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Federal Register / Vol. 51, No. 88 / Wednesday. May 
plasmids are certified for use: pSClOl. 
pMB9. pBR313. pBR322. pDH24. pBR325. 
pBR327, pGLlOl. and pHBl. The 
following E. coli/S. cerevisiae hybrid 
plasmids are certified as EK2 vectors 
when used in £. 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, Ylp30. 
YIp31. Ylp32. and Ylp33. 
EK2 Bacteriophage Systems. The 
following are certified EK2 systems 
based on bacteriophage lambda: 
Vector 
Xgt 
A*t USSXB* 
Xgt Z| 
AgtALO XB 
Cturon 3A 
Charon 4 A 
Charon ISA 
Charon 71 A 
Chiron 23 A 
Chiron 24A 
Host 
DPSOftpF 
DPSOiupF 
£ colt K-IZ 
OPSCUupF 
DPSO or DPSO supf 
DP50 or OPSOjjpF 
DPSO or DPSOjupF 
DPSOaupF 
DPSO or DPIOiupF 
DPSO or DPSOrupF 
E. coli K-12 strains chi-2447 and chi- 
2281 are certified for use with lambda 
vectors that are certified for use with 
strain DPSO or DPSOsopF provided that 
the su * strain not be used as a 
propagation host. 
Appendix E-IV — Neurospora crassa 
HVl. The following specified strains 
of Neurospora crassc which have been 
modified to prevent aerial dispersion: 
Ini (inositolless) strains 37102. 37401. 
46316. 64001. and 896G1. 
Csp-1 strain UCLA37 and csp-2 
strains FS 590. UCLA101 (these are 
conidial separation mutants). 
Eas strain UCLA191 (an "easily 
wettable" mutant). 
Appendix E-V — Streptomyces 
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. plJlOl. actinophage phi 
C31. and their derivatives. 
Appendix E- V ! — Pseudomonas putida 
HVl. Pseudomonas putida strains 
KT2440 with plasmid vectors pKT282. 
pKT283. and pKT264. 
Appendix F — Containment Conditions 
for Cloning of Genes Coding for the 
Biosynthesis of Molecules Toxic for 
Vertebrates 
Appendix F-l— 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 LDio 
of less than 100 nanograms per 
killogram body weight (e g., microbial 
toxins such as the botulinum toxins, 
tetanus toxin, diphtheria toxin. Shigella 
dysenteriae neurotoxin) is covered 
under Section III— A— 1 of the Guidelines 
and requires RAC review and NTH and 
1BC approval before initiation. No 
specific restrictions shall apply to the 
cloning of genes if the protein specified 
by the gene has an LDio of 100 
micrograms or more per kilogram of 
body weight. Experiments involving 
genes coding for toxic molecules with an 
LD*) 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 LD»> 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 the RAC Working Group on Toxins 
prior to inclusion of the molecules on the 
list (see Section IV-C-l-b-(2H e ))- 
Appendix F-ll — Containment 
Conditions for Cloning of Toxic 
Molecule Genes in E coli K-12 
Appendix F-ll-A. Cloning of genes 
coding for molecules toxic for 
vertebrates that have an LD»o in the 
range of 100 nanograms to 1000 
nanograms per kilogram body weight 
(e g., abrin. Clostridium perfringens 
epsilon toxin) may proceed under 
BL2 + EK2 or BL3 + EXl containment 
conditions.. 
Appendix F-ll-B. Cloning of genes for 
the biosynthesis of molecules toxic for 
vertebrates with an LD W in the range of 
1 microgram to 100 micrcgrams per 
kilogram body weight may proceed 
under BLl + 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-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 BLl -t-EKl 
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. 
7, 1586 / Notices 
Appendix F-III — 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 
Working Group on Toxins (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 BLl 
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 suktilis host-vector 
system under BL3 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 BL4 facility. 
Appendix F-TV-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 
BL2 + 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 LDj 0 s in the range 
of one microgram per kilogram body 
weight such as the exfoliative toxin. 
Appendix F-TV-E. Fragments F-l, F-2, 
and F-3 of the diphtheria toxin gene 
(tox) may be cloned in £". coli K-12 
under BLl + EK1 containment conditions 
and may be cloned in Bacillus subtilis 
host-vector systems under BLl 
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; 
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. 
Recombinant DNA Research, Volume 1 1 
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