APPENDIX D— 18 
idea of the location within the molecule 
of: 
(ar) The sites at which DNA synthesis 
originates and terminates, 
(b) The sites that are cleaved by re- 
striction endonucleases, 
(c) The template regions for the 
major gene products. 
(3) It should be well studied genet- 
ically. It is desirable that mutants be 
available in adequate number and vari- 
ety, and that quantitative studies of re- 
combination have been performed. 
(4) The recombinant must be defec- 
tive, that is, its propagation as a virus 
is dependent upon the presence of a com- 
plementing helper genome. This helper 
should either (a) be integrated into the 
genome of a stable line of host cells (a 
situation that would effectively limit the 
growth of the vector to that particular 
cell line) or (b) consist of a defective 
genome or an appropriate conditional 
lethal mutant virus (in which case the 
experiments would be done under non- 
permissive conditions) , making vector 
and helper dependent upon each other 
for propagation. However, if none of 
these is available, the use of a non-de- 
fective genome as helper would be ac- 
ceptable. 
Chirrently only two viral DNAs can be 
considered as meeting these require- 
ments : these are the genomes of polyoma 
virus and SV40. 
Of these, polyoma virus is highly to be 
preferred. SV40 is known to propagate 
in human cells, both in vivo and in vitro, 
and to infect laboratory personnel, as 
evidenced by the frequency of their con- 
version to producing SV40 antibodies. 
Also, SV40 and related viruses have been 
found in association with certain human 
neurological and malignant diseases. 
SV40 shares many properties, and gives 
complementation, with the common hu- 
man papova viruses. By contrast, there 
Is no evidence that polyoma infects hu- 
mans, nor does it replicate to any signif- 
icant extent in human cells in vitro. 
However, this system still needs to be 
studied more extensively. Appendix B 
gives further details and documentation. 
Taking account of all these factors : 
(1) Polyoma Virus, (a) Recombinant 
DNA molecules consisting of defective 
polyoma virus genomes plus DNA se- 
quences of any nonpathogenic organism. 
Including Class 1 viruses (5) , can be 
propagated in or used to transform cul- 
tured cells. P3 conditions are required. 
Appropriate helper virus can be used if 
needed. Whenever there is a choice, it 
is urged that mouse cells, derived pref- 
erably from embryos, be used as the 
source of eukaryotic DNA. Polyoma virus 
is a mouse virus and recombinant DNA 
molecues containing both viral and 
cellular sequences are already known 
to be present in virus stocks grown at a 
high multiplicity. Thus, recombinants 
formed in vitro between polyoma virus 
DNA and mouse DNA are presumably not 
novel from an evolutionary point of view. 
(b) Such experiments are to be done 
under P4 conditions if the recombinant 
DNA contains segments of the genomes 
of Class 2 animal viruses (5). Once it 
See footnotes at end of article. 
has been shown by suitable biochemical 
and biological tests that the cloned re- 
combinant contains only harmless re- 
gions of the viral genome (see Section 
IIIB-2-c-i) and that the host range of 
the polyoma virus vector has not been 
altered, experiments can be continued 
under P3 conditions. 
(2) SV40 Virus. 
(a) Defective SV40 genomes, with ap- 
propriate helper, can be used as a vec- 
tor for recombinant DNA molecules con- 
taining sequences of any non-pathogenic 
organism or Class I virus (5), (i.e., a 
shotgun type experiment) . P4 conditions 
are required. Established lines of cul- 
tured cells should be used. 
(b) Such experiments are to be car- 
ried out in P3 (or P4) conditions if the 
non-SV40 DNA segment is (a) a puri- 
fied “ segment of prokaryotic DNA lack- 
ing toxigenic genes, or (b) a segment of 
eukaryotic DNA whose function has been 
established, which does not code for a 
toxic product, and which has been pre- 
viously cloned in a prokaryotic host- 
vector system. It shall be confirmed that 
the defective virus-helper virus system 
does not replicate significantly more effi- 
ciently in human cells in tissue culture 
than does SV40, following infection at a 
multiplicity of infection of one or more 
helper SV40 vinises per cell. 
