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CTX, a Xenopus thymocyte receptor, defines amolecular family conserved throughout vertebrates

Isabelle Chretien1, Anne Marcuz1, Michele Courtet1, Kaisa Katevuo2, Olli Vainio2,Joan K. Heath3, Sara J. White3 and Louis Du Pasquier1

1 Basel Institute for Immunology, Basel, Switzerland2 Turku Immunology Center, Department of Medical Microbiology, University of Turku, Turku,

Finland3 Ludwig Institute for Cancer Research, Melbourne Branch, Parkville, Australia

CTX, a cortical thymocyte marker in Xenopus, is an immunoglobulin superfamily (Igsf) mem-ber comprising one variable and one constant C2-type Igsf domain, a transmembrane seg-ment and a cytoplasmic tail. Although resembling that of the TCR and immunoglobulins, thevariable domain is not encoded by somatic rearrangement of the gene but by splicing of twohalf-domain exons. The C2 domain, also encoded by two exons, has an extra pair of cyste-ines. The transmembrane segment is free of charged residues, and the cytoplasmic tail (70amino acids) contains one tyrosine and many glutamic acid residues. ChT1, a chickenhomologue of CTX, has the same structural and genetic features, and both molecules areexpressed on the thymocyte surface. We cloned new mouse (CTM) and human (CTH) cDNAand genes which are highly homologous to CTX/ChT1 but not lymphocyte specific. Similar-ity with recently described human cell surface molecules, A33 antigen and CAR (coxsackieand adenovirus 5 receptor), and a number of expressed sequence tags leads us to proposethat CTX defines a novel subset of the Igsf, conserved throughout vertebrates and extendingbeyond the immune system. Strong homologies within vertebrate sequences suggest thatthe V and C2 CTX domains are scions of a very ancient lineage.

Key words: Immunoglobulin superfamily / Evolution / V domain

Received 13/7/98Accepted 1/9/98

[I 18622]

Abbreviations: Igsf: Immunoglobulin superfamily EST:Expressed sequence tag aa: Amino acid(s) CDR:Complementarity-determining region

1 Introduction

About one third of the leukocyte receptors described invertebrates and many of the cell adhesion molecules inother tissues make use of immunoglobulin superfamily(Igsf) domains. These are classified into three major cate-gories: (1) the primitive constant C2 and I-types foundthroughout the animal kingdom in many adhesion mole-cules and co-receptors of the immune system; (2) themore recent constant C1 type present only in speciesthat somatically rearrange their antigen-specific receptorgenes and (3) the variable V type either inherited from thegerm line or obtained after somatic rearrangement. Thedomains are defined by the number and position of theirbeta strands and loops [1, 2].

We recently described CTX, a gene in Xenopus encodinga molecule that lies at the cross-roads between theantigen-specific receptors and adhesion molecules [3]. Acortical thymocyte marker in Xenopus, it contains onedistal V domain with a J-like segment which is, however,generated without somatic rearrangement, one C2domain with four cysteines, one transmembrane seg-ment and a cytoplasmic tail of 70 amino acids (aa). Thisarchitecture is encountered in other Igsf members [4],but some structural features of the CTX protein domainsand of the gene are unique. These specific characteris-tics may provide a vital link in reconstructing the phylo-genetic relationships among members of the Igsf andshed light on the origin of the antigen-specific receptors.To pursue this idea further, we looked for homologues ofCTX in vertebrates. Several chicken molecules detectedby mAb had similar but not identical tissue distributionsand comparable molecular mass [5–8]. ChT1 [7], alsorecognized by the mAb T10A6 [8], turned out to be achicken homologue of CTX [9, 10]. The identification inthis study of a number of related mammalian genesleads us to propose that CTX is the prototype of a newsubset of the Igsf conserved throughout vertebrates.

4094 I. Chretien et al. Eur. J. Immunol. 1998. 28: 4094–4104

While the function of these molecules is largelyunknown, CTX has been implicated in the control of celldivision in a Xenopus tumor cell line [11, 12], and ChT1,which appears to play a role in T cell differentiation [10],is expressed by recent thymus emigrants [13].

