,I. Xol.
Bid.
(1977) 115, 215-235
Polyoma Virus Transcription R. C. COXDIT~. A. COWIE~, R. K.~MEx~
in Vitro ANTI F. BIRG~
1Departnt,ent of Mole&w I’irology and 2Jkpat$tt,ent of Cell Regulatiotb Imperial Cancer Research Fund, Liwolt1’s Inn Fields, Londotc WC2.4 3PX. Etuqlaad
nucleoprotcin complex has been partially A f,ritnscript,ionally a&i\-cb viral pln+ficd from the nuclei of mouse fibroblasts lytically infected wit’h polyoma \.irus. Th(s partially purified material contains polyoma DNA, some of which is complexed with active, proinitiated RNA polymerase II. The act’ive transfaster t’han supercoiled caription complex sediments at 22 to 23 S, slightly ire oitro is viral. Botfh I)olyoma DNA (21 S). At, least 95”,/0 of t,he RNA synthesized virus DNA strands are kanscribed in t)hrir wtirety in, &YI and the R’NA producrd is heterogeneous in size including molecldes larger than genome length. t,o t)hc virus DNA J, strand, and 14”,, Of the in c&o product,, 86Q,, hybridizes ir, vitro is thcrrforc similar by hybridizes to t,hr E strand. The RNA synthesized s,~vcral criteria to viral RNA synthesizecl ill ,ci~rj at thcx time \vhcm t,hc complex is cxtractc~tl.
1. Introduction I’~c DNAs of polyoma virus and simian virus 40 (SV40) provide ideal templates for studying control of eukaryotic t)ranscription. They are small (M, = 3.6 x IO”), and they are genetically and biochemically well-characterized. Furthermore, t’he characterization of polyoma virus and SV40 RXAs present in t’he cell nucleus and cytoplasm during different phases of lytic infection has demonstrated that the growth of bhese viruses is regulated, in part, at, the level of transcripbion (for a review, see .4cheson, 1976). During the early phase of polyoma or SV40 infection, that, is before the onset of viral DNA replication, RNA complementary to only one strand (the E strand) of tht, virus DNA has been found in the nucleus of infected cells. Bfter the onset of viral DNA replication, RNA complementary to the E DNA strand conbinues t)o be synthesized, but RNA is also synthesized from the opposite, or L DNA strand in much higher relative amounts. Thus, late during SV40 or polyoma infect,ion, 90 to 95()/b of the pulse-labelled viral RNA is complementary to the L DEA strand, and 5 to 1oq() is complementary t,o the E DNA strand (Laub $ Aloni, 1975; Have11 & Kamen, 1977). These observations suggest that t)he relative amounts of E and L DXA strand-complementary RNB are regulated atI the level of transcription, but, cwultl be alt’ernatively interpwt’ed as evidence for ra)pitl strand-specific processing of nawcnt t’ranscripts. I,:ltcb nuclear ant1 viral
viral
transcripts
RNA
is complrmentar~
considwably
larger
to all sc’cluenc~s
thaIi
gcn~nio
:: 1.>
lq$h
of bot’h DNA arc present.
strantls.
ln contrast,.
2 16
li.
C. CONI)IT,
z\.
(‘OWII~:.
