating a productive infectious cycle in spheroplasts and that the procedures used to generate site-directed mutations did not lead to unspecific damage of the RNA synthesized in vitro. It is important to bear this in mind when considering that in a similar experiment with RNA containing a G -~ A mutation in position --16 no mutant clones were observed among 120 examined (Sabo et al., submitted for publication).

The RNA with the A-4o -" G transition was as efficient as the wild type in generating infection in spheroplasts and the mutant phage had the same burst size as the wild type when the phages were grown singly. However, when mutant and wild type phage were grown for several cycles under competitive conditions a reduced propagation rate of the mutan~ was revealed. From the data of Fig. 3, the growth rote of the mutant was emhnated to be about 0.9-5 that of the wild type (cf. Appendix). In vitro experiments, in which a mixture of mutant and wild type .Q/~ RNA was replicated by Q~ replicase under conditions of limiting enzyme, gave a similar result as the in vivo experiments with the phage, in that the proportion of mutant RNA was continuously reduced in successive cycles of replication. It should be noted that a cycle of replication in vitro corresponds to about one doubling of the input RNA, while one cycle of infection and lysis in vivo corresponds to about 10 doublings. Thus, the disadvantage of the mutant phage in competitive growth in vivo could be due to slower replication of its RNA by QO replicase. It is of interest to note that the nucleotide in position --40 is part of a sequence ( - 6 3 to --38) to which both host factor I and Sz protein have been shown to bind (Senear and Steitz, 1976). Host factor I is a bacterial protein of unknown function in the host, required by Q~ replicase for chain initiation on plus strands (Franze de Fernandez et al., 1972; Carmichael et al., 1975), while S1 is a ribosomal protein (Inouye et al., 1974; Wahba et al., 1974; Hermoso and Szer, 1974) which, after infection, is recruited as a constituent of QO replicase (Kamen, 1970; Kondo et al., 1970). It has been suggested that the interaction of host factor I with the 3' terminal region of Q/I RNA, as well as with a further sequence (Senear and Steitz, 1976) located in the middle of the RNA, is required to bring the RNA into the proper conformation for the initiation of minus strand synthesis. Conceivably, the nucleotide change may affect the efficiency of these interactions. In contrast to the results with the Q~ mutant A-40 -~ G, ~ RNA with the G-~6 -~ A substitution was propagated slightly b e t t ~ in vitro than wild type RNA, despite its. lack of infectivity (Flavell et ai., 1975; Sabo et al., s u b m i ~ for publication). In vivo experiments in which carefully cloned, pure mutant phage was

23

repeatedly passaged showed that after preparing a lysate from the clone and subjecting the phage to two transfers about 3% wild type phage appeared in the culture and that after further passages the mutant was overgrown by the wild type. Since care had been taken to prevent contamination, and since repeated experiments of this nature gave similar results, we attribute this course of events to a relatively high rate of spontaneous reversion followed by competitive growth. As shown in the Appendix, our data suggest a spontaneous reversion rate at position --40 of about 10 -4 per doubling of the phage. Mutation rates of about 3- 10 -5 and 10 -4 per nucleotide have been estimated by Valentine et ai. (1969), based on the frequency of temperature-sensitive phage in virus stocks and the reversion rote of certain mutations, respectively. The mutation rotes may, of course, be site~lependent. It has been noted earlier that the extracistronic regions cf the related phages R17 and MS2 are identical (AdRmA and Cozy, 1970; de Wachter et al., 1971; Adams et 81., 1972; Robertson and Jeppesen, 1972; Min Jou and Fiefs, 1976) although cistronic sequences differ in about 3--4% of the nucleofide positions, and it was suggested that the biological competence of the virus is dependent on the precise primary structure of the non-translated terminal RNA segments (Min Jou et al., 1972; Min Jou and Fiers, 1976). The results we have obtained with two extracistronic mutants confirm this proposal, and moreover show that the invariance can come about not only by what appears to be unconditional lethality, i.e., an absolute incapacity of the virus to propagate in its host, but also by reduced fitness which becomes apparent under conditions of competitive growth. ACKNOWLEDGEMF~TS

We thank Dr. Donna Sabo for many valuable discussions and for advice. Thanks are due to Mr. T. Weibel for preparing Q~ replicase and host factor, and to Miss Anita Schmid and Dr. M.A. Billeter for supplying us with 3~p. labeled ribonucleoside triphosphates. R.A.F. and E.D. were supported by • EMBO Fellowships during part of this work. The project was supported by grants No. 3.132.73 and 3.475.75 from the Schweizerische Nationalfonds. NOTE ADDED IN PROOF

