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Genome organization of RNA tumor viruses II. Physical maps of in vitro-synthesized Moloney murine leukemia virus double-stranded DNA by restriction endonucleases.

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0022-538X/78/0026-0630$02.00/0

Copyright ©1978 AmericanSociety for Microbiology Printed inU.S.A.

Genome

Organization of RNA Tumor Viruses

II.

Physical Maps of In Vitro-Synthesized Moloney Murine Leukemia

Virus Double-Stranded DNA

by Restriction Endonucleases

INDERM.VERMA* ANDMARIANNE A. McKENNETT Tumor Virology Laboratory, The SalkInstitute,SanDiego,California92112

Received for publication29December 1977

Physical maps of the genome of Moloney murine leukemia virus (M-MLV)

DNAwereconstructedbyusing bacterial restriction endonucleases. The in

vitro-synthesized M-MLV double-stranded DNA was usedas the source of the viral

DNA.Restriction endonucleases Sal I and Hind III cleave viral DNAatonlyone

siteand, thus,generatetwoDNAfragments. ThetwoDNAfragmentsgenerated

bySal I areSal IA(molecularweight, 3.5 x 106) and Sal IB (molecular weight,

2.4 x 106) andby HindIIIareHindIIIA (molecular weight,3.6x 106) andHind

IIIB(molecular weight,2.3x 106). RestrictionendonucleaseBam I generates four

fragments of molecular weights of 2.1 x 106 (Bam IA), 2 x 106 (Bam IB), 1.25 x

106 (Bam IC),and 0.21x

10'

(Bam ID),whereas restriction endonucleaseHpa I

cleaves the M-MLV double-stranded DNA twice to give three fragments of

molecular weights of 4.4 x 106 (Hpa IA), 0.84 x

10'

(Hpa IB), and 0.74 x

10'

(Hpa

IC).

Digestion

ofM-MLVdouble-stranded DNA with restriction

endonu-clease SmaIproduces four fragmentsof molecularweightsof 3.9x 106(SmaIA), 1.3 x 106 (Sma IB),0.28 X 106 (SmaIC), and0.21x

10'

(SmaID). Amixtureof

restrictionendonucleasesBglIandBglII (BglI+ II) cleaves the viral DNAat

foursitesgeneratingfivefragments ofapproximatemolecularweights of 2 x 106

(Bgl+IIA), 1.75 X 106(BglI+IIB), 1.25x 106(BglI+IIC),0.40 x 106(Bgl I

+IID), and 0.31x 106 (BglI+IIE). The order of the fragments in relation to the

5'endand 3' end of the genomewasdeterminedeitherby usingfractional-length

M-MLVdouble-stranded DNA fordigestion by restriction endonucleases orby

redigestion ofSalIA, SalIB, Hind IIIA, and HindIIIB fragments with other

restriction endonucleases.Inaddition,anumber of otherrestrictionendonucleases that cleavein vitro-synthesized M-MLV double-stranded DNAhave also been

listed.

The genomeof Moloney murine leukemiavi- duplex studies (10) and in analogytothe

well-rus (M-MLV) isabout 9kilobases (kb) (1-3,6, studied avian RNA tumorvirus wouldsuggest

21;J. M.Bishop,Annu.Rev.Biochem.,inpress). the geneordertobe5'-gag-pol-env-3' (2).

Itcodesforthe

synthesis

ofatleast threeviral Themostabundant form of viral DNAfound structuralproteins: coreproteins (gag),reverse ininfected cellsislinearduplex,wherethe com-transcriptase

(pol),

and

envelope

(env) (7).It is plementaryDNA(cDNA)strand(minus strand) possible that the M-MLV genome in addition is of genome length but the second or (plus)

also codes forsome asyetunidentifiedprotein. strands are present in small fragments of an

Although the genome of M-MLV sediments at average sizeof0.5 to 1.2kb (22).Smallamounts

38S, analysis of polysomal RNA isolated from of circular and supercoiled forms of viral DNA

M-MLV-infected cells on high-resolution aga- have also been identified in infected cells

rosegels, however, shows 38and24Sspecies of (Bishop, Annu. Rev. Biochem., in press). The

RNA (8). It is currentlythe general consensus molecularweightoflinear double-stranded viral

that the 38S RNA species codes for gag(pol) DNA isolated from M-MLV-infected NIH-3T3

proteins,and the 24S RNA species encodes for cells has beenreportedtobe6.3 x 106(22). The

envproteins (6; Bishop, Annu. Rev. Biochem., amountofviral DNA thatcanbe isolated from

inpress). The order of the genes coding for these the infected cellsby present methodology

ren-proteins,in relationtothe 5' end and 3' end of ders it unfeasible for further manipulations or

the genome, has not been rigorously proven. biochemical analysis.

However, currently available data from hetero- For avariety of reasons, we have been

inter-630

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ested inconstructingaphysicalmapof the viral someexperimentsonly 8- to 10-cm-long vertical 1.2% DNAby employingthe useof bacterial restric- agarose gels were used.Generally, electrophoresiswas

tionendonucleases (14). To obviate the liita- carried out for 18 h at 2.5 V/cm. The portion ofthe

tionofsmallquantitiesof viral

DNA,

wedecided gel

containing

the standards for molecular weights was

tosynteinvdevral stained with ethidium bromidetovisualize the DNA

toA

synhe

sI,

.i

patro,

du

e-setrade siral

fragments. The gel

contaiming

the sample was placed

DNA (20). In this paper we present physical on a Whatman paper I and dried under

vacuum,

maps of the M-MLV genome constructed by followed by autoradiography. When molarratioswere

using in vitro-synthesized viral DNA with re- determined, the DNA fragments were excised and

striction endonucleasesSal I, HindIII,Bam

I,

counted directly in toluene-basedliquid scintillation

Hpa I, Sma I, and BglI + II. In addition, we fluid. DNA fragments for redigestion were eluted from

willdescribe many other restriction endonucle- agarose gels by either of the two followingmethods:

asesthat can cleave viral DNA. (i) the gel slice was dissolved in 5 M sodium

perchlo-rateand the nucleic acidswereboundon an

hydrox-MATERIALS AND METHODS yapatite column as described (19), or (ii)the agarose gel slicewasdisaggregated manually with a spatula to Virus. A cloned isolate of M-MLV (clone 1) was a very fine mesh and incubated at 45°Cfor 6 to 12 h grown, harvested, and purifiedasdescribed (7). The in buffer containing 0.01 M Tris-hydrochloride (pH substrates Escherichia coli DNA polymerase I, ter- 7.4), 0.001 M EDTA, and 0.002 M NaCl,and DNA was minal transferase, etc.,wereobtained from sources as recovered as described (8). Recoveries by either described (20). Adeno type2DNA andpolyoma DNA method were between 50 and 90%.

