11 
activity and the A subunit is presumed to be the biologically active 
subunit that turns off protein synthesis. Dr. Gottesman asked if a method 
exists for detecting either the A or B subunit alone. Dr. O'Brien replied 
that currently no such technique exists. 
Dr - . Levine said Appendix F— II— C already offers a precedent for treating 
toxins on other than strictly pharmacological criteria. Doses of cholera 
toxin administered enterally which will kill an animal will not affect the 
animal if administered parenterally. Cki the other hand, Shiga toxin is a 
powerful toxin if administered parenterally but much less powerful if 
administered enterally. Enteral exposure is, however, for both cholera 
toxin and Shiga toxin the "natural" type of exposure, i.e., the type of 
exposure occurring in nature. 
Dr. Levine then moved that Drs. O'Brien and Holmes and co-workers be per- 
mitted to clone the gene for Shiga toxin under P2 physical containment 
cord it ions in EL_ ooli K-12 with restriction to certified EK2 plasmid 
vectors as the safety of these vectors has been demonstrated. Dr. Joklik 
seconded the motion. 
Dr. Fedoroff asked if EK2 phage vectors might be used. Dr. Levine said his 
motion specified plasmid vectors; however, if a RAC member proposed an 
amendment permitting use of phage vectors, he would accept the amendment. 
Dr. Alexander said there are two "environments" under discussion; one 
between the mouth and the anus, and the other the post-anal environment and 
getting back into the mouth. He knew of no post-anal environment, specifi- 
cally fresh waters, green waters, soils, or sediment, in which the phage 
density for a particular host exceeds the level of 10 4 ph^ge per milliliter. 
Below that critical density there is no evidence phage have an effect on 
the host population or have any significance in the transfer of genetic 
information. Dr. Alexander contended that phage activities in the laboratory 
should not be used as a model to suggest events occurring in the post-anal 
environment. He added that there is no good evidence that plasmids are 
involved in transfer of genetic material in the post-anal environment. 
Dr. Clowes said he would prefer cloning to be restricted to plasmid vectors. 
Phages exist and persist independently in nature whereas plasmids must be 
transferred from cell to cell in order for their DNA to persist. 
Dr. Wens ink offered an amendment to Dr. Levine's motion. Dr. We ns ink's 
amendment would specify that the Shiga-like toxin gene not be put under the 
control of an over-producing premotor. Dr. Clowes thought specifying use 
of the plasmids pBR322 and pBR325 would address that issue as these plasmids 
do not contain strong premotors . No second was offered for Dr. Wens ink ' s 
motion. 
Dr. Gottesman suggested the committee consider the approved EK2 plasmid 
vectors. She thought some of these plasmids were constructed to give 
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