Federal Register / Vol. 46, No. 233 / Friday, December 4. 1981 / Notices 
59423 
vectors provided that an HVl B. subtilis host 
is used. 
2. Unmodified laboratory strains of 
Neurospora crassu can be used in all 
experiments for which HVl N. crassa 
systems are approved, provided that only 
D.NA from Class 1 agents is used. For agents 
other than Class 1, unmodified laboratory 
strains of N. crassa can be used in all 
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. Care must be exercised 
to prevent aerial dispersal of macroconidia, 
in accordance with good laboratory practice. 
Mutationally modified strains of N. crassa 
specified as HVl in Appendix D can be used 
in all experiments for which HV2 N, crassa 
systems are approved, provided that only 
DNA from Class 1 agents is used. 
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. co// 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). However, DNA from plants 
and nonpathogenic prokaryotes may be 
cloned under P2 containment conditions: and 
the Saccharomyces cerevisiae alcohol 
dehydro-genase 1 gene and the gene coding 
for the maize [Zea mays) seed storage 
protein, zein, may be cloned under Pi 
conditions. Transfer into plant parts or cells 
in culture is permitted at the same 
containment level as is ued for the cloning in 
Agrobacterium tumefaciens. 
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 Streptamyces 
coelicolor, S. aureofaciens, S. rimosus, S. 
griseus, S. cyaneus, and s. venezuelae. using 
NIH-approved Staphylococcus aureau 
plasmids as vectors under P2 conditions. 
6. Streptamyces coelicolor. S. aue'-ofaciens. 
S. rimosus, 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 Streptamyces species 
or able to replicate in these hosts by natural 
biological mechanisms. 
7. Certain cloned segments of Aiiaheno 
DNA may be transferred into Klebsiella 
under P2 physical containment. 
8. Permission is granted to clone foot-and- 
mouth disease virus in the EKlCV host-vector 
system consisting of E. coliY.-\2. and the 
vector pBR322, all work to be done at the 
i’lumb Island Animal Disease Center. 
9. Permission is granted to clone the 
Exotoxin A gene of Pseudomonas aeruginosa 
under Pi -(- EKl conditions in Escherichia 
coli K-12 and under Pi conditions in 
Pseudomonas aeruginosa. 
10. Permission is granted to return to the 
host of origin Helminthosporanium mttydis 
(race O) DNA which has been cloned in yeast 
strain SHY2 using the hybrid E. coli — yeast 
plasmid Ylp5. The cloned DNA may be 
returned to, and propagated in, 
Helminthosporanium maydis at the P2 level 
of physical containment. 
11. Permission is granted to return 
Schizophyllum commune DNA (or yeast 
DNA) cloned in Saccharomyces cerevisiae 
with YR or 2 mu circle vectors to 
Schizophyllum commune. The cloned DNA 
may be returned to, and propagated in, 
Schizophyllum commune at the P2 level of 
physical containment. 
12. Permission is granted to return 
Wangiella dermatitidis DNA to Wangiella 
dermatitidis using an HV2 certified 
Saccharomyces /E. coli hybrid vector. The 
Wangiella dermatitidis may be propagated at 
the P3 level of physical containment. 
13. 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 
Genetech, Inc., of South San Francisco, 
California. Further development of the clones 
at Genentech has been approved under PI 
EKl conditions. 
14. Saccharomycopsis Upolytica may be 
used as a host for transformation with 
defined Escherichia coli/Saccharomyces 
cerevisiae hybrid plasmids and the hybrid 
plasmids may be used for cloning S. 
Upolytica DN.4 in E. coli and returning the 
cloned DNA to S. Upolytica. 
15. Conjugative plasmids or transducing 
phages may be employed in recombinant 
DNA experiments when employing E. coli as 
host when a small defined segment of 
Adenovirus 2 DNA is employed as linker 
DNA. 
16. Permission is granted to introduce DNA 
segments from aphid tramsmissible strains 
into non-aphid transmissible strains of 
Cauliflower mosiac virus in order to study the 
factors determining aphid transmissibility. 
