Construction of the genetic map of the polyoma genome.

CopyrightC1976 AmericanSociety for Microbiology PrintedinU.SA.

Construction

of the

Genetic

Map

of the

Polyoma

Genome

LOIS K. MILLER* AND MIKE FRIED

Imperial CancerResearchFund,Lincoln's InnFields,London WC2A3PX, England

Received for publication26January 1976

Sevenearly mutants, three late mutants, andoneplaquemorphology mutant

of polyoma have been mapped by marker rescue using wild-type restriction endonuclease fragments. The early mutants map between 1.0 and 26.4 units from the Eco RI site, a region previously shown to correspond to the 3'-OH

terminal halfof"early"RNA(Kamenetal., 1974). Thelatemutantsaswellas

the plaque morphologymutantmapbetween26.6and 45.4mapunits,aregion

previously shown to correspond to the 3'-OH terminal half of "late" RNA

(Kamen etal., 1974). Analysis of thegenotype ofrescuedvirus demonstrated

that the modificationofthemutantDNAduringmarkerrescuewaslimitedto

the region of the genome covered by the wild-type restriction endonuclease

fragment tested.

Temperature-sensitive mutants of the onco-genic papovavirus, polyoma,havebeenisolated and characterized (2-4, 7, 8). These mutants generally fall intotwoclasses: (i)earlymutants that areblockedinviral DNAsynthesis (6, 7) atthe restrictive temperature, and (ii) late mu-tants that do not form infectious particles al-though viral DNA issynthesizedatthe restric-tive temperature (2-4). Genetic complementa-tionanalysis ofthe mutants has provided some further subdivisions in this classification scheme (2-4), but adetailed knowledge ofthe genetic organization ofpolyoma virus has re-mained elusive due to low recombination fre-quencies (14) and lack of sufficiently distinct

conditional-lethal mutation types for genetic mappingby three-factor genetic crosses.

The genetic mapping technique of marker rescue is anovel one since itinvolves the bio-chemical manipulation of the viral DNA. The

technique relies on the ability of a fragment of

wild-type DNA, which has no infectivity by itself, to produce wild-type virus when an-nealedto afull-lengthmutant DNAand intro-duced to a cell. This will be referred to as a "frescue"p event, but it is important to note that the original mutant DNA is converted to a wild-type DNA by cellular mechanisms.

The rescue ofwild-type virusfrom

transfec-tions with heteroduplex DNA forned by an-nealing wild-type DNA fragments to condi-tional-lethal mutant viral DNA was first ob-served in kX174 studies (13, 28). The marker

rescue technique has been shown to be an

effective method for mapping mutants of sim-ianvirus40(SV40) (16-18). The marker rescue procedure relies on theability to produce

spe-cificfragments of viral DNA by cleavage with

restriction endonucleases, to purify the

frag-ments by gelelectrophoresis, and to know the

physical orderof thefragmentswithin the ge-nome.

Anumberofrestriction endonucleases cleave polyoma DNA into discrete fragments, the physical order of which has been determined (10, 11;B.E. Griffin, unpublished data).Inthis

report, individual restriction endonuclease

fragments(RENFs)were testedfor theirability

to rescue avarietyofearlyandlatetsmutants.

A

genetic.

map of these mutations was

con-structedfrom the results. Inaddition,wetested

the ability of fragments to convert a minute-plaque mutantofpolyoma tothe large-plaque

type,andthe results demonstratethatthis

mu-tation is located in the "late" region of the genome.

MATERIALSAND METHODS

Viruses. All virus stockswerepreparedfrom

sin-gle plaquesand were grown at low multiplicities as

previouslydescribed (9). The early mutant tsa and the latemutanttsCwerederived from the Pasadena large-plaque strain byM.Fried (7; M.Fried, unpub-lisheddata). The CR variant was derived from the Pasadena large-plaque strain (Fried, unpublished

data).Theearly mutants ts25, ts616, ts48,ts609,and

ts697 and the late mutants tslO and ts1260 were providedbyW. Eckhart (4 and references therein). The ts697 mutant was derived from the P16 small-plaque strain, and the others were derived from the Pasadena large-plaque strain. The minute-plaque mutant, 208,wasderived from the Pasadena large-plaquestrainandprovided byM.Vogt.

