Federal Register / Vol. 47, No. 60 / Monday. March 29. 1981 / Notices 
13309 
can be included in Sublist A. Appendix 
A. By a vote of eighteen in favor, none 
opposed, and one abstention, the RAC 
recommended approval of the request. 
I accept this recommendation and 
Yersinia enterocolitica has been added 
to Sublist A of Appendix A of the 
Guidelines. 
F. Proposed EK2 Host- Vector 
Systems. In a letter dated September 25, 
1981. Dr. Roy Curtiss of the University of 
Alabama in Birmingham, requested E1K2 
certiHcation of six different £. coli K-12 
strains in conjunction with various 
virilent and temperate bacteriophage 
lambda, plasmid, and cosmid vectors. 
The request was subdivided into three 
separate requests. The requests were 
published for comment in the Federal 
Register of December 7. 1981 (46 FR 
50734). During the thirty day comment 
period, no comments were received. 
Dr. Curtiss' submission was reviewed 
by a working group of the RAC on 
[anuary 21. 1982. llie working group 
made recommendations on the basis of 
data available, and requested certain 
additional documents. A summary of the 
working group conference telephone call 
was prepared and distributed to the full 
RAC 
The RAC reviewed the submission 
and working group report at its meeting 
on February 9. 1982. It was agreed that 
the First request posed no particular 
problems, and the RAC voted, by 11 in 
favor, none opposed, and 2 abstentions, 
to recommend LK2 certification of E. 
coli K-12 strains chi-2447 and chi-2281 
for use with those lambda vectors that 
already have been certified for use with 
strain DPSO on the condition that the su* 
strain not be used as a propagation host. 
I accept this recommendation and 
appropriate language has been added to 
Appendix F of the Guidelines. 
The RAC then turned its attention to 
the second and third requests which 
proposed EK2 certification of four 
strains of E col. K-12 (chi-1984. chi- 
2705. chi-2001, and chi 2383) in 
conjunction with virulent and temperate 
lambda vectors, plasmid vectors, and 
cosmid vectors. The RAC felt that there 
were no significant problems In 
considering certification of the strains 
specified in requests 2 and 3 with 
virulent lambda vectors However, 
enough data were not available for the 
RAC to recommend their use with 
lysogenizing phage vectors, plasmid 
vectors, and cosmid vectors. The RAC 
voted 11 in favor, none opposed, and 4 
abstentions to recommend KK2 
certification of E. coli strains chi-1984. 
chi-2705, chi-2001, and chi 2363 with 
certified virulent lambda vectors on the 
condition that the su' strains not be 
used as propagation hosts. 
Consideration of the requests for use of 
other vectors with these strains was 
deferred. 1 accept these 
recommendations, and appropriate 
language has been added to Appendix F 
of the Guidelines. 
G. Proposed use of EK2 Host-Vector 
Systems for Cloning DNA from Class 3 
and 4 Etiological Agents. In a letter 
dated September 25. 1981. Dr. Roy 
Curtiss of the University of Alabama in 
Birmingham requested permission to use 
all certified EK2 host-vector systems to 
clone DNA fragments from CDC Class 3 
and Class 4 etiological agents under P2 
containment and under Pi containment 
if the recombinant clones are shown not 
to express a virulence determinant that 
has toxic potential. 
As an alternative to this general 
proposal. Dr. Curtiss requested 
permission to clone DNA from Yersinia 
pestis and Mycobacterium leprae into 
EK2 host-vector systems under P2 
containment, and under Pi conditions in 
the absence of expression of virulence 
determinants by the recombinant clones. 
This request was published in the 
December 7. 1981. Federal Register (46 
FR 59734). During the thirty day public 
comment period, no comments were 
received. 
