22 
" E. coli host vector systems expressing the Shiga-like toxin gene may be 
moved from BL3 to BL2 containment conditions provided that: (1) 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 
0157 :H7, grown and measured under optimal conditions; and (2) tKe - clon- 
ing vehicle is to be an EX1 vector preferably belonging to the class 
of poorly mobilizable plasmids such as pBR322, pBR328 and pBR325. 
"Nontoxinogenic fragments of the Shiga-like toxin structural gene(s) may 
be moved from BL3 + EX1 to BL2 + EK1 containment conditions or such 
nontoxic fragments may be directly cloned in EL_ coli K-12 under BL2 + 
EX1 conditions provided that the Eb_ coli host-vector systems containing 
the fragments do not contain overlapping fragments which together would 
encompass the Shiga-like toxin structural gene(s) . " 
In a letter dated July 26, 1985, Dr. O'Brien subsequently requested permis- 
sion to clone SLT structured genes, defined either by nucleotide sequence 
homology with SLT gene probes from EL_ coli or by antigenic cross-reactivity 
of their gene products with purified E. coli SLT, from bacterial species 
classified in the familes EnterobacterTaceae or Vibrionaceae into EL_ coli 
K-12 under conditions similar to those specified in the previous permission. 
Dr. O'Brien requested permission to broaden the scope of study of SLT 
toxin for the following reasons: (1) to investigate the potential role of 
SLT in the pathogenicity of a variety of bacteria; (2) to compare the SLT 
genes from various pathogenic bacteria; and (3) to facilitate the development 
of an experimental Vibrio cholerae vaccine deleted for SLT genes. 
Dr. Gottesman said the RAC Working Group on Toxins reviewed the July 26, 
1985, proposal on August 16, 1985, and offered three recommendations to 
RAC: (1) The working group first recommended by a vote of six in favor, 
none opposed, and no abstentions that the proposal as it appeared in the 
Federal Register of August 19, 1985 be approved. (2) The working group 
then recommended by a vote of six in favor, none opposed, and no abstentions 
that Campylobacter strains be considered as members of the Vibrionaceae 
for purposes of this proposal. (3) By a vote of five in favor, one opposed 
and no abstentions, the working group recommended that Shigella dysenterlae 
60R strain be substituted in Appendix F-IV-H for 933 E. coli 0157 :H7; as 
the reference strain against which toxin production is measured to determine 
whether the clone can be removed from BL3 containment. 
Drs. Collier and Clowes concurred with the recommendations of the Working 
Group on Toxins. 
Dr. Collier pointed cut that investigators routinely work under BL2 contain- 
ment conditions with pathogenic Shigella dysenteriae strains producing the 
maximum amount of toxin seen in strains in nature. He said it is not 
reasonable to restrict E. coli K-12 host-vector systems expressing the 
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