Federal Register / Vol. 48, No. 6 / Monday, January 10, 1983 / Notices 
1157 
Shiga toxin gene is chromosomal, and he 
questioned what effect introducing that 
gene into a high copy number plasmid 
would have. Finally, questions were 
raised concerning the relationship of 
invasiveness to pathogenicity and to 
toxin toxicity. Most of these questions 
could not be answered as inadequate 
data exist. However, there was general 
agreement that P4 containment would 
be adequate. After hearing the 
arguments, the committee, by a vote of 
twelve in favor, none opposed, and one 
abstention, recommended that the initial 
experiments be performed under 
P4 + EK1 containment conditions. A 
motion to approve the experiments at 
P3-I-EK1 failed by a vote of five in favor, 
seven opposed, and one abstention. A 
motion to approve the experiments using 
P3 laboratory practices and containment 
equipment in a P4 faculty failed to pass 
by a vote of five in favor, seven 
opposed, and one abstention. 
I accept the RAC recommendation 
that P4 + EK1 containment is adequate 
to contain safely the experiments 
proposed by Drs. O'Brien and Holmes 
and appropriate language has been 
added to Appendix F of the Guidelines. 
If the investigators wish to proceed with 
the experiments in the NIH P4 facility, a 
prior review will be conducted by an ad 
hoc group to advise NIH whether the 
proposal has sufficient scientific merit to 
justify the use of the NIH P4 facility. 
C. Request for Permission To Clone a 
Hybrid Gene Involving the Gene 
Encoding Diphtheria Toxin 
Dr. John Murphy of Harvard Medical 
School, in a letter dated October 5, 1982, 
requested permission to construct a 
hybrid molecule in which the gene 
coding for the melanocyte stimulating 
hormone (MSH) is joined to a segment 
of the gene encoding diphtheria toxin. 
The diphtheria toxin gene segment 
would encode the A subunit and 
portions of the B subunit. The segment 
would be devoid of the diphtheria toxin 
binding domain. The MSH gene would 
be a synthetic oligonucleotide. The 
MSH-diphtheria toxin hybrid gene 
would be introduced into poorly 
mobilizable plasmids such as pBR322, 
PUC9, or PUC8, and cloned in E. coli 
EKl host-vector systems. Dr. Murphy 
proposed that work leading up to the 
gene fusion would be conducted under 
Pi + EKl containment. PI + EKl 
containment would be appropriate for 
cloning the diphtheria toxin segment, as 
without a binding domain the 
polypeptide has very low toxicity. Dr. 
Murphy proposed that propagation of 
the hybrid gene in E. coli K-12 be 
conducted in the high containment 
Building 550 at the Frederick Cancer 
Research Facility (FCRF). since the 
specific toxicity of the hybrid gene 
product is unknown. 
Dr. Murphy's request was summarized 
in the Fedoral Register of September 22. 
1982. During the comment period, no 
comments were received. 
This request was discussed at the 
October 25. 1982, meeting of the RAC. 
Immediately preceding this discussion, 
the RAC discussed a previous request 
from Dr. Murphy to clone in E. coli K-12 
restriction fragments of Cor)'nephage 
Beta carrying the structural gene for 
diphtheria toxin (i.c., not joined to 
MSH). This previous request of Dr. 
Murphy's had been recommeded on two 
previous occasions by the RAC 
(meetings of April 24, 1981, and 
September 11, 1981) and approved each 
time by NIH (Federal Register of July 1. 
1981, and October 30, 1981). Because of 
a letter received urging against 
conducting this experiment, it was 
brought back to the RAC for 
reconsideration at this meeting. During a 
long discussion by the RAC, it was 
stated that this letter did not raise any 
issues of risk that were not previously 
considered by the RAC. A motion that 
the previous approval for this 
experiment be rescinded, was not 
seconded. Appendix F, Section F-IV-C, 
has been amended to indicate that the 
previous decision still stands that the P4 
facility is judged to be adequate to 
contain safely the experiment; however, 
if the investigators wish to proceed with 
the experiment in the NIH R4 facility, a 
prior review will be conducted by an ad 
hoc group to advise the NIH whether the 
proposal has sufficient scientific merit to 
justify the use of the NIH P4 facility. 
The RAC now turned to Dr. Murphy's 
new proposal to construct and 
propagate a hybrid molecule in which 
the gene coding for MSH is joined to a 
segment of the gene encoding diphtheria 
toxin. A motion was made that the 
experiment be allowed as requested by 
Dr. Murphy, i.e., work leading up to the 
gene fusion (with the separate fragments 
of the diphtheria toxin gene and of the 
synthetic MSH) could be done at Pi 
containment, but that the propagation of 
the hybrid toxin gene be done at P4 
containment at the Frederick Cancer 
Research Facility. The motion passed by 
a vote of thirteen in favor, none 
opposed, and no abstentions. I accept 
the RAC recommendation as to the 
containment necessary to contain safely 
this experiment and appropriate 
language has been added to Appendix F 
of the Guidelines. If the investigators 
wish to proceed with the experiment in 
the NIH P4 facility, a prior review will 
be conducted by an ad hoc group to 
advise the NIH whether the proposal 
has sufficient scientific merit to justify 
the use of the NIH P4 facility. 
The committee agreed that data on the 
characteristics of the recombinant 
organisms expressing the MSH- 
diphtheria toxin hybrid gene should be 
evaluated before the strains are 
permitted to leave the facility. By a vote 
of thirteen in favor, none opposed, and 
no abstentions, the RAC recommended 
that the ad hoc Working Group on 
Toxins be charged with review of the 
data on the E. coli strains carrying the 
MSH-diphtheria toxin hybrid gene and 
recommend whether the strains may be 
removed from P4 containment; the 
recommendation of the Working Group 
could be acted upon by NIH without 
action necessary by the full RAC 
although a report of the Working Group 
recommendations should be sent to the 
RAC. I accept this recommendation, and 
appropriate language has been added to 
Appendix F. 
D. Request To Field-Test Transformed 
Tomato and Tobacco Plants 
In a letter dated June 9, 1982, Dr. John 
Sanford of Cornell University requested 
permission to field-test tomato and 
tobacco plants transformed with 
bacterial [E. coli K-12) and yeast DNA 
using pollen as a vector. Plants would 
then be screened in the field to detect 
transformation events. Dr. Sanford 
argued that FT containment is 
impractical when the screening of 
thousands of whole seedlings becomes 
necessary. 
The proposal was summarized in the 
September 22, 1982, Federal Register (47 
FR 41925). During the comment period, 
no comments were received. 
The RAC discussed the proposal at 
the October 25, 1982, meeting. There 
was doubt expressed as to whether the 
experiments would actually work, since 
no one has yet reported transformation 
via pollen. There was some discussion 
about the introduction of kanamycin 
resistance into plants. A representative 
of the USDA said that antibiotics are 
used in agriculture to control bacterial 
diseases, particularly in citrus crops. 
Antibiotics are not used in the 
cultivation of tobacco. She noted that 
the plants will be tested in New York 
State under controlled conditions in a 
controlled access field. 
The RAC then voted ten in favor, one 
opposed, with three abstentions, to 
recommend approval of the experiments 
as proposed. 
Final action on this recommendation 
is being deferred pending a review of 
the proposal by the United States 
