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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 [6 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-pg06.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:	142­RANTSTOPMATION of PNEIIWOCOCCAL TYPES The anti-R properties of the serum in the medium cause the R cells to agglutinate during growth, and clumps of the agglutinated cells settle to the bottom of the tube leaving a clear supernatant. 11+hen transformation occurs, the encapsulated S cells, not being affected by these antibodies, grow diffusely throughout the medium.¨n the other hand, in the absence of transformation the supernatant remains clear, and only sedimented growth of R organisms occurs.­his difference in the character of growth makes it possible by inspec- tion alone to distinguish tentatively between positive and negative results. As routine all the cultures are plated on blood agar for confirmation and further bacteriological identification. Since the extracts used in the present study were derived from Pneumococcus Type III, the diflerentiation between the colonies of the original R organism and those of the transformed S cells is especially striking, the latter being large, glistening, mucoid colonies typical of Pneumococcus Type III.Ÿigs. 1 and 2 illustrate these differences in colony form. A typical protocol of a titration of the transforming activity of a highly purified preparation is given in Table IV. Preparative Methods Source 31aterial.-In the present investigation a stock laboratory strain of Pneu- mococcus Type III (A66) has been used as source material for obtaining the active principle. Mass cultures of these organisms are grown in 50 to 75 liter lots of plain beef heart infusion broth. After 16 to 18 hours' incubation at 37°C. the bacterial cells are collected in a steam-driven sterilizable Sharples centrifuge.­he centrifuge is equipped with cooling coils immersed in ice water so that the culture fluid is thor- oughly chilled before flowing into the machine. This procedure retards autolysis during the course of centrifugation.­he sedimented bacteria are removed from the collecting cylinder and resuspended in approximately 150 cc. of chilled saline (0.85 per cent NaCl), and care is taken that all clumps are thoroughly emulsified. The glass vessel containing the thick, creamy suspension of cells is immersed in a water bath, and the temperature of the suspension rapidly raised to 65°C. During the heating process the material is constantly stirred, and the temperature maintained at 65°C. for 30 minutes.¡eating at this temperature inactivates the intracellular enzyme known to destroy the transforming principle. Extraction of Heat-Killed Cells.-Although various procedures have been used, only that which has been found most satisfactory will be described here.­he heat killed cells are washed with saline 3 times.­he chief value of the washing process is to remove a large excess of capsular polysaccharide together with much of the pro- tein, ribonucleic acid, and somatic "C" Polysaccharide. Quantitative titrations of transforming activity have shown that not more than 10 to 15 per cent of the active material is lost in the washing, a loss which is small in comparison to the amount of inert substances which are removed by this procedure. After the final washing, the cells are extracted in 150 cc. of saline containing sodium desoxycholate in final concentration of 0.5 per cent by shaking the mixture me-