Structure linkage : and sequence of the two genes encoding the delta and gamma subunits of the nicotinic acetylcholine receptor

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Reference

Structure linkage : and sequence of the two genes encoding the delta and gamma subunits of the nicotinic acetylcholine receptor

NEF, P., et al.

Abstract

We have cloned and sequenced a fragment of the chicken genome approximately 9 kilobases in length that comprises the genes encoding the delta and gamma subunits of the nicotinic acetylcholine receptor. The two genes are homologous and have identical structures: both consist of 12 exons, some of which precisely correspond to predicted structural domains of the receptor subunits. The delta and gamma subunit genes are encoded by the same DNA strand and are very closely linked, there being only 740 base pairs between the last codon of delta and the initiator codon of gamma. Blot analysis demonstrates that the genes we describe are unique in the genome. Comparison of the predicted protein sequence for the corresponding subunits of chicken and of the elasmobranch Torpedo reveals a high degree of conservation in some but not all of the protein domains.

NEF, P., et al. Structure linkage : and sequence of the two genes encoding the delta and gamma subunits of the nicotinic acetylcholine receptor. Proceedings of the National Academy of Sciences, 1984, vol. 81, no. 24, p. 7975-7979

PMID : 6096871

Available at:

http://archive-ouverte.unige.ch/unige:85382

Disclaimer: layout of this document may differ from the published version.

1 / 1

Proc.Nati. Acad. Sci. USA

Vol. 81, pp.7975-7979, December 1984 Neurobiology

Structure, linkage, and sequence of the two genes encoding the 6 and y subunits of the nicotinic acetylcholine receptor

(acetylcholinereceptorgenes/subunit

genes/linkage/ivolution/protein

^domains)

PATRICK NEF, ALEX MAURON, ROMAINE STALDER, CHRISTINE ALLIOD,

AND

MARC BALLIVET*

DMpartementdeBiochimie, Universitd deGen~ve,30, quai ErnestAnsermet,1211 Geneva 4,Switzerland Communicatedby Norman Davidson, August 20, 1984

ABSTRACT We have cloned andsequenced a fragmentof the chicken genomeapproximately9kilobases inlength that comprises the genesencodingthe 6and 'ysubunitsof the nico- tinic acetyicholine receptor. The two genes are homologous andhaveidenticalstructures: bothconsistof 12 exons, some of whichpreciselycorrespond topredicted structural domainsof thereceptor subunits. The8and ysubunit gehes areencoded by the same DNA strand and are very closely linked, there being only 740 base pairs between the last codon of 8 and the initiator

codon

ofy.Blotanalysisdemonstratesthat the genes wedescribe are unique in the genome.Comparisonof the pre- dicted protein sequence for the correspodding subunits of chicken and of the elasmobranch Torpedoreveals a highde- greeofconservation in Aome but not all oftheproteindomains.

The nicotinic acetylcholine receptor

(AcChok)

mediates synaptic

transmission

atthe vertebrateneuromuscular junc- tion. Locatedin the folds of the postsynaptic membrane,the

AcChoR

is acation channel whose opening is triggered by acetylcholine. AcChoR activation normally resultsinanin- fluxof

Na'

thatdepolarizes the

postsynaptic membrane

and leadstomusclecontraction. The AcChoR isthe best under- stood ofallligand-gated ion channels duetoits relative abun- dance in the

electrocytes

ofTorpedoor

Electrophorus,

from

which

workable

quantities

ofreceptor canbe

purified.

The receptorisapentamerof fourdifferent subunits in the stoi-

chiometty a2f8W.

Allsubunitsare

glycosylated

andspanthe membrane. They assemble into a

barrel-shaped

structure whosecentralcavity is thoughttobe the

gatpd

ion channel

(for

recentreviews seerefs. 1-3). That the four subunitspar- ticipate in the assembly ofasinglefunctional AcChoRmole- cule was elegantly verifiedby showing that

all

fourmRNA speciesmustbe

injected

in Xenopusoocytes toallowdetec- tion of

physiologically active

receptor(4).

Microsequencing

of the aminoendsof the four

purified

Torpedo subunits dem- onstrated thattheywererelatedandsuggested thattheyhad evolvedfrom an ancestral gene

by

a series of

duplications

(5).

Several

laboratories recently

succeeded in

cloning

andse-

quencing

some

(6-9)

or all

(10-12)

of thecDNAs

encoding

the four subunits of Torpedo AcChoR. It was found that thereis

significantly

^more

homology

between the deduced protein sequences of the

a-,8

and

y-8 pairs

than between anyothercombinationsof

subunits, suggesting

^thatan

initial

duplication ofthe ancestralgene had

given

risetotwoproto- genes, oneof whichlateryielded they and8geneswhilethe othergaverisetotheaandpgenes(12). Moreover, allsub- unitswereshowntohavefourvery

hydrophobic

stretches three ofthem closely grouped-of sufficient lengthtospan themembrane. This

finding

suggestedamodelforthetrans- membrane insertion ofthe

receptor whereby

each subunit weavesfour

times through

themembraneto

yield

an assem-

bly

of twenty transmembrane a

helices,

asubset ofwhich constitutes the channelperse

(8, 9, 12).

