Published Papers

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.

Page 05 [141]

< Previous Page     Page 04 [140]

Back to Document Index Studies on the Chemical Nature of the Substan...

Next Page > Page 06 [142]

Title:	Studies on the Chemical Nature of the Substance [5 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-pg05.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 TF AVERY, COLIN MF f ound to be present arid highly active in the autol sates of a nunabcr of different rMY trains. The fact that this intracellular enzyme is released dung autal ss .txplain, in part at lmt, the observation of Dawson and¢s () that it is essential in bringing about transformatioTi in the test tube to use a small ino¥um of young and actively growingœells. The irregularity of the results and often the failure to induce transformation when large inocuia are used may be attributable to th release from autol zin cells of an amount of this enzyme sufficient to destroy the trans- forrning principle in the reaction system. In order to oŸ n c nSist Dt and reproducible rosultS, two facts must be borne in mind: first, that a nœulture can undergo 5pontnneous dissociation and give rise to other variants which have lost the capacity to respond to the traasf o -rnin¬timulus-- and secondly, that pneurnoco ccal cells contain a n intro cells lar enzyme which¡en released destroy­he activity of the trans- forming principle.œonsequently, it i important to select a responsive strain and to pre e n t. a s fax as possible the destructive changes associated with autolvsis. Method of 'ilralt'on of ''ra sfor iftœti t .-Jn the isolation and purifica- tion of the active principle from crude extracts -of pr e u mo co ccal cells it i s desirable to have a method for determining quantitatively the transforming activity of various taiction s. The experimental procedure used is as follows: Sterilization of the material to be tested for activity is accomplished b­he use of alcohol slues it has bem found that this reagent bas no effect on activity.š pea-slared volumeŸ extract: is precipitated in a sterile centrifuge tube by the addition of 4 to 5 volumes of absolute ethyl alcohol, aDd the mixture is llo e-d to staTid 8 or more hours in the refrigerator in order to effect sterilization. The alcohol precipitated material is centrifuged, the supernatant discarded, and the tube containing the precipitate is allowed to drain for a few minutes in. the inverted position to remove excess alcohol. The mouth of the tube is then carefuHy flamed and a dry, sterile cotton plug is inserted.­he precipitate is redis- solved in the on gig a.l volume of saline.¬terilization of active material b­his technique has invariably proved effective. This procedure avoids the loss of active substance which may occur when the,¥ution is passed through a Berkefeld filter or is heated at the high temperatures required for sterilization. To the charcoal-adsorbed broth described above i s added 10 per cent of the sterile a.sc'tic o r ~kural fluid which has previously been heated at 60* C. for 0 minutes, in order to destroy the enzyme knovm to inactivate the transforming principle. The enriched medium is distributed under aseptic§d i tiom in…2e0 cC. amounts in sterile tubes measuring 15‘00 mm. The sterilized extract is diluted serially in saline neutralized to pH 7.2-7.6 bšddition of 0.1 --K NaOH, or it may be similarly dilated in m/40 Phosphate buffer, p1l 7.4..œc. of each dilution is added to a t least¨r 4 tubes of the serum medium. The tubes are then seeded with a 5 to¡our blood bath culture of 136A. 0.05 cc. of a 10-1 dilution o f this culture is added to each tube, and the cultures are incubated at 37'C. for 1­o 4 hours,