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NOTICES 
experiments involving a clone derived 
from a shotgun experiment can be 
done under PI + EK1 conditions if 
the clone has been rigorously charac- 
terized and is free of harmful genes 
and If the foreign DNA was from a 
species that exchanges genes with E. 
Coli in nature. If the foreign DNA was 
from a species that does not do so. 
then P2 + EK1 is required. 
Similarly, when the Initial recombin- 
ation Involves a purified segment of 
the foreign chromosomal DNA. rather 
than a mixture, the potential for 
growth of a hazardous organism will 
be less, since the number of clones 
that must be examined to obtain the 
desired clone is markedly reduced. If 
certain criteria for purity are met. the 
investigator may lower the contain- 
ment conditions from those recom- 
mended for shotgun experiments with 
DNA of the same source by one step 
either in physical containment or bio- 
logical containment. Thus, for exam- 
ple. shotgun experiments with DNA 
from birds require P3 + EK2. A DNA 
fragment from birds that is free from 
harmful genes, and 99-percent purer 
before being joined to a vector, would 
require either P2 + EK2 or P3 + EK1. 
The final group of E. Coli experi- 
ments considered are those in which 
the foreign DNA is itself from an ex- 
trachromosomal element. It is as- 
sumed that such DNA is purified away 
from chromosomal DNA before recom- 
bination. For example. DNA from all 
or part of the genome of an animal 
virus requires P4 physical containment 
and an EK2 host-vector system, or P3 
+ EK3. if the recombinants have been 
purified by cloning and shown to be 
free of harmful regions of the viral 
genome, then experiments can be at 
P3 + EK2 
When complementary DNA's, syn- 
thesized in vitro from RNA prepara- 
tions. are used in recombinant DNA 
experiments, the containment require- 
ments are as described for isolated 
DNA preparations. 
Animal host-vector systems 
Many recombinant DNA experi- 
ments will Involve the use of systems 
In which the host cells are eukaryotes 
grown as single cells In tissue culture. 
Useful vectors may include extrachro- 
mosomal DNA elements, such as the 
DNA of organelles or viruses. The cells 
themselves are fragile and fastidious, 
and there is little or not chance that a 
living cell could escape from a labora- 
tory in the way than an E. coli cell 
might. Therefore, containment consid- 
erations focus on the vectors. 
Animal viruses can escape the labo- 
ratory in a viable form, especially if 
laboratory workers become infected. 
There are now two animal viruses 
whose DNA's are useful as vectors: po- 
lyoma and simian virus 40 (SV40). 
Both viruses are oncogenic— that is. 
they cause tumors in small newborn 
laboratory mammals. Polyoma virus, 
however, does not infect human cells 
grown in the laboratory or. judged by 
the lack of antibody formation, infect 
whole human beings. SV40 does colo- 
nize humans. Indeed, the virus con- 
taminated the early Salk polio vaccine, 
and millions of people were inadvert- 
ently inoculated with in in the 1950's. 
To date, there is no indication that 
the recipients suffered any related dis- 
ease. But under the Guidelines, more 
stringent physical containment is re- 
quired for SV40 than for polyoma. 
The Guidelines require that the 
viral DNA used for recombination 
with a foreign DNA must itself be de- 
fective— that is, its propagation as a 
virus must be dependent on the pres- 
ence of helper virus that supplies the 
genes for the missing functions. This 
helper can be nondefective under cer- 
tain conditions. In some experiments, 
no production of viral particles is re- 
quired and no helper may be needed. 
Biological containment is inherently 
greater in the absence of virus parti- 
cles. since cells themselves are rela- 
tively easy to contain. In experiments 
using a virus as vector, the particular 
levels of physical containment depend 
on the source of the foreign DNA. on 
whether polyoma or SV40 is the 
chosen vector, and whether virus par- 
ticles are produced. 
Plant host-vector systems 
The Guidelines also cover experi- 
ments in which plant cells will serve as 
hosts tor recombinant DNA. The cells 
might be single plant cells grown 
under laboratory conditions, or seed- 
lings. plant parts, or small whole 
plants. This is the only instance where 
the Guidelines address the question of 
recombinant DNA experiments with 
whole organisms. Directions are given 
for modification of the specifications 
for PI. P2. and P3 containment in 
order to provide conditions appropri- 
ate for work with plants. 
Vectors for use in experiments with 
plants Include plant organelle DNA 
such as that of chloroplasts. and DNA 
of viruses of low pathogenicity and re- 
stricted host range. These vectors 
offer moderate levels of biological con- 
tainment. The requirements are orga- 
nized according to the source of the 
foreign DNA and to whether it is a 
species in which the vector DNA is 
known to replicate. P2 conditions are 
required if the source is not dangerous 
and is one in which replication of the 
vector occurs. If the foreign DNA is 
derived from a species in which the 
vector is not known to replicate, then 
requirements range up to P4 depend- 
ing on whether the DNA is purified 
and whether it contains harmful 
genes. 
Other host-vector systems 
Theoretically, there are a variety of 
organisms, both prokaryotes and lower 
eukaryotes such as fungi and yeast, 
which will be useful hosts for experi- 
ments with recombinant DNA's. Some 
may offer the special advantage of not 
infecting humans, animals, or other 
important ecological niches. The 
growth characteristics of such hosts 
indicate that containment problems 
will be like those for E. coli K-12. The 
Guidelines urge development of these 
systems and point out that the de- 
tailed recommendations made for E. 
coli K-12 systems can sene as a guide 
in determining physical and biological 
containment requirements when nec- 
essary. 
Alternatives: RAC-Proposed Revisions 
A major concern for all individuals 
who have participated in establishing 
guidelines for recombinant DNA re- 
search is that any guidelines that are 
drafted and adopted be reassessed pe- 
riodically and changed when warrant- 
ed by new Information. In keeping 
with this responsibility, the RAC has 
compiled additional information per- 
taining to risk assessment in recombin- 
ant DNA-research. The information is 
from the following sources: 
• Consultations with scientists 
expert in the areas of evolution, plant 
biology, bacteriology, virology, and 
human and animal infectious diseases: 
• Reports from scientific meetings 
dealing with the potential biohazards 
of recombinant DNA research (for ex- 
ample. Tenth Miles International 
Symposium on Recombinant Mole- 
cules — Impact on Science and Society. 
Cambridge. Mass., June 1976; National 
Academy of Sciences Forum on Re- 
combinant DNA Research. Washing- 
ton, D.C.. March 1977; Genetic Engi- 
neering for Nitrogen fixation. Brook- 
haven, N.Y.. March 1977; and the 
Workshop on Studies for Assessment 
of Potential Risks Associated with Re- 
combinant DNA Experimentation, Fal- 
mouth. Mass.. June 1977); 
• Results from experiments specifi- 
cally designed to test (1) the survivabi- 
lity and colonizing ability of E. coli K- 
12 and EK2 host-vector systems. (2) 
the transmissibility of plasmids and 
phage vectors, (3) the potential of E. 
coli K-12 for pathogenicity, and (4) 
the potential for genetic exchange be- 
tween diverse bacteria and between 
prokaryotic and eukaryotic organisms. 
Each category of experiments in 
Part III of the 1976 Guidelines was 
then extensively examined, and the 
following criteria were applied to the 
new information: 
• The degree to which the DNA seg- 
ment has been purified away from 
other genes and shown to be free of 
harmful characteristics. 
FEDERAL REGISTER, VOL 43. NO. 144— FRIDAY, JULY 28, 1978 
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