Multiple mRNA species for adenovirus type 2 polypeptides III and pVII.

0022-538X/80/08-0306/08$02.00/0

Multiple

mRNA

Species

for Adenovirus

Type

2

Polypeptides

III

and

pVII

CHARLES B. LAWRENCE

Department ofCellBiology, Baylor College of Medicine, Texas MedicalCenter, Houston,Texas77030,* and

The SalkInstitute, Tumor

Virology Laboratory,

SanDiego,

California

92138

Fractionationofmessengeractivities isolated from thecytoplasmofHeLacells

late in infectionwith adenovirus type 2 reveals that viral polypeptides III and

pVIIareeach synthesizedfrom two different-sized mRNA's. Themajormessenger

activity for each protein has the same sedimentation rate as that previously reported byAnderson et al. (Proc. Natl. Acad. Sci. U.S.A. 71:2756-2760, 1974). The minormessengeractivities for III andpVII sediment morerapidlyandare

notaggregatesof themajormRNA's for theseproteins.Thetwo minormessenger

activities cosediment with two polyadenylated RNA species which are labeled

late in infection with 32Pand whosemolecularweights areestimatedtobe 2.9 x

106 and 2.4 x 106. Both of these species hybridize to adenovirus type 2 DNA

specific for the mRNA family that is 3' coterminal at adenovirus type 2 map

position49.5 and the mRNAfamilythat is 3' coterminalat 62.0. This isconsistent

with the possibilitythat these RNAs have 5'-terminalsequencesidentical tothose

of the normal mRNA's for III andpVIIbutare3' coterminalatmapposition 62,

the normal 3' terminus of the mRNA's forpolypeptidesIIandpVI.Thesespecies

arenotfound inpolyadenylatedRNA isolated from the nucleus,suggestingthat

the minor mRNAspeciesarecytoplasmicRNAs.

Thelate mRNA'sof adenovirus type2 (Ad2)

coded tothe right of Ad2 map position 16 are

arranged in five families whose members have

overlapping 3'-terminal sequencesanddifferent

lengthsof sequencesin the5'regions (6, 16, 19,

24). These mRNA's are synthesized by

differ-ential processing of a single unique transcript

which is initiatedatposition 16and transcribed

rightward to theendof the genome (7, 8, 23, 25).

Cleavage and polyadenylation of the nascent

transcript occur at one of five possible sites which correspond to the 3' termini of the five mRNA families (20). The partially processed

polyadenylated transcript is then further

proc-essedby thesplicingof sequencesat16, 19,and

27 toform a 200-base 5' leader sequence (3, 5,

10) which is subsequently spliced to one of a limited number of acceptor sites coded within

the family that lies to the 5' side of the poly-adenylation site. The slicing of the leader to the mainbody of the mature mRNA may occur by way of a series of intermediates in which the leader is transferred to various acceptor sites along the transcript between the leader and the mainbody of the mRNA (18, 20).

In the course of fractionating cytoplasmic RNAs isolated from HeLa cellsduring the late phase of Ad2 infection, we observed two size classes of messenger activities for each of the viralproteinsIIIandpVII. The major messenger

activities corresponded in size to those

previ-ously reported for these proteins. The minor messenger activities for these proteins were,

however, much larger and may correspond to

molecules which have a 3' terminusatthe site

normallypresentinthemRNAfamily

immedi-ately downstreamfrom thefamily that encodes

IIIand pVII.

MATERIALS AND METHODS

Cells. HeLa S3 cellswere grownin suspension in

Joklik-modified Eagleminimalessentialmedium

sup-plemented

with 5%calfserum.

Virus. Stocks of Ad2were obtained from Gernot

Walter. Cells were infected at a multiplicity of 20

PFU/cell. Virus was adsorbed for 30 min at room

temperatureandataconcentrationof4x10'cellsper

nml. Cellswerethendilutedto aconcentration of4 x

I105/mlwithfresh medium, andinfection was allowed

tocontinuefor24h.

Labelingof cells. Cellswerelabeledat24 hafter

infectionwith32p; (ICNPharmaceuticals, carrier free). Infectedcells (100 ml) were collected by centrifugation at 1,000 rpm and washed once with Eagle minimal essential medium without phosphate. Cellswere

re-suspended in 50 ml of this phosphate-free medium

supplementedwith2%dialyzedcalf serum;25mCi of

32p;wasadded,andthe cells were incubated for 3 h at

370C.

