2790S 
NOTICES 
EK system at the third level of contain- 
ment (EK3 > be more fully explained. The 
Recombinant Advisory Committee was 
asked to consider this suggestion. After 
considerable discussion the committee 
declined to define the procedures more 
fully at tli is time, because development of 
an EK3 system is still far enough in the 
future not to warrant specific testing 
procedures. Further, it is not clear what 
tests are best suited. The language, 
therefore, remains general. The commit- 
tee. however, is aware of the concerns for 
a more completely defined system of test- 
ing. and has considered the possibility of 
organizing a symposium for purposes of 
designating tests. In my view, more fully 
developed protocols for testing EK3 sys- 
tems are warranted, and it is necessary 
that guidelines here be more fully de- 
veloped before the committee proceeds to 
certify such a system. In this regard the 
NTH is prepared through the National 
Institute of Allergy and Infectious Dis- 
eases to support contracts to accomplish 
this task. We will seek the advice and as- 
sistance of the committee to define the 
scope of necessary work. 
These guidelines also include a state- 
ment that for the time being no F.K2 or 
EK3 host-vector system will be consid- 
ered bona fide until the Recombinant 
Advisory Committee has certified it. I 
share the concern of the commentators 
that new host-vector systems require the 
highest quality of scientific review and 
scrutiny. At this early stage of develop- 
ment. it is most important that the com- 
mittee provide that scrutiny. Further, I 
believe that until more experience has 
been gained, the committee should en- 
courage and the NIH support research 
that will independently confirm and aug- 
ment the data on which certification of 
EK2 host-vector systems are based. 
V. CLASSIFICATION OF EXPERIMENTS USING 
THE E. COLI K— 12 CONTAINMENT SYSTEMS 
The guidelines assign different levels 
of containment for experiments in which 
DNA from different sources is to be in- 
troduced into an E. coli K-12 host-vector 
system. The variation is based on both 
facts and assumptions. There are some 
prokaryotes (bacteria) which constantly 
exchange DNA with E. coli. Here it is 
assumed that experimental conditions 
beyond those obtained in careful, routine 
microbiology laboratories are superflu- 
ous, because any exchange experiments 
have undoubtedly been performed al- 
ready in nature. 
In every instance of artificial recom- 
bination. consideration must be given to 
the possibility that foreign DNA may be 
translated into protein (expressed) , and 
also to the possibility that normally re- 
pressed genes of the host may be ex- 
pressed and thus change, undesirably, 
the characteristics of the cell. It is as- 
sumed that the more similar the DNAs 
of donor and host, the greater the prob- 
ability of expression of foreign DNA, or 
of possible derepression of host genes. 
In those cases where the donor ex- 
changes DNA with E. coli in nature, It is 
unlikely that recombination experiments 
will create new genetic combinations. 
When prokaryote donors not known to 
exchange DNA with E. coli in nature are 
used, however, there is a greater potential 
for new genetic combinations to be 
formed and be expressed. Therefore, it 
is required that experiments involving 
prokaryotic DNA from a donor that is 
not known to exchange DNA with E. coli 
in nature be carried out at a higher level 
of containment. Recombination using 
prokaryotic DNA from an organism 
known to be highly pathogenic is pro- 
hibited. 
There are only limited data available 
concerning the expression of DNA from 
higher forms of life (eukaryotes' in E. 
coli (or any other prokaryote) . There- 
fore. the containment prescriptions for 
experiments inserting eukaryotic DNA 
into prokaryotes are based on risks hav- 
ing quite uncertain probabilities. 
On the assumption that a prokaryote 
host might translate eukaryotic DNA. it 
is further presumed that the product of 
that foreign gene would be most harmful 
to man if it were an enzyme, hormone, or 
other protein that was similar (homol- 
ogous) to proteins already produced by 
or active in man. An example is a bac- 
terium that could produce insulin. Such 
a “rogue" bacterium could be of benefit 
if contained, a nuisance or possibly dan- 
gerous if capable of surviving in nature. 
This is one reason that the higher the 
phylogenetic order of the eukaryote, the 
higher the recommended containment, at 
least until the efficiency of expression of 
DNA from higher eukaryotes in pro- 
karyotes can be determined. 
