Size analysis and relationship of murine leukemia virus-specific mRNA's: evidence for transposition of sequences during synthesis and processing of subgenomic mRNA.

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0022-538X/78/0026-0468$02.00/0

Size

Analysis and Relationship of Murine

Leukemia

Virus-Specific

mRNA's: Evidence for Transposition of

Sequences

During Synthesis and Processing of Subgenomic mRNA

HUNG FAN*AND INDERM.VERMA

TumorVirologyLaboratory, The Salk Institute, San Diego, California 92112

Receivedforpublication 28 December 1977

Virus-specific mRNA from purified polyribosomes

of mouse cells infected with

Moloney

murine leukemia virus

(M-MuLV)

was

analyzed by

electrophoresis

in

agarose

gels,

followed

by hybridization

of

gel slices

withM-MuLV-specific

com-plementary DNA (cDNA). The size resolution of the gels was better than that of sucrose gradients used in previous analyses, and two

virus-specific

mRNA's of

38S and

24S

were

detected.

The

24S

virus-specific mnRNA is

predominantly

derived from the 3' half of theM-MuLV genome, since

cDNA,fiaga,po

(complemen-tary to the 5'half

of the M-MuLV

genome) could

not

efficiently

anneal

with this

mRNA. However, sequences complementary to cDNA

synthesized

from

the

extreme 5' end of M-MuLV 38S RNA

(cDNA 5')

are present in the 24S

virus-specific mRNA, since cDNA 5' (130

nucleotides) efficiently

annealed with

this

mRNA.

The annealing of cDNA 5' was not due to repetition of 5'

terminal

nucleotide

sequences at

the 3' end of M-MuLV 38S RNA,

since

smaller

cDNA 5' molecules

(60

to70nucleotides), which

likely

lack the terminal repetition,

also

efficiently

annealed with the 24S mRNA. The sequences in

24S

virus-specific

mRNA

recognized

by

cDNA 5' arenot

present

in 3'

fragments

of virionRNA

that

are the samelength. Therefore, it appears that RNA sequences from the extreme 5'

end

of the M-MuLV genome may betransposed to sequences from the 3'

half

of

the M-MuLV 38S RNA

during synthesis

and

processing

of the 24S

virus-specific mRNA. These results

may

indicate

a

phenomenon similar

to

the

RNA

splicing

processes that occur during synthesis of adenovirus and

papovavirus

mRNA's.

RNA tumor viruses

contain

a

single-stranded

used to

elucidate

the

relationships

of

intracel-diploid

RNAgenome which is

positive

stranded lularpolyadenylic acid

[poly(A)]-containing

vi-and

approximately 9,000

nucleotides

long

(sedi-

rus-specific

RNAs

(19, 35).

In the results

re-mentation value

38S) (3,

4,

10). This

38S

RNA

ported here,

wehave

used

specific cDNA

probes codes for

three classes

of

viral

structural

pro- tostudy the

intracellular virus-specific

mRNA's teins:

the

internal structural

proteins (products

of

mouse

cells infected with Moloney

murine of

the

gag

gene),

envelope

glycoproteins (prod-

leukemia virus (M-MuLV),

anRNA tumor virus uctsof

the

env

gene), and

reverse

transcriptase

which

does not

morphologically

transform

cells.

(product of

thepol gene) (3).

In

addition,

viruses

The

relationship of the viral mRNA's

was

deter-that

morphologically

transform fibroblasts en- mined, and indications of transposition of

virus-code a

protein

responsible

for this transforma-

specific

RNA sequences in

subgenomic

mRNA tion

(a

product

of the src

gene) (3).

No other were

obtained.

viral

proteins

have yetbeen

identified, although

it is

possible

that nonstructural

viral

proteins

MATERLS AND METHODS

could exist. Cells, viruses,andmaterials. M-MuLV clone4A

Studies of the

virus-specific

mRNA's in cells

cells,

alineof NIH-3T3 cells

clonally

infected with

M-productively

infected with RNA tumorviruses MuLV,were grown onmonolayer in Dulbecco-modi-haveindicated that

virus-specific

mRNA exists

fled

Eagle medium supplemented with 5% calfserum. in two

general

size classes: mRNA

equivalent

in In

all

experiments, exponentially growing

cells

from

lengthtothegenomisui

3subconfluent

cultures were used. Purified M-MuLV

lengthtothebgenomic

subunit

(38S2729).

RnA,

aend

was

obtained

from

tissue

culture supernatants of

M-subgenomic

mRNA (11,15,

23,i27,

29).In

recent

MuLV clone 1cells(14),anotherlineofNIH-3T3cells studies on aVian sarcoma viruses, complemen- infected withM-MuLV, as described previously (11). tary DNA

(cDNA) hybridization

probes specific The deoxyribonucleoside triphosphates were ob-for different

regions

of the viral genome were tained from P-L Biochemicals.

[3H]dTTP

(specific

468

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VOL. 26,1978

activity, 60 Ci/mmol) was obtained from Schwarz/ except thatdetergent concentrationwas0.02% Noni-Mann, and

[32P]dGTP

waspurchased from ICN, Ir- detP-40, andapproximately 2 mg of calf thymus DNA vine, Calif. Both oligo(dT)1o and oligo(dT)-cellulose primerper ml wasincluded. After 3 h ofincubation at

(T-3) were purchased from Collaborative Research. 37°C, maximalincorporation had occurred. The re-Purified avian myeloblastosis virus reverse transcrip- sultant cDNAprobe was then processed for hybridi-tasewaskindly givento usby M. T. Lai. zationasdescribedpreviously(11).Specificactivity of Fractionation of cells and preparation of the cDNA was 1.6 x 107 cpm/ug, as calculated from mRNA. For cell fractionation, cells were removed theinput specific activity of the labeled deoxynucleo-fromthe tissueculture dishes by trypsinization on ice, sidetriphosphate (TTP). The cDNA wasquite rep-as described previously (12). Purified polyribosomes resentative of the 38S viralRNA, sinceatanRNA to wereprepared as describedpreviously, be pelleting of DNA ratio of1:1 50% ofthe cDNAcouldhybridize, polyribosomes fromcytoplasmic extracts through1M and ata2:1 ratioall of the cDNAcould hybridize (D. and2M sucrose (13,15). Thepelleted polyribosomes Dolberg, personalcommunication).

weredissolved in2mlof SDS buffer(0.1 M NaCl,0.01 ThecDNA(,gpIoonwaspreared in the following man-MTris[pH

7.4],

1mMEDTA) containing 0.5% sodium ner. 3H-labeled Moloney murine sarcoma

virus-spe-dodecyl sulfate (SDS), and extracted twice with cific cDNA(1.7 x 107 cpm/,ug), which had been pre-phenol-chloroform (26). The extracted RNA was then pared from purified Moloney murine sarcoma virus precipitated with2volumes of ethanol and storedat clone 124(2, 8) using exogenously added calf thymus -20°C until use. In some cases, polyribosomalRNA DNAprimer,waskindly providedby DavidDolberg. was bound to oligo(dT)-cellulose essentially as de- The cDNA(106 cpm) wasannealed with 15 ugof3 scribedpreviously (12),exceptthatbindingwasto 1 poly(A)-containingfragments of M-MuLV virion less ml of oligo(dT)-cellulose T-3 (Collaborative Re- than1,500nucleotideslong per ml inatotalvolume of search), andwashings and elutionswereperformedin 0.1 ml for5 h at68°Cin 0.6MNETES(0.6MNaCl,

acolumn instead ofbycentrifugation. 0.01 M N-tris(hydroxymethyl)methyl-2-aminometh-Preparationof poly(A)-containing size classes anesulfonic acid [pH

