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Studies on the Chemical Nature of the Substance Inducing Transformation of Pneumococcal Types: Induction of Transformation by a Desoxyribonucleic Acid Fraction Isolated from Pneumococcus Type III. January 1944.

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Title:	Studies on the Chemical Nature of the Substance [7 of 23]
Alternative Title:	Induction of Transformation by a Desoxyribonucleic Acid Fraction Isolated from Pneumococcus Type III
Creator:	Avery, Oswald T.
Contributor:	MacLeod, Colin M.
Publisher:	Journal of Experimental Medicine
Date:	1944-01-00
Subject:	Cellular signal transduction
Description:	From the Journal of Experimental Medicine Vol. 79, No. 1.
Type:	Text
Format:	text/plain
Language:	en
Identifier:	avery-pg07.jpg
Source:	Master scanned with Epson GT-10000+ flatbed scanner at 600 dpi.
Rights:	http://osulibrary.orst.edu/specialcollections/coll/pauling/dna/copyright.html
Full Text:	OSWALD T. AVERY, COLIN M. MAcLEOD, AND SIACLYN McCARTY‘43 cbanically 30 to 60 minutes.­he cells are separated by centrifugation, and the ex- traction process is repeated 2 or 3 times. The desoxycholate extracts prepared in this manner are clear and colorless. These extracts are combined and precipitated by the addition of 3 to 4 volumes of absolute ethyl alcohol.­he sodium desoxycho- late being soluble in alcohol remains in the supernatant and is thus removed at this step.­he precipitate forms a fibrous mass which floats to the surface of the alcohol and can be removed directly by lifting it out with a spatula.­he excess alcohol is drained from the precipitate which is then redissolved in about 50 cc. of saline.­he solution obtained is usually viscous, opalescent, and somewhat cloudy. Deproteinization and Removal of Capsular Polysaccharide.-The solution is then deprotcinized by the chloroform method described by Sevag (12).­he procedure is repeated 2 or 3 times until the solution becomes clear.šfter this preliminary treat- ment the material is reprecipitated in 3 to 4 volumes of alcohol. The precipitate obtained is dissolved in a larger volume of saline (150 cc.) to which is added 3 to 5 mg. of a purified preparation of the bacterial enzyme capable of hydrolyzing the Type III capsular polysaccharide (13).­he mixture is incubated at 37°C., and the destruction of the capsular polysaccharide is determined by serological tests with Type III antibody solution prepared by dissociation of immune precipitate accord- ing to the method described by Liu and Wu (14). The advantages of using the antibody solution for this. purpose are that it does not react with other serologically active substances in the extract and that it selectively detects the presence of the cap- sular polysaccharide in dilutions as high as 1:6,000,000.­he enzymatic breakdown of the polysaccharide is usually complete within 4 to 6 hours, as evidenced by the loss of serological reactivity.­he digest is then precipitated in 3 to 4 volumes of ethyl alcohol, and the precipitate is redissolved in 50 cc. of saline.eproteinization by the chloroform process is again used to remove the added enzyme protein and remaining traces of pneumococcal protein.­he procedure is repeated until no further film of protein-chloroform gel is visible at the interface. Alcohol Fractionation =Following deproteinization and enzymatic digestion of the capsular polysaccharide, the material is repeatedly fractionated in ethyl alcohol as follows. Absolute ethyl alcohol is added dropwise to the solution with constant stirring. At a critical concentration varying from 0.8 to 1.0 volume of alcohol the active material separates out in the form of fibrous strands that wind themselves around the stirring rod.­his precipitate is removed on the rod and washed in a 50 per cent mixture of alcohol and saline.šlthough the bulk of active material is re- moved by fractionation at the critical concentration, a small but appreciable amount remains in solution. However, upon increasing the concentration of alcohol to 3 volumes, the residual fraction is thrown down together with inert material in the form of a flocculent precipitate.­his flocculent precipitate is taken up in a small volume of saline (5 to 10 cc.) and the solution again fractionated by the addition of 0.8 to 1.0 volume of alcohol.šdditional fibrous material is obtained which is combined with that recovered from the original solution.šlcoholic fractionation is repeated 4 to 5 times.­he yield of fibrous material obtained by this method varies from 10 to 25 mg. per 75 liters of culture and represents the major portion of active material present in the original crude extract. Effect of Temperature.-As a routine procedure all steps in purification were carried