Arelated model

(13)

predictsthat there isyetafifthtransmembrane helixin each subunit and that its markedamphiphilic character

provides

polarresidues to the channel's interior.

In this report we describe a fragment of the chicken genomecontainingthe8-and

y-subunit

genes.Weshowthat the twogenes are closely linked, encode homologous pro-

teins,

and have identical structures. We note in

particular

that in both cases each of the four hydrophobic stretches predicted to spanthe membrane is encoded by a separate exon.Inaddition,weshow that the 8 and'y genesare

unique

in thechickengenome.

METHODS

GenomicLibrary. Chickenerythrocyte, brain, andmuscle DNA werepreparedfrom the tissues ofday8-12

embryos

by astandard procedure (14). Erythrocyte DNA (thegiftof FranceKeppel)wasusedtoconstructalibraryinthevector phage X L47(15). Adetailed account ofourprocedure has beenpublished (16). Briefly, the target DNA was partially digested with Mbo I and 15- to 25-kilobase (kb)

fragments

generated by severaldegrees ofdigestion were pooledand

ligated

betweentheBamHIsites of the vector. In vitro

pack-

agingwasaccordingtoref. 17. The initiallibrary consisting of5 x 106 recombinants wasamplified

105

times on a

phage

P2

lysogen.

Library Screening. The original procedure (18) has been adapted to permit screening of 5 x

104

phage per 10-cm plate. After transfer of the phage to nitrocellulose mem- branes andprocessing,the filters wereincubatedin5x stan- dard saline/phosphate/EDTA (SSPE)/5x Denhardt's solu- tion/0.1% NaDodSO4 containing sonicated and denatured salmonsperm DNAat100,ug/ml and denaturedprobeat5 x 1 dpm/ml,at580Cfor 18 hr. Afterhybridization thefilters werewashedextensivelyinseveral changes of2x standard saline/citrate (NaCl/Cit)/0.01% NaDodSO4 at room tem- perature. SSPE, Denhardt's solution, and NaCl/Cit (SSC) arestandard hybridizationreagents (19).

Radioactive probes (1-2 x 108

dpm/,ug)

werepreparedby nick-translation (20) of wholeplasmidsorofgel-purifiedre- strictionfragments.

Phage AnalysisandSubcloning. Recombinant phagegiving a positive signal in plaque hybridization were purified by several cycles ofplatingatlow density. Phage stocks were grown in liquid cultureby standardprocedures and purified onequilibrium CsCl gradients.Phage DNAwasprepared by

phenol/chloroform

extraction. The insertswere

mapped

by agarose gel electrophoresis of

appropriate

restriction di- gests.The mappinggelswere blottedtonitrocellulose mem- branes (21) whichwereincubated with labeled cDNAprobes and prepared for

autoradiography exactly

as described

Abbreviations: AcChoR, acetylcholine receptor; kb, kilobase(s);

bp,base pair(s).

*Towhomreprintrequests shouldbeaddressed.

7975 Thepublicationcostsof this articleweredefrayedin partby page charge payment.Thisarticlemusttherefore beherebymarked"advertisement"

in accordance with 18 U.S.C. §1734solelytoindicate this fact.

Proc. Nat. Acad Sci. USA 81(1984)

c106

c102

-4 -."

(t z Q-M M M-

I

E

CID

-i4

Et

4 'a ~

- P-4'r

()0

G Fz O

S 1 I- I I I I I I I II I I 0

t t t pl.6

f

t t -pg t t t

I EfflI I EDI Im

i. nr n ri n fM F mlm Elnm

^---

n-- n n rF m m1

^m

nM---

1 2 3 4 5 6 7 8 9 10 11 12 2 3 4 5 6 7 8 9 10 11 12

500bp 8I

FIG. 1. Physicalmapof the8/ygenepair.Phagec106andc102definea.unique regionof the chicken^genomecontaining the8andygenes.

The region extending between the filled circles has beensequenced.Verticalarrows:PstIsites; p1.6and p9:twoPst Ifragments usedas8and

yprobesingenomicblots. Numbered boxes delineate exons;filled, signal sequences;hatched, hydrophobic regions ItoIV.

above. In this manner we could determine which genomic fragments contained transcribed sequences. Overlapping subclones 400-1600 base pairs (bp) in length andencompass-

ing approximately 15kb ofcontinuous genomic DNAwere

constructed in the vectorpUC 8 (22). Subcloning was rou-

tinely done byligation of thevector totheappropriatetarget DNAfragmentafterthevectorhad been treated with restric- tion endonuclease and alkaline phosphatase.

DNA Sequencing. We used the chemical method (23) as

modified in ref. 24. Thin gelswerefixed and dried onWhat-

man3MM backingpaperbeforeautoradiographyfor 18 hrat

roomtemperature.End-labeling of restriction fragmentswas

effected by filling-in of3' recessed ends with the Klenow fragment of DNApolymerase I in thepresenceof theappro-

priate [a-32P]dNTP.

