59424 
Federal Renter / Vol. 46. No. 233 / Friday, December 4, 1981 / Notices 
M. PermiMion is granted to clone in £ coJi 
K-IZ. in high cooUiiunent Building SSO at the 
Frederich Cancer Research Center, restriction 
fragments of Corynephage Beta carrying tha 
structural gene for diphtheria toxin. 
Laboratory practices and containment 
equipment are to be specified by the CBC 
31. Permission is granted to clone certain 
tubgenoraic segments of Foot and Mouth 
Disease Virus in HVl BaciJlus subti/ia and 
Soccharomycea cenviaiae host-vector 
systems under Pi conditions at Cenentech. 
Inc.. South San Frandsco, California. 
32. Permission is granted in prindpla to 
propagate in mammalian cell culture 
recombinant DNA moieculea consisting of 
segments of Foot and Mouth Disease Virus 
and SV40 delation vectors under P3 
conditions at tha Plum Island Animal Disease 
Center. Approval is subject to review by a 
RAC Working Croup of Individual 
experiments. 
33. A conjugativs plasmid may be used to 
transfer among £ coJi K-12 strains, ondsr P2 
physical containment the qa-2 of Saunepora 
craaaa ligated to a mobilizabla plasmid. 
34. £ coJJ K-12 strain DF214 (or derivatives 
thereof) and plasmid vectors (04.. pBR322. 
pBR323) may be used to clone rat cDNA 
under P2 conditions. After the clone of 
interest has been purified, it may be worked 
with under Pi containment 
33. Permission is granted to Dr. Ronald 
Davis of Stanford University to field lest corn 
plants modified by recombinant DNA 
techniques under specified containment 
condltiona 
30. Permission is granted to clone In £ coti 
K-12. under Pi physical containment 
conditions, subgenomic segments of Rift 
Valley Fsvsr Virus Subject to conditions 
which have been set forth by the RAC 
37. PermiaaioQ is given to transfer a 
recombinant lactose plasmid from 
Streptococcus fa&calis to 5L lactis by 
conjugation. 
30. Attenuated laboratory straina of 
Satmcnella typbimurium may be used under 
Pi physical containment conditions to screen 
hr the Soccharomycm cenviaiae 
peeudouridina synthetase gene. The plasmid 
YEpl3 will be employed as the vector. 
35. Ptrmisslon is granted to clone In 
Haemopbilua parai/iffueniae Moloney 
murine leukemia provirua and mouse cellular 
flanking sequences employing the plasmid 
vector, pRK290. under P2 containment 
conditions. 
40. Permission is granted for the 
development under Pi conditions, of a new 
host-vector system based on the use of 
Corynebocteriuw glutamicum as host and 
non-conjugative poorly mobiliuble plasmids 
as vectors. 
41. Vibrio hamyi DNA may be cloned In 
Vibrio cholera: plasmids may be used to 
transfer the cloned V. harveyi DNA between 
£ co/i K-12. V. cholera, and V. hamyi. P2 
physical containment conditions are required 
for those experiments involving V. cholera. 
Pi containment conditions may be used for 
other phases of the project 
Appendix F of Annex E. — Certified 
Host* Vector Systems 
While many experiments using £ coli 
K-12, Saccharomyces cerevisiae and 
Bacillus aubtilis are currently exempt 
from the Guidelines under Exemption I- 
E-5, some derivatives of these host- 
vector systems were previously 
classified as HVl or HV2. A listing of 
those systems follows. 
HVl — The following plasmids are 
accepted as the vector components of 
certifled B. subtiUs HVl systems; 
pUBlia pCl»4. pSl94, pSA210a pEl94. 
pTl27, pUBll2, pC221, pC223, and 
pAfil24. B. subtiUs strains RUB 331 and 
BGSC 1553 have been certified as the 
host component of HVl systems based 
on these plasmids. 
HV2 — The asporogenic mutant 
derivative of Bacillus subtiUs, ASB 296. 
with the following plasmids as the 
vector component pUBllO, pCl94, 
pSl94. pSA2100. pEl94. pTl27, 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 foUowlng 
plasmids are certified for use; Ylpl, 
YEp2. YEp4. YIp5. YEp6. YRp7, YEp20, 
YEp21, YEp24. Ylp25. Ylp26. YIp27, 
Ylp28. Ylp29, Ylp3a Ylp31. Ylp32 and 
Ylp33. 
EK2 Plasmid Systems. The £ coli K- 
12 strain chk-1776. The following 
plasmids are certified for use; pSClOl, 
pMB9. pBR313. pBR322. pDK24. pBR327, 
pCLlOl. pHBl. The following £. 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; Ylpl. 
YEp2. YEp4. Ylp5, YEp6, YRp7, YEp20. 
YEp21, YEp24. Ylp25. Ylp26. Ylp27, 
Ylp26, Ylp29, Ylp30. Ylp31. Ylp32. Ylp33. 
EK2 Bacteriophage Systems; The 
following are certified EK2 systems 
based on bacteriophage lambda. 
Vactor 
it|lWES AB- 
AftWES AB* 
AftZjrir AB* 
AftALO. AB 
Chxrcm lA 
Cbaran 4A 
Qtaran ISA 
Qiaran ZlA 
Qtaroa Z3A 
Cbaroa 24A 
Hoat 
DPSOVupF 
DPSOfupF 
£ coli K-12 
DPSOxupF 
DPSO oc DPSOvupF 
DPSO or OPSOrupF 
DPSO or DPSOiu^ 
DPSOfupF 
DPSO or DPSOrupP 
DPSO or DPSOrupP 
Appendix G of Annex E. — Containment 
Conditions for Cloning of Genes Coding 
for the Biosynthesis of Toxins for 
Vertebrates 
I. General Information 
Appendix G 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 LOso of less than 100 nanograms 
per kilogram body weight (e.g., 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 LOm of 100 micrograms or more 
per kilogram of body weight. 
Experiments involving genes coding for 
toxins with an LDm of 100 micrograms or 
less per kilogram body weight shall be 
registered with ORDA prior to initiating 
the experiments. A list of toxins 
classifled 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-E-l-b-{3Hl) ) 
Z Containment Conditions for Cloning 
of Toxin Genes in E. coli K-12 
(a) Cloning of genes coding for toxins 
for vertebrates that have an LDm 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- 
EJC2 or P3 4- EKi containment 
conditions. 
(b) Cloning of genes for the 
biosynthesis of toxins for vertebrates 
with an LDm in the range of 1 microgram 
to 100 micrograms per kilogram body 
weight may proceed under Pi -f 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 internally than 
parenterally. The following enterotoxins 
shall be subject to Pi - 1 - EKl 
containment conditions; cholera toxin, 
the heat labile toxins of £ coU, 
Klebsiella, and other related proteins 
that may be identified by neutralization 
with an antiserum mono-speciHc for 
cholera toxin, and the heat stable toxins 
of £ coli and of Yersinia enterocolitica. 
( 313 ) 