were gifts of Charles Lawrence andM. Vogt. Phage Standards for molecular weight determina-lambda DNAwaspurchased from Miles Laboratories. tion. DNA fragments generated bydigestion of phage Restriction endonucleases. Restriction endonu- lambda DNA with HindIII, adeno type 2 DNA with cleaseswerepurchased from Miles Laboratories, New Sma I, polyoma DNA with either Eco RI orBamI, England Biolabs, Bethesda Research Laboratories, and polyoma DNA with Hpa II were usedto construct and Boehringer Mannheim Inc. Restriction endonu- the standard curve of molecular weightsshown in Fig. cleases Sma I and Bgl I + II were gifts from A. 1. The molecular weights cover a range of0.2 x106 to Ohtsuka of theUniversityofCalifornia,San Diego. In 1 x 107. It can be seen that the mobilities of DNA this paperwehave used abbreviatednomenclature of fragments up to molecular weights of 2 x 106 to 3 x the restriction endonucleases(14). 106 are quite linear. However, the curveshowing the Invitrosynthesisof double-stranded DNA. M- molecular weights of larger fragments issteeper. These MLV double-stranded DNAwassynthesizedby utiliz- four DNAs digested with appropriate restriction

en-inggenome-length single-strandedcDNAtranscripts donucleases were included in all gels shown in the

as templates (20). Either the template cDNA tran- paper, but only the molecular weights of relevant

scripts were elongated with (dA)n residues and the fragments are shown in the figures. HindIII-digested second strand was synthesized by employing phage lambda DNA shows six fragmentsof molecular

oligo(dT)1o

asprimer andE.coli DNApolymerase I, weights (X106) of 14.9, 6.1, 4.13, 2.7, 1.42, and 1.14;

or DNase-digested calf thymus DNA primers were Sma I-digested adeno type 2 DNA shows at least 11 usedto prime thesynthesisof the second strand on fragments of molecular weights(x106)of4.6, 4.1, 3.4,

cDNAtemplate with E. coli DNApolymerase I. In 2.8, 1.9, 1.5, 1.4, 1.0, 0.9, 0.7, and 0.4. EcoRI orBam I

thismanuscript, double-stranded DNA synthesized by cleaves polyoma DNA once and generateslinear DNA either methodwas used. Only the peak fractionsof of an average molecular weight of 3.3 x 106;Hpa

II-double-strandedDNA fromneutralsucrosegradients digested polyoma DNA shows eightfragments of

mo-(Fig.6inreference 20)wereused. lecular weights

(x106)

of 0.92, 0.73, 0.57,0.49, 0.24, 0.2, Enzymedigestion.Thesamplesof DNA(contain- 0.18, and 0.06.

ing 200 to 2,000 32p cpm) were digested in a

20-ulR

volume. Whenrestriction endonucleases BamI, Hpa RESULTS

I, Sma I, BglI +II,andHpaII wereused,the reaction Physical maps constructed by using

re-mixture contained 10 mMTris-hydrochloride (pH 7.4), striction endonucleases Sal I and Hindm.

10mMMgCl2,0.5mMdithiothreitol,and 0.01%

gela-tin.Whendigestionswere carriedoutwith restriction

Figure2hows thegrams

of the

gel

endonucleases Sal I and HindIII, the buffer in the

electrophoretic

profiles of n

vitro-synthesized

reaction mixture contained10mMTris-hydrochloride M-MLV double-strandedDNA and its digestion

(pH 7.4),3mMMgCl2,50 mMNaCl,and 0.01%gelatin. byrestriction endonucleases Sal I and HindIII.

Between 1 and 10units of theenzymeswereused (1 The sequence

specificity

ofSalI is G1TCGAC unit=amountofenzyme requiredto digest1.0

jig

of (Arrand, Meyers, and Roberts, unpublished

phagelambda DNA in 60minat37°C). Incubations data), and Hind III specifically recognizes

nu-were carried out at 37°C for 180 to 300 min. The cleotide sequence

A1AGCTT

(12).Both enzymes

reaction wasstopped byaddition of 5,ulofdye (brom- appear to cleave the M-MLV DNA at least once

ophenol blue). In many cases, the samples included and, thus, generate two

fragments.

Panel a

phagelambda DNA to monitor thecompletionof the shows that

the

undigested

M-MLV DNA

has

a

reaction. molecular

the

about 6 D has a

Agarose gel electrophoresis. Sampleswere an- molecular weight of about 6 x 106. In over 12

alyzed on 1.2%agarose gelsas described(17, 20). In different

preparations,

the molecular weightof

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I X 107 - or an inherent

property

of the in

vitro-synthe-sized M-MLVdouble-stranded DNA.

To determine the order of these fragments in

relationtothe 5' end and 3' end of the M-MLV

5 X 106. genomic RNA, we synthesized double-stranded

o DNA from cDNA

transcripts

representing

a

I

\ fraction of the full genome. We

synthesized

dou-\ ble-stranded M-MLV DNA ofan average

mo-lecularweightofabout 4.0x 106

(Fig.

3,

panel

a)

I 2 X

106

and 3.5 x

10'

(Fig.

3,

panel

c).

Figure

3 shows

O i the

patterns

obtained after

digestion

of 4.0 x

3 .

