Synthetic DNA-RNA hybrids and RNA-RNA duplexes as templates for the polymerases of the oncogenic RNA viruses.

TLDR: Six RNA viruses have now been shown to contain DNA polymerase activities directed by single-stranded RNA, double-Stranded RNA and double-stranding DNA, and it is further demonstrated that DNA–RNA hybrids, such as synthetic dC.rG, act as even more effective templates.

Abstract: Six RNA viruses have now been shown to contain DNA polymerase activities directed by single-stranded RNA, double-stranded RNA and double-stranded DNA. It is further demonstrated that DNA–RNA hybrids, such as synthetic dC.rG, act as even more effective templates.

Full text

430
NATURE
VOL. 228 OCTOBER
31 1970
DXA
(unpublished
rosults
of J.
R.). Thus
the
origin
of
tlioso molécules
as
well
as the
DNAdépendent
DNA
polymerase
romains an open question.
G.
S. B. is a visiting
professer
of the
Czeehoslovak
Acudomy
of Sciences, from Orcgon State
Univoraity,
sup]iortod
by the
exchango
programme
between
the
Czeehoslovak
and tho US Academy of Sciences, and
récipient
of a cancer
devolopmeut award.
lUiciveil AuBiist
11;
icviscd SoiitniibtT
10, 1970.
' J'ciiiin,
H. M.,
nml Mlziitniii,
S.,
A"i(/«re,
226, 1211 (1070).
' Iliillimoie, 1)., yature, 228,
1209 (1970).
'
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8.,
Buniy,
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Sclilnm,
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TràvnK'ek,
M.,
niid Walson,
K.,
iVodifp,
227, 563 (1970).
' C rawforU, L.
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t'rawfoid,
E. M.,
Viiologu, 13,
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' Duesberg,
P., and
Itobinson, W.
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219
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• Watiion,
K. F., and
Beaudreaii, O.
S.,
Biochem. Biophus.
Bel. Commun., 37.
925 (1909).
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G. 8., and Becker,
C,
J.
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• Briiiik,
C. F., and
Leick,
V.,
Biochim.
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'• Mccker,
C,
Benudrcaii,
O. S., Castle, W.,
OMwon,
B., Beard, D., and
Iteard,
J. W., J.
Nal.
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M.,
BufiC,
L.,
Kinian,
,T., and
Soiiii,
1'.,
Xeoplutma.ll, 0 (1904).
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O. 8., Sverak,
h.. Zischka,
B., and
Beaid,
J. W., Sut. Cancer
Insl. Monoii.,
17, 791 (1904).
lliiii|ii'it, ,1., Lacour,
F.,
Harcl,
J., and
llarel,
L., Cancer
llft.,2t,
150(1960).
'< lldiiiii. II.
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A. J., Beard, D.,
and Bi'aid,
J. W., Cancer
Bet.,
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(1967).
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Verfek,
L., Biochim.
Biophm.
Acta, 138, 204
' (1907).
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"
Erikaon, K. L.,
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T. L., J.
Yirol.,
4, 454 (1989).
Synthetic DNA-RNA
Hybrids and
RNA-RNA
Duplexes as
Templates
for the Polymerases of the Oncogenic
RNA
Viruses
by
S SPIFGglMAN
M.
R. DAS
J.
KEYDAR
J. SCHLOM,^
M.
TRÂVNICEK
K.
WATSON
Institute
of Cancer Research,
Columbia
University,
Collège
of Ph/sicians and Surgeons,
99
Fort Washington Avenue,
New
York
10032
Six
RNA viruses have now been shown to contain DNA polymerase
activities directed
by single-stranded RNA, double-stranded RNA
and
double-stranded DNA. It is further demonstrated that DNA-
RNA
hybrids, such as synthetic dC rG, act as even more effective
templates.