Blot Analysis.Total DNAfrom chicken tissues, prepared

asdescribed above, wasanalyzed by Southern blothybrid- ization with appropriate nick-translated probes. DNA was

digested with restriction enzymes as indicated in Results.

Samples of the digested genomic DNA (8 pug)aswellasap-

propriate controls (made of roughly single-gene-equivalent

amountsof cloned DNA)wererun on0.7%agarosegels and

...

CRTG6G6GRCRTGGCRGTGCIGCTGGCCTTGITTGGGGCRCTGGTGCTGTCGGgtgogc

^...(399).

ogccccccogGTGGGCTCTGCGTTRRCCRGGRGGRGCGGCTCRTCCRTCRCCTCTTTGRGGRPGR6GGCTRCRRCRRGGRGGIGCG

MetAloVolLeuLeuRloLeuPheGlyAloLeuVolLeuSerG yGlyLeuCysVolRsnGlnG!uGluRrgLeul

leHisHisLeuPhe6luGluRrgG6yTyrAsnLysGluVolRr

-to 1 20

CCCCGTTGCCTCTGCTGRTGRGGTTGTGGRTGTCTRCCTRGCCCrTRCCCTCTCCRRCCTCRTCTCRCTGgtgogc

^....020....

gttottgcogRFRGRGGTGGRCGRGRCACJCRCCRCCCRCGIRTGGGTTGRRCRRgtgogt

^...(163).

gProVolRloSerRlaRspGluVolVolRspVolTyrLeuRlaLeuThrLeuSerRsnLeulleSerLeu LysGluVoIRspGluThrLeuThrThnRsnVolTrpVolG6uGIl

40 SO

....

ctocoRG.

o C (118).GtctGGCGCCCcRaGTTGGGCGCGCGGCCCGCTCTCCGRRGTCG

CGTGCTCCGGCTGCTGCCRGRGgoGCTGgGGCTGCCcG.G...G.CCTGGgG...gtgogc...lGR..

^...

tctcotgCogCco

TGTCG

SerTrpThrRspTyr~rgLeuGlnTrp~snfhrSerGluPheGlyGlyVolRSpVolLeuRrgLeuLeuProGluMetleufrpLeuProGlulleVolLeuGlu~snP~s

n~sn~spG

so

G6CICTTTCiRTGCCTRCTRCTGCRRCGTGCTGGTCTRCFACRCTGGCTACGTCTRCTGGCTGCCCCCCGCCATCT CCGCRGCGCCTGCCCCATCRArGTCRACTTCTTCCCCTTGACTGGCRGRRlCGCRCCCTCARAT CRGgtgtgg...(91 ) I

LeutheGlutaIRloTy~ryrCysRsnVolLeuVolTyr~snfhrGlyryrVolry~rrgLeuProProRlol lePhe~rgSer~loCysProl le~snVol~snPhePheProPhe~sgrrpGln~snCysThrLeuLysPheSe

...

tacgtttcogCTCRTTGGCRTRCRRTGCRCRGGRGRTCRIRCRTGCRCCTGRRGGRGGRGAGTGRCCCRGRGRCTGARRRRGRRrTRCCGGGIGGRGTGGRTCATCATCGRCCCCGFRGGCTTCHCGGgtotcg

^...D...(9) gtcccoocogRR

rSerLevRloTyrRsnRloGInGluIleAsnMetHisLeuLysGluGluSerRspProGluThrGluLysRsnTyrArgVolGluTrpIIe!

lelIeRspProG!uGlyPheThrG lu

150 IS0

RRTGGCGRRTGGGRRTCRTCCRCCGCCCGGCCCGCRRGRRCRTCCRCCCCRGCTRCCCCRCTGRGRGCRGCGRGCRCCRGGRCRTCRCCTTCTRCCTCATCRTCR9GCGCRRGCCRCTCTTCTRCGTCATCPRCRTCGTCRCRCCCTGCGTCCTCAr

TGCCT

X 2Z?Leu~~helyr~alIleAsni leVollhrProCysVolLeul

leRlaP

TCRTGGCCRICCITGTICTCTRCCIGCCIGCTGRCRgtgogt

^.....(247)

tgctctccogGTGGTGRGRlGRTGRCICTGGIGATCTCRGTGCTCCTGGCCCROTCTGTCTTCCTCCTGCTGGTCTCCCRGCGCCTGCCTGCCRCCTCCCRCGCCR

helctRIalIeLeuVolPheTyrLeuProRloRspS

erG1yGIuyMetThrLeuVoI

IIeSerVaIleuLeuRIoGInSerVaIPheLeuLeuLeuVaISerGInRrfLeuProRIaThrSerHisR

AI

---- -254 --- ---27§

TCCCCtTGRggogt. ...( O8

csccicogGTRCCTTCTGTTCRTCRTGCTGCTGGTGnCGGCrGTGGTGGTGRTCTGCGTTGrGGrTcTCRRCITrCCTTrCCGCRC~CCCRGCRCTCRCGTCRrGTCTGRTTGGGTCRGRGGRg

