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Federal Register / Vol. 44, No. 141 / Friday, July 20, 1979 / Notices 
recombinant molecules between 
members of the Actinomycetes group at 
P2 physical containment except for 
species which are known to be 
pathogenic for man, animal, or plants. 
The following notice appeared in the 
Federal Register on April 13, 1979: 
“P2 physical containment shall be used for 
DNA recombinants produced between 
members of the Actinomycetes group except 
for those species which are known to be 
pathogenic for man, animals or plants [2AJ.” 
During the 30-day comment period no 
comments were received. The 
discussion on this proposal was led by 
Dr. Julian Davies, University of 
Wisconsin, an ad hoc consultant at the 
RAC meeting on May 21, 1979. It was 
explained that the Actinomycetes are a 
large group of closely related organisms, 
many of which are used to produce 
therapeutically active compounds. 
Ninety percent of the antibiotics 
produced industrially are derived from 
the Micromonospora and Streptomyces 
genera. They are mainly soil organisms, 
and do not exist in the gut. Genetic 
exchanges occur in almost all cases for 
which it has been looked. The basis for 
exchange includes recombination, 
mating, and fusion (heterokaryosis). 
Plasmid transfer of genetic information 
has also been demonstrated. Substantial 
DNA homology from 20-80% has been 
demonstrated in the Streptomyces 
genus. Although some members of the 
Actinomycetes are known as pathogens 
for man, animals and plants, the 
Streptomyces and Micromonospora 
genera are non-pathogenic for man ano 
animals. An extensive search of the 
literature has revealed no reports of 
pathogenicity. Based on the importance 
of these microorganisms, non- 
pathogenicity, and evidence for genetic 
relatedness, it was proposed that 
recombinant experiments between the 
Streptomyces and Micromonospora 
species be permitted under P2 
containment. The original proposal was 
restricted by the RAC to only the 
Streptomyces and Micromonospora 
genera. 
A motion to accept this proposal, 
amended as follows, was passed with 16 
for, none opposed, and 2 abstentions: 
"P2 physical containment shall be used for 
DNA recombinants produced between 
members of the genera Streptomyces and 
Micromonospora except for those species 
which are known to be pathogenic for man. 
animals or plants [2A]." 
F. Exemption for streptomyces species 
thai exchange genetic information 
The Working Group on Prokaryotic 
Host-Vectors other than E. coli 
recommended that a list of 
Streptomyces species that have been 
shown to exchange chromosomal DNA 
be placed in the exemption category of 
Section I-E-4. This proposal was 
published in the Federal Register on 
April 13, 1979 as follows: 
“ Streptomyces species that have been 
shown to exchange chromosomal DNA are 
proposed to be included under the exemption 
category of Section I-E-4 of the 1978 
Guidelines. Any recombinant DNA molecules 
that are composed entirely of DNA segments 
from one or more of the organisms listed 
below and to be propagated in any of the 
organisms listed below are exempt from the 
Guidelines. (This list is to be separate from 
the other lists of exempt organisms in 
Appendix A.) 
Streptomyces aureofaciens 
Streptomyces rimosus 
Streptomyces coelicolor 
Streptomyces griseus 
Streptomyces cyaneus 
Streptomyces venezuelae. " 
During the 30-day comment period no 
comments were received. The RAC 
considered the criteria for genetic 
exchange that were set forth as a basis 
for placing a proposed list of 
Streptomyces species in the exemption 
category of section I-E-4 of the 
Guidelines. A motion that physiological 
heterokaryosis between intact 
organisms shall be taken as evidence of 
genetic exchange under criterion 2 in the 
discussion of Appendix A of the NIH 
Director’s December 22, 1978 decision 
document was passed 18 to 0 with 1 
abstention. 
A motion was made to divide the list 
of the six proposed Streptomyces 
species into two sublists of three each 
because the evidence for pair-wise 
exchange was not as strong between the 
two sublists as the exchange within 
each sublist. The sublists are as follows: 
Sublist 1 
Streptomyces aureofaciens 
Streptomyces rimosus 
Streptomyces coelicolor 
Sublist 2 
Streptomyces griseus 
Streptomyces cyaneus 
Streptomyces venezuelae 
Any recombinant DNA molecules that 
are composed entirely of DNA segments 
from one or more of the organisms 
within ehch sublist and to be propagated 
in any of the organisms included in that 
sublist are exempt from the Guidelines. 
(This list is to be separate from the other 
lists of exempt organisms in Appendix 
A.) 
The motion was accepted by a vote of 
14 for, none opposed, and 5 abstentions. 
G. Use of agrobacterium tumefaciens 
as a host-vector system 
Dr. Mary-Dell Chilton of the 
University of Washington submitted a 
proposal for approval of Agrobacterium 
tumefaciens and its Ti (tumor-inducing) 
plasmid as a host-vector system for 
recombinant DNA experiments. Crown 
gall tumors caused by A. tumefaciens, a 
ubiquitous inhabitant of the soil, are 
induced by tumor genes located on the 
large Ti plasmids. The Ti plasmid enters 
plant cells and inserts itself in the plant 
chormosomal DNA. The Ti plasmids 
appear promising as vectors for 
introduction of desired foreign DNA into 
higher plants. 
Notice of this proposal was first 
published in the Federal Register, April 
13, 1979 as follows: 
“Non-disabled strains of Agrobacterium 
tumefaciens can be used in combinations 
with the cointegrate plasmid Ti::RP4 as a 
host-vector system at the P3 level of physical 
containment." 
No comments were received by the 
Office of Recombinant DNA Activities 
during the 30-day period following the 
publication of this proposal. On April 25, 
1979, Dr. Chilton submitted a 
supplement to her original proposal 
which represented an alternative 
approach for using the Agrobacterium 
system that would provide greater 
biological containment. The new 
strategy was described by Dr. Chilton at 
the RAC meeting on May 21, 1979. First, 
eukaryotic DNA would be inserted in a 
non-conjugative plasmid, i.e. pBR322, 
that also contains fragments of Ti 
plasmid DNA and an insert of the origin 
of replication of other cryptic 
Agrobacterium plasmids. The plasmid 
would be propagated in E. coli K-12 and 
the recombinant DNA molecules used to 
transform A. tumefaciens. The A. 
tumefaciens host strain would then be 
employed to induce tumors in higher 
plants. The advantage of the newer 
strategy is that it avoids the 
involvement of RP4 which is a wide 
range conjugative replicon. Dr. Chilton 
proposed a one step higher level of 
containment required for the eukaryotic 
insert when the Ti plasmid is used. In 
the RAC discussion, it was pointed out 
that although the Federal Register notice 
of April 13, 1979 cited the use of the 
plasmid Ti::RP4, this new experimental i 
approach was much safer. A two-part 
motion was considered by the RAC: 
a. Approve the cloning of well- 
characterized fragments of eukaryotic 
DNA under P3 conditions, either in E. 
coli K-12 or in A. tumefaciens carrying a 
Ti plasmid, using an EK2 plasmid vector 
coupled to a fragment of the Ti plasmid 
and/or the origin of replication of a 
cryptic A. Tumefaciens plasmid. 
II 
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