13 Nucleic Acids By R. T. WALKER Department of Chemistry Birmingham University Birmingham B 15 2TT THEPAST YEAR has seen a continuing increase in the number of publications dealing with advances in our knowledge of nucleic acids. Several new journals have appeared but have yet to establish themselves except for one-the New Biology edition of Nature-which from the first issue contained and has con- tinued to attract papers dealing particularly with the field covered by the term ‘Molecular Biology’ from leading laboratories all over the world. In an attempt to try to resolve the growing problem of ‘keeping up with the literature’ of such a wide-ranging subject an abstracting journal has started publication. This contains a classified list of the majority of papers in the field together with an abstract as soon as possible after the appearance of the paper in the primary journal.A yearly author and subject index will also be issued and it can now be seen that some 7000 papers a year are being published in this field. Several books have made a welcome appearance this year particularly those dealing with chemical aspects. Among these are books on purines,2 and a second volume in the series of ‘Synthetic Procedures in Nucleic Acid Chemi~try’.~ A translation (edited by Lord Todd and Brown) of a book first published in Russian entitled ‘The Organic Chemistry of Nucleic Acids’ is about to appear4 and another by Hall’ deals with the modified nucleosides. There are also books dealing with physical constants,’ ~tructure,~ and biological effects’ of poly-nucleo tides.’ ‘Nucleic Acids Abstracts,’ ed. E. S. Krudy and A. Williamson Information Retrieval Ltd. London 1971. * J. H. Lister ‘Pnrines,’ Wiley London 1971. ’ ‘Synthetic Procedures in Nucleic Acid Chemistry,’ ed. W. W. Zorbach and R. S. Tipson Wiley London 1972 vol. 2. ‘Organic Chemistry of Nucleic Acids,’ ed. N. K. Kochetkov and E. I. Budovskii (translated by B. Haigh ed. Lord Todd and D. M. Brown) Plenum London 1971. R. H. Hall ‘The Modified Nucleosides in Nucleic Acids’ Columbia University Press London 197 1. B. Janik ‘Physicochemical Characteristics of Oligonucleotides and Polynucleotides,’ Plenum London 1971. ’ ‘Fine Structure of Proteins and Nucleic Acids,’ ed. G. D. Fasman and S. N. Timasheff Dekker New York 1970.‘Biological Effects of Polynucleotides,’ ed. R. F. Beers and W. Braun Springer- Verlag New York 1971. 419 420 R. T. Walker Many other books and over one hundred review articles have been published,' so that it is impossible to mention them all. This review it is hoped covers the areas where really significant advances have been made. Much of the work of greatest significancecontinues to come from the molecular biologist. Crick has started to unravel the eukaryotic chromosome" and has given us another of his now familiar triangles (Figure l) which links the highly specific interactions between proteins and paired and unpaired nucleic acids thus making no distinction between DNA' and RNA. Protein \\ UnDaired / \ Paired Nucleic Acid 4 - - - - - - - +Nucleic Acid (3 Figure 1 A claim to have transduced human cells by transducing derivatives of phage lambda has been made,'* and if substantiated will raise many moral issues as to how this line of research might be developed.A precursor tyrosine tRNA molecule which contains few if any modified bases has been sequenced13 and can be cleaved to give a 4Smolecule by an Escherichia coli crude extract. Contrary to previous e~idence,'~ the -CCA terminus is coded for in the tRNA gene. Khorana continues to synthesize genes and has announced progress in the synthesis of the gene for this same tyrosine tRNA;I5*l6presumably the gene for the precursor molecule will be the next goal. At last the synthesis of poly-ribonucleotides seems to be possible,' with such an elegant method that the use of a polymer support and the accompanying automated methods is in sight.Reverse transcriptase-the unfortunate name given to the RNA-directed DNA polymerase from the oncogenic RNA viruses-has continued to receive much attention," and no doubt with the Americans planning to spend $1600 million in three years to cure cancer much more work on the same lines can be See index of reviews in ref. 1. lo F. H. C. Crick Nature 1971 234 25. Abbreviations used are in accord with the recommendation of the IUPAC-IUA Commission J. Biol. Chem. 1970 245 5171. C. R. Merril M. R. Geier and J. C. Petricciani Nature 1971 233 398. l3 S. Altman and J. D. Smith Nature New Biol.1971 233 35. l4 V. Daniel S. Serid and U. Z. Littauer Science 1970 167 1682. H. G. Khorana I.U.P.A.C.Journal 1971,25,91. l6 P. Besmer K. Agarwal M. H. Caruthers P. J. Cashion M. Fridkin E. Jay A. Kumar P. C. Loewen E. Ohtsuka J. H. van de Sande N. Siderova and U. L. RajBhandary Fed. Proc. 1971,30 1314. J. K. Mackey and P. T. Gilham Nature 1971 233 551. R. C. Gallo Nature 1971 234 194. Nucleic Acids 421 expected. Whether the outcome will be successful remains a matter of opinion but the American politicians in this pre-election year obviously do not agree with Sir MacFarlane Burnet who was quoted as saying that he thought all the brilliant work in molecular biology had made no contribution of any significance to medicine and he was even convinced that it never would.1 Bases Nucleosides and Nucleotides A spray reagent for adenine compounds has been described the result of which sounds like real chromatography”-pink spots on a gold background. Nucleo- sides characterized by a high positive charge such as 7-methylguanosine and/or with strong hydrophobic substituents such as 2-methylthio-N6-isopentenyl-adenosine have been rapidly eluted from cation-exchange columns by incor- porating ethanol in the solvent.20 Bernardi2’ has further developed his poly- acrylamide gel columns so that the eight nucleosides obtained from a digest of RNA and DNA can be separated in five hours. Bio-Gel P2 (<400mesh) which has been fractionated to give particles of uniform (-10-20 pm dia.) size is used. The advantages of the method are speed reusability of the columns and quanti- tative recovery of the nucleosides each of which is eluted in a very small volume which gives a clean spectrum.Analysis of DNA can be achieved on only 0.5 pg DNA. The method has again been refined22 to enable computerized quantitative evaluation of each nucleoside at a 1-10nmol level. Other nucleic acid com- ponents have been separated on Sephadex gels,23 and an investigation of the mechanism of the adsorption of purines onto Sephadex G-10 has been made.24 Purines,” pyrimidines,26 and their derivatives have been separated by g.1.c. and nucleosides nucleotides and dinucleotides have been estimated by pyrolysis gas ~hromatography.~’ Perhaps the time has come to assess the reaction of nucleic acid bases with diethylpyrocarbonate (DEP; ethoxyformic anhydride) which is being widely used as an inhibitor of ribonuclease in the isolation of RNA.As was reported last year,28 Leonard29 showed that DEP reacts with adenine to give 5(4)-N- ethoxycarbonylaminoimidazole-4(5)-N’-ethoxycarbonylcarboxamidine(1). In a recent paper,30 Leonard has shown that 9-propyladenine and adenosine have the imidazole ring opened. Treatment of the adenosine reaction products with l9 M. E. Wright and D. G. Satchell J. Chromatog. 1971 55 413. 2” M. Uziel and C. Koh J. Chromatog. 1971,59 188. 21 G. Piperno and G. Bernardi Biochim. Biophys. Acta 1971 238 388. 22 S. D. Ehrlich J.-P. Thiery and G. Bernardi Biochim. Biophys. Acta 1971 246 161. 23 I. Reifer K.Strzaka and E. Machowicz Bull. Acad. polon. Sci. Ser. Sci. Biol. 1971 19 1. 24 L. Sweetman and W. L. Nylan J. Chromatog. 1971,59 349. 25 C. W. Gehrke and D. B. Lakings J. Chromatog. 1971 61 45. 26 V. Pacakova V. Miller and I. J. Cernohorsky Analyr. Biochem. 1971,42 549. ’’ L. P. Turner and W. R. Barr J. Chromatog. Sci 1971,9 176. 28 G. M. Blackburn Ann. Reports (B) 1970 67 489. 29 N. J. Leonard J. J. McDonald and M. E. Reichmann Proc. Nut. Acad. Sci. U.S.A. 1970 67 93. 30 N. J. Leonard J. J. McDonald R. E. L. Henderson and M. E. Reichmann Bio-chemistry 1971 10 3335. 422 R.T. Walker NHR I Et0,CN Ftn-f"' Ribosyl (2; R = H) (3; R = C0,Et) ethanolic ammonia gave (2) and (3). Adenosine has a half-life of 10min under the conditions used for RNase inhibition (dilute solution pH 7,23 "C).Tobacco mosaic virus (TMV) RNA infectivity was completely destroyed by DEP at 23 "C. Spectroscopic evidence only was obtained for the reaction of DEP with AMP GMP UMP but probably not with CMP;31reaction of radioactive DEP with ribosomal preparations showed that the reaction with nucleic acids was dependent upon the degree of secondary structure and in any case reaction proceeded preferentially with the protein.31 The template activity of TMV RNA,32 and the acceptor activity of several ~RNAs,~~ has been claimed to be normal when DEP has been used in the isolation procedure but the activity of other tRNAs has been affected together with a change in their chromatographic mobility,33 and this change can be obtained by incubating the isolated active tRNAs with DEP.Another report34 states that the presence of RNA interferes with the inhibition of ribonuclease by DEP and it would appear that a fine balance between the reaction of DEP with protein and nucleic acid bases exists which is dependent upon too many variables for the reagent to be used with any guarantee of success. A purine base which has caused the chemist many problems since it was first discovered in tRNA is the base 'Y'. This base occurs adjacent to the 3'-end of the anticodon in tRNAPhe in yeast,35 wheat germ,36 and rat liver37 but the structure was not determined until last year.38 This structure (4) has now been independently confirmed39 with the exception that the proton previously assigned to N-7 is preferred on the oxygen at C-6.The base has now been found in several other tRNAPhe samples4' but it is very labile and easily modified,41 and evidence 31 F. Solymosy P. Hiivos A. Gulyas I. Kapovits 0.Gaal G. Bagi and G. L. Farkas Biochim. Biophys. Acta 1971 238 406. 