IeProLeulleGlLy

sTyrLeuLeuPhellefetLeuLeuVouThrR~aVal

VolVol

IleCysVolValValLeuRsnPheHisPheRrgThrProSerThrHisVo!MetSerRsplrpVolRrgGly

tooog...(t35)...

tcctatgcog6TGTTCCTGGRGRTCCTGCCCCGCTTGTTGCRCRTGTCCRCaCCRGCTGRGG

CCRGCRGGTGCCCCGTGCRTCCGGCGCTGCRG KTCGGCTGGGTRCATCGCCRRGGCRGRGGRGTRCTRCRGCGTCRRGT

VolPheLeuGulIleLeuProRrgLeULeuHisMetSerHisProRloGluSerProRAloGIyRIoProCysi

leRrgRrgCysSerSerloG61yTyrl

eRIaLysRloGluGluyrryrSerVolLysS

34030

CCCGCRG(GRGCTCRTGTTTGRGRRGCRGTCRGRGAGCRGGGCTGGCCRGCCGTGTCRCCCCTGCCCgtgogc

... (83) .ccgccccogGCTTTGCRCCRGCRGCCRCGRGCGRGGRGCRGCTCTRTGRTCRCCTGRRRCCCRCCCTGGRTGR

errgSerGluLeMetPheGluLysGlnSerGIuRrgHisGlyLeuRloSerRrgVolThrProRloR rgPheRlaProRloARloThrSerGluGluGlnLeuTyrRspHisLeuLysProThrLeuRspGI

390 400

GGCCRACTTCRIIGTCW6CRTRIGCGGGRGRRRRWCRGCTRCRRTGRGgtgggg

^.....

cc.c... ccctccccogGRGRRRGRCRRCrGGRRcCGCGrGGCRCGCRCCCrGGRCCGCCTCTGCCTGITCCTCRrCRCCCCCArGCrGGrGGTGGGCRC

uRI1oRsnPhJI_42044t

eVolLysHis11etArg6luLysRsnSerIyrRsnGlu

GluLysRspRsn

rgRsnRrgVaoIRIaRrghrLeu~spRrsLeuCysLeuPheLeulIeThrProMetLeuVoIVo1GlyTh TCTCTGGRTCTTCCTCRFGGGCRTCTRCRRCCRCCCRCCGCCRCTGCCCTTCTCTGGRGRKCCCCTTCGRCTRCCGGGl~iqGRCRRRCGCTRCRTARrRGGCRGCCCCGRGGGTGGGGTGGGGTCCTCTGTGGCRGCCCTTCTRCTCCTTCCTGCCTRCCTG

rLeurrpllePheLeuMetGlyl

leryrRsnHisProProProLeoproPhleSerGly~spProPheRspryrRrgGluGluRsnLysRrgryrl

le

________-_---474 450 45

CTGrGGRGCRRARWATR

CTGGGTGRrTCRCAAIGTGGGGTGCRGRGCTGGCGGITRRGTGRGRCCURRRR...

FIG. 2. Nucleotideanddeduced protein sequence of the chicken 8 gene. Exon 1 and the first 11 bases of exon 2 encode the proposed signal sequence. Lower-casenucleotidesymbolsindicate acceptor and donor sites of intervening sequences. Numbers in parentheses give length of correspondingintron inbp. Underlined symbols represent hydrophobicregionsIto IV. Thecoding sequence contains

43%

A+T(Torpedo:58%

A+T)andcodon usage reflects this considerabledifference in base composition.Acanonical polyadenylylation signal A-A-T-A-A-A is present 77bpdownstream from the terminationcodon.The mature 8 subunit has a calculatedmolecular weight of 57,215 andamino

acid

composition Phe25,Leu56,I1e32,

Met,1,

Val42,Ser3l,Pro31,Thr24, Ala29, Tyr23, His15,

Gln1O,

Asn27,

Lys18, Asp18,

Glu42,Cys8,

Trp11,

Arg26,Gly16. Thereare twopotential N-linked glycosylation sites, the Asn residues at positions 70 and143.

7976 Neurobiology:

NefetaL

Proc.Natl. Acad Sci. USA 81 (1984) 7977 blottedontonitrocellulose filters (Schleicher &Schuell, BA

85). After heating ina vacuum ovenat80°C, the filterswere

treated with 4x SSPE/lOx Denhardt's solution/0.1 mg of heat-denatured salmon sperm DNAperml at65°C for 4-6 hr. Theywerethenincubated overnight inasmall volume of theabove solutiontowhich 10% dextran sulfate (Pharmacia) and a radioactive probe had been added. The filters were

washed in several changes each of 2x NaCl/Cit at room

temperature, then lx NaCl/Cit/0.2% NaDodSO4 at 65°C, andfinally 0.5x NaCl/Cit/0.2% NaDodSO4 at65°C over a

total time of 3-5 hr.

RESULTS AND DISCUSSION

CloseLinkage of the 8and yGenes. Phage c106 and c102 (Fig. 1 and Methods) were originally screened from our

chickengenomic library with a TorpedoycDNA probe (6).

