Proc. Natl. Acad. Sci. USAVol. 89, pp. 12145-12149, December 1992Biochemistry

Cloning and expression of a cDNA encoding the transporter oftaurine and /8-alanine in mouse brain

(neurotransmitters/membrane protein/moecular cloning)

QING-RONG Liu, BEATRIZ L6PEZ-CORCUERA, HANNAH NELSON, SREEKALA MANDIYAN,AND NATHAN NELSON*Roche Institute of Molecular Biology, Roche Research Center, Nutley, NJ 07110

Communicated by Leon A. Heppel, September 8, 1992 (receivedfor review July 30, 1992)

ABSTRACT A taurine/fl-alanine transporter was clonedfrom a mouse brain cDNA library by screening with a partialcDNA probe of the glycine transporter at low stringency. Thededuced amino acid sequence predicts 590 amino acids withtypical characteristics of the sodium-dependent neurotrans-mitter transporters such as sequence homology and membranetopography. However, the calculated isoelectric point of thetaurine/fi-alanine transporter is more acidic (pI = 5.98) thanthose (pI > 8.0) ofother cloned neurotransmitter transporters.Xenopus oocytes Injected with cRNA of the cloned transporterexpressed uptake activities with K. = 4.5 FM for taurine andK. = 56 FM for (3-alanine. Northern hybridization showed asingle transcript of 7.5 kil9bases that was highly enriched inkidney and distributed evenly in various parts of the brain. Insitu hybridization showed the mRNA of the taurine/(3-alaninetransporter to be localized in the corpus callosum, striatum,and anterior commisure. Specific localization ofthe taurine/p-alanine transporter in mouse brain suggests a- potential func-tion for taurine and (3-alanine as neurotransmitters.

Taurine is one of the most abundant amino acids in brain (1).Free taurine is found at millimolar concentrations in excitabletissues, especially those which generate oxidants (2). Taurineis supplied to mammalian brain and other tissues by cysteinemetabolism and diet. It was reported that taurine is taken upinto cells by two transport systems: a low-affinity (Km ofabout 2 mM) but high-capacity uptake system and a high-affinity system (Km of 50-80 AuM) having low capacity foruptake (3-6). The physiological role of taurine in the centralnervous system remains obscure (7-9). It has been proposedthat taurine plays a role in osmoregulation in certain braincells (9-11). Intraperitoneal administration of distilled waterelevated the concentration of taurine in dialysates from therat pyriform cortex (12). This could be due to either increasedrelease or inhibited uptake of taurine. Recent attempts tolocalize the sites of taurine biosynthesis in the brain demon-strated immunostains of cysteine sulfonate decarboxylase inrows like oligodendrocytes and cells around the Purkinje cellsin the cerebellum (7). Activity of the enzyme was alsodetected in glial cell fractions enriched with oligodendrocytesand astrocytes. However, the high taurine concentrations inbrain suggest an important role for transport systems along-side the biosynthesis of taurine. Indeed a decreased Km andVmax of taurine uptake was implicated in retinitis pigmentosa(13, 14).The function of 3-alanine in neurotransmission is also not

clear. It was shown that B3-alanine desensitizes glycine re-sponses and partially reduces y-aminobutyrate (GABA)-evoked currents (15). Application of glycine, .3-alanine, ortaurine onto isolated neurons from rat medulla oblongata and

hippocampus induced an increase in chloride permeability ofthe cellular membranes (16, 17). Responses to all of thosesubstances were blocked by external strychnine, indicatingthat they interacted with the same system. However, nospecific receptors for taurine or f-alanine could be identified.Moreover, /3-alanine and taurine uptake systems could beshared by other neurotransmitters. It was reported that alow-affinity GABA transporter accumulates taurine andf3-alanine (18). The taurine transporter takes up /3-alaninewith high efficiency in neuronal and astroglial cells in culture.In this communication we report on the cloning and expres-sion of a cDNA encoding a sodium-dependent high-affinitytaurine and (3-alanine transporter.t

