Avian myeloblastosis virus transforming gene is related to unique chicken DNA regions separated by at least one intervening sequence.

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Vol.41,No. 1 JOURNALOFVIROLOGY, Jan. 1982,p.250-257

0022-538X/82/010250-08$02.00/0

Avian

Myeloblastosis

Virus

Transforming

Gene Is Related

to

Unique Chicken DNA Regions Separated by

at

Least One

Intervening

Sequence

BERNARD PERBAL AND MARCEL A. BALUDA*

UniversityofCalifornia atLosAngelesSchoolofMedicine andMolecular BiologyInstitute, Los Angeles,

California90024

Received 30July 1981/Accepted 6 October 1981

Identification of several additional restriction endonuclease sites within the

cellular substitution (amv) inserted into the avian myeloblastosis virus proviral

genome haspermittedus toisolate differentregionsof theamvsequence. These

subsets ofthe avianmyeloblastosis virustransforming gene have been cloned in

theplasmidpBR322 and usedashybridization probestoinvestigatethetopology ofhomologous (proto-amv)normal chicken DNAsequences. The results showed

that thecellular proto-amv sequences in C/O chicken DNAareinterrupted by at

least oneinterveningsequence. Apartialarrangement of the proto-amv sequences

ispresented.

Avian myeloblastosisvirus(AMV)contains a

sequence of approximately 1,000 nucleotides

which appears to be responsible foracute

my-eloblastic leukemia in chickens (25, 26). This

sequence, designated amv, is homologous to

cellular DNA sequences(proto-amv) present in

all vertebrate species from amphibians to

hu-mans (5, 24, 27). This AMV leukemogenic

se-quenceis not present in thehelper

myeloblasto-sis-associated virus types 1 and 2 (MAV-1 and

MAV-2) orin Rous-associated virus type 0, the

endogenous virus produced by some chicken

strains (24-26). Previous endonucleasemapping

of the XCharon4Arecombinantclone(XllA1-1)

carrying the AMV provirus has revealed that the

amv sequence lies between the KpnI and

3'-proximalXbaIsites and contains one EcoRI site

but no BamHI or HindIII site (Fig. 1). The

hybridization results obtained earlier with the

cloned AMV HindIII fragment of 3.9 kilobase

pairs (kb)(5) and with other probes (26) (AMV

internal EcoRI-EcoRI and EcoRI-3' HindIII

fragments) indicated that three chicken DNA

fragments generated byEcoRI digestion contain

genetic information homologous to the amv

se-quences. Two of them are adjacent,the 5.4-kb

fragment and either the 2.2- or the8.7-kb

frag-ment, whereas the third one is farther apart or

present at adifferent homologous genetic locus.

The first alternative suggests that the normal

cellularproto-amv DNA may contain an

inter-vening sequence (intron). Mosteucaryotic genes

contain introns, up to approximately 50 in the a-collagen gene (32), whereas some genes have

none, e.g., histone (13) and interferon (15, 18).

The cellular analogs of viral transforming

se-quences analyzed so far also seem to fall into

two categories with respect to introns. Some have introns, e.g., the cellular sequences

ho-mologous to thetransformingregions of Abelson

murineleukemiavirus,felinesarcomavirus,and

Rous sarcoma virus (11, 22), whereassome do

not, e.g., the Moloney-murine sarcoma

virus-related sequences (19). The chicken cellular

DNA sequences homologoustothesrcgene of

thePragueC strainofRoussarcomavirus have

recently been shown to contain fiveorsix

inter-vening sequences (22).

The probes used in the earlier studies

con-tained some viral sequences common to AMV

and MAVinaddition toamv.Therefore,itcould

not be ruled out that some EcoRI or HindIII

chicken DNA fragments which hybridized to

theseprobes containedendogenousproviral

se-quences or that some cellular proto-amv

frag-ments had the same size as the endogenous

proviralfragments. To carry out a more detailed

and accurate analysis of the cellular proto-amv

sequences,weisolated and purified by

subclon-ing inpBR322 amv-containing DNA fragments

with fewer or no proviral sequences and used

themashybridization probes with C/O chicken

DNAdigested with three restriction enzymes.

