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Organization of chicken DNA sequences homologous to the transforming gene of avian myeloblastosis virus. I. Restriction enzyme analysis of total DNA from normal and leukemic cells.

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0022-538X/82/110586-09$02.00/0

Copyright©1982, AmericanSocietyforMicrobiology

Organization

of Chicken DNA

Sequences Homologous

to

the

Transforming

Gene of Avian

Myeloblastosis Virus

I.

Restriction Enzyme Analysis of Total DNA from Normal and

Leukemic Cells

BERNARD PERBALANDMARCEL A. BALUDA*

UCLA School ofMedicine andJonsson Comprehensive Cancer Center,LosAngeles, California90024 Received 19 April 1982/Accepted 29 June1982

Hybridization probes consisting of cloned DNA recombinants whichrepresent

different regions of the leukemogenicsequence(amv) from avian myeloblastosis viruswereusedtocarryoutamoredetailed restriction endonuclease analysis of the homologous sequences (proto-amv) presentin normal and leukemic chicken DNA.The results show that four large introns interrupt the normal cellular proto-amv sequences and that there is no majorrearrangementof these sequences in leukemicmyeloblasts.

The leukemogenic potential of avian

myelo-blastosis virus (AMV) has been associated with

asequence (amv)of approximately 1.2kilobase pairs (kb) whichhasreplacedtheviralenv gene

(18, 19). The amv insert is homologous to a

unique chicken DNA region (proto-amv) which isinterrupted by unrelated DNAsequences(12). Intronsare presentwithinthecellular homologs ofmostviraloncogenes. Forinstance, the

chick-en cellular homolog of the src gene of Rous sarcomavirus (PragueC strain) containsatleast five introns (16), and cat DNA sequences ho-mologoustotheoncogeneof theSnyder-Theilen feline sarcoma virus contain at least three

in-trons(9).

Tofurther characterize the arrangement of the

proto-amv sequences in normalchicken DNA, DNArecombinant clones representing different regions of amv were prepared and used as

hybridization probes with C/O chicken DNA

treated with various restriction endonucleases

eithersinglyorin combinations.Asimilar analy-sis was performed with DNA from leukemic chicken myeloblasts. These studies revealed

that in normal chicken DNA, the proto-amv sequences areinterrupted by atleast four large introns, and that there is no major reorganiza-tion ofthese sequences inleukemic DNA.

MATERIALS AND METHODS

Purification of total DNA. Purification of pBR322

DNAwasperformedasdescribedbyCurtissetal.(8). High-molecular-weight DNA (4.5 kb or larger) was

purified fromC/G andC/E whole chickenembryosor

tissuesasdescribed earlier(1).

Purification of DNAfragments. TheDNAfragments

obtainedby restriction enzymetreatment ofpBR322 hybrid clones were purified by electroelution as

al-ready described(12).

Preparation of Southern blots.The DNAfragments

obtained after electrophoresis in agarose gels were blotted onto nitrocellulose Milliporefilters (Millipore

Corp., Bedford,Mass.) under the conditions described earlier(12, 17).

Nick translation.Conditions used for 32plabelingof DNAfragments by nick translation (13) werereported

earlier(12).

Cloning. Escherichia coli HB101 (4) was the recipi-ent and pBR322 was the vector in transformation

experiments. Ligation conditions and transformation procedures were previously described (12). Screening of the transformant clones for the nature of their

plasmid DNAcontent was performed bya modifica-tionof the method described by Birnboim and Doly (3). Individual transformants were grown overnight at

37°Cin 5 ml of TYE medium(10 g of Tryptone [Difco Laboratories,Detroit, Mich.],5 gof yeast extract, and 5 gofNaCl per liter) containing 100 ,g of ampicillin per ml. A1.5-ml volume of the cell suspensions was thentransferredto anEppendorf tube and spun for30 s inan Eppendorf centrifuge. Afteraspirationof the

supernatant, the cell pelletwas blended inaVortex mixer for 15 s and suspended in 100 ,ul ofafreshly prepared lysozyme solution (25 mM Tris-hydrochlo-ride [pH8.0]-10 mM EDTA-50 mMglucose-5 mg of lysozyme per ml [Sigma Chemical Co., St Louis, Mo.]). After15min of incubationat4°C, 200,ulof0.2 NNaOH-1% sodiumdodecyl sulfatewasadded,and the solutionwasmixedvigorously. Afteranadditional 5minonice, 150,ul of 3 M sodiumacetate(pH 5.0) wasadded, and the mixture was left at4°C for1h. The precipitate which formed after addition of thehigh salt solution was spun down for10 min at4°C, and the

supernatant was extractedonce with400Rl ofa1:1

phenol-chloroform-isoamylmixture

(chloroform-isoam-yl is 24:1) and twice with 400RIof chloroform-isoamyl

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(24:1).Nucleic acids were precipitated at -70°C for 30 min after the addition of 2 volumes of absolute etha-nol,pelietedbycentrifugation,and suspended in 60 ,ul of sterile distilled water. Usually, 5 to 10p,l ofthis suspension was used to run analytical digests in the presenceof 10 ,ug of RNase A.

Colony hybridization. The method described by

Grunstein and Hogness (11) was used. The washing

and sterilization of the filters before seeding with bacteria could be omittedwithout alteringthequality

of thehybridizationobtained ifampicillin plateswere used.

Hybrkzation.The DNAblots and the

colony-bear-ingifiterswerehybridizedasdescribedpreviously (2,

12).

