- 2 - 
II. Dangers Inherent In the Use of Escherichia coli, 
and Its Phages and Plasmids 
A great reliance is placed, in the Guidelines, on the use of ^ coli K-12 
and lambda, because ostensibly these organisms are not suitable for survival in 
vivo . In the case of more potentially dangerous experiments, the survival of 
such organisms is reduced by mutation to only a small fraction of the original 
potential (EK2 vectors). I believe, however, that several problems have been 
ignored : 
(A) EKl Systems . The Guidelines suggest a frequency of either lysogeniza- 
tion by lambda ( att ~ and int ~) or transfer of the nonconjugative plasmids, as 
about 10~^ to 10”^. They also cite evidence that ^ coll K-12 can survive, but 
not reproduce, in the gut of experimental animals and humans. From these argu- 
ments, it is supposed that transfer of recombinant DNA from such plasmids, 
phage, and bacteria would be small if they escaped the laboratory and were 
ingested. Such assumptions minimize the probability of gene transfer by slight- 
ly different mechanisms. It is well known that many ^ coll of the sort that 
populate the gut can carry lambda-like phage, some of which are defective in 
function, and it seems very probable that such defective phage could recombine 
with lambda-carrying vectors. Other effective recombination mechanisms exist; 
for Instance, we have recently discovered a defective transducing bacteriophage 
present in a very common enteropathogenic ^ coli ; this phage transduces at 
extremely high efficiency (as high as PI under laboratory conditions) into E. 
ooli K-12 (W. G. Coleman, Jr., and L. Leive, manuscript in preparation). This 
phage would certainly be capable of recombining genes (chromosomal or plasmid) 
into an accidentally-ingested vector. It might also be capable (although this 
possibility has not been tested) of transferring the recombinant DNA gene into 
other ^ coll strains that inhabit the gut. It seems almost certain that a 
large number of as-yet-undiscovered plasmids and phage exist in free-living E. 
coli and related organisms. By such mechanisms these could easily catalyze 
transfer of genes from EKl organisms. The relevant experiments to test such a 
possibility apparently have not as yet been done. 
The problem is compounded still further since some DNA's that may be hazard- 
ous are permitted for use in conjunction with an EKl system. For instance, 
mitochondrial DNA from primates can be used in an EKl system, even though I know 
of no evidence that mitochondrial DNA is much less likely to be deleterious if 
it escapes from its vector than is chromosomal DNA. Furthermore, continued 
propagation by a vector or related strain might be easier for this DNA if, as 
is thought by some workers, this DNA has greater homology to prokaryote DNA than 
does eukaryote chromosomal DNA. 
Recombination of DNA from virtually all cold-blooded and lower eukaryotes 
(other than known pathogens) is permitted using EKl systems, although markers 
rescued by the above mechanisms, when excreted, might be transferred to other 
hosts. Any damage produced, while not directly lethal to man, might irreparably 
change the environment. 
(B) EK2 Systems . In EK2 systems, great reliance is placed on the vastly 
reduced survival of E. coli K-12 and its plasmids and phage due to wltlple 
mutations. Indeed, the lowered survival, of less than 10 ® to 10 ^ under non— 
permissive conditions, is impressive. However, as admitted in the Guidelines 
Appendix K — 120 
