We have made no attempt to infect any of these tissues in culture in vitro. I am not sure that anyone else has either.

In answer to your question about the myxoviruses, we have not attempted to isolate RNA from the myxoviruses. In assembling this dissertation, I did not consider the report by Maassab, for one reason: It seems to me that to talk about infectious RNA, the number one job is to recover the virus and show that it is identical with the virus from which the RNA was obtained; this Maassab did not do.

Dr. Miroff (University of North Dakota): We have infected HeLa and ascites cells with poliovirus and followed the metabolic alterations with radioisotopes. This cytolytic virus produces a marked increase of nucleic acid and phospholipide synthesis. These alterations have been shown with the use of cytochemical techniques.

To infect ascites cells, which have been grown continuously in agent-free mice with the mammary-tumor agent, produces no detectable effect upon the nucleic acids, but it does alter phospholipide metabolism. This would lend support to the idea that the agent enters the cell, integrates with it, and produces a metabolic alteration, but unlike cytolytic virus does not alter nucleic acid metabolism.

Dr. Rubin (University of California): I should like to say a word about the morphologic alteration of chick cells to sarcoma cells after infection in vitro with Rous sarcoma virus. When we expose cells to a high concentration of virus, we observe characteristic morphologic alterations in some cells in 2 days, and in practically all cells in 3 to 4 days. In studying the multiplication of the virus under the same conditions, we find some cells initiating the production of mature virus in 2 days, but most cells initiating virus production at 3 or 4 days. Therefore, a close association appears to exist between the onset of virus production and morphologic change in the cell. This would suggest that if any analogy with temperate bacteriophages is valid-and it may be that none is-it is more likely to be an analogy with lysogenic conversion which is the consequence of virus multiplication, rather than transduction which results from the integration of new genetic material.

Radiation studies on Rous sarcoma virus infection have been amplified recently. As reported previously, cells irradiated with small doses of X ray lose their capacity to produce Rous sarcoma virus if exposed to the virus after irradiation. This is in sharp contrast to the radioresistance of the capacity of the same cells for the production of cytocidal viruses.

The high degree of radiosensitivity of cellular capacity for Rous sarcoma virus production is obtained only when the cell is infected with a single infectious unit of the virus. If the cell is infected with several infectious units, the capacity is relatively radioresistant. The significance of this multiplicity effect is not yet clear, but a similar observation has been recorded in work on the capacity of bacteria to support growth of the temperate bacteriophage lambda.

Even in single infection with Rous sarcoma virus, the ability of the cell to produce virus becomes radioresistant about 20 hours after infection. Therefore, if we assume that the early radiosensitivity is in fact a reflection of the need for the viral and host functions to become integrated (in the broad sense of the word) we must assume that the integration is completed within 20 hours.

Dr. Beard: Dr. Colter, I understand that it is your idea that the induction of the state of malignancy in these cells is associated with the change in genetic constitution, so that fundamentally the major alteration in the cells, which contributes to their malignant attributes, is this genetic change.

Dr. Colter: I think that is a fair statement of my position. What I have attempted to do is to indicate some of the ways in which that change can be induced, starting with DNA and RNA viruses.

Dr. Beard: There is a great possibility that this cannot be considered as a generalization with respect to the virus-induced tumors. It should be considered, after Dr. Bonar gives his paper, that we have seen no evidence which might indicate a genetic change in the cells of avian myeloblastosis. From this point of view, we feel quite definitely, one should be able to conceive that this process of so-called malignancy, or the exhibition of the attributes of malignancy, does not necessarily have anything to do with the change in the genetic constitution of the cell, or, to put it another way, it is not necessarily associated with mutation of the cell. In this system the myeloblasts would go on multiplying forever if one wished to maintain the cultures. Virus is transmitted from one daughter cell to the other as cell growth continues, and after 2 years the cells still look as they did in the beginning. As far as we can see without doing intimate chromosomal studies, they retain the same characteristics.

It would appear that the apparent neoplastic attributes of the myeloblasts might well be regarded as no more than an interference with the normal processes of maturation of the cells by means of which they are converted ultimately to white blood cells of the blood circulation and tissues.

Dr. Colter: I felt quite sure you were going to raise this question of myeloblastosis. I would say only that you do have a rather unique cell, and, though there is no morphologic evidence of a transformation, the myeloblasts clearly have been transformed with respect to two characteristics: (1) the capacity to support virus replication, and (2) the capacity to grow continuously in tissue culture. These are not characteristics of the normal myeloblast, so there has been, in effect, a transformation of some sort.

Dr. Black (University of Illinois): In connection with this discussion, I think the work of Dr. Armin C. Braun, of the Rockefeller Institute, on the non-self-limiting, autonomous plant tumor, crown gall, is particularly pertinent, and I would like to describe briefly his experiment. A plant tumor, crown gall, grown in culture, is obtained in a state where the cells are separated from one another. Individual cells are isolated, allowed to grow into groups of a certain size, and then grafted onto tobacco plants. In the early stages, under certain conditions, the grafted tissue shows tumor characteristics of a teratoma. If the grafts are treated in a certain way, the tissue reverts to form a normal top which will flower and produce normal seed. Thus, starting from a single crown gall cell (a plant cell that normally remains tumorous) a completely normal plant can be regenerated by a specific treatment. It seems to me that this experiment demonstrates that crown gall is not the result of a somatic mutation. (See Proc. Nat. Acad. Sc. 45: 932-938. 1959.)