'ïHK iiccnt reports
by
Teinin'
and Baltimore' of nn BNA
diicc lcil
DXA
polj'inernso
in oncogenic RNA viruses
havo
ln'eii coiifiriiR'd
and
extendcd'. VVo estoblished
that
tlio
DXA syntlii'sizcd
is complementary to the viral KNA
uiid tlint
DX'ARXA hybrids appear as
early interniedi
uti's
of the reaction.
IJoth fcaturos
argue compcllingly
that
viral RXA
fuiictions
as a
templato
in the
synthesis
of
the DXA.
l'he RX'ADXA
hybrids dotected as presumpti\'e inter
iiiediates
are not
likely
to be
intcgruted
as such
iiito
tho
gonunie,
an event necessary to
oxplain
the
licritably
stable
State of the
transforiration indueed
by
tlie.sc
onco
genic
viruses. The required conversion of the RNA DNA
hybrid iiito
a
DNADNA
duplex
deniands
a DNA
ilirected DN.\
polymerase.
\Vo aiready had^
suggesti\e
évidence
that DNA strands synthcsized lato in tho
réac
tion were
identical rather than complementary to the
viral
RXA. Wc set ont to
fiiid
the DNAdirected DNA
polyniem.ic
implied by
thèse observations
and dotecled'
the
relevant
activitj'
in
ail
six oncogenic viruses
examin(xl.
Tho
DXAdirected
jjolymeraso
activity thus revealed
liad a nuinber
of
interestiiig
properties.
There
was a
cloar préférence
for
doublestranded
DNA and, except for
covalently liiiked
circles, virtually any
doublestraudod
DXA
was
accepted
as a template. In each case, it
could
be
shown by appropriate
hybridizations
that tho DNA
sMithesized
was complementary to the DNA
iiscd
as tho
initiating
template.
We «ère still left with
the
problein
of identifying tho
l>olymerization
reaction
which converts
the DNARNA
hybrids
into the DNADNA
duplex.
To
synthesizo
natural DNARNA
hybrids in
aniounts adéquate
for
enzymatic
test is still a formidable
task.
To a\"oid the
logistio
problems involvod
we
decidcd to use synthetic
polynucleotido
duplexes. There is ample
e\'idence''
of
their iisefulness
in studios of DNA and RNA synthesis,
and
tho DNA polymerase of E.
coli
has been shown to
use
the synthetic ribopolymer
rArU
to synthesizo the
deoxyribopolymer dAdT (réf.
8).
A\'e ha^e
found that
certain
of the synthetic duplexes are not
only
functional,
but ero
superior to natural templates by almost two
ordors
of magnitude in stimulating polymcrization. This
finding leads
to an extraordinarily useful tool for detecting
the
enzyme activity. We
suminarize
hero tho results of
our iin estigations.
Comparison
of Templates
'l'he viiuses
used in the
préparation
of enzymes
\vere
aviau myeloblastosis
virus (AMV),
féline leukaeyiia
virus
(l^'eLV), Molonoy
sarcoma virus
(MSV),
Rauscher
Icuk
aoinia
virus
(RLV),
mouso
maminary
tumour virus (MTV)
and
a rat
inammary
tumour virus
(RMTV).
Table 1
illustrâtes
tho
responses
of the six RNA oncogenic viral
préparations
to the
résident
RNA templates (column 1),
exogenous
DNA (column 2), and to the hybrid duplex
(column ;i)
formed from
polydeoxyadenylato
and
polj'
riboguanylato (dCrG).
Tho
stimulation observed with
CEF—DNA
is not maxi
mal bccttuso
for purposos of comparison it was
held
at the
samo level
(4
(xg/ml.),
at which the
réaction
with the
dCrO
is optimal. For
ail
six viruses there is
clear évidence
of
a hybrid directed DNA synthesis that is
draniatically

NATURE
VOL. 228
OCTOBER
31
1970
431
siipprior
to the reactions
previously studied.
The kinetics
of DN'A synthesis
with
tho threo templates (Fig.
