Vol. 42, No. 2JOURNAL OF VIROLOGY, May 1982, p. 467-4730022-538X/82/050467-07$02.00/0

Sequences of Ribosome Binding Sites from the Large SizeClass of Reovirus mRNA

MARILYN KOZAKDepartment of Biological Sciences, University of Pittsburgh, Pittsburgh, Pennsylvania 15260

Received 4 November 1981/Accepted 14 December 1981

Ribosome-protected fragments from two of the large-sized reovirus mRNAswere recovered from sparsomycin-blocked 80S initiation complexes. The se-

quence of each protected oligonucleotide was determined. The ribosome bindingsite of each message includes the m7G cap and a centrally positioned AUG codon.An adenine residue occurs three nucleotides upstream from the initiator codon,thus conforming to the pattern GNNAUG observed for nearly all eucaryoticinitiation sites.

Although modern sequencing techniques havemade it relatively easy to analyze long stretchesof DNA or RNA, knowledge of a sequence issometimes not sufficient to identify functionallyimportant sites. With respect to the start site fortranslation, the AUG triplet closest to the 5'terminus serves as the initiator codon in morethan 90% of the eucaryotic mRNAs that havebeen examined (9, 10). However, the existenceof a few messages in which translation does notbegin at the 5'-proximal AUG codon cautionsagainst applying the first-AUG rule to identifythe initiator codon in any given message. Thus,to pinpoint the start site for translation in each ofthe reovirus mRNAs, I have used the ribosomeprotection assay introduced many years ago bySteitz (19). In this paper I report the sequencesof ribosome-protected initiation sites from twoof the large-sized reovirus messages.

MATERIALS AND METHODSSynthesis and purification of viral mRNA. The Dear-

ing strain of reovirus type 3 was grown as described byBanerjee and Shatkin (1). Purified virions that weretreated with chymotrypsin to activate the virus-en-coded transcriptase were incubated in the presence ofone a-32P-labeled nucleoside triphosphate (New En-gland Nuclear Corp.) and the remaining three nonra-dioactive nucleoside triphosphates. The standard nu-cleoside triphosphate concentration used in thetranscription reaction mixtures was 2 mM for eachnonradioactive species. To obtain a specific activity ofapproximately 6 x 106 cpm/nLg of RNA, the concentra-tion of the radioactive triphosphate species was adjust-ed to 0.2 mM. This tended to reduce the yield of large-sized mRNA, particularly when the labeled precursorwas ATP or GTP. The other components of thetranscription reaction mixture were as described byBoth et al. (3). The mixture of 10 reovirus mRNAs (4small species, 3 medium species, and 3 large species)was fractionated by sucrose gradient centrifugation(3). The RNA in the region of the large size class

(-25S) was recovered from the first gradient, ethanolprecipitated, heated at 80°C for 30 s in 6 M urea todissociate aggregates, diluted, and centrifuged througha second sucrose gradient. The 25S mRNA recoveredfrom the second gradient contained no trace of thesmall and medium mRNA species.

Characterization of ribosome-protected initiationsites. A mixture of the three large-sized mRNAs wasincubated with wheat germ ribosomes in a reactionmixture containing 30 mM HEPES (N-2-hydroxyeth-ylpiperazine-N'-2-ethanesulfonic acid) buffer (pH 7.4),72 mM KCI, 2.8 mM magnesium acetate, 2 mMdithiothreitol, 8 mM creatine phosphate, 40 ,ug ofcreatine phosphokinase per ml, 1 mM ATP, 0.24 mMGTP, and 200 ,uM sparsomycin. (Sparsomycin was agift from the Drug Research and Development Divi-sion, National Cancer Institute.) After 10 min at 20°Cto allow formation of initiation complexes, 300 U ofRNase T, per ml was added, and incubation wascontinued for 10 min at 20°C. The reaction mixtureswere then chilled and layered onto glycerol gradientsas described previously (12). After centrifugation at39,000 rpm for 3 h at 4°C, gradient fractions werecollected into vials containing phenol. The peak ofradioactive material associated with 80S ribosomeswas located by monitoring Cerenkov radiation. 32P-labeled RNA from the 80S region of the gradient wasextracted twice with phenol, concentrated by ethanolprecipitation, and then applied to a 20% polyacryl-amide gel containing 8 M urea (12). Electrophoresiswas carried out for 16 to 18 h at 130 V. Under theseconditions all of the 32P-labeled material migrated as asingle broad band in the size range of 32 to 38nucleotides. The ribosome-protected fragments wereeluted from the gel and subjected to cellulose acetateelectrophoresis, followed by homochromatography bon DEAE-cellulose thin-layer plates (2). This two-dimensional fractionation resolved three predominantspecies, which were designated fragments A, B, and C(see Fig. la). The radioactive oligonucleotides werelocated by autoradiography, eluted from the thin-layerplate with alkaline triethylamine carbonate, dried, andthen used for digestion with either RNase T1 orpancreatic RNase. The techniques used for finger-printing and sequencing the ribosome-protected frag-

