1158 
Federal Register / Vol. 48, No. 6 / Monday, January 10, 1983 / Notices 
Department of Agriculture Recombinant 
DNA Committee. 
E. Request To Release Strains Of 
Pseudomonas Syringae and Erwinia 
Herbicola 
Drs. Nickolas Panopoulos and Steven 
Lindow of the University of California, 
Berkeley, requested permission to 
construct and release Pseudomonas 
syringae p v. syringae and Erwinia 
herbicola carrying in vitro generated 
deletions of all or part of the genes 
involved in ice nucleation. 
The aim of the experiments is to 
investigate possibilities for biological 
control of frost damage in plants. 
The proposal was summarized in the 
September 22, 1982, Federal Register (47 
FR 41925). During the comment period, 
no comments were received. 
Certain bacteria, such as 
Pseudomonas syringae and Erwinia 
herbicola cause nucleation of ice 
crystals. These bacteria are common 
plant epiphytes. A causal relationship 
has been established between frost 
damage on frost-sensitive crop plants, in 
the temperature range 0° to -5°C, and the 
populations of ice nucleation active 
bacteria present on the plants. There are 
chemically induced mutants of P. 
syringae and and E. herbicola that do 
not cause ice nucleation, and their 
effectiveness under field conditions has 
been demonstrated. The investigators 
now propose to construct deletion 
mutants for ice nucleation activity in 
prototype strains of P. Syringae and E. 
Herbicola and to evaluate their efficacy 
as biological competitors of naturally 
occurring populations of these bacteria. 
The investigators state that the 
advantages of such mutants compared 
to chemically-induced equivalents are 
genetic stability and the absence of 
silent mutations which may adversely 
effect competitive fitness. Prior to the 
field applications, the investigators plan 
to test the mutant strains in a contained 
environment, such as growth chambers 
and greenhouse, to verify that they do 
not induce frost or other •injury to plants, 
and that they are capable of colonizing 
leaves. It was pointed out that fields 
have already been sprayed with the 
chemically induced mutant organisms. 
The RAC reviewed the proposal at the 
October 25, 1982, meeting. Several 
members of the RAC expressed concern 
about the lack of data on these 
organisms, including the host range of 
the constructed organisms, and the 
broad approval being requested. One 
RAC member expressed concern about 
marking the strains with resistance to 
antibiotics such as rifamycin in the 
absence of more information about 
these organisms. The necessity for 
releasing the organisms in six different 
field and experiment stations was 
questioned. In addition, it was noted 
that ice nucleation active bacteria can 
enter the atmosphere as aerosols and 
may be important in atmospheric 
precipitation processes. What effect 
might these experiments have on rainfall 
patterns in California? On the other 
hand, it was pointed out that field 
testing of (non-recombinant-DNA) 
mutants of these bacteria is already 
being done. 
The RAC then passed a motion noting 
that similar (non-recombinant-DNA) 
field tests are already being done, and 
recommending approval of the 
requested field tests, by a vote of seven 
in favor, five opposed, with two 
abstentions. 
Because of concerns raised at the 
RAC meeting, approval of the proposed 
field tests is being withheld. The 
investigators may bring this or a 
modified proposal back for 
consideration at a future RAC meeting 
and may at that time wish to submit 
additional data resulting from 
experiments conducted in the laboratory 
or greenhouse. 
II. Summary of Actions Under the 
Guidelines 
Revision of Appendix F of the 
Guidelines 
A. Appendix F, Section F-I, second 
sentence, is amended to read as follows: 
"The cloning of genes coding for 
molecules toxic for vertebrates that 
have an LD 50 of less than 100 nanograms 
per kilogram body weight [e.g., 
microbial tbxins such as the botulinum 
toxins, tetanus toxin, diphtheria toxin. 
Shigella dysenteriae neurotoxin] is 
covered under Section III— A— I of the 
Guidelines and requires RAC review 
and NIH and IBC approval before 
initiation." 
B. A new Section, Appendix F-IV-H, 
is added to Appendix F as follows: 
"Appendix F-IV-H. The structural 
gene of the Shiga-like toxin from 
clinically isolated strains of E. coli may 
be safely cloned in E. coli K-12 under P4 
-f- EKl containment conditions. If the 
investigators wish to proceed with the 
experiments in the NIH P4 facility, a 
prior review will be conducted by an ad 
hoc group to advise NIH whether the 
proposal has sufficient scientific merit to 
justify the use of the NIH P4 facility." 
C. Appendix F, Section F-IV-C, is 
amended to read as follows: 
"Restriction fragments of 
Corynephage Beta carrying the 
structural gene diphtheria toxin may be 
safely cloned in E. coli K-12, in high 
containment Building 550 at the 
Frederick Cancer Research Facility. 
Laboratory practices and containment 
equipment are to be specified by the 
IBC. If the investigators wish to proceed 
with the experiments, a prior review will 
be conducted by an ad hoc group to 
advise NIH whether the proposal has 
sufficient scientific merit to justify the 
use of the NIH P4 facility." 
D. A new Section, Appendix F-IV-I, is 
added to Appendix F as follows: 
“A hybrid gene in which the gene 
coding for the melanocyte stimulating 
hormone (MSH) is joined to a segment 
of the gene encoding diphtheria toxin 
may be safely propagated in E. coli K- 
12, under P4 containment in high 
containment building 550 at the 
Frederick Cancer Research Facility. If 
the investigators wish to proceed with 
the experiment, a prior review will be 
conducted by an ad hoc group to advise 
NIH whether the proposal has sufficient 
scientific merit to justify the use of the 
NIH P4 facility. Before any of the strains 
may be removed from the P4 facility, 
data on their safety shall be evaluated 
by the RAC Working Group on Toxins, 
and the Working Group 
recommendation shall be acted upon by 
NIH.” 
Additional Announcement of the 
Director, NIA1D 
To correct a typographical error, 
Section Appendix F-IV-B is amended to 
read as follows: 
"Appendix F-IV-B. The pyrogenic 
exotoxin type A (Tox A) gene of 
Staphylococcus aureus may be cloned in 
an HV2 Bacillus subtilis host-vector 
system under P3 containment 
conditions." 
OMB's "Mandatory Information 
Requirements for Federal Assistance Program 
Announcements" (45 FR 39592) requires a 
statement concerning the official government 
programs contained in the Catalog of Federal 
Domestic Assistance. Normally NIH lists in 
Its announcements the number and title of 
affected individual programs for the guidance 
of the public. Because the guidance in this 
notice covers not only virtually every NIH 
program but also essentially every federal 
research program in which DNA recombinant 
molecule techniques could be used, it has 
been determined to be not cost effective or in 
the public interest to attempt to list these 
programs. Such a list would likely require 
several additional pages. In addition, NIH 
could not be certain that every federal 
program would be Included as many federal 
agencies, as well as private organizations, 
both national and international, have elected 
to follow the NIH Guidelines. In lieu of the 
individual program listing, NIH invites 
readers to direct questions to the Information 
address above about whether individual 
programs listed In the Catalog of Federal 
Domestic Assistance are affected. 
Dated: December 22, 1982. 
Richard M. Krause, 
Director, National Institute of Allergy and 
Infectious Diseases, National Institutes of 
Health. 
|FR Doc S3— 441 Filed 1-7-83: 8 45 am| 
BILLING CODE 4140-01-M 