EXPERIMENTAL PROCEDURESCloning and Sequencing. A mouse neonatal brain cDNA

library in the Uni-Zap vector (Stratagene) was screened atlow stringency with a 1-kilobase (kb) cDNA fragment startingat the N terminus of the glycine transporter clone (19, 20).Hybridization was performed at 420C with Denhardt's solu-tion containing 50% (vol/vol) formamide and 10% (wt/vol)dextran sulfate, and washing was at 420C with standard salinecitrate containing 0.1% SDS (20). About 106 plaques yielded56 positive clones from which the pBluescript plasmid (Strat-agene) was excised according to the manufacturer's instruc-tions and sequenced with T3 primer by the dideoxynucleotidetermination method (22). Sequence analysis with the Univer-sity of Wisconsin Genetics Computer Group software indi-cated that most positive clones encoded either a partial or afull-length taurine/f3-alanine transporter. A positive clonewith a 2.6-kb DNA insert was selected for sequencing ofbothstrands, using oligonucleotide primers.RNA Isolation and Northern Blot Analysis. Total cellular

RNA was isolated from mouse liver, kidney, cerebellum,cerebral cortex, brainstem, and the rest of the brain by usingan RNA isolation kit from Stratagene. RNA samples (40 ,ug)from the various tissues were loaded onto a formaldehyde/agarose gel. Equal loading ofRNA was checked by the sameintensity of ribosomal RNA after ethidium bromide stainingand hybridization with proteolipid cDNA of vacuolar protonATPase. Northern blot hybridization was carried out at highstringency as described (19, 20).Expression of the Mouse Brain Taurine Transporter in

Xenopus Oocytes. cRNA was synthesized from a linearizedNTT9 clone by phage T3 RNA polymerase. Microinjection ofXenopus oocytes and uptake measurement were performedas described (21). If not specified 1 ,uM nonradioactivetaurine was present in the uptake experiments.

Abbreviation: GABA, -t-aminobutyrate.*To whom reprint requests should be addressed.tThe sequence reported in this paper has been deposited in theGenBank data base (accession no. L03292).

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The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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12146 Biochemistry: Liu et al.

In Situ Hybridization. A Kpn I fragment at the 3' end ofNTT9 was subcloned into pBluescript and the plasmid wassubsequently digested with Xho I. The linearized plasmid wasused as a template to synthesize the antisense [a-35S~thioJ-UTP-labeled RNA probe by in vitro transcription from thephage T7 promoter. Paraffin sections of fixed mouse braindivided into fore, mid, and hind thirds were obtained fromNovagene (Madison, WI). Hybridization and autoradiogra-phy of the slides were performed according to the manufac-turer's instructions with the SureSite in situ hybridization kit(Novagene, Madison, WI). After the emulsion was devel-oped, the hybridized slides were counterstained with hemo-toxylin and eosin. The positive and negative controls wereperformed by using testis-specific probe hybridized withtestis and brain slices, respectively.

RESULTSTo clone the taurine transporter, a fetal mouse brain cDNAlibrary was screened at low stringency with 32P-labeledcDNA encoding the glycine transporter (19). Numerouspositive clones were isolated and the pBluescript plasmid wasexcised and subjected to DNA sequencing using a T3-promoter oligonucleotide as primer. Two of the clones,NTT9 and NTT15, containing a cDNA fragment of 2.5 kb,had sequence homology with the known neurotransmittertransmitters but were not identical to any of them (21).Expression of these two clones in Xenopus oocytes revealedthat they encoded a high-affinity taurine transporter. Fig. 1shows the nucleotide and deduced amino acid sequences ofthe taurine transporter (TAUT). The predicted initiatorcodon is the first methionine codon in the sequence, whichalso exhibits a strong consensus site for translation initiation(23). The open reading frame encodes a protein of 590 aminoacids with a calculated molecular mass of 65,878 Da. Thehydropathy plot predicts 12 transmembrane helices and alarge hydrophilic loop between helices 3 and 4. This loopcontains three potential glycosylation sites. The predictedstructure is superimposable with all other members of theneurotransmitter transporter family (19, 21, 24-32). The mostunusual feature of the taurine transporter is its calculatedisoelectric point, estimated to be at pH 5.98. The calculatedisoelectric points of all known proteins of the family areabove pH 8. Alignment of the amino acid sequence of thetaurine transporter with other members of the family showed40%o identity with the glycine transporter (19, 32), 42-46%oidentity with catecholamine transporters (26-30), 48% iden-tity with proline transporter (31), and 52%6 and 62% identitywith the GABA transporters GAT1 and GAT2 respectively(21, 33). Similar amino acid homologies among taunne,glycine, GABA, and catecholamine transporters suggest thatall evolved from a common ancestor and diverged from eachother at approximately the same evolutionary time (21). Thepresence of homologous proteins in Drosophila melano-gaster and Caenorhabditis elegans suggests that this eventtook place early in the evolution of the nervous system.The function of TAUT was determined by the expression

