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of an amincacyl oligonucleotide bound to other oligonucleotide material by hydrogen bonding interactions between complementary sequences of bases. It is known that the guanine-cytosine base pair in RNA shows more resistance to alkaline and thermal denaturation than does the adenine--uracil base paire; whether it shows resistance to ribonucleases has not been established. Until these possibilities are investigated, it cannot be decided whether the valine-I and -II components are derived from two chains of valine-acceptor RNA terminating with different sequences. Since the original report of these studiesz ISHIDA AND MIURAT have published preliminary results which are in agreement with those found in this laboratory. This work was supported by grants from the National Science Foundation and from the U.S. Public Health Service.

Departme~ o/ Biology, Massachuse~s Institute o/ Technology, Cambridge, Mass. (U.S.A.)

CHRISTOPHERJ. SmTH* EDWARD HERBERT* CAROLINE W. WILSON

j . TAKAHASHI, J. Biockem. Tokyo, 49 (196I) I. C. J. SMITH AND E. HERBERT, Federation Pvo¢., 22 (i963) 230. E. HERBERT, C. J. SMITH AND C. W. WILSON, J. MoL Biol., in the press. j . APGAR, R. W. HOLLEY AND S. H. MERRILL, f . Biol. CIu~., 237 (I962) 796. M. L. STRPH~NSON AND P. C. ZAMECNIK, Proc. Natl. Acad. Sd. U.S., 47 (1961) x627. * J. R. FRESCO,in H. J. VOGEL, V. BRY$ON AND J. O. LAMPEN, I#t/Ofmatiofgcd Mcwromoleculeso Academic Press, New York, I963, p. x~r. T T. I s m v A AND K. MIURA, J. Biochem. Tokyo, 54 (I963) 378.

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Received March 9th, I964 * Fresent address: D e p a r t m e n t of Chemistry, University of Oregon, Eugene, Oreg. (U.S.A.).

Biockim. Biopkys. Acta, 87 (x964) 34x-343

SC 7146 Guanine oligonucleotides from yeast-soluble-ribonucleic acids In a study of the distribution in yeast-soluble RNA of nucleotide components other than the four common ribonucleotides, useful fractionations of digests produced by Abbreviations: Tp, t h y m i n e ribonucleot~de; 1pUp, uracil-5-ribosyl phosphate; methylated gua~ylic acids; PU, purine; PY, pyrimidine.