7.5],

1 mM EDTA) plus 0.1% of viral RNA. TheproceduredescribedbyWang et SDS. After annealing, the unhybridized cDNA was al. (34) wasused with slight modifications. Purified selected out bybinding tohydroxylapatite in 0.01 M virionsof M-MuLVwerelysedwithSDS (finalcon- phosphate buffer containing 0.3 M NaCl and 0.1% centration1%), and RNAwasextractedbydeprotein- SDS, followed by elution in 0.14 M phosphate buffer ization withphenol-chloroformandprecipitated with plus 0.1% SDS at 60°C. Approximately 70% of the 2volumes of ethanol. Theprecipitatewaspelleted by cDNAwasrecovered in this fraction.ThecDNAwas centrifugation and dissolved in SDS buffer containing then concentratedby ethanol precipitation and freed 0.1%SDS and sedimentedon 15 to30%linear sucrose ofresidualphosphate by passageoveraSephadex G-gradients(11). Material sedimentingat 60 to70Swas 50column. The cDNA was thenagain concentrated pooled andprecipitatedwithethanol. The RNAwas by ethanolprecipitation and finally stored in hybridi-dissolved in buffercontaining0.01MTris-hydrochlo- zation bufferat-20°C.

ride(pH 7.4),0.01M NaCl,and0.001 M EDTA and cDNA 5'. cDNA 5' was synthesized by using puri-incubated at50.5°C for 1 min. A 1.0 M solution of fied M-MuLV virions. The reaction mixture (1.0 ml) sodium carbonatewasaddedtothissolutionto afinal contained50mMTris-hydrochloride (pH 8.3), 10 mM concentration of0.06M(pH 11)and incubated further dithiothreitol,6mMmagnesiumacetate,60mMNaCl,

for 3.5min at 50.5°C.The solution wasneutralized 1 mM each ofdATP, dCTP and dGTP, 50

itM

of withaceticacid,diluted with buffer containing 0.4 M [3H]dTTP (specificactivity of dTTP in the reaction

NaCl, 0.01 M Tris-hydrochloride (pH 7.4), 0.001 M was4,900cpm/pmol),80ug ofactinomycinD,Nonidet EDTA, and 0.5%SDS,and adsorbedto anoligo(dT)- P-40toafinal concentration of0.01%, and2.5mg of cellulose (T-3) columnasdescribed (12). The bound virus. The reaction mixturewasflushedwithN2 and material was eluted with buffer containing 0.01 M incubatedat37°Cfor6h.About5 to6% of theinput Tris-hydrochloride (pH7.4),0.001MEDTA,and 0.5% nucleotides of viral RNA (assuming that 70S viral SDS. The salt concentration of the eluted material RNArepresentsabout 1% of total viralprotein)were wasraisedto 0.4MNaCl and the materialwasread- transcribed. The reactionwasstopped byadditionof sorbed to oligo(dT)-cellulose. Material eluting from SDSto afinal concentrationof1%,andnucleic acids the secondcycleofchromatographywasfractionated wereextractedbyphenol-chloroformasdescribed(26) onneutralsucrosegradients alongwith28S and 18S and precipitated with ethanol. The precipitate was rRNA markers. Varioussize classes of RNAranging suspendedinasolutioncontaining0.01M

Tris-hydro-from 4Sto38Swerepooled,theiramounts werede- chloride (pH 7.4) and0.001 MEDTA, and theviral termined bymeasurementofabsorbancy at260nm, RNAwashydrolyzed byaddition of NaOHtoafinal andtheywerestored under ethanolat-20°C.About concentration of0.3N,followedbyincubationat37°C 15%ofthestarting60 to 70S RNA wasrecovered after for 18h. ThecDNAwasneutralized by acetic acidand twocycles ofoligo(dT)-cellulosechromatography. separated from low-molecular-weight material by Preparation of cDNA probes. 3H-labeled M- chromatography on aSephadex G-75 column equili-MuLVcDNAprobe, whichwasapproximately repre- brated with 0.05 M triethyl ammonium bicarbonate. sentativeof the entire M-MuLV 38SRNA, waspre- Thematerialelutingin thevoidvolumewascombined,

paredbyprimingtheendogenousreversetranscriptase lyophilized to dryness, and suspended in 0.3 ml of reaction ofpurifiedM-MuLV virions withexogenously buffercontaining0.01MTris-hydrochloride (pH 7.4) added random oligodeoxynucleotides obtained from and 0.001 M EDTA.Thematerialwastransferredto DNase-digested calfthymus DNA (32). Briefly, the siliconized1.5-ml conicalpolypropylene tubes (Eppen-reaction mixtures were as described previously (11), dorf), and, after addition of LiClto afinal

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tion of 0.2 M and10,ug of carrierRNA, the cDNA was 30 min at 37°C, and a sample was withdrawn to precipitated with 2volumesof ethanol and stored at determine acid-precipitable material. In this case, -70°C for 15 min. The ethanol precipitatewascentri- about 1.2 to 1.5% of the input nucleotides of viral RNA fuged in a Brinkman centrifuge attop speed for 10 were transcribed into cDNA. The reaction mixture min, and the pellet wassuspended in 20

pl

of 0.01 M was boiled in 0.3 N NaOH for 5 min, neutralized with

Tris-hydrochloridebuffer(pH7.4). HCl, andseparatedfromlow-molecular-weight mate-The cDNA 5' (bands 1 to 7) wasseparated from rial by chromatography on aG-75 column. Material total cDNAbyelectrophoresison10%polyacrylamide eluting in the void volume wascollected and either gels. Included in thesamplewere traceramountsof lyophilizedorprecipitated withethanol.

32P-labeled cDNA 5' bands 1 to 7. The 32P-labeled Analysisof cDNA3'oneitherpolyacrylamidegels cDNA 5'(bands 1 to 7) wasprepared by labeling the oralkalinesucrosegradients revealedtwosize classes. 5' end of the total cDNA with y [32P]ATP and T4 About 30% of the cDNA 3'sedimented with an average polynucleotide kinase. Bands 1to7wereisolated by size of about 150 nucleotides, and the rest of the fractionating the total cDNAon 10%polyacrylamide material sedimented with an average size of about 500 gels, and their nucleotide sequencesweredetermined. nucleotides. Hybridization analysis showed that about Bands2to 7appeartobesubsets of cDNA 5' band1. 30 to 40% of cDNA 3' of small size class could be cDNA 5' band 1 appears to be initiated on tRNA protected bypoly(A),whereasonly about 8 to 10%of primer,asit shows the standarda-32Patomtransfer the large class of cDNA 3' could be protected by fromdAtorA(31). Details of nucleotide sequencing, poly(A). The high degree of hybridization of the small 5'-endlabeling, anda-32P atomtransfer experiments size cDNA 3' topoly(A) suggested that the material will be published elsewhere in collaboration with A. contained largeamounts of polydeoxythymidylic acid

