HLA-DR cDNA

Proc.Nati Acad.Sci. USA

Vol. 79, pp. 1703-1707, March 1982 Biochemistry

Isolation and identification of a cDNA clone corresponding to an HLA-DR antigen ( chain

(hybrid selection/histocompatibility antigens/membrane protein)

K.

WIMAN,

D. LARHAMMAR, L.

CLAESSON,

K. GUSTAFSSON, L. SCHENNING, P. BILL, J. BOHME, M. DENARO, B.

DOBBERSTEIN*,

U. HAMMERLING, S.

KVIST*,

B. SERVENIUS, J. SUNDELIN,

P.

A. PETERSON, AND L.

RASKt

Departmentof Cell Research,TheWallenberg Laboratory, Box 256, S-751 22 Uppsala, Sweden

Communicated by John Kendrew, November6, 1981

ABSTRACT The HLA-D locusinthemajorhistocompatibility complex controls the expression of the genetically polymorphic HLA-DR antigens. mRNAcoding for the A chains of these anti- gens was partiallypurified from the humanlymphoblastoidcell line Raji. The mRNA wascopied into double-strandedcDNAand cloned in Escherichiacoli. One clone,pDR-(3-1, obtained by hy- bridselection, carries a1070-base-pairinsertcomprising all of the coding region except the signal sequence and a substantial portion of the untranslatedregion. Toidentify

pDR-.3-1,

highly purified HLA-DRantigen(3chainsderived from Raji cellsweresubjected to NH2-terminal amino acid sequence determination. This se- quencedisplayedextensivehomology with that deduced from the nucleotide sequence atthe5'endofthepDR-P-Icodingregion.

Takentogether, theaminoacid and nucleotidesequencesstrongly argueinfavor of Raji cellscontainingatleasttwo(-chain loci.

HLA-DRhistocompatibility antigens arecell surfaceproteins present on different subclasses of lymphocytes and macro- phages (1). However,recentlyit wasdemonstrated that expres- sionofthese antigensisnot strictlylimited tocells of theim- mune system: theyalso occur on other types of cells suchas epithelial cells invarious organs (2). The molecular functions oftheHLA-DRantigens, and oftheirmurinecounterpartsthe Iaantigens, arelargelyunknown. However, their roleinseveral immunobiological phenomenaiswelldocumented.Expression ofIaantigens onthe antigen-presenting cellseemstobeapre- requisite forthe proper activation ofT-helpercells (3). Like- wise, the cooperationbetweenT-helpercells and B lympho- cytes iscontrolled bythe HLA-DR(Ia)antigens(4).

HLA-DR antigens are composed of two noncovalently linked, glycosylated, polypeptide chainswith apparent molec- ularweightsof 35,000and29,000,respectively (5).The smaller one, the13chain, displaysmostof the extensive geneticpoly- morphism(6, 7). Such polymorphism is apuzzling, common feature of molecules controlledbythe majorhistocompatibility complex. The other chain, the a chain, is much less poly- morphicthan the1 chain (6, 7).

The murine Ia antigens are controlledbytwodistinct subloci calledI-AandI-E/C (8). LimitedNH2-terminal amino acid se- quenceanalyseshave revealed that

I-Aa,, I-A., I-E/C,,,

and I-

E/Cp

chains display unique primary structures (8, 9). Corre- spondingstructural analysesofhighlypurified HLA-DRanti- gens have revealedonly theexistence ofE/C-like sequences (10). However, recentdata based onserological testsand two- dimensionalelectrophoretic analysessuggest thatHLA-DR an- tigens maybe controlled frommorethanonelocus(11, 12).

Structuralanalyses of HLA-DR antigens lag behindthose of HLA-A, -B, and -C antigens (13). In this communication we describe the isolation andpartialcharacterization ofan HLA- DRantigen1-chain cDNA clone. The insert in the clonecor- respondsto mostif not allof thetranslatedportionofa1 chain.

MATERIALS AND METHODS

Materials. S1 nuclease and terminaldeoxynucleotidyltrans- ferase were obtained from Bethesda Research Laboratories;

DNApolymeraseI wasfrom Boehringer Mannheim. Polynu- cleotide kinase and restriction enzymes were purchased from NewEnglandBioLabs.

