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