of its synthetic RNA in Xenopus oocytes. Oocytes injectedwith the synthetic RNA ofTAUT accumulated up to 500-foldmore [3H]taurine than control uninjected oocytes or thoseinjected with synthetic RNA of the glycine or GABA trans-porters. Except for ,-alanine, none of the other anmino acidstested had any effect on taurine transport. Xenopus oocytesinjected with synthetic TAUT RNA accumulated [3H]-alanine up to 50-fold over control uninjected oocytes. There-fore, TAUT transports both taurine and P-alanine. Fig. 2shows the kinetics of taurine and ,-alanine transport intoTAUT-injected oocytes. Eadie-Hofstee plots revealed anapparent Km of 4.5 ,uM for taurine and 56 p1M for 3-alanineuptake. Thus, the affinity ofTAUT for taurine is greater than

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Proc. Natl. Acad. Sci. USA 89 (1992)

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GGCACACGGCCTGAAGATGAGGCGGACGGGAAGCCCCCTCAGAGGGAGAAGTGGTCCAGCG T R P E D E A D G X P P Q R E K N S SAAGATCGACTTTGTGCTGTCTGTGGCCGGAGGCTSCGTGGGTTTGGGCAACGTCTGGCGTK I D F V L S V A G G F V G L G N V W RTTCCCGTACCTCTGCTACAAAAATGGTGGAGGTGCGTTCCTCATACCGTATTTTATTTTCF P Y L C Y K N G G G A F L I P Y F I FCTGTTTGGGAGCGGCCTGCCTGTGTTTTTCTTGGAGGTCATCATAGGCCAGTACACATCAL F G S G L P V F F L E V I I G Q Y T SGAAGGGGGCATCACCTGCTGGGAGAAGATCTGTCCTTTGTTCTCTGGCATTGCGTACGCAE G G I T C W E K I C P L F S G I A Y ATCCATCGTCATTGTGTCCCTCCTGAACGTGTACTACATCGTCATCCTGGCCTGGGCCACAS I V I V S L L N V Y Y I V I L A W A TTACTACCTATTCCACTCTTTCCAGAAGGATCTTCCCTGGGCCCACTGCAACCATAGCTGGY Y L F H S F Q R D L P W A H C N H S WAACACACCACAGTGCATGGAGGACACCCTGCGTAGGAACGAGAGTCACTGGGTCTCCCTTN T P Q C M E D T L R R N E S R W V S LAGCACTGCCAACTTCACCTCACCCGTCATCGAGTTCTGGGAGCGCAATGTGCTCAGCCTGS T A N F T S P V I E F W E R N V L S LTCCTCCGGAATCGACAACCCAGGCAGTCTGAAATGGGACCTCGCGCTCTGCCTCCTCTTAS S G I D N P G S L K W D L A L C L L LGTCTGGCTCGTCTGTTTTTTCTGCATCTGGAAGGGTGTTCGATCCACAGGCAAGGTTGTCV W L V C F F C I N K G V R S T G R V VTACTTCACCGCTACTTTCCCGTTTGCCACGCTTCTGGTGCTGCTGGTCCGTGGACTGACCY F T A T F P F A K L L V L L V R G L TCTGCCAGGTGCTGGTGAAGGCATCAAATTCTACCTGTACCCTGACATCAGCCGCCTTGGGL P G A G E G I K F Y L Y P D I S R L GGACCCACAGGT TGGATCGACGCTGGAACTCAGATATTCTTTTCCTACGCAATCTGCCTGD P Q V WI D A G T Q I F F S Y A I C LGGGGCCATGACCTCACTGGGAAGCTATAACAAGTACAAGTATAACTcGTACAGGGACTGTG A M S' S L G S Y N K Y K Y N S Y R D CATGCTGCTGGGATGCCTGAACAGTGGTACCAGTTTTGTGTCTGGCTTCGCAATT TTTT CCI L L G C L N S G T S F V S G F A I F SATCCTGGGCTTCATGGCACAAGAGCAAGGGGTGGACATTGCTGATGTGGCTGAGTCAGGTI L G F M A Q E' Q G V D I A -D V A 'E -S GCCTGGCTTGGCCTTCATTGCCTACCCAAAAGCTGTAACCATGATGCCGCTGCCCACCTTTP G L A F I A Y P K A V T M CP L P T FTG.GTCTATTCSGTTTTTCATTASGCTCCTCTTGCTTOSACTGCAGCCAGTTTGTTGAAGTCGAAGGA