Cells and plaqueassay..Monolayersofsecondary wholemouse embryo cellsweregrowninDulbecco modified Eagle medium supplemented with 5% fetal 824

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POLYOMA GENETIC MAP 825

calfserum for viral DNA preparation or 3% horse serum for virus stock production or plaque assay. Plaque assays were performed as previously de-scribed (8).

Preparation of polyoma DNA. All viral DNAs

were prepared by Hirt extraction (12) of infected cells followed by CsCl-ethidium bromide equilib-rium density centrifugation (23). The supercoiled form I (FI) polyoma DNA was further purified by neutral sucrose gradient centrifugation. Random, singly nicked form II (FII) DNA was prepared by X

irradiationof FI tonick approximately 30% of the

molecules. The FII singly nicked DNAs were puri-fiedfrom the remaining FI DNA byCsCl-ethidium bromideequilibrium densitycentrifugation.

PreparationofRENFsofDNA. RENFs were

pre-pared from 3H-labeled FI DNA except the HpaII

fragments, which were prepared from32P-labeledFI DNA. The 3H-labeled fragments were purified by electrophoresis through 1.4% agarose tube gels, 20 cm inlength. The DNA fragments were visualized byethidiumbromidestaining(21, 24), cut individ-ually from the gels, andremoved from the agarose

by electrophoresis. The fragments werejudgedby

gel electrophoresis tobe more than 90% pure. The

32P-labeledHpaIIfragments (a gift of B. E. Griffin) werepurified byelectrophoresison3.3%

polyacryl-amide plus0.5%agaroseslabgels, locatedby

auto-radiography, slicedfrom the gels, andremovedfrom

thegelslicesbyelectrophoresis. Eachfragmentwas

furtherpurified by neutralsucrosegradient

centrif-ugationandcheckedfor puritybygel electrophore-sis.

Restrictionendonucleasesandincubation

condi-tions. Therestriction endonuclease HindIII was

pre-pared by proceduressimilar to thosedescribed

pre-viously (27). The HhaI enzyme waspurified bythe

unpublished procedure ofR. RobertsandP. Myers

(personal communication). TheHpaII and Eco RI

enzymes werekindlyprovided byB. GriffinandR. Kamen. The buffers used for enzyme digestions were: HhaI andHpaII, 10 mM Tris-hydrochloride (pH 7.5)-10 mM MgCl2-1 mM dithiothreitol;

HindIII, sameasHhaIandHpaTI + 50 mMNaCl;

Eco RI, 10mMTris-hydrochloride (pH 7.5)-10 mM

Mg2Cl-100mMNaCl.Incubationswere at 37 Cfor a

sufficienttimeandwithsufficientenzymefor

com-plete digestion. Thecleavage sitesof these

endonu-cleasesareshowninFig. 2(adaptedfrom reference

11).

Transfection of cells with polyomaDNA.

Near-confluentmonolayersofsecondarywhole mouse em-bryocells in 35-mmpetridisheswerewashedonce with 2 ml of Dulbeccophosphate-bufferedsaline

mi-nusMg2+andCa2+ (PBSA).A0.4-mlaliquotof1mg

ofDEAE-dextran per mlof PBSA wasdistributed

overthecellmonolayerandincubated at32C for20

min. The excessDEAE-dextran wasremoved,anda 0.3-ml solution ofpolyomaDNAinPBSAwas dis-tributed over the cell monolayer. The cells were

incubated forapproximately 30minat 32C, after

which 1.5 ml of Dulbecco modifiedEagle medium

supplemented with 3% horse serum and

receptor-destroying enzyme (8) was added. The cells were

incubatedat 32 C forapproximately6days.

Freeze-thawingfollowedby 0.5 min ofgentlesonicationwas usedtobreak open theinfectedcells.