(dT)1218

andoligo (dT)-cellulosewere from Collaborative Research(Waltham, MA). Rabbit reticulo- cytelysate, [3S]methionine, and EN3HANCEweretheprod- uctsof NewEngland Nuclear. [a-32P]Deoxynucleotides (>400 Ci/mmol; 1 Ci = 3.7 x 101° becquerels) and adenosine [y-32P]triphosphate (>2000 Ci/mmol)wereobtained from the Radiochemical Centre (Amersham, England). Nitrocellulose filters (BA 85)werepurchasedfrom Schleicher & Schuell.

Antiserum. An antiserumagainstHLA-DRantigen 13 chains wasraisedin arabbitbyimmunizationwithhighly purified

P

chains. HLA-DRantigenswere purifiedtohomogeneity and aand(3chainswereseparated fromeach otherbypreparative

NaDodSOJpolyacrylamide

gel electrophoresis. Theantiserum obtaineddidnot crossreactwithHLA-DRantigenachains.

Cultivation ofRajiCells.The humanlymphoblastoid cellline Raji(HLA-Dw 3and6) was maintained inroller-flask cultures containing RPMI-1640medium (GIBCO) and10%neonatal calf serum. RNA was isolated from cells in logarithmic growth phase.

Isolation of mRNA. Microsomal mRNA was isolated from Rajicellsaccordingto aprotocol described elsewhere (14). En- richment ofmRNAcodingfor HLA-DR antigen subunits was accomplished by centrifugation ofthe mRNA in a 10-30%

aqueous sucrosegradientfor 11 hr at 39,000 rpm in a Beckman SW 40 rotor at15'C. Distributionof HLA-DR antigen mRNA inthegradientwasdeterminedby cell-freetranslation(15).

Preparation of cDNA Clones. Sucrosedensitygradientfrac- tions enriched for HLA-DR antigen mRNA were transcribed into cDNAwith useof avian myeloblastosis virus reverse tran- scriptase(a generous gift of J. W. Beard, National Institutes of Health). The second strand was synthesized with DNA poly- merase I. The conditions for first- andsecond-strand synthesis describedbyWickens et al. wereused(16). After treatment of thecDNAwith S1nuclease asoutlined byHoeijmakersetal.

*Presentaddress:EuropeanMolecularBiology

Laboratory,

Meyers- hofsstrasse1, Postfach 10.2209,6900Heidelberg, FederalRepublic ofGermany.

tTowhom reprint requests should be addressed.

1703 Thepublicationcostsofthis article weredefrayedinpartbypagecharge payment. This article must therefore be hereby marked "advertise- ment"inaccordance with18 U. S. C.§1734solelytoindicate this fact.

1704 Biochemistry: Wiman etal.

(17), the material was sizefractionated~ona 5-23% sucrose gra- dient(17). Moleculeslongerthan600base pairs weretailedwith deoxycytidine

(18),

and'the tailedcDNA washybridizedto Pst I-digested pBR322 (19) elongated with deoxyguanosine (18).

Theresultinghybrid molecules wereusedtotransform Esch- erichia colistrain294 (20, 21).-Preparation of competentcells was performed accordingto Dagertand Erlich(22).

Purification ofPlasmidDNA.Tetracycline-resistant colonies were grown in Lbroth and amplified by the additionof chlor- amphenicol (200

Ag/ml)

at anopticaldensity of0.6 at 600 nm.

Cleared lysateswerepreparedasdescribed (23). Plasmidswere purified by centrifugationin a

CsCVethidium

bromidedensity gradient.For restrictionmappingandsequencedetermination, plasmidDNAwasfurtherpurified by centrifugationina5-40%

sucrose gradient (24) or by chromatography on a column of Sepharose4B.

mRNASelectionby

Hybridization

onNitrocelluloseFilters.

Plasmid DNA was sonicated into pieces rangingfrom0.3to 1 kilobase. TheDNAwasdenaturedin 0.1 M NaOHfor10min on ice. Afterneutralization with NaH2PO4, the DNA(60

ttg)

was immobilized onto 25-mm-diameter nitrocellulose filters.

Four microgramsofmRNA washybridizedtoeach filter. The conditions for thehybridizationhave been described(25). For rapid screening; plasmid DNAfrom eight different recombi- nantclones wasimmobilizedonthesamefilter. Whenapositive signalwasobtained, the clones wererescreenedindividually.