There is a second, more concrete rea- 
son for scaling containment upward as 
the eukaryote host becomes similiar to 
man. This is the concern that viruses 
capable of propagating in human tissue, 
and possibly causing diseases, can con- 
taminate DNA, replicate in prokaryote 
hosts and infect the experimentalist. 
Such risks are greatest when total DNA 
from donor tissue is used in “shotgun" 
recombinant experiments; it diminishes 
to much lower levels when pure cloned 
DNA is used. 
The commentators were clearly divided 
on the classification of containment 
criteria for different kinds of recombin- 
ant DNAs. Many commentators con- 
sidered the guidelines too stringent and 
rigid. Others viewed the guidelines in 
certain instances as too permissive. And 
still others endorsed the guidelines as 
sensible and reasonable, affording the 
public an enormous degree of protection 
from the speculative risks. Several sug- 
gestions wefe made for the specific 
classes of experiments, and they follow: 
1. Comment on the use of DNA from 
animals and plants in recombinant ex- 
periments varied widely. Some com- 
mentators suggested banning the use of 
DNA from primates, other mammals, and 
birds. Others suggested that higher levels 
of containment be used for all such ex- 
periments. Still' others believed that the 
guidelines were too strict for experi- 
ments of this class. I have carefully re- 
viewed the issues raised by the com- 
mentators and the responses of the com- 
mittee to certain queries concerning use 
of animal and plant DNA in these ex- 
periments. 
In my view, the classification for the 
use of DNA from primates, other mam- 
mals. and birds is appropriate to the po- 
tential hazards that might be posed. The 
physical and _ biological containment 
levels are very strict. For example, bio- 
logical containment levels are at EK2 
or EK3. and will effectively preclude ex- 
perimentation until useful EK2 and EK3 
systems are available. EK2 systems are 
still in the initial stages of development, 
and the first system was only certified at 
the most recent meeting of the Recom- 
binant Advisory Committee. An EK3 
host-vector system has yet to be tested, 
and its certification is far enough in the 
future to place a moratorium on those 
experiments requiring biological con- 
tainment at an EK3 level. The physical 
containment levels of P3 or P4 themselves 
afford a very high degree of protection. 
I am satisfied that the guidelines dem- 
onstrate the caution and prudence that 
must govern the conduct of experiments 
in this category. 
The guidelines allow reduced contain- 
ment levels for primate DNA when it is 
derived from embryonic tissue or germ- 
line cells. This is based on evidence that 
embryonic material is less likely to con- 
tain viruses than is tissue from the adult. 
Obviously, the embryonic tissue must be 
free of adult tissue, and the present 
guidelines so indicate. 
I have also carefully considered the 
special concerns arising from the use of 
DNA from cold-blooded vertebrates and 
other cold-blooded animals, because sev- 
eral commentators questioned the basis 
of lower physical and biological contain- 
ment levels for DNA from these species. 
The Recombinant Advisory Committee 
has debated this extensively, and they 
were asked to do so once again in April. 5 
The committee has now recommended 
high containment levels (P3 + EK2) when 
the DNA is from a cold-blooded verte- 
brate known to produce a potent toxin. 
That recommendation is included in the 
present guidelines. Where no toxin is in- 
volved the committee supported lower 
• A committee member. David S. Hogness. 
Ph. D., Professor, Department of Biochemis- 
try, Stanford University. California, sub- 
mitted a statement in support of lower con- 
tainment levels based on current scientific 
evidence. That evidence is based on certain 
differences between cold- and warm-blooded 
vertebrates. One of the criteria used for the 
evaluation of the relative risk that might be 
encountered with different levels of shotgun 
experiment Is the degree of sequence homol- 
ogy between the DNA of the given species 
and that of humans. Ibis criterion is used 
to estimate the likelihood that segments of 
DNA from the given species might be inte- 
grated into the human genome by recombi- 
nation: the greater the homology, the greater 
the likelihood of integration. Studies of se- 
quence homologies indicate that there is a 
considerable degree of homology between 
human DNA and DNA from other primates, 
much' less homology between primates and 
other mammals, and even lower but detect- 
able homology between birds and primates. 
By contrast, no significant homologies be- 
tween cold-blooded vertebrates and primates 
have been detected. , 
FEDERAL REGISTER, VOL. 41, NO. 131 — WEDNESDAY, JULY 7, 1976 
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