MATERIALS AND METHODS

Cloning. Escherichia coli HB101 (pro leu thilac-4 hsdR endA recA rpsL20 ara-14 galK2 xyl-5 mtl-l

svpE44) was the recipient in transformation

experi-ments(8). The vector used was pBR322. The transfor-mation procedure of Villa-Komaroff et al. (30) was used with minor modifications (S. Horowitz, personal communication). Bacteria were grown in 300 ml of

250

on November 10, 2019 by guest

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INTRON IN proto-amv DNA SEQUENCES

EcoRI 2.6 (4 kb)

5' 7 T zIF ,T ,IY 3'

Hindl2.6 (4 kb)

AMV 5 7 T I wW I T Y

+~~~~~~~~~~~-~

1 s

3'

I

c

C

0.-pBR 322/KX162

(1.3 kb)

r

I.

E 0

co

pBR322/HAX4

(1.0 kb)

pBR322/EX II

(0.8 kb)

Sx1

(0.45 kb)

ES2

(0.35 kb)

-~~ Iu

t

I

t

I t 1(r)0E(c> Tr LU (.')

Q-HaeIl

E I,_

GD

i

It

_

0

x

0I

0

CD

-XTC

_n:

-O

X

in

0

Eal O

[image:2.504.70.429.67.507.2]