Cell-FreeTranslation. The elutedmRNA wastranslated'in arabbitreticulocyte lysatesysteminthepresenceof

dog

pan- creasmicrosomes(15). Analiquot (2

Al)

ofthe translated

prod-

ucts wascharacterizeddirectly by

NaDodSOjpolyacrylamide

gel electrophoresis (15). The remainder(23 Al)was

subjected

toimmunoprecipitation usingan antiserum

specific

forHLA- DRantigen

A

chains. In somecases, proteinstranslatedinthe presenceofmicrosomes were

subjected

toproteinaseKtreat- ment todigestthecytoplasmtailof

membrane-integrated

pro- teins(15). Precipitatedproteinswere

analyzed by

NaDodSOg

polyacrylamide gel electrophoresis

and

fluorography.

Thede- tailedproceduresaredescribedinref. 15.

RestrictionMappingandDNASequence

Analysis.

There-

strictionmapwasconstructedasdescribed

(26).

ForDNA se- quence determination, the DNA was cleavedwith asuitable restrictionendonuclease andlabeledwith

[y-32P]ATP

atthe5' ends (27). DNA

fragments

werethen cleavedasecondtime to

produce fragments with

only

one labeled end. Purification of DNAfragments forsequencedeterminationwas

accomplished

bypolyacrylamide gel

electrophoresis

and electroelution into

dialysis bags. Sequencedeterminationwas

performed

accord- ing toMaxam and Gilbert(27)andMaatandSmith

(28).

AminoAcidSequenceDeterminationsofHLA-DRAntigen

.8

Chains. Rajicellswere

separately

labeled with

[3H]leucine,

[3H]phenylalanine, [3H]tyrosine, and [3S]methionine

(Radi-

ochemical Centre, Amersham,

England) according

to a

pub-

lishedprotocol (29). Radiolabeled HLA-DRantigen 8chains wereisolated andsubjected to amino acid sequence determi- nations. Ineachcasethe ( chainscontained

only

a

single

type ofradioactiveaminoacid. The details of the methodsare out- lined elsewhere (29).

Milligramamountsofhighly purified'HLA-DRantigensfrom Raji cells(5) were

subjected

topreparative

NaDodSOJpoly-

acrylamide gelelectrophoresistoseparate^aand(3chains. The HLA-DR antigen subunits were extracted from the

gel,

di- alyzedagainstdistilled water, and

lyophilized.

The(-chainfrac- tion was subjected to automatic amino acid sequence deter- mination in aBeckman890Csequencer

(30). Phenylthiohydan-

toin derivatives of the amino acids were identified

by high-

pressureliquid chromatography (31).

Biosafety.All workinvolving recombinant plasmidswasdone underconditionsconformingtothe standards outlinedinthe NationalInstitutesofHealthguidelinesfor recombinant DNA research.

RESULTS

Constructionof

PlasmidpDR-l.&3

RNAcoding forHLA-DR antigenchainswasisolatedfromacrudemicrosomefractionof Rajicells. Afterselection for poly(A)-containingmRNA, anali- quot was subjected to cell-free translation. Approximately 0.01%of the[3S]methionine-labeled productwasprecipitated by theantiserumagainstHLA-DRantigen (3chains. Aftersize separationof themRNAbysucrosegradient centrifugation, the purityof the(3chainmRNAincreased10-foldasevidenced by cell-free translation, immunoprecipitation, and

NaDodSOJ polyacrylamide

gel electrophoresis. Fractions containing (3- chainmRNAwerecombined and concentrated.

From the fractions containing HLA-DR antigen

(3-chain

mRNA, 10

gg

was copied into double-stranded cDNA and clonedintoEscherichia coli. Double-strandedcDNAmolecules recombinedwith PstI-digestedpBR322yielded1500 tetracy- cline-resistant, ampicillin-sensitive colonies after transforma- tion. Plasmid DNA that was purified from individual colonies andcombinedintogroupsofeightwasimmobilizedonto nitro-

1 2 3 4 5 6 7 8

53-..

46---- s ^a-..-.

