cdna sequence and expression pattern of the putative

Proc.Natl. Acad. Sci. USA Vol.92, pp.2091-2095, March 1995 Biochemistry

cDNA sequence and

expression

pattern

of

the

putative

pheromone

carrier

aphrodisin

(lipocalin/hamster/vagina/Bartholin's glands)

HANS-JURGEN

MAGERT*t, THORSTEN HADRYS*, ALEXANDRA CIESLAK*, ANJA GROGER*, STEPHAN FELLERt,

AND

WOLF-GEORG FORSSMANN*

*Lower SaxonyInstituteforPeptide Research,Feodor-Lynen-Strasse31, 30 625 Hannover,Germany;andtDepartmentof MolecularOncology, RockefellerUniversity, York Avenue,NewYork,NY10021

CommunicatedbyM. Lindauer, Universitat Wurzburg, Wurzburg, Germany, October 5, 1994

ABSTRACT The cDNA sequence foraphrodisin, a lipoca-lin from hamster vaginal discharge which is involved in pheromonal activity, has been determined. Corresponding genomic clones were isolated and the promoter region was identified. Primer extension analysis revealed an adenosine residue as the main transcription initiation site, located 50 bp upstream of the translation startcodonATG, which is sur-rounded by a typical Kozak sequence. However, data from polymerase chain reactionanalysis suggest the existence ofat

least onealternative transcription initiation site. The aphro-disin cDNA is 732bp long and codesfor the mature 151-aa aphrodisin and an additional N-terminal 16-aa secretory signal peptide. The 3' nontranslated region is 228 bp long. Among the known sequences,the aphrodisin cDNA sharesthe

highest homologywiththe ratodorant-binding proteincDNA

(45%), which verifies the protein data. Vaginal tissue and Bartholin's glands arethe mainaphrodisin gene-expressing tissues ofthefemale hamstergenital tract,as demonstrated byNorthernblotanalysis. Underlessstringent hybridization conditions, RNA isolated from rat Bartholin's glands also showed a signal, indicating the occurrence of

aphrodisin-related mRNA in thisspecies.

The so-called lipocalins (lipocalycins) are a superfamily of structurally related proteins in the size range of 17-20 kDa whichtypicallyconsist oftwo setsof fourantiparallel,3-strands arranged in two orthogonally stranded

(3-sheets

(1-3). The resulting tertiary structure is comparable to a coffee filter containing an apolarpocket for the noncovalent binding of smallhydrophobicmoleculessuchaslipids, steroids, odorants, bilins,and retinoids

(3).

Most of thelipocalinsare knownto serve ascarriers for the mentioned ligands. However,oneof these proteins, prostaglandin D synthase, exhibits enzymatic activity (4, 5). The specificity of the lipocalins for certain ligands is probably determined by the composition of the amino acids within thebinding pocket(6). Nevertheless,some

lipocalins-for instance, ,B-lactoglobulin and mouse major urinary protein-are capable of binding several different ligands(6, 7).The mechanismsof interaction of the

lipocalin/

ligand complexeswith specific receptors have notbeen well investigated. In the case of human serum retinol-binding protein, it has been demonstrated that certain parts of the loops connectingthe

(B-strands

areessential fortheinteraction with an additional protein called transthyretin and a

mem-branereceptorof thetargetcells.

Further,

it is

proposed

that the release of retinol from thebinding pocket istriggered by

some kind of conformational change in the retinol-binding protein after bindingto the receptor

(8).

Aphrodisin, a 17-kDaglycoprotein of the lipocalin family, has been purified from hamster vaginal discharge (9). It

consists of 151 aa and contains two disulfide bonds which connect the cysteine residues at positions 38 and 42 and at

positions 57 and 149. Its N terminus is blocked bya pyroglu-tamateresidue, indicating the occurrence of a largerprimary translationalproduct(10). Actingvia thevomeronasalorgan (11),aphrodisin elicitscopulatory behavior in malehamsters. Atthis time it isnotquite clear whetheranadditional ligand isrequired forbiologicalactivity but, if this should be the case, it must be bound verytightly to the protein(12).On the other hand, proteolytic or heat degradation of aphrodisin-containingfractions leadsto alossofactivity (9).Functionsas

pheromone-bindingproteins have also been discussed fortwo

otherlipocalins, the mousemajor urinary proteinand a-2U globulin (6, 13).

