13 
Dr. Kopecko said F. coll K-12 host-vector systems are not pathogens. He thought 
the worst case scenario would involve the potential transfer of the recanbinant 
DNA plasmid to another organism. Dr. Levine said even if the E. coll K-12 
host-vector system could colonize the gut the rate of transfer of plasmids in 
the qut is extremely low under normal conditions. 
Dr. Murphy said all of the conjugate protein produced by the Eh coll host-vector 
system is exported to the penplasmic compartment. Dr. Habig asked if this vas 
the case even in the stationary phase of bacterial growth. Dr. Mirphy said he 
did not know but reminded the group that experiments involving intra-peri tone al 
injection of viable ard heat killed organisms showed no different response to 
organisms carrying the hybrid gene and the host-vector system without the 
hybrid gene. 
Dr. Levine said he was not sure the observation the protein was not secreted 
is an argument for safety. Bacterial cells are not immortal and will lyse and 
secrete the fusion protein into the immediate environment. 
Dr. Murphy thought a large number of cells would have to lyse to produce protein 
levels lethal to the animal . 
Dr. Dill guestioned whether the toxicity data presented by Dr. Mirphy might have 
been generated using degraded conjugate protein. 
Dr . Habig asked Dr . Murphy whether he had ascertained that the u-MSH-diphtheria 
toxin hybrid protein wes isolated intact. Dr. Murphy .said he had not looked 
at the C terminal seauences to determine whether the hybrid protein had been 
degraded . The active fraction was homogeneous on gels and was close to the 
deduced molecular weight of 50,000, however. 
Dr. Collier asked Dr. Murphy how he knew the fraction injected into the guinea 
pigs contained active a-MSH-diphthena toxin hybrid protein. Dr. Mirphy replied 
that the protein fraction was active against sensitive cells m culture. 
Dr. Gottesman asked how sensitive these tissue culture cells are to diphtheria 
toxin. Dr. Murphy replied that the control cells, African green monkey CV-1 
cells, are sensitive to 10~30 or 10 -11 molar diphtheria toxin. 
Dr. Gill said the important considerations are the toxicity of the Sph l fragments 
and of the conjugate protein. He thought the toxicity of the Sph l fragment is 
very likely to be very lew; however, it is not possible in BL4 conditions to 
obtain sufficient purified material to determine toxicity. 
Hr. Gill sugqested Dr. Murphy be given permission to work m BL3 conditions to 
purify enough material for toxicity testing and to quantitatively assay the 
hybrid protein in more species of animals over a longer period of time. 
Drs. Gottesnan and Collier agreed that Dr. Murphy should be permitted to 
remove the clones frem BL4 contairment for the purpose of obtaining additional 
data. 
[317] 
