APRIL 27-28-MINUTES OF MEETING 
21 
VIII. PROTOCOLS FOR REQUIRED CONTAINMENT 
Below are described the actions taken by the RAC on proposed protocols 
for required containment levels and for compliance with the 1976 Guide- 
lines. 
A. Cloning of Bovine Satellite DNA 
Dr. Susan Gerbi of Brown University requested NIH review of a 
proposal to clone 99% pure bovine satellite CNA under P2-EK2 
conditions. The Guidelines permit a one-step decrease in 
physical or biological containment in relation to the conditions 
required for the corresponding shotgun experiment when the DNA 
to be inserted has been enriched to 99% purity by physical and 
chemical techniques, and is free of harmful genes. Shotgun 
conditions for cloning bovine CNA are P3-EK2. Therefore, 
"purified" bovine DNA can be cloned under P2-EK2 conditions 
if the DNA is 99% pure and free of harmful genes. 
The RAC agreed that the evidence for 99% purity is acceptable. 
There was seme discussion about whether the CNA preparation 
could be said to be free of harmful genes in view of the fact 
that the functions of satellite CNA are unknown. However, it 
was pointed out that, in all cases investigated, satellite DNA 
is not translated. Therefore, it is reasonable to take the 
position that such DNA is free of harmful genes. The RAC then 
voted unanimously to approve the requested containment levels of 
P2-EK2. 
B. Cloning in Pseudomonas putida 
The RAC reviewed requests from Dr. James Shapiro of the University 
of Chicago, and Dr. J. G. Vacca, on behalf of Dr. Chakrabarty, of 
the General Electric Company to use Pseudomonas putida as a host 
for recombinant DNA experiments. 
In one set of experiments, Dr. Shapiro wishes to clone P. putida 
DNA using E. coll as a host. In a second set of experiments. 
Dr. Shapiro requests permission to use P. putida as a host for 
cloning. There are two subsets to the latter experiments. In one. 
Dr. Shapiro wishes to conduct self-cloning experiments. The 
P. putida DNA carried by plagnids R1162 or RP4 1 and previously 
cloned in E. coli would be returned to P. putida for propagation. 
In the second subset of experiments involving P. putida as a host. 
Dr. Shapiro intends to develop cloning vectors with a wide host 
range that could be propagated in either E. coli or P. putida . 
The hybrid vectors would be from Col El and R1162 or RP4 1. 
The vectors could be grown in E. coli under Pi conditions since 
both parental vectors grow in E. coli. 
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