-3. If the studies carried out in 2b indicate that recombinant 
DNA molecules consisting of polyoma oligomers and bacteriophage lambda 
sequences have been generated (which we will designate P-lambda) , an 
aliquot of the reaction mixture will be used to transfect the C600 
strain of E. coli K-12 in the presence of 50 mM CaCl^ under P-A conditions . 
A. P-lambda phage will be purified from infected cultures 
under P-A conditions and the DNA isolated will be incubated with various 
restriction enzymes and analyzed by gel electrqphoresis to ascertain if 
polyoma DNA has replicated in j£. coli . 
C. Biological Activity of Cloned Recombinant DNA (Carried out 
under P-A conditions) 
1. Infection of mouse cells in culture. 
Primary mouse embryo cells in culture will be infected 
with the crude cell lysate, purified P-lambda recombinant DNA, and with 
P-lambda bacteriophage. Polyoma virus will be detected by CPE, indirect 
immunofluorescence of infected cells and a blind passage thereof. 
2. Infection of mice. 
a) Adult and newborn mice will be inoculated subcutaneously 
and intraperitoneally with varying amounts of bacteriophage P-larabda in 
both free and "contained" (i.e. in E. coli freshly infected with the 
phage) forms. 
b) Germ-free and conventional mice will be infected 
intranasally and orally with varying amounts of free and contained 
recombinant P-lambda phage. 
c) In all cases animals will be tested for the development 
of polyoma HI 'Antibody', ' and, possibly,’ tumor formation. It is anticipated* 
that the immunologic assays can be performed under P2-conditions provided 
it can be demonstrated that heat-treated serum (56°-30 min.) is free of 
E. coll or bacteriophage lambda. 
[Appendix C — 83] 
