Irprll 24, 1973 Dr. Ian Macpherson Imperial Cancer Reaearch Fund Lincoln's Inn Fie1 ds London, WC 2, England Dear Dr . Macpherson, I am sorry we have been so long in relaying informatmn about the hamster revertant experiments, but each week it has seemed as though one more bit of data would give as a clearer idea of what to do next. To begin with the DNA vou recently sent: unfortunafely, the DNA was clearly, upon arrival, not sufficiently intact to perform convincing integration studies. It was only minimally viscous at a high con- centration, only 30% of the DNA entered into networks (usually 80- 95% of mammalian DNA forms networks), and in alkaline sucrose gradients (see Figure 1) the sedimentation vaiue proved to be 20-25 S in relation to ailDNA (53s) marker. Thus, the DNA had a single stranded weight of approximately 3 x 106. findings with B77 transformed 3T3 cells (see Lepetit manuscript, Fig. lo), we expected that most of the integrated RSV DNA would have been deteched from reiterated sequence DNA. Thus, the network fractions would be depleted if virus-specific DNA. This is, in fact, observed in Figure 2, and the amount of RSV DNA in transformed and revertant '.- G'Lr''.. DNA seems about the same. UNA is integrated in both cell types and, roughly speaking, in the same pattern, since equal reduction in size of the cell DNA equally reduces the amount of viras-specific DNA covalently linked to re- iterated sequences. However, it would be nice to demonstrate intebration more convinclnglv with very high molecular weight DNA. have more luck, in view of the distance between us, if you could send 2 x 108 or more cells of each type in "DNA buffer" (0.02M Tris, pH 8, i).OlM EmA, 0.1M NaC1) after lysis of 5 x 106-107 cells/ml with 1% SDS. will be all right and we'll finish the extraction tiere, make networks, and assay. Based upon our previous Thus, we have an - indication ____- that the viral I think we might I think air mail shipping at room temperature (ao - not freeze) Things went rather more smoothly in the RNA department. As shown by the experiments in Fig. 3, there is clearly mc;re annealing of labeled single- staanded DNA (synthesized by Schmidt-Ruppin virus palymerase) to RNA from transfc7nned cells than to RNA from revertant cells. The amount of virus specific RNA are slrall - approximately 0.0001% of the cell RNA, or about 1 molecule per cell - but the results are Mac pher son/Varmu s 4- 24- 73 P* 2 reproducible. A reaction with B77 transformed 3T3 ce;ls is shown for cmparison. HeLa RNA, as you can see, does not react at all. At present, this reaction has been pughed to its loyisrical limits: 20 mg/ml of RNA, 0.6 M Na+, 100 hour &&ekbatien. another shipment of RNA from these cells - approximately 2-10 times the amcunt in the first shipment - we could attempt to augment the reaction by running the RNA through a poly dT-cellulose column. This technfque provides a fraction of RNA 3-20 fold enriched for virus-specific sequences, allowing us to carry the reactions to higher ''Cot" values. In addition, we learn whether the virus-specif!c RNA bss poly A attached in these cells. We are, in addition, pursuing several relevantpmatters in R77/3T3 cells: presence of virus-specific RNA in polysomes; nuclear versus cytoplasmic location of RSV RNA; and a competition assay between cell RNA and labeled 70s RNA which permits a precise estimate of the amount of the genome transcribed. (The unequal copying of RSV RNA intc sfngle-stranded DNA, even in the presence of actinomvcin, when the entire genome is copied, albeit assymetrically, forbid equation of the extent of transcriptlvn with the percentage of single stranded DNA hybtidieed t perhaps we cnn pursue them in hamster cells if they seem appr0priate.y If it were possible tc receive cell RNA.) When these techniques are well worked out, In particular, nuclear/cytoplamic fractionation appears promising in regard to B77/3T3 cells, slrnce nuclear RNA seem8 considerably enriched in RSV RNA. The possibility that RSV RNA never gets to the cytkplasm in the revertant wLll is a titillating one, and we would like your opinion about whether yoir'd want to do the cellular fraction there or to send the appropriate living cells to us. I am tentatively expecting to be briefly in London abcwt the middle of July. If this happens, I hope we will have a chance to discuss these matters further. I will send you explicit dates when I have them. Again, thanking you for your efforts in this interesting venture, I am, Yours, Harold E. Vap~us, M.D. P.S. Mike sends his regards. HEV: js encl .