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0022-538X/78/0028-0227$02.00/0

Copyright X 1978 American SocietyforMicrobiology PritedinU.S.A.

Transcription Map for Adenovirus Type 12 DNA

J. R.

SMILEYt

AND S.MAK*

Departmentof Biology, McMaster University, Hamilton,Ontario,CanadaL8S 4K1 Received forpublication 23 March 1978

The regionsof the adenovirus type 12 genomewhich encode 1- and r-strand-specific cytoplasmicRNAwere mapped by thefollowingprocedure. Radioactive, intact, separatedcomplementary strands oftheviralgenome werehybridized to saturatingamounts of unlabeled late cytoplasmic RNA. The segmentsof each DNA strand complementary to the RNA were then purified by

Si

nuclease digestion of the hybrids. Thearrangement of the coding regions of each strand wasdeducedfromthe pattern ofhybridization of these probes to unlabeled viral DNA fragments producedby digestion with EcoRI, BamHI, and HindIII. The resultingmapis

similar,

ifnot identical, to that of adenovirus type 2.The subset of the late cytoplasmic RNAsequenceswhich are expressed at early times were located on the map by hybridizing labeled, early cytoplasmic RNA to both unlabeled DNA fragments and unlabeled complementary strands of specific

fragments.

Early cytoplasmic RNA hybridized to the r-strand toEcoRI-C and

BamHI-Bandtothe

1-strand

of BamHI-E. HybridizationtoBamHI-Cwasalso observed. The relativeratesof accumulation of cytoplasmic RNA complementary to individual restriction fragments was measuredat bothearly and late times. Early during infection, most of the viral RNA appearing inthe cytoplasm was derived from the molecular ends of thegenome.Later (24to26hpostinfection)

the

majority

of thenewly labeled cytoplasmic RNAwastranscribed from DNA

sequencesmappingbetween25and60mapunitsonthegenome.

Theavailability of bacterial restriction endo-nucleases which cleavea

given

DNA molecule to generate a defined set offragments has al-lowedconsiderable progress to be made in the

analysis

of the

transcription

program of the

groupC human adenoviruses.

The DNA sequences encoding the mRNA's

synthesized during

the

early

andlate

phases

of

productive infection,

aswellasthose

expressed

intransformed celllines, have been mappedon theadenovirus type 2 (Ad2) (2,4,6, 28, 31, 39) andAd5 (5,

7)

genomes. The

positions

and po-larities of the sitescomplementaryto

early

and late mRNAaresimilar ifnotidenticalinthetwo

genomes,a

finding

not

expected

in view oftheir extensive nucleotide sequence

homology

(10)

and similar

biological

characteristics.

We areinterested in theanalysis of the

tran-scription

of the genome of

Adl2,

amember of

the highly oncogenic group A human adenovi-ruses. These viruses share very fewnucleotide sequences with the group C adenoviruses (17) anddifferwidelyinanumberof

biological

prop-ertiesfromboth Ad2 andAd5. Wehave

previ-ously shown that early in

productive

infection

t Present address: DepartmentofTherapeutic Radiology, RadiologyLaboratories, YaleUniversitySchool ofMedicine, NewHaven, CT06510.

withAdl2, RNA complementaryto about 30% of theviralgenome,derived from both comple-mentary DNAstrands, is detectable in the cy-toplasm of infectedcells(33).The fractionof the genome

expressed

ascytoplasmic RNA increases toabout 80%atlate times. The majorityof the RNAsequenceswhichappearexclusivelyatlate times are derived from the r-strand of the ge-nome. Further experiments showed

that

the early RNAsequences are asubset of those pres-entlate in infection. Wereporthere experiments which map the physical locations and strand derivation of the DNA sequences expressed as

early

and late cytoplasmic RNA, using a new

method. In addition, the relativeratesof accu-mulation of cytoplasmic RNA derived from dif-ferentregions of thegenome weremonitoredat different times after infection.

MATERIALS

AND METHODS

Virusandcells.PurifiedAdl2 strain1131(24)and human KB cells grown in suspension were used throughout this study. Proceduresfor infection and viruspurificationhavebeen described(12,21).Viral DNAwasextracted frompurifiedvirionsasdescribed by Green and Pina(13).

Separationof the intactcomplementaryAdl2

DNAstrands. Thecomplementarystrands ofAdl2 DNAwereseparated bycentrifugingdenatured viral 227

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SMILEY AND MAK

DNAtoequilibriuminneutral cesiumchloridedensity gradients in the presence ofpolyuridylic acid-poly-guanidylic acid [poly(U,G)], as previously described

(18, 33).

Material pooled from the gradients was further

purified byexhaustive self-annealingat37°C in 0.14 Msodium phosphate buffer, pH 6.8, containing50% formamide,followedbysedimentation in5to20%(wt/ wt) high-saltneutralsucrosegradients (35)at38,000 rpmfor 90mininaBeckmanSW40rotor.Intactsingle strands (sedimenting at 70S) were pooled,

exhaus-tively dialyzed against0.01 M Tris(pH7.3) containing 2%formamide, andused forhybridization.

RNAextraction. RNAwasextractedfrominfected

cells aspreviouslydescribed(33). EarlyRNA(labeled

5.5 to 7.5 h postinfection) wasprepared by

concen-tratinginfected cellsto3 x 106/ml by centrifugation andadding[3H]uridine (New England Nuclear,20Ci/

mmol)toafinalconcentration of 10 MCi/ml.

Restriction endonucleases and electrophore-sis. Endonuclease EcoRIwaspurifiedbythemethod

of Pettersson and Philipson (26). Endonucleases BamHI and HindIIIwerepurchased from Bethesda

Research Laboratories. Digestionbufferswere0.1 M

Tris (pH7.9)-0.01 M MgCl2forEcoRI, 0.02 M Tris (pH 7.4)-0.01MMgCl2-0.06MNaClforHindIII and 0.02 M Tris (pH 7.4)-0.01 MgCl2-0.002 M,B-mercap-toethanol for BamHI.Fragments wereseparated by

electrophoresis through0.7%(EcoRI)or1%(HindIII and BamHI) agarose gels for 11 h at 2 V/cm. The DNA bands were visualized under short-wave UV

illumination after staining the gels with 0.5 Mg of

ethidium bromideperml.

Separation of the complementary strands of DNA fragments by electrophoresis. DNA frag-mentsinagarosegelslicesweredenatured insitu and

subjectedtore-electrophoresisin1.0%agarosegelsas

describedbyHorowitz(16).

