January 2, 1975 Dr. Paul New Division of Oncology Providence Hospital 500 17th Avennre Seattle, Washington 98122 Dear Paul, Thanks for sending us your pretty and thought-provoking data; it has given rise to a frenzy of hypothesis-making to accaunt for the obvious discrepancy in our results, but we are still basically at a loss to explain things. We have re- axamined the possibility that our probe for the deleted sequences (cIHAx) repre- sents in the main a very small portion of the gmane. Our claim that 12% of cloned sarcoma virus RNA annsals to the probe at a 1:l ration of RNA:DNA and 16% at a 1:2 ratio is of course dependent upon the accuracies of the specific activities of the RNA and DNA. In the experiments in question the specific activities of RNB and are computtd to be about the same (20~10t;/ug); this has been independently verified by annealing the RNA to the unfractionated DNA (from which the specific probe was made) and digesting unannsaled nucleic acids with nu leases; the counts remainfng in hybrid form were the sm for RNA (32P) and IN4 ( %l ), demonstrating the equivalence of specific activities. (This result was independent of whether the hybridization was performed in RNA or IlEIA excess.) Therefore we feal obliged to conclude that the probe is a virtually uniform transcript of the deleted region. We, have results of the first thermal denaturations of hybrids formed between cDNAx and avim DNA's/ Stable hybrids are formed with chick and quail DNA's, the Tm's being identical to the Ikn's of hybrids formed with unfractiolrated cLM and chick all IZNA. The Tm is, however, depressed about 8OC for the duplex formed with duck M. This is a pleasing result from the evolutionary standpoint; we are in the process of repeating the experiment and extending it to other birds, transformed cells, etc. We would be very interested in testing whether our cDNAx is capable of annealing to your td-PrC RNA, to exclude the remote but still viable possibility that the dele- tion you are working with is different from the others we have looked at. If, for example, your td virus had a 15% deletion affecting a small region of the transform- ing gene eurd a larger region of other non-transforming gemes introduced into the cell during infection, there wwld be an easy reconciliation of our data. Would you be willing to send us about 1 vg of unlabeled RNA for this test? We wrruld like to test your sarcoma virus RNA at the same time, as I mentionsd in my last letter. Let me know if you have any mer ideas which might resolve our dilemma. It would be a great step forward in this field if we could try to work out this problem in this way without subjecting journal readers to its conEusion d gradual resolution. All of us involved in this project very much appreciate yaur frank discussion of the issues and your pbaklrpt transmission of your data to us. a-brief vacation, I will ask him to send you copies of graphs depicting the exptsri- (When Doainique returns from -2- mmts which bear upon our problem,) Best regards, Harold E. Vannus Associate Professor Department of Microbiology cc: Ik3m Mike