Preparation of amv-spedclc subelones. The pBR

HAX4 clone (12) was used to prepare a subclone

containing the amv sequences located between the

HaeIIandthe EcoRIsites foundwithin the 1-kb amv insert inHAX4.The1.0-kbamvfragmentwasisolated

from pBRHAX4byBamHIdigestion, electrophoresis

in a 0.8% agarose gel, andelectroelution. It wasthen

digested withEcoRIendonuclease,andthe two

result-ingfragments (0.80 and0.20kb)wereseparated on a 2% agarose gel. The 200-base pair fragment

corre-spondingtothe 5'portionwasthen cloned inpBR322

EBpreviously electroelutedfrom a0.8%agarosegel

afterdoubledigestionwith BamHIand EcoRI endonu-cleases. Screening oftheresulting

tetracycline-sensi-tive colonies revealed that sometransformantscarried the expected sizeinsert, and hybridization with

32P-labeledHAX4showed thattheycontained thespecific

amvfragment. Oneof these transformants(pBREB3)

wasused in this work.

Clones SES3 and SX12, which contain the amv sequences located between theEcoRI-Sal sites and theSail-XbaIsites,respectively, wereprepared from

the pBR-EX11 clone(12).

Digestion of pBR-EX11 with Sail generated two DNAfragments(0.75and 4.4kb)whichwere separat-edbyelectrophoresis in a1% agarosegeland recov-ered by electroelution. The 4.4-kb fragment which contained the 3' portionbetween SalIandXbaI also contained thepBR322sequenceslocated between the BamHIandSalIsites(includingthepBR322replicon). Therefore, the electroeluted DNAfragmentwas

self-ligatedand useddirectlytotransform E. coliHB101. Thetransformantswereshowntocontain

amv-specif-ic sequences if screenedbycolonyhybridizationwith

purified 32P-labeled HAX4DNA.One of these clones

(SX12)wasused insubsequent experiments.

The other fragment (0.75 kb) generated by SalI

treatment of pBR-EX11 was subcloned in pBR322 previously digested with Sall and then was treated with alkalinephosphatase under conditions described earlier (12). Tetracycline-sensitive clones were ana-lyzedby bothminiscreeningandcolony hybridization with32P-labeledHAX4DNA.One of the cloneshaving

thecorrect insert(pBRSES3)was used as aspecific probe for the amv sequences located between the

EcoRIandSall sites.

Thederivation of these new amv subclones is sum-marized inFig. 1.

Preparationof theproto-amvE3probe. Probe E3 was derivedfrom theX-chickenrecombinant clone no,111 which contains twoEcoRIfragments (2.0 and 8.7 kb) of proto-amvsequencescorrespondingtothe3'end of

the amv sequence (12). The 8.7-kb EcoRI fragment

waspurified byelectroelution after digestion ofAlll

DNA withEcoRJendonuclease andelectrophoresis in a0.8% agarose gel. The 8.7-kbelectroelutedfragnent

(E3) was 32P labeled by nick translation to obtain

hybridizationprobe.

Physicalandbiological containment. This workwas carriedout atthe P2-EK2containmentlevels

accord-ingtotherevised guidelines of the National Institutes

ofHealth.

RESULTS

Analysis ofnormal C/O chicken DNA. (i) Hy-bridization with HAX4 probe. Todetermine the relativeposition in normal chicken DNA of the EcoRI,HindIII, and BamHI endonuclease sites which generate proto-amv DNA fragments, dou-ble enzymatic digestions were performed, and the resulting Southern blots werehybridized to the 32P-labeled HAX4 probe which contained mostof amv but no viralsequence. Single EcoRI andHindIII DNAdigests wereincluded within the same gel as internal controls in addition to standardsize markers.

The blot from a HindIII + EcoRI digest

AMV Y,T 9

I

w ,

nRR02 /lAY4 _ _'

-pOnIC'In#A-t

(1.0 kb)

pBR322/EXII

IjHoeI1

I H

E o8

C w

111

oE

t

ft

5.41

en x CD

0.8

kb)

ktb

ff

pBR322/EB3 5

(0.2 kb)

t

Hoel t

c t

pBR322/SES3

(0.35 kb) t 1

0 E 76

pBR322/SX12 W

(0.45 kb)

3,

MBomHI linkers

FIG. 1. Derivation of the amv-specific subclones. The restriction endonuclease sites in AMV are mapped as previously reported (12, 18). Symbols: V,

HindIII; *,BamHI; V,XhoI;U,BgllI;C,EcoRI; 0, XbaI; 0, KpnI. Thesubcloning ofHAX4and EX11 from AMV has been described(12). In probes HAX4 andEB3, theHaeIIsiteoriginally present in amv has been removedduring BamHI linker ligation.

44,

1.

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a Gcc

kD 293.

__ .

_4__3a

V~~~~~~~~!A

FIG. 2. Hybridization of restriction enzyme-treat-edC/O chickenDNA withHAX4. C/Ochicken DNA wasdigestedwith eitherHindIII (d), EcoRI (c),

suc-cessivelywithHindlIl andEcoRI (b),orwithHindIlI

andBamHI (a).Thedigested DNAwas

electrophor-esed in 0.8% agarose, transferred to nitrocellulose filters(Southem blots),andhybridizedto32P-labeled

HAX4 DNA. A mixture ofHindIll-digested ADNA and HaeIII-digested 4X174 RF DNA was run in parallel and used asmolecular size markers, expressed inkilobasepairs (kb).

showed four bands of 1.2, 1.9, 2.2, and 4.3 kb (Fig.2,lane b), whereas EcoRI or HindIII alone yielded the band pattern previously reported (12). Threefragments(2.1,5.4, and 8.7kb)were detected in the EcoRIdigest (Fig. 2,lanec),and twofragments (1.3 and 5.2kb)weredetected in the HindIII digest (Fig. 2, lane d). The 1.3-kb HindIlI fragmenthadpreviouslybeenestimated tobe1.4kb (12).