MeGp,

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pancreatic RNAase (EC 2.7.7.I6) were obtained by gel filtrationI. One fraction for which further study and isolation of components seemed of particular interest was that emerging from Sephadex G-25 as a discrete and unretarded peak (Fraction I, ref. I, Fig. 4). This material, which had a guanylic acid content of approx. 60 moles/ Ioo moles of nucleotides, was also markedly enriched in thymine ribonucleotide (7--8 moles/ioo moles of nucleotides) and its behavior on Sephadex G-25 indicated the presence of much larger oligonucleotides than did its purine to pyrimidine ratio of approx. 3 :I. Large oligonucleotides would be expected in the fraction either if RNAase resistant phosphodiester bonds involving thymine ribonucleotide were present or if aggregation of some of the oligonucleotides produced by the enzymic digestion had occurred. The first possibility was suggested by earlier studies in which l~ancreatiC RNAase was found to release only small amounts of the total thymine ribonucleotide present (Io--2o % in one study~ and "traces" in ref. I). The second possibility was suggested by the observations of MARKHA~IAND SMITHs, who demonstrated that the "RNAase resistant" core of RNAs '~ was dialysable in the presence of 2 M sodium chloride, and by recent reports concerning the aggregation of guanine oligonucleotidesS-L In the present paper, it is demonstrated, first, that the oligonucleotides rich in guanine and thymine are aggregates and that it is their aggregation which permitted their separation from oligonucleotides of similar chain length rich in adenylic acid, and secondly, that the thymine ribonucleotides present in Fraction I are in fact in the expected terminal positions following pancreatic RNAase treatment of the RNA. In addition, it is shown that fractionation on Sephadex together with chromatography on DEAE-cellulose in the pres.ence of 7 IV[urea, as described by TOMLINSON AND TENERs, leads to a fractionation of the higher oligonucleotides in the digests which should be of considerable aid in the determination of the sequence of individual amino acid acceptor RNAs'. The methods of preparation of the RNA, its enzymic digestions, isolation of "Fraction I" by chromatography on Sephadex, nucleotide mapping, determination of approximate base composition and electrodialysis in starch gels have been described I. The terminal position of the thymine ribonucleotide in Fraction I was demonstrated as follows. Fraction I was digested with RNAase TI (EC 3.1.4.8) 9 (4 h at 38°, pH maintained at 7.3, enzyme: substrate ratio 4 unitsg/mg of Fraction I). The major spots seen in maps of the digests corresponded to Gp, Gpl Cp, Up, ApGp as well as Tp. The Tp spot was of the same intensity as that observed after alkaline hydrolysis of an equivalent amount of Fraction I. The maps following TI digestion also indicate regularities in sequence of the oligonucleotides comprising Fraction I. Thus, when one considers the specificity of RNAase T I (cleavage of bonds between guanosine 3'-phosphate and other nucleotides), it appears that these oligonucleotides are of the type (PUp), Gp PYp. Those of the type (PUp), ApPYp do not appear to be major constituents of the fraction in that no ApPYp could be detected although small amounts of free Ap were noted, presumably due to a contaminating enz~ne in the RNAase T 1preparation. Further digestion with the alkaline phosphatase (EC 3.L3.I ) of E. co//produced the expected nucleosides. Initial experiments in which digestion with RNAase T1 was omitted prior to digestion with phosphatase and subsequent alkaline hydrolysis did not lead to good yields of nucleosides from the terminal pyrimidine nucleotides and thus indicated aggregation (see also ref. 7). Biochim. Biophys. Acla, 87 (I964) 343-346

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The state of aggregation of Fraction I was investigated in an electrodialysis system in which membranes of calibrated permeability characteristics are placed in starch gels1,1°. Surprisingly, in gels made in o.oIz M sodium acetate buffer (pH 5.o) Fraction I behaved as if it consisted of even larger particles than the original soluble RNA preparations, with passage not occurring until membranes treated with 6o-6I % ZnC1s were used. The results of the electrodlalysis were confirmed by the determination of the sedimentation constants of Fraction I and of the transfer RNA (sj,~, 3.7 and 3.5 respectively). Starch-gel electrophoresis at pH 5.o in gels which were 8 M with respect to urea indicated disaggregation of Fraction I in the presence of urea, in that the band representing Fraction I diffused away very rapidly during the destaining process while that of the soluble RNA remained. These methods in 1.0

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Fig. x. (a) Chromatography of approx. 200 absorbancy units (26o m/~) of Fraction I on a D E A E cellulose-Tris hydrochloride 8 column (0. 9 × 4° cm) in the presence of 7 M urea. - - - represents a gradient of NaCL The qualitative nucleotide compositionof the oligonucleotides (following their recovery and hydrolysis) found in the numbered peaks is as follows, with Gp predominant in each instance: x, Gp, Ap (trace), Cp, Up, Tp; 2, Gp, Ap, Cp, Up, Tp; 3, Gp, Ap, Cp, Up and Tp (trace)~ 4, separated into two ~ractions on Sephadex G-25, one fraction contained Gp, Ap, Cp, Up (trace), the other an unidentified fluorescent material and Gp, Ap, Cp, Up. No ~3n~her material was eluted if the gradient was extended to o. 4 M NaCI. The material emerging before th e numbered peaks is presumably due to occlusion of some mono- and dinucleotides by the aggregates. (b) Chromatography of approx. 52o absorbancy units of Fraction I I A under conditions identical to the above except that the gradient was extended to o. 4 M NaCI. The mononucleotides found in the numbered peaks are as follows with Ap as the predominant mononucleotide or approximately coequal with Gp: x, Ap, Gp, Cp, Up; 2, Ap, Gp, Cp; 3, Ap, Gp, MeGp, Cp, Up, *pUp; 4, Ap, Gp, Cp, pGp; 5, Ap, Gp, MeGp, pGp, Cp; 6, Ap, Gp, MeGp, Cp, Up; 7, Ap, Gp, Cp, Up, *pUp; 8, Ap, Gp, pGp, Cp, Up; 9, Ap, Gp, MeGp, Cp; xo, Ap, Gp, Cp, Up; xx, Gp, i p , Cp.