FormationofheteroduplexDNAs. Heteroduplex

DNAs were formed by combining random, singly nicked, FII mutantpolyoma DNA, RENFs produced by cleavage of FI mutant DNA, and a single, puri-fiedRENF of wild-type polyoma DNA. In the first series ofexperiments(usingHpaHI fragments), the molar ratios of the various DNA types were

1:10:approximately250. In otherexperiments(using

HindIII, HhaI, and HhaI + Eco RIfragments),the molar ratios of the DNAs were 1:4:40 (mutant FII DNA:RENFs containing sequences of the entire mutantgenome:purifiedwild-typeRENF). To a

40-IlI

volumecontaining0.0125 ,ug of mutant FII DNA and the appropriate concentrations ofmutant and wild-typefragments dissolvedin 10 mM Tris-hydro-chloride (pH 7.5) and 1 mM EDTA, 10 ,ul of2 M NaOH was added. Afterstanding at room tempera-ture for 20min, thesolution was neutralized by the addition of 20 ,ul of a buffered HCIsolution (1 vol-ume of 2 MHCI, 1 volume of 1 M sodiumphosphate, pH 7.0). The neutralized solution was incubated at 68C for 30 min. Under these hybridization condi-tions, atleast 80% of thefull-lengthDNAmolecules are inthedouble-strandedform;approximatelyfour outof fivefull-length double-stranded DNA mole-cules contain onefragmented strand.After hybridi-zation, 0.1ml ofdistilledwaterand 0.45 ml of PBSA wereadded, and the solution was frozen until used

fortransfectionofcells. The DNAhybridizedinthis

mannershowedaspecificinfectivity of 2 x 105 PFU

per jig offull-length FU DNA by direct

DEAE-dextrantransfectionassayat 32 C.UntreatedFIor FII DNAhas aspecificinfectivity ofapproximately1 x 106 PFU per ugoTfDNA inDEAE-dextran trans-fection assays at 32 C.

RESULTS

Experimental design. Theexperimental de-sign forgenetic mapping ofpolyoma mutants

bymarkerrescueisschematicallypresented in Fig. 1.Heteroduplexeswereformedby combin-ing,denaturing,and reannealingmutant(ts or minute plaque) FII DNA, a single purified wild-type RENF, and the full complement of

mutantRENFsproduced byrestriction

endonu-clease cleavage of mutant FI DNA. The mu-tantRENFswereaddedfortworeasons: (i) to preventthereannealing offull-length mutant DNA strands of the added mutant FII DNA, and (ii) to minimize the enhancement of

marker rescue by nonspecific fragments that

Lai and Nathansobserved (16). Theresults of

Lai and Nathans demonstrated that a partial

heteroduplex composed ofafull-lengthmutant DNA and one wild-type restriction fragment

wasless effectiveinmarkerrescuethan a par-tialheteroduplex withmorethanonewild-type

restrictionfragment. Thisresultedin an

anom-alous enhancement ofinfectivityby

nonspecific

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Mutant F1I Mutant Fragments One Wild-Type Fragment

Combine Denature

Reanneal

Heteroduplex

I Infect cells at 32 C Virus Stock

I Plaque Assay at 39C and 32 C

Ratio 39C/32C

FIG. 1. Experimentdesign for markerrescue.MutantFII DNA wascombined with thefullcomplementof mutant restrictionendonucleasefragments,produced byrestrictionnuclease digestion of FImutantDNA, andasingle, purifiedwild-type restriction nucleasefragment.The ratiosofthesethree DNA componentswere adjustedtopermit (i)thesinglewild-typefragmenttocompetesuccessfullywith a mutantfragment,and(ii) thefragments to compete successfully with the full-length mutantDNAduring the reannealing reaction. The threeDNAcomponentswere denatured and reannealed to allow the formationof heteroduplex circular DNA composed of one full-length mutant DNA and a complementary strandcomposed of onewild-typefragment andthe remainingmutant fragments. Whole mouse embryo cells were infected with this DNA at 32 C in the presence of receptor-destroying enzyme to inhibit secondary infections. The virus stock obtained from this infection (see Materials and Methods) was assayed for plaque-forming ability at 39 and 32 C. The wild-type restrictionendonuclease fragment resulting in the highest 39C/32C ratiocorresponds to the mutated region of the mutant DNA tested.

which wild-type fragment corresponded to the The molarratiosofthe threeDNA typesused

mutantregion. Weattemptedtominimizethis to form the heteroduplex were balanced in a

enhancement effect by startingwithafullhet- mannerthat (i)permittedthewild-typeRENF

eroduplexDNA. tocompetesuccessfullywiththecorresponding

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POLYOMAGENETIC MAP 827

mutantRENFsothatatleast9outof 10 hetero-duplexes contained the wild-type RENF, and (ii) permitted the RENFs to compete

success-fully with full-length DNA derived from the

mutantFII DNA toenhance formation of het-eroduplexes rather than homoduplexes.

