I. SUMMARY AND CONCLUSIONS 
The proposed candidate strain (and its plasmid containing 
equivalent, X1876) do not satisfy the criteria for EK-2 status outlined 
in the "Proposed Guidelines for Research Involving Recombinant DNA 
Molecules," (January 1976) (see this paper, Section III). 
First, as Curtiss et_ al. state, "there is no available plasmid 
cloning vector which has been specially constructed for safety and/or 
which is dependent on or some other suppressor-containing 'strain 
for its replication" (Curtiss et^ al. , p.3). 
Second, and most important, the data presented by Curtiss concerning 
the survival of X1776 (or x^876) do not and can not promise the reduced 
survival of the same strain carrying a cloned foreign DNA sequence. 
Experiments performed by Dr. Ronald W. Davis (personal communication) 
have demonstrated that cloned DNA from a eukaryotic organism ( Saccharomyces 
cerevisiae) can correct for genotypic defects of Escherichia coli , such 
as auxotrophy for histidine biosynthetic pathway genes or recombination 
deficiency ( recA ) . 
Third, the proposed strains have not been tested in any natural 
environment (environment not specifically peculiar to the laboratory), 
with the exception of the rat gastrointestinal tract. In distilled or 
tap water, the strains show identical survival kinetics with their 
prototrophic parent. Environments not tested include the human gastrointes- 
tinal tract, raw sewage, and- soils, to which the strain may have easy 
access (see this paper, §B22). 
Fourth, the strain xl&76 produces pSClOl-containing minicells 
[Appendix C — 191] 