Restriction endonuclease cleavage map of Adl2 (1131) DNA. The cleavage patterns of Adl2 (1131) DNA by endonucleases EcoRI, BamHI, and HindIIIwereexamined. TheEcoRI and BamHI

frag-mentsof strain 1131DNAwerefoundtobeidentical to those produced from Adl2 (Huie) DNA (Smiley andMak, unpublished data;C.Mulder,personal

com-munication). Since identical sets offragmentswere

produced by digestionof the DNAofbothstrainsof Adl2,we have assumed that the EcoRI and BamHI

cleavage maps of these strains are identical. The HindIII cleavagemapofstrain 1131 DNAwas

deter-mined (Smileyand Mak, manuscriptinpreparation) and is shown with those forEcoRIand BamHI in Fig. 2.

Purification of the segments of each DNA strandcomplementarytolatecytoplasmic RNA. 3H-labeled (leftward transcription)-andr(rightward

transcription)-strands (5 x 105 cpm/,ug, 0.5 Mg/ml)

wereseparately hybridizedtounlabeled late cytoplas-mic RNA (2 mg/ml) for 24 h at 67°C in 0.3 M NaCl-0.01 M Tris(pH7.3),conditionspreviously de-termined to besaturating. The material wastreated

withS1 nucleaseasdescribed previously (33)and then

made 0.2 M sodium phosphate (pH 6.8)-1% sodium dodecylsulfate(SDS),andyeastRNA (1mg/ml)was

added as carrier. The mixture was extracted with phenol saturated with 0.2 M sodium phosphate (pH 6.8), dialyzed against 0.01 M Tris (pH 8.1), and ethanol precipitated.

The pelletsweretaken up in2ml of0.3NNaOH, boiled for 15min,neutralized, bufferedatpH 7.3 with 0.1 MTris, and dialyzed against 0.1x SSC (lx SSC = 0.15 MNaCl plus 0.015 M sodiumacetate) after the addition of1mg of yeast RNAper mlascarrier.

Hybridization of labeled nucleic acidstoDNA fragments generated by restriction endonuclea-ses. Unlabeled DNAfragments separated by agarose gelelectrophoresisweredenatured in situ and trans-ferredtonitrocellulosestrips by the method of South-em(34). Each stripwasprepared fromagel containing 2 ,ug of Adl2 DNA. The labeled nucleic acid was hybridizedtothestrip in2xSSCcontaining0.1% SDS and 500 yg of yeast RNA per mlat65°C for 20 h. The unhybridized nucleic acidwasremovedbyoneoftwo procedures. In [3H]RNA-DNA hybridization, the stripswerewashed and treated withpancreaticRNase as described by Southern (34). In [3H]DNA-DNA hybridization, the unhybridized [3H]DNA was re-movedbywashing for 1h in 500ml of2xSSC plus 0.1% SDSat 65°C followed by1 h in500ml of4 x 103 MTrisplus0.1xSSC (pH 9.4) at room temper-ature (21). Thehybridized radioactivitywasdetected and quantitated by fluorography. Strips were oven

dried,immersed in 20%(wt/vol) Omnifluor (New Eng-land Nuclear) in toluene, air dried, and exposed at -70°C to Kodak RP Royal X-omat (RP R14) film previously exposedto ahypersensitizing flash of white light (19). Preexposure wasdone tobring the back-ground density of the film to 0.15 D above that of unexposed film. Theresulting negatives were scanned withaJoyce-Loeblmicrodensitometer, and the rela-tiveamountofradioactivity hybridized to each band was estimated from the peak areas. In some cases, therewassignificantbackground radioactivityonthe

fluorographs.Estimationof thespecifically hybridized radioactivitywasmoredifficult,and thepeaks were extrapolatedtothe baseline of the microdensitometric trace. It is clear that this procedure results in an overestimation of thespecifically hybridized radioac-tivity where there isasignificant background.

Hybridization of labeled RNA to immobilized 1-andr-strands. To prepare complementary strand-specific RNA, samples containing5 x 106cpm of 3H-labeledlatecytoplasmic RNA (about 150Mgof RNA) werehybridizedto1-orr-strandAdl2 DNA immobi-lized onnitrocellulose filters.Hybridizationwasfor 20 h in 2x SSC containing 0.1% SDS at 65°C. After hybridization, filters were washed with 1 liter of 2x SSC containing 0.1% SDS at 65°C for 20 min with stirring, followed byafurtherwash with 2xSSCunder suction at room temperature. The hybridized RNA waseluted by boiling for5minin0.lxSSC, and yeast RNA(500 ,ug/ml)wasadded ascarrier.After dialysis against 0.01 M Tris (pH 7.3), any contaminating

['4C]DNA was degraded with 100 Mg ofpancreatic DNase per ml in the same buffer containing 0.01 M

MgCl2. The RNA was then extracted by the hot phenol-SDS method, ethanol precipitated, and di-alyzed against0.1xSSC.

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ADENOVIRUS TYPE 12 RNA 229

RESULTS

Mapping the DNA sequences expressed

aslatecytoplasmic RNA. Most of the hybrid-ization mapping studies of adenovirus published

todate have utilized asprobes labeled

comple-mentarystrand-specificsequencesof individual

DNA fragments generated by restriction endo-nucleases. The complementary strands of indi-vidual fragments have been prepared by hybrid-izationtoexcess,intact complementary strands

(39) or by electrophoresis of denatured DNA fragments (7, 31). The complement excess

hy-bridization method (39) required rather large

amountsofintactcomplementary strands,

quan-tities thataredifficulttoobtain with Adl2 DNA because of the relatively low yield of virus and the small density difference between the

com-plements in poly(U,G)-cesium chloride density gradients (18, 33). Some of the Adl2 DNA

frag-mentscouldnotbe separated directlybyeither electrophoresis orbybinding topoly(U,G) fol-lowed bycesium chloride density centrifugation (23;Smiley, unpublished data). We have, there-fore,adoptedamappingstrategywhich obviates

therequirement for strand separating the indi-vidual DNA fragments. Radioactively labeled intact1-andr-strandswerehybridized to

satu-ratingamountsofunlabeled cytoplasmic KB cell RNA extracted at 24 h after infection (late RNA). Late cytoplasmicRNAiscomplementary to65%ofthe r-strand and 15% of the 1-strand of Adl2 DNA (33). In this preparative hybrid-izationtheadded RNA protected 63.8% of the

r-strand and 16.2% of the 1-strand from S1

nu-clease, demonstrating that the hybridization

re-actionwassaturated.Thehybridized portions of

eachstrandwerepurified byS1nuclease

diges-tion ofthe unhybridized nucleic acid, and the RNA was removed by alkaline hydrolysis as

described inMaterialsand Methods. The DNA sequences recovered by this procedure

corre-spondtothesegmentsof1-andr-strands which

are complementary to late cytoplasmic viral RNA andshouldbe inequimolaramounts. We

cannot, however, exclude the possibility that sequencesfromasmallfraction of the genome

whichencodesextremelyrareRNAspeciesare

underrepresented in the DNA selected in this

manner.

