F«dwl Reftotar / Vol. 4fl, No. 54 / Friday. March 20, 1981 / Notices 
experiments for which HVl N. crassa 
systems are approved, provided that only 
DNA from Class 1 agents is used. For agents 
other than Class 1. unmodified laboratory 
strains of N. crassa can be used in all 
experiments for which HVl N. crassa 
systems are approved, provided that these 
are carried out at physical containment one 
level higher than required for HVl. However, 
if P3 containment is specified for HVl N. 
crassa. this level is considered adequate for 
unmodified N. crassa. Care must be exercised 
to prevent aerial dispersal or macroconidia. 
in accordance with good laboratory practice. 
Mutationally modiHed strains of N. crassa 
specified as HVl in Appendix D can used in 
all experiments for which HV2 N. crassa 
systems are approved, provided that only 
DNA from Class 1 agents is used. 
The rationale for the proposed 
changes is stated in a letter to ORDA. 
rv. Request for Approval of Certain 
Streptomyces HVl Host-Vector Systems 
Dr. Stanley Cohen of Stanford 
University has requested that 
Streptomyces coelicolor and the related 
organisms with which S. coelicolor 
naturally exchanges genetic information 
(e.g.. S. lividans, S. parvulus and S. 
griseus). using Streptomyces plasmids 
SCP2. SLPl.2, pIJlOl, actinophage 
phiC31, and their derivatives as vectors, 
be approved as HVl host-vector 
systems. 
V. Proposed Containment for 
Recombinant DNA Experiments 
Involving the Genus Streptomyces 
Dr. Stanley Cohen of Stanford 
University has requested that the 
following entry be added to Appendix E: 
Experiments involving the cloning of DNA 
among members of the genus Streptomyces 
are permitted under Pi conditions. For these 
experiments, no registration document, as 
described in Part 111. is required. 
The rationale for the proposal is 
stated in a letter tp ORDA. 
VI. Containment Levels for Recombinant 
DNA Experiments Involving Bacillus 
Subtilis 
Dr. Donald Dean of the Ohio State 
University has proposed that any 
Bacillus subtilis strain which does not 
revert to a sporeformer with a frequency 
greater than 10’ can be used for cloning 
DNA from any nonprohibited source, 
using vectors indigenous to B. subtilis, 
under the same conditions specified by 
the RAC for E. coli K-12 and 
Saccharomyces cerevisiae host-vector 
systems. 
Dr. Dean has further proposed that the 
following items be added to Appendix E: 
Bacillus subtilis strains that do not carry 
an asporogenic mutation can be used with 
vectors indigenous to B. subtilis for the 
cloning of DNA from any CDC Class 1 
organism under P2 conditions. 
Bacillus subtilis strains that do not carry 
an asporogenic mutation can be used with 
vectors indigenous to B. subtilis under Pi 
conditions for the cloning of DNA from any 
Class 1 Bacillus species. 
VII. Expression of Foot Mouth Disease 
Virus Capsid Proteins in Saccharomyces 
Cerevisiae, Bacillus Subtilis, and 
Mammalian Tissue Culture 
Genetech, Inc. South San Francisco, 
California and the United States 
Department of Agriculture Plum Island 
Animal Disease Center, Greenport. New 
York request permission to extend their 
cooperative studies aimed at the 
eventual production of a viral subunit 
vaccine for Foot and Mouth Disease 
(FMD). They wish to expand studies 
aimed at the expression of the FMD 
capsid protein in host vector systems 
other than E. coli K-12. They propose to 
study the expression of FMD capsid 
proteins in Saccharomyces cerevisiae. 
Bacillus subtilis, and mammalian tissue 
culture systems. 