106-molecular-weight

M-MLV DNA with SalI

(panel b) and Hind III digestion of 3.5 x

106-< I X106 °\ dalton M-MLV DNA

(panel

d). Itcanbe noticed

J .xio6 that in thecaseof Sal I digestion, only the Sal

u \x IA fragment can be identified. In addition, a

2

5X105

MW X

x\~6.0-s*

s

L

|

~~~395-'

S-A

W-*i

x~~~~~~~~zs* * I

0 10 20 2.5

A-B

W-B

*

(cm)

FIG. 1. Standard curvefor determination of mo-lecular weights. Symbols: 0, phage lambda DNA fragments; , adenotype 2DNAfragments;A, linear form of polyomaDNA;x,polyomaDNAfragments.

the undigestedDNA ranges between 5.9 x 106

and6.1x 106.Theaveragemolecular weightsof

thetwofragments, designatedasSalIAandSal

IB,appeartobe3.4 x 106and 2.5x 106,

respec-tively (panel b), and those of Hind IIIA and

HindIIIB (panel c) appeartobe 3.6 x 106and 2.3 x 106, respectively. There is a very slight

difference in theabsolute molecular weights of

fragments ifthe double-stranded viralDNA is

synthesized by oligo(dT) primerorby calf thy-mus DNA primers. The restriction

enzyme-di-gested DNAfragments obtained from M-MLV

DNAsynthesizedby calfthymus DNA primers

runslightly ahead of those fragments obtained

fromoligo(dT)-primedM-MLV DNA(dA). This

ispresumably duetothe fact that in the former

casethe second strand of the DNAis in small

pieces and, thus, migrates more like single-stranded DNA. The difference in mobility

be-comes more apparent with lower-molecular-weight fragments. It can be noticed in Fig. 2

(panels b and c) that some residual DNA re- FIG. 2. Sal I and Hind IIIdigestion of M-MLV

mainsundigested. Thiscanbe due to either less DNA. (a)UndigestedM-MLVdouble-stranded DNA;

thanoptimal conditions employed for digestion (b)Sal Idigested; (c)HindIIIAdigested.

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MW X O6 a D C d

40-3.5- SolI

2.4- *-HindI

1.0

-0.4

-FIG. 3. Digestion of fractional-length M-MLV DNA with Sal I and HindIIIA. (a) Undigested M-MLV DNA ofmolecularweight of4.0x 10'; (b) digestion with Sal I; (c) undigested M-MLV DNA of molecular

weightof3.5x 10'; (d) digestionwith HindIII. Thearrowspointout the bandsdiscussed in the text.

smaller fragment of an apparent molecular then thedigestion of Sal IA fragment and Hind

weight of 0.4 x 106 to 0.5 x 106 can also be III should yieldHind IIIB fragment and,

con-identified. This smallfragmentispresumably a versely, digestion of Hind IIIA fragment with

portionofSalIBfragment. Ontheother hand, Sal I shouldgenerate Sal IB fragment. Figure 4

digestionby Hind IIIgeneratesHindIIIB frag- (panels a and b) shows undigested Sal IA frag-ment (molecular weight, 2.3 x

106)

and smaller ment (molecular weight 3.5 x 106) and Sal IA

fragments that are presumably portions of the fragmentdigested with SalI.Panel cshows Sal

HindIIIA

fragment.

Some residualundigested IAfragmentdigested with Hind III.Asexpected,

M-MLVdouble-stranded DNAcanalso beseen. SalIAfragmentis notfurther digested with Sal The cDNAtranscripts usedtomakesmallerM- I; however, digestion with Hind IIIyields two MLVdouble-strandedDNA representthe3'half fragmentsofapparentmolecular weights of2.3

of the M-MLV genome andlack portions from x

106

and 1.1 x

106.

Thelarger fragment

comi-the 5'halfof thegenome.Thefractional-length grates with the Hind IIIB fragment, and the DNAismoreheterogeneous,because the parent smallerfragment isapparentlyaportion of the cDNA transcript represents a broad size class Hind IIIA fragment. Panels d and e show the

(seeFig. 7of reference 22). Because the 5' ends undigested Sal IBfragment (molecular weight ofthecDNAtranscriptsareunique, the restric- 2.4 x

106)

and Sal IBfragment digestedwithSal tionfragmentoriginating from the 3' end of the I.Panelfshows thedigestion of Sal IB

fragment

genome is quite homogeneous, but the 5'-end withHind III. In all three cases,onlyoneband

fragments are moreheterogeneous. This is why with an average molecular weight of2.4 x 106

aseries of residualSal IB and Hind IIIAfrag- canbe identified. Controlexperimentsin which

ments are obtained, rather than homogeneous fragment Sal IB canbe digested byrestriction

DNA fragments. It appears, thus, that Sal IA endonucleases Bam I (Fig. 7) and Bgl I + II

andHind IIIB fragments originate from the 3' (Fig. 10) were alsoperformed (seebelow). Thus,

half of the genome and SalIB and HindIIIA it appears that the Sal IB

fragment

does not

fragments represent the 5' half of the M-MLV have sequences

specific

forHindIII. Panels g,

genome. If this is the order of the

fragments,

h,and i show the

undigested

HindIIIB

fragment

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and its digestion with Hind III and Sal I restric- X 106-molecular-weightmaterialcorrespondsto

tion endonucleases. In allcases, the size of the Sal IB fragment in

size,

and the smaller

frag-Hind IIIB fragment remains unchanged, sug- mentrepresentsa portionofSal IAfragment.

gesting that it does not containa site foreither Thus, itappears thatHindIIIfragmentA

con-Hind III orSal I.The HindIIIBfragmentcould, tainsasite forSal I. These dataaresummarized

however, be digested with Bam I, Sma I, and in

Fig.

5.

Hpa I (see below). In panel i there is a small

Physical

maps constructed

by

using

re-fragmentof approximate molecularweight of0.8 striction endonucleasesBamI,

Hpa

I,Sma

x 106. This is either an artifact or a digestion I, and

Bgl

I + H.

Figure

6 shows the

gel

product ofsomecontaminating HindIIIAfrag-

electrophoretic

patternsofM-MLV DNA

frag-ment. In other experiments in whichHindIIIB

fragment was free of any HindIIIA

fragment,

SalIB(2.4Xb06)

Sa

SaIIA(3.5X106)

nosuch DNA fragmentwas observed. Panels

JI

HindMA

(3.6X

106)

t

HindMB

(2.3X

106)

3

andkshow thepatterns ofundigested Hind IIIA

HindM

fragment and itsdigestionwithSal I. TheSalI-

FI.(

5. Order of the DNAfragmentsgenerated by

digested sample showstwofragmentsofaverage restrictionendonucleases SalI andHind III, relative

sizes of about2.3 x 106 and 1.2 x 106. The 2.3 to the 5' and 3' ends of the genomic RNA.

MW

O

10

Qo

d

e

f

9

2 5- _ - * 9 |

*.*

LO--~~~~~~r

FcIG. 4. Redigestion ofSalIAand Band

Hi-nd

lllAandB

fragments.

(a) Undigested Sal IA fragment; (b) Sal IAfragment digestedwith SalI; (c) SalIA fragment digested withHind III; (d) undigested Sal IB fragment; (e)SalIBfragmentdigestedwithSalI;

(fi