467

468 KOZAK

ments have been described previously (13, 14), exceptthat the more sensitive chromatographic techniques ofVolckaert and Fiers (20, 21) were used for analyses ofsecondary and tertiary digestion products. Deductionof nucleotide sequences was facilitated by using near-est-neighbor transfer data, as described by Lebowitzet al. (16) and Pieczenik et al. (17).

In later experiments, polyacrylamide gel electropho-resis was carried out for 24 to 26 h. This resolved thelargest fragment (fragment B) from the remainingspecies and permitted ribosome binding site B to beanalyzed without going through the homochromatog-raphy b step. Recovery from the thin-layer plate afterhomochromatography b is often quite low. By avoid-ing the losses associated with that step, I was able todetermine the sequence of fragment B despite its lowyield (see Fig. la) in the initial ribosome protectionstep.

Indirect autoradiography was carried out at -70°Cby using Cronex intensifying screens (15).

RESULTSRibosome binding sites from the three large

messages. The large size class of reovirus mRNA

a

consists of three RNA species (18). A singleregion of each message was selected and pro-tected by wheat germ 80S ribosomes in thepresence of sparsomycin. Figure la shows atwo-dimensional fractionation of the ribosome-protected mRNA fragments. After complete di-gestion with RNase T1, fragments A, B, and Cyielded unique fingerprints (Fig. lb, c, and d),and one oligonucleotide in each fingerprint con-tained the m7G cap, which marks the 5' end ofthe message. Thus, each of the ribosome-pro-tected fragments (fragments A, B, and C) mustbe derived from a different mRNA within thelarge size class. The minor species labeled A' inFig. la is a slightly longer version of fragment A(see below). Similarly, there was some fraying atthe 3' end offragment C, generating two or threeclosely migrating spots in Fig. la. The yield ofinitiation sites B and C was always much lowerthan the yield of site A. Nevertheless, by elutingfragment B directly from a polyacrylamide gel, Iwas able to obtain amounts sufficient for se-

b~

T3 } 2

B A To%:A

C~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~

3 ....5.:T4~~~~~~~~~~

T3 T4AME4 XT2' TiTO~ TO' n

FIG. 1. (a) Fractionation of the 80S ribosome-protected fragments from the large size class of reovirusmRNA. Separation in the first dimension (left to right) was accomplished by electrophoresis on cellulose acetatestrips. Fractionation in the second dimension (bottom to top) was accomplished by chromatography on a DEAE-cellulose thin-layer plate, using homomixture b (2). (b through d) RNase T1 fingerprints of [a_32P]GTP-labeledribosome binding sites A (b), B (c), and C (d). Homochromatography c and polyethyleneimine cellulose thin-layer plates were used for the second-dimension fractionation. The dotted circles indicate the position of thexylene cyanol tracking dye. The capped oligonucleotide TO had a tendency to streak and to generate multiplespots during fingerprinting, thereby reducing its apparent yield in (b) and (c). This is a common problem withcapped oligonucleotides (4, 13).

J. VIROL.

REOVIRUS RIBOSOME BINDING SITES 469

TABLE 1. Analysis of RNase T1 products from ribosome binding site A

RNase T, Products of secondary digestion Products obtainedprimaryewith pancreatic RNaseb with RNase T2 Sequence deduced'producta [ca-32PJGTP [a-32PJCTP [a-32P]UTP ([a-32P]ATP)c

* * * * * *TO C, cape AAU, cap AAU, G, cap U, A m7GpppGmCUAAUCG[UYf

* * * * * * * *Ti AAG C, U AU A, C AUUCCAAG[G]9

* * * *T2 AG U C UCAG[G]

* * *T3 AAG AAG - A AAG[C]