These [3H]DNA preparations were used as

probes to determine the regions of the Adl2

genome complementary to these sequences.

Eachprobewas hybridizedtoexcess (2

,ug)

un-labeled DNA fragments generated by various

restriction endonucleases. The pattern of hy-bridization of these selected sequences to the

fragmentsetsgeneratedbydifferent restriction

endonucleasesprovidesinformationonthe

dis-tribution of thecoding regions of the 1- and r-strands throughout the genome. In addition, within the limits discussed below, the relative amountofradioactivity hybridizedtoeach frag-mentgivesanestimate of thedegree of comple-mentarity of each fragmentto the probe used. This inturnprovidesa measureof the fraction of the DNA fragment transcribed into stable cytoplasmic RNA in the infected cell (see be-low).

To determine how accurately the relative amountofradioactivity hybridizedtoeach frag-mentreflects thedegree of

complementarity

of the probe to that fragment, total 3H-labeled Adl2 DNA, degraded toabout 350nucleotides inchain length byboiling in alkali,was hybrid-izedtoEcoRIfragments AtoE, using the South-erntechnique (34). Theamountofradioactivity bound toeachfragmentwasquantitatedas de-scribed in Materials and Methods. The results are shown in Table 1. It canbe seen that the relative amount of 3H-labeled Adl2 DNA hy-bridizedtothe unlabeled

fragments

is

approxi-mately equaltothe

expected

values of comple-mentarity between the labeled probe and the unlabeled DNA

fragments.

Several

points

con-cerning these data and their

interpretation

are noteworthy. First,

although

thereisclose agree-mentbetweenexperiments, the

degree

of com-plementarity of the

probe

to

fragments

AandC

is

overestimated,

whereas that to D and E is

underestimated. It is

possible

that this result reflects therelative

guanine-plus-cytosine

con-tentsof the

fragments,

since the DNA

probe

was labeled with

[3H]thymidine,

or some other source of

systematic

error. In some cases de-scribed

below,

the

degree

of complementarity between the

r-specific probe

and individual

DNA

fragments

is also

slightly overestimated,

perhaps for similar reasons.

Second,

since the resultsare

displayed

asthe fraction ofthetotal hybridized radioactivitywhich hasannealedto

TABLE 1. Hybridizationof3H-labeledAdl2 DNAto Adl2 DNAfragments generated byEcoRIa

Relativeamtof3H-Adl2 hy-Adl2 DNA

Fragmentisize

bridized

fraction of the

fragment gnm

genome ExptI ExptH

A 0.36 0.39 0.39

B 0.27 0.26 0.30

C 0.165 0.21 0.20

D 0.115 0.096 0.095

E 0.07 0.042 0.047

aFragmented

3H-labeled Adl2

DNAwashybridized

tonitrocellulosestripsbearingelectrophoretically sep-aratedDNAfragments,andthehybridized radioactiv-itywasquantitatedby fluorography.

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AND

each fragment, the determinations within a given

experiment

arenot

independent.

System-atic or spurious errors in the measurement of thefraction

hybridized

to one

fragment

neces-sarily introduceerrorintothe other determina-tions. One of the factors

contributing

tospurious erroris thebackground

radioactivity

on someof thefluorographs (e.g.,

Fig.

1).

To maptheviralDNAsequences complemen-tary tolateRNA,3H-labeled 1- andr-strandviral DNA sequencesselectedby hybridizationtolate cytoplasmic RNAwere

hybridized

toDNA

frag-ments generated by EcoRI, BamHI, and

HindIII, usingtheSouthern technique. The re-sultsareshown inFig. 1and Table2.Since the r-strand probe is derivedfrom0.65 of the total r-strand (33), the fraction of the

probe

hybrid-izedtoeach unlabeledfragmentmustbe multi-pliedby0.65 togive the fraction of the total r-strand transcribed within this

particular

DNA fragment. Only0.15of thetotal

1-strand

is com-plementary to late

cytoplasmic

RNA (33). Therefore, the fraction of the total

1-strand

tran-scribed from each DNA

fragment

can be simi-larly estimated.

Data from Table 2 wereused toconstruct a map of the sites

expressed

aslate

cytoplasmic

RNA (Fig. 2). The map shown wasassembled from the data asdescribed below byassuming

that

cytoplasmic

RNA is

asymmetrically

tran-scribed and

by reducing

the number ofseparate,

contiguously

transcribed regions to the

mini-Eco RI

E D C BA

m r

O

0

mum demanded bythe data. Other, more com-plex arrangements for the coding regions are possible.

Consider first the transcription from the 1-strand. A minimum oftwo separate regions of transcribed DNA were detected by hybridiza-tiontothe EcoRI

fragments;

onewithin EcoRI-A, the other within EcoRI-C. the leftmost re-gion, within

EcoRI-C,

mayextend a small dis-tancebeyond the EcoRI-C-D junction, sincein oneexperiment the probehybridizedto asmall degree with EcoRI-D. This suggestion is sup-ported by the weakhybridizationtoHindIII-C. The data obtained with thefragmentsgenerated by BamHI demonstrate that EcoRI-A contains at least two separate regions of transcribed DNA,onewithin BamHI-C and the other within BamHI-E.

(Although

BamHI-C and -Dare not

separable by gel

electrophoresis,

the failure of

the

1-strand-specific

probe to hybridize with

either EcoRI-B orHindIII-A, which both con-tain BamHI-D, allows the assignment to BamHI-C.) The fragments produced by HindIII allowa more precise localization of these three regions. The probehybridizedtoonly

HindIII-C,

-D, and -E

(however, fragments

J, K, andL were run outof the gels used in the analysis).