TheBamHI + HindIII digest revealed three fragments (1.3, 4.3, and 5.2 kb) with the 32P_

labeled HAX4probe (Fig. 2a). Previously (12), we had reported that BamHI digestion of C/O DNA generates two proto-amv fragments (5.4

and 19.6kb).

A similar hybridization pattern was obtained with DNApurified fromembryonic fibroblasts, spleencells,oryolksaccells obtained fromC/E chickens(datanot shown).

Theproportionofproto-amv sequences

pres-ent within the EcoRI and HindIll fragments

cannot be accurately determined, and

conse-quently the relativeintensityof the bandscannot

beused forquantitative estimations.

Neverthe-less, the strong intensity of the 2.1-kb EcoRI

band suggested thepossibility that itrepresents hybridization of HAX4 with two proto-amv DNA fragments of similar size. Previously, we

had shown thatthe EcoRI 2.1-kb fragment de-tectedwithHAX4included sequences located in

amv beyond the SalI site towards the 3' end. Therefore, an EcoRI + Sail digest was per-formedontotal C/O DNAtodeterminewhether this 2.1-kb band correspondedto adoublet. The resultsshowed that in additiontothe EcoRI 8.7-and5.4-kb bands, twootherbands, of 1.45 and 2.0kb,werepresent(Fig. 3). The lowintensity of the 1.45-kb bandrelative to that of the 2.0-kb bandpresumably resulted from inefficient cleav-age of the 2.0-kb fragment with Sall. In three independent Sall treatments, it was notpossible

to obtain complete digestion if total chicken DNA was used as substrate. The 1.45-kb band also hybridized with the SES3 probe, which consisted of the amv sequences locatedbetween the EcoRI andSail sites(datanotshown).This positioned the Sall site at 1.45 kb downstream from the EcoRI site within the corresponding proto-amv region, thereby revealing an intron between these two enzyme sites. The position of the Sail site was confirmed by analysis of A proto-amv recombinantclones presented in our second paper on this subject (B. Perbal, J. M. Cline, R. L. Hillyard, and M. A. Baluda, submit-tedfor publication). The presence of the intact 2.0-kbfragment and the 1.45-kbfragment is in agreement with the possibility that the 2.1-kb bandgeneratedbyEcoRI alone is adoublet.

To determine the location of the additional EcoRI 2.0-kb proto-amv fragment, in normal chicken DNA, several double enzymatic diges-tions were performed on C/O DNA, and the resulting blots were hybridized with DNA probes representing different portions of the amvsequence. Thepreparationandlocation of

2O -.3.7

4.3

FIG. 3. Doubledigestionof C/O DNA withSall + EcoRI. C/O DNAwasdigestedwithSail andEcoRI endonucleases in the EcoRI running buffer. After

electrophoresisina0.8% agarosegeland transferto nitrocellulose,theresultingblotwashybridizedto

32P-labeledHAX4DNA.HindIll-digestedA DNAwasrun assize markers.

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proto-amv ORGANIZATION IN CHICKEN DNA 589

TABLE 1. HybridizationofrestrictionenzymefragmentsfromC/OchickenDNAwithdifferentamVprobes Fragnents(kb) detected withamv probe:

Enzymes HAX4 EB3 SES3 SX12

HindII + EcoRl 1.2,1.9, 2.2, 4.3 2.2 1.9 1.2, 1.9,4.3

BamHI + EcoRI 2.0,5.4 5.4 2.0 2.0, 5.4

SmaI+EcoRI 2.0, 4.9, 8.7 4.9 2.0 2.0,8.7

Hindlll+BamHI 1.3, 4.3, 5.2 5.2 5.2 1.3,4.3

theseprobes withinamv aredescribed in Materi-als andMethods and shown in Fig. 1.

(ii) Hybridization of double enzymatic digests

with subclones of

HAX4.

Four differentdouble digests were set up. In addition to HindIlI, EcoRI, and BamHI endonucleases, SmaI was used because of the location of its recognition site150 base pairs towards the 3' terminus from the EcoRI site in amv (12, 14), The double digests were electrophoresed in 0.8% agarose gels, transferred to nitrocellulose, and hybrid-izedwith one of the probes

HAX4,

EB3,SES3, orSX12 (forasummary, see Table 1).

(i) Among thefourproto-amv fragments gen-erated in the HindlIl + EcoRI digest (Fig. 4, lanesd), the2.2-kbfragment hybridized with the amv sequences locatedbetween the HaeII and EcoRI sites (probe EB3), whereas the 1.9-kb fragment hybridized with both the SES3 and SX12 probes which include amv sequences

lo-cated between the EcoRI and XbaI sites. The

faintness

ofthe 1.9-kb band detected with the SX12probe indicatesthatonlyashort stretchof amv sequences which layon the 3' side of the Sall site ofamv was present in this fragment.

Thetwoother

fragments

(1.2 and 4.3kb) gener-ated in the EcoRI + HindlIl digesthybridized only with the SX12 probe. These findings indi-cated that the 3'portion ofamvfoundbetween the Sail and XbaI siteswasdistributed in three separate proto-amv fragments generated by EcoRI and HindIll double digestion, i.e., 1.9, 1.2, and 4.3 kb. Therefore, this portiQn of amv mustbelocated inatleast two domains of exon

in proto-amv.