-,

:

- - -

FIG. 1. Identification of

pDR-3-1

byhybridization-selection and cell-free translation. Plasmid DNAswereimmobilizedontonitrocel- lulose filters and hybridized ^with.Raji mRNA. After elution, the mRNAwastranslatedinacell-freesystem.Translationproductswere

characterizedbyNaDodSO/polyacrylamide gel electrophoresisand autoradiography.Lanes:1,totaltranslationproducts,inthe absence ofmicrosomes,ofRajimRNAused forhybridizationtopDR-,-1DNA;

2,aslane1but inthe presence ofmicrosomes; 3,total translationprod-

uctsinthe presence of microsomesofRajimRNAhybridizedto

pDR-

,-1

DNA;4,aslane 3 butafterprecipitationwithananti-HLA-DR antigen 3-chainantiserum; 5,aftertranslation ofRajimRNAhybrid-

izedto

pDR-(-1,

the microsomesweretreatedwithproteinaseK(15) priortoimmunoprecipitationof the

(3-chain;

6, 7,and8,translation productsofRajimRNAhybridizedto twoseparaterecombinantplas-

midscontainingunidentifiedinsertsandtopBR322,respectively.All translationswerecarriedoutinthepresenceofmicrosomesexceptin lane 1. TranslationproductswereprecipitatedwithanHLA-DRan-

tigen (-chain antiserum exceptin lanes 1-3. Arrows, positionsof markers(kilodaltons).

Proc.NadAcad.Sci. USA 79

(1982)

Proc. NatLAcad.Sci. USA 79 (1982) 1705

_

_0

^{_ _ _}

-w 8 w °

n

= _--- -- a_ -

,, r'4 ° '

.< !Z (If &MR(

400 i00 m0

FIG. 2. Restrictionmapof thepDR-3-1 cDNAinsert. The1070-base-pairinsert is drawnas astraightline withflanking pBR322sequences, asitisorientedintheplasmid (withtheEcoRIsite closetotheright-handside of theinsert).The 5' end of thecodingstrand is to the left. Thepart oftheinsert whosesequence wasdetermined is shownas abox,and thestrategy is indicatedbythearrows.The boldarrowindicatessequence

obtained by the methodofMaatand Smith(28).Thinarrowsindicatesequenceobtainedbythe MaxamandGilbertmethod(27). Fragments^were isolated from pDR-,3-1asfollows:⁽ⁱ⁾cutwithPstI,labeled,recutwithEcoRI, 790-base-pair fragment isolated; (ii)cutwith AvaI,labeled,recut with Sac I, 250-base-pairfragmentisolated;(iii)cutwithAvaII,labeled,recutwithAvaI,140-base-pair fragmentisolated.Fragmentiwasanalyzed byboth methods.

cellulose filtersfor the selection by hybridization ofmRNAcod- ingforHLA-DR.antigen chains. ThehybridizedmRNAwas

eluted and then translatedinacell-freesystemcontainingdog

pancreasmicrosomes. Aftertranslation, themicrosomeswere

isolated and analyzed forcontentofnewly synthesizedHLA-DR antigen chains by immunoprecipitation and NaDodSOJ polyacrylamide gel electrophoresis.

Byusingthis selectionmethod, ¹of40filters testedwaspos-

itive (i.e., hybridized to (-chainmRNA). Plasmid DNA from eachoneof theeight bacterial coloniescomprisingthemixture presenton thepositivefilterwasimmobilized separatelyonto

nitrocellulose filters. The selectionprocedure^wassubsequently repeated and ^one positive clone was obtained. The mRNA eluted from thisplasmidDNAdirected thesynthesis of^amajor polypeptide chainwithanapparentmolecularweightof about 28,000(Fig. 1,lane3). This polypeptide^wasprecipitated by the antiserum against HLA-DRantigen chains (lane 4)and^co- migrated with core-glycosylated

P

chains ofRaji cells (not shown). Moreover, proteinase Kdigestion of the microsomes after the translation removedapproximately1000 daltons from theputative chain(lane 5).

This resultis inagreementwith the observation that the ⁽³ chainisatransmembraneprotein. Otherplasmids and thevec-

tor(Fig. 1, lanes6-8) didnotgive risetoanyimmunoprecip- itablepolypeptide chain. The identified plasmid, namedpDR-

,B31,

wasusedtogeneraterestrictionenzymefragments of the insert.Colony hybridization ^wascarriedoutwith such labeled fragments (32). From the original 1500clones, 2 more were

identified ^as positive by this procedure. Detailed analyses of theseclones will be reported elsewhere.