Qualitative

consideration of the data shows that

the leftmost region is located to the right of HindIII-F

(6.3

map

units)

andtothe left ofany of BamHI-D, -H, or -I (25 map units). The region detected within BamHI-C was not

de-MIGRATION

FIG. 1. Hybridization of labeled1-andr-strand-specific probes complementary to late cytoplasmic RNA to unlabeled DNAfragments. Nitrocellulose strips bearing 2 pg of unlabeled DNA fragmentsproduced by EcoRI, BamHI, and HindIII were incubated with 0.02 pg (104 cpm) of r-strand probe or 0.005

pg

(2.5 x 103

cpm)of I-strand probe in2xSSC plus 0.1% SDS at 65°C for 20 h. The hybridized radioactivity was detected byfluorographyto

flash-hypersensitized

X-ray film.Microdensitometer scans of the resulting negatives are shown.

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[image:5.500.60.441.92.435.2]

ADENOVIRUS TYPE RNA 231

TABLE 2. Distribution of the 1- and r-strand sequences complementary to late cytoplasmic RNA among specific DNAfragmentsa

ProbeFragmentFragment size(frac-

Fraction

ofgenome

repre-Probe FragmentFent ntSztion) fa-Fractionofprobe hybridized sented inprobementbwithin

frag-r EcoRI-A r EcoRI-B r EcoRI-C r EcoRI-D r EcoRI-E r EcoRI-F r BamHI-A r BamHI-B r BamHI-C r BamHI-D r BamHI-E r BamHI-F r BamHI-G

r BamHI-H + I

r HindIII-A r HindIII-B r HindHI-C r HindIII-D r HindIII-E r HindIII-F r HindIII-G r HinduII-H r HindIII-I r HindIII-J-L 1 EcoRI-A 1 EcoRI-C 1 EcoRI-D BamHI-A BamHI-C BamHI-E 0.36 0.27 0.165 0.115 0.07 0.02 0.25 0.16

0.131

0.13} 0.115 0.075 0.063 0.065 0.23 0.155 0.13 0.115 0.09 0.063 0.060 0.049 0.029 0.071 0.36 0.165 0.115 0.25 0.13 0.115 0.35 0.36 0.17 0.06 0.06 ND 0.23 0.24 0.11 0.04 0.04 ND 0.13 0.16 0.32C 0.04 0.12 0.10 0.12 0.08 0.11 0.08c 0.13c 0.03 0.08 0.07 0.08 0.29 0.19 0.06 0.02 0.01 0.13 0.12 0.10 0.07 ND 0.56(0.49)d 0.36 (0.51) 0.08(<0.01) 0.39(0.53) 0.31 (0.25) 0.30 (0.22) 0.18 0.12 0.04 0.01 0.006 0.08 0.07 0.06 0.04 ND 0.08(0.07) 0.05(0.08) 0.01(0.00) 0.06(0.08) 0.05(0.04) 0.05(0.03)

1 HindIII-C 0.13 0.09(0.07) 0.01(0.01)

1 HindIII-D 0.115 0.67(0.57) 0.07(0.06)

1 HindIII-E 0.09 0.24(0.36) 0.03(0.04)

a3H-labeled1-and r-strandprobes complementary to late cytoplasmic RNA were hybridized tonitrocellulosestrips bearing

electrophoretically separatedDNAfragments,and thehybridizedradioactivity was quantitated by fluorography (Fig. 1). DNA fragments whichhybridizenondetectable amounts of radioactiveprobe are not presented in this table. ND, Not done.

'Calculatedastheproductof the effective sequencecomplexityoftheprobe used and the fraction of the total hybridized radioactivity annealedtoeachfragment.The effectivecomplexityofthe r-strandprobewas 0.65whenhybridizedtoEcoRI and BamHIfragmentsand0.61when annealedtotheHindIII fragmentsA toI. Effectivecomplexitiesof0.15 and0.106 were

assumed for the 1-strandprobe(see text).

cFragments BamHI-C and -D comigrate. Together they contain sequences homologous to 32% of the r-strand probe (=21%

of thetotalr-strand).The rationale forapportioningthese sequences between thetwofragments is giveninthetext.

dThe two numbersrepresent results from two separateexperiments.

tectable withinHindIII-A to -I and, therefore, resides withinoneor more of theHindIII

frag-ments excluded from the analysis (J-L). This

regioncanbeplacedbetween EcoRI-E(62map units) and HindIII-B (68mapunits).The

right-most 1-specific region maps to the right of 91

mapunits (entirelywithinHindIII-E).The rel-ativesizes of these threeregionswereestimated

fromthe fraction of theprobewhichhybridized

toeach DNAfragment (Table 2). Fromleftto

right these regionsare 6to8,4to 5,and 3to5 mapunitsinlength.If it is assumed thatthe

1-specific segment ofHindIII-C is contiguous to

that of

HindIII-D,

then theleftmost regioncan

be

placed

from about 11.5to18.5mapunits.

The r-strand probe hybridized, tovaried de-grees, with all of the DNA fragments assayed (Fig. 1). Given the above-mentioned constraints onthepositions of thesegments ofthe

1-strand

complementarytocytoplasmic RNA,thisresult demonstrates thatatleastthree separateregions of the r-strandaretranscribed intocytoplasmic RNA.One of theseregionsliestothe leftof the leftmost

1-specific

block. The size of this seg-ment,estimatedfrom theextentofhybridization of the r-probe to EcoRI-C,

BamHI-A,

and

l

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232 SMILEY AND MAK

Eco RI F

C

I

D

I

B I

EUj

A

Bam HI

A I G

I(D.H.I)

I

F

I

C I B I E

Hin dll (G.J,K,L)

F1

D I C

I

A 1 1 B jHjlj E

r---~~~~~-

_E_D

I a

0 0.25 0.5 0.75 1.0

FRACTIONAL LENGTH

FIG. 2. Map of sites of theAdl2genome expressed asearly and late cytoplasmic RNA. The map for late RNAwasdeducedfrom the data shown in Fig.1and

Table2. The mapfor early RNA was deduced from data shown in Table 3 and Fig. 5, together with

information fromOrtinetal. (24).Symbols: (_) late RNA; (o) early RNA.

HindIII-F and

-D,

is8to 11mapunits.Thenext region, which

begins

tothe

right

ofthis

1-strand

block (i.e., some

point

beyond

position

18.5) should encompass all or part of

EcoRI-D,

-B,

and -E,

BamHI-G, -D, -H, -I,

and

-F,

and

HindIII-C,

-A, and -G.