Among the known proteins, rat odorant-binding protein (ROBP)shares thehighestsequencehomology with aphrodi-sin(40%). Itoccurs innasal mucosaofratsandseems tobe responsible forthe bindingof odorant molecules(refs.14 and 15; for review seeref. 16).

Here wereport the nucleotide sequence ofa 732-bp aph-rodisin precursorcDNA§which codes for thematureprotein andanadditional N-terminal secretorysignal peptideof 16aa.

PCRamplificationsof different cDNAfragments indicatethe existence of at least two transcriptional initiation sites. We showby Northern hybridization thatvaginaltissue and Bar-tholin'sglandsarethe mainaphrodisin gene-expressingtissues of the female hamster genital tract. Under less stringent conditions we also obtained a hybridization signal in the expectedsize range fromratBartholin'sglands, suggestingthe synthesis ofarelatedprotein.

MATERIALS AND METHODS

Polymerase Chain Reaction.Total RNAwaspurifiedfrom

one female hamster genital tractby means ofan automatic nucleic acid extractor(model 340; Applied Biosystems). Five micrograms of the total RNAwas subjectedto reverse

tran-scription usingMoloney murine leukemia virusreverse

tran-scriptaseandasyntheticoligo(dT)universalprimer(UNIP-2; Fig. 1).One-thirtieth of the cDNA mixturewasthen used for each of the three-step-cycle PCRs (17) in amodel 480 ther-mocyclerwithAmplitaqDNApolymerase(bothfrom Perkin-Elmer) andsyntheticprimers(Fig. 1).

CloningofPCRFragments. PCRfragmentswereseparated in 3% NuSieve GTG agarose gels

(Biozyme, Oldendorf,

Germany)

and purified with a Qiaex kit

(Qiagen,

Hilden,

Germany).

The ends ofthe PCRfragmentswerecleaved with Xba I,whoserecognition sequencewasincluded in the PCR primers.After the short cleavage productswere removed by

Abbreviation: ROBP,ratodorant-binding protein.

tTowhomreprint requests should be addressed.

I

§The

sequence reported in this paper has been deposited in the

GenBank database (accessionno. X65238). 2091

Thepublicationcostsof this articleweredefrayed in part by page charge payment. This articlemusttherefore behereby marked "advertisement" in accordancewith 18 U.S.C. §1734 solelytoindicate thisfact.

2092 Biochemistry: Magert etal. AP-1 AP-2 AP-3 AP-4 AP-5 AP-6 AP-7 AP-8 AP-9 RAP-1 RAP-2 UNIP-2 +71 +89 CCCTCTAGAATTCARGGNAARTGGTAYACNAT +199 +221

CCTCTA GAATTCTAYGTNATHACNAAYAAYCARTG

+287 +268

AATCTA GAA TCTMNCCYTCRAAYTGNGTYTG

+452 +430

A CTCTA GAATTCTTYTTYTCRTGNGCRAAYTGNAC

+218 +242

AAATCTA GAATTCAGTGCTCCAAGACCACAGTCATTGG

+253 +280

CGGTCTA GAA TTCGGAAATGGAA CYTACCAAACCCA GTTTG

+56 +39 TCCTGAGCATGAGCCAGA +177 +154 GCAG1TTTTATAACAATCAATATG +120 +97 CGGTCTAGAA7TCTATCTM1ICAAGATTGTCAGCAGC -70 -48 GCTTTGTCAGTTGAGTTCTTTTC -21 -1 GGAAAAGCAAAGTCAGGCACC poly(A)tail

CCTGAA TTCTA GAGCTCA(T)17

FIG. 1. Nucleotide sequences(in 5'-- 3'orientation) of primers. Thepositions of theprimers within the presentedcDNAsequenceare indicated.Italic regions donotcorrespondtothecDNAsequence but containrecognition sites for restriction endonucleases(XbaI,EcoRI,

and,inthe case ofUNIP-2,also SstI) which enable easy cloningof the obtainedPCRfragments.

centrifugation in Centricon filters (Amicon), the fragments

were ligatedwith Xba I-cleaved plasmid pUC18 (18). Esche-richiacoliJM109 cells(18)weretransformedwiththeligation productsbythe PEG method (19).

