PAM 
RNA tRNA 
VIRAL CAP BINDING PACKAGING 
ENHANCERS SITE SITE SIGNAL 
ORIGIN OF 
SECOND STRAND POLY (A) . 
DNA SYNTHESIS SITE 
Ctal Ball Pati 
SA 
i 
pol, any 
(Ball) 
pPAM 
pPAM2 
SD ATG 
SA 
r C=TDi v ^=c 
Bag, pol. iny ): 
TAG 
_b- 
n 
Clal 
Pst I 
SO ATG 
*1 
SV4 ° ra»iTl 
POLY(A) 
SITE 
SA TAG 
^= j «•« p«'- •*« 
Jb 
Clal 
Pat I 
BamKl (Bell) 
pPAMS 
SO ATG 
F=®V J=I 
SauSAI Pat I 
SA 
l 
B*9> pot, anv 
TAG 
(•ell) 
pPAM4 
CAP so ATG SA TAG 
i h J 
i. 
jk A 
k. 0*g. pol. ony ) — zi aV\W1 
1 — nr 
SauSAl Sau3AI Pat I 
(Bell) 
l _oaa Lj 
Fig. 10 . pPAM2 The 3' end of pPAM was cleaved with restriction endonuclease 
Rsal at position 7762. Just upstream of the termination coding of env_ (Van 
Beveren et al.. 1985) and a synthetic oligonucleotide was added to duplicate 
the 3' end of env which was removed. A 237 bp BamHI-BclI fragment from SV40 
viral DNA. containing the late polyadenyl atlon signal was ligated to a Bam HI 
site downstream of the terminator codon for env present In the synthetic 
oligonucleotide described above. pPAM2 thus contained a deletion of the 
packaging signal, and a polyadenyl atlon signal. Instead of the 3'LTR. 
pPAM3 pPAM2 was cleaved with Sau3AI at position -352 In the 5'LTR. (Van 
Beveren et al.. 1985) removing the viral sequences 5‘ to the enhancer region. 
This precluded the synthesis of a proper Integration signal, preventing viral 
Integration (Panganiban and Temin. 1983). pPAM3 was Inserted Into pBR322 In 
place of the tetracycline resistance gene (Tc r ) between the Bam HI and the 
PvuII sites (Fig 3). Only the Sau3AI site at the 5' end of pPAM3 remained. 
The site at the 3' end was destroyed by cloning. pPAM and pPAM2 were cloned into 
the pBR322 Clal and PvuII sites In the place of Tc r (Miller and Buttimore. 1986). 
Recombinant DNA Research, Volume 12 (181] 