Cells infected with the heteroduplex DNAs

wereincubatedat32 C inthepresenceof

recep-tor-destroying enzyme, which inhibits

reinfec-tion by the newly formed virus (8). This

pro-vided a stock ofmutant and wild-type virus,

which wasthenplaque assayedatthe

permis-sive and nonpermissive temperatures (32 and 39C). In the case ofthe minute-plaque virus,

208,theplaqueassayswereperformedat37 C,

andthenumber oflarge-plaqueviruses

appear-ing in the presence of minute plaques was

scored. Theratio of PFUassayedat39Ctothe PFU assayed at 32C (referred to asthe 39C/

32 Cratio)indicated which RENFcorresponded

to the ts region of the genome. Similarly, the

ratio oflarge plaqueto minuteplaqueat 37C indicated which RENF correspondedtothe

re-gion of the genome responsible for the plaque

morphology of themutanttested.

Marker rescue with HpaII fragments. In the first experiment for genetically mapping polyoma mutants, the eight HpaII RENFs of wild-type DNA were individually tested for

theirability to "rescue" theearly mutant tsa,

the late mutants ts1O, ts1260, tsC, and the plaque morphology mutant 208 (Table 1). For theearlymutant,tsa,themostactivefragment inmarkerrescue wasHpaII fragment2 (HpaII-2).Forthe latemutantstslO,ts1260and tsCas

wellastheplaque morphologymutant208,the most active fragment for marker rescue was

HpaII-1. Theseresults, however, are not

com-pletely satisfactory since other fragments

showed unusually high ratios (backgrounds).

The results with tsC areparticularly ambigu-ous. The fragments that showed high

back-groundswere usuallythosethatmigrated just

below themostpositive fragment.

For instance,inthetsacase,fragment3gave

aratioapproximatleyone-third the ratio forthe

most positive fragment (HpaII-2), whereas Hpall fragments 2 through4 gave high back-groundswhen tslO was tested. In thecases of tslOandtsC,theHpaII-4 fragmentgaveahigh

background. Although we have no complete

explanation for this observation, our results

using other RENFs demonstrate that the

mu-tantregionof thesetwomutantsis not located in the HpaH-4 region. The problem of high background wasprobably due to impuritiesof

fragment preparations. Since the Hpall

[image:4.503.260.453.57.193.2]

frag-mentsweregreater than90%pureasjudged by

TABLE 1. HpaII

(39 C/32C)or(LP/MP)x 100"

Fragment

tsa talO ts1260 tsC 208

1 1.0 33*b 17* 19* 6.9*

2 7.1* 2.5 6.7 12 2.5

3 2.2 6.7 3.3 2.3 0.5

4 <1.0 7.1 1.7 11.0 0.5 5 <0.5 0.6 0.3 2.0 <1.3 6 <0. 1 0.6 <0.3 2.0 <1.3 7 <0.1 0.5 <0.3 <0.9 <1.6 8 <0.1 <0.7 <0.3 <1.0 <2.0 No <0.1 <0.2 <0.3 <1.0 <0.8

aValuesare averages of at least twoplates for at least

threedilutions over a rangeoftwoordersofmagnitudefor

both 39 C and 32 C. LP, Largeplaque;MP, minuteplaque. bAsterisk indicates themostpositive response.

gelelectrophoresis in the presence of ethidium

bromide andthe method of heteroduplex

forma-tionused wasdesigned to minimize the effect of nonspecific fragment enhancement of marker rescue, weconsidered that the effects of small contaminations in the fragment preparation were accentuated by the high ratios of frag-ments to mutant FII DNA used to form the heteroduplexes. In further experiments (Tables 2-5), we used molar ratios of mutant FII DNA to mutant RENFs to wild-type RENF of 1:4:40 informingheteroduplex DNAs rather than ra-tiosof1:10:250usedintheHpaIIexperiments of Table 1. This change generally improved the differences between the 39 C/32 C ratios of the various fragments (Tables 2-5). However, in

somecases westill observedhighbackgrounds,

andwesuspectthat thiswasduetothe nonspe-cific fragment enhancement effectreported by Lai and Nathans (16), even though we at-tempted to minimize this effect in our experi-mental design.

