20 
level. At this level, a single polypeptide chain having the properties 
of the hormone and the toxicity of diphtheria toxin would be repro- 
duciblv produced. This is stage II of the preposed experiments. 
Dr. Murphy said he would determine in stage I whether Eh coli can 
produce diphtheria toxin and whether the protein is secreted into the 
periplasm or into the culture medium. These data would provide baseline 
information. Dr . Murphy said he wished to use an Jh_ coli host-vector 
system for these experiments because no Corynebacterium diphtheriae 
transformation system has yet been developed. 
Dr. Fedoroff asked why experiments with the intact diphtheria toxin 
gene were necessary. Dr. Murphy replied that whether the toxin 
is secreted into the surrounding medium or whether it is confined to 
the periplasmic space between the two Eh_ coli membranes are important 
considerations. Dr. Murphy said the stability of the product is also 
important; if the toxin is unstable in Eh_ coli , it would make little 
sense bo engineer the hybrid gene. 
Dr. Fedoroff suggested that questions about gene expression could be 
addressed using a fragment of the diphtheria toxin gene. Er. Murphy 
maintained that he wished to determine the stability in Eh_ coli of the 
entire diphtheria toxin molecule. Dr. Gill said such a determination 
would be irrelevant to experiments involving the MSH-diphtheria toxin 
hybrid gene product, and the stability of this hybrid product is the 
only relevant stability. 
Dr. King Holmes, noting that the NIH IBC had requested certain experiments 
be performed, asked Dr. Murphy to outline those experiments. Er. Murphy 
said a sensitive animal species, guinea pigs, would be immunized with 
diphtheria toxoid. Chce high titers of circulating antitoxin are 
obtained, these animals would be colonized with Eh_ coli K-12 carrying 
the diphtheria toxin gene. The animals would be followed for a period 
of time and an histological examination performed. Non- immunized animals 
would also be studied in this manner. Dr. King Holmes asked if these 
experiments would be funded by the NIH. Dr. Murphy replied that they 
would be supported by a grant from a private company. 
Dr. McGarrity asked Dr. McKinney to describe animal procedures in the 
P4 facility at the FCRC, particularly the differences in procedures 
followed under P3 and P4 containment conditions. Dr. McKinney replied 
that under P4 conditions, the animals are maintained in the Class III 
glo/e box. All waste material is autoclaved; the animals only exit the 
glove box in formaldehyde. Under P3 conditions, the animals are housed 
in an open animal roam. Bedding and waste materials are autoclaved. 
Er. King Holmes said he would discourage any superficial attempts at 
risk assessment since no 9olid information will be generated. 
He moved that RAC recommend that no risk assessment experiments be 
[ 26 ] 
