Identification of three novel non-classical cadherin genes through comprehensive analysis of large cDNAs

Molecular Brain Research 94 (2001) 85–95 www.elsevier.com / locate / bres

Research report

Identification of three novel non-classical cadherin genes through comprehensive analysis of large cDNAs Daisuke Nakajima, Manabu Nakayama, Reiko Kikuno, Makoto Hirosawa, Takahiro Nagase, Osamu Ohara* Department of Human Gene Research, Kazusa DNA Research Institute, 1532 -3 Yana, Kisarazu, Chiba 292 -0812, Japan Accepted 17 July 2001

Abstract The terminal sequences of long cDNAs from human brains were subjected to an improved method of motif-trap screening. This process resulted in the identification of three novel genes that encode proteins with 27, 27, and six cadherin domains that we denoted as KIAA1773, KIAA1774 and KIAA1775, respectively. Sequence analysis indicated that the products of these genes were non-classical cadherins. KIAA1773 was found to be a mammalian homologue of the Drosophila dachsous gene but the remaining two genes did not have any likely homologues in public databases. Assessment of their expression in rat tissues indicated that these genes are expressed in highly distinct and tissue-specific patterns. Notably, KIAA1775 is expressed almost exclusively in the olfactory bulb in the rat brain. In situ hybridization further showed that KIAA1775 is strongly expressed by the mitral and tufted cells in the main and accessory olfactory bulbs, suggesting that KIAA1775 may be important in the formation and maintenance of neuronal networks, particularly those in the olfactory bulb. This study clearly shows the importance and usefulness of our cDNA project in search for genes encoding large proteins, as this project has allowed us to identify several novel non-classical cadherin genes that have thus far not been detected by conventional methods.  2001 Elsevier Science B.V. All rights reserved. Theme: Cellular and molecular biology Topic: Gene structure and function: general Keywords: Protocadherin; cDNA analysis; Human brain; Dachsous; Olfactory bulb

1. Introduction The complete genome sequences of a growing number of organisms have been determined and this has resulted in a dramatic expansion of our understanding of genes and their organization in the genome. However, for mammals, the interpretation of such genome information is not as straightforward as it is for prokaryotes and lower eukaryotes. This is largely because mammalian protein-coding sequences (CDSs) are frequently divided by introns that diverge considerably in size and sequence between different mammalian species. Although a number of programs *Corresponding author. Tel.: 181-438-52-3913; fax. 181-438-523914. E-mail address: [email protected] (O. Ohara).

that aim to predict the mammalian CDSs on the basis of the genomic sequence have been developed, none are completely error-free at present. Comparing the genomic sequence with cDNA sequences is thus still important for the accurate assignation of CDSs in a genome, as cDNA sequences convey the sequence information of mature transcripts in which the intron sequences have been spliced out. Consequently, it is necessary to obtain the sequences of entire cDNAs rather than just sequencing their terminal regions (widely known as expressed sequence tags or ESTs). With these considerations in mind, we initiated a human cDNA sequencing project at the Kazusa DNA Research Institute 7 years ago [21]. Many other sequencing projects for human cDNAs have recently been initiated worldwide but our project differs from the others in that we primarily sequence large cDNAs (.4 kb) [22]. In

0169-328X / 01 / $ – see front matter  2001 Elsevier Science B.V. All rights reserved. PII: S0169-328X( 01 )00218-2