A comparison of the sizes of the DNA

frag-ments detected after a single digestion with either EcoRI or Hindlll (12) with the results obtained above allows us to order the

fragments

obtained in the HindlIl + EcoRI digest as fol-lows from the 5' to the 3' end: 2.2, 1.9, 1.2, and 4.3 kb. Therelative order of the 1.2- and 4.3-kb fragments was deduced from the order of the HindlIl fragments: 5.4, 1.3, and 5.4 kb,

assum-ing that the1.2-kbfragmentin thedouble EcoRI

+HindIII digest correspondstotheHindlIl 1.3-kbfragment. Thiswasconfirmed by the results obtained with the HindlIl + BamHI double digestion (see below). The appearance of the 2.2-kb

fragment

fixedthe position ofaHindlIl

a b c d

1-HAX4

FIG. 4. Hybridization ofC/O DNA double digests with different amv probes. C/O DNA was digested successivelyby eitherHindlllandEcoRI(d), BamHI and EcoRI (c),SmaIand EcoRI(b),orHindllland BamHI (a). Afterelectrophoresis in 0.8% agarose gels and transfertonitrocellulose, the blotswerehybridizedto the indicated probes 32Plabeledby nick translation. The size markers were eitherHindIII-digested A DNAor a

mixtureofHindIII-digestedA DNAandHaeIII-digested+X174RF DNA.Thegelused for SX12hybridization waselectrophoresed for a longer period than were the other gels.

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site within the 5'-proximal 5.4-kb EcoRI frag-ment, and the presence of the 4.3-kb fragment fixed theposition ofaHindIII site within the 3'-proximal8.7-kb EcoRI fragment.

Since only two bands (5.2 and 1.3 kb)were

detected if a HindIII digest ofC/O DNA was hybridized to HAX4 (see above), the present resultssuggest that there isasecond 3'-proximal HindIIIfragmentof 5.2 kbwhich

overlaps

with the EcoRI 8.7-kb fragment and includes the HindIII-EcoRI 4.3-kb fragment. This would place mostof the 1.3-kb HindlIl fragmentwithin oneof the two EcoRIfragmentsof 2.0 and 2.1 kb detected with HAX4 and the 1.9-kb EcoRI-HindIIIfragment withinthe other one.

(ii) When a double BamHI + EcoRI digest (Fig. 4, lanes c) ofC/ODNAwas hybridizedto HAX4

32P-labeled

DNA, two very dark bands (5.4and 2.0kb) were detected. The 5.4-kb band was probably a doublet consisting of the 5'-proximal 5.4-kb fragment detected after diges-tion with EcoRI alone and the3'-proximal5.4-kb fragment detected after digestion with BamHI alone (12). The 2.0-kb band was also a doublet consisting of the 2.0-kb fragmentgenerated by EcoRI alone and a 2.0-kb fragment resulting from the presence ofaBamHI sitenear the 3' end of the 2.1-kb fragment also generated by EcoRIalone.

Hybridizationof the BamHI +EcoRI double digest with the probe EB3 revealed the 5'-proximal 5.4-kb EcoRI

fragment,

whereas hy-bridizationwith theprobeSES3 revealedonlya

2.0-kbfragment. TheprobeSX12hybridized as

expectedwith twofragments of 2.0 and 5.4kb, the latter presumably corresponding to the 3'-proximalBamHIfragment

previously

described (12).

(iii) SmaI + EcoRI double digestion (Fig. 4, lanesb) generated three bands(8.7, 4.9, and 2.0 kb) which hybridized with HAX4. The 4.9-kb band hybridized to the EB3 probeand allowed us to localize a SmaI site within the 5.4-kb EcoRIfragment.Thesizes(8.7and 2.0kb)of the two other bands corresponded to the sizes of fragmentsgeneratedbyEcoRIalone.

IfC/O DNA digested with SmaI alone was

hybridized toHAX4, two bands (16 and6.0kb) were detected (data notshown). The existence of the 6.0-kb SmaI fragment and the results given above establish that there isoneSmaIsite

at0.5 kb from the 5' end within the 5.4-kb EcoRI fragment whichhybridizes with the EB3 probe. Also, there must be another SmaI site 1.1 kb outside the 3' endof the same

EcoRI fragment

since a 4.9-kb fragment which hybridizes with the EB3 probe was generatedbyadoubleSmaI

+ EcoRI digestion. Because the 2.0-kb SmaI-EcoRI fragment hybridized with the SES3 probe, it contains amv sequences located

be-tweenthe

EcoRI

andSallsites andmust

corre-spond

to the 1.9-kb

EcoRI-HindIII

fragment

which also

hybridizes

to the SES3

probe.

We

canassumethatasin amv,theSmaI site in this proto-amv

region

islocated150base

pairs

tothe 3' side of the EcoRI site.

Therefore,

thisEcoRI sitewould belocatedat0.95kbonthe 3'sideof theEcoRIsite which is at the 3' end of the

5'-proximal

5.4-kb EcoRI fragment. This was de-terminedasfollows: SmaI-SmaI (6.0kb)minus

SmaI-EcoRI

(4.9

kb)

minus EcoRI-Smal

(0.15

kb)

equals

EcoRI-EcoRI(0.95

kb).

