Vol. 84, No. 3, 1978 October

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATlONS

Pages

16, 1978

J.Nx~jardino* *Divisim

of Cell

Studies,

and A.Janss**

FkyalFree

Hospital

Schoolofmdkine

8 HmlzerSt.m&ImdcmKlN~

*WeLaboratories

forCancerFkseaxch,Lhivex5ityofwiscaMn Madim,

Received

WiscQlsin

53706

May 17,1978

TranscriptionOfpolyaMvirusDNAinlyticallYinfectedcells appears totakeplace

intwostages,before

zmdafterthecnsetof

virdl~synthesis,rmdgivesrisetostablemes~~RNRspecies which axe accordingly

ckscriked

as early

md late

(1).

Stable early

~is~~isedrigfitthroughoutinfedicnbutstablelate~ appearsonlytobesynthesisedaftercpuA~~ishas~place. S~~earlyandlatetrans~~~synthesisedfrandifferent strands ofthe its

length

(2).

labilespecies franthe

ccdingtenplate

andeachcomespauk

ApartfranthestablecytcplasnicmBNAspecies axealsosynt.ksisedinthenucleus

anplem?ntaqtNAstrandbothearly

invariableammts mdlate

asindicatedbythepresenceofasi~fi~t~tofnucleardouble stranded~isolatedaftershortpulsesofradioactiTRprecursor (22) ;

stable

theselabflespe~~extendinlengthbeycndthesizesofthe transcripts

toaboutrnof

andamtha@ktobeprocessedbyspecific

0006-291X/78/0843-0618$01.00/0 Copyright 0 1978 by Academic Press, fnc. All rights of reproduction in any form reserved.

618

during

infect&m

618-626

Vol. 84, No. 3, 1978

endcnucleases

polymerase

togeneratethematuxecyt@asmicMWs.

follcm

in that the cR?Ais codingstrand

BIOCHEMICALAND BIOPHYSICALRESEARCH COMMUNlCATlONS

apatternpartlysimilartothatdxervedinvivo to alargeextentdDuble

(4) andinitiates

--

strakkd,

franalimitednmberof

lmgerthan

the

sites.

Stqercciledpolyana~ (forml)waspreparedfranncn-defective virus, as describedbyFri& (5),andwas foundtobelocrt, sensitive tothe restrictimenzymE . FWLpolymrasewasextractedfrcnl EscherichiaColiMKE600 m Y&n ' gtothemethodof Burgess (6) and f~dtobe~rethan95%~withrmapprmcimatelyequimolar~t of sigma subunit. EWtericphageT4Gene32pro&inwaspui.fiedby theR&hodofAlberts and m (7). Theincubati~mixturefor syntksising RNA cxntained 20 pg/mP. of polyam DNA I, 75 pg/inR of E.Coli RNA polymrase (DNWNA polymrase mlar ratio of 1:23), 5ClM Tris-H31 pH:7.9, la@4 Mgc12,340'1mYM Kcl, 7nMmrcaptce&mol, lnMofATP,CFP. Gm andvrp (or H GFP to a final specific activity of 0.15 Ci/&le). Ikubatimwas carriedout at37oC. 3HcRUwas e&ra&edaccxx&ngtothemthodofMartinandAxelrod anddouble stranded~wasperformdusing (8). Separatimofsingle CFll cellulose as describedby Franklin (9). HcRUwasextrackd fran the incubatim mixture and dissolved in 0.05&l Tris I-BX pH: 6.98 mtaixingO.2MN~landO.oOlMm?A. Single stran&dF@JAhas been shmn to elute in 15% Ethanol md double stranded M in buffer alone Pancreatic FMase digestim (2O@~ml)wasperfarmed forlhour 37OC in 2 x St32 (SSC is 0.15M NaCl, 0.015Z4 triscdiun citrate, pH: DNhse digestion Annealing precipitatedwith Whatman@/C)

(4Oug/hil)was

carriedoutat37°C

(9).

at 7.0).

for 15 minutes.

of &N?kwasperfon1&at65~C in 2 xSSC. 3HcRNAwas cold5% 'RX, filteredthrmghglass fibrediscs andanmtedin a toluenebased scintillator.

Incubatim andpreparatim of samples forvisualisati~ of transcripticmcarplexeswem. carriedautas describsdbyDeliu.s and West@al (10). Afterp~incubati~intheabsenaeofsubstratesand rifaqicinfor 3minutes at37~nuclecsidet+hcephabzs zndrifslpicin (5$Q are addzdandincubati cm is carriedoutat37aC for l5minutes. [email protected] franthe incubatimmixtureare diluted1Otims inO.OlM potassim phosphate (PH: 7.0), O.CO2M WTA axMining 55ug/ml of gene 32 protein andincubatedat37oC for3minutes. Glutaraldehyde is then addedtol~andincubati~ carriedout for15mW.m~ at379: for 3minutes. Thesimple is spnsd f~relec+trmmicmxapyexmi.natim~ tie fleins&midtte&nique as ckscri&dbyDavis et al (11). -tsvaxemckafter Magnificaticmswerecalibratedandl.mgthmeasuremn five-fold phctcqraphic enlargmen tusingamap-mstertype ofdevice. Allreagentsmx-e "ANAIAEV'grak franBritish GlutardldehydewasthespecidlgradeforElectrmMicrosmpy. werecbtainedfranSIGMA. 619

DrugHouses

WH).