Preparationof RNA. At24hafter infection with

Ad2,4 x 10' HeLa cellswerecollectedby centrifuga-tionat 1,000 rpm, washedoncewith cold saline,and

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Ad2 mRNA 307

suspended in 1.7ml of cold isotonic lysis buffer (0.15

MNH4CI, 10 mM Tris[pH6.8],2mMMgCl2, 50

Ag

of

dextransulfate per ml). Then 0.2 ml of 10% Nonidet

P-40(Shell Chemical Co.) was added, followed by brief

blending in a Vortex mixer;0.1 ml of 10% deoxycholate

was added, and the suspension was blended for 30 s.

Nuclei werepelleted by centrifugation at 2,000 rpm for

2min, and then 90

AI

of 0.5 M.EDTA (pH 7.0), 80yd

of 4 MNaCl, 0.2 ml of 10% sodium dodecyl sulfate,

and 20[L of,B-mercaptoethanol were added to the

supernatant. To makecytoplasmic RNA, the

super-natant waswarmed to30°C and extracted with2 ml

of phenol. Chloroform (2 ml) was added, and the

mixture was reextracted. Phases were separated by

centrifugationat 10,000 rpm for 20 min at25°C. The

aqueous phase wasreextracted with 2 mlof

chloro-form, and the phases wereseparated as above. The

aqueousphasewasthencollected, and RNAwas

pre-cipitated by the addition of 2 volumes of ethanol.

Precipitated RNA was stored in aqueous ethanol at

-20°C. Polyadenylic acid [poly(A)]-containing

cyto-plasmic RNA was obtained by chromatography on

oligodeoxythymidylic acid-cellulose (T-3;

Collabora-tiveResearch, Inc.).

To make nuclear RNA, nuclearpellets were

sus-pended in1.0mlof0.5MNaCl-50 mMMgCl2-0.01M

Tris (pH 7.5), 60

A1

of0.5 M EDTA, 50

t1I

of20%

sodiumdodecyl sulfate, and 100 1Iof proteinase K (5

mg/ml)wereadded, and the suspensionwasincubated

at roomtemperature for 15min.Water (1.0 ml) and

BO-mercaptoethanol

(20

tlI)

were added, and the

sus-pensionwasagitated briefly. The suspensionwas

ex-tractedwithphenol and chloroformasdescribed above

forcytoplasmicRNA.

Poly(A)-containing nuclear RNA was selectedby

mixing the aqueous phase from theextracted nuclei

with anequal volume of0.5M LiCl, 1 mM EDTA,

and10mMTris(pH 7.5).Oligodeoxythymidylic

acid-cellulose (0.2 g)wasadded andkept in suspensionby

intermittentagitationat4°Cfor30min, thencollected

bylow-speedcentrifugation and washed three times

with 5 ml ofice-cold0.5MLiCl-1 mM EDTA-10 mM

Tris(pH 7.5). Bound RNAwaselutedbywashingthe

oligodeoxythymidylicacid-cellulosetwotimeswith1.0

ml of sterile water at roomtemperature. The eluted

RNA was adjustedto 0.2 Msodium acetate(pH5.2)

andprecipitated with2volumes ofethanol.

Sucrosegradientcentrifugation.Nuclearor

cy-toplasmic RNAs in a total volume of0.5 ml were

layeredon10.6-mlgradients of10 to30%sucrose(wt/

wt) in 10 mMsodiumacetate(pH5.2)-imM

EDTA-0.1%sodiumdodecylsulfate. Centrifugationwas

per-formed at-20°C for4.5 hat 40,000 rpm for nuclear

RNAs and for 15 h at 30,000 rpm for cytoplasmic

RNAs in aBeckmanSW41rotor.

Agarosegelelectrophoresis. [32P]RNA was

re-acted with glyoxal according to the procedure of McMaster and Carmichael (17). After reaction with

glyoxal,thevarioussamplesweremixed with anequal

volumeof 1 mM EDTA(pH7.0)-50%glycerol-0.001%

bromophenol blue and applied directly to a

4-mm-thick 1%agarosegel(Sigma Type V)containing Leon-ingbuffer Eplus0.1% sodiumdodecyl sulfate.