1)
empha-
size
the magnitude of the
différence
in
tcmplato capabih-
tips.
When plotted on tho same
scale,
tho
Bynthotic
activi-
tie.s obsen-ed
with the
résident
viral
RNA
and CEF-
DXA
are almost trivial comparcd with dC rG.
^Ve
exnmined the template
rapabilities
of
aa inany
synthetic polynticleotides
as possible. Table 2 shows tho
response
to single
homopolymers. It should
be noted
thnt
in this, ns in the tables to
bo discn.ssed, ail mensiire-
meiits
involved examinations over 20-30 min periods with
samplps
taken every 5-7 min. Tho 5 min values are
recorded
to permit better
comparison
with those cases in
uliich
the kinetics diverge from linearity after 10 min.
In ail
cases shown in this and
siibseqtient
tables, both
complementary deoxyribosido triphosphates
wero included
in
the assay. one being
identified
by an
a-"P
label, and
the
other by a 'H label.
Table
2
demonstrates
that of the
ribohomopolymeis,
only
rC possesses
détectable
template activity and here
the
synthesis
lasts
only for 5 min. In the case of the
deoxyribopolymers, poly
dC supports
extonsive
and
prolonged
synthesis of poly dG, and some
later
incorpora-
tion
of dC. Although
much smaller,
both poly dA and
T»ble
1,
Br.spoxsEs
OF
OHCOOEWIO
VIRAI. RNA
POLTMERASKS
TO
ADDED
DXA ASD A SYNTHETIC
ENA-DNA
HYBKID
piTiolen nticleotlde Ineorroritled
In 10
mln/lO tig
of
viral prot«ln
Polymeras»
Template
None CEF-UNA
dCrO
AMV
2-8
6-3
1400
r*tv
21
20
30
7
MSV
1-2 1-2
300
ELV 1-5 4-8
1020
MTV 01
1-0
851
RMTV
01
00 21 4
A "-tniidard incubation inlxtnrc
of
0-25
ml.
conlalns J2-5 /MHOÏCS THs-HCl
3): 3/<molrp MgCl,; 10/imoles KCI;
2
5 ^wmolrs (lilliioflireitol;
n
04
//molc^
of nnch of
tlie (IPoxyrUinniirleosidf» trlphofiplintrs.
To
monltor
the
reactinn, i-"r-(lGÏP wns \ised
nt
a spccinc
activity of
05 c.p.m./pinole.
Rcnrtions
that
were stlnuilnted
by
n<idnd templatcs wcrc
carrlcd
mit
by
iiicliidinff the
template at a
levcl
of 10
/iC
per
0-25 ml.
in the standard
lncul->ation
mixture. The incorporations noted
nbovc represent
thosc
obscrved
in
50 /'I.
aliquotP correspondir.c
to 10
//p
of viral
protcin. Vlnis partirics
Hi'pended
In G 01
M
Tris
(pli 8-:i)--0
1
^! NaCl—0
002 M
KDTA (TNK)
at a
concentration
of
2-3 uni
of viral protein per
inl.
werc
prcincnbatcd
for 10
min
at
0" C lu the presencp of
0-2
per cent'
Nonidci
P-40' nnd 01 M
dithiollircltol.
The incnbated
solution
was
then added to a standard
incubation
mixture at
a
levcI
of 50
/*g
of viral protein per
0-25
ml. and
Inrubated
ai 37° C.
Afier
incubntlon
for 10 min. 50
/d. aliquots wcre withdrawn
and
processed
for
detcrmining
the
arld precipitable radioactivity
as
dc^critrcd nlsewhcre'.