SalIBfragmentdigested with HindIII, (g)undigested Hind IIIBfragment; (h)HindIIIBfragment digestedwith HindIII, (i)Hind IIIBfragment digested with Sal I; 'f HindIIIAfragment digested byHindIII; (k)HindIIIAfragmentdigestedwith Sal L Thearrowspoint

tothefragmentsgenerateduponredigestion (discussedin the text).

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MW

X1lO6

a

b

c

d

e

6.0--A

2.0

-_B

-A

1.7-

ti-B

1.3

-B

--C

4

-C

0.84-

-B

0.74

am-C

0.43

-D

0.31

-

-E

-C-0.20-

-D

FIG. 6. Patterns of DNA fragments of M-MLV DNA generated byrestriction endonucleases Bam I, Hpa I, SmaI,andBgl I+II. (a) UndigestedM-MLVdouble-stranded DNA; (b) Bam I digested; (c) HpaIdigested;

(d) SmaIdigested; (d' enhanced intensities of Sma IC and Sma ID fragments; (e) Bgl I + II digested. The

capitallettersshown inpanelsb to edesignate each fragment generated by the respective restriction enzyme.

ments obtained by using these restriction en- ating four fragments. Although the four

frag-zymes. Theestimated molecular weights of the ments appeartobeinmolarratios

(Table

1), the

fragmentsgenerated byeach enzyme have been sum of theirmolecular weights does not equal calculatedinTable 1.Table 1also includesthe the molecular weight ofundigested DNA. We

molarratiosof eachfragmentasexpectedonthe have analyzed theBam Idigestionproductson basis of their molecular weights and the ob- 10% acrylamide gels tosee whethera series of

served ratios. Forthe sake of

completeness,

the small fragments, undetected inagarosegels,can

molecular weights of the fragments generated befound. Wehave done about 10 experiments

by SalIand Hind III have also beenincluded. and havenever seenfragmentssmallerthan0.18

We shall discuss the ordering of thefragments x

10'

or larger than 2.1 x 106. The two large

in relation to the 5' end and 3' end of the M- fragments generated by Bam I digestion have

MLV genomefor each enzymeseparately. verysimilarsizesand, hence,migrateveryclose

(i) Barn I. Restriction endonuclease Bam I, toeachother. The resolution of these bandscan

which recognizes the palindrome

GIGATCC

be improved by using higher cross-linkedgels,

(14), appears to cleave the M-MLV double- butthen the smallest BamI-digested fragment

strandedDNAatleast threetimes,thus gener- isnotdetectable.Inaddition,wealwayssee two

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636 McKENNETT J.

TABLE 1. Molecularweightestimations and molar band of a molecular weight of 1.2 x 106 to 1.25

ratios of DNA fragments generated by digestion of x 106identical to BamIC.There are also some

M-ML V double-stranded DNAbyrestriction other fragments, presumably either partials or

endonucleases SalI,Hind III,Bam I,

II

Hpa SmaI shorterfragment B. Further analysis of the

Bam

andBgl I +II ICfragment shows that it cannot be digestedby

Restriction Frag- Mol wt of Expected Observed either Sal I or Hind III, but is digested byHpa endonucle- ments fragments molar ra- molar ra- I and Sma I (seebelow). Thus,it would appear

ases gener-ated (xlO6) tios tios thatfragment C originates from the 3' end of the

SalIA3.5 59 62 genome. Panels d to k show the results of

re-S

3.5

41 62 digestion of Sal IA and SalIB, and HindIIIA

B 2.4 41 38 andHind IIIB fragments with BamI. Panelsd,

HindIII A 3.6 60 60 f, h, and j show the undigested SalIA, Sal IB,

B 2.3 40 40 HindIIIA, and Hind IIIB

fragments.

Panelse,

g, i, and k show the digestion patterns of these

BamI A 2.1 37.8 38 fourfragmentswithBam I. Digestion of the Sal

B 2.0 36 34 IA fragment withBam I shows three bands of

C 1.25 22.5 20 apparent molecular weights of 1.9 x 106, 1.2 x

D 0.20 3.6 ND' 106, and 0.21 x 106. Because we know from the

HpaI A 4.40 73 70 previous experiments that the 1.20 x 10"-dalton

B 0.84 14 16 fragment (Bam IC) comes from the 3' end of the

C 0.74 12 11 genome and we assume that the 1.9 x

106-mo-lecular-weight fragmentcorrespondstothe Bam

Sma I A 3.90 IB

fragment,

the molecular

weight

of 0.21 x 106

B 1.35 ND

represents

either the

Bam

ID

fragment

orthe

C 0.29 remainder of Sal IA fragment after digestion

D 0.22 with

Bam

I enzyme. If weassume that the 0.21

BglI + II A 2.0 x 10"-molecular-weight fragment is

Bam

ID

B 1.75

fragment,

then the order of these three

frag-C 1.25 ND ments will be either 3'-C-B-D or3'-C-D-B.

How-D 0.40 ever,digestion of the SalIBfragment with Bam

E 0.30 I

(panel g) gives

rise to two

fragments

of average

"ND,

Not

determined,

molecular weights of 2 x

106

and 0.23 x

106.

Panel g' shows the 0.23 x

106-molecular-frag-ment atenhancedintensity.Ifoneassumesthat

bands of molecularweightsof1.7 x 106and 1.4 the 2 x

106-molecular-weight

fragmentis Bam

x 10'to 1.5 x 106,which appeartobe present in IA and the 0.23 x

106-molecular-weight

frag-less than molaramounts.These could bepartial ment isBam ID, then the order of theBam I

digestionproducts,but addition ofmoreenzyme fragments will be 3'-C-B-D and A. However, if

or alonger incubation period doesnot seem to the0.23 x

10"-molecular-weight

fragmentis the

affect either the ratioorthe size ofthese bands. residualfragment Bam IB, then the order of the

Phage lambda DNA included in the same sys- fragments will be3'-C-D-Band A. Panel i shows tem,however, iscompletely digested byBamI. that digestionofHindIIIAfragmentwithBam

So far we do not have enough of these partial I generates fragments ofmolecular weights of

bandstoallow furtheranalysis. 2.1 x 106, 0.9x

106,

and0.23 x

10".

Inaddition,

The order of thefragmentsgeneratedbyBam it shows the same partially digested fragments

I was determined by two methods: either by ofmolecularweights of1.7x 106 and 1.4 x

106,

using lessthanfull-length double-strandedDNA as seen in panel a. The 2.1 x

106-molecular-fordigestionorbyredigestionofSal Ifragments weight fragment appears to be Bam IA

frag-A and B and/or Hind III fragments A and B. ment, and the 0.9 x

106

fragment is probably

Figure 7 shows the combined results obtained partofBam IB fragment. If the small fragment

from these two methods. Panel ashows thefour ofmolecular weight of0.23 x

10'

istheauthentic

Bam I fragments designated asBam IA, Bam Bam IDfragment, thentheorderof theBam

I-IB, Bam IC, and Bam ID. Panels b andc show digested fragments will be 3'-C-B-D and A-5'.

the undigested M-MLV double-stranded DNA This order seemstobe favored by the results of