Ohtsukaand M. McKennett. The 3H-labeled cDNA- synthesized during the reaction. In the experiments containing 5' 32P-labeled bands 1 to7 weresubjected reportedhere,only the large size classes of cDNA 3' to electrophoresis inTris-borate buffer (90 mM Tris were used.

base,2.5mMEDTA, and 89 mM boricacid) for1hat Gel analysis and hybridization techniques. Size 200 V (about25V/cm). The wet gel was exposed to analysis by electrophoresis in agarose gels was as X-ray film (Kodak NST 54) for several hours. The described previously (9). Briefly, RNA was suspended film was developed and used as a replica toexcise inelectrophoresis sample buffer (4 mM Tris base[pH

bands1 to 7.Thepolyacrylamide gel slices were dis- 7.2], 2 mM sodiumacetate, 2 mM EDTA, 0.2% SDS, aggregated manually with a spatula to a very fine 10%glycerol) and boiled for 1 min, followed by rapid mesh and incubated at 45°C overnight in a buffer cooling in ice.Bromophenol blue dye and 10% glycerol containing0.01 MTris-hydrochloride (pH 7.4),0.001 wereadded, and the sample was layered onto a 10-cm MEDTA,and0.002MNaCl. Thegel suspensionwas 1% agarose tube gel in Tris-acetate buffer.After elec-filteredthrougha0.45-,ummembranefilter(Millipore trophoresis at the times andvoltages indicated, the gel Corp.). The filtratewasadjustedto afinalconcentra- wasstained with ethidium bromide, and the location tionof 0.2 M LiCl and

20,ug

of carrier yeast RNA per of the 28S and18S rRNA's was determined by obser-ml and precipitated with 2.5 volumes of 95% ethanol. vationunder UV light.

TheDNAwascollectedby centrifugationandresus- Hybridization across the agarose gels wasperformed pendedinhybridizationbuffer. essentially as described previously (9). Briefly, the Inatypical experiment, westarted with about12 regions of the gels calculated to contain 15 to 45S mgofpurified virions, and the reaction wascarried RNA were cut out and divided into 1-mmslices. Each outin5.0ml. After6h of incubationat37°C,the total slice (approximately 25

AJ)

was placed in a 1.5-ml amount of cDNA synthesized was 7.2

jig

(about 6% plastic tube(Eppendorf), and 25

p1

of cDNA (100 to

incorporation). Afterfractionationonpolyacrylamide 1,000 cpm) in 0.6 M NETES plus SDS (0.6 M NaCl,

gels,theamountsofcDNA 5' bands(1to7)recovered 0.01 M N-tris(hydroxymethyl)methyl-2-aminometh-were as follows: band 1,0.034,ug (about0.5% of the anesulfonic acid [pH 7.5], 1 mM EDTA, 0.1% SDS) totalcDNA);bands2and3,0.014ug

(0.19%);

band4, was added. After addition of 0.1 ml of mineral oil, the 0.0078

jig

(0.1%);bands5to7,0.008,ug(0.1%). tubes were sealed and boiled for 3min. The tubes were The cDNA 3' was synthesized by using purified thenimmediately transferred to a 68°C water bath, viral 70S RNA,oligo(dT) primer,andpurifiedreverse andannealing wasperformed for the times indicated. transcriptase from avian myeloblastosis virus. The Afterannealing, the samples were digested with

Si

reaction mixture in 0.1 ml contained 50 mM Tris- single-strand-specific nuclease, and trichloroacetic

hydrochloride (pH 7.4), 10mMdithiothreitol, 6mM acid-precipitable radioactivity was determined as

de-Mg2+,15mMNaCl,40

,uM

each ofdATP,dCTP,and scribed before (9).

dTTP, 20 gM of a-[32P]dGTP (specific activity of Solution hybridizations were performed in micro-dGTPinthereactionwas12,000cpm/pmol),10,g of capillarypipettesasdescribedpreviously (11). Reac-actinomycin D, 75 U of avian myeloblastosis virus tion volumeswere 5 to10

pl,

andhybrid assay was by reversetranscriptase (1 U=incorporation of100pmol digestionwithS1 nuclease.

of dGMPin 15minat37°C),0.5,ug ofoligo(dT)1o,and 10

,ug

ofviralRNA.The viral70S RNAwasprepared

frompurified virions byphenol-chloroformextraction RESULTS asdescribed above. The RNAwasboiledin 0.01 M

Tris-hydrochloride (pH 7.4) and 0.005 MNaCl for 2

Identification

of virus-specific mRNA. min and selected forpoly(A)-containingRNAbychro-

sLze

measurements

of

virus-specific

matography on oligo(dT)-cellulose columns as de- mRNAinMuLV-infectedcellsweremadebased

scribedabove. The reaction mixture was incubated for onsedimentationinsucrose

gradients,

since

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VOL. 26, 1978 SIZE ANALYSIS OF MuLV-SPECIFIC mRNA'S

471

sequent

analysis

was

relatively simple (11, 15).

100- ) However, the size resolution of such

gradients

was rather limited. Therefore we performed a

80-more refined sizeanalysis using

electrophoresis

inagarose gels. Purified polyribosomes were

pre-pared

from M-MuLV clone 4A cells (a line of

60-NIH-3T3 cells clonally infected with M-MuLV), 28S

1BS

and extracted RNA wasseparated according to 40-

-80

S'

size by

electrophoresis in an agarose gel. It has

previously

been

shown that

essentially all virus-

a 20

R

40

specific RNA in

a

purified polyribosome

prepa- N

ration

is

functionally mRNA, although

in

total

, R

cytoplasm it represents a minority component 0 K

l,

(13, 15).

After

electrophoresis, the gel

was

frac-

I 6 7

tionated

into 1-mm

slices, and the

amount

of

00- (b)

virus-specific RNA in each slice

was

determined

by

annealing with radioactively labeled

cDNA a.

80-prepared

by the endogenous

reverse

transcrip-tase

reaction of M-MuLV

virions. In

Fig.

1

the

60 l

M-MuLV

cDNA was

prepared by incubation of

60

M-MuLV

virions in

the

presence of random

calf

thymus deoxynucleotide primer; such cDNA

40-contains nucleotide

sequences

complementary

to all

regions

of the M-MuLV 38S RNA

in 20

approximately equal

concentrations

(32). The

20

hybridization

of the

gel

slices indicated

two

ma-jor

virus-specific

mRNA

species (Fig.

la).

The

0o

larger mRNA

comigrated

with

32P-labeled

M- 4 5

6

7 MuLV

38S

virion

RNA and

wasvery

near, if

not .

DISTANCE (cm)

identical,in

size'to38Svo

FIG. 1. Size analysis of

M-MuLV-specific

mRNA.

identical,

i szeto 38Sviron RNA,asreported (a) Purified polyribosomes from 1015-cm tissue

cul-previously (11). The smaller mRNA migrated ture dishes of M-MuL V clone 4A cells at three-quar-between28S and 18SrRNA,and wasdesignated ters confluency were prepared, and the RNA was

24S

(according

to

its sedimentation

in a

neutral

extracted. One twentieth of the RNA was combined

sucrosegradient) althoughitsapparentmobility withapproximately1,OOO) cpm of32P-labeledM-MuLV in the agarose gelwas somewhat larger. Other 38Svirion RNA, denaturedby boiling, and

layered

investigators have reported that even smaller onto a 1% agarosegel. Electrophoresis was for 165

virus-specific

mRNA

might

also be present in mm at 65 V/cm. After staining with ethidium bro-MuLV-infected cells

(15),

but no evidence for mide to visualize the rRNA, the region of thegel suchmRNA'swasfound in the results shown in indicatedwascut outanddivided into 1-mmslices.