2 Results

2.1 Avian and mammalian moleculeshomologous to CTX

As already mentioned, a cDNA from chicken recentlycloned by Katevuo et al. (ChT1), which encodes a mole-cule strongly expressed in the thymus, was found to be ahomologue of CTX [9]. CTX and ChT1 sequences are42 % concordant (Fig. 1).

Human and mouse cDNA clones were isolated asdescribed in Sect. 4.2. They were called CTH and CTM,respectively, by analogy to CTX, to show that theybelong to a family of molecules, even though the tissuedistribution does not always correlate with that of CTX.The deduced aa sequences are 33 % concordant withCTX (Fig. 1).

Two new mammalian molecules [14–16] share structuralproperties with CTX. The human A33 antigen moleculecomprises a V type Ig domain followed by a C2 type, amembrane segment and a 62-aa cytoplasmic tail [14]. Itis expressed only by intestinal epithelial cells and colo-rectal tumors. The A33 antigen is 27 % similar to CTXand 29 % similar to CTH (Fig. 1). The mouse and humanCAR molecules (CARm and CARh) act as receptors forcoxsackie viruses and adenoviruses [15, 16]. They sharethe same structure as CTX though their cytoplasmicdomains are longer (108 aa). CARh is 28 % similar toCTX, 30 % similar to CTH, and 30 % similar to the A33antigen (Fig. 1).

In addition we identified at least 77 expressed sequencetag (EST) sequences homologous to regions of the CTX/CTH, A33 antigen, ChT1 and CAR sequences. Amongthese, 14 sequences were partial sequences of theabove molecules, 12 displayed only a weak homology,and 51 were highly homologous. Re-sequencedselected EST clones or cDNA clones provided longersequences, and we could define five novel types ofmammalian CTX family members: AA386 mouse (m) andits human (h) homologue AA318h, W117m and R336h;AA445m and AA406h; AA497m; and, not strictly belong-ing to the CTX family, T870h. For each pair of sequences,the homology between the mouse and humansequences is 80–90 %. Thus, in the alignments shown inFigs. 1–3, either the human or the mouse homologue is

shown. AA497m is closer to CTX than CTH and CTM. Itcould correspond to the ortholog of CTX.

2.2 Structural definition of the CTX family

CTX, ChT1, A33 antigen and CAR share specific proper-ties at the biochemical, cDNA and genomic levels. Whilethe molecular masses of the predicted proteins are allnear 33 kDa, the molecular masses of the mature mole-cules vary from 43 to 63 kDa [3, 6, 8, 16, 17] due to differ-ential glycosylation. The cDNA encoding these mole-cules (Fig. 1 A) as well as CTH and CTM all predict a sig-nal peptide of about 20 aa, an extracellular Ig-likedomain of the V type followed by a second of the C2type, a transmembrane region devoid of charged resi-dues, and a cytoplasmic tail of 60–120 aa, except forAA405h and T870h where it is only 36 aa.

2.2.1 First extracellular domain: a V-type Ig-likedomain with TCR and Ig features

N-terminal sequences have been obtained for the CTX[3], ChT1 [10] and A33 antigen [18] native proteins,enabling the first position to be assigned. Thesequences, which are of similar size (113–119 aa) with73 to 77 residues between the two cysteines, containbeta strands and complementarity-determining region(CDR) loops in positions typical for a V domain (Fig. 1 B).The key residues of the backbone of Igsf V domains [2]are conserved among all CTX family members (asteriskin Fig. 1B). In some cases the G strands have the fea-tures of the Ig and TCR J segments. Notably, CTX andCTH/CTM contain the typical GXG bulge of most J ele-ments which is thought to be important for dimericreceptors [19]. While only one G is present in ChT1 andW117m, one S is found in the position of the second Gas is sometimes the case [20]. The G strands of all theCTX family members terminate with the motif VLV, com-mon in J segments. Several characteristics place theCTX family members close to TCR/Ig sequences (Fig. 2).For example, in CTH around C22, the sequence is partic-ularly homologous to that of the human TCR TCRAV2.Similarly, as in CTX/ChT1, CTH/CTM and the A33 anti-gen the CTY22-24 motif is present in many human andmouse TCR § or ˇ [21, 22].