of anaverage molecular weight of 3.5 x

106

to the digestion of the Hind IIIB fragment with

4.0x 106and itsdigestion with BamI.The DNA Bam I (panel k). It shows three fragments of

isveryheterogeneous becauseabroad region of apparent molecular weightsof 1.2x

106,

1.05 x

the DNA was collected from aneutral sucrose

106,

and 0.6 x

106.

The 0.6 x

106-molecular-gradient (20).Itcanbe seenthatthere is amajor weightfragment appearstobeeitheranartifact

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VOL. 26,1978

637

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LAdU)

4

CM

co

k

X s) Cq

o\j

Ct

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° O S

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638 VERMA AND McKENNETT J. VIROL.

orcontaminatingDNA as it is not seen inother part of the Hpa IC fragment. Alternatively, if experiments. If the 1.2 x 106-molecular-weight theHpaICfragmentoriginatesfrom the 3'end

fragment is Bam IC fragment and the 1.05 x of thegenome,thenHpaIdigestionof the Bam

106-molecular-weight fragment is part of the ICfragment willgeneratetheHpa IC fragment

BamIB fragment,then theorder will be3'-C-B. and part of the Hpa IB fragment. Figure 8, The data obtained fromredigestion oftheSal panelshandi, shows the undigested and Hpa

I-IA and IB and Hind IIIA and IIIB fragments digested BamIC fragment. Itcanbeseenthat would suggest thatthe order of the Bam L-di- theHpa I-digested Bam IC fragment hastwo

gested fragment is5'-A-D-B-C-3'. However,we additional bands of molecular weights of 0.82 x

would liketoascertain theposition of theBam 106 and0.42 x 106. A

large

amountof theBam

IDfragmentonthe genome more vigorously by IC fragment remainsundigested. We have found using DNA fragments obtained by digestion thatcommercially available preparations of Hpa

with otherrestriction endonucleases. I are often very dilute and salt sensitive.

Fur-(ii)

Hpa

I. M-MLV double-stranded DNA thermore, different batchesof enzyme

prepara-digested with restriction endonuclease Hpa I tions show marked differences in activity. In

generatesthreefragments ofaveragemolecular laterexperiments,wehave increased theamount

weights of4.4x

106,

0.84 x 106, and 0.74 x 106 ofenzymeused fordigestion and have succeeded

(Fig. 6,Table1).Thus, itappears that linear M- inobtainingmorecomplete digestion.Fromthe

MLV double-stranded DNA has at least two datashowninpanels h and i, itappearsthat the sites for Hpa I-specific palindrome,

GTTIAAC

HpaIB fragment originates from the 3' end of

(15). Figure 8, panel a, shows the molecular the genomeand, thus, the order of the Hpa

I-weight of the Hpa IA, Hpa IB, and Hpa IC digested M-MLV DNAfragments is 5'-A-C-B-fragments. Panels b andcshowthe patterns of 3'.

undigested M-MLV DNA ofanaveragemolec- (iii)Sma I. Restriction endonuclease Sma I

ularweight of2.8 x

106

and its digestion with cleaves the M-MLV double-stranded DNA at

Hpa I.

Only

the twosmall fragments, Hpa IB leastthree timestogeneratefourfragments (Fig.

and HpaIC,canbelocated, suggesting thatthey 6dand d' and Fig. 9a). Themolecular weights originatefrom the 3' end ofthegenome.Panels ofSmaIC and Sma IDfragments(Fig. 6d') have

d and e (Fig. 8) show the patterns of Hpa I- not been characterized

rigorously.

The Sma I

digested Sal IA and Hind IIIB fragments. In restriction endonuclease recognizes the

nucleo-bothcases, twoprominent bands,corresponding tidesequence

CICCGGG

(5;Holkers andCollins,

tothe size of theHpaIB and Hpa IC fragments, unpublished data).

Digestion

of

fractional-canbeobserved.We are unableto explainwhy lengthM-MLV double-stranded DNA withSma

wedo notobserve portionsoftheHpa IA frag- I does not produceSmaIAfragment, thus

indi-ment orthepresence ofseveral low-molecular- cating that this fragmentcomes from the 5' end

weight fragments. Because Sal IA and Hind of thegenome (data not shown). Figure 9,panels IIIB fragments represent the 3' end of the ge- b through g, showthe redigestion of theSalIA,

nome, we propose that the order ofHpa

1-di-

Sal IB,andHind IIIB fragments. Panels band

gested M-MLV DNA fragmentsis 5'-A-(BC)-3'. c show undigested and Sma I-digested SalIA

If this order iscorrect, wewouldpredict that Sal fragment. Thedata indicate a major band at a

IBfragment wouldnotbe digestedbyrestriction molecular weight of 1.3 x

106,

a minorband at

endonuclease Hpa I. Panels f and g show the a molecular weightof 1.5 x

106

to 1.6 x

106,

and

undigested and Hpa I-digested SalIB fragment. two very

small

fragments of molecular weights It canbe observed that the size ofthe SalIB of 0.25 x

10W

and0.19x

106.

Ifoneassumesthat fragment before and afterHpa I digestion re- 1.3 x

106-molecular-weight

fragment is SmaIB

mains unaffected, suggesting that the Sal IB fragment, thetwoverysmallfragmentsareSma

fragment does not containasitespecific for Hpa IC andSmaID fragments, and the 1.5 x

106_

to

I. These results, however, do not establish the 1.6 x

106-molecular-weight

band represents the

order of the Hpa IB andHpa ICfragments in part of theSmaIAfragment, then the order of

relation to the 5'-3' ends of the M-MLVgenome. theSmaI-digested M-MLV DNA fragments

will

Todetermine the order of Hpa IB and Hpa be5'-A-(BCD)-3'.Similarly, the HindIIIB frag-ICfragments,wedigestedthe Bam ICfragment ment digested by Sma I (panels d and e) also

with HpaI.TheBam ICfragment is about 1.25 generates four fragments of molecular weights

x 106, whereas Hpa IB and Hpa IC fragments of 1.35 x 106,0.56 x 106, 0.23 x 106, and 0.16x

are 0.84x

106

and 0.74x106inmolecularweight. 106, corresponding to the Sma IB fragment, a

IffragmentHpaIBoriginatesfrom the 3' end, portion of the Sma IA fragment, and perhaps then digestion of the Bam IC fragment with theSmaIC andSmaIDfragments. Panels f and

Hpa I will generate the HpaIB fragment and g show the patternsofundigested and Sma

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26,

@ _S X

X

0

d

x

~ ~

~

~

~

~

+

E

xt

X O O ? . i E 3g~~~~~~~~~~X

:E~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~cQ;

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VERMA AND McKENNETT J.

M

WXO

x

tN

b

c

A

FIG. 9. Ordering ofSmaI-digestedM-MLV DNAfr-agments. (a)SmaI-digested genome-lengthM-MLV

double-strandedDNA;(b)undigestedSal IAfragment (noticeaslightcontamination ofSallBfragmnent);(c)