Marker rescue with HindIII fragments. The twofragmentsproducedbycleavage of polyoma DNA with HindIII (11) were tested for their

abilitytorescue avariety ofpolyomamutants. Every mutanttestedwaspreferentially rescued

by the smaller of the two fragments, which

contained approximately 44% of the polyoma

genome (Table2). These resultsareconsistent with the HpaII results (Table 1) since the HindIII-44% fragment contained large

seg-ments of both the HpaII-1 and HpaH-2

frag-ments, and demonstrate a clustering ofearly and late mutations into a restricted region

(44%) of the polyoma genome. These results have been further confirmed by testing

frag-mentsproduced by cleavagewith HhaI

restric-tionendonuclease (seebelow).

Marker rescue with HhaI + Eco RI frag-ments. Digestion of polyoma wild-type DNA with acombinationof HhaI and Eco RI

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TABLE 2. HindlII (A)

Avg PFU x 10-4/ml

Mutant Fragment(%) (39C/32 C) x 100 44%/56%a

39C 32C

Early

tsa 44 4.0 45

8.9*>

56 <0.05 25 <0.2

ts25 44 3.2 20 16.0* 4.2

56 0.80 21.5 3.8

ts616 44 3.0 24 12.5* 4.8

56 0.6 23 2.6

ts48 44 0.25 6.5 3.8*

56 0.005 10.5 0.05 76.0

ts52 44 1.0 17.5 5.7* 3

56 0.025 15.5 0.16 5.6

ts609 44 1.20 12.5 9.6*

56 0.005 4.0 0.1 96.0

ts697 44 0.50 60.0 0.83*

56 0.005 60.0 <0.008 >100

Late

tslO 44 11.5 70 16.4* 5.1

56 0.8 25 3.2

ts1260 44 2.2 25 8.8* 7.3

56 0.3 25 1.2

tsC 44 4.5 30 15.0*

4.8

56 0.2 6.5 3.1

Controls

Wild-type 44%only <0.005 <0.005

Wild-type56%only <0.005 <0.005

Mutant DNA, no wild- <0.005 <0.005 typeHindIII RENF

(B)

Fragment LPr MP" (LP/MP) x 100 44%/56% Plaque morphology

208 44 6.0 40 15.0* 10.7

56 0.7 50 1.4

aThe wide variation in the 44%/56% ratios reflects, to some extent, variations in the purity of the

differentfragment preparations used to test each mutant. However, differencesinvirus strainsandinthe type orlocation of the mutation may also affect these ratios.

bAsteriskindicates most positive response.

cLP, Largeplaque.

d

MP,

Minuteplaque.

tionendonucleasesproducesfourfragments ap-proximately 46, 28, 14, and 12% (Fig. 2 and reference 10). These fragments are designated HhaI/EcoRI-1,-2,-3, and-4 inorder of

decreas-ing size.

The results of marker rescue using these HhaI+Eco RIfragmentsareshowninTable3. Of theearlymutants,tsa wasrescued by frag-ment 3, whereas ts25 and ts616 were rescued almostequallywellby fragments3and4.Since itisrelatively difficult to purify these two small fragments, another approach was used to

dem-onstrate that the sequences in fragment 4

rather than sequencesinfragment3correspond

to the mutant regions ofts25 and ts616 (see

below).

ThelatemutantstslO, ts1260,and tsC as well

astheplaquemorphologymutant208 were res-cued by fragment 1 preferentially. Combining the resultsthat these latemutants can be res-cued with the HindlII-44% fragment, the

HpaII-1 fragment, and theHhaIIEcoRI-1

frag-ment,the location of themutant regionsof the

genome canbe placed between approximately

26.6and45.4 map unitsfrom theEco RI site.

