IMUA-J8 
-3- 
in those cases where alt°rations have occurred in "late" polyoma genes. 
In such a situation, the MAP test would yield negative results, but the 
potential oncogenic activity of a polyoma containing DNA recombinant could 
still be unmasked if tumors develop in the hamsters. 
In the following paragraphs we outline a series of experiments which will 
utilize polyoma virus in an attempt to answer some of the questions which 
have been raised about potential biohazards of recombinant DNA research. 
Specifically, we intend to generate recombinant molecules consisting of 
either polyoma virus and bacteriophage lambda DNAs or polyoma virus and 
plasmid OTIAs, and clone such molecules in a suitable E. col i host. Progeny 
phage, or plasmid-containing E_. coli will be fed to, and inoculated into 
mice and newborn hamsters in the final step of the proposed experiments and 
the animals tested for evidence of virus infection or tumor formation. 
Although, at first sight, this seems to be a simple, straight-forward 
experiment which could be done cheaply and expeditiously, this is not the 
case. In particular, since we are very concerned that a negative result 
will be misinterpreted as indicating that there is little biohazard in DNA 
recombinant work, we feel that a number of variables must be evaluated, and 
that the experiments should be designed to maximize the chances of a positive 
result. Also, if negative results are obtained with a given system, we feel 
that the experiments should be continued with modified systems until we are 
reasonably convinced that a positive result is just not going to occur. 
Accordingly, our experimental strategy will be to conduct the cloning of 
polyoma DNA in E_. col i in several different stages. 
Our i nitial experiments will be conducted in strict accordance with the NIH 
Guidelines for Recombinant DNA Research. We will employ approved EK2 host- 
vector systems and P4 physical containment to clone polyoma DNA in £. coli. 
Mice and hamsters will be inoculated with bacteria containing polyoma DNA 
recombinants by a variety of routes as well as with the purified recombinant 
DNA; these animals will be maintained in P4 containment. 
In addition to its imoortance for studying transfer of DNA from E_. col i to 
host cells, we feel that the polyoma-JE. col i system may be particularly useful 
for examining the question of expression of eukaryotic DNA in a bacterial 
cell. Accordingly, we will use appropriate nucleic acid hybridization, 
radioimmunoassay and other serologic procedures in an attempt to detect and 
quantitate synthesis of polyoma RNA, polypeptides and proteins. 
III. DESCRIPTION OF THE AGENTS 
A. Polyoma Virus 
1. Strain "H", obtained from Dr. Thomas Benjamin, Harvard University 
(large plaque variant), Cambridge, Massachusetts 
2. Toronto strain (McCulloch et al . , Nature 183 : 1535, 1959) 
[Appendix C — 233] 