SmaI-digestedSal IAfragment; (d) undigestedHind IIIBfr-agment; (e)SmaI-digestedHindIIIBfragment;

(t)

undigestedSallBfragment; (g)SmaI-digestedSallBfragment. Thearrowspointtothefragmentswith molecularweightssimilartoSma I-digestedM-MLVfr-agments.

digested

SallB

fragment.

No

digestion

appears that the order of the Sma I

fragments

is

5'-A-totake

place.

This will be

expected

ifthe Sma (BCD)-3'.

IA

fragment

ofanaverage molecular

weight

of

(iv)

Bgl

I + II. In

experiments

presented

3.9 x

106

originates

from the 5' end of the ge-

here,

wehave used amixture ofrestriction

en-nome. donucleases

Bgl

I+

IL.

Thesequence

specificity

Wehavenot

conclusively

established the or- of

Bgl

I isAGGCNGCCT1 (G.

Magnusson,

per-der of the Sma IB, IC, and ID

fragments

in sonal

communication),

whereas

Bgl

II appears

relation to the 5' and 3' ends of the genome. tocleave hexanucleotideA1GATCT (13).

Diges-From the

preliminary

data obtained

by digestion

tion of M-MLVdouble-stranded DNA with

Bgl

of theBarnIC

fragment

(molecular

weight,

1.25 I + IIgenerates at least five

fragments (Fig.

6,

x

10')

withSma

I,

itappearsthatatleastoneof panele).The molecular

weights

of the

fragments

the smaller

fragments

(Sma IC or Sma ID) are

given

inTable1.The order of the

fragments

originates

from the 3' end of the genome. How- inrelationto the 5' and 3' ends of the M-MLV

ever,moredetailed

analysis

of

redigestion

ofthe genome were determined

by

redigestion

of Sal

Hpa

lB and

Hpa

IC

fragments

with Sma I will I

fragments

A and B andHind III

fragments

A

yield

a moredefinite order ofthe SmaIB,Sma and B

(Fig.

10).Panelsaand b show the

patterns

IC,andSma ID

fragments. Presently,

we assume of

Bgl

I +

II-digested

Sal IA and Hind III B

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[image:11.501.118.403.58.424.2]
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MW

X

106

a

b

c

d

e

MW X

106

2.5

2.0

-1.2-

-1

0.45

"

0.30-4-

<0.25

FIG. 10. OrderingofBglI+II-digested M-MLV DNA fragments. (a) SalIAfragment digestedwithBglI +II;(b)Hind IIIBfragment digestedwithBglI+II; (c) undigestedSalIBfragments;(d) BglI+

Il-digested

SalIBfragment. Theupperarrowpointsto1.2x

HP'-molecular-weight

fragment.Thelowerarrowshows the

1.1 x 1k/-daltonfragment that may beaportion ofBglI+IIfragment B. (e) Hind IIIA fragment digested withBglI+ II. This materialwas runon 1% agarosegels,and, hence, thefragmentshavefastermobility. The molecularweightsforthe bands inpanele areshownontherightsideofthefigure.Thearrows in the figurepointtofragmentswith molecularweightssimilartoDNAfragmentsgeneratedby digestionofM-ML V DNA withBglI+II.

fragments. Digestion of the Sal IA fragment representportionsof eitherBgl+IIfragmentB

shows at least five bands ofmolecular weights or fragmentC, andthe 0.22 x

106-dalton

frag-of2x

106,

0.94X 106,0.47 X 106,0.43 x 106, and mentmay representaportionoffragmentE. It

0.22x 106. If2 x

106-molecular-weight

fragment isnotclearwhywesee twofragmentsof0.47 x

is theequivalent ofBglI +II fragment A and 106 and0.43 x 10' molecular weight. Fromthe

either0.47 x 106or0.43x 106 molecularweight results obtained from

digestion

ofSal IA

frag-is fragment D, then it would appearthat frag- mentwith

Bgl

I+

II,

the order of the

fragments

mentsAand Doriginatefrom the 3' endof the would appear to be

3'-(AD)E(BC)-5'.

Panel b

genome. The 0.94 x 106-dalton

fragment

may shows the data obtained

by redigestion

of Hind

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[image:12.501.96.390.64.479.2]
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IIIB fragment with Bgl I+ TI. A major band of present in M-MLV double-stranded DNA would

approximately 2 x 106 daltons and minor bands be low. Restriction endonucleases Ava I, SstI,

ofmolecular weightsof about 1 x106 and 0.3 x XhoI,XbaI,Bgl I, BglII,HindII,and Hae II

106 can be identified. If the 2 x 106-dalton frag- all cleave M-MLV DNA at least twice.

Restric-ment is Bgl I + II fragment A and 0.3 x 106- tion endonucleases Hpa II,Hin f, and HaeIII

daltonfragment is either fragment E or aportion cleave M-MLV DNA many times (data not

offragmentD,then the order of theBglI+ II- shown).

digested M-MLV DNA fragment will be 3'- DISCUSSION

A(DE)(BC)-5'. To determine the order ofBglI

+II fragments B and C,wedigested Sal IB and Physical maps. We have constructed

physi-Hind IIIA fragments withBgl I + II. Panels c cal maps of invitro-synthesized M-MLV DNA

and d show theundigested Sal IB fragment and by using a variety of restriction endonucleases.

dataobtained afteritsdigestionwithBglI +TI. The DNA fragments obtained have been

or-Twofragments ofmolecularweightsof 1.1 x106 dered with respecttothe 5' and 3' ends of the

to 1.2 x 106 can be identified. If fragment B M-MLV genome. A composite cleavage map of

originates from the 5' end ofthegenome, then M-MLV DNA is shown in Fig. 11. Restriction

we should have seen afragment of 1.75 x

10'

endonucleases Sal I and Hind III cleave the

daltons.However,weobservedafragmentof1.2 viral DNAatleast once, whereasHpaIhastwo

x

10'

daltons andafragment of 1.1x106daltons. cleavagesites.Restriction endonucleases Bam I

If the 1.2 x 106-dalton fragment is Bgl I + II and Sma I cleave the M-MLV DNA at three

fragment C,then the 1.1 x 106-dalton fragment sites, and a mixture ofBglI + II has at least

will represent a portionofBgl I + IIfragment fourcleavage sites.Weare notyetcertain about

B.Thus,the order of the Bgl I+IIfragmentsB theorderof theSmaIB, Sma IC, andSmaID

and C appears to be 5'-C-B.Thisorderis com- fragments. Based upon redigestion ofBam IC

patible with the data obtained by digestion of fragment with Sma I,wethink thatatleastone Hind IIIAfragment withBglI +IT. The Hind ofthetwosmallfragments,Sma ICorSmaID,

IIIA fragment hasa molecular weight of3.6 x originates from the extreme 3' end of the

ge-106andshouldcontain both fragments B and C nome.Thisresult isatvariance with theresults and perhaps portions of fragments D and E. obtained from digestion of clone 124 murine

Panel e shows theresults ofdigestion of Hind sarcoma virus (MSV) DNA with Sma I

(Rob-IIIA fragment with Bgl I + IT. It can be seen erts,McKennett, and Verma,unpublisheddata).

