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Federal Register / Vol. 44. No. 71 / Wednesday, April 11. 1979 / Notices 
Section Ul-C-2-b is being added to 
permit the use of nonviral vectors in 
invertebrate host-vector systems. The 
revised Guidelines permit the use of 
nonviral vectors for plant host-vector 
systems, but do not address their use for 
invertebrate systems. No comments 
were received during the 30-day period 
for comment. The RAC voted 12 to 0 
with 2 abstentions to recommended the 
proposed amendment of Section UJ-C-2. 
H. Addition of Section III-C-1-f 
Vertebrate Host - Vector Systems — Non- 
Viral Vectors 
Similar to the proposal in G above, the 
RAC recommended that a comparable 
section be added to Section M-C-l on 
vertebrate host-vector systems. The new 
sub-section would read as follows: 
"M-C-l-f. Nonviral vectors. 
Organelle, plasmid and chromosomal 
DNAs may be used as vectors. DNA 
recombinants formed between such 
vectors ancfhost 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 nonviral DNA from 
cells other than the host species require 
only Pi physical containment for cells in 
culture since vertebrate cells in tissue 
culture inherently exhibit a very high 
level of containment. Recombinants 
involving viral DNA or experiments 
which require the use of whole animals 
will be evaluated by N1H on a case-by- 
case basis [45].” 
No comments were received during 
the 30-day period for comment. The 
RAC voted 10 to 0 with 4 abstentions to 
accept this proposal. 
I. Addition of Section III-C-6. Return of 
DNA Segments To a Higher Eukaryotic 
Host of Origin 
The RAC considered the following 
proposal to add a new Section (III-C-6) 
to the Guidelines to permit the return of 
DNA segments to a higher eukaryotic 
host of origin: 
“ni-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 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-l]. 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. 
12A]" 
The RAC noted that the revised 
Guidelines do not address the question 
of cloning within a higher eukaryote or 
cultured cells derived therefrom of DNA 
taken from the same species, joined to a 
vector and cloned in Escherichia coli K- 
12, and then returned to cells of the 
species of origin. Analogous clauses 
exist for returning DNA from 
Escherichia coli to a species of origin 
from prokaryotes and lower eukaryotes. 
The experiment is essentially a "self- 
cloning" experiment and therefore does 
not require high levels of physical 
containment. Most such experiments 
would be exempt from the Guidelines 
altogether were it not for the fact that 
the DNA. when returned to the host of 
origin, is joined to the DNA of the 
cloning vector, which is of bacterial 
origin. These vectors comprise a small 
number of well-characterized harmless 
DNA molecules. 
The types of physical containment (Pi 
through P4) defined in the Guidelines 
are applicable to the propagation of 
microorganisms and to tissue cultures, 
but not to whole multicellular 
organisms. Thus, it is impractical to 
require Pi containment, and more 
appropriate to require conditions 
providing a degree of containment 
comparable to Pi. 
The announcement in the January 15. 
1979 Federal Register drew three letters 
of comment prior to the February 15-16, 
1979 RAC meeting. All three comments 
requested that the proposal be changed 
to permit the transfer of DNA segments 
to a heterologous eukaryote instead of 
only to the host of origin. The RAC felt 
that adoption of this proposed change 
would constitute a major change that 
would require an additional period for 
comment. 
The RAC voted 13 to 0 with 2 
abstentions to accept the proposal as 
published in the January 15. 1979 
Federal Register with the following 
interpretations: 
1. the term "host" is interpreted to be 
"species:” 
2. viral DNA from viruses endogenous 
to a species is considered to be DNA 
from the species: and 
3. this does not include exogenous 
viruses at this time. 
II. MAJOR ACTIONS UNDER 
GUIDELINES 
A. N. Crassa and S. Cerevisiae as HV1 
Systems 
Specified strains of Neurospora 
crassa which have been modified to 
prevent aerial dispersion, and 
unmodified laboratory strains of 
Saccharomyces cerevisiae are 
acceptable as HVl systems based on 
[ 85 ] 
their natural containment. The following 
N. Crassa strains can be used: 
(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). 
B. Unmodified Laboratory Strains of N. 
Crassa 
Unmodified laboratory strains of N. 
crassa are approved at the P3 level of 
containment for shotgun experiments 
with phages, plasmids, and DNA from 
Class 1 prokaryotes (1) and lower 
eukaryotes that do not produce 
polypeptide toxins. [34] 
C. Criteria for Saccharomyces 
Cerevisiae as an HV2 System 
The following are criteria for 
Saccharomyces cerevisiae as an HV2 
system. Biological containment of 
Saccharomyces cerevisiae is a product 
of several factors: (a) probability of 
encounter of the host-vector with a wild 
type yeast outside the laboratory: (b) the 
abundance of wild type strains able to 
mate with a laboratory strain; (c) the 
frequency of mating under the dilute 
conditions simulating natural 
environments; (d) the reduction in 
mating frequency conferred by the 
sterility mutations; (e) the stability of 
the cloned segment and its vector; (f) the 
survival and growth ability of the host 
relative to wild types. 
The following data are to be supplied 
in support of a candidate S. cerevisiae 
for HV2 certification: 
(1) Genotype of the strain, description 
of the vectors and selective markers to 
be used, the nature and stability of the 
mutation(s) contributing to sterility and 
the mode of construction of the strain. 
(2) The frequency of mating (in a 
worst case analysis under optimal 
conditions) with a fertile strain of 
opposite mating type. 
(3) Relative growth rates of the 
candidate strain and suitable industrial 
wild types, separately and in mixed 
culture. (The specification of industrial 
strains reflects the rarity of S. cerevisiae 
in nature under non-domesticated 
conditions.) 
(4) Data on the stability and 
maintenance of the cloned segment and 
vector in nonselective media. 
(5) Measurement of the relative ability 
(in a worst case analysis) of fertile 
parents of the sterile HV2 candidate to 
mate at low cell density compared to 
optimal conditions. 
(6) An experimental estimate of the 
frequency of transfer of a model cloned 