Thus,there is

anextraEcoRIsite within the proto-amv

region

under

discussion, establishing

the existence of another intron

consisting

of at least 0.95 kb betweentwo

EcoRI

sites. A similar conclusion

canbedrawn from the results obtainedwiththe Hindlll + BamHI double

digestion

of C/O DNA. Theexistenceof the 0.95-kb EcoRI

frag-ment wasconfirmed

by

analysis ofA proto-amv recombinantclones (Perbal etal. submittedfor

publication).

(iv)

Double

digestion

with HindIII + BamHI

(Fig. 4,

lanesa)andhybridizationtoHAX4gave risetothreebands of

5.2,

4.3, and 1.3 kb. The 5.2-kb

band,

which alsoappearedafter

digestion

with Hindlll

alone,

wasdetected after

hybrid-ization with the EB3 andSES3probes,

confirm-ing

the presence ofthis HindIllfragmentonthe 5' side of the proto-amv sequences. This5.2-kb HindIII

fragment

waspreviouslyshowntocarry proto-amv sequences common to both the 5.4-andthe 2.0-kb EcoRIfragments(12).This obser-vation alsoconfirmsthatthere isnoBamHIsite inthe 5.2-kbHindlIl fragment (12).

The 4.3- and 1.3-kb fragments generated

by

HindIlI + BamHI digestion were detected

by

the probe SX12. This suggests that the 4.3-kb fragmentwasthe result ofaHindlllcutin the 3'-proximal5.4-kb BamHI fragmentwhich ispart of the

EcoRI

8.7-kbfragment. The1.3-kb

frag-ment was generated by HindlIl alone and is

equivalent

to the 1.4-kb HindIll fragment de-scribedpreviously

(12).

These results show that the proto-amv

se-quences corresponding to the portion of amv

delineatedbythe HaeII andSail sitesare locat-edon asingle5.2-kb HindIlI fragment,whereas the proto-amv sequences corresponding to the

amv sequences located between the Sall and XbaI sitesarelocatedontwoadditionalHindlll

fragments.

One of them (1.3 kb) has

already

been characterized (12). The other contains a

BamHIsite such thataHindIII+BamHI

diges-tion generates a4.3-kb fragment which hybrid-izes with the SX12 probe. Since only two

HindIII bands (5.2 and 1.3 kb) were detectedby hybridizationwithHAX4, the 5.2-kb bandmust

beadoublet. Thiswas confirmed by hybridiza-tion of HindIII-digested C/O DNA with the

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CHICKEN 591

a b

-23.7

a, -- 9.5

- 6.6

_W 4.3

- 2.1

1.9

1.35 1.0

-0.87

FIG. 5. Hybridization ofEcoRI-or

HindIII-digest-edC/ODNAwith the proto-amv E3 probe. C/O DNA

digested with either EcoRI (a) or HindIII (b) was

electrophoresedin agarose gel,Southernblotted, and

hybridizedto 32P-labeled E3 DNA,which represents the 3' end of the proto-amv sequences. A mixture of

HindIll-digested A DNA andHaeIII-digested

+X174

RFDNA was used as molecular size markers.

proto-amv E3 probe which corresponds to the 3'-terminalamvsequences(seeabove).

(iii)

Hybridization

ofC/ODNA withproto-wnv E3probe. C/ODNA wasdigested with HindIII

or EcoRI endonuclease, electrophoresed in a

0.8% agarosegel, blot transferred, and hybrid-ized to 32P-labeled E3 DNA. As expected, the 8.7-kbEcoRlfragmentwasdetectable(Fig. 5a). However, the E3probehybridized totwoDNA

fragments

(5.2 and 4.2kb) inC/ODNAdigested with HindlIl (Fig. 5b). This confirms that there are two5.2-kbHindlIl fragments,oneofwhich contains the proto-amv sequences present in the 8.7-kbEcoRI fragment. The 4.2-kb band must

representhybridizationwith non-proto-amv cel-lular DNA sequences presentwithin the EcoRI 8.7-kbfragment. These data allowus toposition the EcoRI, HindIII, SmaI, BamHI, and Sail sites within the region of normal C/O DNA which contains the proto-amv sequences (see Fig. 7).

Analysis of leukemic DNA. To determine whether there was a similar arrangement of proto-amv sequences in leukemic cells, DNA purified from peripheral blood myeloblasts of leukemic C/E chicken no. 21710 was treated withvariousrestriction endonucleases and ana-lyzed with amv probes. This DNA was the

sourceof the Arecombinant library from which the AMV provirus recombinant clone AllA1-1 wasisolated (18). Knowledge of the location of many endonucleasesites within the AMV

provi-rus (12, 14, 18)permits ustoidentifytheAMV

fragmentswhich shouldhybridize to the HAX4 probe.

Hybridization of HindIll-digested leukemic DNA(Fig. 6a) to the HAX4 probegave rise to three bands (5.2, 4.3, and 1.3 kb). The 4.3-kb fragmentwas not generated fromnormal DNA and corresponded to the 3'-proximal HindIII fragment ofthe AMV genome.

AfterEcoRIdigestionofleukemic DNA, two AMV fragments hybridized with the HAX4 probe (4.4 and 3.6 kb) in addition to the three proto-amv fragments (8.7, 5.4, and 2.0kb)(Fig. 6b). One band (3.6 kb) is the internal AMV EcoRI fragment which hybridizes lightly to

HAX4

because it contains only 200 base pairs homologous tothis probe. The otherband (4.4 kb), whichis notdetectedbyhybridization with EB3 (data not shown), probably represents an

AMV provirusjuncture fragment with chicken DNAadjacenttoits 3'end.Thepresenceof this presumptivejuncturefragmentwasunexpected since the DNA wasfromtotalleukemic myelo-blasts. Also, itis different fromthe 3'juncture fragment(2.0 kb) present in the AMV-A clone

hAll-1.