32 M. Denic L. Ehrenberg I. Fedorcsak and F. Solymosy Acta Chem. Scand. 1970 24 3753. 33 B. J. Ortwerth Biochim. Biophys. Acta 1971 246 344. 34 D. G. Humm J. H. Humm and L. I. Shoe Biochim. Biophys. Acta 1971,246,458. 35 U. L. RajBhandary R. D. Faulkner and A. Stuart J. Bid. Chem. 1968 243 575. 36 G. Katz and B. S. Dudock J. Bid. Chem. 1969 244 3062. 37 L. M. Fink T. Goto F. Frankel and I. B. Weinstein Biochem. Biophys. Res. Comm. 1968 32 963 38 K.Nakanishi N. Furutachi M. Funamizu D. Grunberger and I. B. Weinstein J. Amer. Chem. Sac. 1970 92 7617. 39 R. Thiebe H. G. Zachau L. Baczynskyj K. Biemann and J. Sonnenbichler Biochim. Biophys. Acta 1971 240 163. 40 L. M. Fink K. W. Lanks T. Goto and I. B. Weinstein Biochemistry 1971 10 1873. 41 D. Yoshikami and E. B. Keller Biochemistry 1971 10 2969. Nucleic A cids 423 has been obtained that 'Y' in yeast tRNA is different to the 'Y' in beef and wheat germ tRNA.41 The structure of another 'Y'obtained from Torulopsis utilis tRNAPhe is reported to have structure (5)."* The chiral centre in (4)is L."~ A base isolated from rat beef calf and chicken liver tRNAPhe has been named 'peroxy Y' (6)."" The base in rat-liver tRNAPhe appears to be a mixture of 'Y' and 'peroxy Y'.The biosynthetic route and the purpose of this modification remain to be discovered. Me (4;X = H) (5;R = X = H) (6;X = 02H) Another series of purine derivatives which has received much attention during the year is the cytokinins. These again sometimes occur in tRNA adjacent to the 3'-end of the anticodon,"' and the tRNA isolated from tobacco callus grown in the presence of the synthetic cytokinin N6-benzylaminopurine contained small amounts of this unnatural ~ytokinin."~ A stereospecific synthesis of cis-eati in^^ and ribosyl-~is-zeatin,"~ has been described. Silica t.1.c. in chloroform- methanol easily separates the cis- and trans-isomers of the ribosides. The ribosyl- zeatin from tobacco callus and wheat germ tRNA is the cis-isomer whereas that from pea epicotyls is a mixture of cis-and trans-zeatin ribosides."* 3-Methyl-7-(3-methylbutylamino)pyrazalo[4,3-d]pyrimidine(7) has been syn- thesi~ed~~ and found to be a cytokinin inhibitor when used in 10@-200-fold excess by competing with the cytokinin for receptor sites." A series of N6-42 H.Kasai M. Goto S.Takemura T. Goto and S. Matsura Tecrahedron Letters 1971 2725. 43 M. Funamizu A. Terahara A. M. Feinberg and K. Nakanishi J. Amer. Chem. Soc. 197 1,93,6706. 44 K. Nakanishi S. Blobstein M. Funamizu N. Furatachi G. van Lear D. Grunberger, K. W. Lanks and I. B. Weinstein Nature New Biol. 1971 234 107. 4s Y.Yamada S. Nishimura and H. Ishikura Biochim. Biophys. Acta 1971,247 170. 46 W.J. Burrows F. Skoog and N. J. Leonard Biochemistry 1971,10,2189. 47 N. J. Leonard A. J. Playtis F. Skoog and R. Y.Schmitz J. Amer. Chem. SOC., 1971 93 3056. 48 A. J. Playtis and N. J. Leonard Biochem. Biophys. Res. Comm. 1971 45 I. 49 S. M. Hecht R. M. Bock R. Y.Schmitz F. Skoog N. J. Leonard and J. L. Occolowitz Biochemistry 1971 10 4224. so S. M. Hecht R. M. Bock R. Y.Schmitz F. Skoog and N. J. Leonard Proc. Nut. Acad. Sci. U.S.A.,1971 68 2608. 424 R.T. Walker purinyl amino-acids5' and urei~lopurines~~~~~ have been synthesized and their cytokinin activities measured. Reaction of the appropriate isocyanate with a 9-protected adenine followed by removal of the protecting group gave a series of N6-alkyl- or aryl-ureidopurines (8).52 l-Ethoxyethyl was used for N-9 protection owing to its ease of addition and removal and the distinctive contribution it makes to the n.m.r.spectrum. Eight N6-ureidopurines synthesized had lower cytokinin activities than did N6-benzylaminopurine but N6-phenylureidopurine (9) had appreciable activity. Chheda' has synthesized naturally occurring N6-ureidopurines and their nucleosides. Reaction of adenine and ethyl chloro- formate in pyridine in a bomb at 115 "C gave a 52 % yield of N6-ethoxycarbonyl- adenine. Displacement of the ethoxy-group of the urethane or urethane riboside with L-threonine and glycine gave the naturally occurring (10) and (11) and their ribosides. The threonine in the natural compound was confirmed to have the L-configuration.i-0 ) ii NH RNH-C-NH NkN kN Me (7) (8) (9; R = Ph) (10; R = HO,CCH.CHOH.Me) (1 1 ; R = -CH2COZH) Several methods for the incorporation of radioactivity into nucleic acid derivatives have been suggested. Uracil can be quantitatively tritiated to give [5-3H]uracil by electrolytic reduction of 5-bromouracil in tritiated water.54 C-Deuteriated (or tritiated) pyrimidines (in particular [5- and/or 6-3H]uracil) can be made from tritiated water via a 1,3-thiazine (12) which can be rearranged to the corresponding uracil (13).55 Cytidine and cytidine 5'-phosphate when treated with 1M ammonium bisulphite at pH 7.5 and 37 "C for 25 h in tritiated water give a slow incorporation (10%) of tritium via the 5,6-dihydrocytidine-6- sulphonate (14).56 No deamination takes place and the conditions are mild enough for the reaction to be considered for labelling cytosine in polynucleotides particularly as phosphodiester bonds are not cleaved." D. S. Letham and H. Young Phytochemistry 1971 10 23. 52 J. J. McDonald N. J. Leonard R. Y. Schmitz and F. Skoog Phytochemisrry 1971,10 1429. 53 G. B. Chheda and C. I. Hong J. Medicin. Chrm. 1971 14 748. 54 C. Bratu J. Lubelled Compounds 1971 7,161. 55 J. F. B. Mercer and R. N. Warrener Chem. and Znd. 1970 927. 56 K. Kai Y.Wataya and H. Hayatsu J. Amer. Chem. SOC.,1971 93 2089. Nucleic Acids 425 0 0 NH [14C]Methyl iodide has been used in the preparation of ['4C]methyl-thymine57 and -th~midine~~ from the corresponding 5-bromouracil derivative.['"C]-Labelled adenosine guanosine and inosine phosphates can be isolated from human erythrocyte preparations incubated with the corresponding labelled purine bases.59 High incorporation (>80 % for ATP incorporation) is possible. Another more doubtful method for the in vitro labelling of polynucleotides by treatment with NaB[3H] in the presence of u.v. irradiation has been suggested.60 Specific activities of up to 8700c.p.m. per pg DNA have been obtained but degradation and chemical changes in the molecule must be taking place. It is suggested that the DNA would be suitable for physical and hybridization studies. Friedel-Crafts catalysts have been used in the preparation of guanine nucleo- sides." N '-Nonanoylguanine when heated under reflux with a fully acetylated sugar in chlorobenzene in the presence of aluminium chloride for 2 h gave a mixture of the N-7-and N-9-substituted nucleosides in high yield.2'-Deoxy- uridine (1 7) has been prepared6' by the catalytic hydrogenation and debenzoyla- tion of 3'-0-benzoyl-2',5-dibromo-2'-deoxyuridine (16) which can be prepared by the reaction of 2',3'-O-benzylideneuridine (15) and N-bromosuccinimide (2.2 molar equivalents) in a mixture of 1,1,2,2-tetrachloroethaneand carbon tetrachloride. 0 0 0 Ph H OX0 57 L. Pichat J. Deschamps B. Masse and P. Dufay Bull. SOC.chim. France 1971 6 2110. 58 L. Pichat B. Masse J. Deschamps and P. Dufay Bull. SOC.chim. France 1971,6,2102. 59 B. S. Vanderheiden Anu/.vt. Biochem. 1971 40 331.6o V. F. Lee and M. P. Gordon Biochem. Biophys. Acta 1971 238 174. '' W. W. Lee A. P. Martinez and L. Goldman J. Org. Chem. 1971 36 842. 62 M. M. Ponpipom and S. Hanessian Curbnhydrate Res. 1971 17 248. 426 R.T. Walker Deoxyuridine crystallizes to give two independent molecules in the asymmetric unit. The nucleosides are in the anti-c~nfiguration.~~ Dihydrouridine also crystallizes with two independent molecules in the asymmetric unit both in the ~nti-configuration,~~ and it is suggested that the nucleoside can promote loop formation in a sugar phosphate chain.65 The first purine nucleoside analogues containing a bridgehead nitrogen atom (18) and (19),have bzen synthesized by conventional methods.66 One of these (1 8) may be regarded as an inosine analogue.0 0 Ribosyl Ribosyl (18) (19) 6-Selenoguanosine has been prepared by the nucleophilic displacement of the chlorine atom from 2-amino-6-chloro-9-(~-~-ribofuranosyl)purine with either selenourea or sodium hydrogen ~elenide.~ 5-Hydroxyuracils [and their nucleosides (20)]have been converted into 6-alkyluracils via a Claisen rearrange- ment.68 Allylation of 5-hydroxyuridine gives the 5-allyloxy-compound (2 I) which rearranges in 10min at 120°C to the 6-ally1 derivative (22). I I I Ribosy 1 Ribosy1 Ribosyl Diazotization of 2‘,3’,5‘-tri-O-acetyI-8-aminoadenosine with sodium nitrite in 40 % HBF at -20 “C gives 8-fl~oroadenosine.~~ Syntheses of 3’-fluoro- and 3’-chloro-3’-deoxythymidine,70 9-(5‘-amino-5’-deoxy-fi-~-arabinofuranosyl)ade- Z’-amin0-2’-deoxycytidine,~~ nine,71 2’-amin0-2’-deoxyuridine,~~ and a new approach to the synthesis of 3’-amino-3’-deoxynucleosides from a glucopyrano- 63 A.Rahman and H. R. Wilson Nature 1971 232 333. h4 D. Suck and W. Saenger F.E.B.S. Letters 1971 12 257. 65 M. Sundaralingam S. T. Rao and J. Abola Science 1971 172 725. 66 M. W. Winkley G. F. Judd and R. K. Robins J. Heterocyclic Chem. 1971 8 237. ‘’ G. H. Milne and L. B. Townsend J. Heterocyclic Chem. 1971 8 379. 68 B. A. Otter A. Taube and J. J. Fox J. Org. Chem. 1971.36 1251. 69 M. Ikehara and S. Yamada Chem. and Pharm. Bull. (Japan) 1971 19 104. ’O G. Etzold R. Hintsche G. Kowollik and P. Langen Tetrahedron 1971 27 2463. ” M. G. Stout and R. K. Robins J. Heterocyclic Chem.1971,8 515. 72 J. P. H. Verheyden D. Wagner and J. G. Moffatt J. Org. Chem. 1971,36 250. Nucleic Acids 427 side7j have been reported. The previously reported synthesis of pse~douridine~~ has been investigated the intermediates have been isolated and the stereo- chemistry of the reaction has been ~larified.~' Conditions have been found which favour the formation of the 8-isomer. The reaction of chloroacetaldehyde with cytosine,76 or the reaction of chloro-acetic acid with cytosine or cytidine followed by ring closure under basic con- dition~,~ yields [1,2-~]pyrimidines. Chloroacetaldehyde also reacts with adenine to give an imidaz0[2,l-i]purine.