Electro-phoresiswas for 18 h at 75 V. After electrophoresis,

the gels were dried onto Whatman 3 MM paper.

Radioactive RNAspecies werevisualized by

autora-diography enhanced with Du Pont Cronex

Lightning-Plusintensifying screens.

Cell-free translation. RNAs were translated in

the messenger-dependent rabbit reticulocyte lysate

systemdescribedby Pelham and Jackson (21) in the

presenceof[35S]methionine (Amersham Corp., 1,000

Ci/mmol).

Polyacrylamide gel electrophoresis. Proteins synthesized in vitro were resolved by diluting reaction

mixtures with5volumes of sample buffer and applying

5

p1

to eachlane ofa 1-mm-thick,9.5-cm-long

poly-acrylamideslab gel according to the system described

by Laemmli and Maizel (12, 15). Electrophoresis was for4.5hat a constantcurrentof 15 mA.

DNA-paper andhybridization protocols.

Puri-fied plasmid DNA was coupled to

diazobenzyloxy-methyl-paper by the method of Stark and Williams

(22). Each coupling reaction contained 100

,Ag

of

sheared, denatured plasmid DNA and a

1.0-cm-diam-eterpapercircle.

Hybridizationswereperformed in 50%

formamide-0.4 M NaCl-20 mM PIPES

[piperazine-N,N'-bis(2-ethanesulfonicacid)] (pH 6.4)-5 mM EDTA-0.2%

so-diumdodecyl sulfate at37°C for 5h. Each reaction

contained one DNA-paper circle, 200 ,ug of yeast

tRNA, and 100,000 to 500,000 dpm of 32P-labeled

poly(A)-containing RNA in afinal volume of140 Il.

The DNA-paperwaswashed three times (15 min each

time) in50%formamide-0.1 MNaCl-10 mM PIPES

(pH 6.4)-5 mM EDTA-0.2% sodium dodecyl sulfate at

32°C, andtwotimes (5mineach time) in 0.2 M

NaCl-20mM PIPES (pH 6.4)-5 mM EDTA-0.1% sodium

dodecyl sulfateat55°C. Hybridized RNA was eluted

by washing the DNA-papertwotimes with 100ulof

99%formamide-1 mM EDTA at55°C and one time

with 200

pl

of 1.0mM EDTA at55°C. Eluted RNA

wasprecipitatedfrom the pooled washes by the

addi-tion of20 ug of yeast tRNA, 40

pl

of2 M sodium

acetate(pH 5.2) and1ml of ethanolat-20°C.

RESULTS

Two messenger activities for m and

pVH.

Total

cytoplasmic

RNAwasisolated from

Ad2-infected HeLa cellsat 24hafter infection

and fractionated on a sucrosegradient.Aportion

of each fractionwastranslated in the

messenger-dependent rabbitreticulocyte

lysate

in the

pres-enceof

[35S]methionine,

andtheproductswere

analyzed by

polyacrylamide

gel

electrophoresis.

Afluorogram ofthisgel isshown inFig. 1.The

major activitiesfor III andpVII sedimentedat

25S and21S,respectively,aspreviously reported

by Andersonetal. (1). Minormessenger

activi-ties for III and

pVII

werealso

found,

sediment-ing at 32S and30S,respectively (Fig.1, fractions 5, 6, and7).

To examine the possibility that these

repre-sent aggregated forms of the major messenger

activities,

32P-labeled, poly(A)-containing

RNA

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308 LAWRENCE

w 4 O N

15 ;

.iw.*...

...A,.XX

-_

..._____i

_______..v.::^:

_{__w.=E}

_LZ===

_ __ _ _ __

_Fr-111|

9-. X -_ Y ..

k . y*d

SLt-AR&.;&4s..5..ss#

us =33; ze...

aw,."-4w.