The
p;irtirle3
designatrd
Iiere
as
R-MTV were obtalned
from
supernatanl
flulds
of
rat
mammary tumour', R-35 grown
m vitro
ai
Charles
rOzer <k>., Maywood,
New
Jersey. The source of the other
viruses
and
thelr niclhods
of purification
have bcen described prevIoUBly*;
R-MTV wns
pnrlflcd
in the
Bamc
way as
the monltev mammarv-
tumour virus. dC
rO was mnde
by
mlxlng equal
amounts
of poly dC and poly rG (100
/»g
per ml. In 0 01 M
Trte, pH 7-4),
makinç
the solution
0-2
M
with resi>ect
to
NaCl
and
allowinc
the mixture to
«tand
for 15 raln ni room
température.
The
CKF DNA nsed
was madc from
ir^'pslnlzed eelh
from
chick embryos accordlng
to the
procédure
of Glllesple
and 8.8.'».
Tabte
2.
BESPONSE
OF
AMV-POIYMKRASR
TO
SINOLE-BTRAHDMI
HOMO-
POIY.MERS
Incorporation (pmoles/
Template
6 iiiln/10 /ig Tlrnl protein)
rA tIT (<01)
dA (<01)
rC
(10 (3B) dC (<0
1)
rfî
lie
(<01) dG (<01)
ri dC
(<01) dO (<01)
rU
(lA
(<01) dT (<01)
(IT
(1-2) dA
(0'.!)
ilG dG (110)
IK;
(0-8)
dC (<01) dG (<01)
<n
dC
(1-2)
dO (<0-1)
(IT dA (<01)
dT
(<0
1)
The compopitlon
of the
re.icllon mixtures wos .is dcscrihed
for
Table 1,
C'xcept
thnt
In ejtch expcriment
only
Iho iwo deoxyriboalrie triphosphates
were
n*rd whlch correspondcd
to
Ihe tdenllty .md the
compicmrnt of
Iho
bnses in
the
template. The
teniplnte conrentrntlon wa»
1
^/p/O'25 nil.
of the
Inriiba-
tion
mixture. The
labels uscd
were:
'11-dAÏP
(115
e.p.m./nmole),
'H-dCTP
(125 c.p.m./pmole), "P-dOTP
(60
c.p.m./pmole),
nnd
"P-dTTP
(110 c.p.m./
pninle).
60 of the reaction
mixtnre
was withdrawn and
acid
precipitable
radloactlTlty
was determlned as
de8crll>ed'
after 6
min
of incnballon at 37° C.
0
10 20 30
Min
l'"iȕ.
1.
Kliictlrs
of incorporation of
a-'*l*-dGTP
by
HLA' polymerii^c,
A
O
iir» nil. sland.Trd réaction mixture \v:i-; nsed. Ttio spcciflc
radio-
activity
of
o-"l'-dOTP
was 00 c.p.m./pmole.
Methods
of
prcpnrnlion cf
CBF-UNA
and dC • rG are
dc5crll)oa
m the
legend
to Table 1. At
llic>
Indicated
timcs, 60 /d.
sampics
wcre withdrawn and Ihe
acId
precipit-
able radioactivity
was determlned as
described'.
poly dl stiniiilate
the
inrorporation
of the corresponding
complementary miclootidos.
The abilities
of varions
synthetic dotible-fitrnndcd DXA
and
RNA homopolymers to serve os templatcs nrc recorded
in
Table 3.
Exccpt
for
ICTG, ail stimnhite sonie
incorpora-
tion
of one of the complementary
nucleotide
pairs. Of
those
tested,
rl-rC
is clearly the bcst,
followcd
by
dC'-dG
and dC-dl.
It is intercsting to note that,
with
the excep-
tion
of
rC-rG,
the
polyribopolymei-s
are
siiperior
to the
corresponding deoxypolymers
os templates. Table 4
compares
tho template capabilities of a
•variety
of syn-
thetic
DNA-RNA hybrid duplexes.
Somo
of
thèse
are
the
beat templates, notably
dC-rG
and
dl-rC.
Table
3.
