APPENDIX D— 23 
(29) Wilson. Q. A. and P. K. Young. Un- 
published data. 
(30) WUson. G. A., R. Roberts, and P. E. 
Young. Unpublished data. 
(31) Wilson. O. A. and P. E. Young. Re- 
striction and modification In bacilli. In D. 
Schlesslnger (ed.), Microbiology 1976, In 
press. AppsNDDt B to Appendix D 
POLTOMA AND SV40 VJKUB 
Polyoma virus Is a virus of mice, and infec- 
tion of wild mouse populations Is a common 
event, for the virus has often been isolated 
from a high proportion of healthly adult 
animals, both wild and laboratory bred, of 
many colonies (Gross, L., Proc. Soc. E.xp. Biol. 
88, 362-368, 1955; Rowe, W. P., Bact. Rev. 25 
18-31, 1961). As far as Is known the virus 
almost never causes a disease In these 
animals. However, when large quantities of 
the virus are Inoculated Into newborn or 
suckling mice or hamsters, a variety of solid 
tumors Is induced (Gross, L.. Oncogenic 
Viruses, Second Edition, Pergamon Press, 
NY). 
Polyoma virus grows lytlcally in mouse 
cells in tissue culture. Thus mouse cells In 
culture are probably transformed only by 
virus particles that contain certain kinds of 
defective genomes. Cells of other rodent spe- 
cies, however, can be transformed by polyoma 
virus particles that contain complete ge- 
nomes (Folk, W.. J. Virol., 11, 424-431, 1973). 
The virus does not replicate to a significant 
extent In human cells In tissue culture 
(Eddy, B. E.. Virol. Monogr., 7, 1-114, 1969; 
Pollack, R. E. Salas, J„ Wang, R„ Kusano, T., 
and Green. H„ J. Cell Physiol. 77, 117-120, 
197'' ) . The resistance of the cells seems to be 
a consequence of the failure of the virus to 
absorb or uncoat. However even when naked 
viral DNA is Introduced into the cells only 
an abortive cycle of replication ensues: early 
viral proteins are made, there Is Induction of 
cellular DNA synthesis, but no expression of 
late viral proteins Is detectable (Gruen, R., 
Gra.ssmann, M. and Gras-smann, A.. Virology, 
58, 290-293, 1974) . 
There Is no evidence that polyoma virus 
can Infect humans (Hartley, J., Huebner, R.. 
Parker, J. and Rowe, W. P., unpublished 
data). Thus no antibodies to the virus have 
been detected in people living In buildings 
that are Infested with vlrus-lnfected mice, 
nor In laboratory workers who have been ex- 
posed to the virus for a number of years. 
At most, a small segment of polyoma virus 
DNA shows weak homology with a portion of 
the late region of SV40 DNA (Ferguson, J. 
and Davis, R. W.. J. Bol. Biol., 94. 135-150, 
1975). However, there appears to be no gene- 
tic Interaction between the two viruses and 
there Is no Immunological cro.ss-reactlon be- 
tween the gene products of the two viruses. 
SV40 causes persistent but apparently 
harmless Infections of the kidneys of vir- 
tually all adult rhesus monkeys (Hslung, G. 
D., Bact. Revs. 32, 185-205, 1968), It causes 
tumors when Injected Into newborn hamsters 
(Glrardl, A. J., Sweet. B. H.. Slotnlck, V. B. 
and Hlllemann, M. R., Proc. Soc. Exp. Biol. 
Med.. 105, 420-427, 1964) and tran.sforms cells 
of several mammalian species (Including hu- 
man). SV40 Is able to Infect human since 
antibodies to the virus are found In a small 
proportion of the human population (Shah, 
K. V., Goverdhan, M. K. and Ozer, H. L., Am. 
J. Epld. .93, 291-298, 1970) and serum conver- 
sions have been noted In many laboratory 
personnel who have been exposed to the virus 
(Horvath, L. B.. Acta Microbiol. Acta Scl. 
Hung. 12, 201-206. 1966). 