It has recently been found that the mutant oligonucleotide 1" is bound less efficiently by the ribosomal protein $1 (which is also a component of Q~ replicase) than its wild type counterpart, oligonucleotide I (S. Goelz and J. Steitz, personal communication). REFERENCES

A d s s , J.M. and Cory, 8, Un~unkt~d nueleotide sequence at the 5'-end of R17 bscterlophqe RN.4~ Nature, 227 (1970) 570--574.

Adams, J.M,, 8pshr, P.-F. snd Cory, 8., Nudeofide sequence from the 5' end to the first eistron of R17 bs~4~ophqe zibonudeie 8eid, Biochemistry, 11 (1972) 976-988.

24

Bandle, E., Schrittweke 8ynthase vonQ# Minuwtmq RNA, Dimertation (1973) University of Ziirich. • s. nd c., Con on , R N A by Qp•. Experienfia, 28 (1972) 743--744. Banks, G.R., Brown, D.I~L, Streeter, D;G. and Gr~smm, L , Mutqenic analogum of cytosine: RNA polymerase template and substrate studiu, J. Mol. Biol,, 60 (1971) 425-439, . . . . ~ .~ ~ • ~ .... . . .Double-stnmded . Bmeter, LA. and Weimnmm, C., MS2 RNA from ~ 2 -.i.n. .f.e. c. .t.e. .d. .

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edum in Nucle Acid

Research, Harper andRow, New York,'1966; pp. 4 9 ~ 5 1 2 ; ~. . . . " " Budowsky, E.I., The mechanism of the mutagenle action of hydrorylamines, in Cohp, W.E. (Ed.), Progress in Nucleic Acid Research and Molaeul~ Biology, Aeedemie Press, New York, 16, 1976, pp. 125--188. Cermichael, G., Weber, K., Nivelean, A. and Wahha, A.J., The host ~ r required for RNA phage Q~ RNA replication in vitro, J. :Biol. Chem., 250 (1975) 3607--3612. De Waehter, R., Merregaert, J., Vandenberghe, A., Contrems, R. and Fien, W., Studies on the bacteriophage hiS2: The untranslated 5"terminal nueleotide sequence preceding the first cistron, Eur. J. Bioehem., 22 (1971) 400--414. De Wachter, It. and Fiefs, W., Preparative two-dimensional polyaeryhunide gel electrophoresis of 32P-labeled RNA, Anal. Biochem., 49 (1972) 184-197. Domingo, E., Sabo, D. and We,manann, C., Nucleotide sequenos heterogeneity in the ItNA of phage Q~, Experientia, 32 (1976) 792. Feix, G., Pollet, R. and Weissmann, C., Replication of viral RNA, XVI. Enzymatic synthesis of infectious viral RNA with noninfectious QB minus strands as template, Proe. Natl. Acad. Sei. USA, 59 (1968) 145--152. Feix, G., Slot, H. and Weissmann, C., Repliostion of viral RNA, XIII. The early product of phage RNA synthesis in vitro, Proe. Na~l. Aeed. Sei. USA, 57 (1967) 1401--1408. Flavell, R.A., Sabo, D.L., Bandle, E.F. and Weimmann, C., Site-directed mutagenafis: Generation of an extracistronic mutation in haeteriophage QO RNA, J. Mol. BiOl., 89 (1974) 255--272. Flaveli, R.A., Sabo, D.L.O., Bandle, E.F. and Wcilmann, C., 8itedireeted mutaganwia: Effect of an extracistronic mutation on the in vitro propagation of bacteriophage QO RNA, Proc. Natl. Acad. Sci. USA, 72 (1975) 367--371. Franklin, R.M., Purification and properties of the replicative intermediate of the RNA bacteriophage R17, Proe. Natl. Acad. Sei. USA, 66 (1966) 1504--1611. Franze de Fernandez, M.T., Hsyward, W.S. and August, J.T., Bactedsl proteins required for replication of phage Q# ribonuclaic avid, J. BioL Chem., 247 (1972) 824--831. Goodman, H.M., Billeter, M.A., Hindley, J. and Weinmann, C., The nucleotide mquanos at the 5'-terminus of the Q# RNA minus strand, Proe. Natl. Azad. 8el. USA, 67 (1970) 921--928. * Hermoso, J.M., and Szer, W., Replacement of ribosomal protein 81 by interference factor ia in ribosomal binding of phage MS2 RNA, Proe. Natl. Acad. Sei, USA, 71 (1974) 4708--4712. Inouye, H., Pollack, Y. and Petre, J., Physical and functional homology hatween ribosomal protein 81 and interference factor i, Eur. J. Bioehem., 45 (1974) 109-117. Kamen, R., Infectivity of bacteriophage R17 RNA after sequential removal of 5' terminal nucleotides, Nature, 221 (1969) 321--325, ~. Kamen, R., Characterization of the subunits of Q# repliaase, Nature, 228 (1970) 527--533. Kondo, M., Gallerani, R. and Weissmann, C., Subunit structure of Qp replieue, Nature, 228 (1970) 525--527. Kond0, M. and Weissmann, C., Polyethylene sulfonete as inhibiter of initiation by Q~