~~ The N4-acyl group of a tetra-acylated cytidine can be selectively removed under acidic conditions7 and the N4-amino-group can be selectively acylated with an excess of carboxylic acid in dimethylformamide in the presence of N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquin0~ine.~~ The mechanism of the mutagenic action of hydroxylamine with cytosine continues to receive attention.79 It is now realised that adenine also reacts with hydroxylamine80*8 although at 0.005 of the rate of the corresponding reaction with cytidine.Adenosine reacts to give N6-hydroxyadenosine which isomerizes to adenosine-1-oxide," which may also be a primary product of the reaction. A mechanism is postulated and it is emphasized that although the rate of reaction is slow these reactions could occur in uivo and it is interesting to note that whereas hydroxylaminopurines are mutagenic adenine-1 -oxide is carcinogenic.Some purine 1-and 3-N-oxides have also been prepared by oxidation of purine and 6-methylp~rine.'~ In a study of the effect of methylamine on the rate of inorganic phosphate release by periodate-oxidized adenosine 5'-phosphate the optimum conditions of dependence upon the pH solvent and amine concentration have been found.83 Uracil thymine and some derivatives have been oxidized by hydrogen peroxide.84 Near neutrality free-radical pathways are the most important but under alkaline conditions the predominant reaction is that of the anion of the hydroperoxide with the neutral substrate. Thymidine thymidylic acid,*' and the thymine residues in DNA86have been oxidized by permanganate under mild conditions.The former compounds give predominantly the corresponding thymidine glycol at pH 8.6 and the 5-hydroxy-5-methylbarbituricacid deoxy- riboside at pH 4.3. The permanganate oxidation of cytosine and cytidine gives 73 H. H. Baer and M. Bayer Canad. J. Chem. 1971,49 568. 74 D. M. Brown M. G. Burdon and R. P. Matcher J. Chem. SOC. (C),1968 1051. 75 U. Lerch M. G. Burdon and J. G. Moffatt J. Org. Chem. 1971,36 1507. 7h N. K. Kochetkov V. N. Shibaev and A. A. Kost Tetrahedron Letters 1971 1993. '7 R. S. Goody and R. T. Walker J. Org. Chem. 1971,36,727. 78 J. F. B. Mercer and R. H. Symons Biochim. Biophys. Acta 1971,238,27. 79 E. I. Budowsky E. D. Sverdlov R. P. Shibaeva G. S. Monastyrskaya and N. K. Kochetkov Biochim. Biophys. Acra 1971 246 300. no E.I. Budowsky E. D. Sverdlov and G.S. Monastyrskaya Biochim. Biophys. Acta 1971 '' 246 320. D. M. Brown and M. R. Osborne Biochim. Biophys. Acta 1971,247 514. 82 A. Giner-Sorolla C. Gryte M. L. Cox and J. C. Parham J. Org. Chem. 1971 36 1228. 83 A. Steinschneider Biochemistry 1971 10 173. 84 L. R. Subbaraman and E. J. Behrman J. Org. Chem. 1971,36 1256. n5 S. Iida and H. Hayatsu Biochim. Biophys. Acta 1971 228 1. " S. Iida and H. Hayatsu Biochim. Biophys. Acra 1971 240 370. 428 R.T. Walker urea and biuret derivatives but 2’,3’,5’-tri-O-benzoylcytidine gives only urea derivatives as the intermediates formed deaminate more readily than those of the free base or nu~leoside.~’ Cytidine 5’-phosphate cytidine and cytosine are easily converted into the 4-thiouridine derivatives with an isolated yield of 70 % by heating the starting material in a steel container at 60°C for 41 h with liquid hydrogen sulphide solution in pyridine.” The reaction pathways of 4-thiouracil derivatives with nitrous acid,89 bisulphite,” and cyanogen bromide,’ to give the corresponding uracil derivatives have been investigated.The 2‘-0- and 3’-O-methyl nucleosides can be prepared from the nucleoside catalytic amounts of stannous chloride dihydrate and diaz~rnethane.’~ Yields are high and the mono-0-benzyl nucleosides can be prepared from phenyl- diazomethane. The catalyst-solvent system dioxan-acetonitrile-hydrogen chloride converts uridine into 2’,3’-O-ethylideneuridine in 74 % yield and under the same conditions gives 3’,5’-di-O-acetylthymidine in 56 % yield from th~midine.~ In the absence of dioxan uridine gives 5’-O-acetyluridine (46 %) and 2’,3’,5’-tri-O-acetyluridine (23%).Reaction of adenosine with p-nitrobenzal- dehyde ethyl orthoformate and trifluoroacetic acid in dimethylformamide results in two blocking groups being added simultaneously to give N6-(a-ethoxy- ~-nitrobenzyl)-2’,3‘-O-ethoxymethyleneadenosine in 80 % yield.94 Ribonucleo- tides react in the order pG > pA > pU = pC with benzoic or acetic anhydrides in aqueous solution to give selective O-a~ylation.’~ 2,4-Dinitrofluorobenzene reacts with nucleotides in the absence of trialkyl- amines to give a 2,4-dinitrophenyl cster whereas the addition of the base catalyses the reaction of fluoride ion to give the nucleoside phosph~fluoridate.’~ Many physicochemical studies on bases nucleosides and nucleotides have been reported.’ All that can be mentioned here is the growing use of mass spectrometry as a tool for the identification of nu~leotides’~ and a preliminary investigation into the use of chemical ionization mass spectrometry of nucleo- sides,98 where the ion spectrum is formed not by electron bombardment but by low-energy collisions with ions derived from a reagent gas such as methane present in the ion source at relatively high pressure.One advantage over con- ventional techniques appears to be the high relative abundance of the protonated molecular ion. ’’ R. S. Goody A. S. Jones and R. T. Walker Tetrahedron 1971 27 65.** T. Ueda M. Imazawa K. Miura R. Iwata and K. Odajima Tetrahedron Letters 1971 2507. 89 S. Iida and H. Hayatsu Biochem. Biophys. Res. Comm. 1971 43 163. 90 H. Hayatsu and M. Inoue J. Amer. Chem. Soc. 1971 93 2301. ” ’* R. T. Walker Tetrahedron Letters 1971 2145. M. J. Robins and S. R. Naik Biochirn. Biophys. Acta 1971 246 341. 93 J. Zemlicka J. Org. Chem. 1971 36 2383. 94 J. Zemlicka and J. P. Horwitz J. Org. Chem. 1971 36 2809. 95 R. J. Cedergren B. Larue and P. Laporte Canad. J. Biochem. 1971 49 730. y6 P. W. Johnson and M. Smith Chem. Cornm. 1971 379. ’’ A. M. Lawson R. N. Stillwell M. M. Tacker K. Tsuboyama and J. A. McCloskey J. Amer. Chem. Soc. 1971 93 1014. 98 M. S. Wilson I. Dzidic and J. A. McCloskey Biochim. Biophys. Arta 1971 240 623.Nucleic Acids 429 2 Oligonucleotides Polyethyleneimine-impregnated cellulose t.1.c. has been used for the separation of complex mixtures of oligonucleotides obtained during the sequence analysis of RNA and Oligodeoxynucleotides can be sequenced after their limited degradation with snake venom phosphodiesterase into a population of oligonucleotides of successively smaller chain lengths ' ' by labelling these small chains with a [32P]riboadenylic acid residue with terminal deoxynucleotidyl transferase. 'O2 The oligonucleotides are then separated according to chain length and their degradation with spleen phosphodiesterase allows the original sequence of the oligodeoxynucleotide to be determined. Terminal deoxynucleotidyl transferase has also been used in the synthesis of po1ydeoxynucleotides.' O3 A ribonucleoside residue is added by the enzyme to the 3'-terminus of a chemically synthesized hexathymidylate oligomer which is then used as a primer for the acceptance of dAMP residues with the same enzyme.The polymeric product can now be cleaved from the primer by alkaline hydrolysis due to the ribo- nucleotide linkage and the polydeoxynucleotide can be obtained free from primer. Khorana has reported that the chemical synthesis of oligonucleotides which make up the gene for tRNAzh,+ is complete'5 and the enzymatic joining of these is making progress.16 No doubt the synthesis will be modified to make the gene for the precursor molecule,' which 'only' involves modifying the end oligonucleotides to provide overlaps with the precursor and the synthesis of the gene coding for this precursor sequence.The synthesis of oligodeoxynucleotides is now a routine operation (at least in Khorana's laboratory) from where a general method for the preparation in high yield (50%) of all 16 dinucleoside diphosphates (dpXpY) on a large scale has been published.' O4 Intermediate phosphoramidates formed by reaction of a nucleotide with the highly lipophilic aromatic amine p-aminophenyl triphen ylmethane and dicyclohexylcarbodi-imide have enabled the products of the reaction to be isolated by solvent extraction rather than by time-consuming anion-exchange or gel filtration procedures. dpTpT has been prepared by a similar method with NN-dimethyl-p-phenyl- enediamine used to give a phosphoroanilidate which can be selectively absorbed on an ion-exchange column.'O5 Deoxyguanosine-containing deoxyoligonucleo-tides have been prepared on a polymer support using conventional blocking groups and condensation techniques.' 06,' O7 The methodology of oligodeoxy-ribonucleotide synthesis does not yet seem to be at a stage where the insoluble support method offers any advantages. 99 B. E. Griffin F.E.B.S. Lerters 1971 15 165. loo E. M. Southern and A. R. Mitchell Biochem. J. 1971 123 613. R. Roychoudhury D. Fischer and H. Kossel Biochem. Biophys. Res. Comm. 1971,45 430. H. Kossel and R. Roychoudhury European J. Biochem. 1971,22,271. lo' R. Roychoudhury and H. Kossel European J. Biochem. 1971,22,310. '04 K.L. Agarwal A. Yamazaki and H. G. Khorana J. Amer. Chem. Soc. 1971,93,2754. lo' T. Hata K. Tajima and T. Mukaiyama J. Amer. Chem. Soc. 1971 93 4928. 'Oh T. Shimidzu and R. L. Letsinger Bull. Chem. Soc. Japan 1971,44 1673. lo' V. F. Zarutova V. K. Potapov Z. A. Shabarova and D. G. Knorre Dokludy Akad. Nuuk S.S.S.R. 1971 199 1072. 