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Federal Register / Vol. 49, No. 80 / Tuesday, April 24, 1984 / Notices 
the Guidelines (48 FR 245691 under 
Appendix F-IV-H. 
In a letter dated April 4, 1984, Drs. 
O'Brien and Holmes have now asked 
the RAC to address the following 
specific issues: 
1. That the containment conditions 
required for cloning of the intact 
strutural gene(s) for Shiga-like toxin of 
E. coli into E. coli K-12 be reduced from 
P4 + EKl to P3 +Ekl. 
2. If the investigators are successful in 
cloning the structural gene(s) for Shiga- 
like toxin and if they can document that 
the amount of toxin produced by the 
clones is no greater than the amount 
made by highly toxinogenic clinical 
isolates of E. coli (i.e„ approximately 10 1 
50% cytotoxic doses/mg protein in cell 
lysates and 10*50% cytotoxic doses/ml 
in culture supernatants when bacteria 
are grown in iron-depleted glucose 
syncase media), they request permission 
to remove such clones from the original 
containment conditions and to perform 
subsequent work with them under Pi + 
Kl conditions. 
3. If they can identify nontoxinogenic 
fragments of the structural gene(s) for 
Shiga-like toxin, the investigators 
request permission to: 
a. Romove any such cloned nontoxic 
fragments (generated during the search 
for clones that contain intact toxin 
structural genes) from the original 
containment conditions to work with 
them under Pi + EKl conditions; and 
b. Directly clone any such nonioxic 
fragments into E. coli K-12 under Pi -f 
EKl conditions. 
4. If the structural gene for Shiga-like 
toxin is shown to be present in a 
specific bacteriophage genome and its 
physical location is determined, then 
they request permission to: 
a. Remove from the original 
containment conditions any clones of 
fragments of phage genome (generated 
during the process of obtaining cloned 
toxin structural genes) that do not 
correspond to toxin structural genes and 
to work with them under Pi + EKl 
conditions; and 
b. Directly clone any fragments of the 
phage genome that do not correspond to 
toxin structural genes into E. coli K-12 
under Pi + EKl conditions. 
5. If in future experiments the 
investigators can isolate nontoxinogenic 
alleles of the structural gene(s) for 
Shiga-like toxin by transposonmediated 
mutagenesis (insertional inactivation) or 
by chemical mutagenesis, they request 
permission to clone these 
nontoxinogenic alleles of the toxin 
structural gene(s) into E. coli K-12 under 
Pi + EKl conditions. 
Dr. O’Brien and coworkers have 
supplied additional data in support of 
these requests which are available from 
ORDA. 
III. Proposed Guidelines for Submission 
Under Appendix L 
Appendix L of the Guidelines 
specifies conditions under which certain 
plants may be approved for “release 
into the environment” including field 
tests. Experiments in this category 
cannot be initiated without submission 
of relevant information on the proposed 
experiments to NIH, review by the RAC 
Plant Working Group, and specific 
approval by NIH. 
A RAC Working Group has now 
prepared draft submission guidelines for 
individuals preparing proposals under 
Appendix L of the Guidelines. This 
proposed guidance is as follows: 
Items for Consideration to be Included In 
Proposal Submissions Under Appendix L 
These annotated items were presented for 
consideration by prospective proposal 
submitters to facilitate the process of 
approval. The Working Group has found that 
the proposals so far submitted for their 
consideration have omitted information that 
is considered minimal and essential for their 
approval. Basically, the group would like to 
see detailed objectives, materials, and 
methods, including methodology for 
monitoring the experiments, and expected 
results. At a minimum summary data should 
be submitted to support the proposal. A 
check list of detailed requirements should 
include, but is not limited to: 
A. Description of Plant Materials. 
1. Give common and scientific names of 
plants and cultivars, if appropriate. ‘Tomato 
plants will be inoculated" is insufficient. 
2. If appropriate, give data or information 
on the relative homogeneity of the plant 
cultivar, and specific genetic markers the 
cultivar is known to possess. 
B. Vectors and Method of Introduction. 
1. Describe the cloned DNA segment and 
its expression in the new host. 
2. Give the method(s) by which the 
proposed DNA vector will be or has been 
constructed. Diagrams are very helpful and 
may be necessary for adequate 
understanding of the construct. Explain the 
advantages (and disadvantage(s), if 
appropriate) of your vectors, if other 
candidate vectors could be considered. 
3. If microorganisms are used to introduce 
vectors or are vectors themselves, indicate 
how they compare with wild-type strains. If 
disabled pathogens are used to transmit the 
vector, indicate measures that will most 
likely prevent these microorganisms from 
regaining or acquiring pathogenic potential. If 
the vector is likely to survive independently 
of the hosts, refer to this possibility, and 
provide any available data to assess the 
probability of such transfer to likely 
organisms. 
4. If microorganisms are used to introduce 
vectors, the assessment of subsequent 
absence of the microorganisms should be 
specified. Indicate the means of strain 
identification and retrieval. 
C. Characteristics and Monitoring of 
Plants. 
1. Provide data from greenhouse and/or 
growth chamber studies under simulated field 
conditions to support prospective field 
studies. Data should include morphological 
data for at least two generations of plants 
Specify plant monitoring procedures; 
frequency; types of data to be obtained, 
including leaf, seed, fruit, or root 
characteristics. 
2. Provide data for field plot design on the 
following: 
a. Total area; 
b. Location: where, how many; 
c. Plot design: replication, row spacing, 
planting, border rows, etc.: 
d. Name cultivar(s), if appropriate; 
e. Specify plant monitoring procedures: 
frequency; types of data to be obtained, 
including leaf, seed, fruit, or root 
characteristics; abnormalities, such as 
disease; insect population monitoring; 
collection of meteorological data, etc.; types 
of data to be sought, such as yield, resistance 
to stress, lodging, etc.; 
f. specify monitoring of the vector and/or 
introduced DNA; and 
g. specify access and security measures. 
IV. Request To Field Test Genetically 
Engineered Plants 
Cetus Madison Corporation requests 
permission to field test genetically 
engineered plants that have expressed 
disease resistance under greenhouse 
and growth chamber environments. This 
proposal will be reviewed in the portion 
of the RAC meeting closed to the public 
because it contains trade secrets (the 
processes for construction and testing of 
the organisms containing recombinant 
DNA) and/or commercially valuable 
information (the particular plants 
involved, the timing and location of the 
experiments and the details of how the 
experiments would be conducted and 
monitored) the disclosure of which 
cause Cetus Madison Corporation 
substantial competitive harm. 
V. Request to Field Test Genetically 
Engineered Microorganisms 
Advanced Genetic Sciences, Inc., of 
Greenwich, Connecticut, requests 
approval to field test genetically 
engineered Pseudomonas strains. The 
main body of the proposal is available 
from ORDA and will be reviewed in a 
portion of the meeting open to the 
public. This portion of the proposal 
includes information on bacterial ice 
nucleation and frost damage, cloning of 
ice nucleation genes, general 
information on construction of deletion 
mutants, biology of Pseudomonas 
syringae, identification of released 
strains, rationale for field trials, design 
of field trails, crops to be tested, 
biosafety considerations, duration, 
names of involved scientists, locations. 
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