GenomicLibraryScreening.AGoldenhamster

(Mesocrice-tus auratus) genomic library in A Fix II

(Stratagene)

was screened(20) withthe407-bp

AP-1/AP-4

aphrodisin cDNA PCRfragment, which hadbeenlabeledwith

[a-32P]dCTP

by

useofarandom-primer labeling kit

(Boehringer

Mannheim). Primer ExtensionAnalysis.Total RNAwasextracted from Golden hamster vagina and brain as described above. A syntheticantisense oligonucleotide primer (AP-7;Fig. 1)was

5'-end labeled with 32Pbyuseof

[,y-32P]ATP

and T4 polynu-cleotide kinase andhybridizedto5 ,ugof total RNA.Afterthe extension reaction(21),theproductstogether with labeled size markerswereseparatedbyelectrophoresisinadenaturing8% polyacrylamide gel,whichwas

subsequently

exposed tox-ray film (Kodak

XAR-5).

DNA Sequencing. Nucleotide sequences were determined according tothe methodofSangeretal.

(22)

bymeansofan

automatic fluorescence sequencer

(model

373A;Applied

Bio-systems) (23). To avoid sequence errors caused by misincor-poration of nucleotides during PCR, at least three independent clones of each fragment were sequenced. Uncloned PCR fragments were also directly sequenced after agarose gel electrophoresis and purification with Ultrafree-MC filter col-umns(first0.45-,um Durapore, then PTTKpolysulfone

mem-brane; Millipore) using the PCR primers as sequencing prim-ers.

Northern and Southern Blotting. Total RNA was glyoxy-lated and electrophoretically separated as described (24). RNA and PCR fragments were transferred from thegel to Hybond N+ membrane (Amersham) by use of a PosiBlot pressure blotting apparatus (Stratagene). The nucleic acids werefixed to themembranesaccording to the manufacturer's instructions. Northern blots were hybridized to the same 32P-labeled PCR fragment, AP-1/AP-4, that was used for genomic library screening. PCR Southern blots (25) were

hybridized to a synthetic oligonucleotide (AP-7; Fig. 1) that hadbeen 5'-end labeled with 32p.

DNA Sequence Comparisons. Homology searches in the EMBLnucleic acid sequencedatabase, sequence alignments, and other kinds of sequenceanalyseswereperformedon an

Apple SE 30 computerusing the MACMOLLY program package (SoftGene, Berlin).

RESULTS

Aphrodisin cDNA Sequence. Fromthe alreadyknown aph-rodisinprotein sequence, four degenerate PCRprimerswere

constructed (sense, AP-1 and AP-2; antisense, AP-3 and AP-4; Figs. 1 and 2) and used for the amplification of aphrodisin-specific cDNAfragments from total RNA of fe-male hamster genital tract. Four homogeneous PCR frag-mentswere obtained, all ofthem intheexpectedsize range. Thelargest fragment

(AP-1/AP-4)

wascloned inpUC18and threeindependentclones(pPET1-3)weresequenced. Trans-lation of thecDNA intoproteinsequenceandcomparisonwith theaphrodisin aminoacid sequenceverified thecloningofan

aphrodisin-specificpartialcDNA. Bymeansof this sequence,

two highly specific primers (AP-5 and AP-6; Fig. 1) were

constructed and used together with the oligo(dT) primer UNIP-2(Fig. 1)foramplificationof therestof the3'-terminal cDNA(preamplificationwith

AP-5/UNIP-2,

final amplifica-tion with AP-6/UNIP-2). The 500-bp cDNA fragment ob-tained was cloned in pUC18 and five independent clones (pPET11-15) were sequenced. From the partial gene (see below) and cDNA sequence, twoadditional

primers

(RAP-2 andAP-8; Fig.1)werederived for theamplificationoftherest

of the 5'-terminal cDNA.The 198-bpfragmentobtainedwas

sequenced directly and, bymeansofoverlappingregions,the

I-> secretorysignal peptide I> matureaphrodisin AP-1 >

Met Val Lys IleLeu Leu LeuAlaLeuValPhe SerLeu AlaHisAlaGlnAsp PheAlaGlu LeuGln GlyLys Trp Tyr ThrIleVal IleAla Ala AspAsnLeu Glu LysIle

accAMGTAAAGATUCTG CTG CTGGCT TTGGTC mTTAGT CTG GCT CAT GCT CAG GATUTT GCA GAGCUTCAA GGA AAA TGG TAT ACCATUGTCATT GCT GCTGACAATCUTGAA AAGATA

