Federal Register / Vol. 44, No. 73 / Friday, April 13, 1979 / Notices 
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4. A Proposed "cosmid” EK2 vector 
A proposal from Dr. John Collins of 
the Gesellschaft fur Biotechnologische 
Forschung, West Germany, for the use of 
the cosmids pJC75-58, pJC78, and pJC79 
as EK2 cosmid vectors will be 
considered by the RAC. These vectors 
are combinations of EK2 plasmid and 
lambda bacteriophage vectors which 
enable in vitro packaging of DNA. The 
proposal is available from the Office of 
Recombinant DNA Activities. 
5. Amendment of section II-D-l-a-(l) 
The Plasmid Working Group of the 
RAC has unanimously recommended 
that the phrase “when plasmid vectors 
are employed,” be added to Section II- 
D-l-a-(l] of the Guidelines. This section 
would be amended to read: 
II-D-l-a-(l). EK1. The host is always E. 
coli K-12 or a derivative thereof, and the 
vectors include nonconjugative plasmids (e.g., 
pSClOl, ColEl, or derivatives thereof [21-27]) 
and variants of bacteriophage, such as X 
[28-33], When plasmid vectors are employed, 
the E. coli K-12 hosts shall not contain 
conjugation-proficient plasmids, whether 
autonomous or integrated, or generalized 
transducing phages. 
This was proposed to allow for the 
use of EKl bacteriophage vectors in the 
presence of conjugation-proficient 
plasmids. The Working Group had 
concluded that the biological 
containment associated with these 
vectors would not be significantly 
altered. 
6. Proposed Exemption under I-E-5 for 
Experiments Involving EKl and EK2 
Host- Vector Systems 
Drs. Wallace Rowe and Allen 
Campbell, members of the RAC, have 
proposed the following action in accord 
with Section I-E-5 of the Guidelines. 
This action would exempt certain 
categories of recombinant DNA 
molecules in addition to those already 
stated in Sections I-E-l to -4. The 
proposed exemption would read as 
follows: 
Those recombinant DNA molecules that 
are propagated in E. coli K-12 hosts not 
containing conjugation-proficient plasmids or 
generalized transducing phages, when 
lambda or lambdoid bacteriophages or non- 
conjugative plasmids are used as vectors, are 
exempt from the Guidelines. 
Drs. Rowe and Campbell stated that 
this action is being proposed because of 
the large body of information that has 
accumulated concerning the E. coli K-12 
host-vector systems, all of which points 
to the safety of such systems. This 
information includes the extensive 
expert analyses of the biology of E. coli 
K-12 and of molecular segments cloned 
therein, the polyoma risk assessment 
experiments, the negative results of 
monitoring laboratory personnel for 
acquisition of E. coli K-12 and its 
plasmids, and a number of other risk 
assessment studies on the survival and 
pathogenicity of EKl and EK2 host- 
vector systems. (Additional information 
is available from the Office of 
recombinant DNA Activities.) 
Experiments that are presently 
prohibited, including those involving 
more than 10 liters of culture, would 
remain prohibited. 
7. Proposed Exemption Under I-E-5 for 
Cloning in Tissue Culture Cells 
Dr. Wallace Rowe, a member of the 
RAC, has proposed the following action 
in accord with Section I-E-5 of the 
Guidelines. This action would exempt 
certain categories of recombinant DNA 
molecules in addition to those already 
exempted in Sections I-E-l to -4. The 
proposed exemption would read as 
follows: 
Those recombinant DNA molecules that 
are propagated in cells in tissue culture and 
that are derived entirely from non-viral 
components (that is, no component is derived 
from a eukaryotic virus) or that contain no 
more than one-fourth of the genome of a 
eukaryotic virus are exempt from the 
Guidelines. 
As stated by Dr. Rowe, this action is 
being proposed because tissue culture 
experiments that do not involve 
production of competent 
microorganisms containing recombinant 
DNA do not represent a biohazard. 
There are many important experimental 
systems in which recombinant 
molecules are integrated into tissue 
culture cells in order to study gene 
function. Since these experiments do not 
involve the possibility of establishing 
recombinant molecules in the 
ecosystem, there is no need for them to 
be covered by the Guidelines. This 
proposed exemption would not apply to 
whole organisms. 
8. Use of Agrobacterium tumefaciens as 
a Host-Vector System 
Dr. Mary-Dell Chilton of the 
University of Washington has requested 
that the bacterium Agrobacterium 
tumefaciens be approved as an HV 
system for introducing recombinant 
DNA into plants 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. 
The cointegrate plasmid is capable of 
replicating in E. coli in which desired 
genes could be inserted and cloned. It 
can also replicate in A. tumefaciens and 
hence be transferred, by this host’s 
ability to induce plant tumors, to plant 
cells. Since the experimental procedures 
require maintenance of both plasmid 
virulence and the pathogenicity of A. 
tumefaciens for tumor induction, efforts 
to disarm the system will defeat its 
purpose. The proposal is based on the 
premise that P3 physical containment 
will compensate for the less than HVl 
biological containment. [The proposal is 
available from the Office of 
Recombinant DNA Activities.) 
9. Criteria for Characterized Clones 
Footnote 3 of the Guidelines outlines 
the types of data to be considered by the 
Institutional Biosafety Committees (IBC) 
for reducing required containment for 
characterized clones. The rationale for 
reducing containment levels is that 
clones that have been characterized and 
which can be regarded as free from 
harmful genes are considered to present 
a lower risk than shotgun or other 
uncharacterized clones and, therefore, 
justify relaxation of biological and/or 
physical containment. 
A Working Group of the RAC on 
characterized clones requested 
information from members of the 
scientific community which would 
provide guidance to the principal 
investigators and the IBC's for 
determining whether clones are 
sufficiently characterized. The following 
criteria which they have developed are 
intended to amplify Footnote 3 of the 
Guidelines (further information is 
available from the Office of 
Recombinant DNA Activities): 
(a) Absence of potentially hazardous 
genes. Part (a) of Footnote 3 specifies 
examples of harmful sequences which 
are of special concern. In E. coli the risk 
of induced autoimmunity from exposure 
to clones that produce proteins that are 
either human hormones or other 
biologically active molecules is 
considered insignificant. 
(b) cDNAs. These are considered 
characterized by definition. Since their 
functions will be known, judgment of 
potential harm will also be known, The 
cDNA sequences should be shown by 
test of size and hybridization to 
represent a sequence corresponding to 
the specified gene. 
(c) Cloned DNA carrying a specified 
gene. Characterization should include 
data showing that the clone carries a 
specific gene by hybridization and, if 
feasible, by expression. Size 
measurements and restriction maps 
should delineate all other sequences 
including intervening sequences and 
adjacent sequences with or without 
control functions. In accord with current 
knowledge, coding, intervening, and 
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