Provisional N1H Guidelines: 
y!776 and /j.3 76 : 
"In testing the level of biological 
containment afforded by a proposed 
EK-2 plasmid-host system, it is 
important to design relevant tests 
to evaluate survival of the cloned 
DNA under conditions that are 
possible in nature and that are 
also most advantageous for its 
perpetuation." (Guidelines, p.20) 
"Survival of the cloned marker by 
transduction could also be evaluated 
by introducing a known generalized 
transducing phage into the system." 
(Guidelines, p.20) 
"It v.'ould be useful to determine 
the survival of the’ host under non- 
growth conditions such as in water 
and as a function of drying time 
after a culture has been spilled on 
a lab bench." (Guidelines, p.21) 
DNA in the bacterial cell will alter 
its physiological properties. Credence 
for this argument emerges from some of 
the comparative survival data between 
X1776 and yl876, which indicate that 
the presence of additional DNA in the 
form of the pSClOl plasmid does change, 
and sometimes quite radically, the 
properties of the cell (see Curtiss 
et^ £l . , Figures 11 and 22, p. 21, and 
this paper, Section I, second point, 
§ § 1 Ob , 10c, lOd , 14d). 
The above -has not been achieved as 
indicated previously by Curtiss and 
co-workers. Also, the tests that have 
been performed on the "disarmed" 
strains do not contemplate a variety 
of conditions possible in nature and 
in the laboratory (see this paper. 
Section IV-B) . 
This experiment is net reported. 
This test is of importance given that 
the authors obtain multiplication of 
the E. coti, virus To upon infection of 
X1776 under starvation conditions. 
Furthermore, virus growth is more 
prolific in the most deprived medium 
tested (Curtiss et^ :Q. , Figures 29 
and 30, see this paper, §§20a and 20b). 
Survival of these strains was tested 
under the above conditions but was 
observed to be no different than the 
survival of their r.on-weakened parent 
under drying conditions (Curtiss et al. , 
Figure 28). A very large number of 
ceils (0.1 to 1.0% of the initial 
concentration of cells) were able to 
retain viability up to 8 days in tap 
water (Curtiss cpt_ al. , Figures 25, 26, 
and 27, see this paper, §§12 and 13). 
[Appendix C — 198] 
