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Federal Register / Vol. 48. No. 106 / Wednesday. June 1, 1983 / Notices 
pSl94, pSA2100. pEl94, pTl27. pUBH2, 
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, YEp0. YRp7, YEp20. 
YEp21, YEp24, Ylp25, Ylp26, YIp27. 
Ylp28, YIp29. YIp30, YIp31. YIp32. and 
YIp33. 
EK2 Plasmid Systems. The E. coli K- 
12 strain chi-1776. The following 
plasmids are certified for use: pSClOl, 
pMB9, pBR313, pBR322. pDH24, pBR325, 
pBR327, pGLlOl, pHBl. The following E. 
coli/S. cerevisiae hybrid plasmids are 
certified as EK2 vectors when used in E. 
coli chi-1776 or in the sterile yeast 
strains, SHYl, SHY2, SHY3 and SHY4: 
YIpl, YEp2, YEp4, YIp5, YEp6, YRp7, 
YEp20, YEp21, YEp24, YIp25, YIp26, 
Ylp27, YIp28, YIp29, YIp30, YIp31, YIp32. 
YIp33. 
EK2 Bacteriophage Systems. The 
following are certified EK2 systems 
based on bacteriophage lambda: 
AglH'ES.A.B' 
Agt WfS.AB* 
XgtZJwr.AB’ 
AgtALO.AB 
Charon 3A 
Vector 
Charon 4A 
Charon 16A 
Charon 21A 
Charon 23A 
Charon 24A 
L)P5Qsi/pF 
DPSOsupF 
E. coli K-12 
DPSOsupF 
DP50 or DP50st/pF 
Host 
DP50 or DP5QsupF 
DP50 or DPSGsupF 
DP50supF 
DP50 or DPSOsupF 
DP50 or DP50si;pF 
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 DP50supF 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, UCLA101 (these are 
conidial separation mutants). 
Eas strain UCLA191 (an ‘‘easily 
wettable" mutant). 
HVl — The following Streptomyccs 
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, SLP1.2, pIJlOl, actinophage phi 
C31, and their derivatives. 
HVl — Pseudomonas putida strain 
KT2440 with plasmid vectors pKT262, 
pKT2f>3, 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. The 
cloning of genes coding for molecules 
toxic for vertebrates that have an LD M 
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 covered under Section III— 
A-l of the Guidelines and requires RAC 
review and NIH and IBC approval 
before initiation. 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, experiments 
involving genes coding for toxic 
molecules with an LD M 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— 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 LD«, 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 P3 + EK1 containment conditions. 
Appendix F-II-B. cloning 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 
oxygen-lable hemolysins such as 
streptolysin O, and certain neurotoxins 
present in snake venoms and other 
venoms). 
Appendix F-1I-C. Some enterotoxins 
are substantially more toxic when 
administered enterally than 
parenterally. The following enterotoxins 
shall be subject to Pi + EK1 containment 
conditions: 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. 
Appendix F-IU. Containment 
Conditions for Cloning of Toxin 
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)- 
( 0 ) 
Appendix F-IV. Specific Approvals. 
Appendix F-IV-A. Permission is 
granted to clone the Exotoxin A gene of 
Pseudomonas aeruginosa under Rl 
conditions in Pseudomonas aeruginosa 
and in Pseudomonas putida. 
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. 
Appendix F-IV-C. Restriction 
fragments of Corynephage Beta carrying 
the structural gene for 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 to advise NIH whether the 
proposal has sufficient scientific merit to 
justify the use of the NIH P4 facility. 
Appendix F-IV-D. The genes coding 
for the Staphylococcus aureus 
determinants, A, B, and F, which may D° 
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 LDjoS in the range of one 
microgram per kilogram body weight, 
such as the exfoliative toxin. 
Appendix F-IV-E. Fragments F-l, F-2, 
and F-3 of the diphtheria toxin gene 
(tox) may be cloned in E. coli K-12 
under Pi + EKl containment conditions 
and may be cloned in Bacidus subtilis 
host-vector systems under Pi 
containment conditions. Fragment F-l 
and fragment F-2 both contain: (i) Some 
or all of the transcriptional control 
elements of tox. (ii) the signal peptide, 
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