This 4.4-kb fragment was notpreviously detectable with viral probes because it comi-grateswith severalEcoRI

fragments

ofthesame

size which areinternal tothehelper myeloblas-tosis-associated virus and endogenous

provi-ruses.

Five amv-specific bands (1.2, 1.9, 2.2, 2.9,

b d e

-23.7

_ 9.5

- ~ -6.6~

- 4.3

2.1 -' 9

_ _ -- 1.35

-.0

0.87

0.60

FIG. 6. Hybridizationof restriction enzyme-digest-ed leukemic DNA with HAX4 probe. DNA from leukemic myeloblasts of chicken no. 21710 (18) was digested with either HindIll (a), EcoRI (b), HindIII and EcoRI (c),BamHIand

EcoRI

(d), orHindIII and

BamHI(e).The DNA digests wereelectrophoresed in a0.8% agarose gel, Southernblotted, andhybridized to 32P-labeled HAX4 DNA. A mixture of

HindIll-digested)DNAandHaeIII-digested+X174RF DNA wasused as molecular size markers.

44,

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and 4.3 kb)weredetectedafterhybridization of

HAX4 to C/E leukemic DNA digested with EcoRJ + HindIlI. Two fragments (1.2 and 2.9 kb)containingamvsequences wereexpectedto

be generated by EcoRI + HindIII digestion of the AMV provirus. Only the 2.9-kb band was

detected (Fig. 6c) in addition to the pattern

obtained with normal DNA, becausethe 1.2-kb bandcomigrated with the 1.2-kb band character-istic forproto-amv sequences (see above). The

autoradiographic intensity of the 1.2-kb band generated from EcoRI +HindIII-digested DNA

wasinagreementwith thepresenceoftwo

amv-containing fragments.

Ifleukemic DNA digested withboth BamHI

and EcoRi was hybridized tothe HAX4 probe (Fig. 6d), three bands (5.4, 2.0, and 1.8 kb)were

detected. The 2.0-kb band isbroad andintense

inthe autoradiogram because it corresponds to twoproto-amvfragments (see above). The

1.8-kb band corresponds to an EcoRI fragment

generated from the AMV provirus, whereasthe

5.4-kbband isacellular fragment carrying proto-amvsequences.

The BamHI + HindIII digestion of leukemic DNA(Fig. 6e)gaverisetofour bands (5.2, 4.3, 2.8, and 1.3 kb) when hybridized with HAX4 DNA.Onlyoneband(2.8 kb) resulted from the

double digestion ofthe integrated AMV

provi-rus.The other bandscorrespondedtothe

proto-amv-containing fragments. As mentioned

earli-er, the5.2-kbbandconsisted oftwoproto-amv

fragments.

Thesedataare summarized in Table2.

DISCUSSION

A previous preliminary analysis had shown

thatatleastoneinterveningsequenceinterrupts

theproto-amv sequencesin chicken DNA (12).

Theuseofdoubleenzymatic digestions together

withprobes of subcloned definedregions of the

viral amv sequence now establishes that there

are atleast four largeintrons in theproto-amv

sequences.

The double digestion of C/O DNA with

HindIII+EcoRIrevealed that the 2.2-kb EcoRI

bandwhichhybridizestotheHAX4probe (12)is in fact a doublet consisting of two adjacent fragments whosemoreaccuratesizesare2.0 and

2.1 kb. Each of them contains a HindIll site

such that HindIIl digestion generates a single

1.3-kbfragment (12). Hybridizationwith

specif-ic subclonesrepresenting differentamvregions

showed that the 2.0-kb EcoRI fragment

con-tainedproto-amv sequences homologousto the amvsequenceslocated from the EcoRI siteupto 150to200 basepairs beyondtheSallsite.These

EcoRI andSallsitesareseparated by1.45kb in proto-amv, whereasthey areseparated by only

350 basepairsinamv.This revealsanewintron

in proto-amv. The remaining portion of the 3'-proximal amv sequences are carried by the 2.1-and 8.7-kb EcoRIfragments.

The proto-amv sequences corresponding to the amv sequences located between the HaeII and EcoRI sites are present in a 2.2-kb HindIII-EcoRI fragment generated by HindIII + EcoRI doubledigestion. This fragment makes up the 3' endof the 5.4-kb EcoRI fragment. The 5'-proxi-mal5.4-kbEcoRIfragment and theinternal 2.0-kb EcoRI fragment which contain proto-amv sequences are notcontiguous but areseparated by a 0.95-kb EcoRI fragment which therefore defines the minimal length ofan additional in-tronwithin the proto-amv sequences.

The locationof the3'-proximal 5.2-kb HindIII fragment which contains proto-amv sequences homologous to 0.2 kb of 3'-proximal amv se-quences wasestablished (i) by hybridization of single HindIII digests of C/O DNA with the A111-derived E3 probe which contains the 3'-proximal proto-amv sequences (12) and (ii) by hybridization of double BamHI + HindIII and HindlIl + EcoRIdigests with the amv subclone SX12.

