Attachment II - Page 1 
UNIFORMED SERVICES UNIVERSITY 
OF THE 
HEALTH SCIENCES 
4301 JONES BRIDGE ROAD 
BETHESDA, MARYLAND 20814 
TEACHING HOSPITALS 
4 Anrll 1QR4 Walter army medical center 
r NAVAL HOSPITAL, BETHESDA 
MALCOLM GROW AIR FORCE MEDICAL CENTER 
WILFORO MALL AIR FORCE MEOICAL CENTER 
Dr. Elizabeth Milewski 
Office of Recombinant DNA Activities 
Bldg. 31, Room 3B10 
National Institutes of Health 
Bethesda, MD 20205 
Dear Dr. Milewski: 
The NIH Guidelines for Recombinant DNA Research (Appendix F-IV-H) permit 
cloning of the structural gene of the Shiga-like toxin from clinically 
isolated strains of _E. coll into _E. coll K-12 host strains under P4 + EK1 
containment conditions. On 8 Decmber 1983, Dr. Randall Holmes and I requested 
reduction of containment levels for cloning of the structural gene of the E. 
coll Shiga-like toxin from P4 EK1 to P2 EK1. The RAC considered our request 
on 6 February 1984 and recommended that the containment conditions for this 
experiment be reduced to P2 with the stipulation that specific EK2 plasmid 
vectors be used. The vote for their recommendation was 9 in favor, 4 against, 
and 5 abstentions. It is our understanding that because of the split vote 
Dr. Krause will not concur with the RAC recomendation. 
We intend to submit a request in the near future to use the NIH P4 facility to 
clone the structural gene for Shiga-like toxin from _E. coli . In Informal 
discussions, Dr. Malcolm Martin has advised us that technical limitations may 
make it very difficult or impossible to accomplish this goal under P4 
conditions using the reagents and methods that are currently available to 
screen for production of Shiga-like toxin. At best, the experiments will be 
very time consuming. If the cloning can be accomplished under P4 conditions, 
permission from RAC or ORDA will be required before any clones for Shiga-like 
toxin genes or gene fragments can be removed from the P4 facility or used for 
future experiments under lower containment levels. For the reasons listed in 
our December 1983 letter (attached) and because of- available animal toxicity 
data (see attached papers and tables) we continue to believe that P4 EK1 is 
excessively restrictive. To facilitate our work on the cloning and 
characterization of the structural gene for Shiga-like toxin in _E. coll , 
therefore, we are requesting the RAC to consider and vote separately on each 
of the Issues In the numbered list below. 
Finally, several investigators from other countries have informally expressed 
to us their interest in cloning the genes for Shiga-like toxin or Shiga 
toxin. We believe that a significant further delay in reducing the 
containment levels for cloning the gene for Shiga-like toxin in the United 
[ 421 ] 