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addition, we are currently focusing on large cDNAs that encode large proteins (.500 amino acid residues) expressed in the brain [18]. The total number of human cDNA clones characterized to date in our project exceeds 1900 (over 9 Mb of nucleotides sequenced) and these data are accessible in the HUGE protein database at our Web site (http: / / www.kazusa.or.jp / huge [15]). The cadherins are well-known proteins that are involved in calcium-dependent cell–cell adhesion. A number of proteins may be denoted as ‘classical cadherins’ because of sequence conservation in their cytoplasmic domains as described by Nollet et al. [20] and Yagi and Takeichi [32]. These classical cadherins typically have five extracellular domains with Ca 21 -binding sites, a membrane-spanning region, and a cytoplasmic domain that anchors various membrane and cytoskeletal proteins. In addition, there is a large family of non-classical cadherins, sometimes termed protocadherins or CNRs [16], that have more than five extracellular cadherin domains and whose cytoplasmic domains are divergent in sequence. Both the classical and non-classical cadherins have been shown to be important in nervous system function [3,27]. To fully understand the biological roles of the cadherins in the nervous system, a complete catalog of the cadherin genes expressed in the brain must be available. A polymerase chain reaction (PCR)-based approach [26] has previously provided some limited information about cadherins expressed in the nervous system, but other systematic experimental approaches have not yet been applied. One such approach is to employ a computer-based method to identify genes that encode proteins bearing a particular domain / motif (e.g., the epidermal growth factorlike motif [19]). Performing such motif-trap screening [19] on our database of terminal sequences of long cDNAs found in the adult and fetal human brain is likely to allow us to identify the non-classical cadherins in the brain, as non-classical cadherins are usually encoded by long mRNA (.4 kb). We have used an improved method of motif-trap screening to identify which of the cDNA clones in our database contain cadherin motifs. Sequencing of these clones revealed three novel non-classical cadherin genes that we have designated as KIAA1773, KIAA1774, and KIAA1775. The first cDNA clones we identified turned out to contain only part of each gene and thus, the complete sequences of these non-classic cadherin genes had to be determined by additional cloning experiments with rat and human mRNA. KIAA1773 and KIAA1774 were found to encode large non-classical cadherins with 27 cadherin domains, while KIAA1775 seems to encode a totally unknown protocadherin. Interestingly, KIAA1775 is specifically expressed in the olfactory bulb of rat brain, which suggests that this gene might play an important and specific role in the formation and maintenance of the neuronal network in the olfactory bulb.

2. Materials and methods

2.1. Identification of cDNA clones encoding proteins bearing cadherin domain(s) by computer-mediated motiftrap screening cDNA clones encoding proteins with cadherin domains were identified by a modified motif-trap screening approach. Here the motif-trap screening method that has been previously reported was combined with GeneMark analysis that predicts CDSs in cDNAs [11]. This adaptation aimed to reduce the number of false-positive clones. The 59-end sequences of 14 804 long (.4 kb) human brain cDNA clones were thus first subjected to GeneMark analysis. The open reading frames (ORFs) in the 59-end sequences that scored higher than 0.5 in the GeneMark analysis were regarded as potential CDSs [12]. These CDSs were then examined for the presence of the cadherin extracellular repeated domain signature defined in the PROSITE database ([13]; PS00232, [LIV]-x-[LIV]-x-D-x-N-D-[NH]-xP). To minimize the risk of overlooking clones, the translated sequences were also screened for the cadherin domain defined in the Pfam database (PF00028) by HMMER2.1.1 [1]. The cDNA clones selected by either of the PROSITE or Pfam searches were then tested to see whether or not their end sequences were new in GenBank (excluding human genome sequences and ESTs, Release120) by performing a homology search. This work resulted in the identification of three cDNA clones that are likely to encode unknown cadherin genes. These clones, designated as KIAA1773, KIAA1774, and KIAA1775, were entirely sequenced.

2.2. Isolation of cDNAs for KIAA1773, KIAA1774, and KIAA1775 genes from rat mRNA To further characterize KIAA1773, KIAA1774, and KIAA1775, we isolated partial cDNA fragments of each gene in the rat by reverse transcription (RT)-coupled PCR using the following primer sets: for KIAA1773, 59-TGG GAG TTG TGG TGG TGC TTG C-39 and 59-AGC GAG CAG CCA GAG GAG ACA G-3; for KIAA1774, 59-ACA ATG CCA GCG ACC TAC CAG A-39 and 59-TGC CGG CTG CGA TGA AGT AGT A-39; and for KIAA1775, 59-TGA CCG ATG CCA ATG ATG AGG C-39 and 59-GAG GGG GGT GGT CAT TGA CAT C-39. These primer sequences were derived from the corresponding human cDNA sequences described above. Using oligo(dT)-primed cDNAs from total rat brain RNA as templates, we carried out RT-PCR with LA-Taq polymerase (Takara Shuzo, Kyoto, Japan) by performing 30 cycles of denaturation at 948C for 30 s, annealing at 558C for 30 s and extension at 728C for 1 min. The RT-PCR products of the expected sizes were subcloned into pGEMT Easy vector (Promega, WI, USA) and the authenticity of

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these cDNA fragments was confirmed by DNA sequencing.

bearing a 4 to 6 kb insert was obtained by colony hybridization using human KIAA1775 as a probe.