I~t4~~~U g a I

~~~~~Sol

I Sal I (pBR322) Et

M LLJ

a BarmHI linkers

FIG. 1. Derivation of thehybridization probes. The restriction endonuclease sitesin MAVand AMVare

mappedaspreviously described (26). Symbols: V,HindlIl; *, BamHI;V,XhoI; *, BgII,O,EcoRI;0,XbaI;

0,KpnI.Themappingof restrictionsiteswithintheKpnI-XbaI fragmentwascarriedoutbydigestionof the

32P-labeledpurified fragment.DNAfragmentsbearingBamHIlinkersatboth endsweresubcloned afterinsertionat

theBamHI site of pBR322. Arrows indicateknown restriction sites still available aftersubcloning.

tryptone-yeast extract medium (10 g of tryptone [Difco]perliter,5gofyeastextractperliter,and 5 g of NaCl per liter) until the optical density at 600 nm

reached0.3, spundown, suspended in 30ml of cold buffer(25mM sodiumacetatebuffer[pH5.6],70mM

MnCI2, 30 mM CaCl2), andkeptfor 20 min at4°C. Competentcellswerespunat4°Candresuspendedin 10 ml of thesamebuffer,and 200,ulofthesuspension

wasmixed with 25IlIof theplasmidsolution.After 1 h of incubation at4°C, 0.1-ml samples were platedon tryptone-yeast extractagarplates containing100 pLgof

ampicillinperml.Screeningofindividual transformed clones for their plasmid content was performed as

previouslydescribed(6, 14).

DNApurification.Purification ofplasmidDNAwas

performed as described by Curtiss et al. (10) after

amplification in thepresenceofchloramphenicol (9). High-molecular-weightcellular DNA(45kborlarger)

was purified from C/O H&N chicken embryos as

described earlier(3).

Isolation of A DNA recombinant dones containing

proto-amvsequences.A total of 3 x 105 plaquesfroma

VOL. 41, 1982

MAV

251

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252 PERBAL AND BALUDA

ACharon 4Alibrary of leukemic chicken DNA partial-ly digestedwithEcoRJ(27)werescreened by using the in situ plaquehybridizationtechniqueof Benton and Davis (4). Nitrocellulose filters were duplicated and hybridized eithertotheKpn-Xbaprobeor tothe MAV EcoRI 2.6(3.9-kb) probe. Plaques which hybridized

with the Kpn-Xba DNA fragment but not with the MAV-1 DNAfragmentwereconsideredlikelyto con-tain proto-amv sequences. A total of 18plaqueswere picked, and each phage isolate wasreplaqued twice

more. Nineproto-amv-containing recombinantclones were grown tohigh-titer stocks in E. coli DP50 supF (7).The DNAwaspurifiedasdescribed(27).

Purification ofDNA fragments. Restriction enzyme digestions wereperformedin the buffers recommend-ed by Bethesda Research Laboratories, Rockville,

Md. A 10-fold-excess ratio of enzyme to DNA was generally used. Digestion of cellular DNA and gel

electrophoresisprocedures havebeen described(26).

The Kpn-Xbafragment wasisolated from meltedsea plaque gel (Seakemlow-temperature-meltingagarose; Marine Colloids, Rockland, Maine) (26). All other DNAfragments wereelectroeluted fromregular agar-ose(SigmaChemical Co.,St.Louis, Mo.)gelsat150 mA for at least 90 min in 5 ml of TAE buffer(40mM

Tris-hydrochloride [pH 7.6], 5 mM

Na-C2H302 *3H20,1 mMEDTA). DNAfragments were

thenextractedtwice withamixtureof

phenol-chloro-form-isoamyl alcohol(1:24:1),twicewith

chloroform-isoamyl.(24:1),and twice with ether before

precipita-tionwithethanol.

Ligatton ofBamHIlinkersto DNAfraginents. BamHI

linkers (17, 21)were phosphorylated in reaction vol-umes of 30 ,ul containing 0.8 nM BamHI linkers

(Collaborative Research,Inc.,Waltham, Mass.),0.07 MTris-hydrochloride buffer(pH7.4),0.01 MMgCl2,

0.01 M2-mercaptoethanol, 0.06 mM ATP, and 5 U of T4polynucleotidekinase(Bethesda Research Labora-tories). After 2 h of incubationat 37°C, the reaction wasstopped byincubationat70°Cfor 10 min. Restric-tion DNAfragmentswithcohesive ends were incubat-ed in the presence of DNA polymerase I (Bethesda Research Laboratories) to generateblunt ends (1, 2, 12). Inthe case of enzymes generating 3' protruding ends (KpnI, HaeII), DNA fragments (5 ,ug) were incubated in the presence of 60 mM Tris buffer (pH