FIG. 1. Translationof size-fractionated Ad2 mRNA's. A100-,.gsampleofpoly(A)-containingRNA isolated

from the cytoplasmofHeLa cells infected for24hwith Ad2 was fractionated on sucrose gradients as described

in the text. Thegradient was dividedinto 0.45-mlfractions, and RNA in each fraction was collected by

ethanolprecipitation and then dissolved in 50ytlof water; Itldof eachwastranslated in a 10-,Il reaction

mixtureasdescribed in thetext.One-tenthof each reaction mixture was analyzed on a 12.5% polyacrylamide

gel, and35S-labeledproteinswerevisualizedby fluorography.

wasisolatedfrom HeLa cellsat24hafter

infec-tion with Ad2. The RNA was sedimented

througha sucrosegradient, andaportion of each

fractionwas translated and analyzed as shown

inFig. 1.Thefaster-sedimentingmessenger

ac-tivitiesfor III andpVII werepooled, aswellas

themajor peak ofactivityfor III.ThetwoRNA

poolswereprecipitated, dissolvedinbuffer, and

heatdenatured; each was sedimented through a second gradient. The peak fractions of

radioac-tivity from each gradient were again collected

andsedimentedthrough a third gradient as

de-scribed above. The radioactivity profiles from the thirdgardientareshown inFig.2.[32P]RNA

associatedwith thefast-sedimenting messenger activities for III and pVII (Fig. 2A) still sedi-mented faster than did the[32P]RNAassociated

withthemajorIII messengeractivity (Fig. 2B).

The RNAs in the peak fractions from each

gradient were precipitated, and a sample of

eachwastranslated invitro in the presence of

[35S]methionine and analyzed on a

polyacryl-amidegel. The translation productsareshown inFig. 3.The peaks of minor messenger activi-ties for III and pVII (Fig. 3A) and the major

peak of III mRNA activity (Fig. 3B) cosedi-mented with their respective peaks of

radioac-tivity (Fig. 2A and B). The minor messenger

activity for III sedimented slightly faster than

did the minor messenger activity for pVII, sug-gesting the presence of two distinct RNA spe-cies. It was apparent that the minor messenger activities for III and pVIIstillsedimented more

rapidlythan did the major activity for III, even

after3cyclesofheat denaturation and

sedimen-tation.

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[image:3.504.68.446.69.404.2]

Ad2 mRNA 309

C\j

0

x E

a.

CM N

0 x E a.

C-NtL

5

10

15

20

25

molecular-weight speciesais theminormRNA

for

III and thatspecies b is the minor mRNA

forpVII. The peak fraction from Fig. 2B (the

major mRNA for III) contains three labeled

bands(species c,d, ande), whichmigrated more

rapidly than did species a and b (Fig. 4, lanes 2

and5). Since the peak fraction from Fig. 2B is

associated withmessengeractivitiesfor the III,

lOOK, and pVI proteins (Fig. 3B) specified by

4 5 6 7 8 9 O 11 12 13 14 15

A

-III

-PVII

FRACTION NUMBER

FIG. 2. Sedimentation of32P-labeledAd2 mRNA's

afterthree cycles ofsedimentation and heat

denatur-ation.32P-labeled,polyadenylatedRNA was isolated

asdescribed in the text. Minor mRNA's(30S and 32S)

forpolypeptides III and pVII (A) and the major

mRNAforIII(B) were isolatedfrom a sucrose

gra-dient andsubjected to two subsequent cycles of heat

denaturation and sedimentation. The figure shows

radioactivity present in each fraction of the third

sucrosegradient.

4 5 6 7 8 9 10 11 12 13 14 15

-B

m

-100K

- III

Toconfirm that therapidly sedimenting

activ-ities for III and pVII were not aggregates of

smaller RNAs, the

peak

fraction ofradioactivity

fromeachgradientwastreated withglyoxal (17)

andanalyzedon anagarosegel. The peak

frac-tion of radioactivity from Fig. 2A (which has

messenger activity for both III and pVII)

con-tainedtwo

species

of RNA

(Fig.

4,lanes1and4)

designated a and b. Ithas recently been

dem-onstrated that the only polyadenylated RNAs

transported

to the cytoplasm late in infection

are adenovirus specific (2). It is demonstrated

below that the 32P-labeled species a and b are

cytoplasmic and contain sequences from the

mRNA familylocated at map positions 39.0 to

49.5, strongly suggesting that they do in fact

correspond tothe minormRNA's for

polypep-tides III andpVII. Since the minor mRNA for

IIIsedimented

slightly

faster than did the minor

mRNA for

pVII,

it is likely that the

[image:4.504.80.227.66.344.2]

higher-_ _-iUbam -pVI

FIG. 3. TranslationofRNAsisolatedasdescribed in thelegendtoFig.2.RNA ineachfractionofthe

gradientsinFig.2wascollectedbyethanol

precipi-tation and dissolvedin 10,ilofwater,1,ld fromeach

fraction was translatedinvitro, and 35S-labeled

pro-teinswereanalyzedon a10%polyacrylamidegeland

visualizedbyfluorography, asdescribed inthetext.