-(c) A recombinant DNA molecule con- 
sisting of defective SV40 DNA laclung 
substantial segments of the late region, 
plus DNA from non-pathogenic orga- 
nisms or Class I viruses (5) , can be prop- 
agated as an autonomous cellular ele- 
ment in established lines of cells under 
P3 conditions provided that there is no 
exogenous or endogenous helper, and 
that it is demonstrated that no infectious 
virus particles are being produced. Until 
this has been demonstrated, the appro- 
priate containment conditions specified 
in 2. a. and 2. b. shall be used. 
(d) Recombinant DNA molecules con- 
sisting of defective SV40 DNA and se- 
quences from non-pathogenic prokary- 
otic or eukaryotic organisms or Class I 
viruses (5) can be used to transform es- 
tablished lines of non-permissive cells 
under P3 conditions. It must be demon- 
strated that no infectious vinis particles 
are being produced; rescue of SV40 from 
such transformed cells by co-cultivation 
or transfection techniques must be car- 
ried out in P4 conditions. 
(3) Efforts are to be made to ensure 
that all cell lines are free of virus par- 
ticles and mycoplasma. 
Since SV40 and polyoma are limited in 
their scope to act as vectors, chiefly be- 
cause the amount of foreign DNA that 
the normal virions can carry probably 
cannot exceed 2x10’ daltons, the devel- 
opment of systems In which recom- 
binants can be cloned and propagated 
purely in the form of DNA, rather than 
in the coats of Infectious agents is neces- 
sary. Plasmid forms of viral genomes or 
organelle DNA need to be explored as 
possible cloning vehicles in eukaryotic 
cells. 
(b) Plant host-vector systems. For 
cells in tissue cultures, seedlings, or plant 
parts (e.g. tubers, stems, fruits, and de- 
tached leaves) or whole mature plants of 
small species (e.g., Arabidopsis) the Pl- 
P4 containment conditions that we have 
specified previously are relevant con- 
cepts. However, work with most plants 
poses additional problems. The green- 
house facilities accompanying P2 labora- 
tory physical containment conditions 
can be provided by: (i) Insect-proof 
greenhouses, (ii) appropriate steriliza- 
tion of contaminated plants, pots, soil, 
and runoff water, and (hi) adoption of 
the other standard practices for micro- 
biological work. P3 physical containment 
can be sufficiently approximated by con- 
fining the operations with whole plants 
to gi’owth chambers like those used for 
work with radioactive isotopes, provided 
that (i) such chambers are modified to 
produce a negative pressure environment 
with the exhaust air appropriately 
filtered, (ii) that other operations with 
infectious materials are carried out under 
the specified P3 conditions, and (iii) to 
guard against inadvertent insect trans- 
mission of recombinant DNA, growth 
chambers are to be routinely fumigated 
and only used in insect proof rooms. The 
P2 and P3 conditions specified earlier are 
therefore extended to include these cases 
for work on higher plants. 
The host cells for experiments on re- 
combinants DNAs may be cells in cul- 
ture, in seedling or plant parts. Whole 
plants or plant parts that cannot be ade- 
quately contained shall not be used as 
hosts for shotgun experiments at this 
time, and attempts to infect whole plants 
with recombinant DNA shall not be ini- 
tiated until the effects on host cells in 
culture, seedlings or plant parts have 
been thoroughly studied. 
Organelle or plasmid DNAs or DNAs of 
viruses of restricted host range may be 
used as vectors. In general, similar cri- 
teria for selecting host-vectors to those 
given in the preceding section on animal 
systems are to apply to plant systems. 
DNA recombinants formed between the 
initial moderately contained vectors and 
DNA form cells of species in which the 
vector DNA can replicate, require P2 
physical containment. However, if the 
source of the DNA is itself pathogenic 
or known to carry pathogenic agents, or 
to produce products dangerous to plants, 
or if the vector is an unmodified virus 
of unrestricted host range, the experi- 
ments shall be carried out under P3 
conditions. 
Experiments on recombinant DNAs 
formed between the above vectors and 
DNAs from other species can also be car- 
ried out under P2 if that DNA has been 
purified * and determined not to contain 
harmful' genes. Otherwise, the experi- 
ments shall be carried out under P3 con- 
ditions if the source of the inserted DNA 
is not itself a pathogen, or known to 
carry such pathogenic agents, or to pro- 
duce harmful products — and under P4 
conditions if these conditions are not 
met. 
The development and use of host- 
vector systems that exhibit a hl^ level 
of biological containment permit a de- 
crease or one step In the idiyslcal con- 
