UNIVERSITY OF CALIFORNIA SAN FRANCISCO MEDICAL CENTER BERKELEY DAVIS - InVIh'E * LOSANCELES o RIVERSIDE - SANDIECO o S'.dFHANCISCO SANTA nAneAM o SANTA CRUZ SCHOOL OP MEDICINE February 11, 1972. SAN FRANCISCO, CALIFORNIA 94 I 22 Dr. Paul Neiman Oncology Division Depa rtmen t of Med ic i.n e UniversLty of Washington Sea t t 1 e, Wa s h in g t on Dear Paul: Thank you for sending your grant proposal. I have read it with interest and have shown it to Mike Bishop and to Chris Hansen, a graduate student working on the same problem. Needless to say, the results you present appear very promising. One of our concrrns is that you may not bc using sufficiently hidl ratios of DNA/RNA to detect maximal hybridization of the RNA. For examplr-, in the "low CotTf ex- periments, you use a 10/1 ratio of DN1?/INA (the ratio may be higher in Figure 6 but you don't provide the specific activity of the WJA); aq.;uming the mole- cular wcipht of the RSV genome to be lo7 daltons and of the&h genome about 8 x IO" daltons, at a 20/1 ratio the concentration of RSV sequences would be equal to the concentrntfon of cell seqiiences represmted 4000 times per genome. Of course, it is likely that only a small fraction of your RNA is actually re- presented in repetitive sequences, so that the ratio is probably 10-100 fold better. The C0t1/2 of 20 suggests you are detecting a family of about 1000 copie.;; i € 10% of thP Rh" were homologou.: to this DNA, you would have a 2.5/1 ratio o EVA/ A. Since Melli and ishop indicate that DNA-DNA inter- actions are Urr mb,-DNA interactions and since this is particularly so when your DNA is not adequately sheared, it is conceivable that much less than 10% of the RNA would form hybrids. Our view, therefore, is that you may not have maximized the low Cot anncstjng Page 2 expected size of the viral genome. It weald be uqrftil to know when the DNA in yoiir expertments is reannealin&, mainly as a check on your claim that the cRNA is copied from unique sequence DNA. (We would interest4 in learning more of the details of preparntion of the cRNA}. Tt wor~ld also he useftif to know that the riepurinstion procedure, somewhat unusual approach to UNA shearing, was not aEfecting the reassoci-tton kinetips of the DN!. - of course, the stability of your hybri.ds suggests that apprecinhla untoward ef fects have not occurrrd. procedure and its reliability). (Agein, we would br interested in the details of the depurination Chris has not gotten as far as you have wfth this 3pproarh. Hc has heen dcaling with some of the logistical probl~ms, aiming for viril1 i-32 of very h-lgh specific DNA. (It seems to me, by the Wav, thr3t YOU might hc* ~I~JIP to increasc your specific activity by rising more lll-uridine, which ic; relatively cheap). activity (greater than 3 x 10 5 cpm/,tp} and largc amounts of uniqrie sequence cell I have mulled Over the difEcrenccs between your rcsrilts and mine and at pre- sent see no easy reeonciliatibns. If the double-str,~rrded DNA prohe were, in fact representatiweof the whole genome, then T i~0111d calciilatc only 1-2 copics per chick cell. However, then what could T makc. of the slowly re- association product which is 4-5 times rune cornplr'u than the bulk of the DNA? and how would 1 account for the discrepancy hetwet-n our results and classical findings with reassociation kinetics? and hov could 7 explain cell Ifnes from quail or rat which appear to haw 4 or 2 copips witti current calculations and would then appear to haw only fractlions of genome? or genomes in only some of the cells? (Gclb et a1 have this problem wtt-h SV-40 sequence detection in 3T3 cells), -I_ Our most interesting recent finding is an apparent absence of RSV aequencrs in noma1 7T3 cells, with about 7 copies per diploid cell in B77 and Schmidt- Ruppin transformed 3T3 lines. The dctection I.; difficult and we're not saying too much about this until we perfni-m qomf' Pxperimmts to give us qualitatfve (YESINO) results as well as shifting of Cot. curves. Again, thanks for the proposal - let's keep in touch. Have you tried normal chick DNA yet? 1 Yours, Harold E. Varmus Department of Microbiology P.S. We'd like to see reprints of your articles you refer to. HEV : bb