2.3. Isolation of cDNAs containing the complete KIAA1773, KIAA1774, and KIAA1775 non-classical cadherin genes

2.4. DNA sequencing

Since the cDNA clone for KIAA1773 seemed to be only a partial sequence of the gene, we carried out additional cloning experiments to isolate the missing 59-end sequence. Total human lung RNA was reverse transcribed with a KIAA1773-specific primer (59-GGG CGG CCG CAC CGA GTA GGC AGG CAC AGG AAA G-39) and SuperScript II reverse transcriptase. The resulting doublestranded cDNAs were cloned into Not I–Sal I site of pSPORT-1 according to the instructions provided with the SuperScript II plasmid cDNA library construction kit (Life Technologies, Gaithersburg, MD, USA). A cDNA clone with a 4 kb upstream sequence was isolated from this cDNA library and entirely sequenced. Based on this sequence, a new KIAA1773-specific primer (59-GGG CGG CCG CGT GAG GCA GCC GTA GTG ATA AT39) was designed and used to construct another cDNA library as described above. From this second library, a cDNA clone that provided the 59-most sequence information of KIAA1773 was isolated. Thus, the full nucleotide sequence of the entire KIAA1773 gene was derived from three cDNA clones that, respectively spanned its nucleotide residues from 1 to 2016, from 1596 to 5995, and from 5539 to 10 759. A complete cDNA clone for KIAA1774 was isolated from a rat testis cDNA library. Poly(A)1 RNA was isolated from the testes of 6-week-old Sprague–Dawley male rats by using the Quick prep mRNA Purification Kit (Amersham Pharmacia Biotech, Uppsala, Sweden). Double-stranded rat cDNAs were generated by a SuperScript II plasmid library construction kit according to the supplier’s instructions (Life Technologie). The resulting doublestranded cDNAs that were larger than 7 kb were isolated by electrophoresis on a low-melting-temperature agarose gel and then cloned between the Sal I and Not I sites in the pSPORT-1 vector. A pool of cDNA plasmids was recovered from 1.7310 6 primary transformants (ElectroMax DH10B cells, Life Technologies) grown on agar plates containing 50 mg / ml of ampicillin. The resulting cDNA plasmids present in the covalently closed circular form were again selected for size on agarose gels to obtain the cDNA inserts that ranged from 10 to 12 kb. Rat KIAA1774 cDNA clones were isolated from this cDNA pool by colony hybridization using human KIAA1774 cDNA as a probe. A complete cDNA clone of KIAA1775 was isolated from rat olfactory bulb essentially as described above. However, in this case, double-stranded cDNAs larger than 3 kb were first selected and a mixture of the cDNA clones

DNA sequences were determined with the automated DNA sequencers ABI373 and ABI377 (Applied Biosystems, Foster City, CA, USA). Dye terminator cycle sequencing reactions were performed using a kit from Applied Biosystems (BigDye Terminator Cycle Sequencing Kit). The entire cDNA insert sequences were determined on both strands under a shotgun strategy [22]. Any remaining gaps were sequenced by using appropriate primers designed from their flanking sequences.

2.5. Radiation hybrid mapping The chromosomal locations of the novel protocadherin genes were determined with the GeneBridge 4 radiation hybrid panel (Research Genetics, Huntsville, AL, USA) by PCR using the following primer sets: for KIAA1773, 59-GCT GTG GAG GAT GAG AAT GAC-39 and 59-CGA AGC ATG GTA AGG GTC TGG-39; for KIAA1774, 59-CCA AGT CTC GCT ACA TTT CCG-39 and 59-GTG AAA TGG AGA GAA TGC TTG-39; and for KIAA1775, 59-TAC AGT CCC CAA CGT GAA CAG-39 and 59-AAG CAT GAT CCA GCA CAG TCC-39. Thermal cycling conditions were as follows: for KIAA1773 and KIAA1775, denaturation was performed at 958C for 2 min, followed by 30 cycles of 15 s denaturation at 958C and 1 min annealing / extension at 668C; for KIAA1774, the annealing / extension temperature was lowered to 628C.