(Although

the relative

order of

HindIII-G, -J, -K,

and-Lisnot

known,

HindIII-G is

entirely

contained withinEcoRI-E [Smiley,

unpublished

data]).

Therelativeextent

of

hybridization

ofthe

r-specific probe

tothese

fragmentssuggeststhatmost orallof

EcoRI-B,

BamHI-G, -D, -H, -I,

and

-F,

and HindIII-A and

-Gare

complementary

tothe

probe. However,

it

appears that

only

a

portion

of EcoRI-D and HindIII-C is complementary tothe probe. Be-cause ofthe uncertainties inherent in interpret-ingthequantitativedata,aspointedoutabove, it is difficulttoprecisely locatethe endsofthis block. Theavailable data suggest that it starts atorclosetoposition25and ends with EcoRI-E or -F andis,therefore, about 35mapunitsin length. The third r-strand region maps to

EcoRI-A,

BamHI-C,

-B,and-E,and

HindIII-B,

-H, and -I. Thissegment beginsto therightof

the

1-strand

region, which maps to

BamHI-C,

and, basedonthe low levels of

hybridization

to

HindIII-E,

appears to end around

position

92.

Several difficulties were encountered in the interpretation of the data obtained with the r-strand probe. First, because BamHI-C and -D were not separable electrophoretically, we ob-tainedanestimate of the fraction of the r-strand transcribed from within both theseregions. The data obtained with thefragments generated by EcoRIandHindIII indicate thatvirtually all of the r-strand within EcoRI-B and HindIII-A is transcribed ascytoplasmic RNA. The BamHI-Dfragmentmapsentirelywithinthisregion, and consequently wehave assumed total transcrip-tion of the r-strand of this fragment andhave assignedexcesshybridizationtoBamHI-C. Sec-ond, the methodcalculating the fraction of each strand transcribed within each region assumes that all ofthe DNA sequences

complementary

to the probe used are present in the analysis. FragmentsHindIII-Jto-L andEcoRI-Fwere in fact excluded because of their small size. The exclusion of EcoRI-F (2% ofAdl2) hasa negli-gibleeffect, but theexclusion ofHindIII-Jto -L (approximately 8% of Adl2) complicates the analysis. However, asdiscussedabove,aregion comprising 4 to 5 map units located entirely withinfragments HindIII-J to-Lis transcribed from the1-strand andcanbeexcluded from the analysis of r-strand-specific RNA. Effectively, only about3 to4% of Adl2 is then excludedby not examining thesefragments. Assuming that all of this DNAisrepresented as r-strand-spe-cific cytoplasmic RNA, the effective complexity of the r-strand DNA probe becomes 0.61. The effects of this alterationincomplexityaresmall. The values given in Table 2 were calculated using 0.61 as the complexity of the r-strand probe.

Analysis of the RNA sequences actively synthesized at late times. The results out-lined above serve to map the positions and strand specificities of the DNA sequences rep-resented in late cytoplasmic RNA but give no indication of whichsegmentsofthegenome are actively transcribedatlate times.Todetermine whether both strandsaretranscribed, Adl2-in-fectedcellswerelabeled from 21 to 24 h postin-fection with

[3H]uridine,

and the cytoplasmic RNA waspurified.

It wasfoundthat this

[3H]RNA

could hybrid-izetoboth 1- and r-strands of

Adl2

DNA (data not shown). To determine which segments of each strand areactivelytranscribed at 24 h after infection,the3H-labeledviral RNAwasselected byhybridizationto1- and r-strandDNA,eluted, andrehybridizedtounlabeled EcoRI

fragments

(Fig. 3).Itis clear thatmostRNA

species

pres-ent in thecytoplasm atlatetimes are actively synthesizedandtransportedtothecytoplasmat J. VIROL.

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w

u z

rstrand RNA

0 (I)

I strand RNA

MIGRATION

FIG. 3. Hybridization oflabeled late complemen-tary strand-specific RNA to unlabeled EcoRI frag-ments. CytoplasmicRNA labeled with[3H]uridine from21to24 hpostinfection wasstrandselectedas

described in thetextandhybridizedtonitrocellulose strips bearing 1 pg of denatured, separated Adl2

EcoRIfragments. Hybridized radioactivity was de-tectedasinFig.1.

late times. This is in contrast tothe result

re-ported thatatlate times after infection with Ad2 the RNA is transcribed exclusively from the

r-strand of thegenome (26).

It is possible that the labeling times chosen

are not comparable to the 16- to 18-h period

usedby PetterssonandPhilipson (26)with Ad2

and that analysis of RNA synthesized later

would reveal only r-strand transcription. It is

worthnoting, however, that the maximal rates

of viralDNA and RNAsynthesisoccurat 24 h

postinfection with Adl2 (21), whereas this oc-curs at 18 hpostinfection with Ad2 (14). Since

the strand-specific RNA used in the previous experimentwasisolatedbyhybridizing

near-sat-urating amounts of RNA to each strand, the

results of the experiment displayed in Fig. 3 cannotbe usedtoestimate the relative amounts

of radioactivity incorporated in RNA derived

fromdifferentregionsof thegenome.

To obtain estimates of RNA transcription

ratesfrom different regionsof the genome, cy-toplasmic RNAlabeled 24to26 hpostinfection with[3H]uridinewasdegradedin alkalitoabout

500 nucleotides in chainlength (1), and 2,tgof

this RNA (specific activity, 3.4 x 104 cpm/,ug)

was hybridized directly to excess (2 ,tg) frag-ments generated by EcoRI and BamHI. The

relative amount ofradioactivity hybridizing to

eachbandwas taken as a measure of the relative ratesofaccumulation of cytoplasmic RNA de-rived from each fragment. The data obtained (Table 3) indicate tht RNA species derived from different regions of the genome accumulate at widely differing rates. During the labeling period chosen,RNA derived from about 25 to 50 map units onthe genome (EcoRI-B and -D) accu-mulatesat a greater rate than any other class. Mapping of the early regions. At early times,viral RNA forms an extremelysmall frac-tion of the total cytoplasmic RNA. Conse-quently, themethod used to map the late RNA wasdifficultto apply to early RNA, since a large amount of viral RNA is required to saturate substantial quantities of radioactively labeled viralDNA.

