These studies i 
Include: 
1. Titration of polyoma DNA in vivo by the MAP procedure 
using: 
a) oral, intranasal and subcutaneous routes 
b) DNA in: physiological saline, DEAE-dextran, and 
calcium phosphate 
c) conventional and, if possible, germ-free mice of 
veanllng age 
2. Any necessary studies on the ability of the E. coll system 
to colonize germ-free mice and on its pathogenicity following parenteral 
Inoculation. Roy Curtiss (University of Alabama) will play a large role 
in selecting the strain, and will be able to supply much of this information 
STAGE 2. The Preparation of Recombinant DNA Molecules Consisting of 
Polyoma and Bacteriophage Lambda Components and their Subsequent Inoculation 
into Test Animals. 
A. DNA 
1. Polyoma 
Plaque purified polyoma virus (large plaque strain) will 
be propagated as described by Winocour (Virology 19, 158-168, 1963). 
Virus will Be purified by equilibrium density centrifugation in CsCl and 
the superco'iied DNA isolated by dye-buoyant density centrifugation in 
CsCl-ethidium bromide. The homogeneity of closed circular polyoma DNA 
will be assessed by electrophoresis in 1.4Z agarose slab gels and cleavage 
with R Hind . Unique full-length linear molecules of polyoma DNA will be 
prepared with Eco R1 enzyme and Isolated by sucrose gradient centrifugatior 
or agarose slab gel electrophoresis. 
[Appendix C — 81] 
