a , few (oe - ‘ . . nel - x -_ j ay WESTERN RESERVE UNIVERSITY FEE CLEVELAND. OHIO 44106 DEPARTMENT OF BIOLOGY TELEPHONE: 231-7700 AREA CODE 216 jor 7, 1966 bnclier- ts & teqe - Me Aeyt Z£ amen 7 Acie vo Len Lelia . Sevtal ew “ 2 putgt — ~ wr . cai r7 /% , ee . 2 Lue hte ee" g S Lite Ai tawtuy ¥ A t- Lig retardant hy , be Get A “ Lf ete fret we feet ’ eete-of ewe!” ewelsetd the mig ther 8 £ é 2 7” . . a a £. * “ ane mcr Fie ee Pact pray el, eck we he 1 — moreno Sg haewsiee Wt eben reaetter caret 9 wed 2 PP BK Coed ) “nm Aa 4 bruptasant woth tf we ttf HIS. . e . 4 -# 7 . Tow CE a ee LF lt Ceteh. (theta Fitep te tO eben etic th J ; aa i » . : , as ke pb ces te Anat ert Oe bo PEELE pptn’ cat oe OS eee ee 2 . ww 4 thee ret ae . Ve ets ABtal; Poth Ch me “wes pu wef awh ite “1 So ae : ~ ~ Ug , _ , . F ? ot : A af \, EE ee eee Ae Pp Cartt Meteo { ‘ Lt rs f * top , etd tot wie leet Fe - . ~*~ Lo mee ete £O 2mm OPTI M PM ctebe A Leet ett tt Seiten” et 2 cea gr ~ a off r . <7 wy i, oe hao rod ft, 4h —-Woeg > eee ee beh 8 oy . a a v . - tye te po COR RO Cb Ok Oe og ee a ‘? : . wor . eral : a FF “f, a 4 ee A, 7c Pe ct heh DT eee ees cate we v . AN ASSOCIATE IN THE UNIVERSITY CIRCLE a. <> aoe id at sists Naty tey pete ta fo. f an HON RIAA , PROGRAM Ban tap suten hums frnty announcing that the arteve, principle was,in all probability,a desoxyrib- nucleate. The proof brought to bear on the identity of the factor was summarized by Avery in a letter written to his brother,also a medical bacteriologist,in 193: * The active substance is not digested by crystalline trypsin or chymotrypsin,it does not lose activity when treated with crystalline ribonulcease which specifically breaks down yeast nucleic acid. The Type III polysaccharide can be removed by digestion with the spefific Tyep III enzyme without loss of transforming activity of a potent extract, Lipids can be extracted from such extracts by alcohol and ether at minus 12%C without impairing biological activity. The extract can be deproteinized by t the Sevag method (shaking in chloroform and amyl alcohol) until protein free and Biuret negative. When extracts,treated and purified to this extent but still containing traces of probein,lots of C (somatic) cai bohydrate, and nucleic acids of both yeast and thymus types are further fractionated by dropwise addition of absolute ethyl alcohol an interesting thing occurs. When alcohol reaches a concentration of about 9/10 volumes there separates out a fibrous substance which on stirring the mixture wraps itself about a glass rod like thread on a spool and the other impurities stay behind as a granular precipitate. The fibrous material is redissolved and the process repeated several timex, In short this subs:ance is highly reactive and on elementary analysis conforms very closely to the theoretical values of pure desoxyribonucli¢,’é/ acid (Thymus type). (Who could havd guessed it?). This type of nucleic acid has not to my knowledge been re- cognised in pneumococcus before,though it has been found in other bacteria," In the same letter, Avery stated that of a number of crude enzyme preparations from various sources,only those which contained a very active depolymerase dewh ay were able to imactivate the transforming factor. The purification and identification of this polymerase pas to be the > next stepping stone in the advance toward certitude of the identity of the transforming agent. Finally,Avery contuded his letter in this fashion: yet "If we are right,and of Course that is not,proven,then it mean# that nucleic acids are not merely structurally important, but functionally TASOLEAKE/AA/HELL active substances in determining biochemical activites and specific characteristics of cells,and that by means of a known chemical substance}t is possible to induce predictable and hereditary changes in cells. 8 is something that has long been the dream of geneticists. The nuttations they induced by X-rays and ultra-violet are always unpredictable, random,and chance changes. If we prove to be right -=- and of course that - is a big if-- then it means that both the chemical nature of the inducing stimulus is known and the chemical nature of the substance produced is also known,the former being thymus nucleic acid,and the latter,Type ITI polysaccharide,and both are thereafter reduplicated in the daughter cells and after inumerable transfers,Without further addition of the inducing agent the same active and specific transforming substance can be recovered far in excess of the amount originally used to induce the reaction. Sounds like a virus--maybe a gene. But with mechanisms I am not now concemed, One step at a time and the first step is what is the chemical nature of the transforming principle? Someone else can work out the rest, Of course the problem bristles with implications. It touches biochemistry of the thymus type of nucleic acids which are known to constitute the major part of chromosomes,but have been thought to be alike regardless of origin and gf species. It touches genetics,enzyme chemistry,cell metabolism and carbo- hydrate synthesis. But today it takes a lot of documented evidence to convince anyone that the sodium salt of desoxyribonucleic acid,frotein free, could possibly be endowed with such biologically active and specific properties,and that is the evidence we are nowtrying to get. Its lots of fun to blow bubbles but it is wiser to prick them yourself before someone else tries to," | : dxf fus 2 On the basis of these results,a vast, program of research could have 7X been organized. ———~In fact,what was projected was gf the continuation of the strategy which Avery had practised throughout his entire scientific life:that of eliminating the most important d¢f¥%s obstacle in the way of ronan nr —— a generalization. This obstacle was,as he saw it,ythe doubt as to the chemical ———— A —_——— identity of the transforming principle,or TP,as it was now familiarly called. This doubt took two forms: one was the possibility that trace contaminants We «@ the DNA were responsible for biological activiby,and the other was the . dourthakn 4 brelaytet aw possibidity that serum,necessary for successful, biesseey was playing a role of primary imporsance in the reaction,forming a nucleoprotein,for example, which was the active material rather than the DNA itself. With the purification of the enzyme desoxyribonuclease, achieved by MéCarty in 1946,a new and powerful tool was made avallable for transformation studies. The enzyme proved to be activated by Mg and Mn. Minute amounts of oo desoxyribonuclease rapidly destroyed all activity of the purified DNA endowed with the Type III transforming activity,provided appropriate . amounts of the activating ions were present. If they were absent,or with- ~_— — need drawn by a chelating egent,no inactivation of TP occurrred. Although -_ damaged transforming activity of DNA was A¢ft¢oféd before measureable changes in ; —aney viscosity could be detected,the conditions under which inactivation occurred were identical with those under which depolymerization took place.