Construction of a genetic map of human chromosome 17 by use of

Genetics

Construction of

a

genetic

map

of

human chromosome

17

by

use

of

chromosome-mediated gene

transfer

(in situhybridization/acutepromyelocyticleukemia/somatic-celi hybrid/thymidinekinasegene/genemapping)

WEIMING

XU*, PATRICIA A. GORMANt, SUSAN H.

RIDERt,

PHILIP

J.

HEDGE*, GRAHAM MOORE*t,

CATRIN

PRICHARD§¶, DENISE SHEERt,

AND

ELLEN

SOLOMON*

II

*Somatic Cell Genetics Laboratory,tHumanCytogeneticsLaboratory, and§HumanMolecular Genetics Laboratory, Imperial CancerResearchFund, London

WC2A3PX,United Kingdom

Communicated by Walter F.Bodmer, July 28, 1988(received forreviewMay13, 1988) ABSTRACT We used somatic-cell hybrids, containing as

their only human genetic contribution part or all of chromo-some 17, as donors for chromochromo-some-mediated gene transfer.A total of 54 independent transfectant clones were isolated and analyzed by use of probes or isoenzymes for >20 loci located on chromosome 17.By combining the data fromthis chromosome-mediated gene transfer transfectant panel, conventional so-matic-cell hybrids containing well-defined breaks on chromo-some 17, and in situ hybridization, wepropose the following order for these loci:

pter-(TP53-RNP2-DJ7S1)-(MYH2-MYHl)-D17Z]-CRYB1-(ERBAI-GCSF-NGL)-acute

promye-locytic leukemia

breakpoint-RNU2-HOX2-(NGFR-COLIAI-MPO)-GAA-UMPH-GHC-TKI-GALK-qter.

Using chromo-some-mediated genetransfer,wehave alsoregionally localized the randomprobes D17S6toD17S19 on chromosome 17. Structural abnormalities of chromosome 17 are associated withimportant clinical disorders.Forexample,theleukemic cells of nearly all individuals with acute

promyelocytic

leukemia(APL, FABclassificationM3) arecharacterizedby areciprocal translocation involving the long arms of chro-mosomes 15 and 17 t(15;17)(q22;qll.2-12)(1). Deletions of chromosome17havebeen associated withmentalretardation syndromessuchasMiller-Dieker syndrome [del(17)pl3](2) andSmith-Magenis syndrome

[del(17)pll]

(3,4). Inaddition, several genescausingcommongenetic diseaseswereshown to be located on chromosome 17. For example, the domi-nantly inherited disordervonRecklinghausen neurofibroma-tosiswaslinked to markers in thepericentromericregion of chromosome 17(5). Lossof heterozygosity of chromosome 17p markerswasalso recently reported in the progressionof colorectal carcinomas (6).

Construction of genetic maps is central to the study of humangenetics. Physicalmaps canbe used as a basisforthe generation of evenlyspaced restrictionfragment-length poly-morphismsalong chromosomes. Theserestriction fragment-length polymorphismsmay thenallowthedemonstration of positive linkage between human genetic diseases and the polymorphic DNA markers. Here we report the use of chromosome-mediatedgenetransfer(CMGT)in the genera-tion of a physical map of chromosome 17. CMGT is an established method ofconstructing hybrid cells containing subchromosomalfragmentsofaselected donor chromosome (for reviews, seerefs. 7and8). Thegeneration of transfec-tants byCMGTfor chromosome 17 has beenhelped bythe presence of the selectable marker thymidine kinase (TK) located on the longarm. Thistechnique has enabled us to generate apanel ofmore than 50 transfectants containing different transgenomic fragments of chromosome 17. We

used this panel of transfectants to localize most of the published gene markers and sequences onchromosome 17. Byanalyzing cotransfection frequencies,we werealsoable to define groups of locithat areclosely locatedonchromosome 17.Then, by combining CMGT datawith results fromapanel ofhybrids with well-defined breaks on chromosome 17 and fromin situhybridization,weconstructedadetailed mapof thelongarmofchromosome17andalessdetailed mapof the shortarm. Wefound CMGTtobeparticularly useful in the analysis of groups of closely linkedmarkers, suchasGALK, TKI, GHC, UMPH, and GAA, which could not easily be resolved by other means. Our results also confirm that (i) although substantial lengths of DNA may be transferred intact (8, 9), interstitial deletions do frequently occur; (ii) multiple fragments of transfected chromatin canbefoundin the same clone; and (iii) there is also selection for centro-meric sequences(8, 9).

