19 
"Work with recombinant DNA could not be expected to present a more 
severe biohazard than work with the natural pathogens ... recent 
investigations had shewn that a number of bacterial strains related 
to Shigella , in particular _Eh_ coli strains, carried genes homologous 
to the gene for Shiga toxin. . .although Shigellosis is a serious 
disease, it does not represent a serious danger for an epidemic." 
A letter from Dr. Kenneth Tirnnis of the Universite de Geneve said: 
"An ad hoc committee of medical microbiologists specifically constituted 
in Switzerland to evaluate the possible danger of cloning in Eh_ coli 
K12 the gene for Shiga toxin, concluded that the experiment represented 
no greater danger than did work on Shigella itself and, as a result, 
recommended P2/EK1 containment conditions.... A different committee 
of medical microbiologists set up for the same purpose in Western 
Germany arrived at precisely the same conclusion." 
The RAC reviewed the proposal of Drs. O’Brien and Holmes at the February 6, 
1984, meeting. By a vote of nine in favor, five opposed, and four absten- 
tions, the RAC recommended that Drs. O'Brien and Holmes and ccworkers be 
allowed to proceed with cloning the gene for Shiga-like toxin under P2 
physical containment conditions in fh_ coli K-12, restricted to using EK2 
plasmid vectors, commencing first with the use of pBR325 and pBR322 and 
proceeding to other EK2 plasmid vectors only if those are unsatisfactory. 
By a vote of eight in favor, four opposed, and five abstentions, the RAC 
passed the same motion but with the names of the investigators deleted 
from the motion. 
It has been the practice of NIH net to accept RAC recommendations that do 
not indicate a clear consensus. Accordingly, NIH did not accept the RAC 
recommendations offered at the February 6, 1984, meeting. The investigators 
have approval, however, to conduct these experiments at the P4 level of 
containment under their previous permission which appears in the Guidelines 
(48 FR 24569) under Appendix F-IV-H. 
In a letter dated April 4, 1984, Drs. O'Brien and Holmes asked the RAC 
to address the following specific issues: 
(1) That the containment condition required for cloning of the intact 
structural gene(s) for Shiga-like toxin coli into Eh_ coli K-12 
be reduced frcm P4 + EK1 to P3 + EK1 . 
(2) If the investigators are successful in cloning the structural 
gene(s) for Shiga-like toxin and if they can document that the 
amount of toxin produced by the clones is no greater than the 
amount made by highly toxinogenic clinical isolates of ooli 
(i.e., approximately 10^ 50% cytotoxic doses/mg protein in cell 
lysates and 10^ 50% cytotoxic doses/ml in culture supernatants 
when bacteria are grown in iron-depleted glucose syncase media). 
[472] 