Fractions in(A) and(B) arethesameasthosefrom

Fig.2(A)and(B),respectively.

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[image:4.504.262.457.139.567.2]

50.1)

restriction

fragment

immobilized on

dia-zobenzyloxymethyl-paper.

Hybridized

RNAwas

eluted,

treated with

glyoxal,

and

analyzed

on an

agarose

gel.

An

autoradiograph

of this

gel

is shown in

Fig.

5. Two

major

32P-labeled

species

which

migrated

faster than did 28S RNA

hy-bridizedtothisDNA

(Fig.

5,

lane3).

They

most

likely

correspond

to the

major

mRNA's for III and

pVII.

The minor

species

wehave

designated

aand b also annealed

specifically

toHindIIID

DNA, demonstrating

the presence ofsequences

from map

positions

41.0to50.1 in these

species.

Because theradiolabeled

species

a and b

co-sedimented with the minor messenger activities for III and

pVII

and contained sequencesfrom the

region

oftheAd2 genomeknowntocodefor these

proteins,

we conclude that these

species

are in fact the minor mRNA's for these two

proteins.

Since

only

5'-terminal geneson

poten-tially

polycistronic eucaryotic

mRNA's are

translated

(11),

it follows that the

coding

se-quences for III and

pVII

are

likely

tobe onthe 5'terminus of

species

aand b and thatadditional sequencesnot

normally

presentin III and

pVII

mRNA's are present on their 3' termini. One

possibility

isthat theseadditional sequencesare

coded

by

a

region

of the Ad2 genome

immedi-ately

downstream from the III andpVIImRNA

family, namely,

the

region

whichnormally spec-ifies II and

pVI

mRNA's. Totestthis

hypothesis,

32P-labeled,

cytoplasmic,

poly(A)-containing

RNAisolated fromAd2-infectedHeLa cellswas

fft..

FIG. 4. Agarose gel electrophoresis of

[32P]-mRNA's.32P-labeled RNAsweretreatedwithglyoxal

andanalyzedon a1.0%/agarosegel asdescribedin the text. Lane 1, Fraction 8 from Fig. 2A; lane 2,

fraction 11 from Fig. 2B; lane 3, total 32P-labeled,

poly(A)-containing RNA from HeLa cells infected

withAd2for24h;lanes 4to6, same asI to3except

exposed longer; lane 7, ['4C]rRNA's; Lane 8, same aslanes 3 and 6.

adenovirus,

it was not

surprising

to find three

32p-labeled

RNA

species

in this fraction. Total

32p-labeled, poly(A)-containing,

late Ad2 RNA

was also run in a

parallel

lane

(Fig.

4, lanes 3 and 6).

Species

a, b, c, and dareevident inthis RNA.

Species f

and gareidentified belowasthe mRNA forhexon and the 215 mRNAfor

pVll,

respectively.

32S and 30S

32P-labeled species

have

se-quences from Ad2 map

positions

41.0 to

50.1 and 56.5 to

63.7.

Toprovethat the

high-molecular-weight

32p-labeled

RNA

species

con-tained sequences from the

region

of the Ad2

genome

coding

for III and

pVIL, 32p-labeled,

cytoplasmic,

poly(A)-containing

RNA from Ad2-infected HeLa cells was

hybridized

to a

cloned Ad2 HindIII D

(map position

41.0 to

a.

-:*

I.

FIG. 5. Selectionof 32P-labeledcytoplasmic RNAs

on restriction fragments ofAd2 DNA. 32P-labeled

cytoplasmic,poly(A)-containingRNA washybridized

to cloned Ad2restriction fragments asdescribedin

thetext.SelectedRNAwastreatedwith glyoxal and

analyzedon a1.2%oagarosegel.Lane 1,

["4C]rRNA;

lane2,total32P-labeled,

cytoplasmicpoly(A)-contain-ingRNA isolatedfromHeLa cells infected with Ad2

for 24 h; lane 3, 32P-labeled RNA hybridizing to

cloned Ad2HindIIIDDNA;lane 4,32P-labeled RNA

hybridizing tocloned Ad2 PstE DNA; lane 5, same

aslane4exceptlongerexposureofautoradiograph.