IG LTGATCTTTACCCGTCCTTCCTAAGCACGGGTJD L Y P ST L R x G YGTGTAGCATCAGCTACCTGCTGGGCTG-C>'Ilr s e 'VTTt t a! T u

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ACCCGATTGGGCAATTGGGCTGGG:TTTCCTCCATGCTGTGTATCCCCTTGGTCATTGTCATCI

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FiG. 1. Nucleotide and deduced amino acid sequences of cDNAencoding the taurine and P-alanine transporter TAUT. The cDNAwas cloned from the mouse brain library and both strands sequencedby using exonuclease III digestion (22) and oligonucleotide primers.

the affinity of GAT1 for GABA. The affinity of TAUT forp-alanine is close to the published values observed for(-alanine uptake into isolated synaptosomes (34). The effectof anions and cations on taurine and p-alanine transport isshown in Fig. 3. The presence of sodium ions in the externalmedium was necessary for uptake activity of the transporter.Chloride was required for optimal taurine and P-alanineuptake, and only bromide was found to be an efficientreplacement for chloride. Nitrate supported relatively lowactivity of the transporter, and the rest of the anions testedgave 5-10%6 of control activity with chloride. Since bothtaurine and (3-alanine are taken up by the same transporterand changes in medium composition similarly affect theuptake of both substances, the tissue-specific expression ofthe transporter should be important for regulating taunneactivity in different locations of the brain.The pharmacology of TAUT was analyzed with the avail-

able substances reported to inhibit taurine uptake in brainslices and isolated cells. Table 1 depicts the effect of thesechemicals and several other substances on taurine uptakeinto oocytes injected with synthetic cRNA encoding mouse

TAUT. In the presence of 2 ,uM taurine, significant inhibitionwas observed by 100 juM 3-guanidinopropionic acid, L-2,3-diaminopropionic acid, or (3-alanine and 5 ;&M hypotaurine.

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FIG. 2. Kinetics of taurine (Upper) and P-alanine (Lower) uptakeintoXenopus oocytes injected with synthetic RNA ofTAUT. Uptakeof [3H]taurine and [3H]P-alanine into cRNA-injected oocytes wasassayed at indicated concentrations. The oocytes were incubatedwith the radioactive substances for 30 min at room temperature.Values obtained under similar conditions with uninjected oocyteswere subtracted from corresponding injected samples. Eadie-Hofstee analysis is depicted in Insets.

All other substances tested at 100 AM, including GABA,cysteine, and the rest of the common amino acids, as well ascarnitine and other candidates for inhibition, had no effect ontaurine uptake. This observation points out the remarkablespecificity of the TAUT transporter, which can transporttaurine, hypotaurine, and P-alanine but none of the otheramino acids.Northern blot analysis with RNA isolated from various

parts of the brain revealed a ubiquitously distributed singletranscript of about 7.5 kb (Fig. 4). As there is no polyade-nylylation signal in this cDNA clone, we assume that thepoly(A) stretch is in the middle of the 3' nontranslated regionofthe mRNA. Large amounts ofa similar-size transcript werefound in RNA isolated from the kidney. Liver contained avery small but detectable amount of transcript of the samesize. Within the brain the TAUT transcript appeared to bemore abundant in the cerebellum and cerebral cortex. Tran-scripts of some neurotransmitter transporters are readilydetected in the kidney, suggesting additional functions for

Taurine

s sE E w-CD.-Z -L*

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these transporters (19). Recently it has been reported thattranscripts of the glycine transporter are not present inkidney and cerebellum (32). We readily detected highamounts of transcripts in these two tissues (19). Thesefindings were substantiated by cloning and sequencing anidentical glycine transporter from the kidney cDNA library(unpublished work). The functions of neurotransmitter trans-porters in the kidney are not clear, and specific antibodies arerequired to obtain a better understanding of their function.

