with the protein product could be employed. Dr. Randall Holmes said 
the amounts of toxin available for biomedical studies are minuscule. 
Studies of the biological functions associated with the toxin are 
not at the same level of sophistication as those for cholera or 
diphtheria toxin. 
Dr. Fedoroff suggested mutagenesis might be employed in conjunction with 
immuno- identification to isolate an inactivated gene. Dr. Randall Holmes 
replied that approach might be reasonable but pointed out that Shiga-like 
toxin may have an essential function as it appears to be expressed by 
all coli strains. Dr. Fedoroff asked if any Shigella strains are 
Shiga toxin minus. I>r. Randall Holmes said no Shigella strains are truly 
Shiga toxin minus; those termed toxin minus actually produce small 
amounts of the toxin. 
Dr. King Holmes said whether high amounts of toxin are produced by 
bacteria in the gut is a critical question. Dr. Gill added that the 
question of whether the toxin, if produced, crosses the gut into the 
blood stream is equally important. Dr. Randall Holmes said Shigellosis 
caused by strains producing low levels of shiga toxin induces a systemic 
antitoxin response. 
Dr. Kaper suggested a low copy nuifoer plasmid might be employed. 
Dr. Gottesman said more bacteria would then have to be grown to obtain 
sufficient ENA for the studies. She suggested that the most reasonable 
approach is to set high containment for the initial experiments to pro- 
duce a defective probe. She suggested P4 + EK1 containment. If con- 
siderations of inherent toxin toxicity are not of primary importance 
and issues of invasiveness are given more weight, P3 containment 
might be reasonable. 
Dr. King Holmes asked if risk assessment studies would be required before 
the gene could be sequenced or gene regulation and expression studied. 
Dr. Randall Holmes replied that would depend on whether the fully func- 
tional wild type gene would be studied or whether methods for working 
with a mutant toxin could be developed. 
Dr. Maas called the question on his motion (i.e., to allow the experi- 
ments at the P3 + EKl level of containment). Dr. Fedoroff asked if 
Dr. Maas would accept an amendment to raise containment to P4. Dr. Maas 
refused. By a vote of fourteen in favor, none opposed, and no abstentions, 
the question was called. By a vote of five in favor, seven opposed, 
and one abstention, the motion was denied. 
Dr. King Holmes moved to permit the experiment at P4 containment in a 
P4 facility. Dr. McKinney said the P4 facility at Frederick Cancer 
Research Facility (FCRF) is fully equipped for P4 conditions. He asked 
how experiments would be scheduled. Dr. Talbot said scheduling could 
be arranged to permit the experiments on Shiga-like toxin. 
