38060 
Federal Register / Vol. 47, No. 167 / Friday, August 27, 1982 / Notices 
E. coli K-12 strains chi-2447 and chi- 
2281 are certified for use with lambda 
vectors that are certified for use with 
strain DP50 or DPSOsupF provided that 
the su-strain not be used as a 
propagation host. 
Additional certified host-vector 
systems. 
HVl — ^The following specified strains 
of Neurospora crassa which have been 
modified to prevent aerial dispersion: 
Ini (inositolless) strains 37102, 37401, 
46316, 64001, and 89601. 
Csp-1 strain UCLA37 and csp-2 strains 
FS 590, UCLAlOl (these are conidial 
separation mutants). 
Eas strain UCLAlOl (an “easily 
wettable” mutant). 
HVl — ^The following Streptomyces 
species: Streptomyces coelicolor, S. 
lividans, S. parvulus, and S. griseus. The 
following are accepted as vector 
components of certified Streptomyces 
HVl systems: Streptomyces plasmids 
SCP2, SLPl.2, pIJlOl, actinophage phi 
C31, and their derivatives. 
HVl — Pseudomonas putida strain 
KT2440 with plasmid vectors pKT262, 
pKT263, and pKT264. 
Appendix F. — Containment Conditions 
for Cloning of Genes Coding for the 
Biosynthesis of Molecules Toxic for 
Vertebrates 
Appendix F-I. General Information. 
Appmdix F specifies the containment to 
be used for the deliberate cloning of 
genes coding for the biosynthesis of 
molecules toxic for vertebrates. Cloning 
of genes coding for molecules toxic for 
vertebrates that have an LDso of less 
than 100 nanograms per kilogram body 
weight (e.g., microbial toxins such as the 
botulinum toxins, tetanus toxin, 
diphtheria 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 LDso of 100 micrograms or more 
per kilogram of body weight. 
Experiments involving genes coding for 
toxic molecules with an LDso of 100 
micrograms or less per kilogram body 
weight shall be registered with ORDA 
prior to initiating the experiments. A list 
of toxic molecules classified as to LDso 
is available from ORDA. Testing 
procedures for determining toxicity of 
toxic molecules not on the list are 
available from ORDA. The results of 
such tests shall be forwarded to ORDA, 
which will consult with an ad hoc 
working group on toxic molecules prior 
to inclusion of the molecule on the list. 
(See Section IV-C-l-b-{2)-(e).) 
Appendix F-II. Containment 
Conditions for Cloning of Toxic 
Molecule Genes in E. coli K-12. 
Appendix F-II-A. Cloning of genes 
coding for molecules toxic for 
vertebrates that have an LDm in the 
range of 100 nanograms to 1000 
nanograms per kilogram body weight 
(e.g., abrin, Clostridium perfringens 
epsilon toxin) may proceed under 
P2 H- EK2 or P3 + ^1 containment 
conditions. 
Appendix F-II-B. Cloning of genes for 
the biosynthesis of molecules toxic for 
vertebrates with an LDm in the range of 
1 microgram to 100 micrograms per 
kilogram body weight may proceed 
under Pi -|- 1^1 containment conditions 
(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). 
Appendix F-II-C. Some enterotoxins 
are substantially more toxic when 
administered enterally than 
parenterally. The following enterotoxins 
shall be subject to Pi -|- EKl 
containment conditions: cholera toxin, 
■the heat labile toxin 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. 
Appendix F-III. Containment 
Conditions for Cloning of Toxic 
Molecule Genes in Organisms Other 
than E. cali K-12. Requests involving the 
cloning of genes coding for modecules 
toxic for vertebrates in host-vector 
systems other than E. coli K-12 will be 
evaluated by ORDA, which will consult 
with the ad hac working group on toxic 
molecules. (See Section IV-C-l-b-(3)- 
(f).) 
Appendix F-LV. Specific Approvals. 
Appendix F-IV-A. Permission is 
granted to clone the Exotoxin A gene of 
Pseudomonas aeruginosa under Pi 
conditions in Pseudomonas aeruginosa. 
Appendix F-IV-B. The pyrogenic 
endotoxin type A (Tox A) gene of 
Staphylococcus aureus may be cloned in 
an HV2 Bacillus subtilis host- vector 
system under P3 containment 
conditions. 
Appendix F-IV-C. Permission is 
granted to clone in E. coli K-12, in high 
containment Building 550 at the Fredrick 
Cancer Research Facility, restriction 
fragments of Corynephoge Beta carrying 
the structural gene for diphtheria toxin. 
Laboratory practices and containment 
equipment are to be speciHed by the 
IBC. 
Appendix F-IV-D. The genes coding 
for the Staphylococcus aureus 
determinants. A, B, and F, which may be 
implicated in toxic shock syndrome, 
may be cloned in E. coli K-12 under P2 
+ EKl conditions. The Staphylococcus 
aureus strain used as the donor is to be 
alpha toxin minus. It is suggested that, if 
possible, the donor Staphylococcus 
aureus strain should lack other toxins 
with LDsoS in the range of one 
microgram per kilogram body weight, 
such as the exfoliative toxin. 
Appendix F-IV-E. Fragments F-1, F-2, 
and F-3 of the diphtheria toxin gene 
(tox) may be cloned in F. coli K-12 
under PI -|- EKl containment conditions. 
Fragment F-1 and fragment F-2 both 
contain (i) some or all of the 
transcriptional control elements of tox, 
(ii) the signal peptide, and (iii) fragment 
A (the center responsible for ADP- 
ribosylation of elongation factor 2). 
Fragment F-3 codes for most of the non- 
toxic fragment B of the toxin, and 
contains no sequences coding for any 
portion of the enzymatically-active 
fragment A moiety. 
Appendix F-IV-F. The gene(s) coding 
for a toxin (designated LT-like) isolated 
from E. coli which is similar to the E. 
coli heat labile enterotoxin (LT) with 
respect to its activities and mode of 
action, but is not neutralized by 
antibodies against cholera enterotoxin 
or against LT from human or porcine E. 
coli strains and sequences homologous 
to the E. coli LT-like toxin gene may be 
cloned under Pi -F EKl conditions. 
Appendix F-IV-G. Genes from Vibrio 
fluvialis. Vibrio mimicus and non 0-1 
Vibrio cholerae, specifying virulence 
factors for animals, may be cloned 
under PI + EKl conditions. The 
virulence factors to be cloned will be 
selected by testing fluid induction in 
suckling mice and Y-1 mouse adrenal 
cells. 
Appendix G. — Physical Containment 
Appendix G-I. Standard Practices 
and Training. The first principle of 
containment is a strict adherence to 
good microbiological practices [1-10]. 
Consequently, all personnel directly or 
indirectly involved in experiments on 
recombinant DNAs must receive 
adequate instruction. (See Sections FV- 
B-l-e and IV-B-5-d.) This shall, as a 
minimum, include instructions in aseptic 
techniques and in the biology of the 
organisms used in the experiments, so 
that the potential biohazards can be 
understood and appreciated. 
Any research group working with 
agents with a known or potential 
biohazard shall have an emergency plan 
which describes the procedures to be 
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