Localization of the taurine transporter in mouse brain wasexamined by in situ hybridization of its antisense probe inbrain slices (Fig. 5). In the cerebellum TAUT was identifiedin the white matter where the Purkinje cell axons go to thedeep nuclei. The Purkinje cells were not stained for TAUTRNA. The brainstem and the pontine fibers were heavilystained. Ventral and dorsal cochlear nuclei were positive, aswell as the cochlear nerve (data not shown). We also detectedTAUT mRNA in the corpus callosum and the anteriorcommisure. Blood vessels were negative for TAUT RNA. Ingeneral, TAUT mRNA was confined to specific locations in

Table 1. Pharmacology of the taurine transporterAddition

None/3-Guanidinopropionic acidDiaminopropionic acid

DL-Carnitine

Cysteinesulfinic acid

Hypotaurine

P-Alanine

P-tuamdinoethanesuiiomc

Conc., AM % control

100100500100500100500

2102050100

acid 1050100

100 + 1362 ± 1139± 522 ± 190 ± 1585 ± 1790 ± 1279 ± 1083 ± 735 ± 1311 ± 1064 ± 631 ± 1580 ± 1254 ± 629 ± 1

Compounds were tested for their ability to inhibit taurine transportas described in the Experimental Procedures. Compounds that didnot inhibit at a concentration of 100 uM included DL-y-amino-(-hydroxybutyric acid, y-hydroxybutyric acid, DL-a-hydroxybutyricacid, e-aminocaproic acid, 2-mercaptoethylamine (cysteamine), ami-noisobutyric acid, L-anserine, GABA, /3-alanine methyl ester, L-car-nosine, L-cysteic acid, ethanolamine, P-N-methylamino-L-alanine,nicotinic acid, 3-aminobenzoic acid ethyl ester, 3-aminocrotonic acidmethyl ester, O-phosphoethanolamine, L-vinylglycine (2-amino-3-butenoic acid), L-homocarnosine, chlorpromazine, chloroquine, and3-amino-1-propanesulfonic acid (homotaurine).

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-8 kb

FIG. 4. TAUT mRNA in various tissues. Total RNA was pre-

pared from various mouse brain parts and tissues. About 40 ,ug ofRNA was applied to each well in a standard formaldehyde/agarosegel (20). Relative amounts ofRNA in each sample were monitored byamounts of stained rRNA. Lanes: 1, liver; 2, kidney; 3, cerebellum;4, cerebral cortex; 5, brainstem; 6, the rest of the brain.

the white matter and was mostly glial. In contrast with theubiquitous distribution of taurine in the brain, the specificlocalization of the taurine transporter may reveal its specificfunction in the various brain parts. Immunocytochemistrywith specific antibodies against the taurine transporter shouldhelp in the fine localization of the transporter and may revealits function in neurotransmission.

DISCUSSIONWe have isolated a cDNA clone from a mouse brain libraryencoding a high-affinity taurine transporter that also trans-ports (3-alanine. Structural features of the taurine transporterare virtually identical to those of the other cloned sodium-dependent neurotransmitter transporters (24-32). A notabledifference is the low isoelectric point, pI = 6, that wascalculated from the predicted amino acid sequence of thetaurine transporter. Amino acid identity among the variousneurotransmitter transporters sequenced so far is 40-50%o,and among the predicted 12 transmembrane domains onlydomains 1, 2, 5, and 6 are highly conserved. However, onlyabout 100 amino acids were totally conserved in all knownneurotransmitter transporters, and these residues are scat-tered throughout the sequence of the proteins. The proposedleucine zipper in the second membrane domain is not con-served in all transporters, and the function of such an aminoacid organization within membrane domains is not known.Site-directed mutagenesis may shed light on the function ofthe conserved domains in neurotransmitter transporters.Recently we isolated cDNA clones encoding four differentGABA transporters in mouse brain (refs. 21 and 33; andunpublished work). Analysis of the percent identity of theamino acid sequences of the various GABA transporters andthe taurine transporter suggested that all of these genes

evolved from a common ancestor closely related to thecurrent taurine transporter.