* *T4 C, G - CG[G]

* * *T5 AU AU G AUG[A]

a The designations correspond to the oligonucleotides identified in the RNase T, fingerprint shown in Fig. lb.b Secondary digestion products were analyzed by using three separate preparations of mRNA, which were

labeled respectively with [a-32P]GTP, [a-32P]CTP, and [CL-32P]UTP. Each secondary digestion product wassubjected to tertiary digestion with RNase T2; the asterisks indicate the 3'-nucleoside monophosphates whichretained 32P radioactivity after hydrolysis with RNase T2. -, Oligonucleotide was not labeled under theconditions used.

c The amount of radioactive RNA obtained with the [C-32P]ATP-labeled mRNA preparation was insufficientfor a complete analysis. Therefore, the T1 oligonucleotides were treated only with RNase T2. In addition to theoligonucleotides listed, GMP was also detected in the T1 fingerprint of [a-32P]ATP-labeled binding site A.dThe residue enclosed in brackets at the 3' end of each oligonucleotide was determined by nearest-neighbor

transfer.' The designation cap refers to the structure m7GpppGmpCp.f The sequence proposed for capped oligonucleotide TO corresponds to oligonucleotide 6 in the catalog of

reovirus 5'-proximal sequences compiled by Hastings and Millward (5, 6).8 The sequence of oligonucleotide Ti can be tentatively constructed from the results of secondary pancreatic

RNase and tertiary RNase T2 digestions. To confirm the sequence, pancreatic RNase digestion was carried outafter modifying the oligonucleotide with CMCT, a water-soluble carbodiimide which blocks uridine and guanineresidues. With both the [a-32P]CTP- and [a-32P]UTP-labeled preparations, pancreatic RNase yielded a singleradioactive product which comigrated with the marker AUUC.

quence analysis. However, it was not possible todetermine the sequence of ribosome binding siteC.

Nucleotide sequence of ribosome-protected siteA. Complete digestion of fragment A withRNase T1 yielded six oligonucleotides (Fig. lb).The sequence of each T1 oligonucleotide wasdeduced from an analysis of secondary pancre-atic RNase digestion products and from nearest-neighbor analyses (Table 1). The amount ofradioactivity was not sufficient to allow quanti-tation of the secondary digestion products. Theyields appeared to be equimolar, based on visualinspection of the autoradiograms. The proposedsequence of each T1 oligonucleotide is consis-tent with its position in the two-dimensionalfingerprint. The oligonucleotides obtained afterpancreatic RNase digestion of fragment A areshown in Fig. 2, and the deduced sequences aregiven in Table 2. The large pancreatic RNaseoligonucleotides overlapped several of the T1oligonucleotides. These overlaps, together withnearest-neighbor transfer data, permitted thesequence of fragment A to be reconstructed, asfollows.

Oligonucleotide P3 (AAGG) must be derivedfrom the 3' end of the ribosome-protected se-quence, since P3 is the only pancreatic RNase

oligonucleotide that terminates with guanosinerather than with a pyrimidine. (Fragment A wasobtained by digesting ribosome-mRNA com-plexes with RNase T1, which is guanosine spe-cific.) Oligonucleotide P3 was replaced by aslightly larger oligonucleotide in the pancreaticRNase fingerprint derived from fragment A'(Fig. 2d), which was consistent with assigningP3 to the 3' end of the sequence. The only T1oligonucleotide from which P3 can be derived isTi, thus placing Ti at the 3' end of fragment A.The capped oligonucleotide TO obviously is de-rived from the 5' end of fragment A. TO mustbe followed by T2, the only T1 oligonucleotidethat begins with uridine. Oligonucleotide P2(AGGAU[G]) overlaps the 3' end of T2 andreveals that the next T1 oligonucleotide beginswith AU. The only available candidate is T5,since Ti has already been placed at the 3' end offragment A. Thus, oligonucleotides TO-T2-T5are contiguous at the 5' end of the ribosomebinding site. Oligonucleotide Pi (GAAGC[G])overlaps T5 and identifies T3 (AAG) as the nextT1 oligonucleotide, followed by T4 (CG). Thus,the order of T1 oligonucleotides within fragmentA is TO-T2-T5-T3-T4-T1. The complete se-quence is shown in Fig. 3a.

