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Federal Register / Vol. 44. No. 71 / Wednesday. April 11. 1979 / Notices 
The application had been reviewed by 
the Lower Eukaryotic Host-Vector 
Working Croup and recommended to 
the RAC for acceptance. The basis of 
containment in the HV2 systems is the 
inability of cloned DNA in a sterile 
yeast host to be transferred out of the 
host to other laboratory or wild type 
strains. The level of containment 
achieved by the HV2 system is 
acceptable based primarily upon the 
sterility, the probability of encountering 
an appropriate haploid strairt in nature 
which is capable of mating, and the 
inefficiency of mating in dilute 
suspension. 
The following sterile strains of yeast, 
all of which have the s/e-V C9 mutation, 
are proposed as HV2 hosts: SHY1. 
SHY2. SHY3 and SHY4. 
The proposed HV2 pl<0mid vectors 
have in common several characteristics. 
They all contain a bacterial replicon 
derived from the certified EK2 vector, 
pBR322. which makes possible the 
amplification of the DNA in E. coll. 
Another common characteristic is the 
presence of markers which enable 
selection in p coli for the presence of 
the plasmid. Some of these markers are 
drug-resistance genes derived from 
pBR322. Others, however, are yeast 
DNA fragments which are expressed in 
E. coli K-12: these include the ura3. Ieu2, 
his3. and trp 1 genes. 
Most of the vectors also carry one or 
more genes of yeast origin which can be 
used to select the presence of the vector 
in yeast, such as uro3. leu 2. trp\, and 
his3. The SHY strains are arranged to 
make anv or all of these genes 
selectable. All the vectors also carry 
fragments of yeast DNA which provide 
for the maintenance of the plasmid in 
jeast. The following are the proposed 
IIV2 plasmid vectors: Ylpl. YEp2. YEp4, 
Ylp5. YEp6. YRp7. YEp20. YEp21. YEp24. 
YIp25. YIp28. Ylp27. YIp28. Ylp29. Ylp30. 
Ylp31. YIp32. YIp33. 
A complete discussion of the strains 
and the vectors proposed for 
certification is available from the Office 
of Recombinant DNA Activities. 
The RAC voted 17 to 0 with 3 
abstentions to recommend approval of 
the proposed systems because they 
were considered to meet adequately the 
criteria for HV2 S. cerevisiae systems. 
C. Prokaryots Other than E. Coli as 
Host- Vector Systems 
This issue was considered at a 
meeting held at Stanford University on 
September 12. 1978 of the Working 
Group on Prokaryote Host-Vectors 
Other than E. coli. The purpose of that 
meeting was to explore other candidate 
prokaryotic organism* as HVl systems. 
The group considered Bacillus and 
Streptomyces systems as possible 
additions to the presently approved EKl 
and EK2 systems. The Working Group 
report was considered by the RAC at its 
October 30-31, 1978 meeting. The report 
was modified and approved by the RAC. 
The recommendations were summarized 
and published for public comment in the 
Federal Register on January 15. 1979. No 
comments were received during the 30- 
day period for comment. The RAC again 
considered the recommendations at its 
meeting on February 15-16, 1979. 
1. EXEMPTION OF BACILLUS 
SPECIES THAT EXCHANGE GENETIC 
INFORMATION. The RAC considered 
the following proposal for exemption of 
certain Bacillus species on the basis of 
exchange of genetic information: 
" Bacillus subtilis related species that 
have been shown to exchange 
chromosomal DNA will be included 
under the exemption category of Section 
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 already exists 
in Appendix A for exchangers with E. 
coli.) 
Bacillus subtilis 
Bacillus licheniformis 
Bacillus globigii 
Bacillus niger 
Bacillus nato 
Bacillus pumilus 
Bacillus amyloliquefaciens 
Bacillus aterrimus" 
The basis of this list is that these 
organisms exchange chromosomal DNA 
by transformation. 
Th RAC voted 17 to 0 with 3 
abstentions to accept this section of the 
report. 
2. BACILLUS SUBTILIS HVl 
SYSTEM. The RAC considered the 
following proposal for acceptance of B 
subtilis as an HVl system: 
"Asporogenic mutant derivatives of B 
subtilis cam be accepted as the host 
component of an HVl system. These 
derivatives must not revert to 
sporeformers with a frequency greater 
than 10“ 7 ; data confirming this 
requirement must be presented to NTH 
for certification. The following plasmids 
are accepted as the vector components 
of certified B. subtilis HVl systems: 
pUBllO. pCl94, pSl94. pSA2100. pEl94. 
pTl27. pUBll2. pC221. pC223." 
The Bacillus subtilis system was 
recommended by the Working Group as 
a host system in conjunction with 
certain plasmids because it offers 
distinct advantages for genetic 
manipulation and is of little or no 
potential hazard. B. subtilis. an obligate 
aerobe, is not normally indigenous to 
man, is non-pathogenic to man, and is 
generally found in the soil. In Japan, a 
vegetable cheese composed of live 
Bacillus nato. almost identical to B. 
subtilis. is consumed in large quantities 
by an estimated 80% of the population. 
In France, deliberate ingestion of B. 
subtilis known as ''Bactisubtil" is 
similarly widespread and is used 
therapeutically for certain digestive 
disorders. B. subtilis is not a normal 
inhabitant of the gut since it is an 
obligative aerobe. 
The RAC voted 15 to 0 with 4 
abstentions to approve the proposal. 
3. EQUIVALENCE OF BACILLUS 
SUBTILIS HVl SYSTEMS WITH 
THOSE OF E. COLI. The following 
statement regarding equivalency of 
Bacillus subtilis systems that had been 
approved by the RAC at its meeting on 
October 15-16. 1978 was omitted from 
the January 15, 1979 Federal Register 
announcement: 
"HVl Bacillus subtilis systems will be 
equivalent to EKl in the physical 
containment requirements for specific 
experiments. " 
Section III— B— 1 of the Guidelines 
states: 
"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] Other classes of 
recombinant DNA experiments with 
these HVl systems will require prior 
approval and classification by NIH 
The RAC voted 12 to 0 with 6 
abstentions that classes of recombinant 
DNA experiments with B. subtilis HVl 
systems (other than those specified 
above) will be equivalent to EKl in the 
physical containment requirements for 
specific experiments. 
4. ADDITION TO SECTION 1II-B-3. 
NON-HV1 SYSTEMS. The RAC 
considered a proposal in the Working 
Group report to amend Section III— B— 3 
of the Guidelines by addition of the 
following: 
In those cases where genetic 
exchange has not been demonstrated 
between two bacterial species A and B. 
neither of which is pathogenic for man, 
animals, or plants, recombinant DNA 
experiments involving only A and B can 
be conducted under P3 
containment. [2 A]" 
C83J 
