IMUA-18 
-4- 
B. EK2 Lambda Vector System 
1. Certified EK2 bacteriophage: Agt*AB (Tiemeier, T. et al . , Nature 
263 : 526, 1976 and Leder, P. et aJL , Science 196: 175, 1977) 
2. Host bacterium: E. col i Ymel , LE392 
C. EK2 Plasmid Vector System 
1. Plasmids: pSClO'l and pCRl. 
2. Host bacterium: E. col i xl 7 7 6 derived and obtained from Dr. Roy 
Curtiss, University of Alabama, Birmingham, Alabama, and certified 
EK2 by Director, NIH. 
IV. DESCRIPTION OF THE TYPE OF EXPERIMENT 
A. Eco RI cleaved polyoma DNA will be joined to the two largest Eco RI 
fragments of Agt-AB DNA using T4 DNA ligase. Polyoma-lambda (P-lambda) 
DNA recombinant molecules thus generated will be used to transfect 
JE. col i . Infected E. col i , purified phage, or phage DNA will be 
inoculated into mice (adult, newborn, germfree, conventional) by oral, 
nasal, rectal, and parenteral routes after it has been demonstrated 
that P-lambda recombinant DNA is present in the infected cultures. In 
all cases the mice will be tested for the development of polyoma HI 
antibody, and in some cases, tumor formation.' The animals will also be 
examined for changes in the virulence of E. col i such as sudden, 
unexpected death or diarrheal disease. Newborn hamsters will also be 
inoculated i ntraper > toneal ly with infected E_. col i , phage, and purified 
P-lambda DNA and monitored for polyoma induced tumors. Mouse and rat 
cell tissue cultures will be infected with purified P-lambda DNA and 
monitored for the appearance of progeny virus, viral antigens, and 
transformed colonies. 
B. Eco RI cleaved polyoma DNA will be ligated to E co RI digested pSClOl or 
pCRl DNAs and used to transform E_. coli strain yJ776, a certified EK2 
system. Clones transformed to antibiotic resistance will be screened by 
filter hybridization for the presence of polyoma-plasmid (P-plasmid) DNA. 
Bacteria containing P-plasmid DNA as well as purified P-plasmid DNA will 
be inoculated into mice, hamsters, and tissue cultures as described above 
C. Lysates of bacteria containing P-lambda or P-plasmid recombinant DNAs 
will be assayed for the presence of infectious polyoma virus using 
primary and continuous lines of mouse cells. 
D. Bacterial lysates vrth no demonstrable biological activity will be 
examined for the presence of polyoma-specific RNA and proteins. 
[Appendix C — 234] 
