Isolation of specific ribosome binding sites from single-stranded DNA

J. blol. Biol. (1976) 92,363-376

Isolation of Specific Ribosome Binding Sites from Single-stranded DNA HUGH D. ROBERTSON?

Medical Research Council Laboratory of Molecular Biology Hills Road, Cambridge CB2 ZQH, Englund (Received 3 July 1974) The ability of Emherichia coli ribosomes to protect small speoific regions of single-stranded bacteriophage DNA from digestion by pancreatic DNAase has been investigated. A procedure is described by which ribosome-protected fragments can be isolated from the DNA of bacteriophage fl and 4X174. Size determination by polyacrylamide gel electrophoresis or thin layer homochromatography together with fingerprinting analysis following chemical depurination or digestion with E. coli endonuclease IV were employed to show that these fragments represent a small specific portion of these DNAs. The protection reaction is largely dependent upon components necessary for ribosome binding to mRNA, including GTP, formylmethionyl-tRNA, and initiation factors. Thus, ribosomal binding to DNA mimics the ribosome-mRNA interaction. Furthermore, the regions in f 1 and 4X174 DNA which are protected differ in sequence from each other. When E. wli endonuclease IV is substituted for pancreatic DNAase in the ribosome protection reaction, a fragment of 9X174 DNA is obtained about 150 bases in length which contains all of the pyrimidine tracts in the shorter 50-base fragment obtained with pancreatic DNAaae, and a number of additional polypyrimidines. Double-stranded DNAs such as 9X174 replicative form do not bind at all to ribosomes in their native state. Heat denaturation of such double-stranded DNAs allows ribosome binding. Protection of the same specillc regions as those protected in single-stranded +X174 DNA was observed. A similar specific protection was observed following heat denaturation and ribosome binding with DNA from polyoma virus.

1. Introduction A number of promising approaches to the sequencing of DNA have recently appeared (Ling, 1971,1972a; Sadowski & Hurwitz, 1969; Robertson et al., 1973a; Ziff et al., 1973; Wu & Taylor, 1971; Blattner & Dahlberg, 1972; Salser et al., 1972; Sanger et al., 1973). In this paper I will describe a method which allows the isolation of DNA regions of considerable biological interest from a variety of DNA molecules. This method employs specific recognition of single-stranded DNA by ribosomes and should be generally applicable to any prokaryotic DNA molecule, and perhaps also to DNAs from higher cells. The most promising DNA-containing organisms for initial sequencing studies have been the single-stranded DNA-containing bacteriophages of Escherichiu coli such as t Present address: The Roakefeller University, New York, N.Y. 10021, U.S.A. 24

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fl and+X174 (Ling, 1971,1972u,b; Robertson et al., 1973a; Ziff et al., 1973). Several reports analyzing their polypyrimidine sequences have already appeared (Ling, 1972a,b), along with several accounts of direct and indirect sequencing approaches to such molecules (Ling, 1971; Robertson et al., 1973a; Ziff et al., 1973; Sanger et nl., 1973). As has been the case with RNA molecules larger than tRNA, there arc two stages involved in primary structure analysis of phage DNA molecules : first,, reliable methods to reduce the 5000 to 6000 nucleotide molecules to fragments less than 160 bases in length must be worked out; and second, methods for determining the sequence of the isolated fragment must be applied. We will be concerned here primarily with the first of these two stages, and in particular with the isolation of small DNA regions of biological interest to which direct DNA sequencing methods can be applied. The two major approaches used with bacteriophage RNA for fragment isolation were either specific nuclease digestion (Adams et al., 1969) or specific protection by binding of biological agents (Steitz, 1969~; Hindley & Staples, 1969; Bernardi & Spahr, 1972). In an application of the first of these approaches to DNA, Ling has reported that the phage T4-induced E. wli DNAase endonuclease IV can be made to produce specific fragments of DNA from bacteriophage fd (Ling, 1971). This approach was subsequently employed in the isolation of a specific +X174 DNA fragment (Ziff et al., 1973). I have chosen the second of these approaches, selecting among possible biological agents (e.g. repressors, DNA or RNA polymerases, or ribosomes) which might recognize and protect small DNA regions surrounding start signals for protein synthesis from exhaustive DNAase digestion. Ribosomes are already known to protect initiation sites of mRNA from nuclease digestion (Steitz, 1969a,b; Hindley & Staples, 1969). Earlier work has shown that the DNA strand of fl or $X174 corresponding to the in vivo mRNA is encapsulated in the particle, while the other DNA strand is not (Marvin & Hohn, 1969; Hayashi et al., 1963). Furthermore, single-stranded DNA from phages T7 and fd can form initiation complexes with ribosomes in a manner similar in many respects to the same process with authentic mRNA (Bretscher, 1969a,b; McCarthy t Holland, 1965, Ihler & Nakada, 1970,19711. In this paper I will describe the preparation and characterization of specific DNA fragments from both f 1 and $X174 bacteriophages. In addition, I will show that such fragments can be isolated from double-stranded DNA, and that by use of the DNAase endonuclease IV in the ribosome protection reaction, specific fragments up to 150 bases in length can be obtained which contain extensions of the sequences present in the smaller pancreatic DNAase fragments. In the accompanying paper (Barrel1 et at., 1975) the sequence determination of the major DNA ribosome binding site from +X174 DNA is described, and its biological significance discussed. A preliminary account of the isolation and sequence analysis of a ribosome-protected DNA fragment obtained from +X174 DNA using pancreatic DNAase has already appeared (Robertson et aZ., 1973a).

2. Materials and Methods (a) Chemicub and enzymes All chemicals used were of reagent grade. Pancreatic deoxyribonuclease (DPFF), E. coli alkaline phosphatase, snake venom phosphodiesterwe, spleen phosphodiesteraee and crude micrococcal nuclease were obtained from Worthington Biochemical Corp., Freehold, N.J. The bacteriophage T4-induced E. ooli DNAase, endonuclease IV (Sadowski & Hurwitz,

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1969) was kindly provided by Drs P. Sadowski, University of Toronto, and V. Ling. Purified N-formylmethionyl-tRNA from E. coli was the generous gift of Dw M Yoshida and P. Rudland, M.R.C. Laboratory of Molecular Biology, Cambridge, while additional batches were prepared in collaboration with Dr D. Ish-Horowitz of this laboratory according to published procedures (Clark t Maroker, 1966). Crude protein synthesis initiation factors prepared by washing E. wli ribosomes, as well ss factor-free ribosomes themselves, were the gift of Dr D. Ish-Horowitz. (b) Phage and bacteria Bacteriophage f 1, a fllamentous male-specitlc coliphage containing single-stranded DNA (Marvin & Hohn, 1969) was obtained from Dr N. D. Zinder, the Rockefeller University. +X174 was the gift of Dr J. Sedat, M.R.C. Laboratory of Molecular Biology, Cambridge, who also provided the lysis-deficient strain am3cs70 and its host E. coli strain HF4704. Use of this mutant in place of wild-type phage allowed an increase in yield of +X174 single-stranded DNA to a level comparable to that obtained with wild-type f 1. (c) Pancreatic