Marker rescue with HhaI CR fragments. For furtheranalysis of the earlymutants, we measured the markerrescueability of two

frag-mentsproduced byHhaI cleavage of a polyoma

variant,CR. This variantlacks theHhaI

cleav-age site at 26.4 map unitsfrom theEcoRIsite

(M. Griffiths and C. Barry, unpublished 828

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[image:5.503.57.448.76.441.2]

POLYOMA GENETIC MAP

Shalha

el-~

_ - 11h

HhO _

O0t

Hrin l

25 50

2 6 1 3

t

t

Hh al

tu±o11

ECO Rl 75

151

4 181712

0T

EARLY RNA EARLY RNA

LATE RNA

tsa ts609

ts 25 ts48 ts 52 ts616 ts 697

- ts10

ts1260

tsc

[image:6.503.40.452.62.531.2]

208

FIG. 2. PhysicalandgeneticmapofthepolyomaDNAgenome.At thetopisacircularphysicalmapofthe

polyomaDNAgenome(10, 11) showing thecleavagesitesofcertain restriction endonucleasesfrom

Esche-richiacoli (Eco RI), Haemophilus influenzaeD(HinIII), and H. haemolyticus(Hhal). The position of the

originofDNAreplicationisdenotedby (OR). Within the concentric circlesarethepositions ofthecleavage

sites ofa restriction endonuclease fromH.parainfluenzae (HpaII). Thearrangement ofthe eight HpaII

fragments, numbered in orderof decreasing size, is shown. Themapunits,with the Eco RI siteat00,are

marked in thecenterofthecircle;the 100mapunitscorrespondtoapproximately 5,200nucleotides. Below the

circularmapisalinearrepresentationofthesamephysicalmap.Theregionscorrespondingtoearlyand late

mRNAtranscriptsaredesignated by lines,thearrowsdesignatingthe directionof transcription (15). Finally

wehave denoted thepositions ofthemutantsitesoftsa,ts609, ts25, ts48, ts52, ts616,ts697,tslO,ts1260, tsC,

and 208 withrespecttothephysicalmap.

lr,

Ig

RI

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TABLE 3. HhaI +EcoRI (39C/32C) x 100

Mutant

1 2 3 4

Early

tsa 0.2 1.0 7.0*a 1.0

ts25 0.5 1.7 4.6* 6.7*

ts616 0.12 1.5 7.8* 5.0*

Late

tslO 8.7* 2.4 1.4 1.1

ts1260 3.5* 0.3 0.1 <0. 1

tsC 8.5* 3.7 1.4 1.0

Plaque

mor-phology

208 57*W 20 <7 <7

Asteriskindicates themostpositive response. Dataforplaquemorphologymutant208 areexpressed

as(large plaque/small plaque)x 100.

data). Thus, HhaI cleavage of CR generates two fragments, one approximately 58% of the genome (designated HhaI CR-58%) and the other42%ofthe genome (designatedHhaI CR-42%). The results of marker rescue with the

HhaI CRfragmentsareshowninTable4.Some

oftheearly mutantswererescuedby HhaI CR-58% (e.g., ts25, ts48, ts52, ts697, and ts616), whereas others were rescuedby HhaI CR-52% (e.g, tsa and ts609). It is therefore possible to distinguish different mapping regions for early mutants by using these fragments.

Marker rescue byHhaI fragments. To fur-therlocate those earlymutants that mappedin the HhaICR-58% fragment, the marker rescue

abilityofHhaIfragments derived from theA-2

strainofpolyoma wastested. Three HhaI

frag-ments are produced from this strain: (i) a 46%

fragment, HhaI-46%; (ii) a 42% fragment, HhaI-42%; and (iii)a 12% fragment, HhaI-12% (10). To simplify the testing, we combined the

46 and 42% fragments and tested them

collec-tively, since our purpose was primarily to distinguish which region of the HhaI CR-58% region was responsible for marker rescue of certain earlymutants (see Table 4).

