DNA TrenafecOon 
Twenty lour hours tiler 10* cats were seeded an 100 mm plates, me 
cxtjm were nnstecied wm 10 »g of ptesmd DNA by usmg me procedure 
0 * Graham end Vtn der £b (1973) is motkhed by Parser end Sink (1979) 
h uses where MUcton for Bins (acted cels wis aepaed. cets were sc*t 
1 JO nlo teteesve meds 40 nr after nnslecoon UrtsmectiDe DNA wes 
cotransfected n 10 told molar excess over 1 „g of serecsiOe DNA. 
Preparation end Anefysre of RNA 
Toill cefour RNA wes prepared by me memod of Chrgwri et A (1979). 
md ocryeoeryeied RNAs were selected wen oegor dTyceeueose is de- 
•erbed by Ouretel e* d (I960) Vnon RNA was prepared by extraction 
from vt-js pertoes banded n sucrose step gracMnts (Shetds ef d . 1978) 
RNA was sue- fracboneted by erectr o pnoresa througn agarose- formatde- 
hyde gen (Manats tt d . 198?). Bsnstened to mroceAAcse andhybndued 
with ■ftp faceted ONA probes as desotoed (Rgby ef d . 1977) Ouanwenon 
of RNA by dor btots was done as deserted by Thomas (1980) 
Construction of pAAOV-vJ~ 
Three puntod'DNA Segments were igaied together lo conefruct pMOV-f" 
(Fgurt 1) The ftrst was ootaned by Ogestng pMOV^* (Ounanor et d . 
198?) W«I Xho I lo ccmpietcn. folowed by para* ogeseon wrm Eco Rl 
The fragment ertendng lorn me Xho I Die al 2 0 U n fAjLV through me 
3' LTR. 3' mouse flanfcxig sequence, a I of p6R322 and endng at me Eco 
ft see was punted from an agarose gd after atecvopnoretc separanon 
(Vogenten and GAespn. 1979) The second fragment was octaned by 
dgeston of pMOV-iF* with Bat I to compieton folowed by punfeaton of 
me fragment ertenckng from me Baf ! Me n pBR32? mrougn S' mouse 
•antung sequence and S' ITR lo me Baf I Me touted at 0 7 U of SAA.V 
l-tnd ■ rr»es (Coiaborawe Resaarch) were men bkrafrgated to M 
fragment wen T4 ONA agase and me fragment was agested w#i excess 
►and • and Eco f* The LTR-contmwrg fragment was punhed from an 
agarose gel after atectropboretic separeten The mrd fragment present n 
me trial tgetqn reecton was obtamed from pSV2-geg/pof (R MAgan. 
unpubfrshed ptesmrj) where me gag/pol ragon of MjLV had been sub 
Ocned mo pSV? (MAgan and Berg. 1980) pSV? gag/pol was dgasied 
to c o mpiet x y with Xho I and frmd ■ and me (ragmere ertendng from me 
►and (Me (changed from me Rat I Me al 10 U of MA.V) to me Xho I set 
ef ? 0 U o< MjLV was punted from an agaroae gar lotowmg atectropnoretc 
sacaraacn These mree DNA fragments were man raed n eQLeroer 
amounts at a total ONA concentratcn of SO eg/mt n Igase buffer (SO mM 
Tns+O (pM 7 8) 10 mM MgCW 20 mM dlNodrertof 10 mM ATP. 50 »g/ 
mt borne senm dburrwi) and ncubated wen TS ONA tgase lor 18 hr al 
1S*C E cos fretOI wss transfected w«h me Igatad ONA. and mp cl rv 
resrstam franefectants were obfened The ptasnsd DNA obtamed from a 
ramber of Bans ferments was screened tar me Oesred souclure by dges- 
fron nth tppropnale restneton endorucMases end wecBochoress through 
agarose gets (Oevrs et *.. 1980) 
Ack no wfed g men ta 
R C M wrshes to acknowledge me sucoon of Or J. T Potts. Jr., dumg 
me eerfy pan of these cturkes 
The won was lucoorted by gates from me Natrona! Cancer frisotute 
and the Neronel Institutes of Hearth Program Protect Grant (CA 2671203) 
R C M e a McArthur Foundation Prue Feiow D B s an American 
Cancer Sooety Research Ptolesscr 
The costs o< pubfcaiqn of the not were defrayed n pan by the 
payment of page charges Thrs araoe must merefore be hereby maned 
’ advertisement' n accordance wrm 18 U S C Section 1734 safety to 
nduie mo fact. 
Received Jaraiary 4. 1983. revsed March 7. 1983 
References 
Bender W . Chen. Y.-H. Oienopacfriyay. S . Vogl. P K . Gardner M B . 
end Oevidson, N (1978) ►Sgh-motecuAar weght RNAs of AKR N2B. and 
w«d mouse veurses and avian retjCuSoenoomefrosis vrus a* hi^ smear 
Oner structures J Vrol 25. 888-896 
Beryamn. T L (1970) Host range mutants ol potyoms vrus Pnx. Nat. 