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Ad2 mRNA 311

hybridized to aclonedAd2 PstE (map position

56.5 to63.7)restrictionfragment immobilizedon

diazobenzyloxymethyl-paper.

Two major spe-cies of RNA hybridized to this DNA fragment (Fig. 5, lanes 4 and 5), corresponding to the mRNA's for pVI and II. Both species a and b

also hybridizedto this DNA,demonstratingthe

presence of sequences coded inthe 56.5 to 63.7 region in these RNAs. Species a and b do not

hybridizeto DNAspecific for fiber mRNA (data

notshown).

The 32S and 30S mRNA's are not found

in the nucleus. The presence of

high-molecu-lar-weightmessenger activities for III and pVII

among cytoplasmic mRNA's suggested that

some

partially

processed nuclear RNAs may

have been present in our cytoplasmic mRNA

preparations. To determine whether species a

and b werepresent in thenucleus,

32P-labeled,

nuclear, poly(A)-containing RNA was isolated

and sedimented through asucrose gradient. A

sample of each fraction in the 20S-to-45S size

range wastreated wtihglyoxaland analyzedon an agarose gel. Figure 6 shows an

autoradi-ograph of thisgel. A numberof prominent

la-beledbands canberesolved; however, none of

them comigrated with bandsaand b.One

prom-inent nuclear speciesmigratedveryclosetoband

a but was

consistently

found to migrate

just

slower than a in a numberofdifferent

experi-ments.Nonuclear RNAspecies

comigrated

with

band b.Thisexperiment demonstrates that the

presence ofspecies aand b in the

cytoplasmic

RNA is notdue to contamination ofour

cyto-plasmic preparations with nuclear RNA. The

possibilitythatsmall amountsof

species

aand

barepresent inthe

nucleus, however,

cannotbe excluded.

DISCUSSION

The major messenger activities for III and

pVII havepreviously beenshowntosedimentat

approximately 25S and 21S,

respectively

(1).

These mRNA's have been

mapped

onthe Ad2

genome to the mRNA

family

which hasa

com-mon3' terminusatabout map

position

49.5

(4,

6, 13, 14). The 5'endsofthemain

body

of the

mRNA's for III andpVII are

probably

located

at 38.8and 42.8,

respectively (4, 6).

These map

coordinatesareconsistent with theobservedsize

of the mRNA's from these proteins as deter-mined bysedimentation

(Fig. 1)

and

migration

ongels (Fig. 4). Thus,thesetwomRNA's

overlap

in sequence, with III mRNA

having

additional

sequences presentonthe5'portionof the mol-ecule which arelikelytobe those that

actually

code for the IIIpolypeptide.The sequences that code forpVII are present

internally

in III mRNA

but arenot translated.

The present paper describes two minor

mRNA's codingfor III and pVII which are

con-siderably greaterinsizethanthe major mRNA

species for these proteins. Assuming that the

coding region for these proteins is at the 5'end

of these large molecules, then the additional

sequences mustbeatthe3'end of the molecule.

The large messenger activities cosedimented

with two 32P-labeled cytoplasmic

poly(A)-con-taining RNA species (designatedaand b) which

hybridizeto aregion of the Ad2 genome specific

for theIII andpVII mRNA familyand also toa

regionspecificfortheIIand pVI mRNA family.

The molecular weights of a and b have been

estimated by comparison of the migration of

glyoxal-treated species with the migration of

glyoxal-treated 18S and 28S rRNA's (Fig. 4,

lanes 7and9).Themolecularweights of a and

b were thus determined to be about 3.3 x

106

and 2.7 x 106, respectively. Using the same

standards, the molecular weight of hexon mRNA

(speciesf ) wasestimatedtobe about 1.5 x 106.

However, the heteroduplex analyses of Chowet

al. (4, 6) indicate that hexon mRNAspansabout

10.5 map units of the Ad2 genome (molecular

weight,23 x106)(9), whichwouldcorrespond to

amolecularweight of about1.3 x 106, including

leadersequence and poly(A). Thus,use of 18S

and28S rRNAasmolecularweight standards in

this gel system apparently results in a small

overestimation ofthemolecular weight of hexon

mRNA. Ifwe have overestimated thesize of a

and bbyasimilarproportion, their actual

mo-lecularweights would be2.9x 106 and2.4x 106

and would span 24 and 20 map units of the

genome, respectively. Therefore, these

mole-cules are each approximately 10.7 map units

longer than the major mRNA's for III and pVII

(1,4,6). This isquite close tothe

length

of the

mRNA family that lies

immediately

down-stream from the III and pVII

family.

