Tab A - Page 32 
(111) Elimination of the vector carrying the genomic INA* region by growth 
on media that does not select for Its presence within a cell. 
To facilitate population monitoring the researchers will select for the 
deletion bacterial strains carrying spontaneous resistance to certain 
antibiotics. This resistance will be unaffected by the deletion to be 
performed, and will enable the researchers to isolate selectively the bacteria 
released for confirmation of identity and phenotype by Southern blot hybridi- 
zation analysis. This technique facilitates the large-scale monitoring of 
bacterial populations on a routine basis to determine survival and lateral 
dissemination of strains. 
The ONA inserts will be transferred to bacterial plasmid vectors best 
suited for the construction. All plasmid and strain constructions described 
above will be done in the laboratories of Drs. lindow and Panopoulos (not at 
the test site) and will be verified by molecular genetic tests and criteria 
including restriction endonuclease digestions, gel electrophoresis , and blot 
hybridizations. (I) 
5.2.3 Previous Field Tests With INA" Bacteria 
A cruder form of the oroposed experiment has already been field tested. 
In the earlier experiment, Ors. Lindow and Panopoulos used chemicals to mutate 
the natural ly-occurrlng INA* Pseudomonas syrlnoae and £_. herblcola to INA” 
bacteria. Ultraviolet Irradiation and chemical mutagenesis are Imprecise 
mutational procedures as they cause random mutations. Nevertheless, suitable 
INA mutants were Isolated and field tested for their efficacy In preventing 
29 
[ 226 ] 
