326 ERWIN CHARGAFF 



freshly removed thymus were cut into small pieces, mixed with powdered solid CO2 , 

 passed through a meat grinder, and treated in portions with a total of 4 1. of M NaCl 

 in a high-speed mixer for a short time. The mixture then was stirred slowly for several 

 days, until the gel was converted to a very viscous, reddish, turbid solution. The 

 dilution with water to a 6-fold volume (830-cc. portions were poured into 4.2-1. por- 

 tions of water) produced precipitation of the nucleoprotein which was stored in 0.01 M 

 citrate. The combined precipitate was washed by decantation with 8 1. of a 1% NaCl 

 solution and twice more dissolved in M NaCl and reprecipitated, as described above. 

 It was then dissolved in 5 1. of 10% NaCl and the solution, which was being stirred, 

 adjusted with saturated NaCl solution to a volume of 6 1. and saturated by the addi- 

 tion of solid NaCl. Stirring was continued for 4 days and the mixture then kept for 

 14 days. A suspension of 480 g. of Celite 545 in 1.5 1. saturated NaCl was added and 

 the mixture stirred vigorously for 24 hours. Following filtration by suction through 

 two filter papers and a layer of Celiie (and washing of the filter cake) the filtrate was 

 clarified after the admixture of "Hyflo Super-Cel" by filtration as above. It then was 

 poured into 1.5 vol. of alcohol and the fibers were washed with 70% alcohol, squeezed, 

 and dissolved in 4.5 1. of water while being stirred for several days. Precipitation was 

 repeated, this time with 2 vol. of alcohol, and the fibers were washed with 80%, 96%, 

 and 100% alcohol, and ether, and dried in vacuo over H2SO4 . The sodium nucleate 

 weighed 8 g. (1.8% of the tissue). Jt was stored in a desiccator over saturated NaCl 

 solution. 



c. Extraction with Water 



A rapid and simple preparation of deoxypentose nucleic acid, which has 

 the advantage of permitting the isolation of histone at the same time, is 

 based on the work of Crampton et al.^'^ It may start from purified nucleo- 

 histone, as described in Section II.3.a.(l), or be carried out directly. I 

 describe here a typical isolation often performed in our laboratory (based 

 on experiments of Drs. C. F. Crampton and M. E. Hodes and Miss R. 

 Lipshitz). 



(/) Sodium Deoxyribonucleate of Calf Thymus. All operations were carried out at 

 4°. Two hundred grams of fresh, well-trimmed calf thymus were triturated for 30 to 

 45 seconds in 200 cc. of a mixture of 0.14 M NaCl and 0.01 M sodium citrate in a 

 "Waring Blendor." The mixture was centrifuged at 2000 X g for 20 minutes and 

 the sediment washed three more times with the same wash fluid. The supernatant 

 liquids were discarded. The sediment was distributed with rapid stirring in 850 cc. 

 of distilled water (pH 7) and the mixture kept for 15 to 20 hours. It was stirred briefly 

 to reduce its viscosity and passed quickly through a cooled Sharpies supercentrifuge 

 at 33,000 X g. The effluent was diluted to contain about 2 to 4 mg. of nucleic acid per 

 cubic centimeter and brought to a 2.6 M concentration by the addition of solid NaCl. 

 Immediately after the addition of 2 vol. of 95% ethanol the fibers were collected by 

 spooling, pressed free of mother liquor, washed with 66%, 80%, and 95% ethanol and 

 with acetone, and dried in air. The yield corresponded to 80% or better of the phos- 

 phorus present in the original aqueous extract. The sodium nucleate usually con- 

 tained 5% or slightly more of protein. The protein impurity could be removed com- 

 pletely by one treatment with sodium dodecyl sulfate (see the next section). 



For a preparation of histone and of nucleic acid free of protein on a large scale 

 900 g. of fresh calf thymus were minced, washed with citrate - physiol. saline, ex- 