AP-5

AP-2

GluGluGly GlyProLeuArgPheTyr Phe ArgHis IleAsp Cys Tyr Lys Asn Cys Ser Glu Met GluIleThr Phe Tyr ValIleThr AsnAsn GlnCys Ser Lys Thr ThrVal Ile

GAA GAA GGA GGA CCA CTG AGATTC TATUT CGT CATATUGATTGTTATAAA AAC TGC AGTGAAATGGAA ATC ACAUT TAT GTCATTACAAAC AACCAG TGC TCCAAGACC ACA GTCATT

* * 0*

_, AP-6

AP-3

GlyTyrLeuLysGlyAsnGly Thr TyrGln ThrGln PheGluGlyAsnAsn Ile Phe GlnPro LeuTyr Ile ThrSer Asp LysIlePhe PheThrAsn Lys AsnMetAsp Arg Ala Gly GGGTACTUGAM GG AATGGA ACC TAC CAA ACC CAGUT GAA GGT AACMTATAUTT CAACCT TTG TAT ATA ACA TCA GAC AAGATTTUCm ACCAAC AAG AACATG GAT AGAGCTGGC

c - AP-4

Gln Glu Thr Asn MetIle Val Val AlaGlyLysGlyAsnAla LeuThrProGluGluAsnGluIleLeuVal Gln Phe Ala HisGluLys LysIleProValGluAsn IleLeu AsnIle

CAGGAAACGAACATGATTGTTGUTGCT GGA AAAGGTAAT GCTTUGACACCTGAA GAA AATGMATACTT GTG CAA m GCr CAT GAA AAG AAAATTCCA GTGGAAAACATT CTC AAT ATT

* * * 0

LeuAlaThrAspThrCysProGlu STOP

CUTGCT ACAGATACTTGT CCT GAATAAaggcMttccgtaggtgagacaggotggcaottaggcotcatctcttgotcotggoatcagcatcacaeatoootcaccotttcattctocatgaettgtccatctcctgtagogccag

39 120 79 240 119 360 159 480 167 630 732

FIG.2. Nucleotide sequence of the hamsteraphrodisinprecursorcDNA.The deduced aminoacid sequence is shown in the three-lettercode. The translationalstartcodon and thepolyadenylylationsignalareunderlined. Positions ofPCRprimersaremarked witharrows.Distances of 30

bpareindicated with black dots.

Proc. Natl. Acad Sci. USA 92

(1995)

-ap.----.jp.- .- -. .-W--V-. 'IF... 9

Proc.NatL Acad Sci USA 92 (1995) 2093 complete aphrodisin cDNA sequence (EMBL data library,

accession number X65238) (Fig. 2) wasassembled.

The amplified coding region of the cDNA and the 3' nontranslated region together have a size of 732bp. The cDNA codes for the 151-aa mature aphrodisin and a typical N-terminal 16-aa secretorysignalpeptide (26). Nodeviation of the translated cDNA sequence from the published protein sequenceofmatureaphrodisinwasdetected. The 3' nontrans-lated region is 228 bp long and contains atypical AATAAA polyadenylylation signal (27, 28)atits3'terminus. Compari-sons with known cDNA sequences revealed the highest ho-mology (45%nucleotide sequence identity) with the ROBP cDNA (15). This is in accordance with comparisons at the protein level (40% identity).

Identification of the Aphrodisin GenePromoter Region and Transcription Initiation Site. After screening of 400,000. plaques of the Golden hamster genomic library, 4 independent clones wereisolated. As determined by restriction analysis, 1 clone, compared withtheother 3 clones,containedaslightly shifted part of the same genomic region as the insert. PCR amplifications of partial insert fragments using primers (not listed) derived from the incomplete gene sequence and the 3'-terminal end of the cDNA indicated the cloning of the complete gene in all cases. It has a size of

'6000

bp(data not shown).

Sst I andXba I fragments of the gene were subcloned in pUC18 and partially sequenced. One of the obtained se-quences contains a CCAAT-boxmotif (29) in reverse com-plementaryorientation and a TATA-box motif(forreview see refs. 30 and 31) near potential transcription start sites, fol-lowed in the 3' direction by a region that codes for a typical secretory signal peptide (26) and the first 5 aa of mature aphrodisin (Fig.3). These features stronglyfitthose expected for the5' endofthe aphrodisin gene. Thefirst exonis then interrupted by the 448-bp intron

I,

which is followed by a sequenceexpected for exonII. Our sequencedata allow the conclusion that the aphrodisin gene consists of at least five exonsand four introns (datanot shown).