In addition, during the course ofthis study we obtained sometransfectantsthatcontained only the small regions of chromosome17particularly relevanttothestudyofAPLand vonRecklinghausen neurofibromatosis.

MATERIALS AND METHODS

Culture Conditions. The cellswerecultured either inRPMI 1640 or in Dulbecco's minimal essential medium supple-mentedwith10% fetalbovineserum.PCTBA1.8,

PJT2/A1,

and all CMGT transfectants,

PLT,

KLT, and TLT were maintained in HAT medium (100

,uM

hypoxanthine/10

,M

methotrexate/10

,Mthymidine), which selects forthymidine kinase. ThehybridsGPT17.3.2K41 and TRID62 were main-tained inMX medium [mycophenolic acid (25

,ug/ml)/xan-thine (250

,g/ml)].

Back selection ofCMGT transfectants with5-bromo-2-deoxyuridine wasdoneby theaddition of 5-bromo-2-deoxyuridine (50

,g/ml)

to the medium, thus se-lecting for those CMGTtransfectants that have lost the TKI locus. This medium was replenished every 3 days for 2-3 weeks. Resulting colonies were maintained in RPMI 1640/ 10%fetal bovineserumand weredenotedby the letter B after thenameoftheoriginal transfectant.

Cytogenetic Analysis. Chromosomesfrom human lympho-cytes,hybrids,andCMGT transfectantswereallpreparedas described(10).Additional methodsaredescribed in thefigure andtable legends.

SouthernBlots and Hybridization. Twentymicrogramsof restricted DNA was separated on 0.7% agarose gels by

Abbreviations:CMGT,chromosome-mediated genetransfer; APL,

acutepromyelocyticleukemia.

tPresentaddress: Instituteof Plant ScienceResearch,Cambridge, UnitedKingdom.

VPresentaddress: DepartmentofPhysiology, Universityof Califor-nia MedicalCenter,SanFrancisco,CA94143.

I1To

whomreprintrequestsshould be addressed.

Thepublicationcosts of this article weredefrayedinpart by page charge payment. This article must therefore be hereby marked "advertisement"

hybridizedwithprobes forthelociindicated inTable 1. The

presenceorabsence ofthehumanspecific bandsdetected by

theseprobescorrespondstothepresenceorabsenceof these

regions ofchromosome 17in the transfectants. This infor-mation is summarized together with the results of the isoenzyme analysis in Fig. 1. The frequencies with which markers are cotransferred with the selected gene TKI are

indicated in Fig. 2.

Lotalization of Probes by in Situ Hybridiiation. TK1.This locuswasmapped with chromosomesfromboth peripheral

bloodlymphocytes and the cell lineGM0271,which hasthe translocation t(17;19)(q23;p13.3) (36). Chromosomes from 100metaphasespreadsofperipheralbloodlymphocyteswere

analyzed. Atotal of 218 silvergrainswere scored, of which

28 werefound on chromosome 17, and of these, 18 (64%)

obtained by analysis of chromosome 17 in 40 metaphase spreads; ofthe 50grainspresent, 28(56%)werelocatedon

bands 17q21-+q22 (Fig. 3C).

CRYB1. Chromosomes from 70 metaphase spreads of peripheral blood lymphocytes were analyzed; 149 grains

werescored,of which 17werefoundonchromosome 17,and

12(71%)of these werelocatedonbands 17q11.1-*q12(Fig.

3D).