430 R. T. Walker Guanylo-ribonuclease from Actinomyces aureoverticillatus,' O8 ribonuclease T ,Io9 and ribonuclease U,' lo have been used for the small-scale synthesis of oligoribonucleotides. Dinucleotide 2',3'-cyclic phosphates have been prepared in quantitative yield from the corresponding dinucleoside diphosphates in aqueous solution with a water-soluble carbodi-imide.' ' ' These can be converted by the addition of pancreatic ribonuclease and a nucleoside into triribonucleoside diphosphates in 40 yield.' '* Ribonuclease A in solution' '3,1 ' and bound to a polymer support,' ' has been used to synthesize codons containing modified nucleosides in the wobble position" and termination codons;l15 yields are 618%.Gilham '' has synthesized pApApApUpA (from pApApA) in quantitative yield. The method depends upon the enzymatic (polynucleotide phosphorylase) addition of one suitably blocked nucleoside 5'-diphosphate to a growing poly- nucleotide chain such that no further addition is possible until the blocking group is removed. The blocking group needs to be stable under the conditions of the enzymatic addition easily removable without affecting the growing chain and must not alter the nucleoside 5'-diphosphate such that the enzyme can no longer recognize it.The 2'(3')-O-(a-methoxyethyl) group which can be added by direct acid catalysis of the reaction of the 2'(3')-OH group of a nucleoside 5'-diphosphate with methyl vinyl ether has proved to be satisfactory. So far product separation has been effected by ion-exchange chromatography but the method is ideally suited to an automated support procedure as the yields are quantitative and only simple manipulations are required. Apparently we can now look forward to the 'chemical' synthesis of quite long (1&20) oligoribo-nucleotides. Isotactic succinylated polystyrene has been used as a support for the attach- ment of a ribonucleoside via a 5'-ester for the synthesis of triribonucleotides using conventional condensing agents.' ' A hexaribonucleotide albeit a repeating trimer with the same sequence as the 3'-end of yeast tRNAA'" has been synthesized.' ' The trinucleotide was synthesized by conventional means and then polymerized to give the hexanucleotide with a terminal 3'-phosphate.The o-chlorophenyl group has been used to protect the phosphate group in the phosphotriester approach to oligoribonucleotide synthesis. '' The products of the reaction can be quickly separated on silica and a significantly higher yield lo* L. P. Shershneva and T. V. Venkstern Mulekulyarnuyu Bid. 1971,5,480. '09 M. J. Rowe and M. A. Smith Biochim. Biophys. Acta 1971 247 187. lo T. Koike T. Uchida and F. Egami J. Biochem. Japan 1971 69 11 I.'I1 A. P. Kavunenko E. N. Morozova and N. S. Tikhomirova-Sidorova Zhur. obshchei Khim. 1971,41 226. 'I2 A. P. Kavunenko V. Sukharevich and N. S. Tikhomirova-Sidorova Zhur. obshchei Khim. 1971 41 679. 'I3 H. G. Gassen F.E.B.S. Letters 1971 14. 225. E. Ohtsuka H. Tegawa and M. Ikehara Chem. and Pharm. Bull. (Japan) 1971,19 139. 'I5 H. G. Gassen and R. Nolte Biochem. Biophys. Res. Comm. 1971 44 1410. 'l6 K. F. Yip and K. C. Tsou J. Amer. Chem. Soc. 1971,93,3272. 'I7 E. Ohtsuka M. Ubasawa and M. Ikehara J. Amer. Chem. Soc. 1971 93,2296. J. H. van Boom P. M. J. Burgers G. R. Owen C. B. Reese and R. Saffhill Chem. Comm. 1971 869. Nucleic Acids 431 than has previously been claimed (76% for UpUpU) is reported. Necessary intermediates for insertion into oligoribonucleotides in a stepwise synthesis N6-benzoyl-2'-0-tetrahydropyranyladenosine and N4-benzyl-2'-O-tetrahydro-pyranylcytidine and their corresponding 5'-O-p-methoxytrityl derivatives have been synthesized.* Polynucleotide ligase has been shown to catalyse the joining of DNA duplexes at their base-paired ends if a 5'-phosphate and 3'-hydroxy-group are adjacent.I2' The ligase from phage T4 in contrast to the ligase from E. coli catalyses the joining of any combination of ribo- and deoxyribo-oligonucleotides on a ribo- or deoxyribo-nucleotide template except when both strands are ribopoly-nucleotides. 21 Polynucleotide phosphorylase from Micrococcus luteus cata-lyses the synthesis of copolymers several hundred nucleotide residues long containing both AMP and dAMP residues.' 22 The fourth in a series of vinyl analogues of polynucleotides (general formula [-CHX-CH,] where X is N-1-substituted uracil or cytosine or N-9-substi- tuted adenine or hypoxanthine) has been prepared.lz3 These form soluble non-aggregated complexes with the complementary polynucleotide in solution which have broad and incompletely reversible melting profiles and a stoicheio- metry favouring the vinyl component.Electron microscopy has shown that the poly( 1-vinyluracil).poly-A complex is filamentous with the excess of uracil residues (stoicheiometry 9U :1A) looped out from the strand of poly-A which is stiffened by the binding of one or several strands of the neutral polymer. Poly- I.poly( 1-vinylcytosine) shows as high an antiviral activity as poly-1.p0ly-C.'~~ 5'-O-Acryloyl esters of cytidine guanosine and adenosine have been prepared and copolymerized with acrylamide or 6-0-acryloylgalactose to give water- soluble polynucleotide analogues.' 2s Methacryloyl chloride has been used to 0-acylate the four common ribonucleosides,'26 which have been polymerized and used to quantitatively separate several nucleoside mixtures.' 27 The results of the separation are claimed to show that in water nucleosides are paired by base stacking whereas in dimethyl sulphoxide-chloroform base pairing is by hydrogen-bonding.Ribonucleosides after treatment with periodate have been condensed with polyacrylic acid hydrazide to produce polymers containing one base residue per 10 acrylic acid hydrazide residues.The purine-containing poly- mers were retained on a DNA-agar column and exhibited large hypochromic effects when mixed with denatured DNA in Analogues of trinucleo- "'T. Neilson and E. S. Werstiuk Canad. J. Chern. 1971 49 493. 12* V. Sgaramella J. H. van de Sande and H. G. Khorana Proc. Nut. Acad. Sci. U.S.A. 1970 67 1468. ''I G. C. Fareed E. M. Wilt and C. C. Richardson J. Biol. Chem. 1971 246 925. J. Y. Chou and M. F. Singer Biochem. Biophys. Res. Comm. 1971 42 306. J. Pitha P. M. Pitha and E. Stuart Biochemistry 1971 10 4595. J. Pitha and P. M. Pitha Science 1971 172 1146. M. J. Cooper R. S. Goody A. S. Jones J. R. Tittensor and R. T. Walker J. Chem. Soc. (C) 1971 3183. ''' H. Schott and G. Greber Makromol.Chem. 1971 144 333. ''' H. Schott and G. Greber Mukromol. Chenz. 1971 145 11. M. G. Boulton A. S. Jones and R. T. Walker Biochim. Biophys. Acta 1971,246 197. 432 R. T. Walker side diphosphates with the nucleoside units linked by acetate ester (OCH,COO = ~ mand carbonate (OCOO)129b~ ) ~ ~ ~ linkages have been prepared. The former have been shown by n.m.r. and U.V. spectroscopy to have their bases stacked.129" An oligomer (dA cm),A hybridizes with poly-U in solution specifically inhibits the poly-U-stimulated binding of tRNAPhe to ribosomes,' 30 but does not stimu- late the binding of tRNALys. 3 Pairing and Stacking Conformations The conformations in solution as obtained from n.m.r. and c.d. studies of ApA ApU Ap$ ApiPA and iPApA (iPA = isopentenyl) have been compared.The last two are slightly less folded than ApA whereas there is greater base attraction in Apt$ than ApU.' 31 IpA ApI and IpI stack whereas IpU stacks very little.132 Somewhat more surprising is the fact that photochemically reduced UpA in which the uracil ring is reduced to dihydrouracil also shows considerable hypochromicity' 33 which is not affected by heat. If the dihydrouracil is further reduced to give a ureidopropionic acid derivative the hypochromicity is even greater and although the effect can be destroyed by methanol heat has no effect. Similar effects can be obtained with ApU and GpU. The crystal struc- ture of UpA has been obtained.' 34 The asymmetric unit contains two inde- pendent molecules which are hydrogen-bonded together with the purines and pyrimidines bonded to each other.All four bases are in the anti-conformation with all four sugars 3'-endo but in one molecule the sugar residues are oppositely aligned such that a sharp bend can occur in a single strand of RNA without it being necessary to invoke loop formation. A comprehensive examination' 35 of over 70 published solid-state base-stacking patterns in nucleic acid consti- tuents and polynucleotides has been made. Several recurrent stacking patterns were found. The vertical stacking of purines and pyrimidines in polynucleotides is similar to that observed in crystals of nucleic acid constituents with dipole- induced dipole forces largely responsible for the solid-state base packing. ApA forms a triple-stranded structure with poly-U at temperatures below 32 "C.'36 Further investigation of the simultaneous binding of adenosine and guanosine to poly-U previously noted,13' has shown that the binding of guano- sine is a secondary absorption of some kind onto the existing [2 poly(U + A)] com- ~1ex.l~'Poly-A forms both 1 1 and 2 1 complexes with 3-methylxanthine 129 (a)M.D. Edge and A. S. Jones J. Chem. SOC.(C),1971 1933; (6)J. R. Tittensor ihid. 1971 2656. 130 G.J. Cowling A. S. Jones and R. T. Walker Biochim. Biophys. Acta 1971 254 452. 131 M. P. Schweizer R. Thedford and J. Slama Biochim. Biophys. Acra 1971 232 217. 132 C. Formoso and I. Tinoco Biopolymers 1971 10,531. 133 C. Formoso and I. Tinoco Biopolymers 1971 10 1533. 134 N.C. Seeman J. L. Sussman H. M. Berman and S.H. Kim Nature New Biol. 1971 233 90. 135 C. E. Bugg J. M. Thomas M. Sundaralingam and S. T. Rao Biopolymers 1971 10 175. 136 G. P. Kreishman and S. I. Chan Biopolymers 1971 10 159. 137 P. M. Pitha W. M. Huang and P. 0. P. Tso Proc. Nut. Acad. Sci. U.S.A. 1968 61 332. 138 K. S. Schmitz and J. M. Schurr Biopolymers 1971 10 1075. Nucleic A cids 433 in a co-operative process but intermediate species are present during thermal dissociation. '39 Nucleoside binding to single-stranded polynucleotides has been shown to be co-operative and the stoicheiometry in all cases studied was 2 :1 in favour of the polymer even when polymers such as poly-UC and poly-AC were Poly-U adopts a single hairpin structure at -5 "C and on heating branching takes place leading to formation of multi-hairpins which finally melt in a co-operative proce~s.'~' Poly-C and copolymer I,U with differing contents of uridylic acid form double helical structures with 1..-C inter-base pairing and the U residues looped out of the helix.'42 At acid pH values poly-C and poly-G can form a three-stranded poly-G.poly-C-poly-C+ ~tructure.'