Characterization of the genomic structure of the mouse limbic system-associated membrane protein (Lsamp) gene

Genomics 83 (2004) 790 – 801 www.elsevier.com/locate/ygeno

Characterization of the genomic structure of the mouse limbic system-associated membrane protein (Lsamp) gene Aurea F. Pimenta * and Pat Levitt John F. Kennedy Center for Research on Human Development and Department of Pharmacology, Vanderbilt University, Nashville, TN 37232, USA Received 23 June 2003; accepted 17 November 2003

Abstract The Lsamp gene encodes the limbic system-associated membrane protein (LAMP) an immunoglobulin (Ig) superfamily member with three Ig domains and a glycosylphosphatidylinositol anchor. LAMP is expressed by neurons composing the limbic system, is highly conserved between rodents and human, and has structural and functional properties that substantiate its role in the formation of limbic circuits. We report here the genomic organization of the Lsamp gene. The Lsamp gene is composed of 11 exons distributed over 2.2 megabases (Mb). Two exons 1 are separated by approximately 1.6 Mb and contribute to the unusual large size of the gene. Alternative spliced Lsamp mRNAs are generated from distinct promoter regions associated with the two exons 1 that encode distinct signal peptides and thus generate identical native mature polypetides. Additional diversity is created by the use of two small exons to include an insertion of 23 amino acids within the polypeptide Cterminal region of the mature protein. The genomic features of the Lsamp gene described here indicate an intricate mechanism of gene expression regulation that may be relevant in the context of human neuropsychiatric and neurological disorders, where LAMP expression may be altered. D 2004 Elsevier Inc. All rights reserved. Keywords: Cell adhesion molecules; IgLON; Central nervous system; Genomic organization; Glycosyl-phosphatidylinositol anchor; Immunoglobulin superfamily

Introduction Gene regulation in the brain is complex, with regional expression of transcripts and proteins on subpopulations of neurons that are essential for proper functioning of circuits. Guidance molecules that mediate cellular interactions are particularly important in this context. The Lsamp gene encodes the limbic system-associated membrane protein (LAMP) a 64- to 68-kDa glycoprotein expressed on the surface of somata and proximal dendrites of neurons in cortical and subcortical regions of the limbic system [1,2]. Functionally, the structures of the brain composing the limbic system are involved in mediating cognitive, emotional, and autonomic behaviors. These brain regions are thought to be dysfunctional in neuropsychiatric disorders [see 3 –5 for reviews]. The Lsamp cDNA encodes a 338amino-acid polypeptide, structurally characterized by three * Corresponding author. John F. Kennedy Center for Research on Human Development and Department of Pharmacology, Vanderbilt University, 8114C MRB III, 465 21st Avenue South, Nashville, TN 37232. Fax: +1-615-936-3747. E-mail address: [email protected] (A.F. Pimenta). 0888-7543/$ - see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.ygeno.2003.11.013

immunoglobulin (Ig) domains. Two signal sequences, the signal peptide (SP) and the hydrophobic C-terminus, target the protein to the cell membrane where it integrates via a GPI anchor [6,7]. The amino acid (aa) sequence of LAMP is highly conserved among species, with 99% identity between rodent and human [7], indicating strong phylogenetic conservation of protein structure and associated functional properties. Indeed, the expression of Lsamp mRNA from early stages of development [8] through adulthood [9] shows a close correlation with the distribution pattern of the protein in developing [2] and adult rat limbic structures [1]. Anatomical mapping in human [10,11] and nonhuman primates [12,13] also shows conserved distribution. Functional and biochemical studies have shown that LAMP, through homophilic interactions, selectively promotes neurite outgrowth of LAMP-expressing neurons [6,14,15] and mediates proper circuit formation [6,16] of limbic pathways. In addition, recent data indicate that heterophilic interactions between LAMP and unknown counter partners have an inhibitory effect on neurite outgrowth [15,17]. Therefore, these studies suggest that LAMP, through a complex pattern of interactions, is an important determinant of proper limbic system development and function.

A.F. Pimenta, P. Levitt / Genomics 83 (2004) 790–801

Assessment of the genomic structure of the Lsamp gene is necessary to further characterize its function and potential implications in human limbic system development and its association with neuropsychiatric disorders. Here we report the cloning of an alternatively spliced variant of the Lsamp mRNA and describe the complete exon –intron structure of the gene, including additional and unusual splicing events at the 5Vend of the gene leading to alternative exons encoding the signal peptide. The unusual features of the genomic structure of the Lsamp gene reported here may have important implications regarding the regulation of its expression.