17. Permission is granted to return Mucor 
racemosus DNA which as been cloned in 
Saccharomyces cerevisiae host-vector 
systems to Mucor racemosus. In addition, 
permission is granted to transform Mucor 
racemosus with S. cerevisiae vectors with or 
without cloned S. cerevisiae sequences. 
These manipulations may be performed 
under P2 conditions. 
18. DNA from nonpathogenic prokaryotes 
and nonpathogenic lower eukaryotes may be 
cloned into Schizosaccharomyces pombe 
species under Pi containment conditions. 
DNA from higher eukaryotes may be cloned 
in S. pombe species under P3 containment 
conditions. 
19. 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. 
20. A hybrid plasmid composed of, (1) E. 
coli plasmid p>PR.125. (2) the origin of 
replication and transfer genes of 
Agrobacterium tumefaciens plasmid Ti. (3) 
the thiamine gene of E. coli, and (4) 
Arabidopsis DNA, may be transformed into 
Agrobacterium tumefaciens under PI 
conditions. The Agrobacterium tumefaciens 
may subsequently be used to introduce the 
composite plasmid carrying Arabidopsis 
DNA and the E. coli thiamine gene into 
Arabidopsis plants under Pi containment 
conditions. 
21. Chlamydomonas reinhardi can be used 
as a host for cloning defined DNA segments 
derived from E. coli and Saccharomyces 
cerevisiae using E. coli/S. cerevisiae hybrid 
vectors under P2 physical containment. 
22. Candida albicans can be used as a host 
for cloning Candida albicans DNA following 
propagation of the DNA m E. call K-12 or in 
Saccharomyces cerevisiae employing an E. 
coli-S. cerevisiae hybrid plasmid vector or 
the yeast 2 micron plasmid. 
23. The Rd strain of Hemophilus influenzae 
can be used as a host for the propagation of 
the cloned Tn 10 tet R gene derived from E. 
coll K-12 employing the non-conjugative 
Haemophilus plasmid. pRSF0885. under PI 
conditions. . 
24. Zymomonas mobilis may be used as a 
host under P2 conditions for transformation 
by recombinant DNA derived from 
Pseudomonas strains that are non-pathogenic 
for animals or plants, and that has been 
cloned in an E. call K-12 host. 
25. Protoplasts of Streptosporangium 
brasiliense may be transformed with a hybrid 
plasmid containing pBR322 plus a 
Streptosporangium plasmid into which have 
been incorporated specified DNA segments 
from Streptamyces species or an HVl 
approved Bacillus subtilis cloning vector. 
26. Saccharomyces cerevisiae DNA may be 
cloned in Tetrabymena thermoohila using E 
coli/S. cerevisiae hybrid plasmids under PI 
containment conditions. 
27 All members of the nonpathogenic 
Actinomycetes genus Streptamyces and the 
plasmids native to this genus are approved as 
host-vector systems for the cloning under PI 
conditions of DNA derived from other 
nonpathogenic prokaryotic organisms such as 
Streptamyces and other nonpathogenic 
Actinomycetes species, Escherichia coli K- 
12, Bacillus subtilis. Bacillus licheniformis, 
Bacillus circulans, and other nonpathogenic 
Bacillus species, and for the cloning of DNA 
derived from nonpathogenic unicellular 
eukaryotic microorganisms such as 
Saccharomyces cerevisiae and Neurospora 
crassa. 
28. Bacillus subtilis strains that do not 
carry an asporogenic mutation can be used 
under P2 conditions for the cloning of DNA 
from any CDC Class 1 organism, using 
indigenous plasmid and phage vectors whose 
host range does not include Bacillus 
anthracis or Bacillus cereus. 
29. Bacillus subtilis strains that do not 
carry an asporogenic mutation can be used 
under Pi conditions for the cloning of DNA 
from any Class 1 Bacillus species, using 
indigenous plasmid and phage vectors whose 
host range does not include Bacillus 
anthracis or Bacillus cereus. 
[ 312 ] 