Ebymes

BIOCHEMICAL

Vol. 84, No. 3, 1978

AND BIOPHYSKAL

RESEARCH COMMUNICATIONS

~synthesisedafter60minutesincubaticnwas~a~aold analysedby

chrunatography

CnCFllcellulosewhich

separates

single

strrmdedfranpartlyorfullydoubles~~~RNA,asdescribed~~r I--who&

Ccnfinmtimthatthereisasignificmtfractimofthe

(9).

~whichisatleastpartly~lestranded,isprwidedby~e digestimunderamditian~whichexcl~& (Table 1).

thepresencx

Polyana cRNAwas then further

OfDML-RNAhybrids

analysedby

electxm

microscapybyvisualisatianoftranscriptiancarplexes.

Transcriptian

cY3tplexe.s clearlyshowinglcng~dtains~visualisedbythis techni~areshawninFigure1. IMAdmins

mstDNAxr&xlllesobse~carried

althowjhthe

tiich

is likely

shm

inFigure1,

chains varied

significantly

toreflectasynchrmlaus

initiatim.

Frmt the lIF3lecules

it is apparentthatbacterial

transcribingpolyanaDNAis

in length,afinding

RNApolymrasewhen

capable ofmvingpastthe

initiaticm

,squence(s)

~toase~andthird~~ofsynthesisproducingtranscriptsofupto threetixms

thelengthofthe

rifaqicin sites

transcripticllis

initiated

(1-5 with 2-3 pre&ninating)

andproceeds synthesis

at a ratewhich

(10).

tithepresenceof mallnukerof

in Figure

2,

for overall of aFpmxirrately16

frundata&tainedbyDelius

&

~~~chainscnindividualtranscription~l~are

32 canplexes

chains couldnothave thewaysuggestedby

initiation

in the histogram

calculatedtobe

calculated

lengtha

mxxmredtwocmclusicnsbecane

of rifampicin.

fruna as shm

canbe

linedupaccordingtotheir

thatof

strand.

xx&rimicmxliticmswhicharesub@imal

nucleotidesperseamd,as -w-l

template

analysed originated Schaffer

"linearised"

apparenti

DNAxmlecules

Firstly

it has been obsenred

(withcnepossibleexceptim) frana

ccmm

initiation

(12) orbyescapingthe

The seccnd caxlusimis

thatunderthese

620

and

theFC?A side either inhibitory

effect

cakiitims

in

BIOCHEMICAL

Vol. 84, No. 3, 1978

Figure

1.

Visualisaticn

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

ofinvitropolyanaDNAtranscriptim

Ashawsanetranscripti~carplexwithcare~~~. transcriptian carplexwith twovery

ax@exes Bshcwscne

lcng transcriptim&ains.

bothDNAstrandsarebeingtranscribedassuggestedbythefindingthat in sare transcripticns than a transcript

canplexes cRNAchains

of the entire

DNA template.

inothertranscripticr~canplexes transcript&m withourprevious RNA (Table I), the apparent

transcripticn

of a significantprcporkim

"j anming" ofmolecules l),

occurring

Measurmts

carried

out

lkesefindingsareinagreerrwt

Anotherohervaticnwhichmightbe

(canplex D in Figure

inlengthbymre

are alSoirreccPlcilablewith

fran me strand alcne. finding

differ

aroundthe

cctlsequence of symxtrical

mder these ccmditims.

621

strmded

of significance

atcertainpoints

again a possible

of dotile

is ring

Vol. 84, No. 3, 1978

Fig.

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

1 cont.'d.

C-E a?~ transcriptim cxn@Ses With thIX?a = -transcriptim catplexwith 5mchain.9.

chains.

Fshcwsa

DISCUSSICN Cxurresults

shmthat

virus is agtxdtqdate

supercoiledpolyanaLXU&frmncn-defective fortranscriptim

byE.ColiRNApo1ymeraz.e.

622

BIOCHEMICAL

Vol. 84, No. 3, 1978

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

I234

5

no. chains/DNAmolecule

Figure 2. Histograrmrepresentingdistributicnoftrrmscripti~carplexes accordingtonmberofIWAchainspresent. Analysis

OfpolyamacIFJAbycellulose~~~~yand~iligesticn

~~~thatabout50%of~p~~isdoublestrrmdedindicating that transcriptim

is partially

symnetric,

i.e.