Fgorsbeunexet.

HThe

'P

radioactivity in

each

slice

was

determined

by

Fig. 1 or subsequent

experlments.

owever,

an Cerenkovcounting, and then each slice wasannealed mRNA smaller than 105 would not have been with 850 cpm of 3H-labeled calf thymusDNA-primed

detected. M-MuLV cDNA probe. Annealing was for 15 h at

Fig. lb

shows that both

virus-specific

mRNA's 68°C. After annealing, the amount of cDNA

hybrid-contain

poly(A)

sequencesasmeasured

by

bind- ized in each

sample

wasdetermined

by digestion of

ing

to

oligo(dT)-cellulose.

Both mRNA's bound unhybridized cDNA with Sl nuclease. The location

to the

oligo(dT)-cellulose equally

well

(better

of the 28S and 18S rRNA is indicated in the figure.

than 75%). If the

24S

virus-specific RNA was

()

32P

radioactivity;

(0)

percentage of3H-labeled produced by artifactual degradation of 38S M-MuLV cDNA_{polyribosomal} hybridized.

(b)

One fifteenth of the RNA

preparation

described in(a) was

mRNA,

then not all 24S mRNA molecules bound to

oligo(dT)-cellulose,

and

thepoly(A)-contain-would

contain

poly(A).

The fact that

both

ingRNA was eluted and analyzed in a parallel

gel.

mRNA's bound

equally

well to the

oligo(dT)-

No 32P-labeled

385

RNA was added, and only the

cellulose indicates that 24S virus-specific

mRNA hybridization with

3H-labeled

M-MuLV cDNA is didnotarise

by degradation. Furthermore,

since shown. Conditions

of

annealingwerethesame as(a).

poly(A)

sequences are

located

atthe 3' ends of

virion RNA

(4,

33), as

well

asmany hostcellular tionof 38S RNA might also be

polyadenylated.

mRNA's,

theseresults suggest that the

24S

vi-

Relationship of the 24S and 38S

virus-rus-specific

mRNA

might

bederived from the 3'

specific mRNA's.

To investigate the

relation-portion

of the 38S viral RNA.

However,

it is ship of the 24S and 38S virus-specific

mRNA's,

possible

that sequences derived from the 5'por- acDNA specific for the 5' half of the M-MuLV

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472

VERMA J. VIROL.

genome was

prepared,

as

described

in detail in

100 Materials and Methods.

Briefly,

a

cDNA

probe

cDNACT

(a)

was

prepared from

Moloney

murine

sarcoma

)0

virus

clone

124,

which has been shown

tohave 80 sequence

homology with the 5' half of the

M-MuLV

genome, as

well

as somesequences at

the

60-

l

2 3'

end

(8,

20). The

sequences

complementary

to 2

the

3'

end of the M-MuLV

genome were re-

40-moved from the murine

sarcoma

virus

cDNA by

annealing

with short

poly(A)-containing

3'

frag-

a

20-

o

ments

of M-MuLV virion

RNA,

yielding

a

cDNA

N

probe that

would

only recognize

sequences cor-

0 responding

to

the 5' half of M-MuLV RNA.

2 3

4

5 6 7

Heteroduplex

analysis and protein analysis in-

100-dicate that the

gag gene

is 5' terminal for M-

cDNAuggpoV

(b)

MuLV

(20), similar

to

the

gene

order

deduced

S

80-for avian RNA

tumor

viruses

(gag

pol

env src

from 5'

to

3')

(33).

Therefore,

the cDNA

probe

60-described above

was

designated

cDNAg<g,,o)

to

28S

18S

indicate that it likely contained

sequences

from

24S

the

gag

and

possibly

a

portion

of

the

pol

gene

of

404mRNA4

M-MuLV. When

cDNAgog(p,o

was

annealed with

fractions

from

a

gel of M-MuLV clone 4A poly-

20-ribosomal RNA,

hybridization

of the 38S

virus-specific mRNA

was

observed, but

no

hybridiza-

0o

tion

by the

24S

virus-specific

mRNA

was

de-

2

3 4 5

6

7 tected

(Fig.

2b).

Some

annealing with the

DISTANCE (cm)

cDNAg.g(po,

was observed inthe

region

of the FIG. 2. Size analysis of M-MuLV-specific mRNA

gel

corresponding

to

subgenomic

RNA; this

may with specific cDNA. (a) One tenth of the clone 4A

havebeen due todegradd38Sv polyribosomal RNA

preparation

from Fig. 1 was

hRNA boleencdulestohegpradedara38 virunpecic

layered

ontoanagarose

gel,

and

electrophoresis

was

mRNA moleculesinthepreparation, since this performed for 105

min

at 10

V/cm.

Analysis with

3H-RNA was not selected on oligo(dT)-cellulose.

labeled

calfthymus DNA-primed M-MuLV cDNA For

comparison, annealing

across a

gel

run in (1,600 cpm per sample) was

performed

as in Fig. 1. (b)

parallel using

calf

thymus-primed

cDNA

probe

An

equal

amount

of polyribosomal

RNA was

sepa-is shown

in

Fig. 2a;

the

24S

M-MuLV-specific

rated by electrophoresis in a gelparallel to that

mRNA

is

readily

evident. These

results indicate

shown in (a). The gelfractions wereannealed with

that the 24S

virus-specific

mRNA

is

not

derived

3H-labeled

cDNAgagpo

(375 cpm per sample). The

from the 5' half of M-MuLV 38S

RNA,

but location of the

24S

virus-specific mRNA is indicated;

rather from the 3' half. It is therefore

likely

that

no

specific annealing

of

the 24S mRNAwasevident.

the

24S

virus-specific

mRNA contains

sequences

for

only

the

env gene, and

additionally

some

from the

5'

end

of the

38S

RNA, and that sequences which

do

not

code for viral

structural

transcriptionproceeds toward the 5' end of the

proteins.

viral

RNA (31). In conditions of

limiting

deox-Presence

of

transposed

5'

sequences

in

ynucleoside

triphosphate precursor, reverse

24S

virus-specific

mRNA. Recent results

transcription proceeds through

a series of

hesi-studying

mRNA's from avian

sarcoma

virus-in-

tations,

with

a strong

hesitation

as the DNA

fectedcells indicate that

sequences

correspond-

polymerase

reachesthe

5'

end of the

38S

RNA

ing

to

the

extreme5'end

of the 38S viral

genome

(17). The product of such

areaction is a series of

arepresenton

mRNA's

containing

3'

portions

of short DNA

molecules

with overlapping se-the38S

RNA

(35).

The24S

virus-specific

mRNA quences,

which

contain the same 5' end and from

M-MuLV clone

4Acellswasfurther inves-

progressively

moresequences complementary to

tigated

to

determine whether

asimilar

phenom-

theRNA between the tRNA

primer binding

site

enonwas

evident.

and the 5' end of the genome. For the experiment

To

perform

these

experiments,

cDNA corre-

reported here,

cDNA was

prepared

from

M-sponding

totheextreme5'end of

the

M-MuLV MuLV virions in

conditions where

one triphos-38S virion RNA was

synthesized.

