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Federal Register / Vol. 49, No. 227 / Friday, November 23, 1984 / Notices 
HVl or HV2. A listing of those systems 
follows: 
Appendix E-l. Bacillus subtilis. 
HVl. The following plasmids are 
accepted as the vector components of 
certified B. subtilis HVl systems: 
pUBllO, pCl94, pSl94, pSA2100, 
pEl94,pTl27, pUBll2, pC221, pC223, and 
pAB124. B. subtilis strains RUB 331 and 
BGSC 1S53 have been certified as the 
host component of HVl systems based 
on these plasmids. 
HV2d. The asporogenic mutant 
derivative of Bacillus subtilis, ASB 298, 
with the following plasmids as the 
vector component: pUBllO, pCl94, 
pSl94, pSA2100, pEl94, pTl27, pUBH2, 
pC221, pC223, and pABl24. 
Appendix E-II. Saccharomyces 
cerevisiae. 
HV2. The following sterile strains of 
Saccharomyces cerevisiae, all of which 
have the ste-VC9 mutation, SHY1, 
SHY2, SHY3, and SHY4. The following 
plasmids are certified for use: YIpl, 
YEp2, YEp4, YIp5, YEp6, YRp7, Yep20, 
YEp21, YEp24, YIp25, Ylp26, YIp27, 
YIp28, YIp29, Ylp30, YIp31, YIp32, and 
YIp33. 
Appendix E-III. Escherichia coli. 
EK2 Plasmid Systems. The E. coli K- 
12 strain chi-1776. The following 
plasimids 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, 
YIp27, YIp28, YIp29, YIp30,YIp31, YIp32, 
YIp33. 
EK2 Bacteriophage Systems. The 
following are certified EK2 systems 
based on bacteriophage lambda: 
Vector 
Host 
Xgt IV£S.XB' 
DPSOsopF. 
DPSOsupF. 
E cott K-12 
DP 50 supF. 
DP50 or DP 50 sopf. 
DPS0 or DP50supF. 
DP 50 or DPbOsupF. 
DP50supF. 
DP 50 or DPSOsupF. 
DP50 or QP50 sop/F. 
XgttV£S.XB‘ 
XgtALO.XB 
Charon 3A 
Charon 4A 
Charon 16A 
Charon 21 A 
Charon 23A 
Charon 24A 
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. 
Appendix E-IV. Neurospora crassa. 
HVl. The following specified strains 
of Neurospora crassa which have been 
modified to prevent aerial dispersion: 
Ini (inositolless) strains 37102, 37401, 
46316, 64001, and 09601. 
Csp-1 strain UCLA37 and csp-2 
strains FS 590, UCLA101 (these are 
conidial separation mutants). 
Eas strain UCLAT91 (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, SLP1.2, pIJlOl, actinophage phi 
C31, and their derivatives. 
Appendix E- VI. Pseudomonas putida. 
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 
gjfenes coding for the biosynthesis of 
molecules toxic for vertebrates. The 
cloning of genes coding for molecules 
toxic for vertebrates that have an 
LD&o 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— 1 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 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 the RAC Working Group on Toxins 
proior to inclusion of the molecules 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 LDso 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 + EK1 containment 
conditions. 
Appendix F-ll-B. Cloning of genes for 
the biosynthesis of molecules toxic for 
vertebrates that have an LDso in the 
range of 100 micrograms 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-Il-C. Some enterotoxins 
are substantially more toxic when 
administered enterally than 
parenterally. The following enterotoxins 
shall be subject to BLl + 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. 
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 subtilis 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-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 BL2 + 
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