DNAase-free

tibocrornea

Ribosomes were prepsred by grinding 5 g of frozen E. coli MREBOO (grown by the Microbiological Research Establishment, Porton, England) with 10 g of alumina in a chilled mortar, followed by the addition of 10 ml of a buffer containing 0.01 M-Tris*HCl (pH 7.4), 0.01 M-magnesium acetate, 0.06 M-NH&~ and O-006 m-2-mercaptoethanol. Pancreatic DNAase, which is usually added at this point during the conventional preparation of 530 extracts for protein synthesis (Nirenberg & Matthaei, 1961), was omitted here. After a low-speed centrifugation (10,000 revs/mm for 10 min in the SS-34 rotor of the Sorvall RCZ-B centrifuge), the 530 supernatant was prepared by an additional centrifugation at 15,500 revs/min for 30 min. Ribosomes were pelleted from the 530 supernatsnt, still quite viscous with DNA, by a 4-h centrifugation at 40,000 revs/min in the type 65 rotor of a Beckman model L ultracentrifuge. The upper layer of the resulting pellet, consisting almost entirely of DNA, could be removed from the ribosomes themselves following disruption of the upper layer with a spatula and several rinses with the above buffer. The ribosome pellet wss then resuspended in the above buffer, and centrifugation and resuspension were each then carried out 3 more times, and the final pellet was resuspended in about 0.25 ml of the above buffer, yielding a final concentration of about 1000 Aaeo units/ml. Ribosomes were stored on ice at 4°C and used within 1 week. (d) Radioactive DNA The procedure used to prepare DNA from phage f 1 was similar to that described by Ling (1972a). E. coli K38 was grown at 37°C with gentle aeration in two 4OOml portions of a low phosphate medium containing 1.0% Difco Bactopeptone, 0.5% NaCl and O*l% glucose. Bacteria were infected at a multiplicity of 20 phage/baeterium and the culture made 2.5 mM in CaCl, when the cell density reached 2 x 10s cells/ml (10 bacteria per small square of a Petroff-Hauser bacterial counting chamber). After 5 min 50 mCi of [32P]orthophosphate (carrier free, from the Radiochemical Centre, Amersham, England) were added to each 400 mliof culture and incubation continued with gentle:aeration for 3 h at 37°C. The cells were removed by centrifugation (10,000 g for 15 min) and polyethylene glycol precipitation, cesium chloride centrifugation, and high-speed centrifugation of the resulting phage band were carried out upon the supernatant according to the procedure described by Ling (1972a). DNA was extracted with phenol in a buffer containing 0.05 MTrisvHCl (pH 7*0), 0.1 M-Naa, O-005 M-EDTA. Pelleted phage were resuspended in 5 ml of this buffer, 2.5 ml of phenol saturated with the same buffer were added, and extraction and separation of layers by centrifugation carried out at room temperature. DNA was precipitated in the presence of 2% sodium acetate and 2.5 vol. of absolute ethanol at -20°C. Such preparations yielded about 5 mg of DNA of specific activity between 1 and 2 x IO6 cts/min per pg. Bacteriophage 4X174 were grown in a synthetic medium similar to that described by Sedat & Sinsheimer (1964). In 450 ml of TPG salts (Sedat & Sinsheimer, 1964) without

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phosphate, were contained 10 ml of 100-fold concentrated minimum essential medium (Eagle), amino acids (sterile, for tissue culture use from Microbiological Associates, Bethesda, Maryland), 7 pg thymine/ml, 0.4% glucose, and 0.001 M-C&~,. Two 450-ml portions were inoculated with the E. coli strain HF4704 and then grown with aeration at 37°C until a titre of between 4 and 5 x IO8 cells/ml was reached (25 cells/square in the Petroff-Hauser counting chamber). The lysis-deficient 4x174 mutant am3cs70 was added at a multiplicity of 10 plaque-forming units per cell, followed by 50 mCi of [32P]orthophosphate per 450-ml portion as for f 1. Growth was continued for 2 h more, and the cells and phage were then collected by a 15min centrifugation at 10,000 g. The pellets were reborate, followed by addition of 1 ml of egg white suspended in 12 ml of 0.1 M-SOdiUm lysozyme (Sigma) at a concentration of 4 mg/ml. The mixture was incubated at 37°C for 30 min followed by addition of 0.2 ml of chloroform and freezing in a bath of solid CO2 and isopropyl alcohol. The cells were thawed, and the freezing and thawing procedure repeated atotal of 3 times. Then 0.5 ml of 1 M-CsCl, and 3 mg of crude micrococcal nuelease (Worthington) were added. After incubation at 37°C for an additional 30 min, the suspension was subjected to low-speed centrifugation as described above and phage were isolated from the supernatant by column chromatography with glass beads (Gschwender et al., 1969). The peak of phage, which was excluded from the column and eluted first, was extracted with phenol as above for f 1, except that the phenol layer was subjected to a second extraction at room temperature with buffer containing 0.4% sodium dodecyl sulfate. The aqueous layers were pooled and subjected to precipitation in ethanol as described above. 4X174 DNA from the 900 ml amounted to 2 to 4 mg with a specific activity of between 4 and 6 x lo6 cts/min per pg. 32P-labeled nicked 4x174 replicative form DNA was a gift from Dr J. Sedat, M.R.C. Laboratory of Molecular Biology, Cambridge. 32P-labeled DNA isolated from polyoma virus (spec. act. about lo5 cts/min per pg) was the gift of Dr W. Folk, Imperial Cancer Research Fund Laboratory, London. This DNA had been subjected to limited nicking under X-ray treatment so that the strands of double-stranded circles would separate upon denaturation. (e) Ribosme

