Federal Register / Vol. 45, No. 227 / Friday, November 21, 1980 / Notices 
77407 
agalactia of sheep) 
Rickettsia ruminatium (heart water) 
Rift valley fever virus 
Rhinderpest virus 
Sheep pox virus 
Swine vesicular disease virus 
Teschen disease virus 
Trypanosoma vivax (Nagana) 
Trypanosoma evansi 
Theileria parva (East Coast fever) 
Theileria annulata 
Theileria lawrencei 
Theileria bovis 
Theileria hirci 
Vesicular exanthema virus 
Wesselsbron disease virus 
Zyonema 
Footnotes and References of Appendix B 
*A USDA permit, required for import and 
interstate commerce of pathogens, may 
be obtained from the Animal and Plant 
Health Inspection Service, USDA, 
Federal Building, Hyattsville, MD. 20782. 
“Since the publication of the classification 
in 1974 (1), the Actinomycetes have been 
reclassified as bacterial rather than 
fungal agents. 
‘"All activities, including storage of variola 
and whitepox, are restricted to the single 
national facility (World Health 
Organization (WHO) Collaborating 
Center for Smallpox Research, Center for 
Disease Control, in Atlanta). 
1. Classification of Etiologic Agents on the 
Basis of Hazard (4th Edition. July 1974). 
U.S. Department of Health, Education 
and Welfare, Public Health Service, 
Center for Disease Control, Office of 
Biosafety, Atlanta, Georgia 30333. 
2. National Cancer Institute Safety Standards 
for Research Involving Oncogenic 
Viruses (October 1974). U.S. Department 
of Health, Education, and Welfare 
Publication No. (NIH) 75-790. 
3. U.S. Department of Agriculture, Animal 
and Plant Health Inspection Service. 
Appendix C — Exemptions Under I-E-5 
Section I-E-5 states that exempt from 
these Guidelines are “Other classes of 
recombinant DNA molecules, if the 
Director, NIH, with advice of the 
Recombinant DNA Advisory Committee, 
after appropriate notice and opportunity 
for public comment, finds that they do 
not present a significant risk to health or 
the environment. (See Section IV-E-1- 
b-(l)-(d).) Certain classes are exempt as 
of publication of these Revised 
Guidelines.” 
Under exemption I-E-5 of these 
Revised Guidelines are those 
recombinant DNA molecules that are 
propagated and maintained in cells in 
tissue culture and that are derived 
entirely from non-viral components (that 
is, no component is derived from a 
eukaryotic virus). 
Appendix D — HV1 and HV2 Host- 
Vector Systems Assigned Containment 
Levels as Specified in the Subsections of 
Section III-A 
As noted above at the beginning of 
Section III-A, certain HVl and HV2 
host-vector systems are assigned 
containment levels as specified in the 
subsections of Section III-A. Those so 
classified as of publicatioi/of these 
Revised Guidelines are listed below. 
* 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 UCLA37 and csp-2 
strains FS 590, UCLA101 (these are 
conidial separation mutants). 
(3) eas strain UCLA191 (an "easily 
wettable" mutant). 
HVl — Asporogenic mutant derivatives 
of B. subtilis. These derivatives 
must not revert to sporeformers 
with a frequency greater than 10“ 7 ; 
data confirming this requirement 
must be presented to NIH for 
certification. The following 
plasmids are accepted as die vector 
components of certified B. subtilis 
HVl systems: pUBllO, pCl94, 
pSl94, pSA2100, pEl94, pTl27, 
pUBll2, pC221, pC223, and pABl24. 
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, 
ASB298, with the following 
plasmids as the vector component: 
pUBllO, pCl94, pSA2100, pEl94, 
pTl27, pUBll2, pC221, pC223, and 
pABl24. 
Appendix E — Actions Taken Under the 
Guidelines 
As noted in the subsections of 
Sections IV-E-l-b-(l) and IV-E-l-b-(2), 
the Director, NIH, may take certain 
actions with regard to the Guidelines 
after consideration by the RAC. 
Some of the actions taken to date 
include the following: 
1. The following experiment has been 
approved: The cloning in B. subtilis, 
under P2 conditions, of DNA derived 
from Saccharomyces cerevisiae using 
EK2 plasmid vectors provided that an 
HVl B. subtilis host is used. 
2. Unmodified laboratory strains of 
Neurospora crassa can be used in all 
* These follow the assigned containment levels as 
specified in the subsections of Section III-A with 
one exception. This exception is that experiments 
involving complete genomes of eukaryotic viruses 
will require P3 + HVl or P2 + HV2 rather than the 
levels given in the subsections of Section III-A. 
experiments for which HVl N. crassa 
systems are approved provided that 
these are carried out at physical 
containment one level higher than 
required for HVl. However, if P3 
containment is specified for HVl N. 
crassa, this level is considered adequate 
for unmodified N. crassa. For P2 
physical containment, special care must 
be exercised to prevent aerial dispersal 
of macroconidia, including the use of a 
biological safety cabinet. 
3. P2 physical containment shall be 
used for DNA recombinants produced 
between members of the Actinomycetes 
group except for the species which are 
known to be pathogenic for man, 
animals, or plants. 
4. Cloned desired fragments from any 
non-prohibited source may be 
transferred into Agrobacterium 
tumefaciens containing a Ti plasmid (or 
derivatives thereof), using a 
nonconjugative E. coli plasmid vector 
coupled to a fragment of the Ti plasmid 
and/or the origin of replication of an 
Agrobacterium plasmid, under 
containment conditions one step higher 
than would be required for the desired 
DNA in HVl systems (i.e. one step 
higher physical containment than that 
specified in the subsections of Section 
III-A). Transfer into plant parts or cells 
in culture would be permitted at the 
same containment level (one step 
higher). 
5. Bacillus subtilis strains that do not 
carry an asporogenic mutation can be 
used as hosts specifically for the cloning 
of DNA derived from E. coli K-12 and 
Streptomyces coelicolor, S. 
aureofaciens, S. rimosus, S. griseus, S. 
cyaneus, and S. venezuelae, using NIH- 
approved Staphylococcus aureus 
plasmids as vectors under P2 conditions. 
6. Streptomyces coelicolor, S. 
aureofaciens, S. rimpsus, S. griseus, S. 
cyaneus, and S. venezuelae can be used 
as hosts for the cloning of DNA derived 
from B. subtilis, E. coli K-12 or from S. 
aureus vectors that have been approved 
for use in B. subtilis under P2 
conditions, using as vectors any plasmid 
indigenous to Streptomyces species or 
able to replicate in these hosts by 
natural biological mechanisms. 
7. Certain cloned segments of 
Anabena DNA may be transferred into 
Klebsiella under P2 physical 
containment. 
8. Permission is granted to clone foot- 
and-mouth disease virus in the EK1CV 
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. 
9. Permission is granted to clone the 
Exotoxin A gene of Pseudomonas 
aeruginosa under Pi + EKl conditions 
[ 26 ] 