7.5),10mMMgCl2,10 mM2-mercaptoethanol,and1

Uof E. coliDNApolymerase I for 10min at 12°C to

takeadvantageofthe 3' -+5'exonucleaseactivityof

polymeraseI.Thefour deoxynucleotide triphosphates were then added to a final concentration of 0.2mM,

and polymerization was allowed to proceed for 1 h at

12°C. In the case of enzymes generating5'protruding ends(EcoRI,XbaI), the restriction sites werefilledin by polymeraseI.After inactivation ofpolymerase I for 10minat70°C, ATP was added to a final concentra-tion of1mM,and1U of T4DNAligase(New England Biolabs, Beverly, Mass.) was added to the mixture. Ligation was performed at 12°C for 10 h.

Ligation of DNA fragments to pBR322 vector. The pBR322 vector, cleaved with the appropriate restric-tion endonuclease, was treated with calf intestine alkaline phosphatase (Boehringer Mannheim Bio-chemicals, Indianapolis, Ind.) to reduce colony back-ground caused by recircularization of vector DNA (29). Six units of calf intestine alkaline phosphatase as ammonium sulfate precipitate were spun in an

Eppen-dorf microfuge tube and suspended with 5 p.g of cleaved pBR322. The reaction mixture (100 ,ul)was

adjustedto20mMTris-hydrochloride buffer(pH8.0) and incubated for 1 h at 37°C. The reaction was

stoppedby the addition of 10 mMTris-hydrochloride

buffer (pH 7.5)-i mM EDTA (pH 7.5)-0.2 M NaCI-0.5% sodiumdodecylsulfate. The treatedvector was thenextracted twice withphenol-chloroform-isoamyl

alcohol (1:24:1), twice withchloroform-isoamyl alco-hol(24:1),and twice with ether before ethanol

precipi-tation. Ligation of DNAfragmentstotreatedpBR322

was performed in 250 mM Tris-hydrochloride (pH

7.4)-S50mMMgCI2-5mMATP-50 mM

2-mercaptoeth-anolat aDNAconcentration of 200jig/mlwith 1

RI

of T4ligase (NewEngland Biolabs).

Nick translation ofpurified DNA fragments. Nick translation(20) wasperformedin the presence of 200 ,uCi of [a-32P]dCTP (Amersham Corp., Arlington

Heights,Ill.), 30,uMeach ofdATP, dGTP,anddTTP,

5 ,ul of nick translation buffer (Bethesda Research Laboratories), 2 ,ul of DNApolymerase1(2U/,ul),and 2 ,ulof DNase I(WorthingtonDiagnostics, Freehold,

N.J.; DPFFgrade0.2

jig/ml).

DNaseincubationwas started 5 minbefore the additionof,DNApolymerase

I. The 32P-labeled fragments and the remaining [a-32P]dCTPwereseparated by chromatographyon

Se-phadex G-75 equilibrated in 0.1 x SSC plus 0.1% sodium dodecyl sulfate. The specific activity of the 32P-labeled probeswasapproximately2x108cpm/p.g.

Sizing of the 32P-labeled DNA fragments obtained afterdigestion of nick-translated probes with restriction enzymes.Thechainlengthoffragmentsobtained after

singleand doubledigestionsofDNAswasdetermined byelectrophoresis in5%acrylamidegelasdescribed by Maniatisetal.(16). Kodak X-ray filmswereplaced

onthe top of thegels,covered with SaranWrap,and

exposedfor 3 hat4°C.

Preparation and hybridization of DNA (Southern) blots.The DNAfragments separated by

electrophore-sis were blotted on nitrocellulose membrane filters (Millipore Corp., Bedford, Mass.) as described by Southern(23).Hybridizations were performed

accord-ingtoWahletal. (31).Filters were washed twice in 2x SSC plus0.1% sodiumdodecylsulfate atroom tem-peratureandtwo tothreetimesat68°C in 2xSSC plus 0.1% sodiumdodecyl sulfate,rinsedtwice in 2x SSC, dried,andexposed with Kodak X-ray films (XR5) and aDuPontintensifying screen at -700C.

Physical and biologicalcontainment.This work was carriedout atthe P2-EK2containment levels accord-ing to the revised guidelines of the National Institutes of Health.

RESULTS

Derivation of the amv-specific probes. The

preparation of specific amv probes started with

the isolation of the KpnI-XbaI (3'-proximal)