binding

and pro&&on

of DNA

Binding reactions were carried out under conditions similar to those originally described by Kondo et al. (1968) and applied by others (Steitz, 1969a; Hindley & Staples, 1969) in the isolation of RNA ribosome binding sites. The drug neomycin, which has been used to facilitate translation of single-stranded DNA into sizeable polypeptides (McCarthy & Holland, 1965), has been shown by Bretscher not to be required to form GTP-dependent initiation complexes with E. coli ribosomes and phage fl DNA (Bretscher, 1969a,b). Therefore no neomycin was used in these experiments. Reactions were carried out at 37°C in 0.1 to 0.2 ml of a solution containing 0.1 M-Tris.HCl (pH 7.4), 0.05 M-NH&I, 0.005 Mmagnesium acetate, 5 to 50 Aas units of DNAase-free ribosomes containing initiation factors, 0.001 M-GTP, 50 to 500 rg of [szP]DNA or RNA and 6 to 20 pg of purified N-formylmethionyl-tRNA. Single-stranded DNAs were preincubated for 8 min at 37°C in distilled water in the presence of 10e4 M-EDTA in a manner similar to that described by Steitz for RI7 phage RNA (Steitz, 1969a). Double-stranded DNAs were denatured prior to ribosome binding as follows : phage or viral DNAs (10 pg or less) were diluted with water to a volume of 50 ~1, sealed in glass melting-point capillary tubes, and immersed in boiling water for 10 min. The capillaries were withdrawn, rapidly transferred to ice, opened and added to chilled tubes containing the other components of the ribosome binding reaction. Control incubations in which non-denatured double-stranded DNA was utilized were performed in each case. After incubation for 12 nun at 37°C the binding reaction wlks chilled for 1 min on ice and 25 ~1 of a 5 mg/ml solution of pancreatic DNAase in the same buffer were added; after 15 min of incubation at 20°C a further 16 ~1 of DNAase solution were added and incubation continued for 15 min. When digestion was to be carried out with E. coli endonuelease IV rather than pancreatic DNAase, 15 ~1 of & 12,000 units/ml solution in 50% glycerol were added and incubation was for 30 min at 37”C, rather than 20°C. Both sorts of reactions were chilled on ice at their conclusion and the endonuclease IV reactions were diluted 1: 1 with the above buffer to reduce the glycerol concentration.

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Rea&ions were then layered onto &ml, 5% to 20% sucrose density-gradients containing the above buffer and centrifuged for 2 h 45 min at 37,000 revs/mm and 5°C in the SW39 or SW50 rotor of the Beckman model L ultracentrifuge. (f) Collection,

monitoring

and pur$cation

of DNA fragments

Sucrose density-gradients were collected after centrifugation through custom-made 21 gauge, 7 mm long needles which reproducibly gave 66 drops/5 ml gradient. Two-drop fractions were collected into 0.5 in x 2 in siliconized glass test tubes and Cerenkov radiation monitored in a Nuclear Chicago Unilux II liquid scintillation counter. Alternatively, radioactivity was determined after drying a portion on a Whatman GF/A glass fibre filter. The 70 S regions from sucrose gradients were made approximately 8 M in urea by the addition of a number of grams of urea equal to 85% of the number of ml in the pooled fractions. These solutions were applied at 20°C to 0.5 ml DEAE-cellulose columns (Whatman DE23) prepared in Pasteur pipettes equilibrated with 0.05 M-TrisTHCl (pH 7.4), 0.1 M-NaCl, 0.005 M-magnesium acetate and 8 M-urea (Steitz, 1969a). After thorough washing of the columns to remove small fragments of DNA, most of the radioactivity (over 80%) was removed by addition of the above buffer made 0.5 M in NaCl. An intermediate washing in this buffer containing 0.3 M-NaCl removed only a small amount of radioactivity from the column. The DNA was precipitated by the addition of 2.5 vol. of ethanol, centrifuged, and resuspended in 1 ml of water. After extraction with 0.5 ml phenol and an additional ethanol precipitation in the presence of 2% sodium acetate the DNA was dried in vacua and resuspended in 10 ~1 of water. Recovery of fragments ranged from O-2 to 0.5% of original DNA added to the binding reaction. (g) HCl precipitation Since it was often necessary to combine peaks from several gradients in a single subsequent analysis, it was found desirable to remove non-nucleic acid contaminants (probably polysaccharides) present in ethanol precipitates of DNA fragments prepared as described in section (f), above. To do this, an adaptation of a method employed by Ling (unpublished data) was used, in which the DNA or RNA sample to be precipitated was suspended in 1 ml of distilled water on ice, and then made 0.23 M in HCl by the addition of 20 ~1 of concentrated HCl (37% standard solution). After 10 min of stirring on ice in a siliconized 12.ml thick-walled Pyrex Sorvall centrifuge tube, the mixture was centrifuged for 20 min at 15,000 revs/min in the SS34 rotor of the Sorvall RCZB centrifuge, the pellet washed with an additional 1 ml of chilled 0.23 N-HCI, and centrifugation repeated. The resulting precipitate of nucleic acid, while insoluble in water, was readily soluble in 30% triethylamine carbonate, pH 9.0, and was resuspended in 30 ~1 of this substance, dried in vacua, resuspended in 50 ~1 distilled water and evaporated to dryness once again. Fingerprinting analysis before and after treatment showed no changes in the patterns. (h) Separation

of DNA fragments

Two approaches were used to fractionate further the mixtures of DNA fragments obtained by the above procedures. The 6rst of these was polyacrylamide gel separation on 20 cm x 40 cm x 0.3 cm slabs of 10% or 12% polyacrylamide according to procedures similar to those of Adams et al. (1969) or Robertson et al. (1972). Radioactive species in this and subsequent procedures were located by autoradiography on Kodak Autoprocess X-ray 6lm and eluted from the gels according to the methods of Robertson et al. (1972). A second method of separating nucleic acid fragments in the size range expected (i.e. from 40 to 60 nucleotides in length) was by a two-dimensional separation method involving high voltage electrophoresis in the first dimension on cellulose-acetate at pH 3.5 and homochromatography on thin layers of DEAE-cellulose in the second, as originally described by Brownlee & Sanger (1969), and as applied to separation of phage RNA fragments by Adams et al. (1969) and Nichols (1970). It W&B found that after HCl precipitation, it was possible to process fragments made from several mg of input [32P]DNA on a single two-dimensional separation. Separated fragments were detected by autoradiography and eluted with 30% triethylamine carbonate according to standard procedures (Barrel1 1971).