~~ It is quite clear that guanosine (or inosine) can form Hoogsteen pairs with a shared proton in a triple-stranded structure only if this proton is present to bind the second cytidine strand to guanosine. This confirms earlier findings'44 and throws doubt on work which suggested that protonation of poly-G-poly-C involved a changed base pairing with the proton on N-1 of cytidine bound to the keto-group of g~an0sine.l~~ A warning about the chain length of some commercially available samples of poly-G is given.143 Little has been done to clarify the role of the 2'-OH group in the stability and conformation of polynucleotides in the papers published this year which have if anything further confused the issue. 2'-0-Methyl-containing heteropolymers poly-(Cm U ;-1 :5) and poly-(Am C) directly incorporate the correct amino- acids into protein'46 but the methylated homopolymers do not even in the presence of neomycin. Poly-2'-deoxy-2'-chloro-uridylic and -cytidylic acids (prepared from the corresponding nucleoside 5'-diphosphates and polynucleotide phosphorylase) are not bihelical in structure over a temperature range of 4-5OoC and it is suggested that helix formation requires an oxygen or -OH group in the 2'-position of ribose.147 The uracil-containing polymer however forms a comple- mentary structure with poly-A.Apurinic acid has a remarkably similar con- formation to poly-U and behaves like a random coil with no interaction between the bases. 14' The similarity between the deoxyribopolymer and poly-U has been taken as indicating that the interaction between the 2'-OH group of the ribose and the phosphate group is not a cause of rigidity in ribopolymers. Self- association of oligonucleotides occurs at much shorter chain lengths in the deoxy- than in the ribo-series. Trimers of dA and dT will give base-paired com- plexes whereas oligomers of rU and rA shorter than 7 residues do not a~sociate.'~~ Interactions between oligo rA and oligo dT have also been studied.'50 139 R.J. H. Davies and N. Davidson Biopolyrnrrs 1971 10 21. 14' R. J. H. Davies and N. Davidson Biopolymers 1971 10 1455. 141 J. C. Thrierr M. Doulent and M. Leng J. Mol. Biol. 1971 58 815. 142 H. Akutsu and M. Tsuboi Bull. Chem. Sac. Japan 1971,44 20. IJ3D. Thiele and W. Guschlbauer Biopolymers 1971 10 143. 144 G. Green and H. R. Mahler Biochemistry 1970 9 368. 145 A. M. Michelson and F. Pochon Biochim. Biophys. Acra 1969 174 604. 14' B. E. Dunlap K. H. Friderici and F. Rottman Biochemistry 1971 10 2581. 14' J. Hobbs H. Sternbach and F. Eckstein F.E.B.S. Letters 1971 15 345. lJ8J. Achter and G. Felsenfeld Biopolymers 1971 10 1625.J. C. Maurizot J. Blicharski and J. Brahms Biopolymers 1971 10 1429. 15' J. G. Hoggett and G. Mass Chem. Ber. 1971 75,45. 434 R.T. Walker tRNAPhe (anticodon GAA) and tRNAG1" (anticodon probably UUC) form a complex with a much stronger association constantl'l than is found for the binding of UUC to tRNAPhe which emphasizes the effect that a rigid and accep- table conformation has on the stability of base-base interactions. Studies on the interaction of adenylic acid-uridylic acid block copolymers have given enthalpy changes per base pair AH, that increase from -6.4kcal mo1-l for (Ap),(Up),U to -7.6kcal mol-' for (A~),(Up)~u.l~~ When a G or C is inserted between the blocks the stability of the bimolecular helical complex is reduced.C is the more destabilizing resulting in a reduction of the T of 10-1 7 "C,equivalent to the removal of 2 A-U pairs. The duplex apparently rearranges to accommodate G opposite a U residue. When one oligomer con- tains a C between the blocks and the other a G the stability of the bimolecular helix is enhanced. The replacement of A-U by G-C in a decamer raised the T by 8.5 "C.153 The stability constant of the duplex GpGpGpU :GpCpCpC containing the G-U wobble pair is 9.5 fold smaller than that of the perfectly paired GpGpGpC GpCpCpC.' 54 Results obtained for the stability of double-stranded regions loops and bulges in a single polynucleotide chain have been used to calculate the stability of various structures proposed for 5s RNA and sequences of R17 viral RNA."' The stability values used are (i) A-U pairs + 1 ; (ii) G-C pairs +2; (iii) G -U pairs 0; (iv) hairpin loops -5 to -7 ;(v) interior loops -4to -7;(vi) bulges -2 to -6.This calculation of the free energy of single-stranded loops has been criticized by Delisi and Crothers,' 56 who have calculated the size-dependent free energy required to close single-stranded loops by the formation of a base pair in a double helical nucleic acid.The free energy of RNA secondary structures at high salt concentrations can be calculated. No doubt these calculations will be further refined as more data are accumulated and they should be very useful in assessing the thermodynamically most favourable secondary structure for single-stranded polynucleotides. 4 RNA An automated RNA sequenator has been used to determine the 3'-terminal hexanucleotide sequence of a polynucleotide with an average yield at each stage of 98 %.' 57 A modified RNase (~-carboxymethyl-lysine-41-pancreatic RNase A) with a low but specific activity has been used to determine the sequence of oligonucleotides obtained by partial RNase T digestion.' The preference of the enzyme for CpA and UpA can be emphasized by varying the digestion 15' J.Eisinger Biochem. Biophys. Res. Comm. 1971 43 854. 152 F. H. Martin 0.C. Uhlenbeck and P. Doty J. Mol. Biol. 1971 57 201. 0.C. Uhlenbeck F. H. Martin and P. Doty J. Mol. Biol.,1971 57 217. '54 S. K. Podder Nature New Biol. 1971 232 1 15. 155 1.Tinoco 0.C. Uhlenbeck and M. D. Levine Nurure 1971 230 362.156 D. C. Delisi and D. M. Crothers Proc. Nar. Acad. Sci. U.S.A. 1971 68 2682; Biopolymers 1971 10 1809. 15' M. Uziel J. W. Starken J. W. Eveleigh and W. F. Johnson Cfin.Chem. 1971,17,740. 158 R. Contreras and W. Fiers F.E.B.S. Letters 1971 16 281. Nucleic Acids 435 conditions. 3'-Terminal polynucleotides can be selectively bound to columns of cellulose derivatives containing covalently bound dihydroxyboryl groups. 59 A new species of 5s RNA (5sRNA,,,) has been found in Novikoff hepatoma ascites cell nuclei and has been partially sequenced.'60 5s RNA molecules cleaved between nucleotides 41 and 42 by RNase T have been reconstituted to give native 5s RNA,16' showing that the RNA has a specific conformation and that specifically modified molecules are now available for structural and functional studies.Reconstitution experiments have shown that 5s RNA is essential for the ribosomes to have full biological activity and in the absence of this RNA molecule some proteins fail to join the particle.'62 The nucleotide sequences of 5s RNA (from Pseudomonas jluores~ens),'~~ which is similar to but not identical with E. coli 5s RNA of a 6s RNA from E. ~oli,'~~ and of a 3'-terminal sequence of 16s RNA from E. coli of40 nu~leotides'~~ have been reported and secondary structures proposed. The state of the art of determining the sequences of long RNA molecules has been dramatically demonstrated by the discovery that E. coli which is resistant to Kasugamycin has a 16s RNA containing the sequence AACCUG whereas in the sensitive strain both the adenylic acid residues have an N6N6-dimethyl group.'66 The specific methylase present in the wild type E.coli has been identi- fied and shown to methylate 16s RNA from the resistant strain only while present as a 21s ribonucleoprotein methylation being on the expected two adenylic acid residues.'67 This modification then renders the reconstituted ribosome sensitive to the drug. Treatment of sensitive E. coli cells in uiuo with colicin E3 results in a specific cleavage of the 16s RNA ca. 50 nucleotides from the 3'-end. 168 This was originally thought to have been due to a colicin-mediated change of the cell surface but it has now been shown that the in vitro reaction of colicin E3 on ribosomes has the same effect.'69 A remarkable coincidence resulted in the simultaneous publication of four papers concerned with the discovery of a precursor 16s RNA from E.coli. 70-1 73 The precursor has a considerably greater molecular weight differs in the base Is' M. Rosenberg and P. T. Gilham Biochirn. Biophys. Acru 1971 246 337. I6O T. S. Ro-Choi R. Reddy D. Henning and H. Busch Biochem. Biophys. Res. Comrn. 1971 44 963. 16' B. R. Jordan and R. Monier J. Mof.Biof. 1971 59 219. 16' V. A. Erdmann S. Fahnestock K. Higo and M. Nomura Proc. Nut. Acad. Sci. U.S.A. 1971 68 2932. 163 B. Du Puy and S. M. Weissman J. Biol. Chern. 1971 246 747. lh4 G. G. Brownlee Nature New Biol. 1971 229 147. 16' C. Ehresmann P. Fellner and J. P. Ebel F.E.B.S. Letters 1971 13 325.166 T. L. Helser J. E. Davies and J. E. Dahlberg Nature New Biol. 1971 233 12. lh7 T. L. Helser J. E. Davies and J. E. Dahlberg Nature New Biof. 1972 235 1. lhK C. M. Bowman J. E. Dahlberg T. Ikemura J. Konisky and M. Nomura Proc. Nut. Acad. Sci. U.S.A. 1971 68 964. 16' C. M. Bowman J. Sidikaro and M. Nomura Nature New Biol. 1971 243 133. M. Sogin B. Pace N. R. Pace and C. R. Woese Nature New Biol. 1971 232 48. ''I G. G. Brownlee and E. Cartwright Nature New Bioi. 1971 232 50. C. V. Lowry and J. E. Dahlberg Nature New Biol. 1971 232 52. F. Hayes D. Hayes P. Fellner and C. Ehresmann Nature New Biol. 1971 232 54. 436 R. T. Walker sequence of both ends and is under-methylated compared to the mature molecule. The sequence of some of the additional nucleotides has been obtained.E. coli 50s subunits have been assembled in vitro from a single mixture of the three ribosomal RNA species and all the necessary proteins.' 74 The mechanism of the assembly of tobacco mosaic virus from RNA and protein has been ex- plained.' 75 Viral coat protein containing ester linkages' 76 and a polyester of phenyl-lactic acid' have been produced in a cell-free synthesizing system containing aminoacyl-tRNAs the amino-acids of which had been chemically deaminated with nitrous acid. Poly-1,Poly-C has been found to inhibit or stimulate depending upon the age of the animal Moloney sarcoma virus-induced tumours in mice,'74 and it causes the death of mouse bone-marrow and spleen colony-forming cells.' 