Table5presents the results of marker rescue ofearly mutants by either thecombined

HhaI-46% andHhaI-42% fragmentsortheHhaI-12%

fragment. Those early mutants thatpreviously wererescued by HhaI CR-58% (ts48, ts52, ts697, ts616, andts25; see Table 4) were rescued by the HhaI-12%fragmentofA-2. The early mutants thatpreviously were rescued by HhaI CR-46% (tsa andts609;seeTable4)were rescued by the combinedHhaI-46% andHhaI-42% fragments as expected.

Genotypic analysis of the viral DNA pro-duced by marker rescue. The marker rescue techniquepresumably involves only the

conver-sionof the ts region ofthe mutantgenome to

thewild-typeformandshould not affect regions

ofthemutantgenomeoutside theregionofthe

wild-type

fragment

used to rescue the mutant DNA. This hypothesis is testable since tsa DNAismissinganHaeHIcleavage site, which is present in the A-2

wild-type

DNA. The tsa DNA lacks the HaeIII cleavage site between

HaellI-6 andHaeIII-20 that is located at 28.0 map units (in HpaII-1), resulting in a slower

migration of tsaHaeIII-6 and the absence of

HaellI-20whencomparedwith theHaellI

frag-mentsofA-2 (Millerand Griffin,

unpublished

data).

Virusstocks

produced

from theexperimentof

wild-type HpaII-2 fragmentrescueoftsaDNA

(seeTable 1)wereplaque assayedat39 C, and nine plaques were picked. The plaques were

grownat39C toproducevirusstocks,and32P_ labeled viral DNA was prepared from infec-tions with each virus stock. The 32P-labeled

DNAsweredigestedwith theHaeIII and

elec-trophoresed through 7% polyacrylamide

gels.

Nineoutofnineplaques pickedwereshownto have the same HaeIII cutting pattern as the

originaltsaDNA, and yet eachplaque wasno

longer temperature sensitive (results not

shown). This resultdemonstrates that marker

rescue involves only a limited region of the genomeand thatthose virusesplaquingat39C

(see Table 1) were most likely derived from modification of the tsa genome within the HpaH-2 region only.

DISCUSSION

A genetic map ofthe polyoma viral genome hasbeen constructed by using a markerrescue

TABLE 4. HhaICR fragments

(39C/32 C) x 100 Folddifference

Mutant (58%/42%or

58% 42% 42%/58%)

tsa 0.7 3.1* 4.4

ts25 13.8*a 1.1 12.5

ts616 5.5* 1.3 4.2

ts48 36.0* 13.0 3.0

ts52 3.1* 0.67 4.6

ts609 0.1 8.5* 85.0

ts697 1.0* 0.27 3.7

"Asteriskindicates themostpositiveresponse.