The Y24 stabilizes the CDR1 in TCR § [23] and may actsimilarly in the CTX family. Between W38 and R68, theCTX family members have one or two Y residues asfound in Ig and TCR but not other V domains. Specifi-cally, the second Y is found in a CDR loop in TCR § . Pre-ceding C95, CTX/ChT1 have an I/LTISXXQXXDsequence present in all Ig light chains. Finally, the TYLC

Eur. J. Immunol. 1998. 28: 4094–4104 The CTX family in vertebrates 4095

Figure 1. (A) Organization of CTX family members proteins. (B) Alignment of the members. Arrows indicate the location of theintrons. Stars indicate the conserved residues in the Ig V domains. The residues conserved between the CTX family members areboxed; residues specific for the family are in blue; the cysteines and tryptophans conserved in all Ig domains are in red, and Nglycosylation sites are in yellow. The residues in purple are the extra cysteines typical of the CTX C2 domains.


Figure 2. Alignment of the V domains of the CTX family members with other V domains. The residues conserved between theCTX family members are boxed; residues present in the family and the TCR/Ig are in green, and residues conserved in all Igdomains are in red. h, human; m, mouse; Pi, pig; Sh, shark; Dr, Drosophila; Ch, chicken. TCRAV: TCR alpha; TCRDV: TCR delta;IGK: Ig kappa; IGL: Ig lambda. Accession numbers: CD7 m X 06180. Amalgam Dr 113687. Basigin D00611 BG M 61860 Butyro-philin 2062688. CD80h 461606 P0 h 127721. TCRD 22 Pi 1041165. TCRDV105 91761, IgK h U94422. Ig K sh U15143. IgL hD84141. The difference in the alignments between Figs. 1 and 2 is due to the difference in the number of members used in eachalignment. This affects partially the C’, D, F and G strands without changing the output of the comparison.

motif present in CTH is also present in several mamma-lian TCR.

In addition to the residues in common with the Ig/TCR,other invariant positions define the CTX family back-bone. Thus the small hydrophobic residues V, I, or L atpositions 1 and 3, G57, K65/66, G76, A78, V97 and D/E102 are present in the majority of the CTX members.Among these, the most unusual and therefore the besthallmark of the family is A78, which is shared only withthe Ig lambda light chain.

2.2.2 Second extracellular domain: an Ig domainof the C2 type with four cysteines

The C2 domain sequences of the CTX family membersalign easily with each other (Fig. 1 B) and with severalmembers of the N-cell adhesion molecule (N-CAM) fam-ily (Nr-CAM, PANG, fasciclin II, and related moleculescalled titins) (Fig. 3). The most specific characteristic isthe presence of two extra cysteines at positions 122 and199, which align without gaps in all family members.They probably form a second intradomain disulfidebridge [3]. Outside the CTX family, no other C2 domainscontain two extra cysteines in analogous positions,


Figure 3. Alignment of the C2 domains of the CTX family members with other C2 domains. Same color code as in Fig. 2. N gly-cosylation sites are in yellow. h, human; m, mouse; Gh, grasshopper; Ch, chicken; Ray, ray; Dr, Drosophila; C.el, C. elegans.Accession number: N-CAMh U55258. PANG m 1083458. Fasciclin II Gh 462073. V-CAM 137560. Titin Ch 151030. Unc 89 C.el.1160365 RTK Ray 422541. Peroxidasin Dr 630884. Hemolin 1042214.

although extra pairs of cysteines are found in several cellsurface receptors, for example CD2, CD58 and CD48(reviewed in [4]). Other residues specific to the family areP120, Y149 and a conserved potential N-glycosylationsite at N177. AA445m and T107h, distantly related toCTX, do not have N177, but have a more distal potentialN-glycosylation site. Other residues common to all CTXfamily members are also found in other C2 domains(Fig. 3), e. g. P117, G131, G143, R/K153 and G170. Allother common residues are conserved in all C1 and C2domains. While the protein encoded by EST T870h lacksthe extra pair of cysteines, it shares long stretches of aaidentity (E148YTWFKDGI D182TGEYSCEARN) withAA406h (human homologue of AA445m). These mole-cules contain the shortest cytoplasmic tails within thefamily and may define a subgroup.