I ho1nogenization in a Douncc,t,,vl~: homogenizclr in a final volunw of 4 ml buffer A (50 rnM-Tris.HCl (pH 7.5), 75 mM0.1 mM-EDTA, 1 mM-dithiot,hreitjol, O.%GO,, Sarkosyl NL-35, and 25”,, (NH&SO,. f~thylrol: 400 ~1 of t.his 1t1att~rii11 was laycrctl on a l2.8.ml, IOn;, to 30”, glycr~rol gradicnt~ in bnf%r A (rninlls c+hyl~~nc glycol) and ccntrifugcbd in a Beckman SW’40 rotjor i1t 40,WO rcvs/lnin for 15 h a1 4 ‘(I. Fractions of 40 drops \vcr(~ c~olk~cted on ice’ zmtl ;~ssnyc~l fbr transcript,ionnl nrt,ivit,J~. DN,i. ;mtl l)rott>in. ((I) I’ctrification
aurl
restric’tion evzyrne diqestio,c transcription wmp1r.1~
of I)ATz4 from
the
ftcaotion mixtures containing fraction III were brought to 0.2”, sodium dodecyl sl1lph:Lt’e, 50 ,ug yeast RNA/ml, antL 100 pg proteasr K/ml, incubated at 37°C for 30 min, (~xtr.LtGd wit.h phenol, dialyztld overnight l’ersus I 1 of 10 mM-Tris*HCl (pH 7.5), I mu-EDTA, 0.10; sodium dodrcyl sulphate, and pr&pitatctl with ethanol. These sampItas anti a sample of purified virus DNA, were resusprndcd in 10 mM-Tris.HCl (pH 7.5), 10 IIIM-M#CI~, I mM-dithiothrGtol, and digested t,o compk%ion at 37°C with 10 unit,s ‘I’, KNAasr/ml, 25 pg pancreatic> RNAas~/ml anti, \vhcrca indicatrd, with HpaIT, ant1 s~lbmittc~l t,o c,lec*trophoresis on ;L 1.4”;) agarosc~ horizont,al slab gel (Birg rt al., 1977). (‘ontrol c~xpc&llc~ntjs showed t,hat thct ribonllck~asc~ digcdion clots not rcsnlt in convcrsiorr of fhu>r I pol~von~x DNA into for111 11. (TV)RNA Ihl(~ othorwisc 125 mnf-(NH,),SO,,
polyttherase
assays
indicated, rcbactions (0.125 ml) caont,ain 90 m>I-Tris.HCl (pH 7.5), 5 mM-MgCl,, 0.08 mu-EDTA, 1.8 m&f-dithiothreitol, 0.2::) Sarkoqrl
-“IX
I
Bid.
(1977) 115, 215-235
Polyoma Virus Transcription R. C. COXDIT~. A. COWIE~, R. K.~MEx~
in Vitro ANTI F. BIRG~
1Departnt,ent of Mole&w I’irology and 2Jkpat$tt,ent of Cell Regulatiotb Imperial Cancer Research Fund, Liwolt1’s Inn Fields, Londotc WC2.4 3PX. Etuqlaad
nucleoprotcin complex has been partially A f,ritnscript,ionally a&i\-cb viral pln+ficd from the nuclei of mouse fibroblasts lytically infected wit’h polyoma \.irus. Th(s partially purified material contains polyoma DNA, some of which is complexed with active, proinitiated RNA polymerase II. The act’ive transfaster t’han supercoiled caription complex sediments at 22 to 23 S, slightly ire oitro is viral. Botfh I)olyoma DNA (21 S). At, least 95”,/0 of t,he RNA synthesized virus DNA strands are kanscribed in t)hrir wtirety in, &YI and the R’NA producrd is heterogeneous in size including molecldes larger than genome length. t,o t)hc virus DNA J, strand, and 14”,, Of the in c&o product,, 86Q,, hybridizes ir, vitro is thcrrforc similar by hybridizes to t,hr E strand. The RNA synthesized s,~vcral criteria to viral RNA synthesizecl ill ,ci~rj at thcx time \vhcm t,hc complex is cxtractc~tl.