Visualisatimoftmnscripticn

SequenCeSO

fran acmpkkx~tzq

axql~byl+Ihas

m sham

transcriptsthatareasl~gasthreetimesthecodingstrand. theselmg

cRNAmlecules

amaximal

Fran

rate of synthesis

of16

nucleotides

per~dwascdlculated~~ricnicocnditicns~~aresubcptimdl. TheEBlstudieshave

dlsos~that~istrrmscribedinthepresence

of rifaqxicin,

franalimitednukerofinitiatimsites

pxedanimting)

andstrmgly

These findings

agreewithapYxxbus

suggesttrmscriptim

who also found a significant 5 initial&m withthe

siks

(l-5with

reportby

prqcrticn

for shortnascent

fmnbothINAstrands. Lescureetal--

of symnetric RNA&aim

andcorrelatewell

findingof5mi.nA-Trichmgimsmappedm

thepolyma

denaturatim

Kimmetal--

(2) whowere abletosynthesiseasymmtricpolyanacRNA

using E.Coli

lWA polynwase

mderimic

with back&c&age

have clearly

caxiikions

(13)

cFWA (47%) and

genam? after

pramtor

2-3

T4 Gene 32 protein

selected

(14,15).

the "straxgest"

(0,5MKCL) that have keen shun to

623

cFN?i w

8030

23125

1905

3659

Exp.No,

1

2

3

4

3080 (38%)

0% Elu. CTm

1X28 (57%)

10404 (45%)

CFll

mdmAasedigesticmof

DNAase andF@Aasedigestim described under Methods.

777 (43%)

I.2721 (55%)

4950 (62%)

CE'll 15% Elu. cpn

cmlchrcanatography

TAELE 1:

959 (55%)

As inExp.

3 annealledl2

hrs

Incuh. 2 hrs/LBAase/GloO/ mealled67oc2 xss2 7hrs

Incuhatim 90 minutes/DNAase phenolextracticn/precipitaticm

Ihcubatim2hrs/~ograd/ RNA pooled precipitated

cFmbeforechrunatogr~y

carriedoutas

RNAase resist cpn

andcRNAannealingwere

1959 (53%)

745 (45%)

RNAase ens. F

Py CRNA

BIOCHEMICAL

Vol. 84, No. 3, 1978

prevent

even thebindingofthe

its natural

templates

--In vitro

enzymtobactericphage

little

transcription existence

transcripti~

Finally

is kncm products

of at least

T7 IX&me

of

(16). by E,Coli

appear to have sane features Although

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

in camm

RNA polymerase of polyana DNA with the --in vivo process.

about the nmber "in -- viva"

two initiation

and mapping of the original

thefindiqgsreportedrequirethe sites

m cpposite

strands

(2).

the symr&ry

of the --in vitro product which is clearly established in 0~1: experinents parallels the situation "in -- viva" where a significantpqxztim ofmI7NAlabelledwith short pulses of ra%oactiveprecmsors

originates

DNA -late

(3,4).

--in vitro

by the bacterial

theputativemdamclease(s)

frananplemsntarystretches

The double stranded cRNA mlecules enzyme my also prove useful which finallyproducetba

of the synthesised for the study of fmctimal

messenger RNA molecules. ACKNBS Thisworkwas carriedoutin thelaboratoryofMAecularVirologyof the I@pe?Aal Cancer Research Fund, Lincoln's Inn Fields ad, for the facilities provided, we wish to thank Dr Lime1 Crawford. *also wanttothankDrMFriedforsupplyihguswithnm-defectivepolyara virus andDr RKamn foradvicewiththeCF'llcellulosechranatogriq?hy.

1. 2. 3. 4. 5. 6. 2 9. 10. 11. 12. 13.

Hudscq J., Goldstein, D. and Weil, R. (1970) Proc.Nat. Acad. Sci., 65, 226-233. D.M., Sure, H. and Old, R.W. (1974) -, R-I Iindstran, Symposia on Qmntitative Biology, Cold Spring Harbor, XXX=, 187-197. Alani., Y. and Locker, H. (1973) Virology, 54, 495-505. Achesm, N.H., Buetti, E., Scherrer, K. and Wail, R. (1971) Pm&Nat. Acad. Sci., 68, 2231-2235. Fried, M. (1974) J. Viml., 13, 939-946. Burgess, R.R. (1969) J. Biol. Chm., 244, 6160-6167. Alberts, B. and Frey, L.J. (1969) Nature, 227, 1313-1317. Martin, M. and Axelrcd, D. (1969) Proc. Nat. Acad. Sci., 64, 1203-1210. Franklin, R.M. (1966) Proc.Nat.Acad. Sci., 55, 1504-1511. Delius, H., and Westphal, H. (1973) J. Mol. Biol., 74, 677-687. Davis, R.W., S~KKXI, M. and Davidsm, N. (1971) Methods in ~zymology. 2lD, 413-428. Schaffer, R., Kramr, R., Zillig, W. and Iudny, H. (1973) Eur. J. l%iacilem., 40, 367-373. Iscure, B., CXz&?t, P., Chanbm, P. and Yaniv, M. (1976) J.Mol.Biol., 108, 83-97. 625

Vol. 84, No. 3, 1978

14. 15. 16.

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

bknjazdino, J. & Wilde, A. (1975) Nature (Imda~) 255,249-252. Lescue, B. & Yaniv, M. (1975) J.Virol. 16, 72(F724. Richadscn, J.P. (1966) J.Mol.Biol., 21, 83-114.

626