2.2.3 Transmembrane domain

The transmembrane domain is similar in all CTX familymembers and differs from that of the TCR in that no posi-tively charged residue is present. CTH and CTM sharethe sequence LIGVXXGVXXL, which is similar to thedimerization motif LIXGVXXGVXXT of the transmem-brane region of glycophorin A [24].

2.2.4 Cytoplasmic tail

The cytoplasmic tails vary between 36 aa in AA45m and145 aa in AA497m, and the sequences are most diver-gent in this region of the molecule. Nevertheless at themembrane proximal portion of the cytoplasmic domain,CTM/CTH, CAR, W117m and AA386m share the motifDL/IREDA/VA/PP. Strikingly, CAR and W117m share astretch of 21 aa at the C terminus, although they aredivergent in other regions. No obvious pattern was

noticed, hence the absence of “boxes” in Fig. 1 for thisregion. Several potential S/T and Y phosphorylationsites were identified using the Prosite program, but theseare not conserved among all members. Finally, severalmembers contain one or several C at the membrane/cytoplasmic junction. In the A33 antigen, a sequence offour C may be target for palmitoylation [17].

2.3 Gene architecture

ChT1, CTM, CTH and A33 antigen (Fig. 4) have the hall-marks of the CTX gene [3]. All are encoded by sevenexons with each Ig domain encoded by two half-domainexons. The second characteristic feature is the unusualphase of splicing (type 0) between exons Va and Vb (toform the V domain). Most splicing junctions in Igdomains are of type 1. Another rare splicing phase(type 2) is seen at the junction between exons 6 and 7encoding TM/Cy1 and Cy2 (Fig. 4). These genomic fea-tures provide rigorous hallmarks of the CTX family andmay be used to identify family members. For example,the nucleotide sequences of the independent cDNAclones corresponding to AA497m, AA445m and W117misolated from a mouse testis cDNA library are highlyhomologous with CTX family members (Fig. 1) from thesecond half of the V domain to the end of the molecule.In the poorly conserved 5' region of these sequences, asplice acceptor site (type 0) is identified in exactly theposition that would reconstitute the Va/Vb junction.These cDNA clones look like pre-spliced intermediatesof novel CTX family members. The genomic organizationof the CTX family members is thus conserved at leastfrom amphibians to mammals as well as among differentmembers of the family (A33 antigen and CTM/CTH) andthe CTX V domain is on an evolutionary pathway distinctfrom the one leading to somatic rearrangement.


Figure 4. Genomic organization of the CTX family members.

2.4 Phylogenetically related molecules

2.4.1 Molecules sharing the same generalstructural organization

While some molecules [TCR, CD80, CD86 (B7-1 and B7-2), CD2, CD33, CD48, CD58 (LFA-3), CD150, 2B4, OX2(reviewed in [4]), butyrophilin [25]] have the same generalstructural organization (V/C2/TM/Cyto) as CTX, thisdoes not prove that they are phylogenetically related.The V domains of TCR, CD80, CD86 and butyrophilindisplay some homology with those of the CTX family andthe C2 domain of CD80 and CD86 may also be alignedwith those of CTX family members; however, the overallhomology is very low. Strictly speaking, none of thesemolecules has the CTX genomic organization.

2.4.2 Molecules with an Ig domain encoded byhalf-domain exons

The most rigorous criterien for inclusion in the CTX familyis the exon-intron organization. While most members ofthe Igsf contain Ig domains encoded by a single exon, afew molecules resemble CTX family members by contain-ing one or several Ig domains encoded by two exons.These include CD4 [26], RAGE [27], Muc-18 [28], myelinP0 [29], CD83 [30] and members of the N-CAM family,

such as L1 [31] and axonin-1 [32]. However, only P0,RAGE, CD83 and basigin V domains are encoded by twohalf-domain exons with a type 0 splicing junction, consis-tent with these molecules being more closely related toCTX family members (see above and [33]).

