sis. There are in fact current major 
controversies concerning the role of 
natural mutations in evolution, and 
the same questions are relevant to the 
issues raised by recombinant DNA re- 
search. 
Because potentially useful vectors 
such as plasmids and viruses may be 
transferred from the initial recipient 
host cell to other cells, independent of 
the growth and survival of the host, it 
is also necessary to consider survival of 
the vectors. Plasmids and viruses occur 
widely in nature. Any particular plas- 
mid or virus will normally multiply 
only within a limited number of spe- 
cies. Thus, for example, viruses that 
infect particular bacteria neither mul- 
tiply nor cause disease in the cells of 
other bacterial species or complex or- 
ganisms. In many instances, they do 
not even enter the cells of any organ- 
ism other than the particular natural 
host. 
Only limited information concerning 
the effect of foreign DNA insertions 
on the survival or transferability of 
plasmid and viral vectors is available. 
In the case of plasmids, the factors 
contributing to their maintenance or 
loss from cells in natural environ- 
ments, even without insertion of a for- 
eign DNA, are not clearly understood. 
(2) One exception is the selective ad- 
vantage for maintenance provided by 
an antibiotic-resistance gene on the 
plasmid. 
Also, some plasmids are known to 
confer on host cells the ability to man- 
ufacture substances poisonous to 
closely related cells, thus giving the 
poison-producers special advantage in 
a competitive situation. Insertion of a 
foreign DNA fragment into the DNA 
sequence coding for the poison has 
been shown to eliminate production of 
the poison, (5) decreasing the likeli- 
hood that the cells and their resident 
recombinant DNA will survive in 
nature. 
Experiments carried out during the 
last few years have yielded only mini- 
mal information on the stability of 
plasmids containing foreign DNA in 
host cells, or on the stability of the 
foreign fragment itself. For experi- 
mental purposes, cells containing re- 
combined plasmids are generally 
grown under conditions especially de- 
signed to increase the stability of the 
plasmid (called “selective” conditions.) 
For consideration of the loss of the 
plasmids in natural environments— the 
important point for matters of 
safety— the stability of the plasmid or 
recombined DNA under ordinary, or 
nonselective, conditions needs to be 
known. A review of a limited number 
of unpublished observations indicates 
that generalizations as to the rate of 
loss of the recombined plasmid rela- 
tive to the original are impossible. 
NOTICES 
The ability of a plasmid to be trans- 
ferred from the original laboratory 
host to another cell and thereby per- 
petuate itself is also important. In 
short, certain plasmids are incapable 
of being transferred except under par- 
ticular and inferquent conditions. 
Others transfer more readily. Since 
the ability to be transferred depends 
on multiple factors, (2) it is not likely 
to be increased by insertions of a 
single foreign DNA fragment. Other 
than this, no generalization concern- 
ing the effect of a foreign DNA frag- 
ment on transferability can be made. 
The effect of bacteria and veruses con- 
taining recombined DNA on other 
forms of life 
The analysis leading to the guide- 
lines centered on the possiblility of 
deleterious effects, since the concern 
was the health and safety of living or- 
ganisms, including humans, and the 
environment. Agents constructed by 
recombinant DNA technology could 
prove hazardous to other forms of life 
by becoming pathogenic (disease-pro- 
ducing) or toxigenic (toxin-producing), 
or by becoming more pathogenic or 
toxigenic than the original agent. 
There are two basic mechanisms by 
which a recipient micro-organism 
might be altered with regard to its 
pathogenicity or toxicity as a result of 
a resident recombinant: 
(1) The recombinant DNA may result 
in formation of a protein that has un- 
desirable effects. The case in which 
bacterial cells are used as carriers of 
foreign DNA is discussed first. A for- 
eign protein, specified by the foreign 
DNA, might act after being liberated 
from the micro-organism, or it could 
function within the micro-organisi r 
and alter, secondarily, normal micro 
bial cell function in such a way that 
the cell is rendered harmful to other 
living things. Either means depends on 
the expression of the foreign genes; 
that is, the information in the foreign 
genes must be used by the recipient 
bacterium to produce a foreign pro- 
tein. Examples of proteins that might 
prove harmful to other organisms are 
hormones, enzymes, and toxins. 
Present evidence suggests that for- 
eign DNA from bacteria of one species, 
when inserted into bacteria of another 
species, may be expressed in the re- 
cipient, depending on the similarities 
of the protein synthesis mechanisms 
in the two organisms. ( 4 ) For example, 
if the donor of the foreign DNA pro- 
duces a toxic substance, than the re- 
cipient cell may produce such a sub- 
stance, provided the gene for the toxic 
substance is present in the recombin- 
ant. The recipient may or may not be 
more hazardous than the original 
donor organism, depending on the rel- 
ative ability of the two organisms to 
33103 
grow and infect an animal or plant 
species at risk. 
Expression of foreign genes from 
complex organisms (yeast, fruit flies) 
in cloned bacteria has recently been 
demonstrated experimentally. (5) In 
other experiments, insertion of a syn- 
thetic gene into E. coli led to the pro- 
duction of somatostatin, a hormone 
found in the mammalian brain. (6) 
Analogous issues must be considered 
for the case in which animal viruses 
are the carriers of foreign DNA. Inad- 
vertent dispersal of such viruses out- 
side the laboratory might result in 
entry of the recombinant DNA into 
cells of living organisms. The foreign 
genes might be expressed, resulting in 
the uncontrolled synthesis of a normal 
protein or the formation of a protein 
foreign to the infected cell. Currently, 
few relevent experimental data are 
available. (3) 
(2) The recombined DNA may itself 
cause pathogenic or toxic effects. As 
discussed above, foreign DNA inserted 
in a bacterial gene might so alter the 
microbial cell’s properties that it be- 
comes harmful to other organisms. It 
is also necessary to consider situations 
in which DNA molecules themselves 
may escape from the laboratory or 
from the experimental host cell an 
enter cells of living organisms with 
which they come in contact. Free DNA 
molecules are themselves relatively 
fragile, and the probability that they 
would survive, in a significant form or 
for a significant time, in air, water, or 
any medium, is considered remote. 
DNA that is protected in any of a vari- 
ety of ways within cells and viruses 
might be released either into, or close 
to, a living cell. 
When a cell or virus dies, or comes 
close to or invades the tissue of an- 
other living organism, the recombin- 
ant DNA may effectively enter a new 
cell. A hazardous situation similar to 
that described above might ensue if 
foreign proteins were manufactured in 
this "secondary” recipient. The recom- 
binant DNA might survive as an inde- 
pendent cellular component, or it 
could recombine by natural process 
with the DNA of the secondary recipi- 
ent. Various possible deleterious conse- 
quences of such a recombination may 
be considered. 
If the secondary recipient is another 
micro-organism, the considerations de- 
scribed earlier apply. If the secondary 
recipient is one of the cells of an 
animal or plant, the possible effects 
are different. They include alterations 
of normal cellular control mecha- 
nisms, synthesis of a foreign protein 
(such as a hormone), and insertion of 
genes involved in cancer production 
(if, for example, the foreign DNA were 
derived from a cancer-producing 
virus). 
FEDERAL REGISTER, VOL. 43, NO. 146— FRIDAY, JULY 28, 1978 
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