Federal Register / Vol. 44, No. 71 / Wednesday, April 11, 1979 / Notices 
21733 
The RAC voted 19 to 1 to accept an 
amended statement which reads as 
follows: 
“In those cases where genetic 
exchange has not been demonstrated 
between two bacterial species A and B, 
neither of which is known to be 
pathogenic for man, animals, or plants, 
recombinant DNA experiments 
involving only A and B can be 
conducted under P3 containment. [2A]” 
D. Specific Bacillus Subtilis Experiment. 
The RAC considered a request to permit 
cloning in B. subtilis, under P2 
conditions, of DNA derived from either 
E. coli K-12 or Saccharomyces 
cerevisiae using NIH-approved EK2 
plasmid vectors. No comments were 
received during the 30-day period for 
comment. 
The RAC agreed that propagation in 
B. subtilis of E. coli K-12 DNA on an E. 
coli vector would be an exempt 
experiment under Section I-E-3. The 
RAC passed a motion by a vote of 11 to 
0 with 2 abstentions to permit the 
cloning in B. subtilis, under P2 
conditions, of DNA derived from 
Saccharomyces cerevisiae using EK2 
plasmid vectors provided that an HVl B. 
subtilis host is used. 
E. Containment Levels for Shotgun 
Cloning of Primate and Other 
Mammalian DNA in E. Coli K-12 
The RAC considered proposed 
changes in the Sections III— A— 1— a— (1 ) 
and -(2) of the Guidelines so as to 
include the possibility of P3 + EK1 
containment as an alternative to the 
present P2 + EK2 containment 
requirement for the shotgun cloning of 
primate or other mammalian DNA into 
E. coli K-12. During the 30-day period 
for public response, no comments were 
received. 
This proposal had been considered 
during the revision of the Guidelines and 
the issue is discussed in the Decision 
Document which accompanied the 
Revised Guidelines (Federal Register, 
December 22, 1978, page 60088). 
The RAC considered the possibility of 
requiring an EK2 certified vector in EKl 
hosts or even a “nonmobiliza^le vector" 
as had been proposed at the last RAC 
meeting. Two members emphasized that 
there should be no greater containment 
required for general cloning of primate 
or mammalian DNA than is already 
required for tumor viruses. One member 
described at length the physical 
containment provided by a P3 facility 
which under normal medical 
microbiological laboratory conditions is 
used only for serious pathogens. 
The RAC voted 16 to 1 with 4 
abstentions to amend Sections III— A— 1— 
a-(l) and III— A— 1— a— (2) of the Guidelines 
to read as follows: 
“III-A-l-a-(l). Primates. P2 physical 
containment -fan EK2 host-vector or 
P3 + EK1. Any lowering of containment 
below these levels (i.e., for purified DNA 
or characterized clones) cannot be made 
solely by an institutional biosafety 
committee but requires NIH approval. 
(See Section IV— E— 1— b— (3).) 
III-A-l-a-(2). Other Mammals. P2 
physical containment -fan EK2 host- 
vector or P3-fEKl.” 
The RAC also indicated its desire that 
these containment levels for EKl 
systems should apply to other HVl 
systems under equivalence provisions, 
discussed above. 
F. Amendment of Sections lll-B-2 and 
III-C-5 of the Guidelines 
The RAC considered for approval 
changes in the Sections III— B— 2 and III- 
C-5 of the Guidelines so as to include 
the words "into a lambdoid phage 
vector.” This change had been 
previously recommended by the RAC at 
its October 1978 meeting. During the 30- 
day period for public response, no 
comments were received. 
In the publication of proposed 
changes in the Federal Register on 
January 15, 1979, it was noted that the 
greated flexibility afforded with non- 
El^ lambdoid phage vectors does not in 
any way compromise safety or 
containment in the final host. The 
continued requirement for EK2 plasmid 
vectors insures that the final cloning 
back into the host of origin can be done 
with a minimal introduction of E. coli 
genetic information. 
It was emphasized that the subject of 
these sections is essentially the self- 
cloning of DNA with the introduction of 
either lambda or E. coli plasmid DNA. 
For the case of plasmid vectors there 
might be the possibility of the vector 
carrying some E. coli chromosomal 
DNA. This situation is less likely for the 
lambda bacteriophage vectors. 
The RAC voted 19 to 0 with 2 
absentions to amend Sections III— B— 2 
and III-C-5 of the Guidelines to read as 
follows: 
“Section III— B— 2. Return of DNA 
segment to Non-HVl host of origin ' ' * 
* * * For a prokaryote which does not 
exchange genetic information with E. 
coli (Host B), the following type of 
experiment may be carried out without 
Host B having been approved as a HVl 
host: DNA from Host B may be inserted 
into a lambdoid phage vector or into a 
vector from a certified EK2 host-vector 
system and propagated in E. coli K-12 
under the appropriate containment 
conditions [see Section III— A— 1— b— (2)]. 
Subsequently, this recombinant DNA 
may be returned to Host B and 
propagated in Host B under Pi 
conditions. [43] 
III-C-5. Fungal or Similar Lower 
Eukaryotic Host- Vector Systems * * * 
* * * In addition to the experiments 
described above, the following 
experiments may be carried out without 
the eukaryotic host (Host C) having 
been approved as an HVl host: DNA 
from Host C may be inserted into a 
lambdoid phage vector or into a vector 
from a certified EK2 host-vector system 
and propagated in E. coli K-12 under the 
appropriate containment conditions [see 
Section III— A— 1— (a)— (5)]. Subsequently, 
this recombinant DNA may be returned 
to Host C and propagated there under Pi 
conditions. [43] * * * 
G. Amendment of Section III-C-2, 
Invertebrate Host- Vector Systems 
The RAC recommended that Section 
III-C-2 concerning invertebrate host- 
vector systems be divided into two 
parts. The first part, III-C-2-a, would 
retain the present wording of this 
section: the second part III— C— 2— b would 
enable the use of nonviral vectors. 
Section III-C-2 would therefore read as 
follows: 
“III-C-2. Invertebrate Host-vector 
systems. 
III-C-2-a. Insect viral vectors. As 
soon as information becomes available 
on the host range restrictions and on the 
infectivity, persistence, and integration 
of the viral DNA invertebrate and 
invertebrate cells, experiments involving 
the use of insect viruses to propagate 
DNA sequences will be evaluated by 
NIH on a case-by-case basis [45] and 
will be conducted under the 
recommended physical containment 
conditions. (See Section IV-E-l-b-(3)- 
(c))- 
Ill— C— 2— b. Nonviral vectors. 
Organelle, plasmid, and chromosomal 
DNAs may be used as vectors. DNA 
recombinants formed between such 
vectors and host DNA, when propagated 
only in that host (or a closely related 
strain of the same species), are exempt 
from these Guidelines (see section I-E). 
DNA recombinants formed between 
such vectors and DNA from cells other 
than the host species require Pi physical 
containment for invertebrate cells in 
culture since invertebrate cells in culture 
inherently exhibit a very high level of 
containment. Experiments which require 
the use of whole animals will be 
evaluated by NIH on a case-by-case 
basis [45].” 
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