MappingofLocionChromosome17.Inconstructingamap

ofhumanchromosome 17we assumed the following: (i)the

proximity ofmarkers to the selected locus TKI would be indicated by the frequencies with which theywerefoundin

thetransfectants,and (ii)thosemarkerswithsimilar

cotrans-fectionfrequencies with TKI and thataregenerally foundin

thesametransfectants would likely be located close together

Table 1. Loci usedtocharacterize the CMGTpanel of transfectants

Previous*

Gene Name Probe localization Reference

TPS3 Tumorproteinp53 p53-102 17p13 12

RNP2 LargesubunitRNApolymerase pHRpll5.5 17p12-+p13 13

D17S6 DNAsinglecopy cosH17.1 17pter-.p13.3 14

D17S7 DNAsinglecopy cosH17.2 17cen--pter 14

D17SI DNAsinglecopy p12-2 17pter-+p13 15

MYH2 Myosin heavypolypeptide, skeletalmuscle,2adult2 p10-3 17pter-+pll 16 MYHI Myosin heavypolypeptide, skeletalmuscle,1adult 1 p2-3 17pter-*pll 16

DJ7ZI DNAmultiplecopy p17H8 17cen 17

CRYBI 13crystallin pCU14A1 17q21 18

ERBAI Erythroblasticleukemiaviral oncogenehomology pHE-Al 17qll-.ql2.21 19

GCSF Granulocytecolony-stimulatingfactor pBRG-4 17qll-+ql2.21 20

NGLt Neuro/glioblastoma Amprobet 17q21-+q22 2

oncogenehomology erbB2

RNU2 RNA, U2 small nuclear pML2d 17q21-.q22 21

D17S17 DNAsinglecopy cosH17.11 17q22--qter 14

D17S18 DNAsinglecopy cosH17.12 17q22--qter 14

D17S19 DNAsinglecopy cosH17.13 17q22--qter 14

HOX2 Homeoboxregion2 pHU-1 17qll-.q22 22

NGFR Nervegrowth factorreceptor p104 17ql2-+q22 23

COLIAI Collagenal(I) pCG103 17q21.31-+q22.5 24

MPO Myeloperoxidase pMP23 17q11.12-*q24 25

GAA Acida-glucosidase Enzyme 17q23 26

UMPH Uridine5'-monophosphate phosphohydrolase Enzyme 17q 26

GHC Growth hormonecomplex-homologousgenes: pBGHXH01 17q22-*q24 27

GHl-CSPHl-CSHl-GH2-CSHP2

TKI Thymidinekinase pHTK2 17q21-+q22 28

D17S9 DNA singlecopy GERBA1 17qll 14

D17SIO DNAsinglecopy cosH17.5 17qll-+q22 14

D17SII DNAsinglecopy cosH17.6 17qll- q22 14

D17S12 DNAsinglecopy cosH17.7 17q11- q22 14

D17S13 DNAsinglecopy cosH17.8 17qll-.q22 14

D17S14 DNAsinglecopy cosH17.9 i7qll-+q22 14

D17S16 DNAsingle copy cosH17.10 17qll- q22 14

GALK Galactokinase Enzyme 17q21-.q22 26

Isoenzymeanalyses wereall done with starchgelelectrophoresis accordingtoHarris and Hopkinson(26). *Priorlocalizations of the above lociaretakenfromrefs. 31 and 32.

tNGLisnowofficiallyknownasERBB2. tAmersham International.

E

_tv

Ngtv

tTsted

t

MPositive

E]Negative

J3Not.Tested

UnstabLe

FIG. 1. Schematic representationofthe presence orabsence of chromosome 17 markers in the panel of transfectants and somatic-cell hybrids. Markersaredisplayed intheirapproximate order and position on chromosome 17 as determined by this study.DCI,donor cellline:

P,PCTBA1.8;K,GPT17.3.2K41;andT, TRID62.q+,15q+hybrid. TheCMGT protocol has been described in detail (33). Threehuman/mouse interspecificsomatic-cell hybrids were used as chromosome donors. PCTBA1.8 is a mouse3T3TK-/humansomatic-cell hybrid (34) containing achromosome17 as itsonlyhumancomponent.GPT17.3.2K41 isa mouseteratocarcinoma/humansomatic-cell hybrid (35) alsocontaining chromosome 17 as itsonlyhuman component. TRID62isamouse/human somatic-cellhybrid containing as its only humancomponent thelong armofchromosome 17 (17qll.2--qter)(35). AllresultantCMGT transfectantsderived fromPCTBA1.8,GPT17.3.2K41,and TRID62 carry the prefix PLT-,KLT-, and TLT-,respectively.The mouse cell line LMTK- was used as the thymidine kinase-deficient recipient cell line for all theCMGT experiments.ThePJT2/A1 hybrid,sometimesreferredto as the15q+ hybrid, contains the APLtranslocation chromosome with the regionof 17belowthebreakpoint(17qll.2-.qter)(10).

onthe physical map. Bycombining the data from CMGT, somatic-cell hybrids,and in situhybridization, we were able toidentify sixmajorregions of chromosome17covering the longarmthrough thecentromere tothe short arm.