VIII. Proposals Concerning Recombinant 
DNA Experiments With Genes Coding 
for Toxins 
The Recombinant DNA Advisory 
Committee (RAC), at its September 25- 
26. 1980 meeting, requested that an ad 
hoc working group be formed to 
evaluate Section I-D-2 of the 
Guidelines. Section I-D-2 prohibits the 
"deliberate formation of recombinant 
DNAs containing genes for the 
biosynthesis of toxins potent for 
vertebrates (2A) (e.g., botulinum or 
diphtheria toxins; venons from insects, 
snakes, etc.)." An ad hoc working group 
on toxins composed of Drs. Werner 
Maas and Alan Bernheimer of New York 
University, Dr. John Collier of Yale 
University. Dr. Michael Gill of Tufts 
University, Dr. Myron Levine of the 
University of Maryland, and Dr. James 
Mason of the Utah State Department of 
Health, was subsequently convened on 
January 7, 1981 to recommend 
containment conditions for recombinant 
DNA experiments with genes coding for 
toxins. Their recommendations were 
discussed by the RAC at the January 8- 
9, 1981 meeting. The ad hoc working 
group requests that the following 
proposed modifications to the 
Guidelines be published in the Federal 
Register for thirty days of comment. 
A. Section I-D-2 of Section I-D, 
Prohibitions, would be amended to read 
as follows: 
l-D-2. Deliberate formation of recombinant 
DNAs containing genes for the biosynthesis 
of toxins lethal for vertebrates at an LDso of 
less than 100 nanograms per kilogram body 
weight (e.g.. the botulinum toxins, tetanus 
toxin. Shigella dysenteriae neurotoxin). 
Guidelines for the cloning of DNAs 
containing genes coding for the biosynthesis 
of toxins which are lethal to vertebrates at 
100 nanograms to 100 micrograms per 
kilogram body weight are specified in 
Appendix C. 
B. A new Appendix G, would be 
added to the Guidelines as follows; 
Appendix G — Containment 
Conditions for Cloning of Genes Coding 
for the Biosynthesis of Toxins for 
Vertebrates. 
1. General Information 
Appendix G specifies the containment to 
be used for the cloning of genes coding for 
the biosynthesis of toxins for vertebrates. 
Cloning of genes coding for toxins for 
vertebrates that have an LDso of less than 100 
nanograms per kilogram body weight (e.g.. 
the botulinum toxins, tetanus toxin. Shigella 
dysenteriae neurotoxin) is prohibited. No 
specific restrictions shall apply to the cloning 
of genes if the protein specified by the gene 
has an LD» of 100 micrograms or more per 
kilogram of body weight. A list of toxins 
classified as to LD» is available from ORDA. 
Testing procedures for determining toxicity of 
toxins not on the list are available from 
ORDA. The results of such tests shall be 
forwarded to ORDA. which will consult with 
the ad hoc working group on toxins prior to 
inclusion of the toxin on the list. (See Section 
IV-E-l-b-(3Hi)). 
2. Containment Conditions for Cloning of 
Toxin Genes in E. Coli K-12 
(a) Cloning of genes coding for toxins for 
vertebrates that have an LD» in the range of 
100 nanograms to 1000 nanograms per 
kilogram body weight (e.g.. diphtheria toxin, 
abrin. Clostridium perfringens epsilon toxin) 
may proceed under P2 + EK2 or P3-t-EKl 
containment conditions. 
(b) Cloning of genes for the biosynthesis of 
toxins for vertebrates with an LDm in the 
range of 1 microgram to 100 micrograms per 
kilogram body weight may proceed under 
Section Ill-O (e.g.. Staphylococcus aureus 
alpha toxin. Staphylococcus aureus beta 
toxin, ricin. Pseudomonas aeruginosa 
exotoxin A, Bordatella pertussis toxin, the 
lethal factor of Bacillus anthracis. the 
Pasteurella pestis murine toxins, the oxygen- 
labile hemolysins such as streptolysin O. and 
certain neurotoxins present in snake venoms 
and other venoms). 
(c) Some enterotoxins are substantially 
more toxic when administered enterally than 
parenterally. The following enterotoxins, 
whose effects are confined to the stimulation 
of intestinal secretion and whose effects can 
be entirely reversed by administration of 
electrolyte solutions, shall be subject to 
Section III-O. These are cholera toxin, the 
heat labile toxins of E. coli. Klebsiella, and 
other related proteins that may be identified 
by neutralization with an antiserum 
monospecific for cholera toxin, and the heat 
stable toxins of E. coli and of Yersinia 
enterocolitica. 
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