ISOLATION AND COMPOSITION OF PENTOSE NUCLEIC ACIDS 391 



The isolation of pure PNA is made difficult by the presence of large 

 amounts of DXA in bacterial cells. This difficulty seems to have been 

 overcome in a recently described procedure where a mixture of DNA and 

 PNA was extracted from disintegrated cells of Mycobacterium tuberculosis, 

 Mycobacterium phlei, and of Sarcina hitea, and precipitated with cetyltri- 

 methylammonium bromide.^'" PNA could be separated from DNA by 

 fractionation of the cetyltrimethylammonium salts with 0.5 M sodium 

 chloride at 0° C.^^^ In a second method PNA was preferentially adsorbed 

 on charcoal from 0.14 M sodium chloride^' and subsequently eluted by 

 means of 15% phenol at pH 7-7.5.^^^ Very recently the isolation of PNA 

 of Mycobacterium phlei by extraction w^ith 5 % NaCl and precipitation by 

 acid has been described in a short communication.^^ 



(1) Preparation of PNA from Yeast.'** The preparation was made from freshly 

 ground, defatted baker's yeast. The yeast cells (95 g.) were washed with 0.14 M 

 NaCl, and then with 50, 75, 95, and 100% ethanol. Their suspension in equal volumes 

 of 0.14 M NaCl and absolute alcohol was passed through an ice-cooled wet crushing 

 mill for bacteria and 2 vol. (240 cc.) of 70% ethanol was added to the mixture. The 

 precipitate of mostly crushed cells was washed repeatedly with 80 and 90% ethanol, 

 ethanol-ether (1:1), and ether, and dried in vacuo. It was twice extracted with 100-cc. 

 portions of 10% aqueous sodium chloride at 90° for }^ hour, and 2 vol. of ethanol was 

 added to the combined centrifuged extracts. The resulting precipitate, washed with 

 dilute and absolute alcohol and ether and dried, weighed 0.71 g. It was taken up in 

 35 cc. of water, the mixture was centrifuged, and 0.25 vol. of 20% barium acetate 

 solution (pH 7) and 1 vol. of ethanol were added to the supernatant. The precipitate 

 resulting from the centrifugation of the chilled mixture was washed with 5% barium 

 acetate and its aqueous suspension (17 cc.) stirred in a high-speed mixer in the pres- 

 ence of a small excess (150 mg.) of sodium sulfate. The solution, clarified by centrifu- 

 gation, was freed of protein by being stirred six times in a high-speed mixer with 

 chloroform-octanol (9:1) and then was poured into 2}4 vol. of ice-cold ethanol (50 

 cc.) that was made 0.05 A^ with respect to HCl. The mixture was chilled overnight and 

 the precipitate, after being washed with alcohol, was suspended in 20 cc. of water 

 and brought into solution by the cautious addition of dilute ammonia to pH 6. The 

 precipitation with acidified alcohol was repeated and the nucleic acid washed with 

 80 and 100% alcohol and ether and dried, when 0.17 g. of an almost white powder was 

 obtained. 



V. The Nature of PNA 



The final step in the preparation of PNA consists in the precipitation of 

 the material with ethanol at pH 7.0, 4.2, or 1. Consequently, PNA is ob- 

 tained as sodium nucleate, acid sodium nucleate, or free PNA. Recent 

 procediH'es have favored the isolation as sodium nucleate. ^^ In this way 



"» A. S. Jones, Biochim. et Biophijs. Acta 10, 607 (1953). 



"b S. K. Dutta, A. S. Jones, and M. Stacey, Biochim. et Biophys. Acta 10, 613 (1953). 



82 S. Zamenhof and E. Chargaff, Nature 168, 604 (1951). 



"Y. Khouvine, M. Barbier, and L. Wyssmann, Compt. rend. 236, 2118 (1953). 



" B. Magasanik and E. Chargaff, Biochim. et Biophys. Acta 7, 396 (1951). 



