. 5. Unknown factor - suggest&by fact that some sera containing anti-R and enzyme heat inactivated still fail to support transformation. Nature of unknown factor undetermined. 6. Properties of serum - stable; may be stored in refrigerator many months and retain original effectiveness. Becognition of serum factors and properties facilitated standardization of cultural conditions required for consistent and reproducible results. c. K strain (iU6A) - derived from Pneumococcus Type II D. (continued) All cultures plated on blood agar for further identification. i 1 Differentiation of ~colonies of parent E cells and those of 0 ,` )#F' !,' /$ transformed S organisms striking - latter large, glistening ,' i .-/ mucoid-typidal Type III. ..Ac", . 'L. Slide 2 - Colonies 1__--- "^ III. Preparation of transforming principle A. Source material --. .II ^ 1. --- 1. 75 liter lots of culture of Type III pneumococcus. Young, actively growing (16 hr.) - -. 2. Collected on Sharples centrifuge. -_-, .--- - 3. Cells resuspended in saline - heat-killed 65%. 30' This temperature inactivates enzyme known to destroy t. p.' B. Extraction ____ - ..,.-. x_ .-.. . 1. Heated cells washed 3 times with saline, removing large excess capsular pobysaccharide, much protein, ribonucleic acid and.C polysaccharide - lo-15 per cent loss of transforming material. 2. Extr.acted with saline containing 0.5 per cent sodium desoxycholate, by mechanical shaking. Repeated 3 times. 3. Extracts combined , precipitated by excess ethyl alcohol. Ypt - floX'%g fibrous mass. iIedissolved in saiine. C. Deproteinieation and Removal of S polysadcharide -. I_, 1. Preiiminary deproteinization by Sevag chloroform,,method. About 3 times rc -_,, r_..-. .. '1 2. SIIb Enzyme hydrolyzes Type III capsular polysaccharide. Enzymatic l.5i%&down usually complete 4-6 hours - evidenced by loss of serological activity. 3. Heprecipitated by alcohol. Deprcteinization repeated until no further protein-chloroform get at interface. D; Alcohol fractionation ~~"-.."~...,'.,".;..i~-.~.,,,,.~~" ". ),( ..,.,,_;.._ 1. Drapwise addition of absolute ethyi alcohol with constant stirring d. fit critical concentration - 0.8 - 1.0 volume - fibrous strands separate out and collect on rod. 3. Repeated 4-5 times 4. Yield of fibrous material = lo-25 mgm. per 75 liter lot f major portion of active material in crude extract. E. Effect of temperature 1. Extraction less efficient but activity best preserved when procedures are carried out at OO-4%. IV. Analysis of Purified Material A. General properties 1. Saline solutions (1 mg,/cc) - colorless, viscous, clear in diffuse light. In strong transmitted light, silky sheen on stirring. 2. Preservation - shline solutions retain activity 2-40~. at least 3 months. Longer periods in frozen state in CO2 cabinet&-y? "a*' In aqueous solution rapid decrease in activity; completely inert in few days. Material precipitateaxom$aline solutio&byLa&$So&o$ and - w.%-."~,~^_"~II_I.x.Iy_~~ stored under alcohol remain active for long periods. ++.".-....urru"w~~-~ ~~*""~-..vm. .~/) _Y. r..m.---7 3. Effect of temperature - withstands 30-60 min. 65~. Higher temperatures not tested. 4,. Effect of pH - activity rapidly Best preserved at neutral or s B. Qualitative Chemical Tests 1. Biuret and Millon tests negativ - 2. Dische diphenylamine reaction f 3. Orcinol test (Bial) for ribose concentrations pure prep-0 animal origin prepared by diff corresponding intensity. 4. Lipids - no specific tests. Cr with alcohol and other -12oc. Repeated alcohol precipitation results in no decrease in acti c. Elementary Chemical Anaiysis (Dr. El Slide 3 N/P ratio varies from 1.58 - 1 Close agreement with that talc structure of sodium desoxyribl N/P ratio indicates little pro' N or P present, otherwise XA& 4-g i 2, units linked in glycosidic union (22). The presence of the newly formed SC&F irr: .cs + :$ t"ii,neE;v& p capsule containing this,,,type-specific polysaccharide confers on the transformed cells all the distinguishing characteristics of Pneumococcus Type III. Thus, it is evident that the inducing substance and the substance produced in turn A are chemically distinct and biologically specific in their action and that both are requisite in determinina f.ha txrm sner?i:'ic.it.v nf t,ha of which they form a part. *"-,"&---- .A""-- ----"The experimental I I nucleic acids, at least the b specificities as evidenced t J 1 / principle, Indeed, the po: I in biological behavior of r e I but has never been experime lack of suitable biological transformation appear to af , of this hypothesis, and the in favor of this point of B L-....,. ---,-_ _-.-a- I .^1%# If it is ultimate transforming activity of t2 MS. DIV. AI;1 NO. . ! substances, it appears that the techniques employed in the study of transformation are the only ones available at present for testing possible differences in the biological behavior of nucleic acids. ,, Attempts to induce transformation in suspensions of resting cells held under conditions inhibiting growth and multiplication have thus far proved unsuccessful, and it seems probable that transformation occurs only during active reproduction of the cells. Important in this connection is the fact that the R cells, as well as those that have undergone transformation, presumably also all other variants and tvapsof MS. DIV. J,C, NO. principle interacts with the R cell giving rise to a coordinated series of enzymatic reactions that culminate in the synthesis of the Type III capsular antigen. The experimentalfindings have clearly demonstrated that the induced alterations are not random changes but are predictable, always corresponding in type specificity to that oL c the encapsulated cells from which cL- substance was isolated. Once transformation has occurred, the ne characteristics are thereafter transmitted in series through innu transfers in artificial media without any further addition of th agent. Moreover, from the transformed cells themselves, a substa identical activity can again be recovered in amounts far in exces originally added to induce the change. It is evident, therefore, only is the capsular material reproduced in successive generation the primary factor, the transforming principle itselfzdwhich contr occurrence and specificity of capsular development, is also redq the daughter cells. The induced changes are not temporary modifi are permanent alterations which ;persist provided the cultural COT favorable for the maintenance of capsule formation. The transfol can be readily distinguished from the parent R forms not alone b;! reactions but by the presence of a newly formed and visible caps1 MS. DIV. 6. AC. NO. the immunological unit of type specificity and the accessory structure essential in determining the infective capacity of the micro-organism in the animal body. It is particularly significant in the case of pneumococci that the experimentally induced alterations are definitely correlated with the development of a new morphological structure and the consequent acquisition of new antigenic and invasive properties. Equally if not more significant is the fact that these changes are predictable, type specific, and heritable. Various hypotheses have been advanced in explanation of the nature / of the changes induced. In his original description of the phenomenon Griffith (1) ,suggested that the dead bacteria in the inoculum might furnish " `( +$a@+.$$ ~~~~~~~.,~ _ -I some specific protein that serves as a llpabulum" v A' ""r the R form to manufacture a capsular carobhydrate. Bore recently the phenomenon has been interpreted from a genetic point of view (26, 27). The inducing substance has been likened to a gene, and the capsular antigen which is produced in response to it has been regarded as a gf (27) h; mutation - and it is difficult to avoid so describing it - we are dealing with authentic cases of induction of specific mutations by specific treatments . .." Another interpretation of the phenomenon has been suggested by Stanley (28) who has drawn the analogy between the activity of the transforming