2.6. RNA blotting analysis Total RNA was prepared from 6-week-old male Sprague–Dawley rats by using TRIZOL reagent (Life Technologies). This RNA was electrophoresed on a 1% agarose / formaldehyde gel, transferred onto a positively charged nylon membrane (Biodyne B, Pall Biosupport Division, East Hills, NY, USA), and fixed by UV irradiation. Hybridization was carried out as described by Church and Gilbert [4] using a probe labeled with 32 P with a random-primer labeling kit (Rad Prime DNA Labeling System, Life Technologies). The membrane was extensively washed with 0.13 SSC / 0.1% sodium dodecyl sulfate (SDS) at 658C and autoradiographed on X-ray films (XOmat, Kodak, Rochester, NY, USA).

2.7. In situ hybridization Six-week-old male Sprague–Dawley rats were anesthetized with diethylether and decapitated. Brains were rapidly dissected and quickly frozen on dry ice. Transverse, sagittal and coronal sections (7 mm) were prepared

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using a cryostat and these were then thaw-mounted on silane-coated slide glasses and stored at 2808C until use. Before use, the sections were air-dried for 5 min at room temperature and fixed by immersion in 4% paraformaldehyde in phosphate-buffered saline pre-treated with 0.02% diethylpyrocarbonate for 25 min. After the fixation the sections were acetylated with 0.25% acetic anhydrate and then dehydrated with ethanol. In situ hybridization was carried out with complementary RNA (cRNA) probes labeled with 35 S-UTP. The 35 S-labeled cRNA probes were synthesized from subcloned plasmids containing partial rat cDNAs for KIAA1773, KIAA1774, and KIAA1775 by linearizing them by restriction digestion with an appropriate enzyme. These sequences were then used as templates for the synthesis of cRNA probes, which were made with an RNA labeling kit (Roche Diagnostics, Basel, Switzerland) and 35 S-UTP (1250 Ci / mmol) according to the kit instructions. After being fragmented to approximately 200 bp sequences by partial alkaline hydrolysis [23], the 35 S-labeled cRNA probes were diluted to 5310 4 cpm / ml in hybridization buffer containing 50% formamide, 10% dextran sulfate, 0.6 M NaCl, 20 mM dithiothreitol, 0.02% baker’s yeast tRNA, 13 Denhardt’s solution, 0.25% SDS, 10 mM Tris–Cl (pH 7.5), 1 mM EDTA. Hybridization was allowed to proceed in a humidified chamber at 508C overnight. Afterwards, the sections were subjected to successive washing steps with the following solutions: 53 SSC [13 SSC contains 0.15 M NaCl and 15 mM sodium citrate, pH 7.0] at 508C briefly; 23 SSC containing 50% formamide at 508C for 30 min; 23 SSC at 508C briefly; TNE [10 mM Tris–Cl (pH 7.5), 500 mM NaCl, 1 mM EDTA] buffer at 378C for 10 min; TNE buffer containing RNase A (20 mg / ml) at 378C for 30 min; TNE buffer at 378C for 10 min; 0.23 SSC at 508C for 20 min; 0.13 SSC at 508C for 20 min twice. After dehydration in ethanol followed by air-drying, the sections were first autoradiographed with X-ray films (Bio Max film, Kodak) for 3 days. Subsequently, the sections were microautoradiograhed with NTB-2 emulsion (Kodak) for 1 week. The hybridization signals were developed with Kodak D-19 and observed on the sections counterstained with hematoxylin / eosin under dark-field and bright-field microscopes.

3. Results

3.1. Identification of three novel non-classical cadherin cDNAs We screened a database of 59-end sequences of human brain cDNA libraries (14 804 sequences derived from cDNAs that are over 4 kb in size) for genes that encode proteins containing cadherin domain(s) by computer using a modified motif-trap screening approach. The modifications introduced in the motif-trap screening method are as