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(i) Depurination

analysis

of DNA

The procedure described by Ling (1972a) was utilized. 32P-labeled DNA samples were dissolved in 0.1 ml distilled water, and 0.2 ml of 3% diphenylamine in formic acid were added. Tubes were sealed and incubated at 37°C for 18 h, after which 0.3 ml of water were added. The mixtures were extracted 5 times on ice with diethyl ether, and then dried in vacua. Conditions for fractionation of the resulting polypyrimidine tracts were as described by Ling (1972a). The samples, in 2 ~1 of water, were spotted near one end of a cellulose-acetate strip wetted with 5% glacial acetic acid containing 7 M-Wea and 0.005 MEDTA, and subjected to electrophoresis at 6000 V for 20 min. The oligonucleotides were blotted through onto a 20 cm x 40 cm thin-layer plate coated with a mixture of DEAEcellulose and cellulose (Machery-Nagel & Co., W. Germany) at a ratio of 1:7.5. Alternatively, commercially prepared DEAE-cellulose thin-layer plates from Machery-Nagel were utilized. The second dimension was developed at 60°C by ascending homochromatography using a 3% solution of partially hydrolyzed yeast RNA (B.D.H. Ltd, Poole, England) similar to homomix “0” of Brownlee & Sangcr (1969).

(j) Endonuclease IV digestion Preliminary analysis of DNA fragments by fingerprinting after digestion with E. coli endonuolease IV was carried out using conventional techniques. Conditions for digestion of DNA fragments with this enzyme (in contrast to digestion of DNA-ribosome complexes described above in section (e)) werea modification of those also employed by Ziffet al. (1973, and personal communication) as described previously (Robertson et al., 1973a). Specifically, incubations were carried out in volumes between O-1 and 0.2 ml of a buffer containing 0.05 ivr-Tris.HCl (pH 8.4), 0.02 M-magnesium acetate, 0.004 ivr-2-mercaptoethanol, DNA at a concentration of 0.1 mg/ml or less, and an amount of endonuclease IV (12,000 units/ml) in 50% glycerol not exceeding 15% of the total reaction volume and not exceeding 1 unit/IO rg of input DNA. Tubes were sealed and incubated for 3 h at 45% in a constant temperature water bath. Reactions were then chilled, brought to a volume of 1 ml with distilled water and extracted with 0.5 ml of phenol saturated with 0.01 M-Tris.HCl (pH 7.6), 0.001 M-EDTA. DNA fragments were precipitated from the aqueous layer by addition of 2.5 ml absolute ethanol in 2% sodium acetate in the presence of 10 cl@;of phage f2 RNA as carrier. After resuspension of the precipitate in 2 ~1 of distilled water, flngerprinting of these digests was carried out as described in section (i) above, except that the RNA mixture used to develop the second dimensions was 5% in RNA and much less extensive hydrolysis of the RNA was carried out in order to allow separation of the larger fragments (up to 50 nucleotides in length) present in endonuclease IV digests (Sadowski & Hurwitz, 1969; Ling, 1971). Control experiments in which fingerprinting analyses were performed without the ethanol precipitation step following phenol extraction of the endonuclease IV digest showed that this procedure precipitates all fragments, even those which would normally precipitate inefficiently because of their small size in the absence of carrier nucleic acid. by digestion with (k) Compositional analysis of oligonueleotides

venom and spleen phoaphodieaterasea Dephosphorylation of the polypyrimidine tracts from depurination fingerprints was carried out with bacterial alkaline phosphatase as described by Ling (1972a). Base compositions were determined using venom and spleen phosphodiesterases as described by Barrell (1971) and Ling (1972a), with incubation times increased to ensure complete digestion of each oligonualeotide.

3. Results (a) Isolation and characterization of single-stranded bacterio&xge DNA fragnzents protected by ribosomea from pancreatic DNAase digestion As already outlined in a preliminary communication (Robertson et al., 1973a), the most straightforward way to use this approach is to apply it to single-stranded 3aP-labeled DNAs from bacteriophage f 1 or +X174. F’ignre 1 illustrates experiments

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catied out with both of these DNAs. fn FQUFJ l(a), fl DNA was bound to nboeomes, the complexes digested with pancreatic DNAase and subjected to sucrwe densitygradient centrifugation as described in Materials and Methods. It is evident that a peak of protected DNA sediments at 78 S when the complete system is used, but that omission of formyhnethionyl.tRNA or GTP results in respective fivefold or fourfold decreases in protected radioactivity. Figure l(b) illustrates the same phenomenon with +X174 DNA, where protection from DNAase digestion is again observed and again the vatit majority of this protection is dependent on fomylmethionyl-tRNA.

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60 40 20 0

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no

&o. 1 Sucrvse density-Fad%=% ano)y&-+ of 76 S initiation Af9&SYA3 f&m ~U&ES~ digestion. &udcng rvactsons were carrlad adt IR e&oh case as d-s&bed in hlrtattfzk and i\lotao&, se&on (e). (A] Dspendmm of ribosoms prctccticu of fl LJfiA from panorcatic DNAASW drgestron on GTP And formylmethionyl-tRNA. 46 /# cf [Jr PJ-labsled fl DNA (spew. a&. 1.2 x lVcts/mm per pg) were hound to 6 Aaso units of rihasomes. After 12 min of incubation, the tea&on mixture was cooled and subjected to DNAaw digestron, mcrose density-gradient analysis and other procedures described in Ma@rials and Methods , section (f). -O-O--, Complete system; -x-x -, complete system minus GTP; -O-O-, complete system without formyhnethionyl-tRNA. The arrow represents the location of 78 S f2 baoteriophage which sediment& with fraction no. 9 in all gradient profiles shown exoept that shown in (b), where it sedimented with fraction no. 12. (b) Proteotion of $X174 DNA. 27 pg of +X174 DNA (spec. act. 4.6~ 10’ cts/min per pg) were bound, dig&,ed and centrifuged as above. --e--e-, complete system; --o-o-, complete system without N-formylmethionyl-tRNA. (c) Dependence of protection of 6x174 DNA upon m&ration factors. Reactions wera ae m (b) except that 6 &ao units of high salt-washed ribosome irom which initiation factors bad been removed (the gift of Dr D. Iah.Horo~ros) ~vre added to the ~WR~JWW -O-O-, %ACtmn Fpithout added initiation f&ctaTs; -m-e-, reaction to wk$oh Z p( of e sor’ution of cruda E. c0E; i&tatton hctors p**ad thrnugh thv e-oa~xjritti stags (also the gift of Dr Idh-Harawicz) were added. (d) Protectinn of 4x174 DP(A from endonuclea~6 IV digestion. -A-A-, Rcaatlon oarried out as described in Materu& and Methods, ~&on (e), in which endonucleace IL’ DP(UIsubstituted for paoreatic DNAABJ.