During the RAC discussion of the 
proposal on February 9. 1982. it was 
noted that Class 3 agents can currently 
be worked with in EKl systems under P3 
containment conditions (Guidelines 
Section IlI-O-l). It would, therefore, be a 
reasonable alternative also to permit 
DNA from Class 3 organisms to be 
cloned and propagated under P2 -+- EK2 
conditions. Ifowever, the RAC felt that 
recombinant DNA experiments 
involving Class 4 agents should be 
considered only on a case-by-case basis 
under the current Guidelines. By a vote 
of thirteen in favor, none opposed, and 
two abstentions, the RAC recommended 
that DNA from Class 3 etiological agents 
may be propagated in E. coli K-12 under 
P2 + EK2 containment conditions. 
1 accept this recommendation, and 
Section ill-0-1 and Appendix C of the 
Guidelines have been accordingly 
modified. 
II. Summary of Actions Under the 
Guidelines. 
A. Modification of Section llf-O-l and 
Appendix C of the Guidelines. Section 
III-0-1 of the Guidelines is modified to 
read: 
"Ill-O-I Expenwenls Involving Class 3 
Organisms. Experiments involving 
recombinant DN.A from Class 3 organisms (1) 
or from cells known to be infected with these 
agents may be conducted at P3 containment 
in E. coll K-12 EKl Ifosts or at P2 
containment in E call K-12 EK2 hoM.s (see 
Appendix C). Containment levels for all other 
experiments with Class 3 organisms or with 
recombinant DNA which increases the 
virulence and host range of a plant pathogen 
beyond that which occurs by natural genetic 
exchange will be determined by NIH. (See 
Section IV-E-l-b-2-{e))." 
In Appendix C. the second paragraph 
in the language detailing "exceptions" to 
the section "2. Experiments Involving E. 
coli K-12 Host- Vector Systems " is 
modified to read; 
"Experiments involving DNA from Class 3 
organisms (1) or from cells known to be 
infected with these agents may be conducted 
at P3 containment using E. coli K-12 EKl 
host-vectors systems, or at P2 containment in 
E. coli K-12 EK2 host-vector systems. Lower 
containment levels may be specified by NIH. 
(See Section IV-E-l-b-(2)-{e)). Elxperiments 
in this category require prior IBC review and 
approval." 
B. Addition of Yersinia Enterocolitica 
to Sublist A, Appendix A. Sublist A of 
Appendix A of the Guidelines is 
amended to read as follows: 
1. Genus Escherichia. 
2. Genus Shigella. 
3. Genus Salmonella (including 
Arizona). 
4. Genus Enterobacter. 
5. Genus Citrobacter (including 
Levinea). 
6. Genus Klebsiella. 
7. Genus Erwinia. 
8. Pseudomonas aeruginosa. 
Pseudomonas putida and Pseudomonas 
fluorescens. 
9. Serrdtia marcescens. 
10. Yersinia enterocolitica. 
C. Cloning of Subgenomic Segments of 
Foot and Mouth Disease Virus. A net 
item, number 42. is added to Appendix E 
of the Guidelines: 
"42. Permission is granted to transfer 
certain clones of subgenomic segments of 
Foot and Mouth Disease Virus from Plum 
Island Animal Disease Center to the 
laboratories of Molecular Genetics. Inc., 
Minnetonka. Minnesota, and to work with- 
these clones under Fri containment 
conditions. Approval is contingent upon 
review of data on infectivity testing of the 
clones by a working group of the RAC." 
D. Cloning of E. Coli, Bacillus SubtiUs, 
and Staphylococcus Aureus DNA in 
Bacillus Megoterium, A new item, 
number 43, is added to Appendix E of 
the Guidelines: 
"43. Bacillus megoterium can be used as a 
host for cloning DNA segments derived from 
E. coli. Bacillus sublilis. and Staphylococcus 
aureus under P2 containment conditions. 
Local IBCs are authorized to lower 
containment to Pi for specific experiments." 
E. Cloning of Plant DNA in Anacystis 
Nidulans. A new item, number 44, is 
added to Appendix F. of the Guidelines: 
[397] 