Acad So USA 67. 394-399 
Besmer. P . Ofshevsfry. U.. Ba/tmore. 0.. Dotoerg. 0.. and Fan. H. (1979). 
VrusAfre 30S RNA n mouse cats J Vrol 29. 1168-1178 
Canaan. E.. Hefrri. K. V 0 . and CXtesberg. P (1973) Evtoenoe tor 30- 
40S RNA as precursor of me 60- 70S RNA of Rous sarcoma vrus Proc. 
Nat Acad So USA 70. 401 -AOS 
Cheung. K. S.. Smtfh. R. E.. Slone. M P.. and JoW*. W. K. (1972) 
Comparison of rrmeture (rapd harvest) and mature Rous sarcoma vrus 
parades Vrotogy 50. 851-864 
Orgnen. J M . PrryOyfa. A. E.. MacOoneld. R. J.. end Roller. W. J. (1979). 
bofaton of botaguAy active rtxnx3ac ecto from sources enriched n 
rboryxaease Bochemrstry 18. S294. 
Ourakoi. L. Stuhrnam h . Harbers. K.. and Jaerasch. R (1962) Ctonng 
of fwo genetca»y transmmed Moloney teuAenae provesf genome s , can- 
titan between batogul ecnvtfy of ctoned DNA and veal genome acbvs&on 
n me anmaf J Vrol 42. 1088-1098 
Coffn. J (1962) Endogenous Vrusas h Rna Turner Vruses. Wecss. R.. 
Tech. N . Vemxts. H. end Coffn. J . eds (Cdd Spnng Hartxx New Yotfc 
Cc*d Spmg Hertxx Laboratory), pp 1147-1155. 
Oevrs R W Thomas. M . Cameron. X. S» John. T P.. Scherer. S.. and 
Psdgefl R (1980) Ragd DNA odatens tar enrymahe and hybndvaton 
anatysrs Mem Enzymd 65. 404-411. 
Edwards. S A and Fan. H. (1979) gag-ratated pofyprofems of Moloney 
name teuAenae vrus evejerce for ndapendem synthesis of gyoosyfeted 
tnd tmgyoosyfeted toms. J Vrol X). 551-563. 
Edwards. S A. and Fan. H (1980) Sequence refationshc of gyoosytated 
and ungtyooeyfated gag pofyprofems of Motoney name teuAerraa vrus J. 
Vrol 35. 41-51. 
Gerwm B I . and level. J. G (1977) fraeracoons ol Mm* teuAerrae veus 
core components characienzatxYi of reverse trartsenptase packaged in 
me abserce of 70S genome RNA J Vrol 24. 478-488 
Gasmen. Y. (1981) SveOtranstormed srraan cats support me repicatnn 
Of eerty SV40 mutants CM 23. 175-182. 
Goft S . Trsktman. P . and Baftmore. 0 (1981) tootatnn and properties of 
Moloney name teukanaa vrus mutants use Of t rape assay tor release of 
vmon reverse frmscnptase J Vrol 38. 239-248 
Graham. F L. Sm4ey. J.. Russel W C.. end Nam. R (1977) Owsder- 
sties of a haman cei me frinsformed by DNA from raman edenovrus 
type 5 J Gan Vrol 36. 59-72. 
Graham. R . and Van der Eb. A. (1973). A new lechraque tar me assay of 
rrtectrvry of famen edenovrus 5 DNA Vrol 52. 456-467 
Jaerasch. R (1976) Germ trie ntegabon sod Mendeaen Bartsmrsson of 
the exogenous Motoney ieukams veus Proc. Nsl Acad So USA 73, 
1260-1264 
Jeansch. R (1977) Germ tne negation of Moloney leuAema veua eft act 
of homorygosAy al me M-MA.V locus Cel 12. 691-696 
Jeenrsch. R . Jehner. D . Nobs. P.. Srram. L. Lohtar. J.. Hertoera. K.. end 
&olkopp D (1981) Oromoeomal postaon end ectnrafton ol retnjvral 
genomes rsened no me germ tne of naoe Ce* 24. 519-529. 
Level. J . and Seaman J. (1979) Setacbve packegng of host tfiNA s by 
name leuAertae veus parades does not require genome FtNA. J. Vrol. 29. 
328-335 
Lraef. M (1981) Transfer of detective man tumor veus genomes by a 
Rous sarcoma veus RNA packegng mutant J Vrol 38. 380-382 
Lxaal. M Mederos. E . and Hayward W S (1978) An avian onctheus 
mutant (SE 2101b) defioenl n genome RNA bctogcal end brxharraci) 
dieraderuation Cel 15. 1371-1381. 
Lusfcy. M . and Botdwi, M (1981) hhcrtxxi of SV40 repfeatxn n srraan 
exits by p6R322 sequences Nature 293. 79-81 
Magnusson. G . end Nason M -G (1977) Muhpte free vnl DNA oopws n 
poryome vrus transformed mouse cels surviveig productive ntectxm. J. 
Vrol 22. 646-653 
Manats. T . Fntsch. P . and SambrocA J (1982) Moiecutar Oonrig. A 
Recombinant ONA Research, Volume 12 
( 357 ) 