Since a

and b are composed in partofsequences from

this downstream region, it is

likely

that these

RNAspecieshave 5' sequences thatareidentical

to those of the normal mRNA's from III and

pVIIthatare3'coterminal with the mRNA's for

pVI and II

(hexon)

atmap

position

62instead of

the normal 3' terminus for the III and

pVII

mRNA'sat49.5.

Howmightsuchmolecules arise? The

major-ityofmoleculeswith3'termini codedat62 are

destinedtobecomemRNA'sfor

pVI

orII

(4, 6,

20).Thus,it is

likely

that the

large

mRNA'sfor

III and pVII arise

by

incomplete

or aberrant

processingoftranscriptswhich

normally

would

producepVIorII mRNA's.

Processing

of these

transcriptswouldnormally involve the

complete

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312 LAWRENCE

FIG. 6. Analysis of20S to 45S

32p_labeled,

nuclear, poly(A)-containing RNAs on an agarosegel. 32P-labeled, nuclear,poly(A)-containingRNA wasfractionatedon asucrosegradient. RNA in the 20S to45S

regionofthegradient (fractions8to20asindicated) and totalpoly(A)-containing cytoplasmicRNA were

treatedwithglyoxalandanalyzedon a 1.0% agarosegelasdescribed inthetext.

removal of sequences betweenthe3' end of the leader sequence at map

position

27 and the 5' end of the body of pVI mRNA at 49.5. The

processing of the fiber mRNA precursor

appar-ently involves the removal ofRNA sequences

coded between the leader andmain

body

ofthe mRNA in a series of steps (18), which may involve a transfer of the 5' leader to various

acceptorsitesalongthelengthofthe precursor. If theprocessing of the mRNA precursor for II andpVImRNA's occurs in steps in which the 3' end of the leader is first spliced to 5'-mRNA sequences in the familywhich normally codes for III andpVII and then issubsequentlyspliced

to49.5 (pVI mRNA) or 51.2 (II mRNA), then

the large mRNA's for III and pVII might be

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[image:7.504.118.395.74.534.2]

Ad2 mRNA 313

intermediates in this pathway which are

trans-ported to the cytoplasm before completion of

processing.

Alternatively, if removal ofsequencesbetween map positions 27 and 49.5 or 51.2 occurs

nor-mally in a single step, then the large mRNA's for III andpVII could arise byaprocessingstep

in which sequences at map position 27 are

splicedtosequencesat38.8 (III mRNA)or42.8

(pVIImRNA). This would beanabnormal splice

fora transcript witha3' terminusat62butan

entirely normal splice for molecules which have 3' termini at 49.5. In this event, the large mRNA's forIII and pVII would be formed from

anabnormalprocessing eventofa precursorto

pVIorIImRNA resulting inalarge mRNA for

IIIorpVII. Suchamoleculemaynotbe further processed becauseanappropriate nucleotide se-quence or RNA secondary structure which

would allow the leader to be spliced again to map position 49.5 or 51.2 may not be present oncetheleader has beensplicedto38.8or42.8.

Further analysis of the structure of interme-diates in the synthesis of pVI and II mRNAs should distinguish between these two possibili-ties. In theeventthatspeciesa and bproveto

be normal intermediates in thesynthesis of these mRNA's, they may be usefulas substrates for

studying the biochemistry of mRNAprocessing becausetheyarereadily isolated from

cytoplas-micextractsof infectedcells.

ACKNOWLEDGMENTS

I gratefully acknowledge thegenerous supportofTony Hunterand GemotWalter,in whoselaboratoriespartof this workwasperformed,and I thank SueBergetforproviding

cloned Ad2 DNA restrictionfragments.

Thisworkwassupportedin partbyPublic Health Service

grant CA 17096from theNational Institutes of Health to TonyHunter. The authorwassupported byPublic Health Service National Research Service award CA06037,a

fellow-shipfrom the LeukemiaSocietyofAmerica,and Public Health Service grantAI16484 from the National Institutes of Health.

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VOL. 35,1980

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