Partial Characterization of thepDR-,-1 Insert. A restric- tionmapof the pDR-(3-1cDNAinsertisshowninFig. 2. The insert contains 1070basepairs.-Only the leftPst Isitewas re-

constituted. Threecleavage^sitesfor Pvu II,twoforAvaI,and

oneforSac^I,Taq I,AvaII,and EcoRIwerefoundintheinsert.

Nucleotidesequencedeterminationswerecarriedoutfrom both ends of theinsert. No stretch ofpoly(A)residueswasob- served. However,stopcodonswerefoundinallreadingframes inthe^sequenceclosetothe

right-hand

side of theinsert. This isastrongindication that thisportionof thecDNAcorresponds

tothenoncodingregionof themRNA.Consequently, the left- hand side of theinsertcorrespondstothecodingportion.The

sequence of150nucleotidesatthe left-hand side of the insert isdepictedinFig. 3. Following21 or22guanosinesbelonging

tothe poly(G)tail, thesequenceof theinsertmostlikely begins.

Onlyone openreading frameexists. That readingframe pro-

vides unambiguous information for the amino acid ^sequence shown inthefigureapartfrom theNH2-terminal glycine (see below).

NH2-TerminalAminoAcidSequenceDeterminationof Raji HLA-DRAntigen ^f3 Chains. Detergent-solubilized HLA-DR antigenswereisolatedfromRajicells. Duringthe isolationpro-

cedure, which involved several fractionationsteps (5), theoc- currence ofHLA-DR antigens was monitored both by a ra-

dioimmunoassay method and by NaDodSOjpolyacrylamide gel electrophoresis. Whennocontaminating proteinscould be detectedintheHLA-DRantigen preparation, aand

P

chains

wereseparated bypreparativeNaDodSOjpolyacrylamide gel electrophoresis. The (3-chain fraction ^was subjected to auto- maticNH2-terminalaminoacidsequenceanalysis. In33of the 35degradation cycles performed, phenylthiohydantoin amino acidderivatives could be detected (Fig. 4). Forsevenpositions,

morethanasingleaminoacid residue^wasfound, supporting the fact thatRajicells^areheterozygousattheHLA-Dlocus.

Becauseof theknowngeneticpolymorphism oftheHLA-DR antigen chains(6, 7)we wishedtoconfirm that theprotein

sequence was representative for the Raji cells. Accordingly, HLA-DR antigen ⁽³ chains derived from Rajicells separately

25 50 75

pDR-B-1 (G) 22AG GGC AGA GAC TCT CCC GAG GAT TTC GTG TAC CAG TTT AAG GGC ATG TGC TAC TTC ACC AAC GGG Ge q-Arg-Asp-Ser-Pro-Glu-Asp-Phe-Val-Tyr-Gln-Phe-Lys-Gly-Met-Cys-Tyr-Phe-Thr-Asn-Gly-

100 125 0SO

ACA GAG CGC GTG CGT CTT GTG AGC AGA AGC ATC TAT AAC CGA GAA GAG GTC GTG CGC TTC GAC Thr-Glu-Arg-Val-Arg-Leu-Val-Ser-Arg-Ser-Ile-Tyr-Asn-Arg-Glu-Glu-Val-Val-Arg-Phe-Asp

FIG. 3. Nucleotidesequenceatthe 5' end of thepDR-,B-1insert and thepredictedamino acidsequence.

-i

-_ _~~~~~~~~O-

'

a

0V-

Biochemistry:

Wimanetal.

1706 Biochemistry: Wiman et al.

10

-l 0

a

0

1

0.1

10 20 30

Degradation

FIG. 4. Yields ofphenylthiohydantoin derivatives in the amino acidsequencedeterminationon14 nmol of HLA-DRantigen ,3chain.

Theone-letter amino acid codeisused.X,unidentifiedresidue.

labeled

withtUnS]methionine,

[3H]phenylalanine, [3H]tyrosine, and [3H]leucine were subjected to automaticamino acid se- quence analyses. Bythis procedure, ¹¹of 30positionsin the NH2-terminalportion of the Raji ⁽³ chainswere assigned^one

ormoreaminoacid residues(Fig. 5). Seven of the11positions displayedthe^sameresidues^asthose obtainedby analyzingthe unlabeled chains.

