Dr. Stanley (University of California): Dr. Colter has made a beautiful summary of the implications of infectious nucleic acid, and I can add very little. I would like to call the attention of this group to some of the new work that is coming out in the plant virus field, starting with Dr. Gierer and Dr. Mundry's work in Tubingen, Germany, on artificially produced mutants with the use of nitrous acid, in which they showed that through the conversion of cytosine to uracil by nitrous acid treatment they obtained mutants. Other workers in Tubingen found no changes in the protein, which makes you wonder a little bit about the message that is carried in the nucleic acid.

We have made similar investigations in our laboratory and we do find changes. You may remember Dr. Knight's original observation that the last amino acid on the tobacco mosaic virus (TMV) polypeptide subunit is threonine which can be removed very easily With carboxypeptidase. All the TMV strains so far examined have this characteristic.

One of the mutants which has been obtained by nitrous acid treatment does not have this characteristic, and we already know that the changes in the amino acid sequence of a TMV polypeptide is presumably brought about by the conversion of one base into another base-cytosine into uracil. We think this is the first of the unraveling of the code that is contained in the sort of thing Dr. Colter has been talking about.

You need only another hundred or so such relationships between the polynucleotide chain and the polypeptide chain to know why a particular nucleic acid can be self-replicating. I think this will come with time, and that this work on viral nucleic acids is extremely important.

Of course from the standpoint of this group the observation, which I believe was initiated in Dr. Syverton's laboratory, that the nucleic acid will grow to a certain extent in cells which are not susceptible to the intact nucleoprotein is something to be kept in mind.

The implications of the work of Shope and his colleagues with the papilloma virus, as well as certain other work, tend to indicate that the viral nucleic acid probably can move as free nucleic acid. It is quite terrifying to think about, because everybody who is interested in immunology is sort of left behind the eightball, so to speak, until somebody introduces a new technique that will tie something onto nucleic acid and will enable formal immunology to be effective with this particular probable infectious process.

I think the business of integrative and nonintegrative viruses is something that should be kept in mind-this is a good concept, and I was glad to hear Dr. Colter again remind you of it.

Dr. Barricelli (Vanderbilt University): I would like to stress the point that the incorporation of a virus in the genetic material of a cell may indicate the existence of a crossing mechanism by transduction in cancer cells.

According to Dr. R. S. Fisher's results, a crossing mechanism would make it possible to increase the rapidity of evolutionary adaptation of a species by a large factor-a factor roughly comparable to the number of genes involved. It is obvious that the rapidity of evolutionary adaptation, resulting from a crossing mechanism, is essential to explain the rapid formation of a population of cells different from other cells of the body and adapted to overcome the defenses of the organism. The rapid increase in resistance of tumor cells to drugs and other therapy would also be easier to interpret by a crossing mechanism.

The question arises, therefore, whether the primary difference between normal and cancer cells may consist in the acquisition or uncontrolled increase of a primitive ("atavistic") crossing mechanism. Depending on the type of cancer, crossing may occur by any one, or by several, of the three mechanisms which are known in bacteria: (1) transformation; (2) transduction; (3) so-called "regular crossing of K12 bacteria." According to this theory, only cancers which cross by transduction can be transmitted by viruses from cell to cell. Moreover, a latent virus recently evoked by a mutation into a virulent stage could hardly be expected to have developed or restored the ability to survive outside the body and would hardly be infectious. This would explain the lack of infectivity which seems to be a common characteristic of most tumor viruses.

Preliminary work (selection of genetic markers) to investigate the possibility of performing crossings with human synovial cells is being carried on at the New York University-Bellevue Medical Center by Dr. L. N. Chessin under the leadership of Dr. K. Hirschhorn.

Dr. McLaren (University of Minnesota): I would like to compliment Dr. Colter and Dr. Ellem on their excellent paper.

I would like also to comment briefly on Dr. Colter's reference to our work which indicated that RNA from enteroviruses had a wider host cell range than intact virus. Whether our observation has any immediate application to the isolation of viral agents from human tumors is yet to be learned. Infection of various naturally insusceptible cells, such as rabbit cells, with enterovirus RNA did result in production of virus; however, this progeny virus was not distinguishable from the virus from which the RNA was extracted. This strengthened our previous conclusion that enterovirus specificity was dependent upon interaction between viral protein and receptors on susceptible cells. If the receptor mechanism was bypassed by the use of viral RNA, we could infect insusceptible cells. No cytopathic changes were observed, probably because so few insusceptible cells were infected. We had to rely on a susceptible assay system to detect virus multiplication. If these findings have application in studies of the phenomena of tumor viruses, we must have sensitive assay systems for intact tumor viruses and tumor virus nucleic acids.

I have two questions I would like to ask Dr. Colter:

(1) With reference to your statement concerning low levels of intracellular ribonuclease in mouse brain, muscle, and Ehrlich ascites tumor cells, what cells have been found to contain high levels of ribonuclease and are these cells more resistant to infection by viral RNA in vitrol.

(2) You mentioned that infectious RNA has been prepared from only small animal viruses of either neurotropic or myotropic potentialities. This is an interesting correlation which was not obvious to me. Maassab reported last year that infectious RNA was obtained from influenza virus-infected cells. We have tried without success to demonstrate infectious nucleic acid from high titer influenza viruses, and I was wondering what has been the experience in your laboratory with infectivity of nucleic acid from influenza or other myxoviruses.

Dr. Colter: In regard to your question about the intracellular ribonucleases, we have not made a frank attempt to correlate the vulnerability of a cell to infection with RNA with its level of intracellular ribonuclease. A scanning of the literature, however, led me to suggest that there may be this relationship. Certainly it is true that in the mouse only brain and muscle have been successfully infected with naked RNA. There are all kinds of tissues in the mouse that have active intracellular ribonucleases. Nearly all tissues, with the exception of muscle and brain-liver, kidney, salivary gland, lymph nodes, spleen, etc. have relatively high levels of intracellular ribonuclease.