BESPOBSE
or
.V^IV-POLTMEIIASB
TO
SVKTIIETIC DOUBLI-STRANDED
DNA
AUD
RNA
POIY.MEI'.S
Incorpor.ntion (pmolf^s/
Template
5 min/IO /iti
viral protein)
rA rU
dT (160)
dA (04)
rC rO dO
(<01) dC (<01)
rl
•
rC
dG
(1280) dC
(J l)
dA •dT
dT (OS) dA (14)
dC •dG
dO
(20!)) dC (04)
dC
dl dO (•24-9)
dC (20)
The
reaction
mixtures
and conditions of
rr;irtion'. «cre
ns dcscrlbcd
fnr
Table
1. The
dui>lexes
werc
prcpared
by
usinp tlic apj'ro] i i:ile polymcr
com-
poncnts, usiniz
the
niclhoil describcd
for
dC r(;
(sep
icgenit
to Table 1). The
speclflc activities
of tbc
deoxyrlboaidc Iripbospltntcs imed
were the same a»
those
for Table
2,
nnd Ihe
appropriate
complementary
pnlr
of
'H
and
"P-
lat)elled nucleoslde
triphosphates wcre
présent in
each
rcntti'in.
Table
4.
BKsroNSF.
or
AMV-roi.YMi-.insK
TO
SVMHV.TIU HYURIPS
Incorpornlion (i»nu'les/
Template
5
mln/lO /ig
viral protein)
dA-rO
dT
(0-,'<) dA
(<01)
dr>TG
dG
(141(1) dC (10)
dC-rl
dG
(14.-.) lie (l-O)
dGT<!
dC (<01)
(H!
( < 0 1)
dI
rC dC (1 :l)
dG (104 «)
(IT^rA
dA (0
11)
dT
(10-.1)
J'iic ren<-lîf>n mixtures
and conditions
oi réactions
were
llie
same
n-» dus-
cribed
In
(lie lc(!end
to Table 1. The
hybrids
were
picparefi tisini: (lie
melhod
descril)Cd
for
iii:il%inî;
dC •
r(î
In Ihe
leReml
to
Tnblr
1. AU other
détails
nrc ns
dcseribed
in
tlie Ie«cnds
to Tables 2 and 3.
Therc
is a
striking préférence
for
dtîTP
and rGTP as
substrates.
This recalls an
analogoiis sitnotion
obscrved
with
the
QP replicoso»
and the
DNA-depondont PXA
polymeraae',
both of which
synthesize
poly G if provided
with
poly C
Bfl
s template. Tho
préférence
for dG may

432
NATURE
VOL. 228 OCTOBER
31 1970
TIIIIIR
5. TEST
or
AHV
POLYHKRISE
TO
INCORPOBATS RIB0HUCLE0TIDE8
VHTH
BTNTUETIC POLYMUCLEOTIDE TEHPUTES
a
Incorporation (pniolea/OO mIn/IO M
viral
protelii)
S
Teiiiplate Rlbonucleotldes Deoxyrilionucleol
ides
r «.
• rU
rU
(<01)
rA
(<01)
tlT
(73
0)
n
ri • rC rC
(<01)
rG
(<0 2)
dO
(326 0)
«lA •
dT
rU
(<01)
rA (<01)
dT (90)
a
a.
o lie • dO iQ
(<01)
rC
(<01)
dO (lO'J
O)
c
lit' • rO rG
(<01)
rC
(<01)
dO
(322
(1)
c
ilT
rA
rA
(<01)
rll
(<0 1)
dï
(49 7)
a
Tlie i-Oiictioi)
mixtures
niid tlic
conditions of
exi>orlnicntu wrre a»
for
Tnble
1.