Someinformationabout the DNA sequences encoding earlycytoplasmic RNA was obtained by hybridizing cytoplasmic RNA labeled with [3H]uridine from 5.5 to 7.5 h postinfection to

TABLE 3. Distributionof labeled late cytoplasmic RNA sequences among DNAfragmentsa

Fraction of total Specific accumula-Fragment RNA hybridized tion rateb

EcoRI-A 0.07 0.19

EcoRI-B 0.60 2.22

EcoRI-C 0.07 0.42

EcoRI-D 0.20 1.74

EcoRI-E 0.06 0.86

BamHI-A 0.06 0.24

BamHI-B 0.03 0.19

BamHI-C

.05d

0.42C

BamHI-D * 3.93C

BamHI-E 0.02 0.17

BamHI-F 0.07 0.87

BamHI-G 0.16 2.55

BamHI-H+I 0.12 1.80

aLate

cytoplasmic

RNAlabeled24to 26 h

postin-fection with[3H]uridine degradedtoabout500 nucleo-tidesin chainlengthwashybridized toexcess (2,&g)

unlabeled DNAfragments, and the hybridized radio-activitywasquantitatedbyfluorography.

bDefined as the fraction of the total hybridized radioactivity annealing to each fragment divided by thefractionallength of that fragment.

cTherelative distribution ofhybridized

radioactiv-ity between BamHI-C and-D wasestimatedby meas-uring hybridizationtofragmentsproducedby simul-taneouscleavagewithEcoRI andBamHI.The ratio of hybridized radioactivity between BamHI-C/ EcoRIa (within BamHI-C) and BamHI-Dwas0.07.

BamHI-C/EcoRIa represents 0.71 of BamHI-C. As-suming that accumulation within BamHI-C is

uni-form,thisyields aspecific accumulationrateof0.42 forBamHI-C.

d BamHI-C and-D are notseparable

electrophoret-ically.This numberrepresentsthesumof the radio-activityhybridizingtoeachfragment.

E D C BA

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234 SMILEY AND MAK

DNA fragments produced by EcoRI, BamHI, andHindIII. Relativeamountsof[3H]RNA hy-bridizedtoeachfragmentareshown in Table 4. Hybridization was detected with

EcoRI-A,

-B, and-C, BamHI-A, -B,-C, and -E and HindIII-B, -C, -E, -F, and -H. FragmentsEcoRI-F and HindIII-J, -K, and -Lwerenot

analyzed.

These data demonstrate that early RNA is derived fromatleastthree separate

regions

of the viral genome.

PositivehybridizationtoHindIII-E butnotto HindIII-Iindicated thatone

region

is contained entirely within HindIII-E.

Similarly,

positive hybridization to HindIII-F but not -D places anotherregionnearthe left end of the genome. Results from hybridization with EcoRI and BamHI fragments areconsistent with this

con-clusion.Atleastone

region

is located in BamHI-B and -C. The [3H]RNA

hybridized

to the

BamHI-C/D

doubletwas

assigned

toBamHI-C

by the

following experiment.

Adl2 DNA was

cleaved

simultaneously

withEcoRI andBamHI,

and the

fragments

were

separated

by

electro-phoresis (Fig. 4A). BamHI-D isnot cleaved

by

EcoRI,but BamHI-C is cleaved into three

frag-ments

(BamHI-C/EcoRIa

[fragment

VI in

Fig.

4A, lane2]andtwosmallerones). Resultsshown inTable4show that

early [3H]RNA hybridized

onlytoBam-C/EcoRIfa

(designated

as

BamHI/

EcoRI VI in Table 4) but not to BamHI-D (designated asBamHI/EcoRI III). The lack of hybridizationtoHindIII-Aconfirmsthis conclu-sion.

The observed hybridization of early RNA to EcoRI-B (6%ofthetotalhybridized radioactiv-ity; Table 4) does not agree with the data ob-tainedwith BamHI, where no hybridization was

seenwithfragmentsD,H, I, and F and only very small amounts (<1% of the total) were found withG.Similarly,nohybridizationwasobserved

to HindIII-A and -G (<1%), and only small amounts of hybridization were observed to HindIII-C (1%). The rather high levels of hy-bridization ofearly RNA to EcoRI-B can per-haps be explainedby postulating the existence

ofminorityrearrangement genomes bearing se-quencesderived from the normal EcoRI-A

and/

[image:8.500.258.449.95.307.2]

or-Cfragments onfragments of about the size ofEcoRI-B. Such genomes have in fact been

TABLE 4. DistributionoflabeledearlyRNA sequences amongDNAfragmentsa

Fractionof

Maxi-total radio- mumsize Specific

Fragment activityhy- ofregion

accumu-bridizedan- encoding lation

nealed to the early rateb fragment RNA

EcoRI-A 0.47

EcoRI-B 0.06

EcoRI-C 0.47

BamHI-A 0.60 0.067 8.96

BamHI-B 0.11 0.15 0.73

BamHI-C+D 0.09 0.05 1.80

BamHI-E 0.21 0.05 4.20

BamHI-G <0.01

HindIII-B 0.05

HindIII-C 0.01

HindIII-E 0.40

HindIII-F 0.50

HindIII-H 0.13

BamHI+EcoRI-I+IIf 0.63 BamHI+EcoRI-IV 0.28 BamHI+EcoRI-VI 0.08

'CytoplasmicRNA labeled 5.5 to7.5 h postinfection was

hybridizedto2yg of unlabeled Adl2 DNAfragments. The

analyses withEcoRIandBamHIfragmentsweredone with the same RNApreparation,whereasanotherlotof RNAwas usedfor the experimentswiththeHindIII fragments.

Frag-mentsEcoRI-A-E, BamHI-A-I,HindIII-A-I,and BamHI +

EcoRI-I-VIIwere assayed.Onlythosefragmentswhich

hy-bridized detectableradioactivityaretabulated. The limit of detection of the assay is about 1% of the totalhybridized

radioactivity.

b Defined as the fraction of the totalhybridized radioactivity

which annealed to agivenfragment divided bythe maximum size of the regionencodingtheearlyRNA.

'The identityoffragmentsproducedby the double diges-tionare:I,EcoRI-C; II,BamHI-B; III,BamHI-D; IV,

BamHI-E;and VI,Bam-HI-C/EcoRIa.

observed within Adl2 (Huie) populations (I. Mak, H.Ezoe, and S. Mak,manuscript in prep-aration). From data in Table 4, it can be seen thatthedifferent earlyRNA species accumulate atwidely differing rates, with RNA encoded by themolecular ends of the genome accumulating

mostrapidly.These RNA species together com-prise from 75 to 90% of the total viral RNA detected.