It has been phate was

limiting

and then separated on a shown

that

the site of

binding

for

the

tRNA

preparative

polyacrylamide

gel.

Individual

primer molecule

used in reverse

transcription

of cDNAbands

corresponding

todifferent lengths RNAtumorvirus RNA is100 to 150nucleotides of cDNA5' were cutoutand

eluted,

and

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(6)

VOL. 26, 1978

SIZE ANALYSIS OF MuLV-SPECIFIC mRNA'S

473 acterization

of the bands is shown in Fig.

3.

fore

possible

that the

annealing

with cDNA

5'

Seven discrete bands,

varying inlength from 35

band

1 was at

least

partially

due

to

these

re-to 135

nucleotides (the full length between

the

peated

sequences at

the 3' end of the 24S

mRNA.

tRNA

primer binding site and

the 5' end of M-

To

test

this

possibility,

annealing

was

also

per-MuLV 38S RNA)

were

obtained, and the

length

formed with cDNA 5' band 4 (approximately

60

of each

fragment

in

nucleotides

is indicated in

nucleotides). The cDNA 5' band

4

is

approxi-the

figure. DNA sequencing of these

bands has

mately 60

to

70 nucleotides shorter than cDNA

confirmed that they

represent overlapping se-

5' band

1,

and therefore

may

lack

some orall

of

quences

(I. M. Verma,

A. Ohtsuka, and M. the sequences

that

are

complementary

to

the

McKennett, manuscript in preparation).

terminally

redundant 3' RNA

sequences.

Fig.

5

When

cDNA

5'

band

1

(approximately

130

shows

annealing

with cDNA 5'

band

4,

which

nucleotides

in

length)

was

annealed with

frac-

also indicates that

complementary sequences

tions from

a

gel

of M-MuLV clone

4A

polyribo-

were

present in both

38S and 24S

virus-specific

somal RNA, both 38S and 24S

virus-specific

mRNA.

Therefore, it is unlikely that the

an-mRNA's

effectively

annealed the cDNA,

even

nealing observed with the cDNA 5'

preparations

though the 24S virus-specific

mRNA was de- was

exclusively

due

to

terminally repeated

se-rived from the 3' end of

38S viral

RNA (Fig. 4). quencesat

the 3'

end.

These results

suggest

that

nucleotide

sequences

Since

sequences

similar

or

identical

to

those

similar or

identical

to

those found

at the extreme

found

at

the

extreme

5' end of viral 38S RNA

5'

end of M-MuLV 38S RNA

are

also

present in were present in

intracellular 24S

virus-specific

intracellular 24S virus-specific

mRNA.

mRNA,

we

tested

whether similar

sequences are

It

has been

recently demonstrated

that both present

in the

same

location in 38S virion RNA.

avian and murine RNA

tumor virus 38S RNAs

Partially

degraded

RNA

was

extracted from

M-contain

sequences at

the 3' end that

areidentical

MuLV

virions,

and the

portions covalently

at-to

those

at

the 5' end. In

the

case

of

avian

tached the 3'

poly(A)

sequences were

selected

viruses, the terminal repetition

is approximately

by

binding

to

oligo(dT)-cellulose.

The

poly(A)-21

nucleotides, whereas the repetition in MuLV

containing fragments

were

then sedimented in

a

may

be

longer (18, 28, 30; J. Coffin and W.

sucrose

gradient, and

two

size classes of

mole-Haseltine, personal communication). It is there-

cules, corresponding

to

the 400 to 600

nucleo-No.

f

b

d

f

h

Nucleotides

0 -ORIGIN-130 _

AND I

70__

_{70_}

_-BAND

4

60 -50

-40

-35

-FIG. 3. Polyacrylamide gel electrophoretic patterns of cDNA 5' bands1to7. (a-g) Electrophoretic mobility

ofisolated cDNA 5' bands1to 7,respectively. (h)Agel where 3H-labeled cDNA 5'wasmixed with 32P end-labeled cDNA 5' bands 1 and4. The excised bands contained both 32P marker cDNA and 3H-labeled experimentalcDNA. The size of thecDNA5' bands 1 to 7 wasdeterminedbyusing restriction endonuclease HaeIII-digested

4X-174

single-strandedDNAasmarker. Theentire nucleotide sequence of

qLX-1

74 DNA has beendetermined,and hence themarkersgivethe exact numberofnucleotides. The HaeIII-digestedXX-174 DNA wasvisualizedby stainingwith ethidiumbromide.Furthermore, the complete nucleotide sequence of bands2to7hasbeendetermined(Verma,Ohtsuka,andMcKennett, manuscriptinpreparation).

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(7)

100- tides and the 4,000 to6,000nucleotides nearest

cDNA5

(D

to the 3'end, were

pooled

foranalysis. To deter-mine

total virus-specific

RNA concentration in

80- these

preparations,

annealing was performed

* .

with

cDNA

complementary

to

the

3'

end of

M-A

1\ MuLV RNA(cDNA

3').

ThecDNA 3'was

syn-co 60- 28S 18S

thesized

by using

denatured 70S viral

RNA as

I it1 l l

template,

an

oligo(dT)

primer,

and

exogenously

z l l il

added

avian

myeloblastosis

virus reverse

tran-w

-u 40- scriptase. Sequences

complementary

to cDNA

a. l 3' would be present in each

poly(A)-containing

I \ ,. ;'! a

M-MuLV RNA

fragment.

An

appropriate

dilu-20- tion of 400- to 600-nucleotide 3' M-MuLVRNA

/T

'/'wXfragment could

completely anneal

the cDNA 3',

0___________________________________ .

.but

extremely

little

of the cDNA 5'

(bands

2and

2 4 6 3 or band 4) couldanneal to the same

concen-DISTANCE (cm) tration of RNAfragment (Fig. 6). It should be FIG. 4. Annealing of M-MuLV-specific mRNA notedthat cDNA 3' (approximately 500 nucleo-with cDNA 5'. An equal amount ofpolyribosomal

tides)

was

larger

than the cDNA 5'

preparations,

RNA described inFig.2 wasanalyzedinparallelto sothat in fact the cDNA 5'would anneal four-thegels of Fig. 2, exceptthatannealingwaswith3H- to eight-fold slower than the cDNA 3' if3'and

labeled cDNA 5' band1 (160cpmper

sample).

An- 5' sequences were present in the same concen-nealing andprocessingwereasinFig.2. tration (36). However, an even greater difference in

annealing

rate for the two cDNA's was ob-100-

cDNACT

(a) served in Fig. 6a, and it can be concluded that less than 10% of the 400- to 600-nucleotide 3'

80- virion RNAfragments also contained sequences

complementary to cDNA 5'. The results ofthis

60-

figure

also

support

the conclusion that the

28S 18S cDNA 5' fragments (bands 2, 3, and 4) do not 40-

recognize

the

terminally

repeated

sequences at

the 3' end of 38S virion RNA.

20-< t An

experiment

similar to

Fig.

6a was

also

20

performed

with the

4,000-

to

6,000-nucleotide

a

0 poly(A)-containing M-MuLV fragments (Fig.

ta

0-

5 6 7 8 9

6b).

