6738 
Federal Register / Vol. 45, No. 20 / Tuesday, January 29, 1980 / Notices 
proof containers and sterilized. It is not 
necessary to sterilize run-off water from 
the infected plants, as this is not a 
plausible route for secondary infection. 
When the viruses are used as vectors in 
tissue cultures or in small plants in 
axenic cultures, no special containment 
is necessary. Infected plant materials 
which have'to be removed from the 
greenhouse or cabinet for further 
research shall be maintained under 
insect-restrictive conditions. These 
measures provide an entirely adequate 
degree of containment. They are similar 
to those required in many countries for 
licensed handling of “exotic” plant 
viruses. 
The CaMV strain used as a cloning 
vector shall be a mutant that lacks the 
aphid transmission factor. 
The viruses or their DNA may also be 
useful as vectors to introduce genes into 
plant protoplasts. The fragility of plant 
protoplasts combined with the 
properties of the viruses provides 
adequate safety. Since no risk to the 
environment from the use of the DNA 
plant virus/protoplast system is 
envisaged, no special containment is 
necessary, except as described in the 
following paragraph. 
Experiments involving the use of plant 
virus genomes to propagate DNA 
sequences from eukaryotic viruses will 
be evaluated by NIH on a case-by-case 
basis [45) and will be conducted under 
the prescribed physical and biological 
containment conditions. (See Section 
IV -E-l-b-(3 )-( c ) • ) 
III-C-4. Plant Host- Vector Systems 
Other than Viruses. Organelle, plasmid, 
and chromosomal DNAs may be used as 
vectors. DNA recombinants formed 
between such vectors and host DNA, 
when propagated only in that host (or a 
closely related strain of the same 
species), are exempt from these 
Guidelines (See Section I — E). DNA 
recombinants formed between such 
vectors and DNA from cells other than 
the host species require P2 physical 
containment. The development of host- 
vector systems that exhibit a high level 
of biological containment, such as those 
using protoplasts or undifferentiated 
cells in culture, permit (2A) a decrease 
in the physical containment to PI. 
Intact plants or propagative plant 
parts which cannot be grown in a 
standard P2 laboratory because of their 
large size may be grown under the Pi 
conditions described above in Section 
III— C— 3, except that (i) sterilization of 
run-off water is required where this is a 
plausible route for secondary infection 
and (ii) the standard P2 practices are 
adopted for microbiological work. 
Ill— C— 5. Fungal or Similar Lower 
Eukaryotic Host-Vector Systems. 
Certain certified HVl and HV2 host- 
vector systems appear in Appendix D. 
The containment levels for these 
systems are given in the subsection of 
Section III— A. Other systems in the 
future may be certified as HVl and HV2. 
At the time of certification, they may be 
added to Appendix D (and thus the 
containment levels for thdr use will be 
those of the subsections of Section III— 
A). Alternatively, at the time of their 
certification, another classification of 
containment levels for experiments 
using them may be assigned by NIH. 
In addition to the experiments 
described above, the-following 
experiments may be carried out without 
the eukaryotic host (Host C) having 
been approved as an HVl host: DNA 
from Host C 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 C and propagated there under Pi 
conditions. [43) 
Containment levels for other classes 
of experiments involving non-HVl 
systems may be expressly approved by 
the Director, NIH. (See Sections IV-E-1- 
b— (1 j— (b), IV-E-l-b-(2)-(c), and IV-E-1- 
b-(3)-(b).) 
III— C— 6. Return of DNA Segments to a 
Higher Eukaryotic Host of Origin. DNA 
from a higher eukaryote (Host D) may 
be inserted into a vector and propagated 
in E. coli K-12 under Pi containment 
conditions. Subsequently, this 
recombinant DNA may be returned to 
Host D and propagated under conditions 
of physical containment comparable to 
PI and appropriate to the organism 
under study. [2A) 
III-C-7. Transfer of cloned DNA 
Segments to Eukaryotic Organisms 
III-C-7-a. Transfer to Non-human 
Vertebrates. DNA from any 
nonprohibited source [Section I-D], 
except for greater than one quarter of a 
eukaryotic viral genome, which has 
been cloned and propagated in E. coli 
under PI conditions, may be transferred 
with the E. coli vector used for cloning 
to any eukaryotic cells in culture or to 
any non-human vertebrate organism and 
propagated under conditions of physical 
containment comparable to PI and 
appropriate to the organism under study 
[2A). Transfers to any other host will be 
considered by the RAC on a case-by- 
case basis [45). 
III-C-7-b. Transfer to Higher Plants. 
DNA from any nonprohibited source 
[Section I-D] which has been cloned 
and propagated in E. coli under PI 
conditions, may be transferred with the 
E. coli vector used for cloning to any 
higher plant organisms (angiosperms 
and Gymnosperms) and propagated 
under conditions of physical 
containment comparable to Pi and 
appropriate to the organism under study 
[2A). Intact plants or propagative plant 
parts may be grown under Pi conditions 
described under Section III— C— 3. 
Containment must be modified to ensure 
that the spread of pollen, seed or other 
propagules is prevented. This can be 
accomplished by conversion to negative 
pressure in the growth cabinet or 
greenhouse or by physical entrapment 
by "bagging” of reproductive structures. 
Transfers to any other plant organisms 
will be considered on a case-by-case 
basis [45). 
III-D. Complementary DNAs. Specific 
containment levels are given in Section 
III-A-2-a (see also last column of Table 
III) for complementary DNA (cDNA)/)f 
viral mRNA. For the other Sections of 
the Guidelines, where applicable, 
cDNAs synthesized in vitro are included 
within each of the above classifications. 
For example, cDNAs formed from 
cellular RNAs that are not purified and 
characterized are included under III— A— 
1, shotgun experiments; cDNAs formed 
from purified and characterized RNAs 
are included under III— A— 3; etc. 
Due to the possibility of nucleic acid 
contamination of enzyme preparations 
used in the preparation of cDNAs, the 
investigator must employ purified 
enzyme preparations that are free of 
viral nucleic acid. 
Ill— E. Synthetic DNAs. If the synthetic 
DNA segment is, likely to [2A) yield a 
potentially harmful polynucleotide or 
polypeptide (e.g., a toxin or a 
pharmacologically active agent), the 
containment conditions must be as 
stringent as would be used for 
propagating the natural DNA 
counterpart. 
If the synthetic DNA sequence codes 
for a harmless product, [2A) it may be 
propagated at the same containment 
level as its purified natural DNA 
counterpart. For example, a synthetic 
DNA segment which corresponds to a 
nonharmful gene of birds, to be 
propagated in Saccharomyces 
cerevisiae, would require P2 phyical 
containment plus an HVl host-vector, or 
P1 + HV2. 
If the synthetic DNA segment is not 
expressed in vivo as a polynucleotide or 
polypeptide product, the organisms 
containing the recombinant DNA 
[ 30 ] 