Nudeotide sequence of ribosome-protected site

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J. VIROL.470 KOZAK

ba

P7 P7

.* P6

P7 P7

P6

PShP4P2

P1*.

C

P7

diP7

P3 P4pjr ¢ ^iP2

P3-

FIG. 2. Pancreatic RNase fingerprints of ribosome binding site A labeled with [ac-32P]CTP (a), [a-32P]UTP (b),and [a-32P]GTP (c and d). First-dimension electrophoresis was from left to right, and second-dimensionhomochromatography was from bottom to top. The oligonucleotides are numbered as in Table 2. The dottedcircles show the position of the xylene cyanol marker. The faint spots migrating faster than P7 in the seconddimension are 3'-UMP and 2',3'-cycic UMP. The material used in (a) through (c) was the major ribosome-protected fragment designated A in Fig. la. The material used in (d) was the slightly larger fragment labeled A' inFig. la. The only difference between the fingerprints in (c) and (d) is a shift in the position of oligonucleotide P3.In all of these preparations, about one-half of the capped oligonucleotide (P7) underwent ring opening, whichgenerated a second spot, labeled P7'.

B. The five oligonucleotides obtained after com-plete digestion with RNase T1 are shown in Fig.ic. An additional oligonucleotide, labeled TO',

was obtained in variable amounts. Since thecomposition of this oligonucleotide was identicalto that of TO, it probably resulted from ring

TABLE 2. Analysis of pancreatic RNase products from ribosome binding site A

Pancreatic Products of secondary digestionRNase with RNase T,b Products obtainedRnmase with RNaseT1"with RNase T2 Sequence deduceddprimary 3 2jT 2]3 IT)producta [a-32P]GTP [(C-32PCTP [a-32P]UTP ([oI32PIATP)c

* * * * *

Pi AAG, C AAG G, A GAAGC[G]* * * * *

P2 AG,AU AU G AGGAU[G]* * * *

P3 AAG - G,A AAGG[A]* ** *

P4 G AU G GGAU[U]* ~* *

P5 AAU AAU A AAU[C]* *

P6 U G GU[C]P7 cap cap cap m7GpppGmC[U]

a The designations correspond to the oligonucleotides identified in the pancreatic RNase fingerprint shown inFig. 2.

b,c,d See Table 1, footnotes b, c, and d, respectively.

REOVIRUS RIBOSOME BINDING SITES 471

TO T2 T5 T3 T4 Ti(a) m GpppGmCUAAUCGUCAGGAUGAAGCGGAUUCCAAGG

P7 P5 P6 P2 P1 P4 P3

TO T2 T3 Tl(b) m7GpppGmCUACACGUUCCACGACAAUGUCAUCCAUGAUACUG

P3 PiPiP4 Pl P5 P6 P4 P2 P2 P7 P1partial III - 4- partial II -

-4----------- partial I >

FIG. 3. Nucleotide sequences of ribosome bindingsites A (a) and B (b). The products obtained uponcomplete digestion of fragment A with RNase T1 areprefixed with T and correspond to the oligonucleotidesshown in Fig. lb and Table 1. The products obtainedupon complete digestion with pancreatic RNase areprefixed with P and correspond to the oligonucleotidesdescribed in Fig. 2 and Table 2. The oligonucleotidesfrom fragment B are similarly represented (see Fig. lcand 4 and Tables 3 and 4). The pancreatic RNaseproducts released as mononucleotides are not labeled.

opening of the m7G cap. Table 3 summarizes theresults of secondary and tertiary digestions ofeach T1 oligonucleotide from Fig. lc. The yieldsof secondary and tertiary digestion productswere estimated from visual inspection of autora-diograms. TO is the only oligonucleotide forwhich the sequence should be considered tenta-tive. Although the sequence proposed for TO isconsistent with the available pancreatic RNaseand RNase T2 digestion data, confirmation byother techniques is needed. The tendency of TOto generate multiple spots during fingerprinting(see above) made it difficult to obtain adequateamounts for a definitive sequence analysis. (It isnot unusual for a single capped oligonucleotidespecies to generate several spots in two-dimen-sional fingerprints [4, 13]. This heterogeneity is

a b

Al

due in part to ring opening of the labile m7G cap[7] but may also involve conformational alter-ations in the cap structure [4].)The oligonucleotides obtained upon pancreat-