fragment from the AMV HindIII 2.6 fragment

(2.6x 106 daltons [4 kb]) (Fig. 2) already cloned

in pBR322 (5). The Kpn-Xba DNA fragment was

isolated by gel electrophoresis after double

di-gestion of AMV HindlIl 2.6/pBR322

recombi-nant DNA with KpnI and XbaI, ligated to

BamHI linkers, and cloned in pBR322

(pBR322/KX162). This probe still contains

ap-J. VIROL.

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INTRON IN proto-amv DNA SEQUENCES

O DC b C

2"'37-... _

--- _ _

t.-:

fti04Z O#k

4-3

a

K>x 6 i_>P 4

FIG. 2. Hybridization ofresti

ated DNAfragmentswithamv-c4

chicken DNA was digested wit

(b), orHindlll (c), electrophore

gel, and transferredtonitrocellu blots). The three different restri

were runin parllel inthesameg

four separate sets. A sample digested with Hindlll endonuw parallel lane wasused asmolec pressed in kilobase pairs. The shownin Fig. 1wereusedafter dCTPby the nick translationpr(

proximately 200 base pairs

located at the 5' terminus,

previous electron microsc

Therefore, a search was ma

enzyme which could remo'

pairsfrom the 5' end of the j

The 32P nick-translated

was treated with several re

AluI, MspI,Sau3A1, CfoI,

PvuII, Hinfl, HpaI, Sail,

Among the few enzymes w

cleavagein theKpn-Xbafral

at450 basepairs from theX

cleavedat290 basepairsfror

1).A HaeII(XbaI-BamHI)-J isolated afterdigestionof K) nuclease. AfterligationofB

HaeII sitesand digestionw

clease,aHaeII-Xbafragmei

ersatboth endswas cloned

One ofthe

resultinf

cloni

bridized with the 3P nic

EcoRI 2.6 fragment (also 2

kb]), to determine whethe

quencesremainedat the 5'ei

fragment.The results(datan

thathelperviral sequencesv

end oftheamvinsertinHA)

digestedwithSalltoestabli.

the insertinpBR322. Clone,

XbaI DNAfragmentin the t

tionswithin theplasmidDN

a b c a b c The presence of a single EcoRI site in the

HaeII-XbaIfragment and in pBR322 DNA (28)

was exploited to generate an

EcoRI-XbaI

DNA

fragment from pBR322/HAX4 after digestion

_ ~_ with EcoRI. This fragment was cloned into

pBR322 after treatment with DNAPoll,ligation

of BamHI linkers, and digestion with BamHI

endonuclease. The orientation of the

EcoRI-XbaI DNA

fragment

with respect to

plasmid

a DNA in one

(pBR322/EXII)

of the clones

ob-tained was determined by digestion with Sall and EcoRI endonucleases. This clone was then

used to isolate twofragments carrying the amv

sequences located on the 3' side of the EcoRI

riction enzyme-gener-

site:

an

EcoRI-SalI

(ES2) and a

SalI-XbaI

frag-ontaining probes.C/O ment (SX1).

'h BamHl (a), EcoRI

esed in 0.7% agarose The sizes and derivations of the different

ilose filters(Southern hybridization

probes

are shown in

Fig.

1.

iction enzymedigests Hybridization of chicken DNA fragment to

gelbut were blotted in

amv-containing

probes. High-molecular-weight

ofeaseXnd

(0.5

ig)

(larger than 45 kb) DNA from C/O chicken

ularesizemarkners

ex-

embryos

was

digested

with either

EcoRI,

Hin-hybridization probes

dIll,

or BamHI restriction endonuclease,

elec-labelingwith

[a-32p]-

trophoresed in agarose gels, and transferred to

ocedure. nitrocellulose. The blots were

hybridized

with

the

32P-labeled

probes described above (Fig. 2

andTable 1).

(i) Hybridization with the KpnI-XbaI probe.

of viral sequences The Kpn-Xba DNA fragment obtained by

as estimated from BamHIdigestion of pBR322/KX162 was purified

opic

studies (27). by

agarose

gel electrophoresis, electroeluted,

Lde fora restriction and 2P nick translated. The chicken DNA

ve about 250 base EcoRIfragments hybridizing with the Kpn-Xba

Kpn-Xbafragment. probe are 8.7, 5.4, 3.9, and 2.2 kb. The low

Kpn-Xba fragment intensity of hybridization obtained for the EcoRI

.striction enzymes: 3.9-kbfragment suggests thatthis hybridization

