13 
A. Proposal to Clone an E. coli Toxin Gene 
Dr. Gill said Drs. Alison O'Brien and Randall Holmes of the Chi formed 
Services Lhiversity of the Health Sciences request (tab 1083) permission 
to clone the structural gene of the Shiga-like toxin of Escherichia 
coli . Hie JE^ coli Shiga-like toxin has biological activity similar to 
the activity of Shigella dysenteriae neurotoxin. The Shiga-like toxin 
gene would be cloned in Jk_ coli EK1 host-vector systems using plasmid, 
cosmid, or lambda cloning vectors. 
Drs. O'Brien and Holmes argued that these experiments pose minimal 
risks for the following reasons: 
(1) Clinical isolates of Eh_ coli have already been demonstrated 
to elaborate large amounts of a toxin indistinguishable 
from that produced by Shigella dysenteriae 1 (Shiga toxin). 
Therefore, the genes for Shiga-like toxin production are 
present in the Ek coli gene pool in nature. 
(2) Human volunteers fed large numbers of Shigella dysenteriae 1 
organisms that produce Shiga toxin but do not colonize 
the bowel, did not become ill. Therefore, any accidental 
ingestion of the organism to be manufactured, a toxin- 
producing Eh coli K-12 strain that cannot colonize the 
human intestinal tract, should pose little hazard to man. 
(3) Purifications of Shiga toxin and E. coli Shiga-like toxin 
has not identified any excessive risk from the toxin 
aerosol izat ion that probably occurs during the purification 
process. In fact, in one laboratory, toxin was isolated from 
500 liters of culture under PI physical containment conditions. 
(4) Shiga toxin is a potent cytotoxin for a EteLa cell subline 
(a human cervical carcinoma tissue culture cell line), 
but the toxin has no effect on many other human, monkey, 
and rodent tissue culture cells. Therefore, the toxin is 
quite cell-type specific, and this limited spectrum of 
activity suggests that it would be non- toxic for most cells 
in the human body. 
(5) Contrary to the old literature, Shiga toxin is not a neuro- 
toxin. By 1955, it was established that the paralysis 
observed in rabbits and mice (but not monkeys, guinea pigs, 
hamsters, or rats) when toxin is administered intravenously is a 
reflection of the effect of toxin on the endothelium of small 
blood vessels, not a direct effect on nerve cells. 
Dr. Gill said Shiga toxin has three known properties: (1) it is cyto- 
toxic to certain epithelial cells in culture, (2) it is enterotoxic 
in that it causes fluid release from the jejunum, and (3) it causes 
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