Hybridizationof singleSmaIanddouble SmaI

+ EcoRIdigestswith amvsubcloneslocates the proto-amv internal SmaI siteat 1.1 kb outside the 3' end of the 5.4-kb EcoRI fragment. This confirms that a 0.95-kb EcoRI fragment sepa-ratesthe 2.0- and5.4-kbEcoRI fragments which contain proto-amv sequences. The location of the SmaI site within the2.0-kbEcoRIfragment (12, 14) also suggests that the5'-proximalEcoRI site ofthis fragment corresponds to the single EcoRIsite present in amv (12, 14). It isunlikely that in thegenerationof theamvinsert,splicing occurredbetweentwoEcoRI sitestoregenerate

anEcoRIsite (5,6,15).Therefore,it ispossible that avery small stretch (less than 30

nucleo-TABLE 2. Restriction enzymefragmentsfrom leuke-mic and normal DNAs whichhybridizedwith HAX4

probe

Fragments(kb) from thefollowing

Enzymes type of DNA:

Leukemic Normal

HindIIl 1.3, 4.3,a 5.2 1.3, 5.2

EcoRI 2.0,3.6,0a 4.4,0 2.0,5.4, 8.7

5.4, 8.7

HindIlI + EcoRI 1.2,b 1.9, 2.2, 1.2,1.9, 2.2, 4.3

2.9,°4.3

BamHI+EcoRI 1.8,a 2.0,5.4 2.0,5.4

HindIlI + BamHI 1.3,2.8,a4.3, 1.3, 4.3, 5.2 5.2

aFragments originating from AMV provirus

inte-gratedinleukemic DNA.

bThis bandconsists of at least one proto-amv and one amvfragment.

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proto-amv ORGANIZATION IN CHICKEN DNA 593

I

AMV _

Chicken I / I

K,I

r o o E

n

L4: _;-..

;i

I

I

I I

r I % I % /

w

I

It

ii I / /

/I/

//

II / i / /

I I/ /,

BomHI Eco RSrnI H[ndd B )K EcoRI

HindM EcoRIK EcoRI

SmiaI

kb

FIG. 7. Restriction endonucleasemapofthe chicken cellularDNAregion containingproto-amvsequences.

(Top) The amv insert of the AMV provirus has been subdivided into four regions corresponding to the hybridization probes used in this study. (Bottom) The recognition sites for various restrictionenzymeshavebeen mapped in the chicken DNA region containing theproto-amvsequences. Theexactlocations of theproto-amv

sequenceswithin the various DNAsegmentsdelineated by restrictionenzymesitesarenotknown,exceptfor the amvsequenceslocatedbetween the EcoRI andSalI sites (see the text). The question markatthe 5' end ofamv indicates thatwedonotknow whether this5'segmentis contiguouswith theHaeII site inchicken DNA. The size scales (in kilobases) for the viral(top)and cellular (bottom) sequences aredifferent.

tides long) ofproto-amv sequences, not long

enoughtobe detectedby hybridization, is

locat-ed upstream of the corresponding proto-amv EcoRIsite. These fewnucleotides would permit splicingtogeneratetheamvregion within which the internal EcoRI site is located. The present

resultsaresummarized inFig. 7, which showsa

partial restriction map of the chicken DNA

region containing proto-amv sequences. From our previous study (12) with the AMV

KpnI-XbaI probe, we know that the amv sequences

located between the 5' end of theamvinsert and the HaeII site are present within the 2.2-kb HindIII-EcoRI fragment, but we do not know whether their 3' end is contiguous with the HaeIIsite. Theamountofproto-amvsequences

carriedby the 2.1- and 8.7-kb EcoRI fragments

cannotbeprecisely determined. However, from therelativeintensity of the 1.2- and 4.3-kb bands obtained in the doubleHindIII + EcoRIdigest hybridizedtoHAX4, itappearsthat the number ofproto-amvsequencespresentonthe

3'-proxi-mal 4.3-kb fragment (i.e., within the 8.7-kb EcoRI fragment) is slightly greater than the

numberofproto-amv sequencespresenton the

1.2-kb fragment (i.e., within the 2.1-kb EcoRI

fragment). A similar arrangement ofproto-amv sequencesoccurred inallC/O and C/E chicken tissues examined, indicating their stability in development anddifferentiation.

Under theexperimental approach usedinthis

study, we detected the presence of four large

introns which interrupt the proto-amv se-quences, butwedonotruleoutthe existence of

additional smallerintervening sequences. They would notbe detectable if they did notcontain recognition sites for the restriction

endonucle-ases weused. Therefore, the four introns

detect-ed in thisstudyrepresentthe minimumnumber. Also,aswillbe shown inournextpaper onthis

subject (Perbaletal., submitted for publication),

an additional intron was discovered with the

samerestrictionenzymes asthose used here if

proto-amv DNArecombinant clones were

ana-lyzed instead of total chicken DNA.

The restriction endonuclease analysis

per-formed on DNAisolated fromleukemic myelo-blasts didnotrevealanymajorrearrangement of

the cellularproto-amv sequences as aresult of

leukemogenesis. Characterization of chicken-X

DNA recombinants carrying proto-amv se-quences isolated from leukemic cells confirms

that the proto-amv arrangement in leukemic chicken DNA is similartothat innormal DNA.

This implies that the leukemogenic effect of AMVmaybeentirely dependentupon

transcrip-tion of theintegrated AMV provirus. In support

of this hypothesis,the amvtranscript, which is shorter(2.5 versus4.5 kb) than the RNA which

is transcribed from theproto-amv sequencesin

hemopoietic chicken tissues (7, 10, 20; unpub-lished data), is very abundant inleukemic

my-eloblasts, whereas the proto-amv transcript is

absent.