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In Figure 1(c) , the dependence of $X174 DNA binding and protection from pancreatic DNAase digestion upon the presence of protein synthesis initiation factors is demonstrated. Thus, in agreement with earlier observations this effect of single-stranded DNA is showing many properties analogous to ribosome binding with mRNA (Adams et al., 1969; Steitz, 1969a,b; Bretscher, 1969a,b; McCarthy & Holland, 1965; Ihler & Nakada, 1970,1971; Kondo et al., 1968). The first method employed to characterize the fl and +X174 DNA fragments following their isolation as described in Materials and Methods through the HClprecipitation step was chemical depurination. Plate I illustrates autoradiographs of two-dimensional fingerprints of either intact phage DNA or ribosome-protected fragments. Plate I(b) reveals a great simplification of the f 1 DNA pattern shown in Plate I(a). Plate I(a) shows a pattern identical to that previously reported by Ling (1972a) while Plate I(b) shows a much simpler pattern, following ribosome binding and pancreatic DNAase treatment, with no polypyrimidine tracts of a length greater than seven nucleotides in evidence. As shown by Ling (1972a) and in Plate I(a) fl DNA contains a variety of unique oligonucleotides of length between 8 and 20 bases, but all of these tracts are missing from the DNA pattern shown in Plate I(b). Plate I(c) and (d) show that the major species protected by ribosomes within $X174 DNA also represent a very small portion of the entire $X174 genome. In the case of the $X174 polypyrimidine tracts in Plate I(d) sequence analysis has been carried out as summarized in a preliminary communication (Robertson et al., 1973a) using a method described in the accompanying paper (Barrel1 et al., 1975). A second way to characterize these protected fragment populations is by further fractionation. Such analysis should be useful both to determine the number and size of protected fragments present and as a preparative step for subsequent sequencing analysis. Plate II illustrates two such procedures. Plate II(a) shows the autoradiograph of a 12% polyacrylamide gel of fragments from +X174 DNA. Under the electrophoresis conditions employed, the bromphenol blue marker (B) migrates together with nucleic acid fragments about 25 to 35 bases in length. The approximate size of the indicated band 2 could thus be estimated by co-electrophoresis with two fragments of known sequence from phage f2 RNA which are 43 and 57 bases in length (Nichols & Robertson, 1971). Band 2 was observed to migrate midway between these two RNA markers (data not shown). Similar results were obtained with protected fragments from fl DNA, with a somewhat broader size distribution. Plate II(b) shows the depurination fingerprint of band 2 DNA from Plate II(a) following elution and digestion with diphenylamine in formic acid. It is evident that this pattern contains many of the spots prominently visible in Plate I(d). In Plate II(c) and (d), a second fractionation was applied to the $X174 protected DNA fragments. In an experiment carried out with Dr H. L. Weith, a portion of the DNA analyzed in Plate II(a) was also fractionated by two-dimensional thin-layer analysis as described in Materials and Methods, section (h). It is evident again that the indicated spot 2 contains over half of the total radioactivity migrating as a single prominent oligonucleotide after this two-dimensional analysis, In Plate II(d), it is evident that this spot 2 contains the same subset of polypyrimidine tracts previously shown in Plate II(b), and that therefore both of the fractionation methods applied here suggest that the protected DNA from +X174 contains at least half of its total radioactivity in a single oligonucleotide sequence about 50 bases in length. This hypothesis was later confirmed by application of sequence analysis (Robertson et al.,

PI,ATES

I-V

PLATE I. Two-dimensional fractionation c,f dopurinate(1 I)NA. “21’-lab~~led DN:1 samplfv ww dissolved in 0.1 ml of distilled wrater and subjected to depurination and fingerprinting rtnaly~~as described in Materials and Methods, section (i). (a) Aut,oradiograph of two-tlimensional f’ractt~,n aCon of depurinated f 1 DNA (2 x 10’ ct,s/min sprc. act,. 2 x 106 cts/min per pg) prrparr~~ as ~11 section ((1) of Materials and Methods. In this and all subsequent autoradiographs of such tbvodimensional separations, the origin is at thr lower right-hand corner. The first dimension (high voltage electrophoresis at pH 3.5 in 7 wurra) in from right to kft,, while t,he second (thin-lay<‘r Tho circle of hfark ~lr,t J homochromatography) is from the bottom of t,he illust,ration to th(L top. in the upper right of this and subsequent, ~tut,r)radiogla~,hs rrpwsents an outlirw in radioart 1~’ rnarkw ink of thr final position of the blue marker tiyv normall,y a(ltlrvl bt~for~~ the first (linr~~lwll~rt of such two-dimensional separat,ions (Barrell, 1971). (b) ‘~~0.tlilnt:nsional fractionatioli q~f’ (11, purinated ~ibosome-protected f 1 DNA preparcvl a~ in Fig. 1 (a). Raw compositions iwlwat~~tl I;,r the major spots were determined by comparison with thv sys!cwx&o pat,tcwl 01 migratiotr
I’LAT~: 11. l+wt,her fract,ionation of ,ibo~~)n~c~-l~~otc~ctc~cl DNA l’ragnwnts. 4-X 174 rlt1oso1111.. protc&oll 1)NA fragment,s were prepared as shown in Fig. l(h) amI vst~ractcd ant1 pwificvl aro~w~l~~r~ to Matcrialri and Methods, section (f). (a) 1.2 x 106 cts/min of C&X 174 1)N.i fmgmcnts ww~~ I;r>~-cwl ont,o a 2.cm indentation of a 20 cm X 40 cm 12”/b polyacrylamicl~~ slab gel prepared a~ ~l~wwtx~l 111 Materials ant1 Methods, section (h) and according to Adams et rrl. (1969) ant1 run for 20 1~ at ;I current of 20 mA. In this time the bromphrnol blue dye markw (“B”) migrated 25 cm. 0111~. t lrla 12 cm above the blue marker are shown in the autoradiograph tlepictcvl hew, as thaw xva~ IIO significant radioactivity between this point and the origin (not shown). (b) Autr~ra~liograph <)I’ 41 depurination analysis (Materials and Method. Y, section (i)) carried out on band 2 (‘.2”) from (a), which was eluted from the polyacrylamide gel as outlined in Mat,erials and M&hods, n(sction (h). (c) 1.2 x 10fi cts/min of $X174 ribosome-protected DSA fragments were subjrct~ecl to t\vo-dirnt%Ilsional fractionation by high-voltage electrophorevis at pH 3.5 in 7 wurea on cc~llulox-awt atr* followed by thin-layer homochromatography on DEAE-cellulose using homomis “b” of 13r1~wnl~v~ & Sanger (1969). As in other such autoradiographs, t)he origin hew is again at lhv Iowc~r right. (d) Autoradiograph of a depurination analysis of spot I (“2”) f ram (c) which was vlltitc(l I’r~~ttl tlrt, pwparatiw~ thin-layer platt, as tlnscribrtl in M&vials and Mvthrxls, wrtirw (h).