7s Despite this the double-stranded polynucleotide has been injected into humans with advanced cancer.It has failed to show a toxic effect but unfortunately has failed to show anti-tumour effects.' 76 The uptake of polynucleotides by cells has been demonstrated. In the poly-A- poly-U or poly-A-2-poly-U systems both strands enter Ehrlich ascites tumour cells but with poly-I-poly-C the poly-C remains on the cell surface while the poly-I enters the cells.'77 The uptake of polynucleotide is stimulated by the addition of deoxyribonuclease to the cell culture. ' DEAE-dextran increases the infectivity of many viral RNA molecules'79 and has been shown to increase the antiviral activity of poly-I-poly-C,' 80,18 ' probably by protecting the nucleic acid from ribonuclease attack and also increasing the binding of the poly- nucleotide to the cell surface.Poly-I has been covalently bound through its terminal 5'-phosphate to Sepharose'82 and then annealed with poly-C to give an insoluble matrix-bound poly-1-poly-C which has a similar activity to that of the normal molecule. Both the toxicity and biological activity of poly-I.poly-C are dependent upon the homopolymer molecular weights' and are both drastically reduced if these fall below lo5. It seems unlikely that a signficant separation of antiviral activity and toxicity can be made by simple manipulation of the homopolymer molecular weights. Other double-stranded RNA molecules synthetic' 84 and naturally occurring,' 85 also stimulate interferon production but evidence is accumulating that the stimulation of synthetic and natural inducers is not by the 1'4 E.de Clercq and T. C. Merigan Proc. Soc. Exp. Biol. Med. 1971 137 590. P. Jullien and J. de Maeyer-Guignard Internat. J. Cancer 1971 7 468. C. W. Young Med. Clin. North Amer. 1971 55 721. P. L. Schell Biochim. Biophys. Acta 1971 240 472. P. L. Schell and W. Muller Biochim. Biophys. Acta 1971 247 502. R. Hall J. Gen. Virol. 1971 11 111. lE0 P. M. Pitha and W. A. Carter Virology 1971 45 777. 181 F. D'ianzani S. Baron C. E. Buckler and H. B. Levy Proc. Soc. Exp. Bio. Med. 1971 136 1111. A. F. Wagner R. L. Bugianesi and T. Y. Shen Biochem. Biophys. Res. Comm. 1971 45 184. J. F. Niblack and M. B. McCreary Nature New Biol. 1971 233 52. E. de Clercq and T.C. Merigan J. Gen. Virol. 1971 10 125. 85 W. F. Long and D. C. Burke J. Gen. Virol. 1971 12 1. Nucleic Acids 437 same mechanism. However much work remains to be done since a lack of correla- tion between interferon production and anti-tumour effect is claimed,' 86 and the anti-tumour effects of synthetic polynucleotides have been explained as due to a stimulation of classical immune response rather than to interferon effects.'87 Successive administration of poly-I followed by poly-C has as great an effect if not greater than when both are administered together,188 and addition of poly-C-oligo-I under conditions above the T of the complex also stimulates the induction of interfer~n.'~~ 5 Viral RNA Significant progress has been reported in the sequence determination of the RNAs of the phages MS2 R17 f2,and QB.The A protein cistron is closest to the 5'-terminus in MS2 with the initiator codon starting at position 130.'90*191 The previous 129 nucleotides have been sequenced and have been shown to be identical with those of R17192 and RNA whereas in the next 237 nucleo- tides which can be compared for MS2 and R17 nine differences in sequence have been detected although the phages produce identical A proteins. These differences only occur because of code degeneracy and the identity of the leader sequences suggests that the tertiary structure of this part of the molecule is important. This sequence defines the sequence at the 3'-end of the complementary minus strand which may serve as a recognition site for the viral RNA polymerase complex.'94 The 5'-terminal sequences can be arranged in a cloverleaf-like fashion with a CCA end but with longer loops and one additional loop compared to the conventional tRNA cloverleaf (Figure 2).Other nucleotide sequences corresponding to amino-acid sequences of the coat protein cistron" and other parts of the MS2 molecule'96 and further sequences of R17 RNA have been obtained. 197 With the determination of the sequence of the ribosome binding sites for the initiation of translation of the Qp assembly protein'98 and the replicase gene,'99 the sequences of all six sites (for the three protein cistrons in QB and R17) are A. J. Weinstein A. F. Gazdar H. L. Sims and H. B. Levy Nature New Biol. 1971 231 53.lS7 W. Braun 0. J. Plescia J. Raskova and D. Webb fsraef J. Med. Sci. 1971 7 72. E. de Clercq and P. de Somer Science 1971 173 260. P. M. Pitha and W. A. Carter Nature New Biol. 1971 234 105. 190 R. de Wachter A. Vandenberghe J. Merregaert R. Contreras and W. Fiers Proc. Nut. Acad. Sci. U.S.A. 1971 68 585. 191 W. Fiers R. Contreras R. de Wachter G. Haegeman J. Merregaert W. Min Jou and A. Vandenberghe Biochimie 1971 53 495. 192 R. de Wachter A. Vandenberghe J. Merregaert R. Contreras and W. Fiers Arch. Internat. Physiol. Biochim. 197I 79 199. 193 V. Ling Biochem. Biophys. Res. Comm. 1971 42 82. 194 R. de Wachter J. Merregaert A. Vandenberghe R. Contreras and W. Fiers European J. Biochem. 1971 22 400. 195 W. Min Jou G. Haegeman and W. Fiers F.E.B.S.Letters 1971 13 105. 19h G. Haegeman W. Min Jou and W. Fiers J. Mol. Biol. 1971 57 597. 19' P. G. N. Jeppesen Biochem. J. 1971 124 357. 198 D. H. Staples J. Hindley M. A. Billeter and C. Weissmann Nature New Biol. 1971 234 202. 199 D. H. Staples and J. Hindley Nature New Biol. 1971 234 21 1. 438 R. T. Walker A/A \ \/ U-A U-A A-U C-G A-G A-A G-c G' A A-A \/ A-U C-G A-U C-G U-A C-G C-G C-G G-c A-U c-u G-c &''A U-C G-c G C-G A' \G \ U-A A c' A, ,C-G A\ \U-Ac-G-c 'A G G A/\G U U G A-G' C-C-C-AOH IG I A UAGCC IIIIIIIII C C U C A A C U-G I IIIII A/ U I C AUCGG C-A I 'G, 'A 'c-G' 4 \"JC C-G U-A U-A A-U /\ A@ Figure 2 Proposed secondary structure of the 3'-terminal sequence of MS2 RNA comple-mentary strand now known.Despite the fact that the ribosome specifically binds at these sites even in alkali-treated RNA and ignores the many other initiator codons present in internal sites of the molecule it is still not possible to define the specific recog- nition signal which tells a ribosome to initiate translation. Comparing this with the analogous situation of protein-nucleic acid recognition in the tRNA field this result is perhaps not really very surprising. So much work has been published on the RNA-directed DNA polymerase of the oncogenic viruses that two long and comprehensive reviews have already and the reader is referred to these. The status of the enzyme after a year of conflicting reports is that definite evidence is still needed to prove the requirement of the enzyme for initiation of neoplastic transformation and more evidence is needed before it can be concluded that it is not required for maintenance of transformation.Early reports that the enzyme was present in most cells have been shown to be due to the use of incorrect assay procedures for the enzyme. 6 tRNA Many reviews of the subject have been published.' A rapid dialysis method2" for the assay of tRNA's is claimed to be accurate over a wide range of tRNA concentration (0.5-200pg) enables the tRNA to be reused and is adaptable *"" S. Spiegelman Proc. Roy. Soc. 1971 B177 87. 201 H. C. Chen C. H. O'Neal and L. C. Craig Analyt. Chem. 1971 43 1017. Nucleic Acids 439 to a continuous and automated assay procedure.A statistically designed series of experiments to establish optimum assay conditions for several tRNAs by changing each of a dozen qualitative and quantita$ve variables and evaluating the results by computer,202 confirms that optimum charging conditions for different tRNAs vary widely; it will be of great use if the range of tRNAs covered can be extended. In uivo transcription of a tRNA gene and the isolation and nucleotide sequence of a precursor tRNA have all used the 680 phage carrying various E. coli tRNATy' genes in the phage genome. Smith203 has reported the isolation of a tyrosine tRNA-tsDNA hybrid by allowing tRNATy' to hybridize with the denatured DNA from phage @3Opsu& a phage which carries two structural genes speci- fying tRNATy'.Subsequent treatment with Neurospora crassa endonuclease which is specific for single-stranded nucleic acids releases regions of reannealed DNA and hybrid molecules composed of tRNATyr and the complementary DNA (tsDNA) which can be separated on Sephadex G-100. The locations and orientations of the Su& gene in transducing phages 480 psu& and the defective phage +80dsuil suithave been determined.204 Labelled E. coIi tRNA2' hybridized with the r strand of the former phage. As SuittRNA had previously been found to hybridize with the 1 strand of the defective phage,205 it was concluded that an inversion of the E. coEi DNA fragment carrying the Suit gene occurred before its incorporation into the +8Opsu& genome.These phages have been used to isolate the tRNATyr gene206 by a method similar to that used by Beckwith for the isolation of the lac ~peron.~" The separated heavy strands of the phage DNAs containing the tRNATY' genome inserted into their DNA in opposite directions were annealed the single-strand tails of the resulting hybrid removed with N. crassa endonuclease and the resulting DNA was used for the in uitro transcription of tRNATYr-like molecules. The assay was based on their ability to compete with tRNATy' in hybridization experiments with complementary DNA. The DNA-directed cell-free synthesis of biologically active tRNATJ:tI has been reported.'08 The DNA from ~8Opsu& was transcribed by E. coli RNA polymerase. The product was assayed by taking advantage of the specific suppressor properties of the mutant SU;~ tyrosyl-tRNA which stimulated DNA- directed /?-galactosidase synthesis using a DNA with an amber triplet in the /?