The varying yields of the radioactive phenylthiohydantoin derivatives clearlydemonstrated that the Raji chains were

heterogeneous in amino acidsequence. Forinstance, the ty- rosine inposition7gavealoweryieldthan thatinposition10.

Likewise, the phenylalanine in position 11 wasrecovered in smalleramountsthan thatinposition 13. These datatogether with the fact that4 of the 11 positions analyzed by the radi- ochemical methoddisplayedaminoacid residuesnotfound in

pDR-(3-1

the sequenceof theunlabeled 3 chains strongly indicate that the(3chain family of Rajicells is greater than revealed by the sequenceanalysis of theunlabeled material.

Identification of pDR-fi-1As an HLA-DR Antigen 3-Chain- Like Clone. Fig. 5 shows the NH2-terminal amino acid se- quences of HLA-DR antigen (3 chains as determined on the unlabeled(middle row) and onthe biosynthetically labeled sub- unit(bottom row). The amino acid sequence deduced from the nucleotide sequence ofthepDR-f3-1 insert isalso shown (top row). In the region available for comparison (i.e., 33 positions), pDR--3-1 displaysanoverallhomologytothe combinedprotein sequencesof64%. To maximizethe homology, thepenultimate arginineof pDR-/3-1 hastobe aligned withthe NH2 terminus of the protein sequences. This. suggeststhattheNH2-terminal glycine of pDR-f3-1 representsthe last residue of the signal se- quence. However, the glycine residueshould be regarded as tentative because a single adenosine separates the poly(G)tail from three additional guanosines, two of which are part of the glycinecodon.

Thus,

theadenosine may be an artifact which aroseduringthe tailing reaction.

The homology between the protein sequence of the unla- beled material and

pDR-,8-1

is 48%whereas the homology be- tweentheradiochemicalsequenceandpDR-(3-1is 64%.Those positionsin the unlabeled(-chainsequencecorresponding to theknownones intheradiochemicalsequence show only18%

homologywith

pDR-,(31.

DISCUSSION

Toclone HLA-DR antigen 3-chain cDNA, we usedmethods thatprovedsuccessful incloningH-2 antigen cDNA (14).There seemstobe threekey features related to using this protocol.

First, themRNAhas to beenriched becauseRajicellscontain relativelysmall amounts of

P-chain

mRNA. Althoughsizesep- arationbysucrosegradientcentrifugationis anefficientmeans forenrichment ofthe mRNA, the use ofmicrosomal mRNA rather than totalmRNA asthe startingmaterialwasprobably just as important. Second, cell-free translations were always carriedout inthe presence ofdogpancreas microsomes(15).

Inourexperiencethe microsomes promote thetranslation,pro- tect the protein against proteolyticdegradation, and, by re- movingthesignalsequenceandby addingthe core sugars,allow the expressionofmostifnotall antigenicdeterminantspresent onthe cell surface form of theprotein...Third, theavailability ofaspecificantiserumstronglyreactiveagainstthe.microsomal form of the

P

chain was important. It seems that the use of NaDodSOjpolyacrylamide gel electrophoresis,. which dena- tures the

protein,

to some extent, toisolate 3chains for im- munization.provideduswithanantiserumthat reactedbetter with the isolated (3 chains than with the intact HLA-DR antigens.

The cDNA clone

pDR-,8-1

contains an insertof 1070 base

5 10 15 20 25 30 35

R

oJS[iJE

DjV

rY

^{F K G}

CM

^{C Y} F1^{T N}

CG

^{T E R V R L V S JS I}

Y[i RE]J

Raj i (3 chain

Raji (3 chain radiochem.

XWJTIE3P RWDL E[EO S[KJF

R V T

T A

L

l [

F

iR JY

F F

By

LfH[F

^{X G T E R V R Y L}

Df[Y

^V

HF3Qf

Q T Q F

S L

L By

FIG. 5. Predicted amino acidsequencesofpDR-,3-1,RajiHLA-DRantigen,achainsequence determinedonunlabeledmaterial,andradiochem- ical aminoacidsequenceofthe HLA-DRantigen chainsbiosyntheticallylabeled withleucine,phenylalanine, tyrosine,and methionine. Amino acidsequencehomologiesaredenotedbyboxes.

