11 
Provisional NIH Guidelines : 
"The combination of the dapD8, 
deletion bioll-asd and deletion 
gal-chl nutations in E. coll K12 
results in no detectable survivors 
in feces of rats foll^ying feeding 
by stomach tube of 10 cells in 
milk and similarly leads to complete 
lysis of culture suspended in broth 
medium lacking diaminopimelic acid 
(DAP)." (Guidelines, p.19) 
"Mutations can also be introduced 
into the plasmid to cause it to be 
dependent on a specific host, to 
make its replication thermosensitive 
and/or to endow it with a killer 
capability such that all cells 
(other than its host) into which 
it might be transferred will not 
survive." (Guidelines, p.20) 
yl776 and y]876 : 
The fate of stomach tube fed cells 
is confirmed (Curtiss £t^ al^. , Table 
17), although there are questions as 
to the statistical validity of these 
data, as the sample size is very 
small (only 3 to 9 rats per sample 
average, see this paper, §l4a). 
Nevertheless, in the case of yl876 
(the complete host-vector system), 
survival upon passage through the 
rat gut occurs when the antibiotic 
tetracycline is administered concur- 
rently with the feeding of cells. 
Tetracycline appearr to "override" 
all the "safety" properties of the 
strain (Curtiss ej^ . 1. , Table 17, 
see this paper, §§l-a, 14b, and 14c). 
On the other hand, complete lysis of 
a culture suspended- in a minimal 
medium lacking DAP does not occur 
(Curtiss et al. , Figure 22). In fact, 
one million cells ( Jf, of the total 
initial cells) are able to survive 
180 hours of DAP starvation. This 
experiment demonstrates that the 
presence of the pSCMl plasmid in 
yl876 allows an eno;nous survival 
advantage over the strain without the 
plasmid in very poor media. This 
particular experiment can be con- 
sidered one of the ultimate tests of 
biological containment, since survival 
of yl87 6 is tested in poor media lacking 
both D/VP and thymine (see this paper, 
§§10a, 10b, 10c, anc lOd). * 
Such a plasmid is not available 
(Curtiss et_ aj^.., p.3). Furthermore, 
it is not definite that yl77 6 has a 
suppressor mutation at all (see this 
paper, §§5a and 5b). A "safe" plasmid 
would depend, for its replication, 
on the existence of a suppressor mutation 
in the host. In addition, Stewart and 
Levin (in press) demonstrate that 
plasmids will establish themselves in 
a bacterial population even when their 
introduction into the host markedly 
reduces its Darwinian fitness. 
[Appendix C — 199] 
