Federal Register / Vol. 47, No. 102 / Wednesday, May 26, 1982 / Notices 
23127 * 
subtilis HVl systems; pUBllO, pCl94, pSl94, 
pSA2100, pE194, pTl27, pUBll2, pC221, 
pC223, and pABl24. B- subtilis strains RUB 
331 and BGSC 1S53 have been certified as the 
host component of HVl systems based on 
these plasmids. 
HV2. The asporogenic mutant derivative of 
Bacillus subtilis, ASB 298 with the following 
plasmids as the vector component: pUBllO, 
pCl94, pSA2100, pEl94, pT127, pUBll2, 
pC221, pC223, and pABl24. 
HV2 — The following sterile strains of 
Saccharomyces cerevisiae, all of which have 
the ste-VC9 mutation, SHYl, SHY2 SHY3, 
and SHY4. The following plasmids are 
certified for use: YIpl, YEp2, YEp4, YIp5, 
YEp6, YRp7,YEp20, YEp21, YEp24, YIp25, 
YIp26, YIp27, YIp28, YIp29, Ylp30, Ylp31, 
YIp32, and YIp33. 
EK2 Plasmid Systems. The E. coli K-12 
strain chi-1776. Tlie following plasmids are 
certified for use: pSClOl, pMB9, pBR313, 
pBR322, pDH24, pBR327, pGLlOl, pHBl. The 
following E. coIi/S. cerevisiae hybrid 
plasmids are certified as EK2 vectors when 
use in E. coli ch'i-1776 or in the sterile yeast 
strains, SHYl, SHY2, SHY3, and SHY4: YIpl, 
YEp2, YEp4, YIp5, YEp6, YRp7, YEp20, YEp21, 
YEp24, YIp25, YIp26, YIp27, YIp28, YIp29, 
YIp30, YIp31 YIp32, YIp33. 
EK2 Bacteriophage Systems. The following 
are certified EI^ systems based on 
bacteriophage lambda; 
Vector 
Host 
XgtWES B' 
DPSOsupF 
XgtWES, B* 
DPSOsupF 
XgtZJv/r. B' 
E CO// K-12 
XgMLO. B 
DPSOsupF 
driaron 3A 
DPSO or DPSOsup/^ 
Charon 4A 
DP50 or DPSOsupF 
Charon 16A 
DPSO or DPSOsupF 
Charon 21 A 
DPSOsupF 
Charon 23A 
DPSO or DPSOsupF 
Charon 24A 
DPSO or DPSOsupF. 
E. coli K-12 strains chi-2447 and chi-2261 
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. 
E. coli K-12 strains chi-1984, chi-2705, chi- 
2001, and chi-2363 are certified for use with 
lambda vectors that are certified for use with 
strain DP50 or SP50supF provided that the 
su- strains not be used as propagation hosts. 
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 UCLA 37 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. 
Appendix 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 LDu is available 
fi'om 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-1- 
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 LDbo in the range of 100 nanograms to 1000 
nanograms per kilogram body weight (e.g., 
abrin, Clostridium perfringens epsilon toxin) 
may proceed under P2+EK2 or M+EKl 
containment conditions. 
Appendix F-II-B. Coloning of genes for the 
biosynthesis of molecules toxic for 
vertebrates with an LD» in the range of 1 
microgram to 100 micrograms per kilogram 
body weight may proceed under pl-(-EKl 
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 oxgen- 
labile hemolysins such as streptolysin O, and 
certain neurotoxins present in snake venoms 
and other venoms). 
Appendix F-U-C. Some enterotoxins are 
substantially more toxic when administered 
enterally than parenterally. The following 
enterotoxins shall be subject to Pl-l-EKl 
containment conditions; cholera toxin, the 
heat liable 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. 
Appendix F-IIl. Containment Conditions 
for Cloning of Toxic Molecule Genes in 
Organisms Other than E. coli K-12. Requests 
involving the cloning of genes coding for 
molecules toxic for vertebrates in host-vector 
systems other than E. coli K-12 will be 
evaluated by ORDA. which will consult with 
the ad hoc working group on toxic molecules. 
(See Section IV-C-l-b-(3)-(f).) 
Appendix F-IV. 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 Frederick Cancer 
Research Facility, restriction fragments of 
Corynephage Beta carrying the structural 
gene for diphtheria toxin. Laboratory 
practices and containment equipment are to 
be specified 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-I-EK1 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 LDmS in the range of one 
microgram per kilogram body weight, such as 
the exfoliative toxin. 
Appendix F-IV-E. Fragments F-1 and F-2 
of the diphtheria toxin gene (toK) may be 
cloned in E. coli K-12 under Pl-t-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). 
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 
Pl-|-Ekl conditions. 
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 IV-B-I-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 followed if an accident 
contaminates personnel or the environment. 
The principal investigator must ensure that 
everyone in the laboratory is familiar with 
both the potential hazards of the work and 
the emergency plan. (See Sections IV-B-3-d 
and rV-B-5-e.) If a research group is working 
with a known pathogen where there is an 
effective vaccine it should be made available 
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