Dr. Mary Clutter 
Page 3 
The intact Ti-T37 plasmid could be used, but its large size 
would present potential problems for cloning. The foreign 
gene to be inserted into the T-DNA is chicken ovalbumin DNA t 
available as a clone in the laboratory of Dr. Savio Woo. 
The ovalbumin gene excised from this clone will be recloned 
into the shrunken Ti plasmid (E. col i host, selection for 
the ampicillin resistance of the HP 4 component) . Because 
the shrunken Ti plasmid will possess multiple cleavage sites 
for the cloning enzyme, a partial digest will be used. 
Subsequent screening by fingerprint analysis will .reveal 
v/hich clones have the ovalbumin DNA inserted into the T-DNA 
of the shrunken Ti plasmid. This recombinant' DNA -will be 
transferred to Agrobacterium by conjugation, and tumors 
will be incited on tobacco or potato plants in a high con- 
tainment greenhouse. Tumor tissues will be put into tissue 
culture and can be tested for the presence of ovalbumin DNA, 
RNA transcripts from this DNA, and the protein (immunological 
assay). By Braun's grafting technique, teratoma plants will 
be obtained. We will attempt to propagate these vegetatively 
in order to obtain more experimental material. The resulting 
plants will be tested for production of ovalbumin or immuno- 
logically related protein, using various plant parts (especially 
the tubers, in the case of potato plants). 
The advantages of the approach outlined above are several. 
We propose to use a eukaryotic gene, v/hich should have the 
best chance of expression in the plant cell, for the engineering 
experiment. Vie do not require that an active enzyme be 
produced. The detection system is very sensitive, and should 
have low background. (We do not expect to find in normal 
plant tissue any protein serologically related to ovalbumin.) 
All of the steps in the experiment are feasible and appear to 
have a high probability of success. The only disadvantage 
of the experiment is that it involves recombinant DNA manipu- 
lations not allowed under the present N.I.H. Guidelines. This 
disadvantage could be overcome by v/orking under conditions 
of high physical containment. 
It has been pointed out by one member of our research group 
that the proposed experiment might be viewed as the. creation 
of an egg-plant. Although it is stated with levity, this is 
indeed the purpose of the experiment. It is more promising 
to alter the nutritional quality of plant tissues by intro- 
ducing genes coding for high quality storage protein than 
genes coding for synthesis of an amino acid. The amino acid 
is soluble, and thus is likely to be lost in cooking. In 
addition, expression of a foreign biosynthetic pathway in the 
plant promises to be a difficult process. 
Sincerely yours, 
- .//si.", cOY/ 
C 
7 . 
? 
> 
-7 — 7 ^ 
[Appendix A — 305] 