Ilerc, however, riboside trlpliosiiliates wurc uscd toccllicr wltli llicir
deoxy aniilugues. Iteiictions
in
llie absence
of
Ucoxyribosido triiiltosphutes
(liive liie Banie
reaults.
a-"l'-lttbellcd
dcoxyriboside
tri)ilioa|)liiilcs
and
'H-labelted
riboside
tripitosphatcs were used
to
inonitor ttiepoiyribnnucicotidc
s.x ntliesis,
ami the
spécifie activilies
for the
rATI', rOTP, rCTl'. and rt'Tl'
were
nii
at
120
c.p.ni./pniolc. Tlio o-"P-dGTP
and
a-"l'-dTTl'
used to
fiillow tlie DNA syntliesis
were at 60
c.p.m./iniiole.
indicate thnt guanosinc
is
tlio
5'
toimintis
nnd is therefore
ilie
first nucleotide
inscrted. This
is in fact the case
with
the Q[3 rephcQse.
Asymmetric
Copying
Tables
3 and 4 show a
marked nsymrnetry
in the use
of
the substrates provided. In no case are the two
com-
pli-mentaiy
nucleosido triphosphates
incorporated
with
eqiial
facility. The asymmetry,
howovor,
is
net absoliite,
e\-cii
in the 5 min
results
rocorded in Tables 3 and 4.
^Ve
fonncl
that
although
thcre
was
an initial
iJreferonco foi' tho
incorporation
of
'^P-dT, "H-dA
is
polyniorized.
A very
siinilar
phenomenon is observed (Fig. 2) with
'T-dG
and 'H-dC
in
polymerizations
by
dl-rC.
In somo cases,
synthesis
prolonged for 90
inin
or more
load»
to
eventual
equalizatioii
of thc incorporation of tho two
siibstratcs.
It shoukl
bc noted that the use of the prefcrrcd
substrate
(foi' « xamplo,
dTTP by
dT-rA
or dGTP by
dI
rC) is not
strikiiigly
influenced by the
présence
of the
coniplenientary
nucleoside
triphosphate in the
réaction
mixture. As
expccted, thero
is symmctrical use and a
requirement
for
the présence
of both
compleiniîntary
substrates when an
alternating
copolymer is used as a
template (Kig.
3).
Here
both
compleinentary
nucleotides are incorporated
from
the very onset at almost
equal
rates and molarities.
Further,
unlike tho
homopolymerio
duplexes,
leaving eut
oither
one of the complementary substrates
leads
to thc
éoniplete loss
of
sj'nthetie
aetivity.
,
100
S 60
32
PdT
'H
dA
Fiu.
2.
Kinetics
of incorporation of
a-"P-TTP
and
'H-dATP
by
A!«V
polyinerasc
with dT rA template. A
0-25
ml. standard
réaction
mixture
was
used.
Tlie spccillc radioactivities of-a-"P-TTl'
nnd
'JI-dATP
were
110 c.p.m./pmole
and 115
c.p.in./pmoic. resiwctiveiy.
The method of
préparation
of the
hybrid template
is
described
In
tlie ieecnd
to Table 1.
At
the
iodicated timea,
60
fil. samples wero
withdrawn and
tlie acid
precipitable radioactivity
waa determined as described
earlIeH.
C"^ ''HdC
"PdG(Singly)
•H
dC(Singly)
10
15
2U
Time (min)
Fig.
3. Kinetics of incorporation of
a-"P-dGTP
and
'li
dCTP by
A51V
polymcrase
with d(I-C) as template. A 0 25 ml. standard
rcadion
mixture
waa used. The
apecific radioactivlliea
of
a-"P-dGTP
and
'H-
dOTP
were 60
c.p.in./pinolc
and 110 c.p.m./pmole respecllvely. At the
iiuiicatcd
limes, 50
/il.
aliquots were
wittidrawn
and
tlie TCA
precipitable
radioactivity
was
determined
as described
earlier*. Kesuits
are
expressed
as
pmoics of nucleotide
iiicoriM>rated/iO /ig
viral protein. O and x
indicate
the incorporation
oljtained «heii
both
deoxyrihoiiiicIoïKidB
triphosphates
are
présent. A
and
O
refcr to
tlie incorporaiiun ul)t;tiiied
when
one nucleotide
oiily
is
présent.
