pFR400 
amp 
| AT( 
ATQ TAA (A) n 8 
r 
t 
f XBXPM 
| 8V40 f* DFHR | HBV(A) n I *— 
pML * — ~ 1 1 pML 
* ° 8fct> I 
,:LDL1 tv M 6 'ltr -ll 
E j-^ P 
P-DL2 lv ^ S’LTR 1 j 
E !»► PXB 
pLSOL »tTR | ^ 
OHFR 
<*>n 
l U»«n 
-( a'LTR> 
Bgt 1/B 
DFHR 
-{ 3'LTR H 
Bal I 
El 
SV40 -OHFR 
CA)„ 
■ \ » I-TB > 
Ball 
pLHDSL 
f r LJ /8 * HN N 
tw ( 6'LTR I - jpMtl HBV(A)n [ f DFHR. j SV40~j 
r 
«A) n 
<A)« 
i L.E4I 
DHFR-virus constructs. Restriction enzyme sites arc indicated (E. EcoRI; H. //mdlll: S. So/1; F. Frrl; X, Xhol, B. fomHl; N. 
A/col). DHFR expression plasmid pFR400 (29) is shown at the top. DNA sequences between the £VoRI and Pul sites at Ihe left ends of the 
viral constructs, including the S' LTRs. were derived from a cloned MoMSV provirus' pMSV r lL (31. 32). The wavy lines indicate mouse 
genomic sequences adjacent to the provirus integration site. Viral sequences at the right ends of the constructs, including the 3' LTRs, ware 
derived from a cloned unintegTated MoMLV DNA. pMLV-1 (22 . 28). pMLV-1 Is a circularly permuted clone, to sequences to the right of 
the 3' LTRs art identical lo sequences to the right of the MoMLV S' LTR. These EroRI to Bull fragments were inserted between the EcoRJ 
and Pvoll sites in pBR322 in place of the Tet' gene (Bal 1 and Pvull both leave blunt-end DNA and thus can be ligated directly). For construct 
pLDLl the mutant DHFR cDNA was excised from expression plasmid pFR400 by using //mdlll and A/roI (position 736 in the DHFR cDNA) 
(29). This fragment was inserted between the Pul site of Moloney murine sarcoma virus (MoMSV) and a A/roI site (position 7229) (28) is 
MoMLV by using a piece of DNA from MoMSV (positions 4.936 (Arrl) lo 3.023 (//mdlll)) (31) to join the Pul and //mdlll sites. This piece 
of DNA from MoMSV contains no ATC initiation codohs that might interfere with DHFR translation. A DNA fragment consisting of 
restriction enzyme linkers in the order Pul-Xhol-BamHl-Xhol-Pjll (provided by Paul Kaplan) was inserted at the Pul site;. For construct 
pLDL2 a Pul linker was added at the />*<HI Site (position 43) in mutant DHFR cDNA done pFR*00-12 (29), and this Pjrl-lo-Bf/U (position 
731) fragment containing the entire mutant DHFR coding region minus the polyadenylation site was inserted between the retroviral LTRs at 
the Pul site of MoMSV (position 996) (31) and the BomHl site of MoMLV (position 6.339) (28). For construct pLSDL the early promoter 
region of SV40 was excised with Pvull and //mdlll, a BomHl linker was added to the Pvull site, and the resultant Ba/nHl-//mdlll fragment 
Was inserted into pLDLl at the unique RorriHI and //mdlll sites, replacing some MoMSV sequences. For construct pLHDSL (he entire 
expression unit from mutant DHFR expression plasmid pFR400 (29) was excised with Sail and A/rol (nucleotide position 333 in SV40), and 
P/col partial digestion was bsed to obtain a fragment including the complete SV40 early promoter and inserted between the kho\ and Ncol 
sites of pLDLl. kb. Kilobasc. 
Fiq. 8. (Miller and Verma, 1985) 
Recombinant DNA Research, Volume 12 
[1791 