Region 1. Thisregionincludes theselectable marker TKI andfourother genes (GALK, GHC, UMPH,and GAA) that havecotransfection frequencies withTKIof>50%.GHC has the highest cotransfection frequency with TKI and can therefore be assumed to map closest to the TKI locus. Because thetransfectantPLT7only has

TKJ

and GALK and transfectantPLT4only has TKI and GHC,it is probable that GHC and GALK arelocatedon eithersideofTKI. UMPH cotransfects with the GHC genes with a high frequency, indicating that it liesadjacent to GHC.

8

0-o

Tac40 SIS< SMDtEm- Q(

C.~~~~~~~~~~~~~~M C

Gene

FIG. 2. Overall genefrequenciesofchromosome 17 markers in thePLT and KLT transfectant series.Frequenciesareexpressedas apercentageof the total numberof transfectants thatacquiredthe humanthymidinekinase gene. TLT transfectantsarenotincluded,

as the donor chromosome for this series was not a complete

chromosome 17.

In situ hybridization has localized the thymidine kinase genetothe bands17q24-*q25. This positionismore telome-ric than that previously reported (17q21--q22) (40). It

is,

however,consistent with themorerecentfindingsof

others,

where TKI was mapped to17q23-*qter with a hybrid derived from GM0271, by showing that thymidine kinase

activity

remainedonlyin thepresence of the derivative 19 chromo-some(41). The GHC locus has been assigned tochromosome 17 and localized by in situ hybridization to chromosome bands 17q22--q24 (42).Herewereport confirmation of this assignment and have further localized it to17q23->q24.This regional localization of GHC is consistent with the GHC locus being closer to the centromere than TKI. Region 1 probably spans 17q23-}q25 withthe geneorder being

cen-tromere (cen)-GAA-UMPH-GHC-TKI-GALK-qter. We have also used this CMGT transfectant panel with several other probesisolated fromahuman chromosome-17-only library. D1759, D17SJO, D17SII, D17S12, D17S13, D17S14,andDl7S16(14)weredetected on the transfectant PLT8 and have therefore been assigned to region 1, 17q23-*qter.D17S9 andDJ7510havecotransfer frequencies with TKI of>90%, indicatingvery close proximity to TKI. It should be noted that although D17S9 has also been localized by in situ hybridization to 17q23-,qter, it also cross-hybridizes to sequences just below the APL break-point.

Region 2. The loci HOX2, NGFR, COLIAJ, and MPO compose the second group, as they are commonly found together in thesametransfectants. TheMPO probe hasonly been used with a more limited CMGT transfectant panel. However, MPOisalways presentwith COLIAJ, indicating the closeproximity of these loci; for example, theyareboth presentin the transfectantsPLT6, TLT2, TLT8, TLT10, and TLT20. COLIAJ is probably closertoTKIthan eitherNGFR or HOX2, as it has ahighercotransfection frequency with TKI. HOX2 is likely to be located more proximal to the centromere than the other loci in region 2, as it is not cotransferred with them inthe transfectants PLT6,PLT15, KLT13, and TLT8. However, HOX2 is found in KLT3, TLT10, and TLT20,suggestingthat itislocatedinthisregion. Thishypothesisis substantiatedbythelossof HOX2together with MPO, COLJAJ,andNGFR in thetransfectantPLT20, which is deleted for this region. The close proximity of

17 17q 19 19p

FIG. 3. Insituhybridizationtolocalize the loci TKI, GHC, HOX2,andCR YB). Thedistribution of grainsonchromosome 17 isrepresented

schematicallyfor thefollowingloci: TK) (A), GHC (B), HOX2 (C), andCRYB) (D). (E) Amoreprecise localization of TKIwasdoneusing

thehumanfibroblast cell line GM0271, which is characterized byareciprocal translocationt(17;19)(q23;pl3.3) (37). Beforein situhybridization, cellsweretreated with5-bromo-2-deoxyuridine (37).Insituhybridizationsweredonewiththe methodof Harper and Saunders (38). The probes for the TKI,GHC, HOX2,andCRYBI lociwereallradiolabeled using oligoprimers and[3H]deoxyribonucleotidestoaspecific activityof1x