WaeIII,

TaqI,HaeII, resultsfrom the 200 base pairs of viral sequences

EcoRl, and SmaI. present at the 5' end of the KX162 probe.

ihich gave a

single

However, this probe no longer detects the

gment,

Sallcleaved EcoRI20.8-kbfragmentwhichhybridizedtothe

'bal

site,

andHaeII HindIll2.6probe (5).Thechicken DNAHindIII ntheKpnIsite

(Fig.

fragments detected are 5.4, 3.3, and 1.4 kb.

ffaeII

fragment

was Hybridization was notdetected with the 2.6-kb

(162byHaeII endo- fragmentwhich hybridizedwith theHindIII2.6

tamHIlinkers to the probe. The intensity of the HindIII3.3-kb

frag-rithBamHIendonu- mentrelativetothe othertwois

greatly

reduced,

nthavingBam link- suggesting that this may also be due to the intopBR322. presence of viral sequences

remaining

in the

es

(HAX4)

was

hy-

probe. The detection of the 5.4-kb

fragment

Sk-translated

MAV might be due either to the viral sequences

re-.6 x 106 daltons

[4

maining

in theKX162

probe

or to the existence

sr helper viral se- ofanamvfragmentwhichhasthesame

molecu-ndof theHaeII-Xba lar

weight

as the

endogenous proviral

DNA

iotshown)indicated

fragment

which

hybridized

to the MAV EcoRI

vereabsentatthe5' 2.6

probe

described earlier

(5).

The patterns

K4.HAX4wasalso obtained with the C/O DNA BamHI digest

sh theorientationof showed that four

fragments hybridize

with the

sbearingtheHaeII- AMV

Kpn-Xba

probe

(19.6,

12.4, 5.5, and 4.8 :wopossibleorienta- kb) and four

fragments

hybridized

with the

{Awereidentified. MAV

probe

(12.4,

5.8, 4.8,

and 1.9

kb).

There-253

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TABLE 1. Hybridization of C/O chicken DNA fragments with different amv-containingprobesa

Cellularfragments(kb)generatedwith:

DNAprobes EcoRI HindIII BamHI

MAV-1(EcoRI2.6) 3.9 5.4, 3.3, 2.6, 1.9 12.4, 5.8, 4.8,1.9

AMV

HindIl 2.6 20.8, 8.7, 5.4, 3.9, 2.2 5.4, 3.3, 2.6, 1.4 NDb

KpnI-XbaI 8.7, 5.4, 3.9,2.2 5.4, 3.3, 1.4 19.6, 12.4, 5.5, 4.8

HaeII-XbaI 8.7, 5.4, 2.2 5.4, 1.4 19.6,5.5

EcoRI-SalI 2.2 5.0 19.6

SalI-XbaI 2.2, 8.7 5.4, 1.4 19.6,5.5

aThe MAV-1 (EcoRI 2.6) and AMV

(HindlIl

2.6) probes were obtained from pBR322 clones

previously

described(5). The sizes of thefragmentsweredeterminedbycomparisonwith those ofXDNAmarkers. bND,Notdetermined.

fore, it appears that the 19.6- and 5.5-kb

frag-ments are amv specific and that the 12.4- and

4.8-kb fragmentsare detectedby KX162owing

to thepresenceofviral sequences in thisprobe.

(ii) Hybridization with the HaeHI-XbaI probe.

The HaeII-XbaI DNA fragment obtained by

BamHIdigestionofpBR322/HAX4wasisolated

by

adarose gel electrophoresis, electroeluted,

and

3

P nick translated. Three bands were

ob-tained after hybridization with C/O chicken

DNAdigested with EcoRI:8.7, 5.4, and 2.2 kb.

Thus, there is no EcoRI 3.9-kb bandcontaining

amv-related sequences. Hybridization of this

probe toC/O chicken DNA digested with

Hin-dIIIrevealed the presenceof onlytwofragments

complementary to the amv sequences, the

5.4-kb band being much darker than the 1.4-kb

band. Therefore, the detection of the HindIII

3.3-kb fragment by the Kpn-Xbaprobe resulted

from the

presence.

ofendogenous proviral

se-quences.Hybridization of theHaeII-XbaIprobe

tochicken DNA digested with BamHI confirms

theconclusions reached above from the

compar-isonof the bandpatternsdetected withthe MAV

EcoRI 3.9-kb and the AMV Kpn-Xba fragments.

Only two amv-related BamHI bands were

de-tected:19.6 and 5.5 kb, the latter being relatively

darker than the 19.6-kb band.

(iii) Hybridization with Sall-XbaI and

EcoRI-Sail probes. These probes were generated by

Sall digestion of pBR322/EX11, purified by

agarose gel electrophoresis, electroeluted, and

32P nick translated. The SalI-XbaI probe