ic RNase digestion of fragment B are shown inFig. 4 and identified in Table 4. Three prepara-tions, which were labeled with [a-3zP]GTP,[a-32P]CTP, and [a-32P]ATP, were analyzed.The pancreatic RNase oligonucleotides were nothelpful in establishing the order of the T1 oligo-nucleotides. Instead, the T1 oligonucleotideswere ordered by analyzing several large partialdigestion products. Partial products I, II, and III(Fig. 3b) were obtained by hydrolyzing fragmentB with a low concentration of RNase T1, asdescribed previously (14). The oligonucleotidesreleased by complete RNase T1 digestion ofeach partial product were then identified. Partialproduct I lacked only oligonucleotide T4, indi-cating that T4 lies at the 3' end of fragment B.Partial product III revealed that TO and T2 arecontiguous at the 5' end. T2 is followed by a T1oligonucleotide that begins with adenine, andthe only available candidate is T3. Partial prod-uct II confirmed that T3, Ti, and T4 are contigu-ous. All of the pancreatic RNase oligonucleo-tides from fragment B are accounted for by thesequence proposed in Fig. 3b.

DISCUSSIONThe sequences of the ribosome-protected ini-

tiation sites from two of the large-sized reovirusmRNAs are shown in Fig. 3. Each ribosomebinding site includes the m7G cap. Binding siteA contains a single AUG triplet, which is almostcertainly the initiator codon. Although site Bcontains two AUG codons, the first (in position19 to 21) can be identified fairly confidently as

c

Pi P2 P3 P4

P5 P6 P7

FIG. 4. Pancreatic RNase fingerprints of ribosome binding site B labeled with [a-32P]CTP (a) and [a-32P]GTP(b). In (a), oligonucleotides PI through P5 are evident. In (b), oligonucleotides P1, P2, and P3 (migrating veryclosely) and P6 are evident. (c) Combined tracing of (a) and (b). The extra spot in (c) (labeled P7) was detectedonly with an RNA preparation labeled with [a-32P]ATP. Oligonucleotides P1 through P7 are identified in Table 4.Cellulose acetate electrophoresis was used for the first dimension (left to right), and homochromatography c onDEAE-cellulose was used for the second dimension.

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TABLE 3. Analysis of RNase T1 products from ribosome binding site B

RNase T, Products of secondary digestion Products obtainedpfimary with pancreatic RNaseb with RNase T2 Sequence deduced'product0 [a-32P]GTP [a-32P]CTP [a-32PJUTP ([i-32PJATP)c

* * * * *TO AC, cape AC, cap G, cap U, C m7GpppGmCUACACG[U]

* * * * * * *Ti AU AU, C, U AU G, C UCAUCCAUG[A}f

* * * * * * *T2 AC AC, C, U U G, C UUCCACG[A]9

* * * * * *T3 AAU AC AAU, G A, C ACAAUG[U]

* * * * * *T4 U AC AU, AC G, U AUACUG[A]I

a The designations correspond to the oligonucleotides identified in the RNase T1 fingerprint shown in Fig. ic.b,c,d,e See Table 1, footnotes b, c, d, and e, respectively.f From the results obtained with pancreatic RNase and RNase T2, it is possible to construct tentatively the

sequence shown for oligonucleotide Ti. The sequence was confirmed by pancreatic RNase digestion on theCMCT-blocked oligonucleotide labeled with [a-32P]CTP; the products obtained were A(Jt and UJC. Digestion ofthe [a-32P]CTP- and [a-32P]UTP-labeled oligonucleotide with RNase U2 yielded two products, the compositionof which was consistent with the sequence proposed for Ti.

g The sequence of oligonucleotide T2 can be tentatively constructed from the results of secondary pancreaticRNase and tertiary RNase T2 digestions. With both the [k-32P]CTP- and [a-32P]UTP-labeled preparations,secondary digestion with RNase U2 yielded a single labeled oligonucleotide, confirming that oligonucleotide T2contains a single internal adenine residue.

h The sequence of oligonucleotide T4 was deduced from the results of secondary pancreatic RNase andtertiary RNase T2 digestions and was confirmed by digestion with RNase U2.