-galactosidase gene.202 I. B. Rubin T. J. Mitchell and G. Goldstein Analjlr. Chem. 1971 43 717. 203 A. Marks E. Keyhani S. Naono F. Gros and J. D. Smith F.E.B.S.Letters 1971 13 110. 204 R. C. Miller P. Besmer H. G. Khorana M. Flandt and W. Szybalski J. Mol. Bid. 1971 56 363. '05 H. Lozeron W. Szybalski A. Landy J. Abelson and J. D. Smith J. Mol. Biol. 1969 39 239. '06 V. Daniel J. S. Beckmann S. Sarid J. I. Grimberg M. Herzberg and U. Z. Littauer Proc. Nat. Acad. Sci. U.S.A.,1971 68 2268. '07 J. Shapiro L. MacHattie L. Eron G. Ihler K. Ippen and J. Beckwith Narure 1969 224 768.20R G.Zubay L. Cheong and M. Gefter Proc. Nat. Acad. Sci. U.S.A.,1971 68 2195. 440 R.T. Walker Tyrosine tRNA precursor molecules have been isolated by a rapid phenol extraction technique without collection of the cells from E. coli infected with $80 phage carrying a mutant Su,, gene which results in a much higher yield of precursor than can be obtained from the original strain with the Su& gene.209 In addition to the expected tyrosine tRNA sequence these molecules contained 41 additional bases at the 5'-end and 3 at the 3'-end.13 The 5'-terminal residue was found to be pppGp which indicates that this end of the molecule is as initially transcribed. Nucleotide modification is absent and as the precursor molecule can be enzymatically cleaved by an E.coli crude cell extract to a 4s molecule this modification is apparently not required for this step in the tRNA biosynthesis. Moreover the cleavage points in the molecule when subjected to partial enzy- matic hydrolysis are the same as those in the 4s molecule indicating that the precursor probably contains the normal cloverleaf configuration. The CCA 3'-terminus is coded for in the DNA genome. As a result of experiments with other mutants the significance of the lengthy 5'-segment is thought to be con- cerned with defining the specificity of the cleavage point and to protect the molecule during transcription by hydrogen-bonding to the molecule near the cleavage point (Figure 3 a) until transcription is complete when the cloverleaf configuration (Figure 3,b) is assumed and the 4s molecule is enzymatically cleaved from the precursor molecule.The precursor molecule of normal tRNA may be even longer but will differ only at the 3'-end. An in oitro synthesis of tRNATy' precursor and its conversion into 4s RNA have also been described.210 A tryptophan tRNA suppressor has been isolated from a UGA suppressor strain of E. coli21 and the sequence differs from that of the normal tRNAT'p by a G +A mutation at position 24 in the dihydrouridine loop.212 The anticodons for both molecules are CCA which would only be expected to code for tryptophan (codon UGG). This is the first reported instance of a tRNA molecule reliably recognizing a sequence other than its complementary one. The suppressor tRNA has been shown to stimulate the synthesis of lysozyme in ~,itro~'~ by trans- lation of the phage T4 lysozyme mRNA bearing a UGA mutation.An E. coli leucine suppressor tRNA which can be used in the in oitro poly-leucine formation from poly-r (AUG) has been purified.214 Five leucine tRNAs have been purified,2 '5-2 ' and there is evidence that they can all be amino- acylated by the same en~yme.~'~,~'' Two of these tRNAs have a chain length of 87 nucleotides but differ in 22 positions of their primary sequence.218 Yeast 209 S. Altman Nature 1971 229 19. H. Ikeda Nature 197 1 234 198. 211 T. S. Chan R. E. Webster and N. D. Zinder J. Mol. Biol. 1971,56 101. 212 D. Hirsh J. Mol. Biol. 1971 58 439. D. Hirsh J. Mol. Biol. 1971 58 459.'14 H. Hayashi and D. Soll J. Biol. Chem. 1971 246,4951. 215 D. W. Holladay R. L. Pearson and A. D. Kelmers Biochim. Biophys. Acta 1971 240 541. 216 J. Kan and N. Sueoka J. Biol. Chem. 1971 246,2207. '17 H. U. Black and D. SOH J. Biol. Chem. 1971 246 4947. 218 H. U. Black and D. Soll Biochem. Biophys. Res. Comm. 1971 43 1192. Nucleic Acids 441 AU G A C A C-G U C-G G U U-G G-C U-A G-C C-G G-C G.*. PPPG-C A G G-U C-G C-G/ A-U GC G-C C U-A A-U A-U A A AGC A U G A C A C-G . . C-G U-A Ll-G G-C Ll-A \G-C C--G U-A pppG-C A G G C C A G U A A A A G C A U U A C CC G-C C A Figure 3 Possible conjiguration for tyrosine precursor tRNA during transcription (a) and after completion of transcription(b).I3 The arrows indicate the beginning of the 5'-end of the tRNA moiety 442 R.T. Walker tRNA has been labelled with 32Pin viv0219 with sufficient activity to enable a tRNAL"" to be purified and its sequence determined22o by the Sanger method. This advance enables the sequence of small amounts of yeast tRNAs to be quickly determined but before the method can be applied to tRNAs from other sources media have to be developed in which practically all the phosphate is [32P]-labelled. Among the many tRNA sequences reported this year are the following:- E. coli tRNAyd,221 tRNAVd 222 tRNAVd 222 tRNA%r223 tRNAIle 224 2A 3 2B 9 2,226 tRNA7'y,225 and tRNAgz;225 brewers yeast tRNAVa' I tRNAASp3.22 7,22 8 rat liver tRNAS"';229 Staphylococcus epidermidis tRNAy'y.230 These sequences can all be written in the cloverleaf form but they do little to clarify the structure-function relationship of a tRNA and its amino-acylating enzyme.Two of the three tRNAVal now sequenced222 have similar sequences differing only in three base pairs located in double-stranded regions. Their affinity for the synthetase is an order of magnitude greater than that of the tRNA:"' species whose sequence differs in 22 positions. It is suggested that the synthetase recognition site may be formed by the interaction of two or more of the sequences common to all three tRNAVal. The E. coli tRNApr can be efficiently charged by the yeast and rat liver synthetase but comparison of the sequences of these three tRNAS"' ~pe~ie~~~~,~~~,~~~ shows nothing which can give any definite indication of the recognition site.The sequence of tRNA;"' from brewers yeast226 differs in four positions from a previously published sequence,233 and in the few cases where tRNAs have been sequenced independently by more than one group there is an uncomfortably high percentage of cases where the results do not agree. Complexes (1 1)of tRNAG1" from E.c01i234and its synthetase and of a purified methionyl-tRNA transf~rmylase~ have been reported. It is hoped that even- tually one of these complexes will be obtained crystalline so that the enzyme recognition sites can be located. 21y S. Kowalski and J. R. Fresco Science 1971 172 384. 220 S. Kowalski T. Yamane and J.R. Fresco Science 1971 172 385. 221 F. Harada F. Kimura and S. Nishimura Biochemistry 1971 10 3269. 222 M. Yaniv and B. G. Barrell Nature New Biol. 1971 233 113. 223 H. Ishijura Y. Yamada and S. Nishimura F.E.B.S.Letters 1971 16 68. 224 M. Yarus and B. G. Barrell Biochem. Biophys. Res. Comm. 1971 43 729. 225 C. Squires and J. Carbon Nature New Biol. 1971 233 274. 226 J. Bonnet J. P. Ebel and G. Dirheimer F.E.B.S.Letters 1971 15 286. 227 J. Gangloff G. Keith J. P. Ebel and G. Dirheimer Nature New Biol. 1971 230 125. 228 G. Keith J. Gangloff J. P. Ebel and G. Dirheimer Compt. rend. 1970 271 D 613. 229 T. Ginsberg H. Rogg and M. Staehelin EuropeanJ. Biochem. 1971,21,249. Also see preceding two papers. 230 T. S. Stewart R. J. Roberts and J. L. Strominger Nurure 1971 230 36.231 H. G. Zachau D. Dutting and H. Feldman Z. Physiol. Chem. 1966 347 212. 232 M. Staehelin H. Rogg B. C. Baguley T. Ginsberg and W. Wehrli Nature 1968 219 1363. 233 A. A. Bayev T. V. Venkstern A. D. Mirzabekov A. I. Krutilina L. Li and V. D. Axelrod Molekulyarnaya Biol. 1967 I 754 (English Edn. p. 63 1). 234 W. R. Folk Biochemistry 1971 10 1728. 235 H. W. Dickerman and B. C. Smith J. Moi. Biol. 1971 59 425. Nucleic Acids 443 The tRNAE',Y (ins = insensitive to tryptophan)225 is of interest as it is a new species of tRNA which appears as a result of a mutation causing the loss of tRNA:'Y. The tRNAy'y compensates for this loss (which is normally lethal) with a G +U mutation in the 5'-end of the anticodon triplet so that the tRNA can recognize GGA/G instead of GGU/C and thus all glycine codons can still be translated.The tRNAs tRNA?lY and tRNATi' have a 78 %sequence homology with the differences located in hydrogen-bonded stems suggesting that these may be of key importance in the synthetase recognition process. This sequence similarity further confirms that three base pairs of the CCA-stem do not constitute the synthetase recognition point because the three are identical in tRNAyIY and tRNAzi' but different in tRNATi' and tRNA7"'. tRNAyi, from S. epiderrnidi~~~' fails to participate in protein synthesis and functions only in peptidoglycan synthesis. The tRNAs contain few modified bases and have several other pecularities (Figure 4),236 including the replacement of the common sequence GTrl/C by the sequence GUGC.The tRNA will not bind to ribosomes even non-specifically and this may well be due to the lack $OH C U pG-C C-G G-C G-C A G-U f A-U <:p C G-CUAUCCU A u 4tU A IIIII !A ?- AUAGGu c A C U UIGjA G U I I I jIj '<:&,-A v A G A A LCJA 'D / AG C-G r---L'I32-C JU-A G U-A C-G CG U C uCc Figure 4 Structure of S. epidermidis tRNAYAy and tRNA7; (alternative sequence indicated) 236 R. J. Roberts paper submitted to Nature. 444 R.T. Walker of this common sequence which has previously been implicated in ribosome binding. The enzymatic modification of tRNA has been reviewed237 but the function of the modified bases is still not understood.238 4-Thiouridine has still only been found in bacterial tRNA (and in mouse and chick mitochondria1 tRNA239) and its biosynthesis has been clarified.240 An E.coli tRNA lacking ribothy- midine behaves normally241 and tRNAs containing only 16% of their normal 5,6-dihydrouridine 4-thiouridine pseudouridine and ribothymidine content appear to have the same stability as normal ~RNAs.