CM 
1S4 
■w 1 
Fgui 1. Constructor! ot p*OV-^~ 
P6R322 md SV40 tequancm *r« r«pm«ntacj by nr lr*s UiV m- 
Ousnces art rsjrtuniKt by bo tries, and mouse sequences ftanfcng me 
proweus are 'eoresenied by wavy tries The long tarmnel repeats o' luk/LV 
are ndcaled by open tmes VS marvenrig ugutocc ot SV40 imal 
Umor aregan. 
Fg/i 2. Scnemabc Aepresersaton o< me Prowua Presere r pMOv-y* 
The tong lemriat repeats are represented as do sed Oo«es and reamat 
tajLV sequences are represented as a mn tne The 350 bp presere 
between me Bel I and Pet I sues were dented and a i-tno ■ sre wes 
raoducsd at me tee o< detetor The new tend I sue ■ u rvoeonoes 
hm me apparere donor space sue tor me anv mPHA and 55 rudaoedes 
trom me ATG lor Pr65^ (Sr»»sca at *. 1961). 
crease in activity as a result of efficient spread of vrus 
from the nrtiaity transfected cells to an cells on the plate. 
In contrast, for 4 to 6 days foflowng transfecton of pMOV- 
no detectable reverse transcriptase activity was ob- 
served in the medium, suggesting that virus spread was 
nitiaJty defective Uninfected cells had less than 5% of the 
activity of MOV-1. 
Within 9 days of transfecton of pMOV-^ - . large amounts 
of reverse transcriptase actn/ity developed n the culture 
supernatant (Figure 3). suggesting that vrus spread even- 
tually occurred The reverse transcriptase activity present 
by day 9 could have been due either to the slow, atten- 
uated spread of mutant vrus or to the generation of a 
nondefective virus by recombination with a cellular se- 
quence capable of providing the missing function. To 
differentiate between these possibilities, various dilutions 
of culture supernatants from cells transfected 9 days 
previously with pMOV-^* or pMOV-^" were used to infect 
Recombinant DNA Research, Volume 12 
Fg/I 3 Revarwt TranscnpUi* Ac**** c* CxttJt Suwmatants as • 
Fircton et TVna Anar Tranalacton wan Enfiar pMOV-rf* or pMOV-rF" 
Scad ao is rapraaant baognxnd acov*y o< iraranslaciad F*V3T3 o«h 
ThcAtna pMOV-^" Thri ma pMCV -»* Ai ravaraa tranacnpUM acnvitoa 
ara rapraaaraaq as a parcaraaoa at ma acbvay produced by a standard 
IAjLV producer oM na MOV 1. 
NIH/3T3 cells High levels (3-4 times MOV-1 activity) of 
reverse transcnptase actrvity were observed 2 days after 
rlecton with only 10 iti of both supernatants (data not 
shown), demonstrating the presence of nondefective virus 
in the ctiture supernatants of celts transfected with pMOV- 
9 days previously. 
pMOV-V' - Provide* All Viral Function* Required In 
7 rant 
To determine whether the deletion made in pMOV-^ - 
a fleeted functions required only in ds by the virus, helper 
actrvity was measured by using a recombinant retrovirus 
genome derived from Moloney sarcoma virus (MSV) ca- 
pable of expressing the E. edi gene encoding xanthne- 
guanme phosphonbosyttransferase (XGPRT) (R. Cone. R. 
Mann. D. Baltimore, and R. C. Mulligan, unpublished data) 
(Figures 4A, 4B). This gene, designated gpt. is a dominant- 
acting selectable marker in mammalian cells (Mulligan and 
Berg. 1980). The plasmid (pMSVgpt) encodes no intact 
retroviral proteins yet retains the region deleted in pMOV- 
pMSVgpt also has the polyoma vims early region to 
increase its copy number in NIH/3T3 cells early after 
transfection. To assess the helper activity of pMOV-^". 
we cotransfected pMSVgpt and a 10-fold excess of either 
1353 ] 