Strand assignment of the early regions. To determine directly the strand derivation of

FIG. 4. Electrophoreticstrandseparation of someAdl2DNAfragments.Adl2DNA wascleaved simulta-neouslywith BamHI and EcoRI, and the resulting fragments were separated in 1% agarose gels. Fragments EcoRI-C, BamHI-B, BamHI-E, andBamHI-C/EcoRIa were isolated in gel slices, denatured in situ, and again subjectedtoelectrophoresis.(A)Electrophoreticseparation of fragments produced byEcoRI(1),EcoRI plusBamHI (2, 3), and BamHI (4) in 1% agarose gels,allowing theidentificationof theorigin of some of the fragmentsproduced by doubledigestion. (B)Electrophoresisofdenatured DNAfragments in 1% agarose gels. 1,EcoRI-CDNA;2,BamHI-BDNA; 3,BamHI-E DNA; 4,BamHI-C/EcoRIaDNA.Electrophoresis was for 8hat 2V/cm.

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B

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235

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236 SMILEY AND MAK

the regions coding for the early RNA, we at-tempted to resolve the strands of the DNA fragments(i.e.,EcoRI-C, BamHI-B and -E, and the BamHI-C/EcoRIa) by gelelectrophoresis. Themethod used is that used by Horowitz (16). Figure 4B shows that fragments EcoRI-C and BamHI-B and -Ewere resolved into doublets, whereasdenatured

BamHI-C/EcoRIa

migrated as asingleband. To determine the strand deri-vationof the fast- and slow-migrating bands of EcoRI-C, BamHI-B, and BamHI-E, the rele-vantportion of each gel wasexcised and com-positesweremade. TheDNAfromthe compos-ite gels was transferred to nitrocellulose strips by the Southern method (34) without further denaturation. These stripswerethenhybridized

to purified,

3H-labeled,

intact l- and r-strands

and

fluorographed (Fig.

5).Thelabeled

comple-mentary strands

hybridized

primarily

to one band of the

doublet, allowing

strand

assignments

to each band to be made. The

fast-migrating

band of

EcoRI-C

is derived from the intact

1-strand, whereas the fast bands of BamHI-B and -E are part of the intact r-strand. The strand derivations of

early

RNAs were then

directly

determined

by

hybridizing

labeled RNAtosuch

strips (Fig. 5). The results demonstrate that early

cytoplasmic

RNA is derived from the r-strand of

EcoRI-C

and BamHI-B and the 1-strand ofBamHI-E. The strand derivation of early RNA with BamHI-Cwas not

directly

de-termined.

A

.. ;.I

.-EP.i

r

8

r'

41 M i

R.-I~~~~~~~~,s

a...

.. ffY"

T.

U

I

I

RI

..

A.

A.

41

Pl

Bam

F

I

kI

It#

i

i i -kiI

[image:10.500.115.397.254.575.2]

Zi

FIG. 5. Hybridization of H-labeledintact1-andr-strand DNA and early cytoplasmic RNAto complemen-tarystrandof EcoRI-C,BamHI-B,andBamHI-E DNA.Each DNA fragmentwasdenatured and subjected

toelectrophoresisinagarosegels.The gelswereexaminedunderUV illumination after staining with0.5 pug ofethidiumbromideper mLTheregion of eachgel containing the double bandswasexcised, andcomposite

gels werecreated byend-to-end alignment of the excisedsegments. The DNA in the composite wasthen

transferredtoanitrocellulose membrane andhybridizedto10i cpmof 3H-labeled r-strandAdl2 DNA (A)or

1-strandAdl2DNA(B) andto5x106cpmof 3H-labeledearly cytoplasmic RNA (C). Hybridized radioactivity wasdetectedbyfluorography.Aschematicrepresentation of the positionsof each of the DNA bands in the

composite gelsis shown.

.^

_ _

'SF

_

_ _ :.

t.2

I

I

f.

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12 237 DISCUSSION

We havedevelopeda newmethod of mapping

viral genes expressed late during infection. It

involves theisolation of radioactively labeled l-and r-strl-and DNAsequencescomplementaryto

latecytoplasmic RNA and, subsequently, rehy-bridization of these sequences to DNA

frag-ments generated by restriction endonucleases. The method has several advantages over the

standardones. First, the requirement for strand

separating the individual DNA fragments is by-passed. Second, the transcribed segments of each strand can be hybridized to any set of ordered DNAfragmentstorefine themapwith a minimum of additional effort. Drawbacks of

the method include the lack ofinformation on

the abundance of different RNA species, the requirement for rather large quantities of viral RNA toprepare the DNA probes, and the

re-duced precision of the method ascompared to

saturationhybridization. The ends of blocks of transcribed DNAcanbe locatedonlytowithin

several map units by this procedure, and it is likely that small gaps withinwhatwe have

as-sumedareregions of contiguous

complementar-itytocytoplasmic RNA wouldgounnoticed.

Using this new method and the DNA

frag-mentsgeneratedby three restriction

endonucle-ases, we have mapped the sites of the Adl2

(strain 1131)genomewhichareexpressedaslate

cytoplasmic RNA. The map is similar to the Adl2(Huie)mapobtained by saturation

hybrid-ization of labeled, complementary strand-spe-cificsequencesgeneratedby EcoRI and BamHI (29). The closeagreementofthetwo transcrip-tionmaps indicates thatourmethod is suitable

for mapping late mRNA. The Adl2 transcrip-tional map for late cytoplasmic RNA is very

similar to those previously obtained with Ad2 (27, 31) and Ad5, with the exception that a

region of Adl2 DNA extending from approxi-mately 17 to 25 map units is apparently not

transcribed into stable cytoplasmic RNA. The corresponding region of Ad2 and Ad5 DNA is transcribed asstable RNA (27, 31).

Well before the onset ofviral DNAsynthesis,

we have detected cytoplasmic RNA derived

fromaminimum ofthreeseparateregions of the

Adl2 genome. Ortin et al. (23) have detected

fourseparateblocks of DNAsequencesencoding

early Adl2 cytoplasmic RNA, mapping within the r-strand of EcoRI-CandBamHI-B, and the 1-strand of BamHI-C and BamHI-E. We were

not able to separate the regions mapped in BamHI-B and -C. The data ofOrtin etal. (23) and those obtained in this studyallow a quite

precise mapping of the four major early regions.