the functional initiator codon since it is morecentrally located. Wheat germ 80S ribosomesconsistently protect 12 to 14 nucleotides on eachside of the initiator codon (8), although theprotected region to the left or right of the AUGtriplet obviously will be longer than 14 nucleo-tides if there are no nuclease-sensitive residues(guanine in the case of RNase Tj) nearby. Al-though there was limited fraying at the 3' end ofthe protected region in some experiments, whichgenerated the extra spots in Fig. la, the finger-prints revealed no contamination with se-quences from the interior of the mRNA chain.This is rather remarkable since the -30-nucleo-

tide initiation site represents <1% of the radio-activity in each -3,500-nucleotide chain.The sequences of ribosome binding sites from

9 of the 10 reovirus mRNAs have now beendetermined (8, 13, 14; this paper; Kozak, J. Mol.Biol, in press). In eight of the reovirus messages,ribosomes protect (and presumably initiate at) asingle site which encompasses the 5'-proximalAUG codon. The only exception is the sl mes-sage, which yields two ribosome-protected sites-centered at the first and second AUG codons(Kozak, in press). I have speculated elsewherethat flanking nucleotides might modulate theefficiency with which the AUG initiator codon

TABLE 4. Analysis of pancreatic RNase products from ribosome binding site BPancreatic Products of secondaryRNase digestion with RNase T1b Products obtainedprimase digestion with RNaseTwith RNase T2 Sequence deduceddproduct' [x-32P]GTP [a-32P]CTP ([a.32PJATP)c

* * *Pi AC AC C AC[G,A]P2 AU AU AU[G,C]P3 cap cap m7GpppGmC[U]P4 U GU[C]P5 AC G,C GAC[A]P6 AAU A AAU[G]p7 ~~~~~~~~~**P7 - G,U GAU[A]

a The designations correspond to the oligonucleotides identified in the pancreatic RNase fingerprint shown inFig. 4.

b,c,d see Table 1, footnotes b, c, and d, respectively.

472 KOZAK J. VIROL.

REOVIRUS RIBOSOME BINDING SITES 473

is recognized (11). The sequence GNNAUG,which occurs in -95% of the eucaryotic initia-tion sites, including the two sequences describedhere, identifies an "efficient" initiator codon. Inthe reovirus sl message, on the other hand, apyrimidine occurs three nucleotides upstreamfrom the first AUG triplet; this may explain whysome ribosomes bypass that site and initiate atthe next AUG downstream. The nucleotide im-mediately after the AUG codon also seems tomodulate the efficiency of ribosome binding.The sequence AUGG is found in more than 60%of the eucaryotic initiation sites, including thethree medium and four small reovirus messages.The ribosome binding sites from the two largereovirus messages deviate from this pattern. The5'-proximal initiation sites from nine reovirusmRNAs have little in common beyond the con-served sequence GNNAUG(G) and the 5Y-termi-nal sequence m7GpppGmCUA. The distancebetween the cap and the first AUG codon variesfrom 12 to 31 nucleotides. (The Gm residueadjacent to the cap is counted as residue 1.)Whereas the AUG triplet is always centrallypositioned within the 80S ribosome-protectedsequence, the m7G cap is included only if it liesclose to (i.e., within 15 to 20 nucleotides of) theAUG codon.Only one reovirus message from the large size

class remains to be analyzed. Hastings andMillward (5, 6) have identified a capped oligonu-cleotide (m7GpppGmCUAAAAG) from one ofthe large-sized messages. Since that sequencewas not part of ribosome binding site A or Bdescribed above, it must be derived from thethird mRNA species. The limited analyses that Iwas able to conduct on ribosome binding site C(Fig. ld) revealed that the cap was included inthe ribosome-protected portion of that message.Thus, translation must begin at an AUG codonthat lies within the first 15 to 20 nucleotides fromthe 5' end.

ACKNOWLEDGMENTSThis work was supported by Public Health Service grant AI

16634 and Public Health Service Career Development AwardAl 00380, both from the National Institutes of Health.

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17. Pleczenik, G., P. Model, and H. D. Robertson. 1974.Sequence and symmetry in ribosome binding sites ofbacteriophage ft RNA. J. Mol. Biol. 90:191-214.

18. Schuerch, A. R., W. R. Mitchell, and W. K. Joklik. 1975.Isolation of intact individual species of single- and double-stranded RNA after fractionation by polyacrylamide gelelectrophoresis. Anal. Biochem. 65:331-345.

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20. Vdcdert, G., ad W. Flers. 1977. A micromethod forbase analysis of 32P-labeled oligoribonucleotides. Anal.Biochem. 83:222-227.

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