I’I,ATE 111. (‘haracterization of I)N.i\s by enclonucleas~~ IV (liglastion. I )NA sampl(ks QY>I’C% Ilig~~*i (~1 wit,h rndonucleaae 1V as describotl in Materials and Methotls, wcti
PLATE IV. lsolation and characterization of $X174 DNA fragments protected by ribosornt>s from endonuclease IV digestion. Protected fragments prepared as in Fig. l(d) were purifiedaccortling to Materials and Methods, section (f) and (g). (a) Polyacrylamitle gel analysis of 4X174 1)N.A digested by endonuclease IV in t,he presence of ribosomes. Two reactions were carried out as described in tho text) and fractionated on a lo%, 20 cm x 40 cm polyacrylamitle slab gel pronarcvt according to Robertson et&. (1972) and run 18 hat a current, of 25 mA. In this time the bromphr~mll blue marker dye (“R”) had migrated 24 cm from the origin (“0”). On the left, a 2.cm indentation at the top of the polyacrylamide slab contained DNA digested by endonuclease IV in the prc~sr~nnv of ribosomes and other components of the binding reaction but in the absence of formylmethionyltRNA under the conditions described in Materials and Methods, section (e). This reaction was processed according to Materials and Methods, section (f), and resuspended in 25 ~1 of a solution containing 6% sucrose and bromphenol blue marker dye. On the right, DNA which had sediment,ed at 70 S following the reaction analyzed by sucrose density-gradient analysis depicted in Fig. l(d) was layered onto another 2.cm indentation at the top of the same polyacrylamide slab gel following processing according to Materials and Methods, section (f). (b) Radioautograph of a two-dimensional separation of the depurination products of polyacrylamide gel band 3 of DNA protected by ribosomes from endonuclease IV digestion. The prominent band shown in the right,.hantl sample lane of Plate V(a) in the position marked “3” was eluted and subjcctrd to tlrpnrmat~ion analysis according to Materials and Methods, sections (h) and (i).

PLATE V. Depurination analysis of polyoma virus DNA and DNA fragments following riboxomc protection. (a) 2 x lo5 cts/min of polyoma virus DNA (spec. act. approx. 2 x 10” cts/min 32P/pg) the gift of Dr W. Folk, were subjected to chemical depurination and two-dimensional analysis as described in Materials and Methods, section (i). (b) 1.2 x 10s cts/min of polyoma virus DNA were heat denatured, bound to ribosomes, and subjected to pancreatic DNAase treatment and sucrose density-gradient centrifugation as in Materials and Methods, section (e) and Fig. 2. DNA fragments sedimenting at 70 S were processed according to Materials and Methods, section (f) and subjected to depurination analysis.

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RIBOSOME

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1973a; Barrel1 et al., 1975). A similar conclusion has also been reached with regard to the protected f 1 DNA fragments as illustrated in Plate I(b). Both polyacrylamide gel and thin-layer analysis revealed components migrating in a size range of 40 to 50 bases which had depurination fingerprints containing the same specific subset of the spots shown in Plate I(b) (data not shown). Sequence analysis of this major fl DNA ribosome binding site has not yet been carried out. The third method used here to characterize these populations of DNA fragments was two-dimensional fingerprinting analysis following digestion with endonuclease IV as described in Materials and Methods, section (j). Plate III(a) shows the pattern obtained following digestion of intact +X174 DNA under the conditions described here. By comparison, the pattern observed in Plate III(b) was obtained following endonuclease IV digestion of +X174 DNA fragments protected by ribosomes from pancreatic DNAase digestion. As was the case in the depurination analyses shown in Plate I, the protected fragments give a much simpler pattern containing only a few prominent spots. Plate III(c) shows a fingerprint obtained following endonuclease IV digestion of the prominent band (band 2) eluted from an acrylamide gel of +X174 protected DNA fragments such as that shown in Plate II(a). This very simple pattern also suggests that the major sequence in these preparations is a very short, specific one containing the major spots from the pattern in Plate III(b). As described elsewhere, digestions such as this provided one of the means by which the sequence of this+X174DNA fragment was elucidated(Robertson et al., 1973aJ ; Barrel1 etal., 1975). (b) Isolation and preliminary characterization of longer $X174 DNA fragments protected by ribosomes from e&on&ease IV digestion It seemed likely that a longer specific fragment might be obtained by using endonuclease IV in place of pancreatic DNAase in the ribosome binding site isolation. Figure 1(d) illustrates the sucrose density-gradient centrifugation profile obtained when such a procedure is carried out. A prominent peak is observed at 78 S which comprises about l-5 to 2% (versus less than 0.5% with pancreatic DNAase) of the total DNA added to the reaction. Furthermore, as expected with endonuclease IV digests (Ling, 1971), the unprotected DNA now sediments detectibly under these conditions (fractions 26 to 32 of Fig. l(d)) in contrast to the pattern of digested DNA shown at the tops of the gradients depicted in Figure l(a) to (c). Plate IV(a) shows polyacrylamide gel analysis of endonuclease IV digests of $X174 DNA in the presence of ribosomes. On the left is the pattern of a control reaction in which ribosomes were added to 3aP-labeled #X174 DNA at the same time as endonuclease IV, in a standard binding reaction as described in Materials and Methods, section (e) but in the absence of formylmethionyl-tRNA. It is evident that a variety of bands are observed throughout the gel with the majority migrating in the size range of 20 to 100 bases in length in agreement with previous reports (Ling, 1971; Ziff et al., 1973). In contrast, when DNA from the 70 S peak of the gradient illustrated in Figure l(d) was elec6rophoresed in the same gel (as shown in the right-hand lane of Plate IV(a)) several prominent bands were observed migrating in a size class of between 120 and 200 bases in length. In particular, the band from the right-hand sample lane at the position indicated by “3” was eluted and subjected to depurination and fingerprinting analysis as shown in Plate IV(b). Comparison with Plate II(b) shows a similar but not identical pattern. Subsequent characterization of these spots following elution and treatment with alkaline phosphatase showed that band 3

372

H.

D. ROBERTSON

contained all of the prominent spots shown in Plate II(b) and at least five new ones containing at least 31 additional pyrimidines. The new tracts identified have the compositions PA, (‘L), P’S), V-&J, CLCd, and (T,,C,). Coelectrophoresis of band 3 on polyacrylamide gels with RNAs of known size, including the tyrosine tRNA precursor of E. coli (129 nucleotides) (Robertson et al., 1972) and E. coli 5 S RNA suggests that band 3 is about 150 nu&otides in length.

