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Federal Register / Vol. 40, No. 126 / Wednesday, July 1, 1981 / Notices 
V. Request for Permission To Clone the 
Vibrio Cholerae Enterotoxin Gene in E. 
Coli K-12 
Dr. J. J. Mekalanos of Harvard 
Medical School requested permission to 
clone in E. coli K-12 the Vibrio cholerae 
enterotoxin gene. In support of his 
request. Dr. Mekalanos noted that E. 
coli elaborates a heat-labile enterotoxin 
(LT), which has been shown to share a 
high degree of structural, antigenic and 
DNA sequence homology with cholera 
toxin. 
Dr. Mekalanos proposes to perform 
the investigation in three stages; i.e., (1) 
the cloning in E. coli K-12 of DNA 
restriction fragments of V. cholerae 
known to contain sequences 
homologous to the A subunit of the LT 
gene: (2) the cloning in E. coli K-12 of 
DNA restriction fragments of V. 
cholerae containing sequences 
homologous to the B subunit of the LT 
gene, and (3) the cloning in E. coli K-12 
of restriction fragments of V. cholerae 
containing sequences homologous to 
both the LT A and B subunit genes. 
Dr. Mekalanos’ request appeared in 
the Federal Register of March 20, 1981 
(46 FR 17997). During the thirty day 
comment period, no comments were 
received. 
RAC discussed Dr. Mekalanos’ 
request at the April 23-24, 1981 meeting. 
RAC reviewers indicated that Pld-EKl 
containment would be adequate for all 
three stages of the proposed 
experiments. By a vote of 15 in favor, 0 
opposed, with 1 abstention, the RAC 
recommended approval of the proposal 
at PH-EKl containment. 
This recommendation is in agreement 
with part 2-a of Appendix G dicussed in 
the previous item of this decision 
document and is permitted under that 
part. 
VI. Cloning and Expression of DNA 
Coding for Diphtheria Toxin 
Dr. John Murphy of Harvard 
University, in a letter dated March 11, 
1981, proposed to clone in E. coli K-12, 
the 3.9 )(b Bam restriction fragment and/ 
or the ks kb Eco fragment-of 
Corynephage Beta carrying the stuctural 
gene for diphtheria toxin. 
Dr. Murphy advanced the following 
arguments in support of his proposal: 
(1) Studies of diphtheria toxin- 
producing organisms would yield 
valuable data for risk assessment: 
(2) The localization of diphtheria toxin 
in E. coli would be determined as well 
as whether the toxin might be secreted 
by E. coli; 
(3) Mutagenesis of the tox gene would 
be used to elucidate (a) diphtheria toxin 
interaction with the eukaryotic cell toxin 
receptor, an’d (b) the mechanism of 
fragment A translocation into the 
eukaryotic cell cytosol. 
Dr. Murphy proposed to perform the 
experiments under P4 conditions at the 
Frederick Cancer Research Center or at 
the NIH if the RAC recommended the P4 
level of containment. 
A Federal Register announcement of 
Dr. Murphy’s requet appeared on March 
20, 1981 (46 FR 17997). During the thirty 
day comment period, no comments were 
received. 
The RAC discussed the proposal at 
the April 23-24, 1981 meeting. Members 
of the ad hoc Working Group on Toxins 
presented to the RAC the data gathered 
by the group on diphtheria toxin. The 
data showed that the LDso in the most 
sensitive animal tested, the guinea pig, 
was 160 nanograms per kilogram body 
weight. The LDjo in humans is estimated 
to be equal to or less than 100 
nanograms per kilogram body weight. 
This figure was estrapolated from an 
incident in Japan in which children were 
inadvertently injected with diphtheria 
toxin rather than diphtheria toxoid. By a 
vote of 15 in favor, 0 opposed, with 1 
abstention, the RAC recommended 
approval of Dr. Murphy’s proposal 'at P4 
containment. 
I accept this recommendation, and the 
following entry, number 30, is added to 
Appendix E: 
Permission is granted to clone in E. coli K- 
12 under P4 containment conditions, 
restriction fragments of Corynephage Beta 
carrying the structural gene for diphtheria 
toxin. 
VII. Proposals Involving the Expression 
of Foot and Mouth Disease Viral Coat 
Proteins in Saccharomyces Cerevisiae, 
Bacillus Subtilis, and Tissue Culture 
Systems 
The RAC at its April 23-24, 1981 
meeting reviewed proposed experiments 
jointly submitted by Genentech, Inc., 
South San Francisco, California, and the 
United States Department of 
Agriculture, Plum Island Animal Disease 
Center, Greenport, New York, to study 
the expression of Foot and Mouth 
Disease Virus (FMDV) sequermes in 
Bacillus subtilis, Saccharomyces 
cerevisiae, and mammalian tissue 
culture systems, with the goal of 
production of a viral subunit vaccine for 
Foot and Mouth Disease. This proposal 
was an extension of their prior NIH 
approved proposal for the study of the 
expression of the capsid protein of 
FMDV in£’. caliK-\Z. 
A. summary of the request was 
published for comment in the Federal 
Register of March 20, 1981 (46 FR 17996). 
During the 30 day comment period, no 
comments were received on the 
proposals. 
The RAC recommended that the work 
be permitted at Pi physical containment 
with both the B. subtilis and S. 
cerevisiae aproved host-vector systems 
if the subgenomic FMD segments were 
restricted to those sequences which map 
between 500 and 4100 of the FMD viral 
genome. Separate motions to approye 
these experiments at Pi physical 
containment under the conditions set 
forth above were passed by votes of 16 
in favor, 0 opposed, with 2 abstentions. 
I accept these recommendations with 
the added stipulation that HVl 
hostvector systems be used, as specified 
in the Genentech request, and an 
appropriate entry has been made in 
Appendix E. 
In the RAC discussion on the 
proposed experiments using a SV40 
virus vector for cloning the FMD capsid 
segments in mammalian tissue culture, 
concern was raised about the potential 
for recombination between an 
adventitious picornavirus in the cell 
culture with the SV40 and FMD viral 
sequences. The RAC recommended by a 
vote of 17 in favor, 0 opposed, with 1 
abstention, that cloning of FMD capsid 
protein sequences employing a SV40 
deletion vector in a mammalian tissue 
culture system at the Plum Island 
laboratories by approved in principle 
under P3 physical containment. The 
approval is subject to review by the 
Working Group of individual 
experiments. 
I accept this recommendation, and cn 
appropriate entry has been made in 
Appendix E. 
Vill. Contaiiunent Levels for 
Recombinant I>NA Experiments 
Involving Neuroepora Crassa 
Dr. David Perkins of Stanford 
University proposed that entry 2 in 
Appendix E be amended to read as 
follows; 
Unmodified laboratory strains of 
Neurospora crassa can be used in all 
experiments for which HVl N. crassa 
systems are approved, provided that only 
DNA from Class 1 agents is used. For agents 
other than Class 1, unmodified laboratory 
strains of N. crassa can be used in all 
experiments for which HVl N. crassa system 
are approved, provided that these are carried 
out at physical containment one level higher 
than required for HVl. However, if P3 
containment is specified for HVl N. crassa, 
this level is considered adequate for'^ 
unmodified N. Crassa. Care must be 
exercised to prevent aerial dispersal of 
macroconidia, in accordance with good 
laboratory practice. 
Mutationally modified strains of N. crassa 
specified as HVl in Appendix D can be used 
[146] 
