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 Icleaves 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 x10'
(Hpa
IC).
Digestion
ofM-MLVdouble-stranded DNA with restrictionendonu-clease SmaIproduces four fragmentsof molecularweightsof 3.9x 106(SmaIA), 1.3 x 106 (Sma IB),0.28 X 106 (SmaIC), and0.21x
10'
(SmaID). AmixtureofrestrictionendonucleasesBglIandBglII (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),
andenvelope
(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 gelcontaining
the standards for molecular weights wastosynteinvdevral stained with ethidium bromidetovisualize the DNA
toA
synhesI,
.i
patro,
due-setrade siral
fragments. The gelcontaiming
the sample was placedDNA (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 scintillationHpa 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 thegel
endonucleases Sal I and HindIII, the buffer in the
electrophoretic
profiles of nvitro-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 digest1.0jig
of (Arrand, Meyers, and Roberts, unpublishedphagelambda 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 enzymesreaction 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 aphagelambda DNA to monitor thecompletionof the shows that
the
undigested
M-MLV DNAhas
areaction. molecular
the
about 6 D has aAgarose 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
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I X 107 - or an inherent
property
of the invitro-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
aI
\ 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 x10'
(Fig.
3,
panel
c).Figure
3 showsO i the
patterns
obtained afterdigestion
of 4.0 x3 .
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 noticedJ .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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[image:3.501.62.254.54.367.2] [image:3.501.269.458.239.627.2]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 IAfragments 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 x106.
Thelarger fragmentcomi-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 IBfragment
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 notfragments 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 theundigested
HindIIIBfragment
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[image:4.501.111.392.67.347.2]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 smallerfrag-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 constructedby
using
re-fragmentof approximate molecularweight of0.8 striction endonucleasesBamI,Hpa
I,Smax 106. This is either an artifact or a digestion I, and
Bgl
I + H.Figure
6 shows thegel
product ofsomecontaminating HindIIIAfrag-
electrophoretic
patternsofM-MLV DNAfrag-ment. In other experiments in whichHindIIIB
fragment was free of any HindIIIA
fragment,
SalIB(2.4Xb06)
SaSaIIA(3.5X106)
nosuch DNA fragmentwas observed. Panels
JI
HindMA
(3.6X106)
tHindMB
(2.3X106)
3andkshow thepatterns ofundigested Hind IIIA
HindM
fragment and itsdigestionwithSal I. TheSalI-
FI.(
5. Order of the DNAfragmentsgenerated bydigested 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
f9
2 5- _ - * 9 |
*.*
LO--~~~~~~r
FcIG. 4. Redigestion ofSalIAand Band
Hi-nd
lllAandBfragments.
(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 Thearrowspointtothefragmentsgenerateduponredigestion (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), thefragmentsgenerated 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,canmolecular 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 largein 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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[image:6.501.107.390.71.448.2]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 theBam
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 HindIIIAB 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 molecularweight
of 0.21 x 106B 1.35 ND
represents
either theBam
IDfragment
ortheC 0.29 remainder of Sal IA fragment after digestion
D 0.22 with
Bam
I enzyme. If weassume that the 0.21BglI + II A 2.0 x 10"-molecular-weight fragment is
Bam
IDB 1.75
fragment,
then the order of these threefrag-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 twofragments
of average"ND,
Notdetermined,
molecular weights of 2 x106
and 0.23 x106.
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 Bamx 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 theaffect 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 x10".
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 probablyFigure 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'
istheauthenticBam 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 with4.0x 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 xthe DNA was collected from aneutral sucrose
106,
and 0.6 x106.
The 0.6 x106-molecular-gradient (20).Itcanbe seenthatthere is amajor weightfragment appearstobeeitheranartifact
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[image:7.501.66.256.87.392.2]VOL. 26,1978
637
49~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~:
+~~~~~~~~~~~~~~~~~~'.- t _
t_
_R t-|~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~x'
*>X>eQ~~~~~~
zoS S
.0
-s
§-
Sx
°
LAdU)
4
CM
cok
X s) Cq
o\j
Ctc\j
° O SE~
rciy
--E-0
x
04 Q6
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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 theBamIDfragmentonthe 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 enzymeprepara-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 atHpa 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 Idigested 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
offractional-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 bandgested 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 atendonuclease Hpa I. Panels f and g show the a molecular weightof 1.5 x
106
to 1.6 x106,
andundigested and Hpa I-digested SalIB fragment. two very
small
fragments of molecular weights It canbe observed that the size ofthe SalIB of 0.25 x10W
and0.19x106.
Ifoneassumesthat fragment before and afterHpa I digestion re- 1.3 x106-molecular-weight
fragment is SmaIBmains unaffected, suggesting that the Sal IB fragment, thetwoverysmallfragmentsareSma
fragment does not containasitespecific for Hpa IC andSmaID fragments, and the 1.5 x
106_
toI. These results, however, do not establish the 1.6 x
106-molecular-weight
band represents theorder 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, aIffragmentHpaIBoriginatesfrom 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;
on November 10, 2019 by guest
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VERMA AND McKENNETT J.
