Federal Register / Vol. 46, No. 126 / Wednesday. July 1, 1981 / Notices 
34455 
Prohibition l-D-6 concerning large-scale 
applicable to these experiments. 
• The entry in Appendix C which 
would effect the exemption under 
Section I-E-5 would contain a statement 
that although exempt, for these 
experiments "PI physical containment 
conditions are recommended." 
• No prior review of experiments 
involving attempts at deliberate 
expression of a eukaryotic gene in E. 
coU K-12 would be required. 
• Experiments involving the DNA of 
CDC Class 3 agents would not be 
exempted. Containment might be 
specified at Pi. P2 or P3, and review by 
tite IBC would be required. If there is 
any chance that the original Class 3 
agent can be regenerated from the 
cloned DNA. the containment level shall 
be no lower than that appropriate for 
the agent itself. 
I accept these recommendations. The 
laet recommendation dealing with Class 
3 agents is unclear as to the appropriate 
physical containment level, and as to 
how this level is to be estabUshed. To 
clarify this, the following language 
regarding DNA from CDC Class 3 agents 
in E. coh K-12 is being added to 
Appendix C; 
"Experiments using DNA from Class 3 
organisms [Ij or from cells known to be 
infected with these agents will be 
conducted at P3 containment. Lower 
containment levels may be speciHed by 
NIH (See Section IV-E-l-b-(2He))- 
Expermiments in this category require 
prior IBC review and approval." 
These decisions regarding E. coli K-12 
and Saccharomyces cerevisiae host- 
vector systems are being promulgated 
under Exemption I-E-5. and appear in 
Appendix C of the revised CuideHnes. 
Although the movement of 
experiments currently under Section 111- 
0 to the exempt category leaves no 
entries in Section lU-0. 1 am retaining a 
Section IIl-O in the Guidelines. In the 
future, experiments may be placed 
I under Section Ul-0 by their listing in 
Appendix H to the Guidelines. These 
experiments may be performed at Pi 
physical containment, and IBC review 
i prior to initiation of the experiment will 
not be required. 
n. Containment Levels for Recombinant 
DNA Experiments Involving Bacillus 
Subtilis 
Dr. Donald Dean of the Ohio State 
University requested that the status of 
recombinant DNA experiments 
invqlving BacHtus subtifis as host-vector 
systems be reevaluated. Currently, two 
asporogenic B. subtilis strains, RUB331 
and BGSC 1S53 have been certified, 
with the plasmids pUBMO. pCl94. pSl94. 
pSA2100. pEl94. pTl27. pUBll2. pC221, 
pC223. and pABl24. as HVl host-vector 
systems. 
The asporogenic mutant derivative of 
B. subtilis. ASB298. has been certified 
with the above plasmids as HV2 host- 
vector systems. 
Dr. Dean proposed that any Bacillus 
subtilis strain which does not revert to a 
sporeformer with a frequency greater 
than 10-^ can be used for cloning DNA 
from any nonprohibited source, using 
vectors indigenous to B. subtilis. under 
the same conditions specified by the 
RAC for E. coli K-12 and 
Saccharomyces cerevisiae host-vector 
systems. 
Dr. Dean further proposed that the 
following items be added to Appendix E: 
“Bacillus subtilis strains that do not 
carry an asporogenic mutation can be 
used whh vectors indigenous to B. 
subtilis for the cloning of DNA from any 
CDC Class 1 organism under P2 
conditions." 
“Baciltas subtilis strains that do not 
carry an asporogenic mutation can be 
used with vectors indigenous to B. 
subtilis under Pi condition* for the 
cloning of DNA from any Class 1 
Bacillus species." 
In support of his proposal Dr. Dean 
noted that B. subtilis is not a pathogen 
and that the organism is well 
characterized, and a great deal is known 
of its genetics. 
An announcement of Dr. Dean's 
proposal appeared in the Federal 
Register on March 20, 1981 (46 FR 
179961. During the thirty day comn^nt 
period seven letters supporting the 
proposal were received. One of the 
letters cited data showing that (1) 
Bacilhts subtilis spores either cannot 
germinate, or upon germination in the 
ileum or colin die very quickly, arrd (2) 
the viable coimt of B. subtilis in soil or 
water decreases much more rapidly than 
that of E. coli. 
The RAC evaluated Dr. Dean's 
request at the April 23-24, 1981. meeting. 
Noting that the Federal Register 
language would permit use of 
"in^genous vectors," the RAC 
questioned the advisability of permitting 
use of all Bacillus vectors, and 
reoonunended excluding from use as 
vectors any that have as hosts the 
pathogens. Bacillus cereus or Bacillus 
anthracis. 
By a vote of 12 in favor, 0 opposed, 
with 5 abstentions, the RAC voted to 
recommend that any Bacillus subtilis 
strain which does not revert to a 
sporeformer with a frequency greater 
than 10-’ can be used for cloning DNA 
from any nonprohibited source at the 
same containment conditions as used 
for E. coli K-12 and Saccharomyces 
cerevisiae host-vector systems, using 
indigenous plasmid and phage vectors 
whose host range does not include 
Bacillus anthracis or Bacillus cereus. 
I accept this recommendation, and 
this decision is being promulgated under 
Exemption l-E-5. and appears ia 
Appendix C of the revised Guidelines. 
Information about Bacillus subtilis HVl 
and HV2 systems has been accordingly 
removed from Appendix D and placed in 
Appendix F. 
By the same vote, the RAC 
recommended approval of Dr. Dean's 
additional two specific proposals 
concerning Bacillus subtilis host-vector 
systems, with the limitation that 
Bacillus plasmids or phages whose host 
range includes Bacillus cereus or 
Bacillus anthracis may not be used as 
vectors. 
I accept these recommendations and 
these decisions are promulgated as new 
entries in Appendix E of the revised 
Guidelines. 
III. Streptomyces HVl Host-Vector 
Systems 
Dr. Stanley Cohen of Stanford 
University requested that “Streptomyces 
coelicohr and the related organisms 
with which 5. coelicolor naturally 
exchanges genetic information (e.g., S. 
lividans. S parvulus. and 5. griseus]. 
using Streptomyces plasmids SPC2, 
SLPl.2, pljlOl, actinophage phiC31, and 
their derivatives as vectors, be approved 
as HVl host-vector systems." 
During the 30 day comment period, no 
comments were received on this 
proposal. 
The RAC reviewed this request at the 
April 23-24. 1981. meeting, and noted 
that these organisms are not pathogenic. 
There has been extensive experience 
with large-scale culture of these 
organisms with no known hazards. 
By a vote of 10 in favor. 0 opposed, 
with 8 abstentions, the RAC 
recommended approval of this proposal. 
I have reviewed this recommendation, 
and I am certifjnng the four specific 
Streptomyces species cited in the 
proposal as HVl host-vector systems. 
These host-vector systems have been 
added to Appendix D of the Guidelines. 
IV. Guidelines for Recombinant DNA 
Experiments with Genes Coding for 
Toxins 
The RAC, at its September 25-26, 
1980. meeting, requested that an ad hoc 
working group be formed to evaluate 
Section I-D-2 of the Guidelines. Section 
I-D-2 prohibits the "deliberate 
formation of recombinant DNAs 
containing genes for the biosynthesis of 
toxins potent for vertebrates (2A) (e.g., 
botulimum or diphtheria toxins; venoms 
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