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Feddral Renter / Vol. 46. No. 46 / Thursday. March 12. 1961 / Notices 
coupled to a fragment of the Ti plasmid 
and/or the origin of replication of an 
Agrobacterium plasmid, under 
containment conditions one step higher 
than would be required for the desired 
DNA In HVI systems (l.e., one step 
higher physical containment than that 
specified In the subsections of Section 
lO-A). Transfer into plant parts or cells 
in culture would be permitted at the 
same containment level (one step 
higher)." 
Dr. Clarence Kado of the University of 
California. Davis, proposed in a latter 
dated October 2. 1960 that the fourth 
entry In Appendix E be modifled to read 
as follows; 
**4. Cloned desired fragments from any 
non-prohibited source may be 
transferred into Agrobacterium 
tumefaciena containing a Tl plasmid (or 
derivatives thereof), using a 
nonconjugative £. coU plasmid vector 
coupled to a fragment of the Tl plaamid 
and/or the origin of replication of an 
Agrobacterium plasmid, under 
containment conditions that would be 
required for the desired DNA in HVl 
systems (Le.. that specified in the 
subsectioiu of Section lU-A). Transfer 
into plant parts or cells In culture would 
be permitted at the same containment 
level" 
An announcement of Dr. Kado's 
proposed amendment was published in 
the Federal Rsflster of November 26. 
1860 (45 PR 79387). No comments were 
received during the thirty day comment 
period. 
During the discussion of the proposal 
at the January g-g. igei RAC meeting, it 
was noted that this proposal would 
effectively designate Agrobacterium 
tumefaciena an HVl system. 
It was pointed out that while 
Agrobacterium tumefaciena is a plant 
pathogea plant tissue must be injured to 
permit Infection. Further, the Mnetic 
information Introduced into plant cells 
by the Agrobacterium tumefaciena Tl 
plasmid Is not stably integrated into the 
plant genome and is not transmitted at 
meiosis into seeds. Reservations were 
expressed, however, about approving A. 
tumefaciena as an HVl system. A 
motion to permit cloning of DNA from 
plants and nonpathogenic prokaryotes 
in Agrobacterium tumefaciena with 
subsequent transfer to plants or plant 
tissue under P2 containment conditions 
was recommended by a vote of 
eeventeen in favor, none opposed and 
two abstentions. 
1 accept this recommendation, and 
entry 4 of Appendix B. la amended to 
read as follows: 
"4. Cloned desired fragments from any 
non-prohibIted source may be 
transferred Into Agrobacterium 
tumefaciena containing a Ti plasmid (or 
derivatives thereof), using a 
nonconjugative E. coU plasmid vector 
coupled to a fragment of the Ti plasmid 
and/or the origin of replication of an 
Agrobacterium plasmid, under 
containment conditions one step higher 
than would be required for the desired 
DNA In HVl systems (i.e.. one step 
higher than that specified in the 
subsections of Section III-A). However. 
DNA from plants and nonpathogenic 
prokaryotes may be cloned under P2 
containment conditions. Transfer into 
plant parts or cells in culture is 
permitted at the same containment level 
as is used for the cloning in 
Agrobacterium tumefaciena. 
l-F. Request for Lowering of 
Containment Under Entry Four of 
Appendix E 
Dr. Mary-Dell Chilton of Washington 
University in SL Louis, in a letter of 
September 10, I860, requested a 
reduction in physical containment, to 
the P2 level for the manipulation in 
Agrobacterium tumefaciena of (1) the 
Saccharomycea cereviaiae alcohol 
dehydrogenaaa 1 gene and (2) the gene 
coding for the maize [Zea mays) seed 
storage protein, zein. The cloned DNA 
and the cloning vectors will be 
introduced into tobacco plants. These 
experiments are c\irrently covered under 
entry 4 of Appendix E which stipulates 
use of P3 containment. Dr. Chilton's 
proposal was published for comment In 
the November 26, 1960 Federal Register 
(45 FR 79367). No comments were 
received during the thirty day comment 
period. 
Noting that the cloned DNA 
sequences to be manipulated are well 
characterized, the RAC at the January 6- 
9. 1961 meeting recommended, by a vote 
of fifteen in favor, none opposed and 
four abstentions. Pi containment 
conditions for the described 
experiments. 
1 accept this recommendation, and 
text has been added to entry 4 of 
Appendix E, so indicating. 
f-C. Propoaed Procedurea for Previoualy 
Approv^ Large-Scale Recombinant 
DNA Experimenta 
1. Application Procedurea for Minor 
Modifications of Previously Approved 
Large-Scale Recombinant DNA 
Experiments. The following procedures 
for handling minor modifications of 
previously approved large-scale 
recombinant DNA experiments, 
generated by a Working Croup of the 
Recombinant DNA Advisory Committee 
(RAC), were published in the November 
26. 1960 Federal Register (45 FR 78367): 
"Procedures have been developed for 
considering applications to grow more 
than ten liters of an-organism containing 
recombinant DNA. These procedures 
Include consideration of the request by a 
working group of the NIH Recombinant 
DNA Advisory Committee, submission 
of the request to the full RAC after 
consideration by the working group, and 
subsequent submission to the Director. 
NIH. for Hnal review. This procedure 
has taken a minimum of two months. 
Therefore, the following procedures are 
proposed to expedite consideration of 
requests to grow more than ten liters of 
recombinant DNA-containing organisms 
when these proposals represent minor 
modifications of previously approved 
experiments. Modifications indude 
deletion of sequences from the 
recombinant DNA. changes in 
promoters, addition of short seqments 
not affecting the nature of the expressed 
products, and minor changes In the 
properties of the host Changes are 
considered minor if they do not affect 
either the containment properties of the 
vector or the host, or the natiire of 
products made, or add new products. 
Therefore, the procedures for dealing 
with minor modifications have the 
objective of determining that the change 
is indeed minor. To determine whether a 
change is minor, two levels of review 
will take place: 
"(1) By ORDA. which will decide upon 
receipt ol a request to process it as a 
new request or as a minor modification, 
and in the latter case 
"(2) by a working group of at least two 
members of RAC. 
"The Working Group has proposed 
that the following language be added to 
the 'Application Procedures for Large- 
Scale Recombinant DNA Experiments'; 
"6. Proposals that the submitter 
considers to represent minor 
modifications of already approved 
experiments will be handled by an 
expedited procedure. A request must be 
submitted to ORDA. This request should 
include the changes made, the way in 
which these changes were made (e.g., 
mutagenesis, recloning), and the nature 
and results of any tests done to 
determine that no major change has 
occurred (e.g.. restriction enzyme 
analysis, tests for produced products, 
tests of vector mobilization). 
"ORDA will determine whether the 
submission represents a minor 
modification of an approved experiment 
If so. the request will be submitted 
promptly to a working group of at least 
two RAC members. If possible, these 
members should have been present at 
the RAC discussion of approval of the 
original experiment. If any member of 
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