3 
The second experimental advantage of the P3 + EK1 option would be the 
application of "cosmid" cloning systems to vertebrate shotgun experiments. 
Cosmids are bacterial plasmids bearing bacteriophage X cohesive ends. The 
presence of the X cohesive ends makes it possible to clone exceedingly 
large fragments of DNA (U 5 kilobases) using bacteriophage X in vitr o 
packaging systems to introduce the hybrid DNA into bacterial cells. X1776 
is the only EK2 host presently available and the strain is X-resistant. 
It would therefore be necessary to develop, test and certify a new EK2 
host before cosmids could be used to clone vertebrate DNA. 
In conclusion, I urge you to reconsider your decision against the 
P3 ♦ EK1 option for vertebrate shotgun experiments. There is no evidence 
that this containment level is unsafe and the availability of this option 
provides flexibility in designing experimental strategies. 
Sincerely yours 
Tom Maniatis 
Assoc. Professor 
of Biology 
TM: JCh 
[A-85] 