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starch gels with and without urea thus appear to be excellent for rapid and easy determination of aggregation of-otigonueleotides. From the above results and those of othersS, e it appears that the guanine content of oligonucleotides must determine in large measure whether aggregation occurs. It has been shown that the material emerging from Sephadex columns immediately after Fraction I was relatively enriched in adenine x and thus the separation of large oligonucleotides on Sephadex may be considerably more specific than if it were dependent on chain length alone. To investigate this point further, Fraction I (which comprises 2- 4 % of the total absorbancy units in a digest) and the fraction representing the next 6 % of material to emerge from the Sephadex (referred to hereafter as Fraction II A) were chromatographed b y the method of TOML1NSONAND TENERa. The results given in Figs. Ia and zb show very different patterns for the two fractions but demonstrate the presence of large oligonucleotides in both. By comparison with the data of TOMLINSON AND TENERs the size range of most of the material in Fraction I is from pentanucleotides to a possible mardmurn of octa- or nonanucleotides. The size of samples chromatographed on the DEAE-cellulose columns varied from 5oo-5o absorbancy units and the nucleotides were recovered from the ureacontaining buffer by a scaled down version (columns of DEAE-cellulose o.2 × Io cm were used) of previously described methods 8 except that I M triethyl ammonium bicarbonate 11 was used as the eluent. Cleaner maps for determination of base composition were obtained if, before alkaline hydrolysis, the desalted, urea-free nucleotides (about IO absorbancy units) were passed through o.2 × ro cm columns of Sephadex G-z5 with water as ehient. The nucleotide compositions of the oligonucleotide peaks shown in Fig. z are listed in the legend. In each instance guanylic acid was by far the major constituent of the oligonucleotides in Fraction I and in Fraction IIA adenylic acid content was predominant or approximately equal to that of guanylic acid. The authors are indebted to Miss D. MCNALLAND Mrs. J. I-LCBEN~-Rfor assistance and to Dr. C. LEWNTHAL for a gift of phosphatase and to Dr. R. HOLLEY for a gift of soluble RNA. This investigation was supported in part b y a U. S. Public Health Service Grant C-2z9o.

Department o/ Biological Chemistry U.C.L.A. Center /or the Health Sciences Los Angeles, Calif. (U.S.A.)

JOHN G. PIERCE PAUL L. ZUBKOFF

a V. M. INHRAM AND J. G. PLBRCE, Bioch6mistry, x (I962) 580. s F. F. DAvis, A. F. Cx~J.trcct AND I. F. ROUBBn% J. Biol. Clan., 234 (I959) x525. • R. MARKHAM AND J. D. SMITH, Bioc~m. J,, 52 (I952) 565. • B, MAOas~Ix AND E. CHaJtOAVV, l ~ i m . Biopl~s. Acta, 7 (x95x) 396. m H. IsHrKtmA, J. Bioci~m~. Tohyo, 52 (x962) 324. • R. K. I~LPX, W. J. CONDORS AND H. G. K ~ A , ] . A m . C~nm. 5oc., 84 (x962) 2265. 7 M. N. LXPSSTT AND L. A. HZPPBL, J. Am. C/~m. Soa,, 8 5 (I963) xI8. s R. V. TOMLINSON AND G. M. T~NER, B~oall~m~al~'/, 2 (I963) 597. o K. TALUIaswr, J. BioMwtm. Tokyo, 49 (x96x) z. xo j . G. I~BTtce ANn C. A. l ~ t n , ltioahim. Biopllys. A~a, 48 (xgax) 436. xl M. SMITH, D. H. RAMMLBR, i. H. GOLDaZlt¢~ ~ H. G. KIIOItkNA, J . Am. C/Jem. $oa., 84 (x962) 45o.

Received J a n u a r y z4th, x964 Biochim. Biophys. Acta, 87 (x964) 343-346