follows: (1) CDS prediction by GeneMark analysis was combined with a search for the cadherin protein motif and (2) searching was performed using the cadherin signature motifs identified by the Pfam and PROSITE databases [13]. This modified motif-trap screening method allowed us to detect cDNA sequences derived from three novel cadherin genes in addition to six previously identified ones (KIAA0279 / MEGF3 [19], KIAA0811 / MEGF1 [19], KIAA0726, KIAA0588 / pcdhg [30], KIAA0345 / pcdha [30], and pcdh7 [33]). Interestingly, we could also identify two independent cDNA clones that contained two ectodomain exons of the pcdhg gene arranged in tandem and that missed an exon encoding a constant domain (data not shown [30]). It is likely that these cDNA clones are generated by internal priming with the oligo dT primer from poly (A) stretches in the immature pcdhg transcripts. These clones are of interest with regard to helping us understand how the transcription of the pchdg gene, which has an extraordinary genomic structure, is regulated [30,31]. The various cDNA clones derived from the pcdhg gene have thus been structurally characterized in detail and will be described elsewhere. The three novel cDNA clones that were selected were entirely sequenced, and the structures of their gene products were predicted. This analysis showed that the cDNA clones contained only partial sequences of the genes. We thus carried out additional cloning experiments to isolate cDNA clones that would provide the complete transcribed sequences of these genes in rats and humans (see Materials and methods). The resulting cDNA sequences were judged to cover the complete gene CDSs because: (1) the cDNA sequence sizes were consistent with the mRNA sizes of the respective genes as revealed by RNA blotting analysis described in the next section, and (2) the predicted aminoterminal sequences were consistent with the notion that they were secretory signal sequences that would allow the cadherin domains to extrude into the extracellular space. The latter point was confirmed by a computer program, TargetP [6]. Full-length cDNA clones could be isolated for rat KIAA1774 and KIAA1775 but the complete sequence of the human KIAA1773 gene product was finally obtained by assembling the sequences of three partial cDNA clones (the nucleotide sequences of these clones are available upon request). The nucleotide sequences of KIAA1773, KIAA1774, and KIAA1775 cDNAs have been deposited into DDBJ / GenBank / EMBL DNA databases under the following accession numbers: human KIAA1773, KIAA1774 and KIAA1775 as AB053446, AB053445 and AB053448, respectively; rat KIAA1774 and KIAA1775 as AB053447 and AB053449, respectively. Radiation hybrid mapping of these genes on the human genome showed that the most closely linked sequencetagged site markers for KIAA1773, KIAA1774 and KIAA1775 were D11S922, CHLC.GATA101E02, and CHLC.GATA81F06, respectively. From these markers, the cytogenetic loci of KIAA1773, KIAA1774, and

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KIAA1775 were determined to be 11p15.5, 10q21.2-21.3, and 10q22.1-22.3, respectively.

3.2. Predicted structures of the gene products of KIAA1773, KIAA1774 and KIAA1775 Fig. 1 shows the physical maps of the three full-length cadherin cDNAs found in rats and humans together with the predicted domain organization of their genes (panels A and B). The actual nucleotide and protein sequences of KIAA1773, KIAA1774 and KIAA1775 are accessible at http: / / www.kazusa.or.jp / huge. As shown in Fig. 1B, according to the domain search program HAMMER2 [1], the number of cadherin domains found in the predicted gene products of KIAA1773, KIAA1774 and KIAA1775 are 27, 27, and 6, respectively. All bear possible secretory signal sequences at their amino termini [6] and a SOSUI program [10] predicted that they contain single transmembrane regions downstream from clusters of cadherin domains. Homologues of these genes were identified by carrying out a homology search characterized by certain limitations. That is, the length should range between 80 to 125% of the query sequence, the length ratio of the aligned region to the original query sequence should be 0.8 or greater, and the sequence identity should be 30% or higher. This search showed that KIAA1773 is a homologue of the Drosophila dachsous gene. In particular, the cytoplasmic domain of the KIAA1773 protein was similar (38% sequence identity) to that of the dachsous protein, which is known to contain part of the b-catenin-binding site of the classical cadherins at its carboxy terminus (Fig. 1). In addition, a region between the last cadherin domain and the membrane-spanning region of KIAA1774 exhibited significant local sequence identity (40%) to a corresponding region of a Drosophila non-classical cadherin encoded by CG3389. The evolutional relationship between these two proteins is unclear at present. Apart from this region and their cadherin domains, the gene products of KIAA1774 and KIAA1775 did not show any additional local sequence similarity to any other protein sequences in the public protein database.