The leftmost block, transcribed from the r-strand, maps between 0 and 6.3 map units (withinHindIII-F). Ortinet al. have estimated thesize of this region as about 7.5%ofAdl2.If itis assumed that the earlyRNA derivedfrom BamHI-C is transcribed entirely from the 1-strand (23), this segment mustlie betweenthe EcoRI-E-F junction (62 map units) and the leftmost end of the HindIII-B (68 map units). The maximum size ofthis region is 4 to 5 map units. The total lack of hybridization of early cytoplasmic RNA to HindIII-I allows placing the right terminus of the early region within BamHI-B to the left of 88.1 map units (the HindIII-H-I boundary).Themaximumpossible size ofthisblockisthen 15mapunits,which is identicaltothe estimates obtainedby saturation hybridization (23). The rightmostearly region, transcribed from the

1-strand,

mapsentirelyto the right of91 mapunitsand is

probably

iden-ticaltotherightmost late

1-specific

region. The sites of these early regions are also similar to those of Ad2 (27, 31). Itwas ofinteresttonote that the extreme left r-strand early region of Adl2 (at most 6.3 map units) is considerably smaller than the analogous region of Ad2 (11 mapunits) (27, 31).

At both early and late times postinfection, cytoplasmic RNAs complementary to different segments ofthe genome accumulate at

widely

differentrates.Figure6showsthe

specific

rates of accumulation in the

cytoplasm

of RNA-de-rived different regions of the genome at

early

and late times. The data obtained with

early

RNA have been normalized to the maximum

possible

sizeof each

early coding

region,

whereas

thelate data have been normalizedto

fragment

size. Ifnotall DNAsequencesare a

template

for

the

synthesis

of late

cytoplasmic

RNA

(for

ex-ample,

the

region

from18to25map

units),

this

procedure willunderestimate the

specific

accu-mulation rate of RNA derived fromsequences

flanking

the nontranscribedsegments.The

ma-jor RNAspecies

synthesized

and

transported

to

the

cytoplasm during

the interval between 5.5

and 7.5 h postinfection are derived from the molecular ends of the genome

(HindHII-E

and -F).Smalleramounts aredetected from BamHI-B and -C. By 24 to 26 h after infection the patternhas

drastically

changed,with RNA de-rived from between 25 to about 60 map units being byfar themost

rapidly

accumulating

class. This RNA constitutes the major class

synthe-sizedexclusivelyatlate times. The

correspond-ingregionof Ad2 encodes themajorvirion pro-teins(20).Ifsimilar

regions

of theAd2 andAdl2 genomes encode

analogous

proteins,

thisresult

isnot

surprising.

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238 SMILEY AND MAK

class in the nucleus and cytoplasm. At 32 h postinfectionRNAmappingbetween 66 and 80 map units comprises over half of the total viral cytoplasmicRNA. Our data indicate thatduring the interval for 24 to 26 h postinfection with

Adl2,

RNA derived from 25 to 60 map units accumulates most rapidly in the cytoplasm. Since the labeling time chosen corresponds to the time at which the maximal rate of viral RNA synthesisoccurs(14),this RNAwillpresumably eventually constitute the most abundant cyto-plasmic class. This may indicate a difference in the fine control of Ad2 and Adl2 RNA accu-mulation.

The distribution ofcontiguous regions encod-ing early and late cytoplasmic RNA is virtually identical in Ad2, Ad5, Ad7 (37), and Adl2. The gross homology of genome organization of the group A, B, and C adenoviruses may indicate that genes with similar functions share common I _ t v map

positions.

This

supposition

is

strengthened

by the finding that the gene(s) responsible for

0 0.25 0.5 0.75 1.0 the maintenance of in vitro transformation in

bothgroupsof viruses maptothe left (guanine FRACTIONAL LENGTH pluscytosine rich) end of thegenome (7, 9, 11; Correlationof the rates of accumulation of Mak et al., manuscript inpreparation). The ob-tic RNAcomplementary to DNAfragments served

simflarity

further impliesthat during the

D position. The data obtained with the evolutionary divergence of the group A, B, and

'ragments, shown in Tables 3 and 4, are Cadenoviruses theoveralllayout of the genome

l.Specificaccumulationratesfor early RNA

ere calculated by dividing thefraction of the has beenpreserved,whereastheDNAsequences !y RNApresent in each class by the maxi- of the idividual genes have diverged to the sible fractional size of the corresponding pointwhere very little sequence homology can

ling region. (Upper) Cytoplasmic RNA la- be detected (17). beled5.5 to 7.5 hpostinfection. (Lower) Cytoplasmic

RNAlabeled24 to 26hpostinfection.

These results clearly show that both the "early" and "late" cytoplasmic RNAs can be subdivided accordingtotheirrates of accumu-lation.Byexamining onlytwoperiods postinfec-tion we are able to distinguish three classesof RNA onthisbasis: 1, RNA whichaccumulates rapidly early (derived from HindIII-E and -F); 2, those early RNA species which accumulate moreslowly (withinBamHI-B and-C);3, RNA not detectableearly which accumulates rapidly late (derived from EcoRI-B, -D, and -E). It is possiblethat an examination of moretime inter-valswill reveal furthercomplexity. It istempting tospeculatethat these RNA classesdiffer in the timesof their maximalrate ofaccumulation.

Flint andSharp (8) have quantitated the lev-els of Ad2 nuclear andcytoplasmic RNA derived from different segments of the viral genome at varioustimespostinfection. At both 18 and 32 h postinfection, RNA derived from theregion of thegenomeextendingfromapproximately65 to 90 map units constitutes the most abundant

ACKNOWLEDGMENTS

This work was supported by grants from the National

Cancer Institute of Canada and National Research Council of Canada. J.R.S.isaResearch Student of th-e National Cancer Institute of Canada.

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6 4

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Figure

FIG.1.EcoRI,unlabeledcpm)by fluorography Hybridization oflabeled 1- and r-strand-specific probes complementary to late cytoplasmic RNA to DNA fragments
TABLE 2. Distribution of the 1- and r-strand sequences complementary to late cytoplasmic RNA amongspecific DNA fragmentsa
FIG. 2.RNA;RNAdataasinformationTable early Map ofsites of the Adl2 genome expressed and late cytoplasmic RNA
FIG. 3.fromments.stripsEcoRIdescribedtarytected Hybridization of labeled late complemen- strand-specific RNA to unlabeled EcoRI frag- Cytoplasmic RNA labeled with [3H]uridine 21 to 24 h postinfection was strand selected as in the text and hybridized to nit
+4

References

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