Fromthepartial gene sequence an antisense primer (AP-7; Figs.1 and3)wasderived andused for primerextension analysis of GoldenhamstervaginalandbrainRNA. Inthefirstcase we obtained a 103-nt extension product (Fig. 4),whereas we ob-tainednonein the second case(negative control).Theextension product correlates withapair of adenosines located29and 30bp downstream from the secondthymidineof theabove-mentioned TATA-boxmotif.Forty-nine base pairs downstream of the first potential transcription startsite, anATG in the correctframe appears, surrounded by nucleotides matching the Kozak se-quence(CCRCCATGG) (32) typicalforhighly efficient eukary-otictranslational start codons(Fig. 1).

Brain agin S Size (Nucleotides) so -0--153 . -_--105 103 * ~- 78 * -4--- 75

FIG.4. Primerextension analysis of hamster aphrodisin mRNA.

Thesize of the extensionproduct obtained by using the primerAP-7 andvaginal totalRNAis calculatedas103bp. Thus, themostprobable transcriptionstartsite is the adenosine inposition 1 of Fig. 3.

Toverify the detected transcription start site by an alter-nativemethod,weperformedcDNA PCRs with twodifferent sense primers located upstream

(RAP-1)

and downstream (RAP-2; Fig. 1) of the identified transcriptionstart site. To enable us to distinguish between amplified cDNA and gene fragments,weused antisenseprimers(AP-8andAP-9;Fig. 1) located behind thefirst intron(Fig.3). Surprisingly,allprimer combinations led tothe amplification of fragmentswhich fit thesizesexpectedfor cDNA.UsingtheinternalprimerAP-7 as aprobe,weverifiedtheamplification ofaphrodisin-specific fragments by Southern blotanalysis(Fig. 5). Despite the fact that we obtainedjust one signal from the primer extension analysis, these results indicate an alternative transcription initiation ofthe aphrodisingene.

Distribution ofAphrodisinmRNA Within the Female Ham-ster Genital Tract. To identify tissues expressing the aphro-disin gene, we analyzed total RNAfrom variousparts ofthe female hamstergenitaltract

(ovaries, fallopian tubes,

uterus, vaginal tissue, and Bartholin'sglands) byNorthern blot anal-ysis. Understringent conditionsweobtainedsingle

signals only

from hamstervaginal tissue and Bartholin'sglands (Fig.

6),

both incomparable intensity.

CAAT-Box gctaatcacattagtttttgagatatgaggtcatacatcatttggcagggccaaagaggaaacactagacagatgtagcttagtgtagatcaaactgattottccgtgaaactccctct * * * 0 -160 -41 +1 TATA-Box **Exn I > RAP-i >* aactatctg±ttaaaagctttgtcagttgagttcttttcggccactcacctcttcgagcttctgt +103 <-Ex2n II < 'AP-7 (ILi

11 Phe Ser LeuAla His AlaGlnAsp Phe Ala Glul

+81 AGT CTG GCTCAT GCT CAG GATTTT GCA GAGgtaaattcatttgct.

000

RAP-2 > MetVal Lys Ile LeuVal Leu Ala LeuVal tggaaaagcaaagtcaggcarccac_ATG.GTAAAGATT CTG GTG CTGGCT TTG GTC

0 O6

tronI 448bp) - I

ILeu Gln Gly LysTrp Tyr Thr Ile Val

...tgttgcttttttcag CT CMA GGA MA TGG TAT ACC AU GTC

.

<- AP-9 AP-8

31 Ile Ala Ala Asp Asn LeuGlu Lys Ile Glu Glu Gly Gly Pro Leu Arg Phe Tyr Phe Arg His Ile Asp Cys Tyr LysAsn Cys Ser Glu Met Glu 62 +141 ATTGCT GCT GAC AAT CUT GAA AAGATA GA GAA GGAGGA CCA CTG AGATTCTATmCGTCATATT GAT TGT TATAAAAACTGC AGTGMAATG GAA +236

FIG. 3. Part of the Golden hamster(Mesocricetus auratus) aphrodisingeneshowingthetranscriptionstartsiteand thepositionof theprimers used forprimerextensionanalysis (AP-7)andPCRamplificationsof5'-terminalportionsof the cDNA(RAP-1, RAP-2, AP-8, AP-9).Theputative CAAT box inreversecomplementary orientation,theTATAbox,thetranscriptionstartsite,andthe Kozaksequencesurroundingthe ATGstart codonareunderlined. Thetwoadenosines whichcorrespondtothetranscriptionstartsiteareadditionallymarkedwith asterisks(themoreprobable

startsite is marked withtwoasterisks).The codon forvaline, differingfromthe codon for leucine in thecaseof hamster(Cricetus cricetus),ismarked withdiamonds. Distances of 30bpareindicated withblack dots. Intron Iwasnotincluded in the numeration.