2.4.3 Molecules with homologous C2 domains

The CTX C2 domain resembles those of the N-CAM fam-ily. Moreover, C2 domains encoded by two exons arefound in N-CAM, L1 and axonin (Fig. 3). In one C2domain of the Drosophila molecule, peroxidasin, thesequence of amino acids between positions 158 and 188is identical to that of CTX in 18 out of 31 positions;also from C188, CEARNSLG is almost identical toCEARNSVG found in AA406h. None of these moleculescontains a C2 domain with an extra pair of cysteines thatperfectly align with those of the CTX family members.However, a Drosophila gene, isolated from a DNA seg-ment with the accession number HTG ac003577sequence and related to the titin family, has a C2 domainwith a characteristic extra pair of Cys. (VFLKKIGD C DIY-EGMVAKFTA C ATGYPEPEVEWFKNDQKLFPSDRFLI-DIEPNGLLRLTIKNVTEYDVGRYS C RIFNPYGDDI CHAELFYD). This reinforces our argument that CTX mole-cules have a number of ancient characteristics, featuresnot found in typical TCR or B cell receptor molecules.


Figure 5. Northern and dot blot analysis of CTM and CTHexpression. (A) The Northern blots with the indicated mousetissue RNA were hybridized with a CTM probe. Lower box:ethidium bromide staining. (B) The Northern blot and the dotblot were hybridized with a CTH probe. The mRNA from theindicated tissue comes from either a tumoral (T) or normal(N) tissue. Lower box: control hybridization with g actin. Onthe dot blot, each position corresponds to a different tissuemRNA: A1, whole brain; A2, amygdala; A3, caudate nucleus;A4, cerebellum; A5, cerebral cortex; A6, frontal lobe; A7, hip-pocampus; A8, medulla oblongata; B1, occipital lobe; B2,putamen; B3, substantia nigra; B4, temporal lobe; B5, thala-mus; B6, subthalamic nucleus; B7, spinal cord; C1, heart;C2, aorta; C3, skeletal muscle; C4, colon; C5, bladder; C6,uterus; C7, prostate; C8, stomach; D1, testis; D2, ovary; D3,pancreas; D4, pituitary gland; D5, adrenal gland; D6, thyroidglands; D7, salivary gland; D8, mammary gland; E1, kidney;E2, liver; E3, small intestine; E4, spleen; E5, thymus; E6,peripheral leukocyte; E7, lymph node; E8, bone marrow; F1,appendix, F2, lung; F3, trachea; F4, placenta; G1, fetal brain;G2, fetal heart; G3, fetal kidney; G4, fetal liver; G5, fetalspleen; G6, fetal thymus; G7, fetal lung; G8, H1, yeast totalRNA 100 ng; H2, yeast tRNA 100 ng; H3, E. coli rRNA100 ng; H4, E. coli DNA 100 ng; H5, Poly r(A) 100 ng; H6,human cot1 DNA 100 ng; H7, human DNA 100 ng; H8,human DNA 500 ng.

2.4.4 Molecules containing V domainshomologous to CTX V domain

V domains are rare in invertebrates. Exceptions are the Vdomains of Drosophila amalgam [34] and lachesin [35]which have similarities with the CTX V domain. Moreover,the Drosophila amalgam has, from position 9, thesequence DVVASVGDSV like the DVLRASQGKSV of theA33 antigen. Some vertebrate molecules such aschicken BG, mammalian myelin P0 and CD7 (allreviewed in [4]) contain a CTX-like V domain resemblingthat of Ig and TCR. In CD7, the sequence from position73 to 96 is highly homologous to that of CTX (15 aa iden-tical out of 23), and in P0 the sequences from positions57 to 69 and from 74 to 92 are highly homologous tothose in ChT1 and CAR, respectively (Fig. 3). The emerg-ing concept is that these V domains represent the ances-tral V domain prior to divergence between adhesion mol-ecules and Ig/TCR.