that there aretwoprominentbands ofapproxi- Fromtheheteroduplexstudies (10) and

hybrid-matemolecularweightsof1.75 x 106and 1.2 x izationexperiments (4), it has been shown that

106and a smallerfragmentof about 0.25 x 106 about 600 to 700 nucleotides at the 3' end of

daltons. As expected, the Hind IIIA fragment both M-MLV and clone 124 MSV RNA are

containsboth theBglI+ II B andCfragments common. IfSma ICorSmaIDfragments

origi-anda portionof D orEfragments. Thus, from nated from the 3' end of M-MLV genome, we

thecombined data obtained bythe redigestion would have expected that full-genome-length

ofthe Sal IA, Sal IB,HindIIIA, and Hind IIIB clone 124 MSV DNA should have at least one

fragments,theorder of theBgl I+IIfragments SmaI site. However, the data indicate that clone

appearstobe5'-C-B-(DE)-A-3'. 124 MSV DNA is not digested by restriction

Other restriction endonucleases. There endonuclease Sma I(Robertsetal., unpublished

were three main purposes of our attempts to data).If SmaICorSma ID fragments originate

screen several other enzymes as follows: (i) to from the 3' end of the genome, we would predict

find a restriction enzyme that does not cleave that ifcircular M-MLV DNA is digested with

M-MLVdouble-stranded DNA;(ii)tofind those Sma I, fragment A will be only slightly larger

that cleaveno morethanonceortwice; and (iii) thanthat obtained from linear viral DNA. There

tofind those enzymes that cleave the M-MLV isalso uncertainty about the order of Bgl I +II

double-stranded DNA many times to generate fragments D and E. We now have access to

a finephysical map. So far the only restriction isolated Bgl I and BglII restriction

endonucle-endonuclease that doesnotappear tocleave M- ases and would like to constructphysical maps

MLV double-standed DNA is Eco RI (Wein- using each enzyme separately. If fragments D

berg, personal communication; Bacheler, per- and E are generated by the sameenzyme, then

sonal communication; and Verma and Mc- theorder of these fragments can be determined

Kennett, unpublished data). We used several by their redigestionwith Hind III. The physical

restriction enzymes that were known to cleave mapsshown in Fig. 11 indicate that if fragment

hexanucleotide sequences, hoping that the prob- D iscontiguous with fragment A, then fragment

abilityofa hexanucleotide of a given sequence D will have HindIII site, generating two

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(14)

9 8 7

*

6 5 4 3 2

i

13'

(kb)MMLVgenome

S'

B

A

13'

SalI

M

A

B

13'

Hindm

5,1

A

4

B

C

13'

BamI

S'l

A

4,c4,

B

1

3'

Hpa

I

S'

A

4,

(B,C,D)

3'

Sma

I

S'l

c

4,

B

4(D,E)4

A

13

BglI+1I

SoaI

HindX SmaIHpaI HpaI

5S

J

JPJ1

P 13'

t

tt

t

t

t

BglI+.I

BomI BamI

Bf,II+4I

BgII Baml

VI

gag

Po/

envI

5'1

Ia

o

||3'

1

Fi(.. 1 1. Proposed restriction endonuclease cleavage maps of invitro-synthesizedM-ML Vdouble-stranded DNA.

ments of 0.3x 106 and 0.1 x 106 daltons.Onthe genome-length cDNA transcripts containa

mix-otherhand, fragmentEwill not bedigested by ture of two size classes, one of which is about

HindIII. 600nucleotides shorter than thefull-length

ge-Aspointed out in theresults, BamI-digested nometranscripts. The datashown in this paper

M-MLV DNAfragments donot addup to the do notdistinguish between these sizeclass mol-size of the undigested DNA. When linear M- ecules and could

presumably

affect the size of

MLV DNA isolated from infected cells is di- the DNA fragments. If the shorter molecules gestedwithBamI,alarge fragment of molecular representa deletion at the 5' end of the RNA weight of2.5 x 106 isobserved (Yoshimura and genome, thenitwould

explain why

the restric-Weinberg,

personal

communication, and Bach- tion fragments obtained from the 5' end of the eler,

personal

communications). Furthermore, if genomic RNA are somewhatheterogeneous in circular viral DNAisused, a fragmentof 3.7 x size.

106daltonsis observed (Bacheler, personal com- In the experiments reported in this

manu-munication),which iswhat would beexpectedif

script,

the parent strand was labeled. Because

BamI

fragment

C

(molecular weight,

1.2 x 106) the

synthesis

of the parent strand

requires

a

originates fromthe 3'endof thegenomeand the very

high

concentration of precursor

deoxyri-2.5x

106

fragment

originates

from the5' end of bonucleoside

triphosphates

(20),

it is difficultto

thegenome.The

largest fragment

thatwehave

synthesize high-specific-activity

material. We

observed is

only

2.1x 106daltons. It is

unlikely

have

recently

synthesized

double-stranded DNA that M-MLVDNAused for

digestions

isincom- from

AKR,

where the second strandwaslabeled. plete and lacks a portion of the 5' end ofthe The data were identical to those obtained

by

genome.If thiswerethe case, then the

fragments

using

double-stranded

DNA,

when the parent

obtainedby digestion with otherrestrictionen- strandwaslabeled. For thepurpose of

construct-donucleases shouldalso not add up to the ge- ing physical maps, the use of

DNase-digested

nomelength.It ispossiblethatourBam I prep- calf

thymus

DNA

primers

appears to be

quite

aration is contaminated withsomeother restric- effective to

synthesize

the double-stranded

tion endonuclease that cleaves the 2.5 x 106- DNAs.

daltonfragmentto2.1x 106-dalton

fragment.

It

Preparation

of

specific

DNAprobes. The

has been

reported by

Rothenberg

etal.

(16)

and gene order of murine RNA tumor viruses has

confirmedbythe

accompanying

paper

(20)

that notbeen established

unambiguously.

However,

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the data obtained fromheteroduplex formation ADDENDUMINPROOF

between M-MLV cDNA and clone 124 MSV

Yoshimura

and

Weinberg

haveused

in

vivo-synthe-RNA (10) and M-MLV cDNA and AKR RNA sized proviral DNA to construct physical maps. The

(Chien, Davidson, and Verma, unpublished resultsobtained by the two approaches are generally

data) suggest the gene order to be 5'-gag-pol- ingood agreement.

env-3', analogous to that described for avian

sarcoma viruses(2,21; Bishop,Annu. Rev. Bio- LITERATURE CITED

chem., in press). It appears that 38S M-MLV 1. Baltimore, D. 1974. Tumor viruses: 1974. Cold Spring

viral mRNA codes forgag(pol) protein, whereas HarborSymp.Quant. Biol.39:1187-1200.

the 24S mRNA codes forenv proteinand pos- 2. Beemon, K. 1974.Thecomplexityof tumorvirusRNA.

