390 B. MAGASANIK 



to diminish above 0.3 M, owing probably to the rapid coagulation of virus before 

 cleavage of the nucleic acid. Presumably for the same reason, salts of polyvalent 

 metals, such as magnesium or aluminum chloride, gave low yields of nucleate. Heat- 

 ing times from 15 seconds up to 10 minutes at 100° were investigated and it was found 

 that 1 minute was optimum, the yield of nucleate being virtually quantitative at this 

 point. The yield of nucleate was greatly reduced when, owing to the size and shape of 

 the reaction vessel or to a diminution of the heating, the temperature within the 

 reaction mixture failed to rise above 99°. Hence, the optimum temperature seems to 

 be about 100°, although temperatures higher than 100° were not studied, and the 

 critical temperature between 96-100° was not ascertained. The concentration of virus 

 in the heated mixture affected the cleavage, as previously noted by Cohen and Stan- 

 ley." Moreover, when the concentration was high, there appeared to be more nucleo- 

 protein in the final preparation than when the final virus concentration did not exceed 

 20 mg. per cubic centimeter of salt-virus mixture. 



4. Isolation of PNA from Microbial Tissues 



PNA was first isolated from yeast, and yeast is still the preferred start- 

 ing material for PNA preparations. However, in most procedures for the 

 isolation of yeast PNA alkali is used, and the preparations are of low 

 molecular weight and have lost a portion of their pyrimidine nucleotides. 

 It is questionable whether any of the procedures described so far for the 

 isolation of PNA from yeast can result in the production of a preparation 

 that has escaped major degradation. (3ne reason for the failure to obtain 

 better preparations from a material as rich in PNA as yeast, is the diffi- 

 culty of breaking the wall of the yeast cell. Unless the cell is dried by 

 extraction with organic solvents such as ethanol or acetone, it is impossible 

 to isolate the major portion of the nucleoproteins. This rough treatment is 

 in all likelihood responsible for an extensive degradation of the PNA. The 

 nucleoproteins may be extracted with saline, purified, and fractionated by 

 isoelectric precipitation, and PNA prepared from the nucleoprotein frac- 

 tions by treatment with 10% NaCl.-^-^^ Another method consists of 

 grinding the defatted yeast in a bacterial mill, followed by extraction of 

 the PNA with 10% NaCl. This method is given in detail below.^^ Loring 

 and his collaborators used' short treatment with dilute alkali in the cold to 

 extract PNA from yeast. ^^ 



Little is known about bacterial PNA. Bernheimer^^ was able to show 

 that pentose nucleic acids from Streptococcus 'pyogenes, Clostridium welchii, 

 and Escherichia coli were inhibitors of group A streptococcal deoxyribo- 

 nuclease; PNA preparations from mammalian tissues, wheat germ, and 

 yeast failed to show inhibition, while tobacco mosaic virus PNA was 

 inhibitory, but only in relatively high concentrations. 



*^ Y. Khouvine and H. De Robichon-Szulmajster, Bull. soc. chim. hiol. 34, 1056 



(1952). 

 <>» H. S. Loring, J. L. Fairley, and H. L. Seagran, J. Biol. Chem. 197, 823 (1952). 

 " A. W. Bernheimer, Biochem. J. 53, 53 (1953). 