In the processof subcloning restriction fragments from the

genomeofc106,wenoticed that in additiontofragments giv- ing a strong signal there were fragments yielding faint but reproducible signals when hybridizedtothe probe. We then decidedtosubclone and sequencethe whole regioncovered by thetwophages and found ittoencode the entire 8 and y genes ofchicken AcChoR.

In controlexperiments, aTorpedo 8 subunit cDNA probe (25) hybridized stronglytochicken³genomic fragmentspre-

sentin c106.Moreover, ascreenof the chicken library with the Torpedo probe yielded a set of phage identicalto or

overlapping c106. Restriction analysis of the regionencom-

passed by phage c106 and c102 yielded the map shown in Fig. 1. We madeuseof the high frequency of Pst I sites (an

averageofonesite in 900bp)toconstruct^abattery of contig-

uoussubclones. Overlapping subcloneswerealsoconstruct-

ed from the other restriction sites shown in Fig. 1 (see Meth- ods).

We sequenced 9000 bp of continuous DNA, comprising the3andysubunitgenesfromapoint located about 900bp 5'tothe (putative) initiator codon of the3genetoapoint 240 bp 3' tothe ultimate triplet of theygene. Assummarized in Fig. 1, we found that bothgenes are encoded by the same

DNA strand and that thereareonly 740 bp in the intergenic region separating the last codon of the geneand the initia- torcodon of the ygene.

Structure of the and yGenes.The ^3-andy-subunitgenes arequitecompact(about 3300 and 4000bp,respectively) and have very similar structures (Figs. 1, 2, and 3). Bothgenes

consist of 12 exons. Exons of like rank have extensive se- quence homology andare nearly identical in size. In addi- tion, the splice sites between homologousexons areexactly conserved: atall 11pairs of splice sites the codingsequences

of thetwogenesareinterrupted atthesamelocation within

orbetween theaffected codons. Thus all homologousexons

either have the same number ofnucleotidesordiffer by 3n nucleotides. Interveningsequences areflanked byacceptor anddonor sites that closely conformto thecanonical splice

sequence(26). Introns of like rank in thetwogenes,howev-

er,differ sharply in length and insequence(intronsequences

notshown, full sequenceprintouts available on request).

The above features and the overall homology of the de- duced proteinsequencestothe corresponding Torpedo³and ysubunits

(Fig. 5)

make it

likely

thatour

assignments

of cod- ing and intervening sequences areentirelycorrectfor those

exons(2-12) that encode thematureproteins. The signalse- quences wesuggest(exons 1,Figs. 2 and 3)areconjectural.

Wenote,however, that nowhere in the intergenic regioncan

...CRGCTCCARCRTGCCTGCTCCGRTCICCICCICCTC6TCCTCCTCGCRCTCTGTGTCCTGCCRGgtaogg^...(237). ctcccoccagGCRTRRGCTGCRGGRCCAGGRGGRGRRGCTGCTCCRRGRCCTGRTGRCCRRCTRTRRTCGTCR MetfrgCysSerRspLeuLeuLe euPheLeuLeuRldeuCysYalLeuProG lylleSerCysfrglsnGlnGluGluLysLeuLeuGlnRspLeuMetThrfsnTyrfsnRrgHi

-22 1

CCTGCGCCCGCGCTGCGTGGGGRCCRGTCRATGRCGTCCUCRRRC TCACCCTCRCCRACCTCATCTCCTTGgtgoga ... 18) tccctgtcagRRTGRGCGGGRGGRGRCCCTCRCCRCCRATGTTIGGRTCGRGRTGgtgoga.

sLeuRrgProAloLeurgGlyRspGlnVallleRspValThrLeuLysLeuThrLeuThrRsnLwlleSerLeu RsnGluRrgGluGluThrLeuThrThrfsnValTrpl leGluMet

20 40

(149)

t.atccttcccogCRcTGGCGaCTTCGCCTGCGGTGGGRCCCCGATRRTRCGHTGRCRTCCRCRGCTGCGGGTGCCTTCCGCCRTGGTCTGGCTGCCCGRCRTTGTCTTGGRGRRCR~gtgagg

^...(84)...

ttccccgcogCRT

GInTrpSerRspTyrfkrgLeuRrgTrpRspProRspLysTyrRspRspIleGInGInLeuRrgValProSerRIafMetValTrpLeuProRsplIeValLeuGIuAsnRs nil

80 80

CGRTGGGIRCRTTTGRTCRCRCTCTRTRCCRRCGTGCTRGTGTRCCCTGATGGCRGCRTCTRCTGGCTGCCCCCTGCCRTCTRCCGCRGCTCCTGCTCCRTCCRCGTCRCCTRCTTCCCCrTTGRCTGGCRGRRCTGCRCCRTGGTCTTCCngtgogG

....

eRspGlyThPheGlulleThrLeuTyrThrRsnValLeuValTyrProfspGlySerlleTyrTrpLeuProProRlal

leTyrirgSerSerCysSerlleHisValThrTyrPheProPheRspTrpGlnfsnCysThrMetValPheGI

t00 120 140

.(332).(6).