Results Alternative spliced forms of the Lsamp mRNA Nucleotide insertion The nucleotide sequence of the cDNA encoding LAMP was originated from four independent clones, isolated from

791

an adult rat hippocampus library [6]. An additional clone containing an insert of 1276 basepairs (bp) was isolated from the same library and sequenced. The cDNA contains an open reading frame of 1083 bp identical to Lsamp, except for an additional stretch of 69 bp that results in alteration of one codon and insertion of 23 amino acids within the Cterminal region of LAMP (Fig. 1). Consensus sequences at the putative splice sites of the Lsamp –6c cDNA suggest a splicing mechanism for the introduction of the 69-nucleotide (nt) insertion that is confirmed in mouse, rat, and human genomic DNA (Fig. 2). Indeed, the 69-nt insertion is encoded by two exons (Fig. 3, Table 1). The predicted pre-pro-protein sequence derived from the open reading frame produces a 361-aa polypeptide with a calculated molecular mass of 34 kDa after cleavage of the signal peptide and attachment of the GPI anchor. The expression of the recombinant isoform in CHO cells is characterized by punctate immunoreactivity (Fig. 4A) that is identical to the expression pattern of LAMP on the surface of cultured neurons [2] and the recombinant LAMP on the surface of

Fig. 1. Alternative splicing generates an isoform of Lsamp mRNA that encodes a polypeptide with a 23-aa insertion at the C-terminus. (A) Nucleotide and deduced aa sequence of the rat Lsamp cDNA [6] encoding the C-terminus of LAMP (GenBank Accession No. U31554). (B) The nucleotide sequence of the rat Lsamp-6c cDNA includes an insertion of 69 nt (underlined) encoding 23 aa (bold) at the C-terminus of LAMP (GenBank Accession No. AY326256). In A and B, the putative splice site for the 69-nt insertion is indicated by an arrow. The proposed cleavage/attachment site for the GPI anchor [7] is denoted by the arrowhead. A conserved cysteine residue (C) in the third Ig domain is shown in bold. (C) Alignment of the alternative spliced nucleotide sequence expressed in rat, mouse, human, and chicken. Rn indicates Rattus norvegicus; Mm, Mus musculus; Hs, Homo sapiens; and Gg, Gallus gallus. Accession numbers for nucleotide sequences identified in the GenBank database are the chicken Lsamp ortholog [18], g11-isoform (Z94719), and mouse ESTs from hypothalamus (BE943615), corpus striatum (AI837405), visual cortex (BY646092), and brain stem (BI134406) libraries. (D) Species comparison of the deduced amino acid sequence encoded by the 69-nt insertion. Identical residues are highlighted in C and D.

792

A.F. Pimenta, P. Levitt / Genomics 83 (2004) 790–801

Fig. 2. Comparison of the intron – exon structures between Lsamp orthologs in mouse, rat, and human.

CHO cells [6]. Consistent with the attachment to the membrane by a GPI anchor, treatment of the transfected cells with PI –PLC results in release of the protein and loss of immunoreactivity (data not shown). Structural analysis of

the deduced amino acid sequence, in silico, using the GCG package (University of Wisconsin, Madison, WI), suggests an elongation of the C-terminal region and an additional putative flexibility domain close to the GPI attachment site

Fig. 3. Genomic organization of the mouse Lsamp. (A) Schematic representation of the LAMP structure, derived from the predicted amino acid sequence. The three Ig domains are represented by the globular structure, the site for the attachment of the GPI anchor is indicated by an arrow, and the putative site for the insertion of 23 amino acids is represented by a thick open bar. Splice sites defining the Lsamp exons are indicated by short arrows. (B) Physical map of Lsamp. Exons are numbered from the 5Vend and depicted as boxes. Sizes of introns are indicated in kilobases. CpG islands are indicated by thick bars. (C) Schematic representation of the splicing events that generate the Lsamp cDNAs isoforms. Use of the alternative first exons encoding distinct signal peptides in the Lsamp transcripts does not alter the LAMP protein structure.

A.F. Pimenta, P. Levitt / Genomics 83 (2004) 790–801

793

Table 1 Exon/intron junctions on the mouse Lsamp gene Exon

Domain

1a 1aV

5V UTR/SP SP

1b 2 3 4 5 6 7 8 9

5V UTR/SP IgI IgII IgII IgIII IgIII insert insert C-terminus 3V UTR

Exon size (bp) 365a 60 722 232 125 134 120 148 36 33 >253**

Acceptor splice site

N\A *ttttctttag TCTTGGC *cttcccacag GACTGCC N\A gctctgttag GTGTGTG ccatctacag TTCCACC *tttttttcag GAAGAGA cttgctacag ATCCACC ctactcttag GATAAAC *tccccttcag AGCGTGT *tcttttttag AAATTGG tgctctgcag GACCCGG

Scores

86.0 94.7 85.6 86.7 99.5 89.8 79.4 90.0 87.5 96.1

Donor splice site

Scores

Intron Size (kb)

Phase

TTGCAAG gtagggggag* ATCCCAG gtgggtcacc*

84.7 83.1

799 838

1 1

TCCTCAG gtagggcttg GTACAAG gtaaggggga CCACTAG gtaagcaact GTGAACT gtgagtatag* ACACCAG gtacatgcta CTTTTCA gtaagtatgc ATTCAAG gtcagtatgg* CCAAAAG gtatggttac* —