Isolations of SV40 have been reported from 
humans, twice from patients suffering from 
the rare demyellnatlng disease, progressive 
multifocal leukoencephalopathy (Weiner, L., 
Herndon, R„ Narayon, O., Johnson, R. T., 
Shah, K., Rubinstein, L. G., Prezozisi, T. J. 
and Conley, F. K., New England J. Med. 286, 
385-390, 1972) and apparently from a tumor 
of a person with metastatic melanoma (Sori- 
ano, F., Shelburne, C. E. and Gokcen, M„ 
Nature, 249, 421-424, 1974). In other studies 
a non-structural antigen characteristic of 
papovavlruses, T antigen, has been detected 
In the nuclei of cells cultured from 2 menin- 
giomas, while another SV40-rpeclfic antigen, 
U antigen, has been found in the cells of a 
third tumor of the same type (Weiss, A. F., 
Portman, R., Fisher, H., Simon, J. and Zang. 
K. D.. Proc. Nat. Acad. Scl. USA 72. 609-613, 
1976). Furthermore new papovavlruses have 
been Isolated from the brains of patients 
with PML (JC virus — Padgett, B. L.. Walker, 
D. L., zuRheln, G. M., Eckroade, R. I. and 
Dessel, B. H., Lancet 1. 1257-1260, 1971) , from 
the urine of a patient carrying a renal allo- 
graft (BK virus — Gardner, S. D., Field, A. M.. 
Coleman, D. V. and Hulme, B. Lancet 1, 1253- 
1257, 1971) and from a reticulum cell sar- 
coma and the urine of patients with the sex- 
linked recessive disorder, Wiscofct-Aldrlch 
syndrome (Takemoto, K. K., Rabson, A. S„ 
Mullarkey, M. F., Blaese, R. M. Garon, C. F. 
and Nelson, D. J., Nat. Cancer Inst., 53, 1205- 
1207, 1974). All of these viruses which are 
distributed widely throvighoiit human popu- 
lations share antigenic and biological prop- 
erties with SV40; the virus particles are 
Identical in size and architecture (Madeley, 
C. R., In Virus Morphology, Churchlll-Llv- 
Ingstone, London, 134-135, 1972); the non- 
structural Intracellular T antigen, which ap- 
pears to be coded by the A gene of SV40 cross 
reacts extensively with antigens found In 
cells Infected or transformed by BK or JC 
viruses; both JC or BK viruses Induce tumors 
In newborn hamsters (Walter, D. L., Padgett. 
B. L. zuRhein, B. M., Albert, A. E. and Marsh, 
R. F., Science 181, 674-676, 1973; Shah, K. V., 
Daniel, R. W. and Strandberg, J., J. Nat. Can- 
cer Inst. 54, 945-950, 1975) ; BK virus causes 
transformation of hamster cells In culture 
(Major, E. D., and DlMayorca, G., Proc. Nat. 
Acad. Scl. US, 70, 3210-3212, 1973; Portolanl, 
M.^ Barbantl, A., Brodano, G. and LaPiaca, 
M. J., Virol. 15, 420-422, 1975) and Is able to 
complement the growth of certain tempera- 
ture-sensitive mutants of SV40 (Mason, D. H. 
and Takemoto, K. K., submitted for publica- 
tion) . 
FURTHER WORK 
At present, a potential eukaryotic vector 
of choice Is polyoma virus. And while avail- 
able Information Indicates that It fulfills all 
the necessary criteria, we recommend that 
the following subjects be further Investi- 
gated: 
1^ The molecular mechanism of resistance 
of human cells to the virus. 
2. The extent of homology between polyoma 
virus DNA and the DNAs of human papova- 
vlruses. 
3. The ability of human papovavlruses to 
complement defective polyoma virus ge- 
nomes. 
Report of a working group consisting of: 
Dr. Bernard Fields, Harvard University School 
of Medicine; Dr. Thomas J. Kelly, Jr., Johns 
Hopkins University School of Medicine; Dr. 
Andrew Lewis, National Institute of Allergy 
and Infectious Diseases: Dr. Malcolm Martin, 
National Institute of Allergy and Infectious 
Diseases: Dr. Robert Martin, National In- 
stitute of Arthritis, Metabolism, and Dlgest- 
tlve Diseases; Dr. Elmer Pfcfferkorn, Dart- 
mouth Medical School; Dr. Wallace P. Rowe, 
National Institute of Allergy and Infectious 
Diseases; Dr. Aaron Shatkln, Roche Insti- 
tute of Molecular Biology; Dr. Maxine Singer, 
National Cancer Institute. Rapporteur; Dr. 