Again,

it can

be concluded

that less

than

10% of the virion RNA molecules of

this

length

;-100-z cDNA54 (b)

contained

sequences

complementary

to cDNA

ir 5'. These

poly(A)-containing

M-MuLV virion

X80- RNA

fragments

were, in

fact,

as

large

or

larger

than

the

24S intracellular

mRNA.

Therefore,

60- 28S 18S the presence of sequences

complementary

to I X 1 cDNA 5' in intracellular

24S

virus-specific

40- mRNA cannot be

explained by

thepresence of

thesesequences in a similar location in M-MuLV

20- virion RNA. Rather, it appears that these

se-quences must be

transposed

ontothe 24S

virus-0

specific

mRNA

during synthesis

and

processing.

5 6 7 8 9

DISTANCE (cm)

DISCUSSION

FIG. 5. AnnealingofM-MuLV-specificRNA with Two major

M-MuLV-specific

mRNA's. shortcDNA 5'.(a) One tenthoftheclone 4Apolyri- These

experiments

indicate that there are two

bosomalRNA

preparation of Fig.

1was

layered

onto

major

virus-specific

mRNA's in cels infected anagarosegel, andelectrophoresiswasfor2.5hat majo

virus-speifimRnasin cellsifecte

6.5V/cm. Thegelwasprocessedand annealed with w

3H-labeledcalf

thymus-primed

M-MuLVcDNA(950 and 24S. The 38Svirus-specific mRNA, which

cpm per sample)asabove, exceptthatannealingwas is extremely similar if not identical in size to

for16.5 h.(b)Agelparalleltothatof(a)wasanalyzed virion 38S RNA, contains the nucleotide se-withcDNA 5' band 4(110 cpm per sample). quencesfor all three M-MuLV structuralgenes.

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(8)

475

0.4 - 0.6 Kb

poly

A+(a)

Several lines of evidence indicate that the

38S

0-

virus-specific

RNA is

responsible

for

synthesis

cDNA 5

89 of

the

internal

structural

proteins (products

of

cDNA5sE)

the gag

gene). Immunoprecipitation

of

polyri-CDNA\ED

bosomes with antiserum

monospecific for the

internal structural protein p30 selectively

en-z 50-

riches the

38S

virus-specific mRNA (23).

Fur-0

thermore, in vitro translation

experiments of

intracellular mRNA

as

well

as

isolated virion

N \ RNA indicate that

38S

viral RNA can code for

the

large-molecular-weight

precursors

(pr78

and

100- \cDNA3' pr65) of the internal structural proteins, while

RNAs

of

smaller

size do not efficiently code for

these

proteins

(10, 16).

The

experiments

re-_

4-6 Kb poly

A+

(b)

ported here also support this notion, since the

x

24S

virus-specific

mRNA

lacks

sequences

of the

>~cDNA5a() Ogag

(and

possiblypol)

gene.

Thus the 38S

virus-clNAV

specific

mRNA appears to be the only

virus-z

'-cDNA5s

specific

mRNA

that

can

code for the

internal

o

structural

proteins.

W 50

*

Although the 38S virus-specific mRNA also

contains the

sequences

for

the

env gene, it is

likely

that these sequences are not translated in

the 38S

virus-specific

mRNA. This is because in

vitro

translation studies of

intracellular

RNA

100-

-

cDNA3

indicate that

38S

virus-specific RNA directs the

l0

l

synthesis of the gag

gene-related

precursor

0O

100 000o

polyproteins,

but

no

synthesis

of

envelope

gly-TIME (min.)

10coprotein

polypeptides

occurs

from RNA of this

TIME

(min.)

size

(16,

25).

This

hypothesis

can

be

directly

FIG. 6. Annealing of cDNA 5' with

3'

fragments Of

tested

by analyzing nascent chains of polyribo-virion RNA.

(a)

Anappropriate dilution ofM-MuLV

somes

immunoprecipitated

with

anti-p30

anti-virion

poly(A)-containing

fragments, 400to600nu-

serum,

or

by

determining

the size of

virus-spe-cleotides

long,

which could anneal

'P-labeled

cDNA cificmRNA from

polyribosomes

immunoprecip-3',

was

empirically found.

This concentrationcould itated with

anti-glycoprotein

antiserum. be calculatedto be

equivalent

to

approximately

2.5

iLg

offull-length

38SRNA

per

ml

(from

the

tll2

value).

The 24S

virus-specific

mRNA is

likely

trans-3P-labeled

cDNA3'

(360 cpm per reaction)

was an- lated to

give

the

envelope glycoproteins,

since nealedwith the RNA

for different lengths of

time. 3H-

the

only

M-MuLV structural gene contained is

labeled cDNA 5' bands 2 and 3

(140 cpm per reaction)

probably the env gene. In addition, in vitro

was alsoannealed with the same concentration

of

the

translation of intracellular RNA from

Rauscher

RNA. Percentage of

maximal

annealing

is shown as

MuLV-infected

cells indicated that envelope

gly-a

function of

incubation at the

annealing

tempera-

coproteins are coded by an mRNA of

approxi-ture.Maximnal annealing forthe cDNA 3'was51% a

and

for

the cDNA 5' was 55%. At the maximum

mately

this

slze

(16). Cell

fractionation

experi-annealing

time,

cDNA5' band 4

(80 cpm)

was also

ments

also support this

hypothesis,

since

mem-annealed withthe RNA.

(0)

Annealing of

cDNA

3';

brane-bound polyribosomes, which are

respon-(0) annealing of

cDNA 5' bands 2 and

3; (A)

an- sible for the

synthesis

of membrane

glycopro-nealing of

cDNA 5' band 4.

(b)

An

experiment

similar

teins,

are enriched for the 24S

virus-specific

to that describedin

(a) wasperformed, usingpoly(A)-

mRNA (15). It is also possible that additional

containing

M-MuLVvirion RNA

fragments of 4,000

nonstructural viral proteins coded by the

24S

to

6,000

nucleotidesin

length.

virus-specific mRNA might also be synthesized,

but no such

proteins

have

yet

been

identified.

The24S

virus-specific

mRNA is

predominantly

Athird

virus-specific

mRNA

likely

tocode for

derived

from the 3'

end

of

the

38S viral

RNA,

the

viral

reverse

transcriptase

wasnot

identified

and

likely

contains sequences

forthe env

gene

in these

experiments.

The

amount of reverse

as

well

as3'RNA

sequences which

donotcode

transcriptase synthesized

in

infected

cells

is

ap-for known virus

structural

proteins.

Sequences

proximately

10-fold less

than the

internal

struc-transposed

from theextreme5'

end

of

38S

virion

tural

proteins

or

envelope glycoproteins,

and it RNA are

also present

in the 24S

virus-specific

is therefore

likely

thata

putative

mRNA

coding

mRNA. forreverse

transcriptase

alone

would

be present

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(9)

476

in an

approximately 10-fold lower

amountthan

mRNA's.

In these

viral

RNAs it has been shown

either the 38S

or24S

virus-specific mRNA's. An

that short

nucleotide

sequences arepresent at

mRNA

present at

these levels would

very pos- the

5'

ends

of the mRNA's

which are derived

sibly

have

been missed in these

experiments,

fromregions

of the viral

genome

physically

sep-especially if its size

werevery

close

to

the size of

aratefrom

the region coding

themRNA, and it one

of the

major

virus-specific mRNA's. There-

has beenproposed that these sequences may be

fore these

experiments

cannot

be taken

as

evi-

transposed by RNA

ligation

during processing

dence that

a

unique mRNA coding only for

of viral RNA

molecules into functional

mRNA reverse

transcriptase does

not

exist.