con-tains amv sequences complementary to the

chicken DNAEcoRI8.7- and 2.2-kb fragments,

totheHindlIl5.4- and1.4-kb fragments, and to the BamHI 19.6- and 5.5-kb fragments. The

hybridizationintensityof theEcoRI8.7-kb, Hin-dIII 1.4-kb, and BamHI 5.5-kb bands was

great-ly increased when the

SalI-XbaI

probewas used

instead of the HaeII-XbaI probe, reflecting the

increasedconcentrationofamvsequences in the

former probe. Only one chicken DNA EcoRI

fragment (2.2kb),oneHindIII fragment (5.4kb),

and one BamHIfragment (19.6 kb) were found

tocontainsequences homologoustothe

EcoRI-SalIprobe.

Analysis ofchicken-A DNA recombinants

car-rying proto-amv sequences. Nine DNA

recombi-nant clones carrying cellular proto-amv

se-quencesidentified as describedin Materials and

Methods were digested with EcoRI and

fraction-ated by electrophoresis. They fall into three

different categories whose prototypes areX111,

X533, and X1041 (Fig. 3A). In addition to the

19.8- and 10.9-kb XDNA fragments (7), clone

Xlll contains three EcoRIfragments (8.7, 2.2,

and 1.8 kb), clone X533 contains three EcoRI

fragments (8.7, 4.1, and 1.8 kb), and clone X1041

contains four EcoRI fragments (15.1, 8.7, 5.4,

and 2.6 kb) (Table 2). Theinternal EcoRI

frag-< -.

_ _

--* _

E. .g.. <... _ ....-. v,.'

8£

Y, dsx:.3n': ':'.:.se'.!: #':: ,...

FIG. 3. Analysis of chicken-A recombinant DNA clones containing proto-amv sequences. Chicken-A Charon 4A recombinants carrying proto-amv se-quences were isolated and their DNA was digested with EcoRI as described in the text. (A) a, X+; b,

X1041; c,X533; d, Xlii.Paralleldigests were blotted on nitrocelluloseand hybridized with the 32P-labeled Kpn-Xba probe. (B) a, X1041; b,X533; c, Alii. The marker A DNA fragments with sizes in kilobase pairs were run in the same gel.

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TABLE 2. Size of the DNAfragmentsgeneratedby

EcoRIdigestion of chicken-A DNA recombinants

IN

Recombinant clone Fragmentsize (kb) kil1 19.8,a 10.9,a8.7,b 2.2,b 1.8 X533 19.8 a10.9a87,b4.1, 1.8 X1041 19.8,9 15.1,10.9,a 8.6,5.4,b2.6

aACharon 4A EcoRI fragments.

bFragments which hybridize with the AMV

(KpnI-XbaIorHaeII-XbaI)probes.

mentsof A Charon 4A can be replaced by foreign

DNA inserts in the size range of 8.2 to 22.2 kb

(7). Also, the leukemic chickenDNAfragments

used to prepare our chicken-A library ranged in

A

_I-I

'-4 ~ 4 c

C 4) 0

Q. Oa

-Y rI L1J

i

j

0.22

1

[TRON IN proto-amv SEQUENCES 255

sizebetween 16 and 22 kb (27). Consequently,

from the summation (approximately 32 kb) of

the fragment sizes obtained with X1041, it

ap-pears that the 15.1-kb EcoRI fragment might represent an intramolecular recombinant, e.g.,

from looping out during amplification of the

DNAstock.

Hybridization of Southernblots of the

EcoRI-digested DNA with the 32P-labeled Kpn-Xba

probe (Fig. 3B) shows that Xl1 contains two

DNA fragments(8.7 and 2.2 kb)carrying

proto-amv sequences, and X533 and X1041 contain

onlyoneeach, 8.7 and 5.4 kb, respectively.The

same results were obtained with the HAX4

probe. Thus, the proto-amv-containing EcoRI

0

c)

0.35

1

AMV

0

-0 x 0.45

/ / LI

B

LI IS L2

t

2.2

o

0

T5.4

0 I 19.6

A+----i

1-u--o 0

o 0

llJ LLI

1.4l

c C c

l t 5.4

5- -r

I I

E E

m 0

en e

FIG. 4. Partial arrangementof the proto-amv sequencesin C/O chicken DNA.(A)Theproto-amv sequences

areinterrupted byatleastoneinterveningsequence(IS)in C/O chicken DNA.(B)The relativepositionofthe

EcoRI, Hindill, and BamHI fragments carrying proto-amv sequences was established from hybridization patterns obtainedwith theprobesdescribedin thelegendtoFig.1. Itis not known whether the2.2-and8.7-kb EcoRlfragments arecontiguous.Thesameuncertainty appliesto the twoHindIIIand BamHIfragments;i.e., theremaybeone ormorerestrictionenzymesites within the intronshown.Also,as discussedin thetext,there maybeasecondintron between the5.4- and 2.2-kb EcoRIfragments.