107-1x 108cpm per

jzg

of DNA. In situhybridizationsweredoneovernightat37°C withafinalprobe concentration of 0.1-0.02,tg/ml.Slides werethenwashedat39°C, dehydrated, and dippedinIlfordK-5emulsion.All slidesweredeveloped after 1-3 weeks and then G-banded(39);

the slideswerealso stained withWright'sstain.

COLIAI, MPO, and NGFR is substantiatedby the loss of these threegenes, together with theregion-1 markers, in the

back-selected transfectant PLT6.B. The transfectant PLT6 contains two fragments of chromosome 17 integrated into separate regions of the mouse genome. Selection with

5-bromo-2-deoxyuridine results in the loss of the fragment containing thethymidinekinasegeneand,consequently,loss

of theother locionthisfragment.

Insituhybridization confirmsthelocalization ofHOX2to

17q21-*q22 (43), andCOLIA) has beenpreviously mapped to 17q21-*q22 (44). It therefore appears that this second

groupis located in theregion 17q21-+q22,mostprobablyin theordercen-HOX2-(NGFR-COLIAI-MPO)-qter.

Region 3. The genefor small nuclear RNA U2 (RNU2)

mapsbelow the APLbreakpoint. Although this genehas a

similar cotransfection frequency with TKI to the region-2 loci, RNU2 doesnotbelongtothesamelocalizationgroup,

asit is absent from cell lines KLT3 andTLT10and is retained

incell linePLT6.B, whichhas lostregions1 and 2.RNU2 has therefore beenplacedinaseparateregion,which liescloser tothecentromerethanregion 2. ThelociDI7S)7,DI7S18, andDI7SI9 (14)have been localizedto17q11.2-+q22,below the APLbreakpointand above theregioncoveredbycell line PLT8,butasyetthese loci havenotbeenfurtherlocalizedto eitherregion2or3.

Region 4. Using the hybrids TRID62 and PJT2/A1, we

havemappedthelociERBAI, GCSF,andNGL closetobut below thecentromereonthelongarmofchromosome 17 and above the APLbreakpoint 17q11.1- q12. The GCSFprobe hasonly beenused withamorelimitedCMGT transfectant

panel. However,GCSF isalwayspresentwithERBAI-for example, in the transfectants KLT8, KLT12, and TLT2. NGL isalso present in transfectants KLT8 and TLT2butnot KLT12. As KLT12 alsocontains thecentromericsequence

D17ZI, it is likely that ERBAI and GCSFarelocated closer

tothecentromerethanNGL,theorderbeingasfollows:

cen-(ERBAI-GCSF)-NGL-qter.

Region5. The centromericregionisrepresented by D17ZI and CRYBI. Interestingly, the human centromere has a

relatively high cotransfection frequency with TKI compared withadjacent loci, suggesting that there is selection for these

sequences.We found that all the transfectantsthat contained the locus D)7ZI also retained the CR YBI locus. In situ hybridizationhadpreviously mapped CRYBI to17q21 (18).

This positioning is inconsistent with our more proximal

localization of CR YBI at17qll.1-+ql2.

Region6.Many transfectants containing thecentromereof chromosome 17 alsocontainmarkers from the shortarm.The

probes from the shortarmarerather far from the TKI locus,

but we dosee these genes intransfectants such as PLT6,

PLT15,PLT20, KLT3, and KLT12. TP53, RNP2, and D17S5

are always associated with each other, as are MYHI and

MYH2 (except in transfectant PLT6). We therefore tenta-tivelydividethisregion into thesetwosubregions.

D17S6andD17S7 (14)arealso locatedonthe shortarmof chromosome 17, 17pter--cen.

