Dr. Donald S. Fredrickson 
September 23, 1978 
Page 9 
of the propagating host. Although the lambda vectors constructed by the Blattner 
and Leder groups require use of either the DP50 or DP50 supF host, it is evident 
from the data provided by those laboratories and now substantiated by other in- 
vestigators that the lambda vectors alone come very close to satisfying the level 
of containment required for EK2 certification all by themselves. Thus the use of 
a lambda vector that has been certified as a part of an EK2 host-vector system 
provides biological containment that would be equivalent to EK1.8 to EK2. On the 
contrary, with EK2 plasmid-host systems, almost all biological containment is pro- 
vided by the host and little, if any, by the plasmid vectors which do not have 
mutations causing them to be dependent on the host. The plasmid vectors pMB9, 
pBR322 and pBR313 have defects which cause them to be mobilization-defective 
(Mob") but this only decreases their probability for transmission 1000 to 10,000 
fold when compared with the mobilization of unmodified Mob + Col El-derived vectors. 
It should be noted that the Mob" defects of pMB9, pBR313 and pBR322 can be com- 
plemented by the ColK plasmid (Dougan et al , J. Inf. Dis. Vol . 137:676, 1978). It 
is also known that the low frequency of transmission of these plasmid vectors from 
xl776 is also due to the conjugation defectiveness of xl776, to the fact that 
xl776 rapidly dies under nonpermissive conditions leading to its inability to trans- 
mit plasmid vectors and to the presence of a mutation causing a requirement for thy- 
mine or thymidine which leads to a marked reduction in the probability of transmission 
of plasmid vectors under nonpermissive conditions. These nonconjugative plasmid clon- 
ing vectors can also be transmitted from one microorganism to another by generalized 
transduction and this is diminished substantially in x\71E> which is partially or 
totally resistant to the generalized transducing phages PI, D108, Mu, etc. and which 
possesses a thyA mutation that reduces transductional transmission of plasmid vectors 
under nonpermissive conditions. I should indicate that James Robeson in our labora- 
tory has shown that the generalized transducing phage, PI, can infect 70% of over 500 
wild-type strains of E. col i tested and there is a substantial amount of literature 
to indicate that PI has a very broad host range and is able to infect strains of 
Salmonella , Shigella , Klebsiella , etc. These results suggest that transductional 
transmission of nonconjugative plasmid vectors (which are not dependent on their 
propagating host) may contribute to plasmid transmission. It is therefore evident 
that the use of plasmid vectors that have been certified as components of EK2 
plasmid-host systems provide only nominal biological containment compared to the use 
of lambda vectors which have been certified as components of EK2 phage-host systems. 
I should also point out that the reference to these plasmid vectors that have been 
certified as components of EK2 host-vector systems as "non-mobil izable" in Appendix 
F to the Proposed Environmental Impact Statement (p. 33168) is erroneous. It is thus 
evident that experiments classified in Section III-A-2-a as requiring either P3 + 
EK1 or P2 + an EK1 host and a vector certified for use in an EK2 system can actually 
be done under P2 + EK1.1 if the investigator chooses to use a plasmid vector certified 
as a component of an EK2 host-vector system. Furthermore, the actual level of bio- 
logical containment might even be less if the investigator selected an £. col i 
K-12 host like some of those used in the feeding experiments mentioned above. 
Although the consequences of transmission of vectors containing viral DNA to other 
microorganisms is not discussed in Appendix E or F to the Proposed Environmental 
Impact Statement (p.33159-33169). Dr. Wallace Rowe has informed me that some con- 
sideration was given to this issue. Although I concur that working with viral DNA 
[A-316] 