M
WXO
xtN
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
SallBfragment.
Nodigestion
appears that the order of the Sma Ifragments
is5'-A-totake
place.
This will beexpected
ifthe Sma (BCD)-3'.IA
fragment
ofanaverage molecularweight
of(iv)
Bgl
I + II. Inexperiments
presented
3.9 x
106
originates
from the 5' end of the ge-here,
wehave used amixture ofrestrictionen-nome. donucleases
Bgl
I+IL.
Thesequencespecificity
Wehavenot
conclusively
established the or- ofBgl
I isAGGCNGCCT1 (G.Magnusson,
per-der of the Sma IB, IC, and ID
fragments
in sonalcommunication),
whereasBgl
II appearsrelation to the 5' and 3' ends of the genome. tocleave hexanucleotideA1GATCT (13).
Diges-From the
preliminary
data obtainedby digestion
tion of M-MLVdouble-stranded DNA withBgl
of theBarnIC
fragment
(molecularweight,
1.25 I + IIgenerates at least fivefragments (Fig.
6,x
10')
withSmaI,
itappearsthatatleastoneof panele).The molecularweights
of thefragments
the smaller
fragments
(Sma IC or Sma ID) aregiven
inTable1.The order of thefragments
originates
from the 3' end of the genome. How- inrelationto the 5' and 3' ends of the M-MLVever,moredetailed
analysis
ofredigestion
ofthe genome were determinedby
redigestion
of SalHpa
lB andHpa
ICfragments
with Sma I will Ifragments
A and B andHind IIIfragments
Ayield
a moredefinite order ofthe SmaIB,Sma and B(Fig.
10).Panelsaand b show thepatterns
IC,andSma ID
fragments. Presently,
we assume ofBgl
I +II-digested
Sal IA and Hind III Bon November 10, 2019 by guest
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[image:11.501.118.403.58.424.2]MW
X
106
ab
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 the1.1 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. It0.22x 106. If2 x
106-molecular-weight
fragment isnotclearwhywesee twofragmentsof0.47 xis 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 IAfrag-is fragment D, then it would appearthat frag- mentwith
Bgl
I+II,
the order of thefragments
mentsAand Doriginatefrom the 3' endof the would appear to be
3'-(AD)E(BC)-5'.
Panel bgenome. The 0.94 x 106-dalton
fragment
may shows the data obtainedby redigestion
of Hindon November 10, 2019 by guest
http://jvi.asm.org/
[image:12.501.96.390.64.479.2]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 thedaltons.However,weobservedafragmentof1.2 viral DNAatleast once, whereasHpaIhastwo
x
10'
daltons andafragment of 1.1x106daltons. cleavagesites.Restriction endonucleases Bam IIf 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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9 8 7
*
6 5 4 3 2i
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 HpaI5S
JJPJ1
P 13't
tt
t
t
t
BglI+.I
BomI BamIBf,II+4I
BgII BamlVI
gag
Po/
envI
5'1
Ia
o
||3'
1Fi(.. 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 ofMLV 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. BecauseBamI
fragment
C(molecular weight,
1.2 x 106) thesynthesis
of the parent strandrequires
aoriginates fromthe 3'endof thegenomeand the very
high
concentration of precursor deoxyri-2.5x106
fragmentoriginates
from the5' end of bonucleosidetriphosphates
(20),
it is difficulttothegenome.The
largest fragment
thatwehavesynthesize high-specific-activity
material. Weobserved is
only
2.1x 106daltons. It isunlikely
haverecently
synthesized
double-stranded DNA that M-MLVDNAused fordigestions
isincom- fromAKR,
where the second strandwaslabeled. plete and lacks a portion of the 5' end ofthe The data were identical to those obtainedby
genome.If thiswerethe case, then the
fragments
using
double-strandedDNA,
when the parentobtainedby 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
DNAprimers
appears to bequite
aration is contaminated withsomeother restric- effective to
synthesize
the double-strandedtion endonuclease that cleaves the 2.5 x 106- DNAs.
daltonfragmentto2.1x 106-dalton
fragment.
ItPreparation
ofspecific
DNAprobes. Thehas been
reported by
Rothenberg
etal.(16)
and gene order of murine RNA tumor viruses hasconfirmedbythe
accompanying
paper(20)
that notbeen establishedunambiguously.
However,
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http://jvi.asm.org/
the data obtained fromheteroduplex formation ADDENDUMINPROOF
between M-MLV cDNA and clone 124 MSV
Yoshimura
andWeinberg
haveusedin
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
withphysical 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. Tworestriction-ofendogenousviruses
.21
ishop,Aies
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 sequencecomplexityofclonedMoloneymurineleuke-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 fromHemophilusparainfluenzae.
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. Recognitionthesized 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 adeoxyhomopolymeric
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
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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.