~~~ The search for the synthetase recognition site in tRNA continues by the conventional methods of the dissected molecule243 and chemical modifica- tion244,245 approaches. Methoxylamine failed to react with a cytidine residue at the 5’-end of the anticodon in a tyrosyl-tRNA despite reacting with several other cytidine residues in the Moreover the residue was still unreactive in a dissected anticodon loop and stem.The results can only be explained in terms of a model which differs from the Fuller-Hodg~on~~~ model for the anticodon loop. The covalent bond formed by U.V. irradiation between the 4-thiouridine residue at position 8 and a cytidine at position 13has continued to excite Work on model compounds suggests247 that the two bases must be close together and oriented geometrically so that a thietan and ultimately a covalently linked can 5-(1’-~-~-ribofuranosyl-4’-pyrImidin-2‘-one)cytidinebe readily formed. The covalently linked tRNA molecule has a reduced (i)affinity for the synthetase complex but the bond can still be formed by irradiation of the tRNA-synthetase tRNAPhe,249 and tRNAArg,249 complex.248 tRNAVa1,248 containing the link have been shown to function in all steps of protein synthesis albeit at a somewhat reduced rate.Results from X-ray experiments on crystalline tRNA continue to be dis- appointing. It has been suggested that conformations of tRNA molecules are too dependent upon the influence of counterions and hydration to enable anything other than the overall shape of the molecule to be deduced from a comparison of experimental curves with scattering curves calculated from atomic co-237 D. Soll Science 1971 173 293. 238 A. Peterkofsky M. Litwack and J. Marmor Cancer Res. 1971,31,675. 239 Y. Lalyre and E. B. Titchener Biochem. Biophys. Res. Comm. 1971 42 926. 240 J. W. Abrell E. E. Kaufman and M. N. Lipsett J. Biol. Chem.1971 246 294. 24’ I. Svensson L. Isaksson and A. Henningsson Biochim. Biophys. Acta 1971 238 33 1. 242 I. I. Kaiser Biochemistry 1971 10 1540. 243 A. D. Mirzabekov D. Lastity E. S. Levina and A. A. Bayev Nature New Biol. 1971 229 21. 244 For example M. A. Q. Siddiqui and J. Ofengand F.E.B.S. Letters 1971 15 105; M. Krauskopf and J. Ofengand ibid. 1971,15,111;Z. Kucan K. A. Freude I. Kucan and R. W. Chambers Nature New Biol. 1971 232 177. 245 A. R. Cashmore D. M. Brown and J. D. Smith J. Mol. Biol.,1971 59 359. 246 W. Fuller and A. Hodgson Nature 1967 215 817. 247 N. J. Leonard D. E. Bergstrom and G. L. Tolman Biochem. Biophys. Res. Comm. 1971 44 1524. 248 M. Yaniv A. Chestier F. Gross and A. Favre J. Mol. Biol. 1971 58 381. 249 L. J. Chaffin D.R. Omilianowski and R. M. Bock Science 1971 172 854. Nucleic Acids 445 ordinate^.^" Only results which are consistent with the cloverleaf form~lation~~ ' and show the presence of short parallel double-helical segments in tRNA molecules252 have been obtained. Other physical techniques have been used temperature-jump relaxation,254 n.m.r.,255*256 including fl~orescence,~~ and ~.d,~~~9~~~ the last showing curves reminiscent ofa globular protein rather than of a polyn~cleotide.~~~ fdr people to demolish have been Two new models258~259 suggested. 7 DNA The secondary structure of DNA depends upon the base composition.260 DNA molecules with a high A + T (366%) content give distinctly different X-ray scattering patterns in solution from other DNA molecules and do not seem to adopt the B conformation.This may provide regions of DNA which are specific markers for control and recognition.26 ' A circular bihelical synthetic DNA has been prepared for use as a substrate in enzyme recognition studies.262 Single- stranded complementary deoxyribopolynucleotides d(T-G) and d(C-A) were mixed under dilute condition in the presence of T4-polynucleotide ligase and treated with DNA polymerase the four deoxyribonucleoside triphosphates and more ligase. Density gradient centrifugation of the products digested with exonuclease I11 enabled double-stranded circular DNA to be isolated in 4% yield. The chromosomes of higher organisms have been st~died,'~*~~~ and it has been suggested by Crick that DNA is of two types globular DNA (which con- tains unpaired regions for control purposes) and a much smaller fraction consist- ing of fibrous DNA which alone codes for proteins.The forces and energy needed to unpair the recognition stretches of the DNA are provided for by the combination of DNA with chromosomal proteins. It has been shown that histones are not evenly distributed in chromatin,263 and extensive contiguous regions of the DNA helix are completely free of chromatin protein. Satellite DNA has been the subject of two and the location of satellite and homogeneous DNA sequences on human chromosomes has been 250 I. Pilz 0. Kratky F. Von Der Haar and F. Cramer European J. Biochem. 1971 18 436. 2s1 S. H. Kim G. Quigley F. L. Suddath and A.Rich Proc. Nut. Acad. Sci. U.S.A. 1971 68 841. 2s2 T. Sakurai S. T. Rao J. Rubin and M. Sundaraiingam Science 1971 172 1234. "' B. Robison and T. P. Zimmerman J. Biol. Chem. 1971 246 110. 254 M. Dourlent M. Yaniv and C. Htlene European J. Biochem. 1971 19 108. 2s5 D. R. Kearns D. J. Patel and R. G. Shulman Nature 1971 229 338. 25f' S. I. Chan and M. P. Schweizer Biochem. Biophys. Res. Comm. 1971,44 1. 257 G. Melcher D. Paulin and W. Gruschlbauer Biochimie 1971 53 43. 258 D. J. Abraham J. Theor. Biol. 1971 30 83. 259 A. Danchin F.E.B.S. Letters 1971 13 152. 26') S. Bram Nature New Biol. 1971 232 174. 261 K. Shishido and Y. lkeda Biochem. Biophys. Res. Comm. 1971,44 1420. 262 V. H. Paetkau and H. G. Khorana Biochemistry 1971,10 1511. "' P.J. Clark and G. Felsenfeld Nature New Biol. 1971 229 101. 26J P. M. B. Walker Nature 1971 229 306. 26s R. J. Britten and E. H. Davidson Quart. Reti. Biol.,1971 46 11 1. 446 R.T. Walker determined by the in situ RNA-DNA hybridization technique.266 Satellite I1 appears to be close to the centromeres of three pairs of chromosomes. A new method for RNA-DNA hybridization which can be used to analyse for any defined fraction of cellular RNA in terms of the reiteration frequencies of the complementary DNA sequences has been described.267 The DNA is added in such a vast excess that the DNA withdrawn into DNA-RNA hybrids does not significantly reduce the concentration of denatured DNA sequences and so the renaturation rate is essentially independent of the hybridization reaction.DNA polymerase has been used to determine the sequence of the termini of two phage [32P]-Labelled T7 phage DNA was subjected to exonucleolytic degradation with T4 DNA polymerase in the presence of each of the deoxynucleoside triphosphates in turn. The nucleotides released were quantitatively estimated and the results were consistent with the previously determined sequence.268 1 DNA has the 5’-terminal strands longer than the 3’-terminal strands. The sequence in both protruding single strands has been determined by partial and complete repair with DNA polymerase followed by sequencing of the isolated oligonucleotides. Each of the two oligonucleotides was confirmed to be 12 units long and they had complementary sequences.269 The conclusions of a paper2” on the intercalation of ethidium bromide into the DNA molecule are going to cause a drastic rethinking of the subject of intercalation and the conformation of DNA in solution if as is claimed inter- calation of ethidium bromide does cause the winding of the DNA helix by 13 f4” instead of unwinding it as has previously been assumed.A protein with similar properties to the gene-32 protein discovered last year in T4 phage has now been found in meiotic cells of lili~rn~~’ and the spermatocytes of rat bull and man.272 It would now appear that this protein which binds co- operatively to single-stranded DNA and catalyses DNA denaturation and renaturation in uitro may occur universally. It is probable that this protein plays an important part is meiosis and also in genetic recombination.It is reassuring to realise that occasionally even molecular biologists cannot find instant answers to problems or even to problems which have been recognized since the birth of the subject. DNA duplication is an outstanding example of this and despite a proliferation of theories-mostly conflicting-little progress has been reported this year. Kornberg’s enzyme DNA polymerase I has definitely been shown to have a repair function in uivo273 and has now by (al- most !274) universal consent been removed from the list of acceptable candidates for the DNA replication enzyme in uiuo. This leaves two candidates polymerases 266 K. W. Jones and G. Corneo Nature New Biol. 1971 233 268. 267 M. Melli C.Whitfield K.V. Rao M. Richardson and J. 0.Bishop Nature New Biol. 1971 231 8. 268 P. T. Englund J. Biol. Chem. 1971 246 3269. 269 R. Wu and E. Taylor J. Mol. Biol. 1971 57 491. 270 J. Paoletti and J. B. Le Pecq J. Mol. Biol. 1971 59 43. 271 Y.Hotta and H. Stern Develop. Biol. 1971 26 87. 272 Y.Hotta and H. Stern Nature New Biol. 1971 234 83. 273 W. S. Kelly and H. 0. Whitfield Nature 1971 230 33. 274 V. H. Paetkau and A. R. Morgan Nature New Biol. 1971 234 36. Nucleic Acids 447 11275 and 111,275*276 and the claims of both have been ad~anced.’~~.’~~ There is also a pertinent report which suggests that as most people insist on using Kornberg’s assay system for detecting new repair enzymes it is not surprising that enzymes with similar properties to his enzyme are found which may well have no more to do with in vim replication than the original p~lyrnerase.’~~ Evidence is provided which suggests that deoxyribonucleoside triphosphates are not the precursors used by the replicating enzyme in uivo and that there may be some activated form of nucleoside monophosphate instead.For all those who like to see their molecular biology Kornberg has provided some superb electron microscope photographs of E. coli DNA polymerase I attached to nicks in the helical structure formed between p01y-dA.oligo-dT.’~~ 275 T. Kornberg and M. L. Gefter Proc. Nut. Acad. Sci. U.S.A. 1971 68 761. 276 V. Nusslein B. Otto F. Bonhoeffer and H. Schaller Nature New Biol. 1971,234 285. 277 G.V. R. Reddy M. Goulian and S.S. Hendler Nature New Biol. 1971 234 286. 278 R. Werner Nature New Biol. 1971 233 99. 279 J. Griffith J. A. Huberman and A. Kornberg J. Mol. Biol. 1971 55 209.