Dr. Donald S. Fredrickson 
September 23, 1978 
Page 10 
inserts in £. col i K- 12 is probably safer than working with the intact infectious 
virus, I do have reservations based on the lack of appreciation by the various 
groups that studied and reviewed the cloning of viral DNA in E_. col i of the differ- 
ences between phage and nonconjugative plasmid vectors and the consequences of trans- 
mission of recombinant DNA to wild-type microorganisms in nature. The recent re- 
sults of experiments with polyoma in £. col i K-12, conducted by Dr. Michael Fried 
in Great Britain and by Drs. Malcolm Martin, Wallace Rowe and their colleagues in 
the U.S., also contribute to my concern. As you will recall at the Falmouth 
Workshop, while it was the consensus that one could not likely convert £. col i K-12 
into a pathogen, much less an epidemic pathogen, there was considerable concern 
and debate about the consequences of transmission of vectors containing recombinant 
DNA to microorganisms indigenous to various natural habitats. I am also informed 
that certain viral ly specified mRNAs need not be processed to lead to synthesis of 
fully functional viral proteins and that some viral ly-specified proteins can be 
made using IE. col i RNA polymerase with an col i generated translation system. 
I thus believe that this section of the Revised Guidelines dealing with the cloning 
of viral DNA in £. coli K-12 host-vectors needs to be examined not only by virolo- 
gists but by some of the types of experts who were in attendance at the Falmouth 
Workshop Meeting. 
In view of what I have read and learned and based on some of the preceding com- 
ments, I would not have voted in favor of lowering the containment levels for 
cloning viral genetic information in E. col i K-12 to the levels now stipulated in 
the Revised Guidelines if I were stilT a member of the RAC. This is particularly 
true in view of the fact that the risk assessment experiments which were to have 
defined what levels of containment are fitting have yet to be completed. 
17. Section III-B-2. Return of DNA segments to non-HVl host of origin, (p. 33080). 
The provisions of containment for a prokaryote which does not exchange genetic 
information with £. col i , the so-called Host B, are poorly thought out and in need 
of refinement. For example, if Host B is a Class 1 agent, the cloning into £. col i 
would require P2 + EK1 or PI + EK2 and the return of the potential double vector 
to Host B would only require PI containment. If Host B were a Class 2 agent, and 
this is not precluded, then the nonsymmetry of the containment required and the 
problems associated with double vectors become even more exaggerated. Obviously, 
these points need to be addressed and further refinement of this section is needed. 
Based on comments in the Decision of the Director to Issue Revised Guidelines 
statement, I surmise that it was intended to use the cloning in £. col i K-12 to 
purify a genomic segment from Host B which would then be separated from the E_. col i 
vector, purified and returned to Host B in the absence of the E_. col i vector. I 
should add that it might be wise to note in this section that P2 containment would 
be advisable if Host B is a Class 2 agent or a plant pathogen. 
18. Section III-C-5. Fungal or Similar Lower Eukaryotic Host-Vector Systems 
(p. 33084). The stipulations for cloning DNA from a non-HVl host into E_. col i K-12 
and return to that host (designated Host C) result in the same problems detailed in 
the immediately preceding comment. 
[A-317] 