ACKNOWLEDGMENTS

This researchwas supported by researchgrantCA-10197 from the National Cancer Institute, National Institutes of

0.1 kb

i I I

I I

I II

II II

HindM BamHI EcoRI VOL. 44,1982

7z1

_i

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Health. B.P. waspartlysupported by the Centre National de la Recherche Scientifique and was therecipient of a travel fellowship from the North Atlantic Treaty Organization.

LITERATURE CITED

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deoxyribonucleic acid complementarytothe ribonucleic acid of avian myeloblastosisvirus in tissues ofnormaland

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VertebrateDNAs containnucleotidesequencesrelatedto

the transforminggene ofavian myeloblastosis virus. J. Virol. 40:450-455.

3. BIrboim, H. C., and J. Doly. 1979. A rapid alkaline

extractionprocedurefor screening recombinant plasmid

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4. Boyer, H. W., and D.RoulandDussoix. 1969.A

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DNA inEscherichia coli.J.Mol. Biol.41:459-472.

5. Breathmach, R., C. Benoist, K. O'Hare, F. Gannon, and P. Chambon. 1978. Ovalbumingene: evidence foraleader

sequence in mRNA and DNA sequences at the exon-intron boundaries. Proc. Natl. Acad.Sci. U.S.A. 75:4853-4857.

6. Catteral, J. F., B. W. O'Malley, M. A. Robertson, R.

Staden,Y.Tanaka,and G.G.Brownlee.1978.Nucleotide

sequence homology at 12 intron-exonjunctions in the

chick ovalbumingene.Nature(London)275:510-513. 7. Chen, J.H.1980. Expressionofendogenous avian

mye-loblastosis virus information in different chicken cells. J. Virol.36:162-170.

8. Curtss,R., M. Inoue, B. Pereira, J. C.Hsu, L. Alexander, and L. Rock.1977.Constructionand use ofsafer bacterial host strainsfor recombinantDNAresearch, p.99-111.In

W. A. Scott and L. Rock (ed.), Molecular cloning of

recombinantDNA.AcademicPress, Inc., NewYork.

9. Franchini,G., J. Evan, C. J.Sherr,and F. Wong-Staal.

1981. onc sequences (v. fes) of Snyder-Theilen feline

sarcomavirusarederived fromnoncontiguous regionsof a catcellular gene(c-fes).Nature(London)290:154-157.

10. Gonda, T. J., D. K. Sheiness, L. Fanshier, J. M.Bishop,C.

Moscovci,and M. G.Moscovid.1981. The genome and theintracellularRNAsof avian myeloblastosisvirus.Cell

23:279-290.

11. Grunstein, M., and D. S. Hogness. 1975. Colony hybrid-ization: a methodfortheisolationof cloned DNAs that contain a specificgene. Proc. Natl. Acad. Sci. U.S.A. 72:3691-3695.

12. Perbal, B., and M. A. Baluda. 1982. Avianmyeloblastosis

virus transforming gene is related to uniquechicken DNA regions separated by at least oneinterveningsequence. J. Virol. 41:250-257.

13. Rlgby,P. W., M. Dieckmann, C. Rhodes, and P. Berg. 1977. Labeling deoxyribonucleic acid to high specific

activity in vitro bynick translation withDNApolymerase

I. J.Mol. Biol. 113:237-251.

14. Rushlow, K. E., J. A.Lautenberger, T. S.Papas,M. A.

Baluda, B. Perbal, J. G. ChirlkJlan, and E. P. Reddy. 1982. Nucleotide sequenceofthetransforming geneof

avianmyeloblastosis virus.Science216:1421-1423. 15. Self, I., G. Khoury, and R. Dhar. 1979. BKV splice

sequences based on analysis of preferred donor and

acceptorsites.NucleicAcidsRes.6:3387-3398.

16. Shalloway, D., A. D. Zelnetz,andG. M. Cooper. 1981. Molecular cloning and characterization ofthe chicken

genehomologous to thetransforminggeneofRous sarco-mavirus. Cell 24:531-541.

17. Southern, E. M. 1975. Detection ofspecific sequences among DNAfragments separatedbygelelectrophoresis.

J.Mol. Biol.98:503-517.

18. Souza, L. M., M. J.Brlskin,R.L. Hillyard, and M. A. Baluda.1980. Identification of theavianmyeloblastosis

virus genome. II. Restriction endonuclease analysis of

DNAfromAproviral recombinantsandleukemic myelo-blast clones. J.Virol. 36:325-336.

19. Souza, L. M., J. N. Stroumer, R. L. Hillyard, M.C. Komaromy, and M. A.Baluda.1980.Cellularsequences arepresent in thepresumptive avianmyeloblastosis virus

genome. Proc. Natl. Acad.Sci. U.S.A. 77:5177-5181.

20. Westin, E. H., R. C.Gaflo, S. K. Arya, A. Eva, L. M.

Souza,M. A.Baluda, S. A. Aaronson, and F.Wong-Staal.

1982.Differential expression of the amv gene in human

hematopoietic cells. Proc. Natl. Acad. Sci. U.S.A. 79:2194-2198.

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Figure

FIG. 1.XbaI;fromTheHindIII;beenandmapped Derivation of the amv-specific subclones.restriction endonucleasesitesin AMV are as previously reported (12, 18)
FIG. 3.EcoRI.endonucleasesnitrocellulose,labeledaselectrophoresis size Double digestion of C/O DNA with Sall + C/O DNA was digested with Sail and EcoRI in the EcoRI running buffer
TABLE 1. Hybridization of restriction enzyme fragments from C/O chicken DNA with different amV probesFragnents
Fig. 7).whetherAnalysis of leukemic DNA. To there was a similar arrangement
+3

References

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