r

T

t.

05 Min Jou, W. and Fiefs, W., Studies on the bacteriophage MS2, XXXIII. Comparison of the nueleotide sequences in related bacteriophage RNA's, J. Mol. Biol., (1976) in press. Nichols, J.L. and Robertson, H.D., Sequences of RNA fraL~nents from the bacteriophage f2 coat protein eJstron which differ from their R17 counterparts, Biochim. Biophys. Acta, 27,8 (1971) 676--681. Patterson, M.S. and Greene, R.C., Measurement of low ener{ff beta-emitters in aqueous solution by liquid scintillation counting of emulsions, Anal. Chem., 37 (1965) 843--857. Poller, R., Knolle, P. and Weissmann, C., Replication of viral RNA, XV. Purification and properties of QI~ minus strands, Proc. Natl. Aead. Sei. USA, 58 (1967) 766--773. Reining, U. and August, J.T., The 3"terminus and the replication of phage RNA, Nature, 224 (1969) 853--856. Robertson, H.D. and Jeppesen, P.G.N., Eztent of variation in three related bacteriophage RNA molecules, J. Mol. Biol., 68 (1972) 417--428. Sabo, D.L., Bandle, E. and Weissmann, C., Site-directed mutagenesis: Effect of an extraeistronie mutation in phage Q# RNA, Ezperientia, 31 (1975) 746. 8anger, F. and Brownlee, G.G., A two~limensional fractionation method for radioactive nueleotides, in Gromman, L. and Moldave, K. (Eds.), Methods in Enzymology, Academic Press, New York, 12A, 1967, pp. 361--381. Senear, A.W. and 8teitz, J.A., Site-directed intmaction of Q# host factor and ribosomal protein $1 with Q# and R17 bacteriophage RNAs, J. Biol. Chem., 251 (1976) 1902-1912. Strauss Jr., J.H. and Sinsheimer, R.L., C b a r a ~ a t i o n of an infectivity assay for the ribonueleic acid of bacteriophage MS2, J. Virol., 1 (1967) 711--716. Valentine, R.C., Ward, R. and Strand, M., The replication cycle of RNA bacteriophages, Adv. Virus flea., 15 (1969) 1--59. Wahbs,/LJ., Miller, M.J., Nivelean, A., Landers, T.A., Cannichael, G.G., Weber, K., Hawley~ D.A. end 81obin, L.I., 6ubunit I of Q# replicase and 30 S ribosomal protein 61 of F,~herfehm eoli, J. Biol. Chem., 249 (1974) 3314--3316. Weber, H. and Weismmmn, C., The 3' termini of bacteriophage Q~ plus and minus strands, J. Mol. Biol., 51 (1970) 215--224. Weilmann, C., Colthart, L. and Libonati, M., Determination of viral pluz and minus ribonueleie acid strands by an isotope dilution assay, Biochemistry, 7 (1968) 865--874. Weimmann, C., Billeter, M.A., Goodman, H.M., Hindley, J. and Weber, H., Structure and function of phal~e RNA, Annu. Rev. Bioehem., 42 (1973) 303--328 Weith, H.L., ~ , G.T. and Gilham, P.T., Comparison of RNA terminal sequences of phslge f2 and Q#: Chemical and sedimentation equilibrium studies, Science, 160 (1968) 1459--1460. Communicated by W. Szybalski.