UNA 
J L 
i SL 
t*t r 
CcoRI 
N'X'7'/ 
9*1 
pol 
-6-3kbf>— 4 
•n^ 
Mind III 
— fc= 
pMLV*46 
Clal Xhol 
pMLV-1 
•nv 
Hindli 
=± 
EeofB 
jji ‘ f 
— J.5kbp 
tit 5 
nu ? 1 
D=± 
9*9 
pol 
1 1 *** 
<□ 
XC ptaquM^igAOP cotta 
Ctal Hindli Xhol /Sol I 
[Clal] Xhol Hind* fXhol/SoIl] 
1 1 "0 =1- 
Fig. 5. Construction of pMLV-K . A nonpermuted clone of pMLV-1 was first 
constructed. The Clal-HIndlll fragment and the Hindli I-Xhol fragment of pMLV-1 
were mixed and ligated with the bacterial alkaline phosphatase (BAP)-treated 
3.7 Kb Sall-Clal pBR322 fragment containing the amp r gene, forming construct I. 
Next, the 5 ' Cl al-XhoI fragment of pMLV-1 and the 3' XhoI-EcoRI fragment of 
pMLV-48 were cloned Into the BAP treated Clal-EcoRI fragment of pBR322 
containing the amp r gene, forming construct J. Construct K was made by BAP 
treating and ligating the larger of the two fragments derived from the 
XhoI-HI nd I I I cleavage of construct I with the 3.3 Kb Internal fragment derived 
from the Hi nd I I I-Xhol cleavage of construct J. pMLV-K was active by XC assay, 
yielding 2700 XC plaques/ g/10 6 cells compared to 0 for pMLV-1 (Miller and 
Verma. 1984). 
[176] 
Recombinant DNA Research, Volume 12 