3.3. Expression of KIAA1773, KIAA1774 and KIAA1775 in the rat In order to examine the mRNA sizes and the expression patterns of the novel genes, we performed RNA blotting analysis using rat samples prepared from eight tissues and five brain regions. The mRNA of KIAA1773, KIAA1774 and KIAA1775 were estimated to be 11, 11, and 4.5 kb, respectively, which agrees well with their corresponding cDNA sizes (Fig. 2). KIAA1773 and KIAA1774 were found to be most abundantly expressed in lung and testis, respectively (Fig. 2). With regard to the brain, while the mRNA level of KIAA1773 in the whole brain was rather

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low, the olfactory bulb was found to contain a relatively large amount of this mRNA. In contrast, the KIAA1774 mRNA levels were very low in both the whole brain and the regions of the brain that we examined (Fig. 2). The expression profile of KIAA1775 differed completely from that of KIAA1773 and KIAA1774 as KIAA1775 mRNA was not detected in any of the tissue RNA samples examined. However, when RNA samples isolated from various brain regions were tested, KIAA1775 mRNA was found to be expressed specifically in the olfactory bulb (Fig. 2). Since the hybridization signal of KIAA1775 already appeared as an intense band after 16 h of exposure, KIAA1775 is probably expressed in the olfactory bulb at much higher levels than KIAA1773 in the lung and KIAA1774 in the testis. As the KIAA1775 hybridization signals in the other tissues and other brain regions could barely be detected on the same blot, it may be that KIAA1775 is expressed only in a limited type of cells that are specific to the olfactory bulb.

3.4. Identification by in situ hybridization of the rat brain cells that express KIAA1775 To characterize the cells that express KIAA1775 in the rat brain, we performed in situ hybridization using a 35 S-labeled KIAA1775 probe and rat brain sections sliced in different directions [25]. Fig. 3 shows X-ray film images of the hybridization signals on transverse (panels A and B), sagittal (panels C and D), and coronal (panels E, F, G, and H) sections of whole rat brain using the sense-strand probe (panels B, D, F, and H) and the antisense-strand probe (panels A, C, E, and G) of KIAA1775 cDNA. The signals shown in Fig. 3 are clearly specific for KIAA1775 mRNA because only the antisense-strand probe gave strong signals. In accordance with the RNA blotting data, the strongest hybridization signals were observed in the olfactory bulb (panels A, C, and E). The septum and olfactory cortex also expressed KIAA1775 but at lower levels than in the olfactory bulb (arrows in panels A, C, and G). Expression of KIAA1775 in the olfactory bulb, septum and olfactory cortex was further examined by microautoradiography of the in situ hybridization signals. Fig. 4 shows that the mitral cells in the mitral cell layer and the tufted cells in the external plexiform layer predominantly express KIAA1775 in the main and accessory olfactory bulbs. Faint expression could also be seen in the triangular septal nucleus and piriform cortex.

4. Discussion The number of cadherin superfamily members is rapidly expanding as genome analysis proceeds. According to a recent review, there are 17 and 13 cadherin homologues in Drosophila and Caenorhabditis, respectively [7,8,14].

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Fig. 1. Structures of the full-length KIAA1773, KIAA1774 and KIAA1775 cDNAs and their products. Physical maps of the KIAA1773, KIAA1774 and KIAA1775 cDNAs are shown in panel A. The horizontal scale represents the cDNA length in kb. The ORFs and untranslated regions are shown by solid and open boxes, respectively. Panel B indicates the domain organization of the KIAA1773, KIAA1774 and KIAA1775 genes. Panel C displays the amino acid sequence comparison between the cytoplasmic domains of the Drosophila dachsous and KIAA1775 proteins. The underlined text indicates a pair of beta-catenin binding sites.

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Fig. 2. Expression patterns of KIAA1773, KIAA1774 and KIAA1775 in the rat. Total cellular RNAs from eight tissues and five brain regions (10 mg each) were run in individual lanes. To confirm the amount and integrity of each RNA sample, hybridization with a glyceraldehyde-3-phosphate dehydrogenase probe was done as shown in the lower panels. Numbers on the left of each panel indicate the RNA size markers.

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Fig. 3. Expression of KIAA1775 in the rat brain. Autoradiograms of in situ hybridization assays on sections of the rat brain are shown. Transverse (panels A and B), sagittal (panels C and D), and coronal (panels E, F, G, and H) sections of rat brain were subjected to in situ hybridization using a KIAA1775 sense-strand (panels B, D, F, and H) or an antisense-strand RNA probe (panels A, C, E, and G). Although weak signals were observed in the septum (arrows in panels A and C) and the piriform cortex (arrow in panel G), prominent and strong signals appeared only on the olfactory bulb (panels A, C, E). Scale bar53 mm.