Chicken DNA

A

/is

\

_\.

L2

/

Chicken DNA

5.4

0

C.)

LUJ

8.7

I

cr

0

LUJ

I

E

I

E

m

im

http://jvi.asm.org/

[image:6.504.67.431.248.602.2]

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DNAfragments from thesechicken-A

recombi-nant clones havethe same sizes as those

detect-ed in hybridization studies between the

Kpn-Xba probe and normal chicken DNA digested

withEcoRI.

DISCUSSION

Previous studies performed with different

AMV internal restriction fragments as

hybrid-ization probes indicated that theamv sequence presentin the AMVgenome washomologousto

a setof normal chicken DNAsequences

(proto-amv)from which itmighthaveoriginated (27).A

complex organization of the proto-amv

se-quences inchicken DNA was suggested by the

existence of several Hindlll and EcoRI

frag-ments complementary to the amv sequence (5, 27).

In this study, DNA fragments representing

different regions of the amv sequence having

little or no homology with MAV helper

se-quences were used as hybridization probes to

maptheproto-amv sequencesinchickenDNA.

Three EcoRI fragments (8.7, 5.4, and 2.2 kb),

twoHindIIIfragments (5.4 and1.4 kb)andtwo

BamHI fragments (19.6 and 5.4 kb)in chicken

DNA are homologous to amv. Since the amv

sequence contains only one EcoRI site but no

HindIII orBamHI site (26), these results

indi-cate that proto-amv information might not be

contained in asingle setof DNAsequences.

Hybridization with the AMV EcoRI 3.3-kb

fragment and with the AMV 1.3-kb fragment

locatedbetween the 3'-proximal EcoRI and

Hin-dIII sites had shown (27) that the 5.4-kb EcoRI

fragment was adjacent toeither the 8.7- or the

2.2-kb fragment. The hybridization patterns

ob-tained with the SalI-XbaI and EcoRI-SalI

probes now place the 2.2-kb EcoRI fragment

adjacenttotheEcoRI5.4-kbfragmentand

indi-cate that approximately one half of the amv

sequences located between the SalI and XbaI

sites originate fromtheEcoRI 2.2-kb fragment.

Therefore, it appears that the 3' region of the

proto-amv chicken sequences are interrupted by

atleastone interveningsequence.

The isolation of a X Charon 4A recombinant

(Xlii)

DNA carrying the two

EcoRI

8.7- and

2.2-kb fragments in the same insert indicates

thatthesetwo fragments are relatively close to

each other and certainly are not located on

different chromosomes. Comparison of the

in-serts of

Alii

and X533 reveals that the

EcoRI

8.7-and 2.2-kb fragments are either contiguous or separated by 1.8 kb of chicken DNA at the most.

Sincewedidnot find achicken-A recombinant

carrying both the EcoRI 5.4- and 2.2-kb

frag-mentsamong the 18 clones obtained so far, the possibility exists that another intervening

se-quence might be present between these two

proto-amv fragments.

These results, along with those obtained

pre-viously (5, 27), permitapreliminary arrangement

of theproto-amv sequencesin H&NC/O

chick-en DNA (Fig. 4). Two different domains of

proto-amvsequences(Li andL2)areseparated

in chicken DNA by an intervening sequence

which contains at least one BamHI site, one

EcoRI site, and one HindIII site. The relative

positions of the EcoRI, BamHI, and HindIlI

sites with respect to each other within the

in-tron, and the size of this intron, are being investigated.

ACKNOWLEDGMENTS

We thank L. Souza for helpfuldiscussions.

This research was supported by Public Health Service research grantCA-10197from the National Cancer Institute. B.P. was partly supported by the Centre National de la Recherche Scientifique and was the recipient ofa travel fellowshipfrom the North AtlanticTreaty Organization.

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