2.5 Tissue distribution

CTX and ChT1 [3, 7, 8] are expressed by thymocytesand, in the case of ChT1, by recent T cell emigrants [13].CTM mRNA is detected in stomach and prostate(Fig. 5 A). Upon longer exposure of Northern blots, aCTM mRNA signal is also detected in the colon. CTHmRNA is detected at relatively high levels in stomach,colon, prostate, trachea and thyroid glands and at lowerlevels in bladder and lung (Fig. 5 B) and CTH could beamplified from a thymus cDNA library but was notdetected on Northern blots. The A33 antigen is restrictedto the gastrointestinal tract [14] and CAR has a broaderspectrum of expression with some differences betweenmouse and human [16]. As with the A33 antigen [14],cancerous forms of these tissues expressed less of theCT mRNA (Fig. 5B). Given those results, a more system-atic survey for CTX expression has been done in Xeno-pus and the stomach indeed stained positively with themAb (Fig. 6), whereas the intestine was negative.

3 Discussion

In CTX family members the presence of V domains simi-lar to those of Ig/TCR, the ability to form dimers of threeof the molecules described (CTX, ChT1, A33 antigen),and the presence of a G strand with J segment featuresmay suggest an interaction of the extracellular domainswith ligands similar to those of Ig and TCR. Moreover, thepresence of the same conserved Y stabilizing the CDRloop in TCR suggests that CTX family members may alsointeract with their ligand via their CDR1. Other membershave similar features: CTM/CTH do have a J element


Figure 6. Expression of CTX in Xenopus. Immunofluores-cence in (A): Xenopus thymus section showing the cortexstaining. (B): Xenopus stomach section with anti-CTX mAb1S9-2.

and we already suggested that this feature is correlatedwith the formation of dimers [20]. On the other hand,many molecules with Ig domains are homophilic adhe-sion molecules or interact with Ig domains on other mol-ecules. It is conceivable that CTX family members maybe cell adhesion molecules, maintaining the cohesive-ness of the thymic cortex in the case of CTX/ChT1, orthe structural integrity of the intestinal crypts and villi inthe case of the A33 antigen and CTM/CTH. In theimmune system, examples of CTX-like molecules inter-acting with other Igsf members, for example CD80 and86 (B7) with CD152 (CTLA4; [36]) could give an indicationof the type of binding partner involved.

The common structural organization of many genesencoding a number of CTX-related cell surface mole-cules and the identification of distinguishing amino acidresidues allows us to propose a novel family within theIgsf. Its members do not have the same tissue distribu-tion, some are expressed on lymphocytes, others not,but they all seem to have a relationship with the controlof cell proliferation. Although a cortical thymocyte formhas yet to be isolated in mammals, the existence of mol-ecules with as much homology to CTX as AA497 leaves

the door open to the discovery of such molecules. Thethymocyte marker, CTX has the features of both antigenreceptors and cell adhesion molecules and may haveoriginated from a family of molecules with a broader tis-sue distribution. CTX itself and ChT1 may be representa-tive of an ancestral lymphocyte receptor before the intro-duction of somatic rearrangement but after its targetingto lymphoid tissues. The genetic organization encodingthe CTX V domain reinforces the idea that the original Vdomains had no intron and that the introduction of vari-ous introns created different families of molecules lockedinto specific lines of evolution by the exon-intron struc-ture of their genes.

4 Materials and methods

4.1 Cloning of the ChT1 gene

A library of chicken genomic DNA in the lambda-FIX II vector(Stratagene) was screened with the ChT1 cDNA probelabeled with [ § -32P] dCTP using the Prime-It II RandomPrimer labeling kit (Stratagene). Three overlapping clones,which encompassed all the exons, were isolated (accessionno. AF061023).

4.2 Cloning a human homologue of the CTX gene

A colon tumor clone 6H4 (accession no. T24595) [37]) identi-fied by screening EST data bases with the BLAST programhad strong homology to CTX; it was used to screen a humangenomic library in lambda-FIX II (Stratagene). The completegene (CTH) was obtained (accession no. AF061022). Theclone 6H4 sequence was also used to design primers(CCAGTAATCCCTTATGC and TCAGTGCAGTAGAGCCTCC)to amplify cDNA fragments by PCR from a human thymuscDNA library in Q gt11 (Clontech). Paired with the gt11 vectorforward and reverse primers they yielded a single, not full-length cDNA sequence from 3 × 108 recombinants. 6H4 wasin fact a pre-spliced intermediate lacking the second cyto-plasmic exon.

