CM 
156 
T*OM 2 Propemea of cM Irw* uady transfected w«h pMOV-4' 
Hatparlne 
Reverse 
Transcnpuse 
Activity n 
Supernatant* 
Wecovey* 
gprt (ciu/mf) 24 
hr liter 
Transiecbon w«h 
pMSVgpf* 
MOV-1 
100.0 
♦ 
5x 10* 
4-1 
£0 
- 
60 
4-2 
35 0 
- 
10* 
4-3 
1314 
♦ 
10* 
K*V3T3 
09 
- 
<10 
•h irbArary mss w«i Vw wed-type kAjLV producer In*. MOV-1, uftgua 
to 100.0. 
* hfectrvey Ml MdgaO by nlectng NP-I/3T3 ce*s wen ifflolln ClAn 
supernal ant and munmg reverie banacrptase ecbvey lor at least two 
*hm lotoMig bt nlecbon. 
* pcT ootony tarmng inns par mfleter at culture luoemetart of ca4s 
transfected wan pMSVgpf. 
•clrvity and p30 protem by measuring thev concentration 
r portions ot the punfied particles just poor to RNA 
extraction. Both cellular and wal RNAs from MOV-1 and 
i-2 were fractionated by electrophoresis through a 1% 
agarose-formaldehyde gel. transferred to a nitrocellulose 
filter, and probed with a rack-translated plasmid contaxang 
the entxe M-MuLV genome (Rgure 5A). Full-length ge- 
nome RNA could be detected r MOV-1 celts (8.2 kb. lane 
1 ) and in ^-2 cells (7.9 kb. lane 4). as could the subgenonac 
2.9 kb env mRNA (lanes 1 and 4). The 8.2 kb genome 
RNA could be readfly detected n RNA preparations from 
MOV-1 wens (lanes 2 and 3). but no hybnduzabie RNA 
was detected r RNA preparations from an equivalent 
number of f-2 particles (lanes 5 and 6). In figixe 5A. lane 
6 contains RNA from 10-fold more f-2 particles than were 
used for the MOV-1 particles n lane 3. Also, exposure of 
the autoradiogram shown in Figure SA for a 10-fold longer 
tone stil revealed no hybndizabie bands n lanes 5 and 6 
(data not shown). Thus particles released from f-2 cells 
contain at most 1% of the wal RNA found in MOV-1 
particles. 
Further quantitative data were obtained by spotting 2- 
fold dilutions of MOV-1 and ^-2 wal RNA preparations 
onto a rvtrocelulose filter and probing it with a nek- 
translated plasmid containing the entire MuLV genome 
(Figure 58). The amount of RNA spotted was again nor- 
malized to the number of particles present prior to RNA 
extraction by measuring reverse transcriptase activity and 
p30 concentration From this analysis, it appeared that 
wal RNA was packaged nto wus particles with an effi- 
ciency much less than 1 % that of wild-type wal RNA. 
f-2 Can Efficiently Package Other Retroviral 
Genomes into Infectious Particles 
We next examined whether the pMOV-^" -containng cell 
Ines could package a highly defective retrovirus genome 
nto wus particles and bud the particles nto the culture 
supernatant without also budding helper wus. To this end. 
pMSVgpt was transfected into ^-1. f-2. ^-3. MOV-1 and 
Fgurs s Hybndizsbon Arefys* of CafUar and Vraf tvU-V RNAs From 
MOV-1 and 4-2 CM» 
(A) Potyadenytstad ce* Jar RNA and wal RNA from MOV-1 and 4-2 •*« 
bacbonated by etactrophrratri through • 1% agaroee- formaldehyde gal. 
transferred to a nurocefcAos* War and probed with a ncx-bsnsiaied pfasrrvd 
oontsnng the enare IAjLV genome (Lane 1) 3 *g potyadanyteted MOV-1 
oeAAar RNA. (lane 2) wal RNA bom purified MOV 1 parbelea. (lane 3) 10- 
tald Via MOV-1 vraf RNA preeant n lane 2: (lane 4) 3 *Q pofyaderrylated 
4- 2 cafkiar RNA. (lana 5) vraf RNA bom punted 4-2 particles: (lane 6) 10- 
Icfd the 4-2 vraf RNA present n lane 5. Lanes 2 and 5 conta*i RNA 
exvacted bom agirvalerX numbers of MOV-1 and 4-2 particles as deter- 
mned by bom reverse trsnacnplsse acbvibes and p30 cu n c enga tc n a 
(6) Sequential 2 kXd dlubona of vraf RNA ofxaned bom punfied MOV-1 
vna or 4-2 parades were molted onto a mtrocefcioee War and probad 
wan a nc* translated plasma centering the enbre IAjLV genome. As in 
(A) the amount of vraf RNA ueed was normalized to reverie banscnptaae 
adMtee and p30 concern a bona present r the parbefes prior to RNA 
exvactoru 
NIH/3T3 cells, and 24 hr later the culture supernatants 
were used to infect fresh NIH/3T3 cells. Two days after 
the infection, selection pressure for XGPRT was applied, 
and 8-10 days later gpT colonies were counted. Cell line 
^-1 produced only 60 gpT cfu/ml ot culture supernatant 
Recombinant DNA Research, Volume 12 
[355] 
