6 
Dr. Gill suggested the working group approve the third item of the request 
with the added clarification that under PI + EK1 conditions the modified 
organism will not contain overlapping fragments which together would encompass 
the structural gene(s). This specification will eliminate the possibility 
that the structural gene might be regenerated through recombinational events. 
Dr. Gill so moved. Dr. Levine seconded the motion. 
By a vote of five in favor, none opposed, and no abstentions, the working group 
approved the motion to offer this recommendation to RAC. 
Dr. Gottesman asked the working group to consider the second item in the request 
from Dr. O'Brien and her colleagues which reads as follows: 
"2. If we are successful in cloning the structural gene(s) for Shiga-like 
toxin and if we can document that the amount of toxin produced by the 
clones is no greater than the amount made by highly toxinogenic clinical 
isolates of E. coli (i.e., approx imately 10' 50% cytotoxic doses/mg 
protein in cell lysates and 10^ 50% cytotoxic doses/mg in culture 
supernatants when bacteria are grown in iron-depleted glucose syncase 
media), then we request permission to remove such clones from the 
original containment conditions and to perform subsequent work with 
them under PI + EK1 conditions." 
Dr. Levine suggested that under the conditions specified by Dr. O'Brien, P2 
containment would be acceptable. He felt highly toxigenic isolates should be 
handled under conditions equivalent to P2 in the clinical laboratory. 
Dr. Holmes felt clinical isolates are often handled in the clinical laboratory 
at conditions comparable to PI. Seme discussion occurred on the appropriate 
clinical laboratory conditions for handling clinical isolates of Shigella . The 
consensus appeared to be that P2 was preferrable for organisms such as Shigella , 
because the infectious dose of the organism is low. 
Dr. Levine moved that the working group recommend to RAC that the second item 
of the request be approved with the specification that the organisms be handled 
as pathogenic EL_ coli or Shigella are to be handled under the NIH/CDC Guidelines 
entitled "Biosafety in Microbiological and Biomedical Laboratories." He said 
he would accept a friendly amendment to specify containment at P2. Dr. Gill 
said he preferred that P2 containment conditions be specified. Dr. Levine 
accepted this modification of his motion. 
Dr. Gill said some novel considerations arise when a gene is cloned in a new 
genetic background. These include: (1) has the potential for genetic transfer 
of the gene to other organisms been increased; and (2) hew does the cloning 
affect the amount of gene product expressed? He felt these issues should be 
considered in evaluating the second item in the proposal. 
[ 415 ] 