4.3 Cloning a mouse CTX homologue

HindIII digested BALB/c genomic DNA was fractionated onan agarose gel, and the region containing DNA size 4.4–6.6kb previously bound to hybridize with CTH Vb region wasexcised. A 200-bp PCR product was obtained from theeluted material with CTH Vb primers, subcloned intopBluescript, sequenced, and designated CTM. New primersbased on this sequence were used in rapid amplification ofcDNA ends (RACE)-PCR with mouse prostate cDNA usingthe Marathon kit (Clontech) and yielded the complete CTMcDNA. Total RNA from BALB/c organs was prepared withthe TRIzol reagent (Gibco-BRL).


In parallel two 129 SVJ mouse genomic libraries in thelambda-FIX II vector (Stratagene and a kind gift from Dr.G. K. Sim) were screened with the 200-bp CTM PCR prod-uct and the CTM gene was isolated (accession no.AF061024).

4.4 Characterization of the human A33 antigen gene

Approximately 106 clones of a human genomic DNA libraryin lambda-FIX II (Stratagene) were screened with a 2.8-kb(full-length) A33 antigen cDNA probe [15] labeled with[ § -32P] CTP using the Megaprime DNA labeling system(Amersham International, Little Chalfont, GB). Three partiallyoverlapping genomic clones were isolated which togetherencompassed exons 1–4 (total 33 kb). PCR of genomicDNA with primers designed to amplify each intron were car-ried out using the EXPAND 20kbPlus system (BoehringerMannheim).

4.5 Nucleotide sequencing

The human CTH and chicken genomic clones weresequenced on both strands directly from the bacteriophageDNA without subcloning, as previously described [38]. Thesequences of the mouse CTM and the human A33 antigengenes were obtained using the thermo Sequenase fluores-cent labeled primer cycle sequencing kit (Amersham), andthe samples were analyzed on a Li-cor DNA sequencer or anApplied Bio Systems automated DNA sequencer.

4.6 EST sequences

I.M.A.G.E Consortium cDNA clones were obtained from theUK HGMP Resource Center in Cambridge and re-sequenced. For each sequence the name is followed by theI.M.A.G.E Consortium ID and by the Genbank accessionnumber: AA 386m/ID0775456/AA386948; W117m/ID329666/W11712; R336h/ID0135840/R33685; AA445m/ID848010/AA445150; AA406h/ID0753386/AA406389;T870h/ID115348/T87045. For AA497m we designed prim-ers based on the published EST sequence (accession no.AA497966) and amplified a probe from a prostate cDNAlibrary that led to the isolation of several clones whenscreening a mouse testis cDNA library (Stratagene). cDNAclones AA445m and W117m probe yielded independentclones from the same library. AA318h was generated by twooverlapping EST (AA318060 and AA37342).

4.7 Northern blot analysis

Total RNA from BALB/c mouse organs was prepared usingthe TRIzol reagent (Gibco-BRL) and Northern blotting wasperformed as described [3]. Human Northern and dot blotmembranes were purchased from Clontech.

4.9 Homology search and alignment

Similarity searches of several NCBI databases were con-ducted with the advanced BLAST Sequence SimilaritySearching algorithm. Multiple alignments were done usingthe DNA STAR MegAlign program with the Clustal methodand with the PAM 250 weight table then corrected manually.

4.10 Immunohistology

Whole tadpole or adult Xenopus organs were embedded inOCT on dry ice, sectioned at − 20 °C (6 ? m), fixed for 30 s inacetone and processed for immunofluorescence with anti-CTX mAb, IS9 and X71 [3].

Acknowledgements: The authors thank Thomas Brockerfor his help with the PCR to isolate CTH cDNA, MarcoColonna and Charley Steinberg for critical reading of themanuscript and D. Jenne for pointing out the first ESThomologue to us. The Basel Institute for Immunology wasfounded and is supported by Hoffmann La-Roche, Basel,Switzerland, K.K. and O.V. were financially supported by theAcademy of Finland and the Human Frontier Science Pro-gram grant (RG-366/96).

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Correspondence: Louis Du Pasquier, Basel Institute forImmunology, Grenzacherstraße 487, CH-4005 Basel, Switz-erlandFax: +41 61 605 12 22e-mail: dupasquier — dial.eunet.ch