An

alterna-

(1, 5, 6, 22). The results reported here suggest

tive

explanation of the mechanism of synthesis

that

a

similar situation

may exist for M-MuLV

of

reverse

transcriptase has been

suggested by

24Svirus-specific mRNA, although the location

the

finding of

small amounts

of

a very

large

of the

transposed

5'sequences

in

the 24S

RNA

molecular

weight (approximately 180,000)

pro- has not yet

been identified.

It is interesting to

tein, pr180, with

antigenic determinants for both

note

that

alkali

degradation of 38S virion

RNA

internal structural

proteins and

reverse tran- to

the

samesize as

24S intracellular

mRNA does

scriptase

(7,

21).

It

has been

proposed that pr180

not

produce

an mRNA

active

in

translation

of

arises

by

read-through

translation of the

gag

the 3'

terminal

env gene (K. Beemon and T. gene

into the pol

gene,

the

prl80

protein being

Hunter,

personal communication).

It may be

cleaved

to

produce the

reverse

transcriptase.

that

transposition of the 5'

sequences to the

24S

Recent

evidence

supporting this

hypothesis has

virus-specific

mRNA may be necessary for

acti-been

reported in which increased

amounts

of the

vation of the initiation site for

envgene

trans-prl80

protein

were

synthesized

in

vitro if

ayeast lation.

amber

suppressor

tRNA

was

added

to

the

reac-

Quantification of mRNA's. In

the gel

hy-tion mixture

(L. Phillipson, P. Andersson, R.

bridization

experiments shown here,

data were

Weinberg,

D.

Baltimore,

and

R. Gesteland, sub-

presented

as percentageof cDNA hybridized. In

mitted for

publication). Synthesis

of the prl80

these

values

were

converted

to

protein would

not

require another virus-specific

relative

virus-specific RNA

concentration (11).

mRNA in addition

to

38S

mRNA,

so

that

only

For

this

conversion

to

be

performed, it

was nec-two

virus-specific mRNA's could

account

for

essary to know the

maximum

hybridization level

synthesis of all of

the

viral structural

proteins.

of

cDNA for each

RNA

sample;

in sucrose gra-However, it

has

still

not

been

rigorously

estab-

dient

analyses,

every

fraction maximally

hybrid-lished that

prl80

is the in vivo

precursor

for

ized the cDNA

probe. However,

in

the

higher-biologically

active

reverse

transcriptase.

resolution

experiments

reported here, it is likely

We

recently proposed

a

model

of

M-MuLV-

that

the

relatively

pure

24S virus-specific

specific mRNA

organization

and function in

mRNA could

not

maximally hybridize

a

rela-which the

virus-specific

mRNA's consisted of

tively

representative calf thymus-primed

cDNA.

three

(or

possibly only

two) molecules with

se-

Therefore,

conversion

to

relative

virus-specific

quences

overlapping

from the

3'

end

(24).

In RNA

concentration could

not

be

readily

per-each

case,

only

the

5'

terminal

gene

would

be

formed. The

hybridization

of the

gel with

cDNA

active

in

protein

synthesis,

and the

internal

5'

band

4 in

Fig. 5b

was

noteworthy

in

this

genes

would

not

be

translated. The results

pre- respect.

The

cDNA 5'

represented only

about 60

sented

here,

showing

that the 24S mRNA

rep-

nucleotides,

whichare

perhaps completely

pres-resents sequences

from the

3'

end of

the

M- entin both 24S

and 38S

virus-specific mRNA.

MuLV genome,

support this

model.

A

similar

Therefore,

the

relative concentrations of the

two

organization

for

the

virus-specific

mRNA's

in

mRNA's could be inferred from the levels of

avian

sarcoma viruses has

also

recently

been

hybridization

in the 38S and 24S

peaks. Thus,

proposed (35).

Fig.

5b indicates that the concentrations of 38S

Transposition of

RNA sequences in

24S

and 24S

virus-specific

mRNA in

purified

poly-mRNA. Our results indicate

that sequences

sim-

ribosomes

are

approximately equal.

This

result

ilar or

identical

tothoseat

the

extreme5'

end

of was not

immediately

obvious from the

gel

hy-the

viral 38S

RNAarepresent in the

24S

virus-

bridization

of

Fig.

5a, in which calf

thymus-specific

mRNA,

similar toresults

reported

for primed cDNAwasused. This result is in agree-avian RNA tumor viruses. The data of

Fig.

6 ment with the observation that

the

amountof

futhermore

indicate that

equivalent

sequences

synthesis

of gag and envrelated

protein

is ap-arenotpresent in the 3'

half

of

38S

virion RNA.

proximately

equal,

as

measured

by

pulse-label-Thepresence of

RNA

sequences fromnoncon-

ing

and

immunoprecipitating

viral

proteins

from

tiguous

regions

of the viral genome in 24S virus-

infected

cells

(S.

Edwards,

unpublished

obser-specific

mRNA may be

similar

tothesituation

vation).

observed

for adenovirus and

papovavirus

Results similar to these have also been

on November 10, 2019 by guest

http://jvi.asm.org/

(10)

477 tained

by

Rothenberg

et

al.

(E.

Rothenberg,

D. sequence complexityof cloned Moloney murine

leuke-J.

_J.

Dngu,and D. Baltimore, Cell, in press). miavirus60 to

708

RNA: evidence forahaploid ge-Donoghue,

and D.

Baltimore,

Cell, in

press).

nome. J.Virol.14:421-429.

In

addition, these workers have obtained elec-

15.

Gielkens,

A.L J.,M. H.

L.

Salden,

and H.

Bloemen-tron

microscopic

heteroduplex

evidence for the

dal. 1974.Virus-specificmessengerRNA onfreeand

transposition

of virion RNA 5' sequences in membrane-bound polyribosomes from cells infected subgenomicvirus-specific

polyAcontainwith

Rauscher leukemia virus. Proc. Natl. Acad. Sci.

suRgenomic

virus-specic

poly(A)-containing

U.S.A. 71:1093-1097.

RNA. 16.Gielkens, A. L. J., D. van Zaane, H. P. J.Bloemers,

ACKNOWLEDGMENTS and H. Bloemendal. 1976. Synthesis of Rauscher mu-rineleukemiavirus-specificpolypeptides in vitro. Proc. Wethank PaulMacIsaac, Marianne McKennett, and Rich- Natl. Acad. Sci. U.S.A.73:356-360.

ard Swanson for excellent technicalassistance. Mei-Huei Lai 17.Haseltine, W. H., D. G.Kleid,A.Panet, E. Rothen-and DavidDolberg kindlyprovided purified reversetranscrip- berg, and D.Baltimore. 1976. Orderedtranscription tase and Moloney murine sarcoma virus cDNA. We thank ofRNA tumor virusgenomes. J. Mol. Biol.106:109-131. CarolynGoller fortypingthemanuscript,and themembers of 18. Haseltine, W. A., A. M. Maxam, and W.Gilbert.1977. the TumorVirologyLaboratoryforsuggestions and discus- The Roussarcoma virusgenome isterminally

redun-sion. dant: the 5' sequence. Proc. Natl. Acad. Sci. U.S.A.