DISCUSSION

We describeour results using CMGT methods togenerate transfectantscontainingdifferentfragments ofhuman

chro-mosome17.OriginallyCMGTwaslimitedtotheavailability ofendogenous dominantly actingselectable markerssuchas

TKIand HPRT,which enabled selection for chromosomes 17 andX, respectively.Thetechniquehasnowbeen extended

tootherregionsofthegenomebytheuseof other forms of selection, suchas selection of humantransforming genes-e.g., HRASI (45)-or by cotransfecting with a selectable marker-e.g., pSV2neo (33). In our study, by selectingfor the thymidine kinase gene we obtained a panel of >50

transfectantscoveringalarge region of chromosome 17. We

observed thatourtransfectantsfrequently havesome inter-stitialdeletions.Sometimes these deletionscoveronerather largeregion, butmanytransfectantsare deleted for several

different shortregions,clearly showingseriousdrawbacksto using CMGT alone forgene mapping. However, when we

combinedourCMGTdata withdata from bothachromosome

17 hybrid panelthat contained well-characterized constitu-tionalrearrangementsof human chromosome 17 andin situ hybridization, thedeletedregions could be detectedand the

genescould bemappedinfiner -detail thanwould have been possible usinganyone of these methods. Usingthese three

methods, we constructed the following order ofmarkerson

chromosome 17: pter-(TP53-RNP2-D17SI)-(MYH2-MYHI)-D)7Z)-CRYBI-(ERBAI-GCSF-NGL)-APL

break-point-RN U2-HOX2-(NGFR-COLIAl-MPO)-GAA-UMPH-GHC-TKI-GALK-qter.

Recently

some

family

studies have demonstrated the

genetic

distance between some of the markers usedin this

study.

These studiesshowthat DI7ZJandCOLIAIareabout 20

centimorgans

apart, GHCandDI7ZJ areabout 28 centi-morgans apart,and NGFR and HOX2are

closely

linked(46,

47)-results

thatare consistentwith ourdata.

Possibly

the most useful aspect of CMGT is that it generates

hybrid

cell lines thatareenriched for certainsmall

regions

of the human chromosome. Thisresultis of

particular

value when the genes of interest are located near the selectable marker. CMGTcanalso be used whenthe select-able marker is distantfrom the

region

ofinterest duetothe

high frequency

of interstitial

deletions,

which allows

frag-ments

normally

distant from the selectable marker to be transferredon

relatively

short

pieces

of the chromosome.For

example,

the transfectant KLT8 contains the loci from

regions

1and

4,

butnotthosefrom

regions

2and

3,

and could therefore be useful in the

study

of von

Recklinghausen

neurofibromatosis and APL. In

addition,

the transfectant

PLT6.B,

whichhas

only

:'13% of chromosome17

according

toourDNAdot blot

analysis

(W.X.,

unpublished

data)

and which has been deleted formostof the

long

arm,couldalso be of valueforthis purpose.

Whilethis

manuscript

wasin

preparation,

another group

published

data on a

regional mapping

panel

for human chromosome17

(40).

Their

approach

wasto construct

inter-specific

somatic-cell

hybrids

from human cells that had well-defined breaks on chromosome 17. Our

approach

was different inthat itwas

largely

based ondatafroma

panel

of CMGTtransfectants with some somatic-cell

hybrids.

There

arenoinconsistencies forthelocalization of locion

chromo-some 17inboth studies. Our data

give

amoredetailedmap of the

long

armofchromosome

17,

and their data

supply

a moredetailed map of theshortarmofthesamechromosome. Note Added in Proof. Morerecentdata make theorderingofERBAI, GCSF,and NGL ambiguous.

We thankthefollowing peopleforgenerously providingprobesfor theloci used in thisstudy:G. Matlasheski(TP53),N.Spurr (RNP2,

D17SJ),

H. Willard

(DJ7ZJ),

B.Vennstrom (CERBAI), N. Shige-kazu(GCSF), A.Weiner(RNU2),C. Hauser(HOX2), K.Heubner

(NGFR),G.Barsh(COLIAI),K.Chang(MPO),and Y. Kan(TKI). We thank Sue Povey and Annabel Kearney for the isoenzyme

analysis.

We are grateful to Peter Goodfellowfor his advice on

CMGT and his valuable discussions onthe manuscript. We also thank Mrs. P.Miller for herhelpin thelaboratory.

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