ctc.tcatagGgATCTRCCTRCRCGCGRTCGPTCRIRCCTGCTGCTGRCRGTGGRGGRR CRGRCCGTRGRGTGGRTCRRTTAGTCCCCGRGGCTTTRCRGgtoocc

^...(661)

cgcotggcogRGRATGGRGRATG

nSerGlnThrTyrSerRlaRsnGlulleRsnleuLeuLeuThrValGluGluGlylnThrlleGluTrpilePhelleRspProGluRlaPheThrG luRsnGlyGluTr

180 181

GGCCRTCR209RCCGCCCTGCRCGGRRGRTC-TC-RTTC-CGCTTC-CCCC-RTG-C-TCCRGTRCCRGC-GGTC-TCTTCTRCCTCRTC-TCC-RCGC-RGCCGCTCTTCTRC-TC-TCR-C-TC-TCGTGCCCTGTGTTCTC-TCTCCTCC-TGGCT

pfilalleLysHis~rgProfilo~rgLysllelleflsnSerGly~irglheThrProRspRspl

leGlnTyrGlnGlnVa~lIlePheTyrleullelleGln~irglysProleuPheTyrllellefisnllelleValProCysValLeulleSerSerMet~la

200 2t9

GTGCTGGTCTFCTTTCTGCCTGCCRRGgtocct...(k140).

ValLeuVaTyrPheLeuProRlolysR

4---7

...

g2ttt0tcogCGGGGGCRGPRTGCRCCGTCTCCATCRRTGTCCTCCTGGCCCRGRCTGTCTTCCTCTTCCTCRTTGCCCRGRRGGTGCCCGRGRCCTCCCRGGCRGTGCC loGly6lyGlnLys~ysThrValSerl

leRsnVolLeuLeuRloGlnThrValPheLeuPheLeulleRlaGlnLysValProGluThrSerGln~loVolPr

252-- -273 280

RCTCRTTGGGRRgtoogt

...(231).

cctccgcogGTRCCTGRCCTTCCTCRTGGTGGTGRCRGTGGTGRTTGTGGTGRRCGCCGTCRTCGTCCTCRRCGTCTCRCTGR6RRCCCPACRCTCRCTCCRTGTCTCRGRGGGTGCGCCRGgtocc OLcullellyLy

slyrLeuThrPheLeutetVol VolThrVal ValIleVolValRsnRloValleValLeLu1snValSerLeuRrgThrProAsnThrHisSerMetSerG1nArgValRrgGln

-311----320---3t320

t... (227)^...

tttccogGTGTGGCTGCRCCTCCTGCCCCGCTRTCTGG6CRTGCRCRT6CClUGIGCCCGGGGCCRCCGCRRGCCRCCCGGCGRCGCRGTTCCCTGGGGCTTGATGGTGffRRGCTGRTGRGTRCRTGCTRTGGPRGRCCCGGRC

Vol

TrpLeuHisLeuLeuPro~krgTyrLeu~lynetHis~etProGluGlu~loProGlyProProGlnRlaThrRrgflrgflrgSerSerLeu6lyLeunetValLysRloRspGluTyr~etLeuTrpLys~loargTh

340 360

CGRGCTTCTCTTTGflGRAGC GRRGGRR TGRCTGRGRRRCCGTGCTGGRGRAGRTTGgtgugt

.....(70).

ogttgtgcogGRCGTGGCCTGGRGRGCARCCCTGCGCRGARCTTCT6CCG6RGCCT6GRGGAGGCGRGCCCRAGRGTC

rGluLeLeuPheGIuLysGlnLysGluRrgispGlyLeuMetLysThrValLeuGluLyslleG lyF'rg6lyLeuGluSerAsnCysAlaGlnRspPheCysGlnSerLeuGluGluRlaSerProGlul

le

380 400

CGGGCRTGCGTTGRGGCCTGCPRCCRCRTCGCCARCGCCRCGCGGGRGCRGRATGRCTTCRGCRGCgtgogt

...(80).

gcccoocogGRGAACGRC6RGTGGRTCCTGGTGGGGR6RTGRTCA

RCCGCGTCTGCTTCTTCRTCRTGGCCTCGC

FRgRlaCysValGhLAlaCysAsnHisl leRla~snAlaThrRrgGluGlnRsnRspPteSerSer GluAsnGluGluTrplleLeuValGlyArgVal IleRspRrgValCysPhePhelleMetRluSerL

420 440 451---

TCTTCGTGTGCGGTRCCRTTGGRRTCTTCCTCRTGGCTCRCTTCFIRCCRGGCGCCCGCCCTGCCCTTCCCTGGGGRCCCCRRGiRCGTRCCTGCCCCCGTGRCRGGTCCRRRTGRCRGGGTCTGGGGGTCCCTRGRCCTGGCTCCCRGTGCCRIRGTGGGGTGRC euPheValCys6lyThrlleGlyl

lePheLeuMetRlaHisPheRsnGlnRlaProfllaLeuProPheProGlyAspProLysThrTyrLeuProPro

_-____ --_-________--- 473 480 490

GGRGGCQCGGGGTGGCTGTGGTTGTGGTTGTGC...