Ph.). thesis, tJniversitvofCalifornia.Berkelev.

sibly some nonstructural protein (6). From the 3. Beemon, K. 1977. Oligonucleotide

fingerprinting

with

physical maps (Fig. 11) we would predict that RNA tumor virus RNA. Curr.Top.Microbiol.

Immu-fragments SalIB, HindIIIA, Bam IA, and Bgl nol. 79:73-110.

I +IIC willonly annealto38Sviral mRNA and 4. Dina,D., and K. Beemon. 1977. Relationship between

notto24Sviral mRNA. Thus, these fragmeiits Moloneymurine leukemia and sarcomavirus RNAs:

not to 24S viral mRclNA. Thnus, these fragments purification andhybridization map of complementary

canbe very useful foridentification,quantifica- DNAs from defined regionsofMoloneymurinesarcoma

tion, and isolation ofspecificviralmRNA's. virus124.J.Virol. 23:524-532.

Mousegenomescarry manyintegrated

copies

5. Endow, S. A., and R. J. Roberts. 1977. Two

restriction-ofendogenousviruses

.21

ishop,A

ies

likeenzymesfromXanthomanosmalvacearum.J. Mol.

of endogenous viruses (21; Bi51shnop, Annu. Rev. Biol.112:521-529.

Biochem.,inpress;R.Jaenisch andA.Berns, In 6. Fan,H. 1977.Expression of RNA tumor viruses at

trans-M.Sherman, ed., ConceptsinMammalian Em- lation and transcription levels. Curr. Top. Microbiol.

bryogenesis, inpress). It is very difficult to de- Immunol. 79:1-41.

terminethsites ofintegration

.ofagiven

V 7. Fan, H., and M. Paskind. 1974. Measurement of the sequencecomplexityofclonedMoloneymurine

leuke-by currenttechniquesofSouthernblotting (18) mia virus 60 to 70S RNA: evidence for a haploid

ge-because of nucleic acid sequence homology nome.J. Virol. 14:421-429.

among endogenous and exogenous viruses 8. Fan, H., andI.M. Verma. 1978. Size analysis and

rela-(Jaenisch and Berns, In M.Sherman, ed., Con- tionship of murine leukemia virus-specific mRNA's: evidence fortranspositionofsequencesduring synthesis

ceptsinMammalianEmbryogenesis, inpress). and processing of subgenomic mRNA. .J. Virol. Availability of restriction endonuclease frag- 26:468-478.

mentsspecificfor various virusesshouldhelp to 9. Garfin, D. E., and H. M. Goodman. 1974. Nucleotide

obviate the problem of nucleic acid sequence sequences atthecleavagesites of two restriction

endo-obit.hpolmi

sence.nucleases fromHemophilus

parainfluenzae.

Biochem.

cross-homologies. Wehave foundthatdigestion Biophys. Res.Commun. 59:108-116.

of in vitro-synthesized AKR double-stranded 10. Hu, S.,N.Davidson,and I. M.Verma. 1977. A

hetero-DNAwithrestrictionendonucleasesHaeIIand duplex study of thesequence relationship betweenthe

Hind II shows some DNA fragments not ob- Moloneymurinesarcoma virus RNA andMoloney

mu-rine leukemia virus complementary DNA. Cell

served by digestion of M-MLV DNA by the 10:469-477.

same enzymes (Verma andMcKennett, unpub- 11. Maxam,A.M.,and W. Gilbert.1977. Anewmethod for

lished data). It thus appears possible to make sequencing DNA. Proc. Natl. Acad. Sci. U.S.A

specifiDNAfrgmentsby usin in vito-syn- 74:560-564.

specific DNA fragments by using in vitro-syn-

12.

Old, R., K.Murray, and G. Roizes. 1975. Recognition

thesized viral DNA. The isolated specific frag- sequence of restrictionendonuclease III from

Hemoph-ments can then be made highly radioactive ilusinfluenzae.J. Mol. Biol.92:331-339.

either by labeling the 5' end with

[y-32P]ATP

13. Pirrotta, V. 1976. Two restriction endonucleases from

(11) or

by

addition of a

deoxyhomopolymeric

Bacillusglobiggi.Nucleic AcidRes. 3:1747-1760.

11) or

eby

add

of a d 14. Roberts,R. J. 1976. Restriction endonucleases. Crit. Rev.

tract at the3' end (20). Biochem.4:123-164.

15. Roberts,R.R., G.A.Wilson,andF. E.Young. 1977. Recognitionsequence ofspecificendonucleaseBamHI ACKNOWLEDGMENTS from Bacillus amyloliquefacieus H. Nature (London)

265:82-84.

We thank M.Vogtforhelpintheearlystages of some of 16. Rothenberg,E., D.Smotkin,D.Baltimore,and R. A. theseexperiments.We aregratefultoLeslieJerominski,Lisa Weinberg.1977.In vitrosynthesisofinfectious DNA Dee, Billie Green, and Christine Roberts for their help at of murine leukemia virus. Nature (London) 269: variousstages. We appreciate the generous gifts of restriction 122-126.

endonucleases fromTonyOhtsuka and John Abelson. It is a 17. Sharp,R., B. Sugden,and J. Sambrook. 1973. De-pleasure toacknowledge thanks to members of the Tumor tection of two restriction endonuclease activities in Virology Laboratoryfor constant helpand encouragement. Haemophilus parainfluenzae using analytical ag-We thank J. T. Simon for theillustrations andCarolynGoller arose-ethidium bromideelectrophoresis. Biochemistry

fortypingthemanuscript. 12:3055-3063.

This work wassupported by Public Health Service research 18. Southern, E. M. 1975. Detection of specific sequences grants CA 16561 and CA 21408 from the National Cancer among DNA fragments separatedby gel electrophore-Institute and Core Grant CA 14195 from theNational Cancer sis.J. Mol. Biol. 98:503-517.

Institute. 19. Southern, E. M. 1975. Long range periodicities in mouse

on November 10, 2019 by guest

http://jvi.asm.org/

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satelliteDNA.J.Mol. Biol.94:51-69. 21. Vogt, P.K.,and S. S. F. Hu.1977.Thegenetic structure 20. Verma, I. M.1978. Genomeorganization of RNAtumor of RNAtumorviruses. Annu. Rev. Genet. 11:203-238. viruses.I. Invitrosynthesis offull-genome-lengthsin- 22. Weinberg, R. W. 1977. Structure of the intermediates gle-stranded and double-stranded viral DNA tran- leadingtotheintegrated provirus. Biochim. Biophys. scripts. J. Virol. 26:615-629. ActaRev. Cancer473:39-55.

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Figure

FIG.1.lecularfragments;form Standard curve for determination of mo- weights. Symbols: 0, phage lambda DNA , adeno type 2 DNA fragments; A, linear ofpolyoma DNA; x, polyoma DNA fragments.
FIG. 3.weightDNA Digestion of fractional-length M-MLV DNA with Sal I and Hind IIIA. (a) Undigested M-MLV of molecular weight of 4.0 x 10'; (b) digestion with Sal I; (c) undigested M-MLV DNA of molecular of 3.5 x 10'; (d) digestion with Hind III
FIG. 6.capital(d)Sma Sma Patterns ofDNA fragments ofM-MLVDNA generated by restriction endonucleases Bam I, Hpa I, I, and Bgl I + II
TABLE 1. Molecular weight estimations and molarratios ofDNA fragments generated by digestion ofM-ML V double-stranded DNA by restriction
+3

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