TABLE 5. HhaIfragments

(39C/32C) x 100 Folddifference

Mutant

~~~~~~[(46%

+42%)!

46% +42% 12% (46% +42%)/

ts48 <0.014 6.15*a 439

ts52 0.75 10.6* 14.1

ts609 23.6* 2.5 9.4

ts697 <0.03 2.8* 93

"Asteriskindicates themostpositiveresponse.

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POLYOMA GENETIC MAP 831

technique. The map is diagrammatically pre-sented in Fig. 2. Since high backgrounds were noted in certain sets ofRENFs, we tested the ability of at least three different sets of RENFs to rescue each mutant. The results of each of thesesets were consistent, and the results are

summarizedinFig. 2.

Of the seven early mutants tested, two (tsa

and ts609)arerescued by fragmentscontaining the DNA regionbetween the HindIII site at 1.0 mapunitsand the HhaI site at 14.0 map units. The other five early mutants (ts25, ts616, ts48, ts52, and ts697) arerescuedbyfragments con-taining theDNA region between the HhaI site at 14.0mapunitsand the HhaI site at 26.4 map units. Thus theearly mutants show a cluster-ing effect in the DNA region between 1.0and 26.4 map units,whichcorresponds to the 3'-OH terminal half of the "early" RNA (15). These mutants are of interest since most ofthem have beendemonstrated to bedefective in transfor-mation attherestrictive temperature (4, 8).

Of the late mutants, tslO and ts1260 have

beenclassifiedintodifferentgroupsbygenetic

complementationtest (2-4). Thethird late mu-tant, tsC (Fried and Griffith, manuscript in preparation), efficiently complements early mutantsbutappears toshowpoor

complemen-tation with mutants of the other two late groups (W. Eckhart, personalcommunication)

andisthereforereminiscent of theBCgroupof

SV40. Our results indicate that ts10, ts1260, and tsC all map between 26.6 and 45.4 map units. This is the region specifiedby the 16S late stable mRNA that has been shown to

encode the majorcapsid proteinVP1 (26). Thus itwould appearthatthe complementation

ob-servedbetween tslO and ts1260 represents

in-tragenic complementation and not intergenic complementation. This finding is similar to

that observed for SV40, where the mutants from the twolate complementing groups (Band C)and thethirdlate group(BC,which

comple-ments early mutants but not other late

mu-tants)all mapinaregion of the SV40genome

that apparently encodesthe SV40 majorcapsid protein VP1 (17, 19, 22).

The plaque morphology characteristics of208 aredetermined by thesame regionof DNAin

which thelatetsmutantsts10,ts1260, andtsC map (26.6 to 45.4 map units). Hence the

mi-nute-plaque morphology of 208 is determined

bythe majorcapsid proteinVP1rather thanan

earlygeneoraltered sequenceatthe origin of DNA replication. Our results with HindIII

fragments have been confirmed by an

inde-pendent mappingtechnique (20) that involves

the in vitro recombination offragments from

large-plaqueA2andminute-plaque208parents

(Miller and Fried, unpublished experiments). Intheseexperiments, the hybrid DNA contain-ing the 56% HindHI fragment from large-plaque viral DNA and the 44%HindIII frag-ment from 208 DNA produced minute-plaque virus, whereas the hybrid DNA containing the 56% HindIII fragment from 208 and the 44%

HinduI

fragment from large-plaque virus pro-duced large-plaque virus upon infection of mousecells with the hybrid DNAs.

The genetic organization of polyoma virus appears to be verysimilar to the genetic organi-zationof SV40determinedbyLai and Nathans (17). Mutants of both viruses show a distinct clustering effectinthesense that no mutations have been mapped inapproximately 55% of the genome, which comprises roughly half of the early region and half of the late region

sur-roundingtheorigin of DNAreplication. In the caseofpolyoma, we have yet to test two types of mutants, the host range mutants isolated by Benjamin (1) and the ts-3 mutant described by Eckhart and Dulbecco (5). This latter mutant

has similar properties to the class D mutants that Lai and Nathans have mapped in SV40

(17),

which laytowards the 5' terminus of the late RNA.

Themarker rescue technique is a directand

relatively easy method for constructing a rough genetic map ofsmall, DNA-containing animal

viruses.Although more precisedeterminations

of the genetic map positions can be made by using smaller restriction endonuclease

frag-ments, such fragments are more difficult to

purify and would be time consuming to test. Moreprecise measurements of map locations of certain mutantsof SV40have beenprovidedby the

S,

nuclease technique ofShenketal. (25). The

S,

technique, however, can suffer from two majordisadvantages: (i) itis purely biochemi-cal and cannot relate base changes to pheno-typic differences; and (ii) the mapping proce-dure apparently cannot distinguish all

single-basemismatchesand is therefore the method of choice for mappingdeletionmutationsonly.

We have studied certain aspects of the marker rescuetechnique ingreaterdetail. We

havedemonstrated that the region ofthe mu-tantgenome coveredbyawild-typerestriction endonuclease fragment is converted to wild-type, whereas sequencesinanother regionnot coveredby thewild-type fragment remain

un-changed. The cellular events involved in the marker rescue technique are presently being

studied.

Ourresults demonstratenotonlythe useful-ness of marker rescue for thegenetic mapping oftsandplaque morphology mutants of

poly-oma but alsothe usefulness ofthis method in VOL. 18, 1976

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transferring genetic markers from one virus strain toanother.

ACKNOWLEDGMENTS

We wish tothank Beverley E. Cooke for her excellent technical assistance. We also thankBeverly E.Griffinfor helpand information on thephysical order of restriction endonuclease fragments during the course ofthiswork.

L.K.M. wassupported by Public Health Service Postdoc-toral Fellowship no. 1F22 CA 01198-01 from theNational Cancer Institute.

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