FIG.3. Nucleotide and deducedproteinsequenceof the chicken ygene.Symbolsare asinFig.2. Thecodingsequencecontains42%A+T (Torpedo:56%A+T). Thematureysubunit hasacalculated molecularweightof56,484and aminoacidcomposition Phe22,Leu53, Ile39, Met14, Val39,Ser25,Pro29, Thr30,Ala28, Tyr17,His8, Gln26,Asn25,Lys17,ASP21,Glu32,

Cys1o, Trp11,

Arg27,Gly1g.TherearethreepotentialN-linked glycosylation sites,the Asnresiduesatpositions 141, 307,and 427.

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Proc. Natl. Acad. ScL USA 81 (1984)

A B

2 2

kb b

11.0-

4.8- 0

kb

C D

2 3 4 5 6 2 3 4

11.0- 9.8-3

.5-_6.0

4

go 5.4-*

4.8- 4.3- 3.8- 3.4- .

FIG..4. Genomic Southern blot analysis of8and y subunit genes in the chicken genome. (A and B) Total DNA extracted from chicken erythrocytes was digested withEcoRI (lanes 1) and double-digested with EcoRI/HindIII (lanes 2), separated by electrophoresis on du- plicate agarose gels, and blotted onto nitrocellulose. The blotofone gel, A, was hybridized to a

y-specific

probe, p9, whereasblot Bwas hybridized to pl.6, a 8-specific probe. (C and D) A similar experi- ment was performed with muscle DNA digestedwith BamHI (lanes 1) andHindII1(lanes 2). The patterns in C result from hybridizing with the y probe; those inD were obtained with the 8 probe, asinA andB above. In addition, the figure shows the hybridization pattern obtained from the control lanes in the gel, in which appropriate quantities of digested DNA from the recombinant phagesc102and c106 had been loaded as follows: C3 andD3, c106 digested with BamHI; C4 and D4, c106 digested with HindIII; C5, c102digested with BamHI; C6, c102 digested with HindIII.The sizes of thevari- ous bands were obtained by calibration with HindIII-digested X phage DNA.

another sequence be found with the following required fea- tures:

(i)

an initiator ATG flanked by a purine at -3 (27), (ii)

followed

by

a sequence encoding ahydrophobic core, and

(iii) interrupted by

anacceptor

splice

siteconnecting the

sig-

nalsequence to exon 2inthe appropriatephase. The same argument

applies

totheproposedexon1of the 8 gene(Fig.

2),but inthat case we cannot exclude that another sequence with therequired features ispresentfurther upstream. The intergenicregion contains asingle canonical polyadenylyla- tionsignal 77bp downstream from the termination codon of the8 gene(Fig. 2). Nosuchsite ispresentinthe 240 bpwe sequenced downstreamfrom the terminatorof the y gene.

The 8 and y Genes Are Unique in the Chicken Genome.

Chicken DNA was digested by several restriction enzymes and the fragments were separated by electrophoresis on agarose gel, blotted on nitrocellulose, and hybridized tola- beled probes encoding known 8 and ygenomic sequences.

These probeswereproduced by nick-translationof thepuri- fiedeukaryotic inserts of the relevant subclone plasmids. As indicated in Fig. 1, the 8 probe (pl.6, 1650 bp) and the y probe (p9, 1280 bp)respectivelyencompass exons4-10 and 2-6of the correspondinggenes.Asevidenced bytheautora- diographsofFig. 4(A and

B,

lanes 1) both probes hybridize to a verylarge(>23kb)EcoRIfragment. When thisfragment iscutfurtherby HindIII(Fig. 4 Aand

B,

lanes 2)the 8 and y probes hybridize to distinct fragments 11.0 and 4.8 kb in length, respectively.This resultestablishes thecolinearityof the two genes on an EcoRI fragment bearing at least one HindIII site between the probedsequences. Inaddition, the simple hybridization pattern seen in this experiment indi- cates that the 8 and y genes areunique in the genome.

The

blots presented in Fig. 4 CandDconfirmthisfinding and demonstrate that theorganizationof the two genes is the same in the genome and in the recombinant clones. When BamHI and HindIII blots ofmuscle DNA are probed with p1.6 and p9, simple hybridization patterns areobtained. The y probe (Fig. 4C) is seen to hybridize with 5.1- and 4.8-kb fragments that migrate with the corresponding cloned frag- ments. Fainter signals are also detectable at 4.3 and 3.4 kb (BamHI) and 3.8 kb (HindIII); fromtheir variableintensity

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S I

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SGE-KMTLYISYllFSVFLLLVSRLPRIS

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ENGEVRIKHRPRRKIINSGR[IP0DIOYMOVIFYLIIORKPLFYIINIIVPCVLISSfLVYFLPRK RGGKCIVSINVLLRQTVF[FLRKYVPETS

6 T CSLVFR

SOTYNRHfVNLOL--SfE-E-G[--RVEVIHIIPEOFT ENGEVTIII KKNYNOLTK-IO[OF[1

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IQRKPLFYIINIIRPCVLISS.LVYLYFLPRl R8GGKCILSISVLlR0TIFLUIRO TPEIS

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YLMFIMSlVTGVIVNCGIVLNFHFRTPSTHVLSTRVKQ IFLEKLPRPILHYSRDESEl-PDwgNDlK-LRRSSSVGYISKA0EYFNIKSRSELMFEK0SELH6[VP-RVTPR IGFGNENIRSHO---CI

