Federal Register / Vol. 47, No. 77 / Wednesday. April 21, 1982 / Notices 
17197 
subtilis HVl systems: pUBllO, pCl94, pSl94, 
pSA2100, pEl94, pT127, 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. 
HV2 — The asporogenic mutant derivative 
of Bacillus subtilis, ASB 298, with the 
following plasmids as the vector component; 
pUBllO, pCl94, pSl94. 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: Ylpl, YEp2, YEp4, Ylp5, 
YEp6, YRp7, YEp20, YEp21, YEp24, Ylp25, 
Ylp26, Ylp27, Ylp28, YIp29, YIp30, YIp31, 
Ylp32, 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, 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: Ylpl, 
YEp2, YEp4, Ylp5, YEp6, YRp7, YEp20, YEp21, 
YEp24, YIp25, YIp26, YIp27, YIp28, Ylp29, 
YIp30, YIp31, Ylp32 and Ylp33. 
EK2 Bacteriophage Systems. Tlie following 
are certified EK2 systems based on 
bacteriophage lambda: 
Vector 
Host 
K^WESkB ’ 
DPSOsupF 
)igtW£S\B' 
np50st/pF 
\gtZJwrxS ' 
T3Eco// K- 12 
XgtALO^B 
DP50st/pF 
Charon 3A 
DP50 or DP505f/pF 
Charon 4A 
OP50 or DPSOsi/pF 
Charon 16A 
DPSO or DPSOsupF 
Charon 21 A 
DPSOsypE 
Charon 23A 
DPSO or DPSOsf/pF 
Charon 24A 
DPSO or DPSOsupF 
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. 
'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 SPSOsupF provided that the su® 
strains not be used as propagation hosts. 
Additional certified host-vector systems 
are as follows: 
HVl — The following specified strains of 
Neurospora crassa which have been modified 
to prevent aerial dispersion: 
(1) ini (inositolless) strains 37102, 37401. 
46316, 64001, and 89601. 
(2) csp-1 strain UCIA37 and csp-2 strains 
FS 590, UCLAlOl (these are conidial 
separation mutants). 
(3) eas strain UCLA191 (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 Toxins for Vertebrates 
1. General Information. Appendix F 
specifies the containment to be used for the 
deliberate cloning of genes coding for the 
biosynthesis of toxins for vertebrates. 
Cloning of genes coding for toxins for 
vertebrates that have an LDw of less than 100 
nanograms per kilogram body weight (e.g., 
the botulinum toxins, tetanus toxin, 
diphtheria toxin. Shigella dysenteria 
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 toxins 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 toxins 
classified as to LDm is available from ORDA. 
Testing procedures for determining toxicity of 
toxins not on the list are available from 
ORDA. The results of such tests shall be 
forwarded to ORDA, which will consult with 
the ad hoc Working Group on toxins prior to 
inclusion of the toxin on the list. (See Section 
IV-F,-l-b-(3)-(e).) 
2. Contaiment Conditions for Cloning of 
Toxin Genes in E. coli K-12. (a) Cloining of 
genes coding for toxins 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 P2-I-EK2 or 
P3-)-EKl containment conditions. 
(b) Cloning of genes for the biosynthesis of 
toxins for vertebrates with an LDso in the 
range of 1 microgram to 100 micrograms per 
kilogram body weight may proceedunder 
Pl-l-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). 
(c) Some enterotoxins are substantially 
more toxic when administered enterally than 
parenterally. The following enterotoxins shall 
be subject to Pl-t-EKl contairunent 
conditions; cholera toxin, the heat labile 
toxins of E. coli, Klebsiella, and other related 
proteins that may be identified by 
neutralization with an antiserum mono- 
specific for chlorea toxin, and heat stable 
toxins of E. coli and of Yersinia 
enlerocolitica. 
3. Containment Conditions for Cloning of 
Toxins Genes in Organisms Other than E. 
coli K-12. Requests involving the cloning of 
genes coding for toxins for vertebrates in 
host-vector systems other than E. coli K-12 
will be evaluated by ORDA, which will 
consult with the ad hod working group on 
toxins. (See Section IV-B-l-b-(3)-(f).) 
4. Specific Approvals, a. Permission is 
granted to clone the Exotoxin A gene of 
Pseudomonas aeruginosa under Pi conditions 
in Pseudomonas aeruginosa. 
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. 
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. 
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 P2-1-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 LDsoS in the range of one 
microgram per kilogram body weight, such as 
the exfoliative toxin. 
e. Fragments F-1 and F-2 of the diphtheria 
toxin gene (tox) 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). 
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 LTrlike toxin gene may be cloned under 
Pl-t-EKl conditions. 
Dated: April 12, 1982. 
Bernard Talbot, 
Acting Director, National Institute of Allergy 
and Infectious Diseases. 
Note. — OMB’s “Mandatory Information 
Requirements for Federal Assistance Program 
Announcements” (45 FR 39592) requires a 
statement concerning the official government 
programs contained in the Catalog of Federal 
Domestic Assistance. Normally NIH lists in 
its announcements the number and title of 
affected individual programs for the guidance 
of the public. Because the guidance in this 
notice covers not only vitually every NIH 
Program but also essentially every federal 
research program in which DNA recombinant 
molecule techniques'could be used, it has 
been determined to be not cost effective or in 
the public interest to attempt to list these 
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