April, 1951
DESOXY-5-METHYIXYTIDYLIC
a reflection of the general lability of purine desoxyribosyl compounds rather than an indication of the position of esterification of phosphate on the desoxyribose residue for, as shown in Fig. 4, the phosphoester linkage of desoxy-
[CONTRIBUTIONPROM
THE
ACID FROM THYMUS NUCLEICACID
1539
adenylic acid is more labile in 1 N sodium hydroxide than yeast or muscle adenylic acids.
6, OHIO CLEVELAND
RECEIVEDAUGUST28, 1950
BIOLOGY DIVISION,OAK RIDGE NATIONAL LABORATORY]
The Isolation and Identification of Desoxy-5-methylcytidylic Acid from Thymus Nucleic Acid’ BY WALDOE. COHN Ion-exchange fractionation of enzyniatic digests of DNA from thymus has resulted in the isolation of a desoxynucleotide of 5-methylcytosine in amounts small compared to the amount of cytidylic acid present. This conlirms the earlier reports on the isolation of 5-methylcytosine from acid digests of DNA and suggests that this base occurs in desoxyribonucleic acids as a nucleotide similar to the others, being released upon enzymatic digestion. new nucleotide present in the original desoxycytidylic acid. Introduction The desired fraction was concentrated by reabsorption and Since the time that Johnson and Coghill re- elution with 0.1 M formic acid. All subsequent tests were portedza the isolation, from an acid hydrolysate of carried out upon this formic acid solution which contained tuberculinic acid, of a substance crystallographi- approximately 2 mg. per ml. Phosphate’ and desoxypendeterminations8 upon the nucleotide and spectrophocally identical with synthetic 5-methylcyto~ine~~~ it tose tometric comparison of the derived base with synthetic 5has been an open question as to whether this sub- methylcytosine, kindly supplied by Dr. Hitching9 of Wellstance was a bona fide constituent of nucleic acids come Laboratories, were carried out in this Laboratory, as in the usual combination with a pentose phosphate. were all ion-exchange tests. Paper chromatographic of the nucleotide, the derived base, and the enJohnson and H a r k i d demonstrated that none of analyses zymatically deaminated1@derivative of the base (thymine), this material could be isolated from “yeast nucleic as well as spectrophotometric comparison of both bases with acid.” Aside from this observation, there was synthetic substances, were most generously made by Drs. no follow-up of the original finding; it remained an Erwin Chargaff and Jacob Kream of Columbia University. uncorrelated oddity until very recently when Results Hotchkiss o b s e r ~ e din , ~ the base mixtures derived (a) Identification of the Base.-Hydrolysis of from desoxyribonucleic acid preparations, a small the nucleotide was accomplished by heating for amount of a cytosine-like base which he termed 40 minutes on a steam-bath in 72% perchloric “epicytosine.” Although it seemed likely to him acid.loa The digest was diluted tenfold with water that this substance was 5-methylcytosine, the and poured through a 1 cm. X 1sq. cm. bed of sulamounts available for study were too small to per- fonic-acid type cation-exchanger to recover the mit identification. pyrimidine base which was then eluted with amIn the course of isolating gram amounts of the de- monium hydroxide. This substance had the specsoxyribonucleotides from enzymatically degraded trophotometric properties, compared to synthetic DNAJ6the presence of a hitherto unobserved sub- 5-methylcyto~ine,~ shown in Fig. 1. It could not stance was detected in the desoxycytidylic acid be absorbed upon a strong base anion-exchanger exfraction.6 From approximately 800 mg. of the cept in the hydroxy form, which is characteristic of crude desoxycytidylic acid, enough of this sub- cytosine alone among other nucleic-acid bases. l1 stance (ca. 20 mg.) was isolated to permit identifiHydrolysis12in 99% formic acid a t 175’ for 2 cation of it as the desoxypentosyl phosphate of 5- hours followed by paper chromatographic analysisla methylcytosine. The isolation and identification in butanol-water for 16 hours gave but one compoof this nucleotide are described in this communica- nent detectable by ultraviolet fluorescence. l 4 This tion. component lay in the same position as 5-methylExperimental cytosine (RF 0.28), which differs from cytosine The source of 5-methylcytidylic acid was the desoxycyti- (0.22), uracil (0.36) and thymine (0.52). When dylic acid fraction prepared from thymus DNA by Volkin, eluted from the paper, it had a spectrum identical Khym and Cohn.6 The separation was performed on the same ion-exchange column (33 sq. cm. X 12 cm. 200-500 with that of synthetic 5-methylcytosine. Another mesh strong-base anion exchanger, in formate form) with portion of the formic acid hydrolysate, containing 0.003 M formic acid. The 5-methylcytidylic acid fraction 159 pg. of the pyrimidine, was incubated with puriwas reabsorbed on a smaller column and refractionated to remove the last traces of desoxycytidylic acid; this was necessary because of the very low amount (ca. 3%) of the
(1) Work performed under Contract W-7406-eng-28 for the Atomic Energy Commission. (2) (a) T. B. Johnson and R. D. Coghill, THIS JOURNAL, 47, 2888 (1925); (b) H. L. Wheeler and T. B. Johnson, A m . Chcm. J . , 31, 591 (1904); cf. ref. 10. (3) T. B. Johnson and H. H. Harkins, THISJOURNAL, 61, 1779 (1929). la R. D. Hotchkiss, J . Bid. Chem.. 176, 315 (1948). (5) E. Volkin, ‘J.!X.Khym and W. E. Cohn, THISJOURNAL, 71, 1633 (1951). (6) W.E. ‘Ahd, .t
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1.0
0
Q,
E 0.8 0.6
G1 7.0
-
7.5
8.0
8.5
P H-
Fig. l.-hwer half; acetyl-L-tryptophsnamide, [SIo = 10 X 10-8 M, [El 0.208 mg. protein-nitrogen per ml.: upper half; nicotinyl-ctryptophanamide, [SI0 10 X 10-8 M, [g] 0.069 mg.protein-nitrogenper ml.
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