Joe Sambrook, Cold Spring Harbor Labora- 
tory. 
Appendix C to Appendix D 
SUMMART OP THE WORKSHOP ON THE DESIGN 
AND TESTING OF SAFES PBOKARTOTIC VEHICLES 
AND BACTERIAL ROSTS FOR RESEARCH ON RE- 
COMBINANT DNA MOLECULES 
TOar.EY PINES INN, LA JOLLA, CALIFORNIA 
The development of techniques for the 
cloning Of DNA from both prokaryotic and , 
eukaryotic organisms in bacteria has had | 
great impact on research in biology and 
medicine and promises extraordinary social 
benefits. The biohazards involved In the use 
of this technology in many Instances are 
very difficult to assess. For this reason codes 
of practice are being formulated In the 
United States and other countries for the 
conduct of those e.xperiments that present i 
a potential biohazard. One of the require- | 
ments for conducting certain cloning ex- | 
periments Is the use of safer vector (bac- 
teriophage or plasmid) -host systems, l.e., ! 
vector-bacterium "systems that have re- 
stricted capacity to survive outside of con- , 
trolled conditions in the laboratory. Ap- 1 
proximately sixty scientists from the United i 
States and several foreign countries partici- 
pated In a workshop on the Design and Test- 
ing of Safer Prokaryotic Vehicles and Bac- 
terial Hosts for Research on Recombinant 
DNA Molecules at La Jolla, California, on 1 
to 3 December, 1975. The workshop was spon- 
sored by the Research Resources Branch of 
the National Institute of Allergy and In- 
fectious Diseases. The purposes of the meet- 
ing were the exchange of recent data on the 
development of safer prokaryotic host-vector 
systems, devising methods of testing the ’ 
level of containment provided by these sys- i 
terns and exploring the various directions 
that future research should take In the con- 
struction of safer bacterial systems for the 
cloning of foreign DNA. i 
The first session of the workshop, chaired 
by W. Szybalskl (University of Wisconsin) , 
was devoted to bacteriophage vectors. Szybal- 
skl outlined the main safety features of the 
two-component, phage-bacterlal system, in 
which the host bacteria offer the safety fea- , 
ture of not carrying the cloned DNA, and the 
phage vectors cannot be propagated In the ' 
absence of an appropriate host. There are 1 
two primary escape routes for the clones of I 
foreign DNA carried by the phage vector: 
(1) establishment of a stable prophage or 
plasmid In the laboratory host used for 
phage propagation, and subsequent escape 
of this self replicating lysogen or carrier 
system, and (2) escape of the phage vector j 
which carries the cloned DNA and Its sub- 
sequent productive encounter with a suit- 
able host In the natural environment. The 
general consensus was that to ensure safety, 
both routes should be blocked by appropri- 
ate genetic modifications. For phage \, route i 
( 1 ) can be blocked by phage mutations that 
Interfere with lysogenlzatlon (aff-. Inf-, cl-, 
cIII-, vir) and plasmid formation (N*, ninR, 
vS, ri’, cl 7, Ots, crots), and by mutations on , 
the Escherichia coli host that affect these 
processes (affB-, dncAts) and host survival. 
Route (2), [which Is of low probability since 
X phages do not survive well In natural envi- 
ronments (no Xcl phage was recovered after 
Ingestion of 10*-10" particles), are killed by 
desiccation, and have a low chance to en- j 
counter a naturally sensitive host] can be 
blocked further by the following phage 
modifications: (a) mutations which result 
In extreme Instability of the Infectious 
phage particles under all conditions other 
than those specially designed for phage pro- 
pagation In the laboratory (e.g., high con- 
centrations of putresclre or some other com- 
pound), or (b) employing phage vectors in 
which the tall genes a*e deleted and wblcU 
permit propagation of only the DNA-packed 
heads; only ' under laboratory oonditlona 
could such heads be made transiently Infee- 