Expression of TAUT synthetic RNA in Xenopus oocytesinduced taurine uptake at relatively high affinity. The affinityof the taurine transporter was greater than that observed forGABA and glycine by their corresponding transporters (19,21, 24). The Km of 5.6 juM observed with the expressedtransporter is close to the value reported for the high-affinitytaurine transport activity in brain slices, cultured neurons andastrocytes, and synaptosomes (2, 18, 35). The taurine trans-porter also transports /3-alanine at relatively high affinity,with a Km of 56 ,uM. This value is also close to the reportedaffinity for /3-alanine uptake into neurons or astrocytes andsynaptosomal preparations (18, 34). Competition betweenGABA and f-alanine and between taurine and /3-alanine wasreported. It was concluded that one of the GABA uptakesystems, presumably the glial transporter, transports .3-ala-nine (34). The cloned high-affinity GABA transporter (GAT1)does not transport /-alanine, but the recently cloned GABAtransporter (GAT2) transports (3-alanine at very low rates(33). Therefore, it can be concluded that P-alanine transportis shared by both GABA and taurine transport systems.Northern hybridization showed the presence ofTAUT in all

brain parts tested and in the kidney. Similar distribution wasobserved for other amino acid transporters, including GAT1,GAT2, and the glycine transporter (19, 25, 33). In situ hybrid-ization revealed the presence of high levels ofTAUT mRNAin specific locations in the brain. The significance of thelocalized high-affinity taurine transporter is not apparent.Taurine is present at millimolar concentrations in most of thebrain. The presence of the transporter may have a dualfunction, either to decrease its concentration in certain areasor to accumulate taurine at high concentrations in particularcells.The physiological role of taurine in the central nervous

system is not clear, mainly because it is present in all of thebrain tissues at relatively high concentrations (2). Recently,the sites of taurine production in various parts of the brainwere studied by immunocytochemistry with an antibodyagainst cysteine sulfinate decarboxylase (7). In the cerebel-lum relatively high concentrations of the enzyme were foundin the white matter in typical structures of oligodendrocytes,a few cells in the granular layer, and around the Purkinjecells, presumably in Golgi epithelial cells. It was concludedthat the glial localization of the taurine biosynthetic enzymedoes not support its involvement as a neurotransmitter.However, due to taurine's abundance in the brain, the sitesof taurine biosynthesis might not reveal its function. Aconventional neurotransmission is based on the synthesis ofthe neurotransmitter in presynaptic cells, accumulation intosynaptic vesicles, quantal release following stimulation,binding to a specific receptor in postsynaptic cells, andreuptake into the presynaptic cells by a sodium-dependentsystem. However, there may be alternatives for the conven-tional mechanism. For example, the neurotransmission bynitric oxide lacks a specific membrane receptor and reuptakesystem (36). The 6-sec half-life of nitric oxide presents aproblem in its function as a neurotransmitter. In its lifetimenitric oxide may diffuse rapidly to relatively long distances,in contrast with conventional neurotransmission operating atshort distances. The combination of this property and thepotential toxicity of nitric oxide should be controlled byneutralizing substances in cells that are located near produc-tion sites of nitric oxide but have to be protected from itstoxicity. It was shown that taurine concentrations are high incells and tissues that produce oxidants and radicals (2, 37,38), and it protects against radiation damage (39). We proposethat taurine may function as a scavenger of nitric oxide in thebrain, acting as an inhibitory neurotransmitter coupled tonitric oxide action.

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Proc. Natl. Acad. Sci. USA 89 (1992) 12149

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FIG. 5. In situ localization ofRNA hybridizing to TAUT syn-thetic antisense RNA of TAUT.Mouse brain sections were hybrid-ized with an 35-labeled syntheticRNA complementary to the 3'nontranslated region of TAUTcDNA. Hybridization with thecorresponding "S-labeled senseRNA gave no positive signals. Pic-tures of bright (Left) and dark(Right) fields were taken. (A)Coronal section of corpus cok..sum. (B) Sagittal section of cere-bellar lobes. (C) Sagittal section ofcorpus collosum and caudate-putamen. (x45.)

A~~~~~A%

We thank Dr. R. Huxtable for his generous gift of 3-guanidino-ethanosulfonic acid. We thank Dr. Delia I. Lugo and Ms. Ying Qinfor their help with the in situ hybridization. B.L.-C. is a NorthAtlantic Treaty Organization fellowship recipient.

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20. Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989) MolecularCloning: A Laboratory Manual (Cold Spring Harbor Lab., ColdSpring Harbor, NY), 2nd Ed.

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