Federal Register / Vol. 44, No. 71 / Wednesday, April 11, 1979 / Notices 
21731 
only for certain experiments. 
Accordingly, this section will now read 
as follows: 
"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 net produce 
polypeptide toxins. [34]" 
3. S. CEREV1SIAE AS AN HV2 
SYSTEM. The RAC considered the 
following criteria for the use of S. 
cerevisiae as an HV2 system: 
"S. cerevisiae as an HV2 system. 
Biological containment of yeast is a 
product of several factors: (a) 
probabilily 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 fequency 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 
mutations(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 
segment (in a worst case analysis, again 
with a fertile strain) to a non-mating, 
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." 
This section addresses the criteria for 
the acceptance of S. cerevisiae as an 
HV2 system. One member of the RAC 
raised a question as to whether 
numerical values should be assigned to 
the containment criteria. It was slated 
that the combined effect of various 
containing features (the product) is the 
important consideration. Explicit in the 
Guidelines is the factor of 10~ 8 (See 
Guidelines, Section Il-D-l-b). 
Investigators will have to present data 
that will at least satisfy this value. 
The RAC voted to accept this section 
by a vote of 19 to 1 with no abstentions. 
4. EQUIVALENCE OF LOWER 
EUKARYOTE HV SYSTEMS WITH 
THOSE OF E. COLI. The RAC 
considered a proposal for the 
equivalence of lower eukaryote HV 
systems with those of E. coli. The 
proposal appeared in the January 15, 
1979 Federal Register as follows: 
"Equivalence of lower eukaryote HV 
systems with those of E. coli. In accord 
with Section III-B-1, 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. 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. [45]" 
This section contained a typographic 
error as it appeared in the Federal 
Register of January 15, 1979. The next to 
last sentence in the paragraph should 
have read: 
“Experiments involving complete 
genomes of class 1 eukaryote viruses 
will require P3 + HVl or P2 + HV2 
containment levels." 
The RAC decided to treat this 
proposal in two sections. Accordingly, 
the RAC first considered the following 
part of the proposal: 
“Equivalence of lower eukaryote HV 
systems with those of E. coli. In accord 
with Section III-B-1, 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 sysems and S. cerevisiae 
HV2 systems may be used at the 
physical containment levels applicable 
to EKl and EK2 systems, respectively." 
In order to use the approved systems 
in various experiments it is necessary to 
assign containment levels. One RAC 
member pointed out that if equivalence 
was not adopted it would be necessary 
to go through the Guidelines and 
individually assign containment levels 
for each set of experiments. 
The RAC proposed to equate HVl to 
EKl and HV2 to EK2 for the S. 
cerevisiae and N. crassa systems. 
The RAC voted to accept the proposal 
by a vote of 18 to 2 with no abstentions. 
The RAC then considered the second 
part of this section which was 
incorrectly printed in the Federal 
Register of January 15. 1979. The RAC in 
its deliberations took a less restricted 
position than either that of the Federal 
Register announcement or the report of 
the Working Group, and recommend 
that the following wording be 
substituted for the previous proposal: 
"Experiments involving complete 
genomes of eukaryote viruses will 
require P3 + HV1 or P2 + HV2 
containment levels.” 
This recommendation passed by a 
vote of 17 to 1 with 2 abstentions. Since 
this is a major change in the proposed 
action as it appeared in the Federal 
Register on January 15, 1979, additional 
opportunity for public comment is 
appropriate. Accordingly, this proposed 
action will be published for an 
additional 30-day comment period and 
will be reconsidered by the RAC at its 
next meeting. 
B. Saccharomyces Cerevisiae HV2 
Systems 
The RA.C reviewed an application 
submitted by Dr. David Botstein et al. 
for certification of sterile yeast hosts 
(SHY strains of Saccharomyces 
cerevisiae) as HV2 containment systems 
when used with a variety of composite 
vectors capable of replication and/or 
integration in yeast. 
During the 30-day period for comment, 
no comments were received. Prior to 
consideration of this proposal, the RAC 
had recommended the criteria for 5. 
cerevisiae as an HV2 system described 
in A above. 
[82] 