-40 10 +80 30 +140

Biochemistry:

Mdgert

etaL

Proc.NatL Acad Sci USA 92 (1995) qj Size

_'

- fe'

(bp)

247 ~~~_

~

~

J"'203

154

FIG. 5. PCRSouthernblotanalysis of the transcription start site of the hamsteraphrodisin gene with differentsetsofprimers (seetextand Fig.3). After35PCRcycles, allprimer combinations revealedDNA fragmentsinthe size rangeexpected forcDNA(RAP-1/AP-8,247 bp;

RAP-2/AP-8,198bp;RAP-1/AP-9,203bp;RAP-2/AP-9, 154bp). Southern blotting and hybridization with theinternaloligonucleotide AP-7 verified the amplification of aphrodisin-specific cDNA frag-mentsin allcases.

Under less stringent conditions we performed Northern hybridization experiments with the corresponding tissues of several other species, including rat, pig (data not shown), guineapig (datanotshown), tupaia, andhuman (Fig. 6).We obtained a signal in the expected size range only from rat

Bartholin'sglands. Tupaia vaginal and Bartholin's gland tissue (mixed) and human Bartholin's glands showed long signal

smearsbetween the 28S and 18S rRNA andbelow the 18S rRNA.

DISCUSSION

Since the presented cDNA sequence was determined by sequencing PCR products, we had to identify the region of

Hamster Rat Human Tupaia

0 47Z3

e

-_*--28S

---18S _- AP

FIG. 6. Northernhybridizationwith the radioactive-labeled

aph-rodisincDNAPCRfragmentAP-1/AP-4. Fivemicrograms of total RNA was applied per lane. Hybridization was performed under conditions ofhigh stringency (hamster)

[5X

standardsaline citrate

(SSC)/5x Denhardt'ssolution/0.1% SDS/50% formamideat42°C,

with washes in2x SSC/0.1%SDS twicefor 10min at room temper-ature and 0.5x SSC/0.1% SDS once for 10 min at 65°C] or low

stringency (rat, human, tupaia) (1 M NaCl/50 mM Tris-HCl pH

7.5/0.1%SDS/10%dextransulfateat45°C,withtwo5-min washes in 6xSSC/0.1%SDSat roomtemperature).RNAsamplesfrom hamster

vaginaand Bartholin'sglandsshowastronghybridization signaleven underhighly stringentconditions. Under conditions of lowstringency

asignalintheexpectedsize rangewasobtained fromratBartholin's

glands.RNAfrom mixed tissue oftupaia vaginaandBartholin'sglands

andfrom human Bartholin'sglands gave long signalsmears.Under conditions of low stringency rRNA from all tissues showed

cross-hybridizationwith the aphrodisin probe. Positions of 28S and 18S rRNAand aphrodisin (AP)RNAareindicated.

transcription initiation by primer extension analysis. For this purpose,and in order to clone the corresponding gene, we have screened a Golden hamster genomic library in A phage. Assuming that the differences between aphrodisin gene-specific sequences of hamster (Cricetus cricetus) and Golden hamster (Mesocricetus auratus) are only slight, we havechosen a library of the latter species because of its easy commercial availability. Indeed, during the not yet completed

sequence

determination of the gene, we have detected just one deviating nucleotide in the region coding for the putative secretory signal peptide of the aphrodisin precursor. This deviation leads to a predicted valine in position 6 of the Golden hamster aphrodisin precursor instead of leucine as in the case of the hamster (Fig. 3).