I I

_ .

D0

* F

r Ret

0 *

F H F

K* *. G

E F 0

0 0

L E

0

* R

0v

L R

R * L 0

* S R -

* 0 H

oc V ^ve

00 0 * Q

VT ~~~~~~~~0

yr.. D F

S F0

1 YL E

* 0*0

A N

0 0

S 0

* 0

x I xl I

Proc.Natl.

Acad.

Sci. USA79

(1982)

Proc.Natl. Acad. Sci. USA 79(1982) 1707 pairs. Because pDR-(3-1 comprises the nucleotide sequence

corresponding to the NH2 terminus of a

P

chain and because

P

chains should becomposed of about230 aminoacids,ascal- culatedfrom the apparentmolecular weight of nonglycosylated (3-chains, pDR-j3-1 should provide the entire nucleotide se- quence of thetranslated portion of a

P

chain. Inaddition, ap- proximately 300base pairs of untranslated sequence should be obtained. This is obviously less than the complete untranslated region because our preliminary nucleotide sequence analysis at theright-hand side of the pDR-(3-1 insertfails to reveal a poly(A) site. Apartfrom providing the protein sequence for a

,(

chain, the

pDR-,8-1

clone shouldproveusefulinanalyses of thestructureandorganizationofthe HLA-D locus and its genes.

Data on HLA-DR antigen

/3-chain

aminoacid sequences are scarce. Infact, 15 positions in theNH2-terminalregion appear tohave beenidentified (10,33,34).Thissituationtogetherwith the fact that

,(

chains displayanextensive geneticpolymorphism promptedus toelucidatetheNH2-terminalsequenceof HLA- DR antigen (3chainsderived from Raji cells. The amino acid sequencefoundforunlabeled (3chainsdemonstrated thehet- erogeneityof the materialbecausemultiple residueswereob- tainedin some positions. Weexpectedtofindtwo aminoacid residues in some positions because the Raji cells express two alleles, DO3 and Du6, atthe HLA-D locus. However, three aminoacid residueswereobtained for fourpositions.This sug- geststhat the HLA-DR antigens may comprisemorethantwo types

of(3

chains. Infact, recentdatastronglyindicate that the HLA-Dregion may contain twoloci (11, 12). The presentob- servations arecompatible with such an idea because in three ofthe four positionswhere the aminoacidsequencedetermi- nationrevealedthree aminoacidresidues,

pDR-,(31

contained yetafourth aminoacid residue. Consequently, Raji cells may beheterozygous also at asecond HLA-D locus.

It iswellestablishedthat the murine counterpart ofthe HLA- Dlocus, the I region, contains two subloci. A similar situation has beennotedinthe rat (35) and in the chicken (36). Unfor- tunately, notenoughaminoacidsequenceinformationisavail- able relative to murine I-Aand I-E/C (3 chains to allow any conclusion as to whether

pDR-3-1

maybe the equivalent ofan I-Aor anI-E/C(3chain.

The NH2-terminalaminoacid sequence determinations were carriedoutbytwoentirelydifferent techniques.This had tobe done because theisolationprocedures usedmayhaveprovided highly purifiedHLA-DRantigensthat were not quantitatively representative ofthe Raji cell HLA-DR antigens. Indeed, the two protein sequences only displayed 64% homology at the positionsavailablefor comparison. It is interesting to note that the four positions in the radiochemical sequence that differed fromthecorrespondingamino acid sequence of theunlabeled (3 chains were identical to the

pDR-,(31.

Therefore, the two protein sequencesfirmly establish that the isolated cDNA clone corresponds tothe mRNA of an HLA-DR antigen 3 chain.

Thegenerous adviceofDr. T. Edlund proved invaluable. Expert technicalassistance wasprovided byMs. A. Moron, I. Schenning, I.

Sj6quist, andMr. K. Anderson. M.D. istherecipientofaEuropean MolecularBiologyOrganization Long-TermFellowship. This workwas supported bygrantsfromthe SwedishCancer Society, KingGustafV:s 80-yearsfund,andCentralaForsoksdjursnamnden.

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Biochemistry:

Wimanetal.