Federal Register / Vol. 47, No. 167 / Friday. August 27. 1962 / Notices 
38059 
conditions specified in Section IU-B-2 
with prior review and approval 
Large-scale experiments (e.g., more 
than 10 liters of culture) require prior 
LBC review and approval. (See Section 
ni-B-S.) 
Experiments involving the deliberate 
cloning of genes coding for the 
biosynthesis of molecules toxic for 
vertebrates. (See Appendix F.) 
Appendix C-IV. Experiments 
Involving Bacillus subtil is Host-Vector 
Systems. Any asporogenic Bacillus 
subtilis strain which does not revert to a 
sporeformer with a frequency greater 
than 10'* can be used for cloning DNA 
with the exception of those experiments 
listed below. Indigenous Bacillus 
plasmids and phages, whose host-range 
does not include Bacillus cereus or 
Bacillus anthracis. may be used as 
vectors. 
For these exempt experiments Pi 
physical contairunent conditions are 
recommended. 
Exceptions 
Experiments described in Section 01- 
A which require speciFic RAC review 
and approval before initiation of the 
experiment. 
Experiments involving Class 3. 4. or 5 
organisms |1| or cells known to be 
Infected with these agents may be 
conducted under containment 
conditions specined by Section Ul-B-2 
with prior IBC review and approval 
Large-scale experiments (e.g.. more 
than 10 liters of culture) require prior 
IBC review and approval. (See ^tion 
UI-B-5.) 
Experiments Involving the deliberate 
cloning of genes coding for the 
biosynthesis of molecules toxic for 
vertebrates. (See Appendix F.) 
App«f>dU C-V. — FootnolM and Rsfaraocea 
of Appendix C 
1. The original reference to organisms aa 
Oass 1. 2. 3. 4. or 5 refers to the cJasallication 
In the publication Classification of Etiologic 
Agents on the Basis of Haxard. 4lh Edition. 
)uly 1B74: U.S. Department of Health. 
Education and Welfare. Public Health 
Serv ice. Centers for Disease Control. Office 
of Biosafety. Atlanta. Georgia 30333 
The Director. NIH. with advice of the 
Recombinant DNA Advisory Committee, may 
revise the classification for the purposes of 
these Guidelines (see Section rV-C-l-b-{2)- 
(d|). The revised list of organisms in each 
class is reprinted in Appendix B to these 
Guidelines. 
2. A subset of non-con|ugative plasmid 
vectors are also poorly mobilizable (e.g.. 
pBR32Z pBR313). Where practical these 
vectors should employed. 
3. Defined as observable under optimal 
laboratory conditions by transformation, 
transduction phage infection, and/or 
conjugation with transfer of phage, plasmid. 
and/or chromosomal genetic information. 
Note that ths definition of exchange may be 
less stringent than that applied to exempt 
organisms under Section UI-D-4. 
Appendix D. — Actions Taken Under the 
Guidelines 
As noted in the subsection of Section 
rV-C-l-b-(l). the Director. NIH. may 
take certain actions with regard to the 
Guidelines after the issues have been 
considered by the RAC. 
Some of the actions taken to date 
include the following: Appendix D-l 
Permission is granted to clone Foot-and- 
Mouth Disease Virus In the EKl host- 
vector system consisting of E. coli K-12 
And the vector pBR322. all work to be 
done at the Plum Island Animal Disease 
Center. 
Appendix D-II. Certain speciFied 
clones derived from segments of the 
Foot-and-Mouth Disease Virus may be 
transferred from Plum Island Animal 
Disease Center to the facilities of 
Cenentech, Inc., of South San Francisco. 
California. Further development of the 
clones at Cenetech has been approved 
under Pi + EKl conditions. 
Appendix D-lIl. The Rd strain of 
Hemophilus inPuenzae can be used as a 
host for the propagation of .the cloned 
Tn 10 tet R gene derived from E. coli K- 
12 employing the non-conjugative 
Hemophilus plasmid. pRSFOBSS. under 
Pi conditions. 
Appendix D-IV. Permission is granted 
to clone certain subgenomic segments of 
Foot-and-Mouth Disease Virus in HVl 
Bacillus subtilis and Saccharomyces 
cerevisiae host-vector systems under Pi 
conditions at Cenetech. Inc.. South San 
Francisco. California. 
Appendix D-V. Permission is granted 
to Dr. Ronald Davis of Stanford 
University to field test com plants 
modified by recombinant DNA 
techniques under speciFied containment 
conditions. 
Appendix D-VI. Permission is granted 
to clone in E. coli K-12. under Pi 
physical contairunent conditions, 
subgenomic segments of Rift Valley 
Fever virus subject to conditions which 
have been set forth by the RAC. 
Appendix D-VU. Attenuated 
laboratory strains of salmonella 
typhimurium may be used under PI 
physical containment conditions to 
screen for the Saccharmoyces 
cerevisiae pseudouridine synthetase 
gene. The plasmid YEpl3 will be 
employed as the vector. 
Appendix D-VUI. Permission is 
granted to transfer certain clones of 
subgenomic segments of Foot-and- 
Mouth Disease virus from Plum Island 
Animal Disease Center to the 
laboratories of Molecular Genetics, Inc., 
Miiuietonka. Minnesota, and to work 
with these clones under Pi containment 
conditions. Approval is contingent upon 
review of data on infectivity testing of 
the clones by a working group of the 
RAC. 
Appendix E. — Certified Host-Vector 
Systems 
While many experiments using E. coli 
K-12. Soccharomyces serevisiae and 
Bacillus subtilis are currently exempt 
from the Guidelines under Exemption 
Ill-D-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 
certified B. subtilis HVl systems: 
pUBllO, pCl94. pSl94. pSA2100, pEl94. 
pTl27. pUBll2, pC221. pG223. and 
pAfil24. 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 296, 
with the following plasmids as the 
vector component: pUBllO, pGl94, 
pSl94. pSA2100. pEl94. pTl27. pUB112, 
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. YIp5. YEpd. YRp7, YEp20. 
YEp21. YEp24. Ylp25. Ylp26. YIp27, 
Ylp28. Ylp29. Ylp30, Ylp31. YIp32. and 
Ylp33. 
EK2 Plasmid Systems. The E. coli 
K-12 strain chi-1776. The following 
plasmids are certified for use: pSClOl, 
pMBg. 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: 
Ylpl. YEp2, YEp4. YIp5. YEp6. YRp7, 
YEp20. YEp21. YEp24. Ylp25. Ylp20. 
Ylp27. Ylp28. YIp29. Ylp30, YIp31. YIp32, 
YIp33. 
EK2 Bacteriophage Systems. The 
followirig are certified EK2 systems 
based on bacteriophage lambda: 
Vector end Host 
Xgt WES.AB— DPSOsupF 
Xgl WfiS.XB*— DPSOsupP 
XgtZ)V'/r.XB’ — E. coli K-12 
XgtALO.XB— DPSOsupF 
Charon 3A — DP50 or DPSOsupF 
Charon 4A — DP50 or DPSOsupF 
Charon 16A — DP50 or DPSOsupF 
Charon 21 A — DPSOsupF 
Charon 23A — DPSO or DPSOsupF 
Charon 24A — DPSO or DPSOsiipF 
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