COVTMUNIC~TIONS TO THE EDITOR
4052
to the limit of the solvent migration. Consequently, an elution chromatogram of a portion of Solution I was made by diluting with acetone and running on a silica gel column (0.9 X 30 cm.) using acetone-water mixtures. The radioactivity of the fractions was measured by counting evaporated drops of the eluate on aluminum discs with a C.M. counter. One fraction (Solution IX) behaving in the column in a manner similar to authentic B12, was then subjected to further study. This solution exhibited absorption maxima a t 2780 and 3610 A. No radioactivity could be detected in the organic phase when IX was extracted with dithizone in chloroform. X paper chromatogram of IX revealed a concentrated spot with little tailing, coincident for both the radio- and bioactivity. Tlie Rf of this spot (0.79-0.SO) was in agreement with :I BIZ standard run simultaneously. The radioactivity of the original and purified samples was shown to be Co60 (5.3 y) by measurement of the gamma ray spectrum in a scintillation spectrometer. On a G . X . counter (geometry -9%), Solution I had 6813 =t 16 cpm./y, solution IX 567 i 30 cpm./y, giving a retention value of -80%. By employing serial dilutions of I and IX against standard B12, i t was determined that the bio-activity in Solution I was 80 i 20% and in 15’3 of the standard by Solution I X was 100 weight.
1701. 73
TABLE I Wheat Germ DNA; Intact Preparation and Enzymatically Produced Cores (as moles per 100 moles P ) Constituent
Adenine Guanine Cytosine 5-Methylcytosine Thymine
Total Purine to pyrimidine ratio
Intact DNA
19%; Core
8% Core
26.3 21.8 16.2 5.8 26.1 96.2
33.2 20.0 11.8 4.3 26.2 95.5
35.4 19.8 10.3 3.6 23.4 92.5
1 ,00
1.26
1,4X
TABLE I1 Liberation of Adenine (as moles per 100 moles P) Agerit
1 iV HeSol, 100”, 1 hr. 98% HCOOH, 175”, 2 lir. 7.5 AT HClOd, loo”, 1 hr.
Intact DNA
19% Core
8% Core
26.5 26.3
31.2 33.3 33.2
31.6 35.4
Several points appear of interest. The ratio of cytosine to 5-methylcytosine remained constant in all stages, viz.,2.8. I n the intact DNA, the sum of these two pyrimidines equalled the molar concentration of guanine, a relationship observed with respect to the ratio of guanine to cytosine in almost all DNA specimens ~ t u d i e d . ~As judged from the extent of its liberation by various hydrolyzing agents, adenine seems to occur in two types of linkage, one of which is enriched in the cores (Table CHEMISTRY DEPARTMENT BROOKHAVEN XATIONAL R . CHRISTIAN ANDERSON 11). The procedures employed for the isolation LABORATORY YVETTEDELABARREand purification of the DNA will soon be discussed UPTON,L. I., N. Y. in detail. The enzyme used was supplied by the RECEIVED MAY14, 1951 Worthington Biochemical Laboratory, Freehold, N. J. The analytical methods have been described before. ENZYMATIC DISINTEGRATION OF WHEAT GERM This work was supported by research grants DESOXYPENTOSE NUCLEIC ACID from the U. S. Public Health Service and the RockeSir : feller Foundation. Previous work in this Laboratory1 has shown that IS. Chargaff, Enpericntia, 6, 201 (1050); J . Cell. Coirzp P h v i o l . , the degradation of calf thymus desoxypentose nuc- i n (3) press; Fedel alioii Pioc., 10, in press. leic acid (DNA) by crystalline pancreatic desoxy(4) E . Vischer and E. Chargaff, J . B i d . Chem., 176, 703, 715 (1948); ribonuclease proceeded according to a complex E. Chargaff, E. Vischer, R. Doniger, C. Green and F. Misuni, ibid., pattern, resulting in the formation of dialyzable 177,405 (1949); E. Chargaff, R. Lipshitz, C. Green and M. E. Hodes, fragments and of a non-diffusible core which was i b i d . , 192, in press. OF BIOCHEMISTRY characterized by greatly increased ratios of adenine DEPARTMENT OP PHYSICIANS AND SURGEONS to guanine, thymine to cytosine, and purines to COLLEGE COLUMBIA UN~VERSITY GEORGEBRAWERMAN pyrimidines. ERWINCHARCAFF XEIVYORK32, N. Y. A study of the generality of this phenomenon RECEIVED JUNE 18, 1951 appeared important, since it offers an additional tool for the differentiation between DNA preparations of different origin and for the understanding of ON THE SYNTHESIS OF CORTISONE ACETATE the relationship between nucleotide sequence and Sir : enzyme action. ’CVheat germ DNA4,highly polyWe wish to report the synthesis of cortisone merized and entirely free of pentose nucleic acid, was employed. This DNA contains an appreciable acetate from allopregnane-3P-ol-l1,20-dioneacequantity of a third pyrimidine, 3-methylcytosineI2 tate, I, made available recently from As,6 steroids, and provides thereby one more marker, useful for devoid of functional groups in ring C.’j2 Hydroxylation of the allopregnane I a t the the study of the enzymatic attack. The results 17-position by conversion into its enol acetate and summarized in Table I indicate the trend of degradation; “19Y0 core” and “Sy0core” refer to the treatment with perbenzoic acid followed by caustic dialvsis residues recovered after 81 and 927* of saponification3 yielded allopregnane-3/3,17cr-diol(1) Chamberlain, Ruyle, A. E. Erickson, Chemerda, Aliminosa, the DNA, respectively, had been convertei to R. L. Erickson, Sita and Tishler, THISJOURNAL,73,2396 (19.51). dialyzable products.
*
_I
(1) S. Zamenhof a n d E . Charraff, J . A i d . Chem., 178, 531 (1949);
187,1 (19.50). ( 2 ) G . R. Wyatt. F % x h ~ m. I , , 48, 6Xi ~1Cl;l).
(2) Stork, Romo, Rosenkranz and Djerassi, i b i d . , 73, 3546 (1951). (3) Kritchevsky and Gallagher, J . Biol. Chrm., 179, 507 (1949); Marshall, Kritchevsky, Lieherman and Gallagher, THISJOURNAL, 70, 1837 (1948).