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Fru. 2. Sucrose density-gradient analysis of ribosome binding reaction8 involving single- and double-stranded DNA. Ribosome binding ma&ions were oarried out in a volume of 60 ~1 as described in Materials and Methods, eeotion (e). -O-O--, Sedimentation profile of 9X174 eingkstranded DNA (10s &s/mm, spew. act. 2.4~ IO6 cts/min per pg) bound to 2 Aaeo unit8 of DNAase-free ribosomes prepared as in Materials and Methods, se&ion (d). Reactions were run and analyzed as in Fig. 1 exaept that no DNAase was added following the 12 min incubation at 37°C. -A-A-, Sedimentation analysis of ssP.labeled 9X174 replicative form DNA (2x 10s cts/min, spec. act. in the range of lo8 ats/min per pg) after inoubation in the presence of 2 Asso units of DNAase-free ribosomes as above. -@-a--, Sedimentation profile of 32P +X174 repliaative form DNA (2 x lo6 ots/min) incubated as above after heat denaturation carried out as described in Material8 and Methods, section (e).

(c) Application of ribosmne binding and protection techniques to heat-denatured double-stranded DNAs Figure 2 shows sucrose density-gradient analysis of several ribosome binding reactions carried out as in Figure 1 except that no DNAase was added after the reactions were completed. The profile obtained with +X174 single-stranded DNA is thus an example of what Figure l(b) would look like in the absence of pancreatic DNAase (about 60% of the single-stranded DNA binds to ribosomes and sediments at 70 S in the complete reaction system). Figure 2 also shows that if $X174 replicative form DNA is added in the double-stranded form to a ribosome binding reaction, it sediments at its native position (about 18 S) without any radioactivity detectible in the 70 S region. However, if the $X174 double-stranded replicative form DNA is first heat-denatured according to Materials and Methods, section (e), about one-third of the radioactivity sediments at 70 S. When replicative form DNA containing 32P-label only in the “plus” or viral strand was digested with DNAase and the resulting radioactivity sedimenting at 70 S isolated, it gave a fingerprint upon depurination identical to that illustrated in Plate I(d). Preliminary experiments with such DNAs labeled in both strands suggest that “minus” strands do not bind to ribosomes (H. D. Robertson, unpublished experiments). In any case, the major point illustrated here is that

RIBOSOME

BINDING

SITES

IN

DNA

373

ribosomes are able to bind to the same sites following heat denaturation of doublestranded DNA as they do in the single-stranded DNA from phage particles, regardless of the possible existence of additional sites on the complementary strand. Plate V shows an experiment in which this approach was extended to [32P]DNA isolated from the animal virus, polyoma. Plate V(a) shows the depurination fingerprint of double-stranded, nicked polyoma DNA which was denatured as above. This pattern has a complexity comparable to that observed for bacteriophage DNAs (Plate I(a) and (c)). However, if the procedure described above and in Figure 2 is used to seek ribosome binding sites in polyoma DNA, an amount of DNA is protected comparable to that obtained using denatured #X174 replicative form, and the depurination pattern illustrated in Plate V(b) is obtained. As was the case with fl and $X174, a much simpler pattern is observed, suggesting that this combination of ingredients may be protecting specific regions within the DNA of this animal virus. These specific regions may or may not correspond to ribosome binding sites used in viva by mammalian ribosomes.

4. Discussion (a)

Speci$city of DNA-ribosorne interactions

The results reported here show that ribosomes will bind to sequences within singlestranded DNAs and protect them from pancreatic DNAase digestion (Fig. 1) yielding specific DNA fragments (Plates I to III). A number of the properties of this protection make it analogous to ribosome binding to mRNA (Fig. l(a) to (c)). Furthermore, larger specific fragments related to those protected from pancreatic DNAase digestion can be obtained using endonuclease IV instead (Fig. l(d), Plate IV). In addition, protection of specific sequences within double-stranded DNA molecules can be obtained following heat denaturation, suggesting that specific fragments can be prepared from a wide variety of DNA molecules in this manner (Fig. 2, Plate V). The data illustrated in Plates II and III make it clear that a small number of specific fragments about 50 bases in length are protected by ribosomes from pancreatic DNAase digestion within both fl and $X174 DNAs. The ribosome binding sites of $X174, while similar in base composition to those of fl, show major differences (Plate I(b) and (d)). These differences, together with sequencing analyses described in the accompanying paper (Barrel1 et al., 1975), show that ribosomes select different specific regions from fl and $X174 DNA. Further analyses of the fl DNA fragment and comparison to known sequences off 1 mRNA ribosome binding sites (G. Pieczenik, H. D. Robertson & P. Model, unpublished observations) are being carried out. The results reported here and a number of earlier studies indicate that N-formylmethionyl-tRNA and GTP-dependent ribosome binding to single-stranded DNA takes place in a manner analogous to such binding to mRNA (Bretscher, 1969qb; McCarthy & Holland, 1965; Ihler & Nakada, 1970,1971).However, results reported by Bretscher and by Condit et al. (Bretscher, 1969a,b; Condit et al., 1973) suggest that fMet dipeptides synthesized from a variety of natural single-stranded DNA templates under conditions of protein synthesis initiation constitute a preferred set (fMet-Ile, fMet-Val, fMet-Thr). It is not possible to explain this difference at present, although it is likely that the majority of fMet dipeptide synthesis under these conditions may be occurring at sites which, while capable of binding ribosomes, do not render the DNA resistant to DNAase treatment. A similar difficulty in interpretation

374

H. D. ROBERTSON

has sometimes arisen in analysis of ribosome binding and initiation with mKI\;X;; (St,eitz, 1.969b; Webster et al., 1969; Steitz et al., 1970; Goldman & Lodish, 1972). It is important ribosomes

to keep in mind that both the formation of nuclease-resistant complexes I,y acting together with a single tRNA species (tRNA,,,,) (Kondo et al., 1968) and the formation of fMet dipeptides in the a’bsence of protein synthesis (Lodish, 1969), are somewhat artificial processes, neither of which occur in viz’o.

(b) Potential applications of this approach to other systems The data in Figure 2 and Plate V suggest that specific DNA fragments could be obtained from a variety of DNA molecules using this approach. The isolation of a specific fragment of DNA from lambda transducing phage carrying the E. coli lac region has recently been described (Gilbert & Maxam, 1973). Indirect sequence analysis of the region surrounding this DNA fragment suggests that an RNA polymerase start point, an operator region, and a ribosome binding site are located in close proximity to each other (Maizels, 1973). Heat-denaturation of large DNA molecules containing such regions, followed by ribosome binding and digestion with endonuclease IV might produce a fragment of single-stranded DNA encompassing the various signals of interest, which could be identified by comparing mutant and wild-type

sequences.

