9 
Request to Remove Clones Carrying the a -MSH-Piphthena Toxin Gene From 
High Containment 
Hr. Gottesman sa id Dr. John Murphy, then of Flarvard Medical .School and now with 
the University Hospital of the Boston University Medical Center, in a letter 
dated October 5, 1982, requested permission to construct a hybrid molecule in 
vhich the qene coding for a -melanocyte stimulating hormone (cx-MSH) is joined to 
a segment of the gene encoding diphtheria toxin. The gene segment would encode 
the A subunit and portions of the B subunit of the diphtheria toxin gene. The 
segment would not contain the diphtheria toxin binding danam. The a-MSH gene 
would be a synthetic oligonucleotide. The a-MSH-diphthena toxin hybrid gene 
would be introduced into poorly mobilizable plasmids such as pBR322, PUC9, or 
PUC8 and cloned in Fh coll EKl host-vector systems. The goal of these experi- 
ments is to construct a family of chimeric toxin genes whose products have the 
potency of diphtheria toxin and the receptor binding specificity of a-MSH. 
This reouest was discussed by the RAC at the October 25, 1982, meeting; RAC 
reccmmended these experiments be permitted at RM containment at the Frederick 
Cancer Research Facility. The RAC also reccmmended that data on the character- 
istics of the reccmhinant organisms expressing the ot -MSH-diphthena toxin 
hybrid gene should be evaluated before the clones are permitted to leave the 
RL4 facility. The RAC charged the Working Group on Toxins with reviewing the 
data on the F. coll strains carrying the hybrid toxin; the group also was 
charged with offering a rec emendation to the Director, NIH, on vhether the 
strains may be removed fron RL4 contairment . The recommendation of the Working 
Group on Toxins could be acted upon by the NIH Director without RAC review of 
the data. A report of the Working Group on Toxins would, however, be sent to 
the RAC. 
Dr. Gottesman said Dr. Murphy made the following three separate requests con- 
cerning removal from the BI4 facility of the F. coll K-12 host-vector systems : 
(1) that fragments of the diphtheria toxin structural gene (expressed 
from either the tax or lambda phage Pp premotor) which include 
fragment A and fragment B up to, but not beyond , the Sph I site 
be classified at BL1; 
(2) that the diphtheria toxin structural gene modified by the introduction 
of a C-terminal cysteine residue up to, but not beyond, the Sph I site 
(pABC508) be classified at Bid; 
(3) that the diphtheria toxin-related a-MSH fusion genes _up to , but not 
beyond, the Sph I (pABM508, or pABM1508) site be classified at BL1 . 
Dr. Gottesman said Dr. f\irphy had supplied data in the form of two tables. 
Table 1 contains data on intra-peritoneal injection challenge of guinea pigs 
with viable and boiled Escherichia coll host-vector systems and _E^ coll pABM1508 
carrying the a-MSH diphtheria toxin fusion gene. Table 2 contains data on 
guinea pig survival after injection with partially purified conjugate protein 
at araded doses up to 1000 minimal lethal dose equivalents (based on 160 nano- 
grams diphtheria toxin per kg body weight as minimal lethal dose.) 
[ 313 ] 
