Federal Register / Vol. 44, No. 71 / Wednesday, April 11, 1979 / Notices 
21735 
segment (in a worst case analysis, again 
with a fertile strain) to a nonmating, 
industrial strain, after prolonged 
cultivation in mixed culture. 
Changes of auxotrophic selective 
markers in host and vector, or change-in 
mating type allele are trivial 
modifications not requiring certification, 
provided the level of sterility is not 
decreased. Certified EK2 E. coli vector 
DNA or fragments thereof can be used 
in HV2 S. cerevisiae. 
D. Equivalence of Lower Eukaryote HV 
Systems with those of E. Coli 
In accord with Section III— C— 5, host- 
vector systems which have been 
approved as HVl systems may be used 
under P2 containment conditions for 
shotgun experiments with phages, 
plasmids, and DNA from nonpathogenic 
prokaryotes which do not produce 
polypeptide toxins. [34] For other 
classes of recombinant DNA 
experiments with these HVl systems, 
except for the cloning of complete 
genomes of eukaryote viruses, the S. 
cerevisiae and N. crassa HVl systems 
and S. cerevisiae HV2 systems may be 
used at the physical containment levels 
applicable to EKl and EK2 systems, 
respectively. 
E. Certified Saccharomyces Cerevisiae 
HV2 Systems 
The following sterile strains of 
Saccharomyces cerevisiae, all of which 
have the ste-V C9 mutation, are certified 
as HV2 hosts: SHY1, SHY2, SHY3, and 
SHY4. The following plasmids are 
certified for use in an HV2 system: YIpl, 
YEp2, YEp4, YIp5, YEp6, YRp7, YEp20, 
YEp21, YEp24, YIp25, YIp26, YIp27, 
Ylp28, YIp29, YIp30, YIp31, Yip32, and 
YIp33. These plasmids can be 
considered EK2 vectors when 
propagated in xl776. 
F. Exemption of Bacillus Species that 
Exchange Genetic Information 
The following Bacillus subtilis related 
species that have been shown to 
exchange chromosomal DNA are now 
included under the exemption category 
of Section I-E^l of the 1978 Guidelines. 
Any recombinant DNA molecules that 
are composed entirely of DNA segments 
from one or more of the organisms listed 
below and to be propagated in any of 
the organisms listed below are exempt 
from the Guidelines. (This list is to be 
separate from that which already exists 
in Appendix A for exchangers with E. 
Coli.) 
Bacillus subtilis 
Bacillus licheniformis 
Bacillus pumilus 
Bacillus globigii 
Bacillus niger 
Bacillus nato 
Bacillus amyloliquefaciens 
Bacillus aterrimus 
G. Bacillus Subtilis HVl System 
Asporogenic mutant derivatives of B. 
subtilis can be accepted as the host 
component of an HVl system. 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 the vector components 
as certified B. subtilis HVl systems: 
pUBllo, pCl94, pSl94, pSA2100, pEl94, 
pT127, pUBll2, pC221, pC223. 
H. Equivalence of Bacillus Subtilis HVl 
Systems with Those of E. Coli 
Host-vector systems which have been 
approved as HVl systems may be used 
under P2 containment conditions for 
shotgun experiments with phages, 
plasmids, and DNA from nonpathogenic 
prokaryotes which do not produce 
polypeptide toxins. [34] Other classes of 
recombinant DNA experiments with B. 
subtilis HVl systems will be equivalent 
to EKl in the physical containment 
requirements for specific experiments. 
I. Modification of Section III-B-3 
Section III-B-3 of the Guidelines is 
replaced with the following modified 
text: 
III-B-3. Non-HVl Systems. 
Containment levels for other classes of 
experiments involving non-HVl systems 
may be approved by the Director, NIH. 
(See Sections IV-E-l-b-(l)-(b), IV-E-1- 
b— (2)— (c), and IV-E-l-b-(3)-(b).) 
In those cases where genetic 
exchange has not been demonstrated 
between two bacterial species A and B, 
neither of which is known to be 
pathogenic for man, animals, or plants, 
recombinant DNA experiments 
involving only A and B can be 
conducted under P3 containment. [2A] 
]. Specific Bacillus Subtilis Experiment 
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. 
K. Modification of Sections III-A-l-a- 
(1) and III-A-l-a-(2), Containment 
Levels for Shotgun Cloning of Primate 
and Other Mammalian DNA E. Coli K- 
12 
Sections III-A-l-a-(l) and III-A-l-a- 
(2) of the Guidelines are replaced with 
the following modified text: 
[86] 
III— A— 1— a— (1). Primates. P2 physical 
containment + an EK2 host-vector or P3 
+ EKl. Any lowering of containment 
below these levels (i.e., for purified DNA 
or characterized clones) cannot be made 
solely by an institutional biosafety 
committee but requires NIH approval. 
(See Section IV— E— 1— b— (3).) 
Ill— A— 1— a— (2). Other Mammals. P2 
physical containment + an EK2 host- 
vector or P3 + EK1. 
L. Modification of Section III-B-2 
Section III-B-2 of the Guidelines is 
replaced with the following modified 
text: 
III-B-2. Return of DNA Segments to 
Non-HVl Host of Origin. Those 
prokaryotes that exchange genetic 
information with E. coli by known 
physiological processes will be exempt 
from these Guidelines if they appear on 
the "list of exchangers” set forth in 
Appendix A (see Section I-E-4). For a 
prokaryote which can exchange genetic 
information [35] with.#, coli under 
laboratory conditions but which is not 
on the list (Host A), the following type of 
experiment may be carried out under PI 
conditions without Host A having been 
approved as an HVl host: DNA from 
Host A may be inserted into a vector 
and propagated in E. coli K-12 under Pi 
conditions. Subsequently, this 
recombinant DNA may be returned to 
Host A by mobilization, transformation, 
or transduction and may then be 
propagated in Host A in any desired 
vector under Pi conditions. 
For a prokaryote which does not 
exchange genetic information with E. 
coli (Host B), the following type of 
experiment may be carried out without 
Host B having been approved as an HVl 
host: DNA from Host B may be inserted 
into a lambdoid phage vector or into a 
vector from a certified EK2 host-vector 
system and propagated in E. coli K-12 
under the appropriate containment 
conditions [See Section III— A— 1— b— (2)]. 
Subsequently, this recombinant DNA 
may be returned to Host B and 
Propagated in Host B under PI 
conditions. [43] 
M. Modification of Section III-C-5 
Section III-C-5 of the Guidelines is 
replaced with the following modified 
text: 
III-C-5. Fungal or Similar Lower 
Eukaryotic Host- Vector Systems. The 
containment criteria for DNA 
recombinant experiments using these 
host-vectors most closely resemble 
those for prokaryotes, rather than those 
for the preceding eukaryotes, since the 
host cells usually exhibit a capacity for 
dissemination outside the laboratory 
