NOTICES 
3227 
such vectors and DNA from cells other 
than the host species require PI physi- 
cal containment for invertebrate cells 
in culture since invertabrate cells in 
culture inherently exhibit a very high 
level of containment. Experiments 
which require the use of whole ani- 
mals will be evaluated by NIH on a 
case-by-case basis. [451 
4. Similar to proposal 3. above, the 
RAC recommended that a comparable 
statement be added to Section III-C-1 
on vertebrate host-vector systems. 
This new sub-section would be as fol- 
lows: 
III-C-1-f. Nonviral vectors. Organ- 
elle. plasmid, and chromosomal DNAs 
may be used as vectors. DNA recom- 
binants 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 nonviral DNA from 
cells other than the host species re- 
quire only PI physcial containment 
for cells in culture since vertebrate 
cells in tissue culture inherently ex- 
hibit a very high level of containment. 
Recombinants involving viral DNA or 
experiments which require the use of 
whole animals will be evaluated by 
NIH on a case-by-case basis (451. 
5. Lower eukaryotes as host-vector 
systems. (A complete report concern- 
ing these proposals is available from 
the Office of Recombinant DNA Activ- 
ities.) The fungi Ateurospora crassa 
and Saccharomyces cerevisiae have 
been examined by the RAC for their 
suitability as host-vector systems 
under Section II-D-2 and III-C-5 of 
the Guidelines. The following de- 
scribes conditions for their proposed 
use as HV1 and HV2 systems: 
a. At. crassa and S. cerevisiae as HV1 
systems. Specified strains of At. crassa 
which have been modified to prevent 
aerial dispersion and unmodified labo- 
ratory strains of 5. cerevisiae are ac- 
ceptable as HV1 systems based on 
their natural containment. The follow- 
ing At. 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, ULCA101 (these are 
conidial separation mutants) 
(3) eas strain UCLA191 (an "easily 
wettable” mutant) 
Unmodified laboratory strains of At. 
crassa can be used in experiments that 
require the HV1 level of containment 
provided that these are carried out at 
physical containment one level higher 
than required for HV1. However, if P3 
physical containment is specified for 
HV1, this level is considered adequate 
for unmodified At. crassa. For P2 phys- 
ical containment, special care must be 
exercised to prevent aerial dispersal of 
macroconidia. 
b. S. cerevisiae as an HV2 system. 
Biological containment of yeast is a 
product of several factors: (a) prob- 
ability of encounter of the host-vector 
with a wild type yeast outside the lab- 
oratory; (b) the abundance of wild 
type strains able to mate with a labo- 
ratory 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 sup- 
plied in support of a candidate S. cer- 
evisiae for HV2 certification: 
(1) Genotype of the strain, descrip- 
tion of the vectors and selective mark- 
ers to be used, the nature and stability 
of the mutation(s) contributing to ste- 
rility and the mode of construction of 
the strain. 
(2) The frequency of mating (In a 
worst case analysis under optimal con- 
ditions) with a fertile strain of oppo- 
site mating type. 
(3) Relative growth rates of the can- 
didate strain and suitable Industrial 
wild types, separately and in mixed 
culture. (The specification of industri- 
al strains reflects the rarity of S. cer- 
evisiae in nature under non-domesti- 
cated conditions.) 
(4) Data on the stability and mainte- 
nance of the cloned segment and 
vector in non-selectlve media. 
(5) Measurement of the relative abil- 
ity (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 segment (in a worst case analy- 
sis, again with a fertile strain) to a 
non-mating, industrial strain, after 
prolonged cultivation in mixed cul- 
ture. 
Changes of auxotrophic selective 
markers in host and vector, or change 
in mating type allele, are trivial modi- 
fications not requiring certification, 
provided the level of sterility is not de- 
creased. Certified EK2 E. coli vector 
DNA or fragments thereof can be used 
in HV2 S. cerevisiae. 
c. Equivalence of lower eukaryote 
HV systems with those of E. coli. In 
accord with Section III-B-1, host- 
vector systems which have been ap- 
proved as HV1 systems may be used 
under P2 containment conditions for 
shotgun experiments with phages, 
plasmids, and DNA from nonpatho- 
genic prokaryotes which do not pro- 
duce polypaptide toxins.[34] For other 
classes of recombinant DNA experi- 
ments with these HV7 systems, except 
for the cloning of complete genomes 
of eukaryote viruses, the S. cerevisiae 
and N. crassa HV1 systems and S. cer- 
evisiae HV2 systems may be used at 
the physical containment levels appli- 
cable to EK1 and EK2 systems, respec- 
tively. Experiments involving complete 
genomes of class 1 eukaryote viruses 
will require P3 + HV2 containment 
levels. Other eukaryote viruses are to 
be handled on a case-by-case basis.C451 
6. Proposed S. cerevisiae HV2 system. 
A proposal from Dr. David Botstein of 
the Massachusetts Institute of Tech- 
nology and Dr. Ronald Davis of Stan- 
ford University for a S. ceretnstae HV2 
system based on the mating type non- 
specific sterile mutation VC9 w-ill be 
considered by the RAC. The proposal 
is available from the Office of Recom- 
binant DNA Activities. 
7. Prokaryotes other than E. coli as 
host-vector systems. (Complete reports 
concerning these proposals are availa- 
ble from the Office of Recombinant 
DNA Activities.) Various bacterial spe- 
cies have been examined by the RAC 
for their suitability as host-vector sys- 
tems under Section II-D-2 of the 
Guidelines. The following describes 
conditions for their proposed use as 
HV1 systems or for experiments in- 
volving species that exchange DNA. 
a. Bacillus subtilis related species 
that have been shown to exchange 
chromosomal DNA will be included 
under the exemption category ol Sec- 
tion I-E-4 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 al- 
ready exists in Appendix A for ex- 
changers with E. coli.) 
Bacillus subtilis 
Bacillus licheniformis 
Bacillus pumilus 
Bacillus globigii 
Bacillus niger 
Bacillus nato 
Bacillus amyloliquefaciens 
Bacillus aterrimus 
b. Streptomyces species that have 
been showm to exchange chromosomal- 
DNA will be included under the ex.- 
emption category of Section I-E-4 of 
the 1978 Guidelines. Any recombinant 
DNA molecules that are composed en- 
tirely of DNA segments from the fol- 
lowing pairs of species listed below and 
to be propagated within the pair are 
exempt from the Guidelines. 
■S. aureofaciens x 5. rimosus 
S. aureofaciens x 5. coelicolor 
S. grisues x S. coelicolor 
S. grisues x S. cyaneus 
S. grisues x S. venezuelae 
S. lividans x S. coelicolor 
S. rimosus x S. coelicolor 
S. parvulus x S. coelicolor 
S. fradiae x S. bikiniensis 
FEDERAL REGISTER, VOL 44, NO. 10— MONDAY, JANUARY 15, 1979 
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