432 MILLER. LAW. AND VERMA 
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PFR400 
pLDLI 
pLDL2 
pLSDL 
pLHDSL 
r?° 
Mol. Cell. Biol. 
s-y3/ ( 
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— | SV40 I- DFHR' I HBV(A)nt — 
, 0.5Kb , 
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SV40 DHFR 
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FIG. 1. DHFR-virus constructs. Restriction enzyme sites are indicated (E, EroRl; H. Hindlll: S, Sail ; P. Prrl; X, Xhol: B. BamHl: N, 
Nco I). DHFR expression plasmid pFR400 (29) is shown at the top. DNA sequences between the EroRl and Pul sites at the left ends of the 
viral constructs, including the 5' LTRs. were derived from a cloned MoMSV pro virus, 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 cohstnicts, including the 3' LTRs. were 
derived from a cloned unintegTated MoMLV DNA. pMLV-1 (22. 2 8). pMLV-1 Is a circularly permuted clone, so sequences to the right of 
the 3' LTRs are identical to sequences to the right of the MoMLV S' LTR. These EroRl to Bui 1 fragments were inserted between the EroRl 
and Pvull sites in pBR322 in place of the Tefgene ( Ball and Pvull both leave blunt-end DNA and thus can be ligated directly). For construct 
pLDLI the mutani DHFR cDNA was excised from expression plasntid pFR400by using Hindlll and Ncol (position 736 in the DHFR cDNA) 
(29). This fragment was inserted between the Pul site of Moloney murine sarcoma virus (MoMSV) and a Ncol site (position 7,229) (28) in 
MoMLV by using a piece of DNA from MoMSV (positions 4.936 [Ps/I] to $.023 (///ndlll]) (31) to join the Pul and Hindlll sites. This piece 
'of DNA from MoMSV contains no ATG initiation codons that might interfere with DHFR translation. A DNA fragment consisting of 
restriction enzyme linkers in the order Pstl-Xhol-BamHl-Xhol~Psll (provided by Paul Kaplan) was inserted at the Pul site. For construct 
pLDL2 a Pst I linker was added at the E#iu4HI kite (position 43) in mutant DHFR cDNA clone pFR400-12 (29), and this Psil-to-Bg/ll (position 
738) fragment containing the entire mutant DHFR coding region minus the pofyadenylation Site was inserted between the retroviral LTRs at 
the Pst 1 site of MoMSV (position 986) (31) and the BamHl site of MoMLV (position 6.339) (28). For construct pLSDL the early promoter 
region of SV40 was excised with Pvull and Hindlll , a BamHl linker was added to the Pvull site, and the resultant Ba/nHI-/f/ndIII fragment 
was inserted into pLDLI at the unique BamHl and Hindlll sites, replacing some MoMSV sequences. For construct pLHDSL the entire 
expression unit from mutant DHFR expression plasmid pFR400 (29) was excised with So/1 and Ncol (nucleotide position 333 in SV40), and 
Ncol partial digestion was Used to obtain a fragment including the complete SV40 early promoter and inserted between the Xhol and Ncol 
sites of pLDLI. kb. Kilobasc. 
[198] 
Recombinant DNA Research, Volume 12 
I 1 
