8 
cytotoxic doses/mg protein for cell lysates, and 10^ 50% cytotoxic 
doses/ml in culture supernatants); and 
"(3) the vector should preferably be an EK1 plasmid of the class of poorly 
mobilizable plasmids such as pBR322 and pBR328." 
Dr. Gottesman noted that this motion would permit the investigators to work 
with organisms expressing an average amount of toxin. 
Dr. Gill asked how cytotoxic doses would be defined under the motion. Dr. Levine 
suggested a positive control be designated; the modified organisms should not 
express more toxin than a specified standard organism. Dr. Gottesman said that 
specifying a biological standard would serve to standardize the characteristic 
toxicity of the clones in all laboratories. 
Dr. Levine asked Dr. O'Brien to suggest a standard strain. Dr. O'Brien suggested 
strain 933 Eb_ coli 0157H7 be used. She said that this strain usually produces 
1()6 50% cytotoxic dose/mg protein in cell lysate; when all conditions are good, 
the strain will produce 10' 50% cytotoxic doses /mq protein in cell lysates. 
Dr. Gill suggested the language of the motion specify that the clones, and the 
test strain are to be grown and measured under optimal conditions. Dr. Levine 
agreed to include language specifying use of strain 933 EL_ coli 0157H7 grown 
and measured under optimal conditions in his motion. 
Dr. Levine's amended motion read as follows: 
"The working group recommends to RAC that Eb_ coli host-vector systems 
expressing the Shiga toxin gene may be removed from P3 to P2 conditions 
under the following conditions: 
"(1) that the amount of toxin produced by the modified host-vector systems 
be no greater than that produced by the positive control strain 933 
E. coli 0157H7, grown and measured under optimal conditions; and 
"(2) the cloning vehicle is to be an EK1 vector preferrably belonging to 
the class of poorly mobilizable plasmids such pBR322, pBR328 and pBR325." 
By a vote of five in favor, none opposed, and no abstentions, the working group 
accepted this motion. 
Dr. Gottesman then asked the working group to consider the fourth item of the 
request of Dr. O'Brien and her coworkers. That item reads as follows: 
"4. If the structural gene for Shiga-like toxin is shown to be present in 
a specific bacteriophage genome and its physical location is determined, 
then we request permission to: 
[ 417 ] 