These numbers are likely to be even greater in vertebrates as cell–cell adhesion is probably controlled in a highly complex manner in mammals. As the draft sequence of the human genome is now available, one might believe that a complete catalog of human cadherins can be easily obtained by using computer programs that predict CDSs. However, because of the complex exon / intron organization of human genes, these programs are prone to error. Consequently, comprehensive human cDNA analysis is important for the accurate assignation of the CDSs in the genome. To this end, our

cDNA project aims to comprehensively analyze large cDNAs (.4 kb) in humans. Our work has particularly focused on the accumulation and analysis of large cDNA clones obtained from the human brain [22]. As nonclassical cadherin mRNAs are frequently larger than 4 kb, it is reasonable to expect that the non-classical cadherin genes expressed in the human brain are fully represented in our cDNA libraries. The results in this study show that this is the case. We have identified three novel cadherin genes and designated them as KIAA1773, KIAA1774 and KIAA1775

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Fig. 4. Cellular expression of KIAA1775 in the septum, piriform cortex, and main and accessory olfactory bulbs of the rat. Enlarged pictures of micro-autoradiograms of in situ hybridization assays are shown. The in situ hybridization results are displayed as a set of eosin / hematoxyline staining, hybridization data using sense- and antisense-strand probes, which are horizontally aligned as indicated. The in situ hybridization signals were detected under dark-field microscope. The regions examined are shown at the left side of the set of pictures; main olfactory bulb (MOB), accessory olfactory bulb (AOB), septum, and olfactory cortex. Strong hybridization signals were seen at mitral and tufted cells in mitral cell layer (MCL) and external plexiform layer (EPL), and relatively weak signals were detected on cells in triangular septal nucleus (Ts) and piriform cortex (Pir). These observations are consistent with the results shown in Fig. 3. Scale bar5200 mm.

in this study. As authorized gene names, the HUGO gene nomenclature committee has denoted KIAA1773, KIAA1774, and KIAA1775 as CDH25, CDH23, and PCDH21, respectively. Interestingly, after completion of this work, independent two groups have very recently identified CDH23 as a gene causing Usher syndrome type 1D [2,24]. According to the cadherin classification described by Hynes and Zhao [14], the gene products of KIAA1773, KIAA1774 and KIAA1775 are non-classical cadherins. To date, many human non-classical cadherin

genes have been characterized but none have been located at either of the genomic loci to which KIAA1773, KIAA1774 and KIAA1775 were mapped. Sequence homology searches revealed that KIAA1773 could be a mammalian homologue of the dachsous gene in Drosophila [5]. This means that homologues of all the large protocadherins (.3000 amino acid residues) found in Drosophila ( fat [17], fat-like [14], flamingo [29], dachsous [5]) are now confirmed to be present in mammals [19,31]. In addition, some local sequence similarity was

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observed between the gene products of KIAA1774 and the Drosophila CG3389 gene, although how they are related in evolutionary terms is at present unclear. No KIAA1775 homologue was found in Drosophila as is the case of the clustered protocadherin genes [30]. Cadherins have recently been suggested to be important in governing the establishment and maintenance of synaptic association [32]. In this respect, KIAA1775 is the most interesting of the genes identified in this study as its expression was almost completely restricted to the mitral and tufted cells in the rat olfactory bulb. Although other cadherins have also been reported to be produced in the olfactory bulb [9,16,28], the KIAA1775 protein is to our knowledge the first non-classical cadherin that is expressed specifically in the mitral and tufted cells in the olfactory bulb. The in situ hybridization patterns suggest that KIAA1775 may play a special role in the establishment and maintenance of the synaptic junctions in the olfactory bulb. This conclusion needs to be verified by further histochemical analysis. This study has demonstrated the utility of our comprehensive analysis of human large cDNAs in identifying genes like the non-classical cadherins that have transcripts longer than 4 kb. In addition, the human cDNA information that we are accumulating will form an important complement to the human genome sequence information as it will allow more accurate prediction of the complete primary structures of gene products. It will also be an invaluable tool in aiding our understanding of how genes are transcribed and processed.

Acknowledgements We thank Dr. Reiko Ohara for technical advice about the in situ hybridization assay. We also thank Ken-ichi Ishikawa and all our colleagues from the Kazusa cDNA project team for their cooperation.

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