Finally, the detailed application of this approach to eukaryotic DNA has awaited the development of ribosome binding systems involving eukaryotic ribosomes. It is now probable that systems which allow protection of eukaryotic mRNA from nuclease digestion have been worked out (Darnbrough et al., 19’73; Jackson & Hunter, 1970), and therefore radioactive, heat-denatured DNA such as that from polyoma virus can be tested for its ability to catalyze formation of specific complexes. Once these have been obtained, protected fragments can be studied both by the application of direct techniques, and also by the application of additional indirect ones employing, for example, chemical iodination of DNA (Commerford, 1971; Robertson et aZ.. 19733) or reverse transcriptase copying (Salser et aE., 1973). Most of this work was carried out under a Helen Hay Whitney postdoctoral fellowship at the Medical Research Council Laboratory of Molecular Biology, Cambridge, England, August 1969 to June 1972. I thank Dr F. H. C. Crick for initial encouragement and continued support of this work, Dr J. D. Smith for use of laboratory facilit,ies and Dr F. Sanger for his generous provision of laboratory facilities and discussion time. The initial stages of this work were carried out in collaboration with Dr V. Ling during his tenure as a postdoctoral fellow of the Medical Research Council of Canada, September 1969 to July 197 1 and I thank him for communication of experimental techniques and a gift of endonuclease IV. I thank B. G. Barrel1 for encouraging utilization of this approach in sequence analysis, Dr M. S. Bretscher for advice, and Drs P. Sadowski, E. Ziff, J. Sedat, D. Ish-Horowitz and W. Folk for helpful discussions and generous gifts of materials. REFERENCES Adams, J. M., Jeppesen, P. G. N., Sanger, F. & Barre& B. G. (1969). Nature (London), 228, 1009-1014. Barrel& B. G. (1971). In Proceduw.s in Nucleic Acid Research (Cantoni, G. L. & Davies, D. R., eds), vol. 2, pp. 751-779, Harper & Row, New York. Barrell, B. G., Weith, H. L., Donelson, J. E. & Robertson, H. D. (1975). J. Mol. Biol. 92, 377-393. Bernardi, A. & Spahr, P-F. (1972). Proc. Nat. Acad. Sci., U.S.A. 69, 3033-3037. Blattner, F. R. & Dahlberg, J. E. (1972). Nature New Biol. 237, 227-232.

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Bretscher, M. S. (1969a). J. MOE. BioZ. 42, 595-698. Bretscher, M. S. (19693). Cold Spring Harbor Symp. Quant. Biol. 34, 651-663. Brownlee, G. G. & Ssnger, F. (1969). Eur. J. Biochem. 13, 395-399. Clark, B. F. C. & Marcker, K. A. (1966). J. Mol. BioZ. 17, 394-406. Commerford, S. L. (1971). Bioohemiatry, 10, 1993-2000. Co&it, R. C., Goldberg, M. L. & Steitz, J. A. (1973). J. Mol. Biol. 75, 449-454. Darnbrough, C., Legon, S., Hunt, T. & Jackson, R. J. (1973). J. Mol. Bill. 76, 379-403. Gilbert, W. & Maxsm, A. (1973). Proc. Nat. Ad. Sci., U.S.A. 70, 3581-3584. Goldman, E. & Lodish, H. F. (1972). J. Mol. BioZ. 67, 35-47. Gschwender, H. H., Haller, W. & Hofschneider, P. H. (1969). Biochim. Biophys. Actu, 190, 460-469.

Hayashi, M., Hayashi, M. N. & Spiegelman, S. (1963). Proc. Nat. Acad. Sk., U.S.A. 50, 664-672. Hindley, J. & Staples, D. H. (1969). Nature (London), 224, 964967. Ihler, G. & Nakada, D. (1970). Nature (London), 228, 239-242. Ihler, G. & Nakada, D. (1971). J. Mol. BioZ. 62, 419-421. Jackson, R. J. & Hunter, A. R. (1970). Nature (London), 227, 672-676. Kondo, M., Eggerston, G., Eisenstadt, J. & Lengyel, P. (1968). Nature (London), 220, 368-371 Ling, V. (1971). FEBS Letters, 19, 50-58. Ling, V. (1972a). J. MoZ. Bid. 64, 87-102. Ling, V. (1972b). Proc. Nat. Acud. Sci., U.S.A. 69, 742-746. Lodish, H. F. (1969). Biochem. Biophye. Rea. Commun. 37, 127-136. Maizels, N. (1973). Proc. Nat. Acad. Sci., U.S.A. 70, 3585-3689. Marvin, D. A. & Hohn, B. (1969). BacterioZ. Rev. 33, 172-209. McCarthy, B. J. & Holland, J. J. (1965). Proc. Nat. Acad. Sci., U.S.A. 54, 880-886. Nichols, J. L. (1970). Nature (London), 225, 147-151. Nichols, J. L. & Robertson, H. D. (1971). Biochim. Biophys. Actu, 228, 676-681. Nirenberg, M. & Matthaei, J. (1961). Proc. Nat. Ad. Sci., U.S.A. 47, 1588-1602. Robertson, H. D., Altmsn, S. & Smith, J. D. (1972). J. BioZ. Chem. 247, 62435251. Robertson, H. D., Barrel& B. G., Weith, H. L. & Donelson, J. E. (1973a). Nature New B&Z. 241, 38-40.

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Sadowski, P. & Hurwitz, J. (1969). J. BioZ. Chem. 244, 6192-6198. Sslser, W., Fry, K., Brunk, C. & Poon, R. (1972). Proc. Nat. Acud. Sci., U.S.A. 69,238-242. S&or, W., Poon, R., Witcome, P. & Fry, K. (1973). In l’im Research (Proceedings of the 2nd ICN-UCLA Symposium on Molecular Biology) (Fox, C. F. & Robinson, W. S. eds), pp. 545-572, Academic Press, New York & London. Sanger, F., Donelson, J. E., Conlson, A. R., Kbssel, H. & Fischer, D. (1973). Proc. Nat. Acad. Sk., U.S.A. 70, 1209-1213. Sedat, J. L%Sinsheimer, R. L. (1964). J. Mol. Biol. 9, 489-497. Steitz, J. A. (1969a). Nature (London), 224, 957-964. Steitz, J. A. (1969b). Cold Spring Harbor Symp. Quant. Biol. 34, 621-633. Steitz, J. A., Dube, S. K. & R&land, P. S. (1970). Nature (London), 226, 824-827. Webster, R. E., Robertson, H. D. & Zinder, N. D. (1969). CokZ Spring Harbor Symp. Quunt. Biol.

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Wu, R. & Taylor, E. (1971). J. Mol. BioZ. 57, 491-511. Ziff, E., Sedat, J. & Galibert, F. (1973). Nature New Bill.

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