By primer extension analysis, we obtained an extension product which correlates with a pair of adenines (see Figs. 3 and4), identifiedaspotential transcription start sites by both theiridealdistancefrom a TATA-box motif and their location within a polypyrimidine tract. In two independent experi-ments, weobtainedasingle extensionproduct which has been determined as 103 nt by means of logarithmic regression. These resultssuggestthat the first adenine of the mentioned pair represents the real transcription initiation point. A further indicationforthe correct detection of this locus isthefactthat thefirst ATG which follows in the 3' direction is surrounded byaKozak sequence (see above) which is typical for highly efficient eukaryotic translational start points (32). It is the beginningof the region codingfor aputative 16-aa secretory signal peptide,which ends before the codon for the first amino acid(Gln) of matureaphrodisin. Comparison ofthe deduced signal peptidetothat from ROBP revealedaslight homology, especiallywithin the first 9 aa (Fig. 7). However, the PCR experiments indicated the existenceof atleastoneadditional alternative transcription initiation site. This phenomenon is alsoknownforsomeother genes-for instance, the renin gene (33). Since no alternative transcript has been detected by primer extension, itmustappear in a much lower concentra-tion thanthe predominant transcript.

Northern blot analysis revealedvaginaltissue and Bartho-lin's glands (glandulae vestibulares majores) as the main aphrodisin mRNA-synthesizingtissuesofthe female hamster genitaltract.Taking intoaccountthat weused totalRNAand that only relative short exposure times were necessary to obtain strongsignals,theexpressionofthegene in thesetissues wouldseem tobe abundant.Whetherthe expressioninvaginal tissue is due to an activity of the glandulae vestibulares minores, whicharehomologoustoBartholin'sglands butmuch smaller, has to beinvestigated byin situ hybridization exper-iments orimmunocytochemistry.To totally clarifythe tissue distributionof aphrodisin-specificmRNA, other tissues also have to be analyzed, preferably by using the more sensitive poly(A)+ RNA Northern blot or the nuclease S1 protection assay. Becauseaphrodisinactsvia thevomeronasal organ(34) and is probably a carrier for an additional ligand, salivary glandsarealso potential candidatesasloci of synthesis.

Less stringent Northern hybridizations revealed signals from Bartholin'sglandsofotherspecies, includingrat,tupaia

(mixed

vaginal and Bartholin'sgland tissue), and human. In the rat,the signal obtainedwasinthesamesize range as for aphrodisinmRNA,suggestingthesynthesisof an aphrodisin-related

protein.

We nowhavetodeterminebycDNAcloning and sequence analysis whether the signal is due to a novel mRNAor to anexpression of the genefor ROBP normally

Aphrodisin I7V LLA|VFsL- A

ROBP FFLLI V.:A L G V S CH E N L

FIG. 7. Comparisonof theproaphrodisin signal peptidewiththe ROBPsignal peptide(15).Identicalregionsareboxed.

Proc. NatLAcad Sci. USA 92 (1995) 2095 occurring in nasal mucosa (14, 15). In the case of Tupaia

belangeri (a putative primate) and humans,weobtained long signalsmearsfromBartholin's glands instead of distinctsignal bands. We first supposed this to be due to experimental artifacts, but this stands incontradiction to the fact that only specimens which contained RNA from Bartholin's glands behaved in this way. Further, the RNA used was absolutely undegraded and the signals cannot be explained by effects of concentrationasin the caseof28S and 18S rRNA, becauseno

additional dominant bands corresponding to the signalswere

visiblein the ethidiumbromide-stainedgel(datanotshown). Signals from vaginal tissue could not be obtained from species other than hamster or Golden hamster.

Insummary, the results of Northern hybridization indicate highexpression of theaphrodisingene in hamster Bartholin's glands and vaginal tissue and suggest the synthesis of mRNA species with at least a slight homology to the aphrodisin mRNA in Bartholin's glands of rat, tupaia, and human. Whetherthese mRNAspecies are related tolipocalins acting aspheromone carriers has to beinvestigated. Indicationsfor the occurrence ofprimatesexpheromonesinvaginal discharge already exist (35). Also, the occurrence of the vomeronasal organ in humans (36, 37) and its ability to perceive putative pheromones (38) have been demonstrated. The cloned aph-rodisin cDNAfragmentsaswell asthegene will now serve as potentially useful tools for the investigation ofaphrodisin/ lipocalin-related pheromone systems and their regulation in mammals.

Wethank G.Schmeding,H.Stober,H.Schindler, andW. Posselt for theirexcellenttechnicalassistance;I.Kunstyr,A.Bub,M.Meyer,and E.Fuchs for tissue specimensand helpful advice; and G. Klock for the partialsequenceanalysis. Thiswork wassupportedbyagrantfrom the DeutscheForschungsgemeinschaft (Ma1605/1-1).

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