Dr. Donald S. Fredrickson 
September 23, 1978 
Page 8 
I learned that 4 of 24 human volunteers who had been fed strains of col i K-12 
in a risk assessment experiment being conducted in Great Britain excreted low 
titers of the IE. col i K-12 strain for periods of up to four weeks. These results 
as well as those obtained by Rolf Freter (J. Inf. Dis. Vol . 137:624, 1978) in which 
prototrophic K-12 strains were found to persist in some animals under some conditions 
implies that the use of K-12 hosts without mutations to diminish their survival and/ 
or ability to transmit DNA cloned on nonconjugative plasmid vectors will result in 
a higher cumulative amount of transfer of such recombinant plasmids to other micro- 
organisms in nature than would occur if the K-12 hosts possessed mutations to dimin- 
ish their survival and/or transmission ability. 
13. Section 1 1 -D-l-c- ( 2 ) (p. 33075). The requirement that "Reversion to host-inde- 
pendence must be less than 1/10^ per vector genome per generation. 11 is operationally 
difficult, if not impossible, to define since there is no meaningful operational 
definition of the term generation when applied to plasmids or to bacterial viruses. 
Quite possibly this should be clarified and put into some terminology of frequency 
starting from single plaques or cells containing plasmids. 
14. Section II-D-2-a. Responsibility (p. 33075). The statement that "HV1 systems 
other than £. col i K-12 and HV2 and HV3 host-vector systems may not be used unless 
they have been certified by the NIH", in view of the lists not contained in Appen- 
dix A, effectively precludes already approved recombinant DNA research with the 
B. subtil is and Saccharomyes cerevisiae cloning systems except for self cloning. 
It thus becomes important that NIH through the RAC begin to develop procedural 
means to inform the scientific community of exactly how to get approval of HV1 
systems and to begin to develop appropriate guidelines for the performance of ex- 
periments that are permitted under the Current Guidelines but which will not be 
initially permitted under the Revised Guidelines. 
15. Section III. Containment Guidelines for Covered Experiments (p.33076-33084). 
For the most part, I think that containment specifications are adequately justified 
by the available data on the increased safety of using Z. col i K-12 host-vector 
systems but I do have a few minor points to make and one major reservation as 
detailed below. I should note that the statement on page 33058 (Col. 1) enumera- 
ting the criteria used to classify experiments is extremely good. One point which 
was omitted from the list concerns the potential consequences of transfer of the 
vector containing recombinant DNA to some other natural host or vector. 
16. Section III-A-2-a. Viruses of Eukaryotes ( p . 33077-33080 ) . There are numerous 
instances in which the stipulated containment is P3 + EK1 or P2 + an EK1 host and a 
vector certified for use in an EK2 system. The specified use of a vector certified 
for use in an EK2 system in the absence of its certified host is inconsistent with 
other sections of the Revised Guidelines which consider hosts and vectors as in- 
separable components of EK1 and EK2 systems. More importantly, this allowance 
permits wide differences in the actual level of biological containment dependent 
upon the selection of the vector that has been certified as a component of an EK2 
system. All bacteriophage lambda vectors certified as components of EK2 systems pos- 
sess mutations which cause their replication to be dependent on the propagating 
host, block lysogenization and cause death of all host cells infected. Indeed, 
the lambda vector constructed by Donoghue and Sharp meets EK2 standards irrespective 
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