1. Introduction I’~c DNAs of polyoma virus and simian virus 40 (SV40) provide ideal templates for studying control of eukaryotic t)ranscription. They are small (M, = 3.6 x IO”), and they are genetically and biochemically well-characterized. Furthermore, t’he characterization of polyoma virus and SV40 RXAs present in t’he cell nucleus and cytoplasm during different phases of lytic infection has demonstrated that the growth of bhese viruses is regulated, in part, at, the level of transcripbion (for a review, see .4cheson, 1976). During the early phase of polyoma or SV40 infection, that, is before the onset of viral DNA replication, RNA complementary to only one strand (the E strand) of tht, virus DNA has been found in the nucleus of infected cells. Bfter the onset of viral DNA replication, RNA complementary to the E DNA strand conbinues t)o be synthesized, but RNA is also synthesized from the opposite, or L DNA strand in much higher relative amounts. Thus, late during SV40 or polyoma infect,ion, 90 to 95()/b of the pulse-labelled viral RNA is complementary to the L DEA strand, and 5 to 1oq() is complementary t,o the E DNA strand (Laub $ Aloni, 1975; Have11 & Kamen, 1977). These observations suggest that t)he relative amounts of E and L DXA strand-complementary RNB are regulated atI the level of transcription, but, cwultl be alt’ernatively interpwt’ed as evidence for ra)pitl strand-specific processing of nawcnt t’ranscripts. I,:ltcb nuclear ant1 viral
viral
transcripts
RNA
is complrmentar~
considwably
larger
to all sc’cluenc~s
thaIi
gcn~nio
:: 1.>
lq$h
of bot’h DNA arc present.
strantls.
ln contrast,.
2 16
li.
C. CONI)IT,
z\.
(‘OWII~:.
I ho1nogenization in a Douncc,t,,vl~: homogenizclr in a final volunw of 4 ml buffer A (50 rnM-Tris.HCl (pH 7.5), 75 mM0.1 mM-EDTA, 1 mM-dithiot,hreitjol, O.%GO,, Sarkosyl NL-35, and 25”,, (NH&SO,. f~thylrol: 400 ~1 of t.his 1t1att~rii11 was laycrctl on a l2.8.ml, IOn;, to 30”, glycr~rol gradicnt~ in bnf%r A (rninlls c+hyl~~nc glycol) and ccntrifugcbd in a Beckman SW’40 rotjor i1t 40,WO rcvs/lnin for 15 h a1 4 ‘(I. Fractions of 40 drops \vcr(~ c~olk~cted on ice’ zmtl ;~ssnyc~l fbr transcript,ionnl nrt,ivit,J~. DN,i. ;mtl l)rott>in. ((I) I’ctrification
aurl
restric’tion evzyrne diqestio,c transcription wmp1r.1~
of I)ATz4 from
the
ftcaotion mixtures containing fraction III were brought to 0.2”, sodium dodecyl sl1lph:Lt’e, 50 ,ug yeast RNA/ml, antL 100 pg proteasr K/ml, incubated at 37°C for 30 min, (~xtr.LtGd wit.h phenol, dialyztld overnight l’ersus I 1 of 10 mM-Tris*HCl (pH 7.5), I mu-EDTA, 0.10; sodium dodrcyl sulphate, and pr&pitatctl with ethanol. These sampItas anti a sample of purified virus DNA, were resusprndcd in 10 mM-Tris.HCl (pH 7.5), 10 IIIM-M#CI~, I mM-dithiothrGtol, and digested t,o compk%ion at 37°C with 10 unit,s ‘I’, KNAasr/ml, 25 pg pancreatic> RNAas~/ml anti, \vhcrca indicatrd, with HpaIT, ant1 s~lbmittc~l t,o c,lec*trophoresis on ;L 1.4”;) agarosc~ horizont,al slab gel (Birg rt al., 1977). (‘ontrol c~xpc&llc~ntjs showed t,hat thct ribonllck~asc~ digcdion clots not rcsnlt in convcrsiorr of fhu>r I pol~von~x DNA into for111 11. (TV)RNA Ihl(~ othorwisc 125 mnf-(NH,),SO,,
polyttherase
assays
indicated, rcbactions (0.125 ml) caont,ain 90 m>I-Tris.HCl (pH 7.5), 5 mM-MgCl,, 0.08 mu-EDTA, 1.8 m&f-dithiothreitol, 0.2::) Sarkoqrl
-“IX
I
