Federal Register / Vol. 49, No. 179 / Thursday, September 13, 1984 / Notices 
36053 
d. Phages for two clinical isolates of E. 
coli have been shown to control high- 
level production of Shiga-like toxin in E. 
coli K-12 host strains by phage 
conversion. Thus, either the structural 
gene(s) for the Shiga-like toxin or 
regulatory genes that control high-level 
production of the toxin are present on 
wild-type phages from clinical isolates 
of E. coli. In this sense, “cloning” of 
genes that affect production of Shiga- 
like toxin onto phage genomes has 
already occurred in nature. 
In addition, the U.S. Cholera Panel of 
the National Institute of Allergy and 
Infectious Diseases (NIAID) 
recommended that NIH reconsider the 
ban: 
... on Shiga toxin cloning experiments in 
containment facilities other than P4. This 
strict requirement will prevent most 
laboratories from deleting the Shiga gene 
from candidate V. cholerae and ETEC 
vaccine strains. Shiga toxin is now found in 
many nonpathogenic E. coli, including the 
common vector host, E. coli K-12. 
The request for reconsideration was 
published in the January 5, 1984, Federal 
Register (49 FR 696). During the 
comment period, a letter was received 
from Dr. Werner Arber, the chairman of 
the Swiss Commission for Experimental 
Genetics, which is in charge of questions 
related to research involving 
recombinant DNA molecules. Dr. Arber 
wrote that a Swiss ad hoc committee of 
experts requested by the Commission 
for Experimental Genetics had reviewed 
proposed research involving cloning of 
the Shiga toxin gene in an E. coli host- 
vector system. Dr. Arber wrote this 
committee had concluded that: 
Work with recombinant DNA could not be 
expected to present a more severe biohazard 
than work with the natural pathogens . . . 
recent investigations had shown that a 
number of bacterial strains related to 
Shigella, in particular E. 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 Timmis 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 E. coli K-12 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. A full account 
of the RAC discussion, including the 
request by Mr. Jeremy Rifkin for an arms 
control impact statement, is found in the 
Minutes of the meeting available from 
ORDA. By a vote of nine in favor, five 
opposed, and four abstensions, the RAC 
recommended that Drs. O'Brien and 
Holmes and coworkers be allowed to 
proceed with cloning the gene for Shiga- 
like toxin under P2 physical containment 
conditions in E. 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 is those are unsatisfactory. 
By a vote of eight in favor, four 
opposed, and five abstensions, the RAC 
passed the same motion but with the 
names of the investigators deleted from 
the motion. 
It has been the practice of NIH not to 
accept RAC recommendations that do 
not indicate a clear consensus. 
Accordingly, it was announced in the 
Federal Register of April 25, 1984 (49 FR 
17846), that NIH did not accept the RAC 
recommendations offered at the 
February 6, 1984, meeting relating. to the 
cloning of the Shiga-like toxin gene. The 
investigators had 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: 
a. That the containment conditions 
required for cloning of the intact 
structural gene(s) for Shiga-like toxin of 
E. coli into E. coli K-12 be reduced from 
P4 + EK1 to P3rbEKl. 
b. 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 E. coli (i.e„ approximately 10 7 
50% cytotoxic doses/mg protein in cell 
lysates and 10 8 50% cytotoxic doses/ml 
in culture supernatants when bacteria 
are grown in iron-depleted glucose 
syncase media), they request permission 
to remove such clones from the original 
containment conditions and to perform 
subsequent work with them under 
Pi -f EKl conditions. 
c. If they can identify nontoxinogenic 
fragments of the structural gene(s) for 
Shiga-like toxin, the investigators 
request permission to: 
(1) Remove any such cloned nontoxic 
fragments (generated during the search 
for clones that contain intact toxin 
structural genes) from the original 
containment conditions to work with 
them under Pi + EKl conditions; and 
(2) Diiectly clone any such nontoxic 
fragments into E. coli K-12 under Pi + 
EKl conditions. 
d. If the structural gene for Shiga-like 
toxin is shown to be present in a 
specific bacteriophage gename and its 
physical location is determined, they 
request permission to: 
(1) Remove from the original 
containment conditions any clones of 
fragments of phage genome (generated 
during the process of obtaining cloned 
toxin structural genes) that do not 
correspond to toxin structural genes and 
to work with them under Pi + EKl 
conditions; and 
(2) Directly clone any fragments of the 
phage genome that do not correspond to 
toxin structural genes into E. coli K-12 
under Pi -f EKl conditions. 
e. If in future experiments the 
investigators can isolate nontoxinogenic 
alleles of the structural gene(s) for 
Shiga-like toxin by transposon mediated 
mutagenesis (insertional inactivation) or 
by chemical mutagenesis, they request 
permission to clone these 
nontoxinogenic alleles of. the toxin 
structural gene(s) into E. coli K-12 under 
Pi + EKl conditions. 
Dr. O'Brien and coworkers supplied 
additional data in support of these 
requests. 
I-A-2. Comments on the Proposal in 
Response to the April 24, 1984, Federal 
Register Notice 
This proposal containing the five 
specific issues raised by Drs. O'Brien 
and Holmes in their April 4, 1984, letter 
appeared in the Federal Register of April 
24, 1984 (49 FR 17672). During the public 
comment period, three communications 
were received concerning this proposal. 
These communications were from Mr. 
Jeremy Rifkin of the Foundation on 
Economic Trends, Dr. Jay P. Sanford, 
President, Uniformed Services 
University of the Health Services 
(USUHS), and Ms. Patricia Campbell, 
Public Affairs Officer, USUHS. 
Mr. Jeremy Rifkin in a letter dated 
May 15, 1984, proposed that: 
. . . the RAC postpone its consideration of 
the Shiga-like toxin experiment to be 
conducted by the Uniformed Services 
University of the Health Sciences until an 
adequate mechanism is developed and 
applied that will enable RAC to determine 
the potential military applications of this and 
certain other types of recombinant DNA 
research. I am particularly concerned about 
the relationship between the development of 
vaccines and other prophylactic and 
defensive uses of recombinant DNA 
technology and the potent' al convertibility of 
this research for military purposes. 
Once again, I am asking that the RAC send 
a formal request to the Department of 
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