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YLIFVYFVSILIVMNCVIVLNVSLRTPNTHSLSEKIKH LFLGFLPKYLGMO-LCPSEETPC---KPI-P SSFGIMIKIEEYILKKPRSELYFEOKDRHGLKRVNKMTS DIIIGTTVILYKILANFR--PEI

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KPTlOERNFIVKHHR[KNSYNE EKDOlNRIVRTlORLCLFLITPHL-VVGTLIFlMGIYNHPPPLPFSGDPFDYREENKRYI

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FIG. 5. Alignment of the mature 8 and ysubunits from chicken and Torpedo. Vertical arrows, exon boundaries as determined on the chicken genes.Overlined, proposedtransmembrane regions I-IV. Garnier analysis of the four presented sequencespredictsextendedsecondarystruc- ture inexon 5(/1/)anda-helical structure in exon 11 (XXXX). Asterisks identify those positions in thealignment where threeorfour residuesare identical. Numbers are positions of last residue of chicken 8 subunit encoded by successiveexons.The 8 andypairsbothhave63%homology and allothercombinations of paired subunits have

49-50%o

homology.

7978

Neurobiology:

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Proc. NatL Acad Sci USA 81 (1984) 7979 onthis and otherblots it islikely that theyrepresentdistinct

relatedsequences-perhapsencoding another AcChoRsub- unit. The 8 probe(Fig. 4D)yieldsanunequivocalpattern of unique BamHI and

HindIII

fragments. Fig. 4 Cand D also rules out the formal possibility that the 8 and y genes are tandemly repeated in the chicken genome, because similar signal intensities are obtained with chickenDNA and with cloned DNA loadedatapproximatelyone gene copyequiva- lent.

Fromthese observationsweconcludethat the genomic or- ganization ofthe8 and y genesis faithfully conservedinthe cloned sequences and that the gene pair is unique in the chicken genome.

Recent reports (25, 28) have established that the 8 and a subunit genes areunique in the Torpedo genome.

Homologyof the6 andyProteins. The predicted primary sequences of the mature 8 and y subunits of chicken Ac- ChoRarerespectively 495 and492residues inlength(calcu- lated molecularweightsare 57,215for8and 56,484for y). As shown in Fig. 5, they can easily be aligned with the corre- sponding Torpedo subunits and so can the two sets of sub- units. The 8 and y subunits of chicken AcChoR have an overall homology of63% with the corresponding Torpedo subunits andof 49% between themselves. Most of the fea- tures detected in the analysis of Torpedo AcChoR subunits arealsofound in the chicken subunits. In particular, thehy- dropathy profiles (29) of all subunits that have been se- quenced are essentially superimposable (not shown). Con- servationisnotevenlydistributedthroughout themolecules:

it is highest in the extracellular aspect of the subunits, partic- ularly in the region thought to encompass the acetylcholine bindingsite on the a subunit (encoded by exons 4 and 5) and in the main transmembrane region containing hydrophobic stretches

1-III

(encoded by exons 7-9). The high degree of conservation between the main transmembrane regions of such distantly related vertebrates as chicken and Torpedo strongly suggests that those sequences are part of the chan- nel per se and that DNA probes encoding them may detect kindred channel genes throughout the animal kingdom.

Wefound that the four transmembrane regions thought to anchor the subunits in the membrane and to assemble into a channel are each encodedby a separate exon inthe 8and y genes. This finding constitutes a clear example of the fact that particular structural domains in aprotein often are en- coded by separate exons.

The segments of the8 and y subunits that show the poor- est homology are those encoded by exons 11. It is worth noting, however, that the COOH-terminal halves of those segments are predicted to be a-helical by Garnier analysis (30) and that the a helices are clearly amphiphilic (wheel dia- grams notshown) in this region, as wasfound tobethe case in the corresponding regions of the four Torpedo subunits.

This observation supports but does not constitute a test of the model (13) wherebyin each-subunit afifth, amphiphilic, a-helix traverses the membrane and supplies

hydrophilic

side chains to the channel.

In conclusion, we note that thereis soundimmunological and biochemical evidence for the presence in chicken brain of a nicotinic AcChoR with properties similar to but distin- guishable from those of the AcChoR at the neuromuscular junction (31, 32). It will be interesting to see if our set of chicken AcChoR probes detects related sequences in brain cDNA libraries. In addition, the compact and closely linked 8 and 'y subunit genes offer a potentially fruitful model sys- tem for transcriptional studies, as their expression is tissue specific and probably coregulated.

Wethank Norman Davidson andco-workers for Torpedo cDNA clones, Bernard Fulpius for constant support,FrancoisVeuthey and AmosBairochfor computer programs, and manyundergraduates- particularly Carole Oneyser-for diligent screening of phage librar- ies. This research was supported by grants of the Swiss National Fund to Bernard Fulpius (3.685.0.80) and M.B. (3.432.0.83).

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