Inabllity
to Incorporate
Ribonucleotides
It lias
already
becn
noted'that the oncogenic
polymer-
ases
do not synthesize
ribopoljiners
using
tlie
\ iral
RXA
as
a template. In view of the vastly superior template
capabilities
of
some
of tho
syiithetic polyiner.s,
this ques-
tion
was ro-examined and the results are recordetl in
Table
5. Evcn after 90 min, no signs of
ribomicleotide
incorporation eoiild
be dotected with auy of the templates
diiring
a period in which excellent
poljTnerization (last
cohimii
of Table 5) was oecurring with the
deoxyribo-
nucleotides.
Uses
for Synthetic Templates
A
detailed oxamination of the
interinediates
in tho
reactions
mediated by
thèse synthetic
duplexes
slioiitd
be
very useful
for the study of the chcmistry of
the polynier-
ization
and tho factors which
détermine
its asymmetry.
Even
a casual inspection of Table 1 and Fig.
l
revculn the
ob\
ious
advantago
of using templates
such
as dC rCi fur
dotccting
enzyme in
eells
or virus particles
and following
aetivity
during purification
procédures. We
ha\ o
iiow
established
that oncogenic
\
iruses contain
DXA pul.v-
mera.se
activitiea directed by
single-stianded 1ÎN.\,
doublo-stranded RNA, double-stranded
DXA, and
DX.\-
RN^
hybrids. Further,
ail
oncogenic RXA
viiiisea
examined
contain
ail thèse
activities. The
miiiiber
of
différent
kinds of protein
molécules
required to
handlo
this
variety
of templates remains to be determined.
We thaiik
Drs F.
Bolhun,
R. D.
AVclls and
D. Brutlag
for deo.xyribonucleotido
polymers; Dr J. W. Beard for a
siipply
of AMV; Drs J.
Moloney,
F. Rauscher and T.
O'Connor
of tho
Spécial
Virus Cancer Program (SVC'P)
of
tho National Cancer Institute for their
assistance,
and
personnel of
tlie laboratories
associated with the SVC'P
for coopération.
Tho work was
supported
by a contract
with
the SVCP and n grant from the National Cancer
Institute.
Hecelvcd Alignât 24,1070.
' Temln,
II. M., nnd
Mlzutniii,
S.,
A'ndi/r,
226, 1211 (1970).
• Haitimore,
D.,
•V««ure,226.
1200 (1070).
• Spiegelman.
S.,
Jiurny,
A.,
Das.
M. R.,
Keydar,
J.,
Schloni.
J.,
TrAvnlfek,
M.,
and Watson, K.,
Nalure.W,
583 (1970).
' Spicgoiniaii,
S.,
Ilurny,
A., Das,
K.
E.., Keydar, J.,
Schloin,
J.,
TriviiliVli,
M.,
and Watson, K.,
Hature. 227. 1020
(1070).
• Kornbcrg,
A.,
Enzymalic Synlhesii
of
DNÀ,
103
(Wiley,
Xew York nnd
London,
1061).
• Klkhom,
T. S., and
Splegelman,
3.,
Proe.
US Not.
Arad.
Sei., 57,
1832
(1907).
'
Fox. C. F.,
Ilobinson,
W. 8.,
Haseikorn,
R., and
Welss,
S. B., J.
Biol. Chtm.,
239,
180 (1904).
' Lec-Huang,
S., and Cavallerl, L. F., Proe. US
A'a<. Acad.
Sei., 81, 102*
(1964).
• Chopra,
H. C, Bogden, A. E.,
ZelIJadt,
J., and Jensen, C. H.,
Eiirop.
J.
Cnneer
(In the
preas).
Gllleaple,
D., and
Bplegehnan,
B.,
/.
Mol.
BM., U, B29
(1963).