This workwassupportedbyresearch grants no.CA-16561 74:989-993.

andCA-21408toI.M.V. from theNational Cancer Institute, 19. Hayward, W. S. 1977. Size and genetic content of viral grantno.CA-15747toH.F. from theNational CancerInstitute, RNAs in avian oncovirus-infected cells. J. Virol. and core grantno. 14195from theNationalCancerInstitute. 24:47-63.

LITERATURE CITED 20. Hu,S.,N.Davidson,and I. M.Verma.1977.A hetero-duplexstudy of the sequencerelationships between the 1.Aloni, Y.,R. Dhar, 0. Laub, M. Horowitz, and G. RNAs ofM-MSV and M-MLV. Cell10:469-477.

Khoury.1977.Novel mechanism for RNAmaturation: 21. Jamjoom,G.A., R. B. Naso, and R. B.Arlinghaus. theleader sequences of simian virus40mRNA are not 1977.Furthercharacterizationofintracellular precursor transcribed adjacent to the coding sequences. Proc. polyproteins of Rauscher leukemia virus. Virology Natl. Acad. Sci.U.S.A.74:3686-3690. 78:11-34.

2. Ball, J.K., J. A.McCarter, andS. M. Sunderland. 22. Klessig, D. F. 1977. Two adenovirus mRNA's have a 1973.Evidence forhelper independentmurine sarcoma common 5'terminal leader sequence encoded at least virus.1.Segregationofreplication-defectiveandtrans- 10 kB upstream from their main coding regions. Cell formation-defective viruses.Virology56:268-284. 12:9-22.

3. Baltimore, D. 1974.Tumor viruses: 1974. ColdSpring 23. Mueller-Lantzsch,N., and H.Fan. 1976. Monospecific HarborSymp. Quant.Biol. 39:1187-1200. immunoprecipitation ofmurineleukemia virus polyri-4. Beemon, K. 1977. Oligonucleotide fingerprinting with bosomes:identification ofp30protein-specific

messen-RNAtumorvirus RNA. Curr.Top.Microbiol.Immu- gerRNA. Cell9:579-588.

nol.76:73-110. 24. Mueller-Lantzsch, N., L. Hatlen,and H. Fan. 1976.

5. Berget, S.M.,C.Moore,and P.A.Sharp.1977.Spliced Immunoprecipitationof murineleukemiavirus-specific segmentsatthe 5'terminus ofadenovirus2late mRNA. polyribosomes: identificationofvirus-specific messen-Proc. Natl.Acad. Sci. U.S.A. 74:3171-3175. gerRNA, p. 37-53. In D.Baltimore, A. S. Huang, and 6. Chow, L. T.,R.E.Gelinas, T. R.Broker, and R. J. C.F.Fox(ed.),Animalvirology. Academic PressInc.,

Roberts. 1977.Anamazing sequence arrangementat NewYork.

the5'ends ofadenovirus2messenger RNA. Cell12:1-8. 25. Pawson,T.,G. S.Martin,and A. E.Smith. 1976. Cell-7.Dina, D.,and K.Beemon. 1977.Relationship between free translation of virion RNA fromnondefectiveand Moloney murine leukemia and sarcomavirus RNAs: transformation-defective Roussarcoma viruses. J. Vi-purification andhybridization map ofcomplementary rol.19:950-967.

DNAsfrom definedregionsofMoloneymurine sarcoma 26. Penman, S. 1966. RNA metabolism in the HeLa cell virus124.J. Virol. 23:524-532. nucleus. J. Mol. Biol. 17:117-130.

8. Dina,D.,K.Beemon,and P. H.Duesberg.1976.The 27. Schincariol, A.L,and W. K. Joklik. 1973.Early syn-30S Moloney sarcoma virus RNA containsleukemia thesis ofvirus-specific RNA and DNA incellsrapidly virusnucleotide sequences. Cell 9:229-309. transformed with Rous sarcoma virus. Virology 9. Fan,H.1977.RNA metabolism of murineleukemiavirus: 66:532-548.

size analysis of nuclear pulse-labeled virus-specific 28. Schwartz, D. E., P. C. Zamecnik, and H. L Weith. RNA. Cell 11:297-305. 1977.Rous sarcoma virus genome isterminally redun-10. Fan,H.1977.Expressionof RNAtumorvirusesattrans- dant: the 5' sequence. Proc. Natl. Acad. Sci. U.S.A.

lation and transcriptionlevels. Curr. Top. Microbiol. 74:994-998.

Immunol.79:1-41. 29. Shanmugam, G.,S.Bhaduri,and M.Green.1974.The 11. Fan,H.,and D.Baltimore. 1973.RNA metabolism of virus-specific RNA species in free and membrane-murine leukemia virus:detection ofvirus-specificRNA bound polyribosomes oftransformed cell replicating sequences ininfected and uninfectedcellsand identifi- murinesarcoma-leukemia viruses. Biochem. Biophys. cation ofvirus-specific messenger RNA. J. Mol. Biol. Res. Commun.56:697-702.

80:93-117. 30. Stoll,E.,M. A.Billeter,A.Palmenberg,and C.

Weiss-12.Fan, H., and P. Besmer. 1975. RNA metabolism of mann. 1977.Avian myeloblastosis virus RNA is ter-murine leukemia virus. II. Endogenous virus-specific minallyredundant: implicationsforthemechanism of RNA in theuninfected BALB/ccell line JLS-V9. J. retrovirusreplication.Cell12:57-72.

Virol.15:836-842. 31. Taylor, J. M. 1977. An analysis ofthe role oftRNA 13. Fan, H.,andH.Mueller-Lantzsch. 1976.RNA metab- speciesas primersforthetranscription into DNA of olism of murineleukemiavirus. III.Identificationand RNA tumorvirus genomes. Biochim. Biophys. Acta quantitationofendogenous virus-specificmRNAinthe 473:57-71.

uninfected BALB/c cell line JLS-V9. J. Virol. 32. Taylor, J. M., R. Ilmensee, and J.Summers. 1976.

18:401-410. Efficient transcription ofRNA into DNA by avian

14. Fan, H., and M. Paskind. 1974. Measurement ofthe sarcoma virus polymerase. Biochim. Biophys. Acta

on November 10, 2019 by guest

http://jvi.asm.org/

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442:324-330. tive viruses are at the poly(A) end. J. Virol. 16: 33. Vogt, P. K., and S. S. F. Hu.1977.Thegenetic structure 1051-1070.

ofRNAtumor viruses.Annu. Rev.Genet. 11:203-238. 35. Weiss, S. R., H. E. Varmus, and J. M. Bishop. 1977. 34. Wang, L.-H., P. Duesberg, K. Beemon, and P. K. The size and genetic composition of virus-specific Vogt.1975.Mapping RNase T,-resistant oligonucleo- RNA's in the cytoplasm ofcells producing avian sar-tides of aviantumor virus RNAs: sarcoma-specificoli- coma-leukosis viruses. Cell 12:983-992.

gonucleotides are near thepoly(A) end and oligonucle- 36. Wetmur, J. G., and N. Davidson. 1968. Kinetics of otides commontosarcoma andtransformation-defec- renaturation of DNA. J. Mol. Biol. 31:349-370.