84.7 95.4 94.2 75.7 74.1 89.8 95.4 84.1

355 66 179 9 0.5 6 17 6

2 1 1 1 2 1 1 1

a

Include predicted size for the 5V UTR. * Splice sites sequences identified in the mouse genome database. ** Polyadenylation sites are present in the mouse genome in sequences downstream of the termination codon of Lsamp. Several ESTs map to the 3VUTR up to 2.0 kb downstream of the termination codon.

as a result of the 23-aa insertion. No consensus motif has been associated with the 23-aa sequence. Therefore, a functional assertion for the insertion of 23 amino acids in the LAMP isoform is not clear, although an increase in flexibility in addition to the possibility of raising the molecule from the plane of the neuronal membrane could be advantageous for molecular interactions. Lsamp sequences containing the 69-bp insertion also were amplified and cloned from human cerebral cortex cDNA (Fig. 1). In addition, a PCR product of the predicted size was amplified from an E18 rat brain cDNA library (data not shown). By RT-PCR we were able to identify this Lsamp variant in several areas of the rat brain including hippocampus, prefrontal cortex, cerebellum, brain stem, and olfactory bulb (Fig. 4B). In addition, searches of the mouse Expressed Sequence Tags (EST) database identified Lsamp cDNA sequences containing the 69-nt insertion cloned from hypothalamus, corpus striatum, brain stem, and visual cortex (accession numbers cited in Fig. 1). An insertion of 36 nt at the same position, with conserved homology, has been reported on the g11 isoform of the chicken ortholog of Lsamp [18]. Alternative signal peptides Sequence comparisons with the mouse and human EST database, using the entire Lsamp cDNA as query, revealed additional sequences that are mouse and human orthologs of the previously described g9 chicken isoform of Lsamp [18]. These Lsamp cDNAs encode an alternative signal peptide with no homology to the cloned rat [6] and human [7] LAMP signal peptides (Fig. 5A). The alternative signal peptide is encoded in mouse by two exons, an additional first exon (exon 1a) and exon 1aV, and in human, chicken, and pig by exon 1a (Figs. 3 and 5; Table 1). The sequence encoding exon 1aV is present in the rat genome (Figs. 2 and 5B), suggesting a possibility of alternative splicing in this

species. The sequences and genomic structure are highly conserved among species, indicating a complex splicing mechanism to generate Lsamp mRNAs encoding the prepro-protein. The processed polypeptides after cleavage of the signal peptide, however, contain identical N-termini, indicating that this complex alternative splicing process may serve regulatory mechanisms without altering the functional properties of the protein. Isolation of genomic clones Eighteen Lsamp-positive clones containing inserts of 13 – 18.5 kb were isolated from a 129/ReJ mouse genomic library screened with the rat Lsamp cDNA. The identity of these positive clones were determined by subsequent dot blot and southern blot analyses using rat Lsamp cDNA probes specific for the regions encoding the individual Ig domains of LAMP and the 5V UTR. Initial orientation and alignment of the clones was done by restriction mapping and PCR amplification using exon –exon and exon –T3/T7 (vector) primers. Exon boundaries were defined by comparing the genomic sequences with the rat and mouse Lsamp cDNA sequences. Ten of the splice junctions were identified using this strategy. As the mapping progressed, however, it became clear that several introns of this gene were unexpectedly large. The availability of the mouse genome sequences provided the remaining junctions as noted in Table 1. General features of the Lsamp gene To characterize the mouse Lsamp gene, all the identified exons and genomic sequences from isolated Lsamp-positive E-clones were used in alignments with the mouse genomic sequence of chromosome 16 using the Blast algorithm. We had previously assigned the Lsamp gene to the mouse

794

A.F. Pimenta, P. Levitt / Genomics 83 (2004) 790–801

Fig. 4. Expression of Lsamp-6c transcripts and translated polypeptide. (A) LAMP-6c recombinant protein is targeted to the membrane of CHO cells and attached by a GPI anchor. Live CHO cells stably transfected with Lsamp-6c cDNA are immunoreactive with the monoclonal antibody that recognizes the native form of LAMP. Treatment with PI-PLC eliminates the immunoreactivity (data not shown). (B) Specific amplification of the Lsamp-6c insertion indicates the presence of the transcript in CNS regions. Agarose gel showing the RT-PCR product (237 bp) amplified from brain stem (lane 1), prefrontal cortex (lane 2), cerebellum (lane 3), hippocampus (lane 4), and olfactory bulb (lane 5).

chromosome 16B5 by fluorescence in situ hybridization [19]. The mouse Lsamp gene spreads over approximately 2.28 megabases (Mb) and contains 11 exons (Fig. 3). In view of the fact that the mouse Lsamp mRNAs identified on Northern blots are 1.6, 3.3, and 8 kb long (Fig. 6), introns represent approximately 99.65% of the gene. This is an unusually large gene to encode the LAMP polypeptide isoforms of 338, 355, and 361 aa. Our current searches on the GenBank publicly available mouse genome and Celera Mouse Genome databases have localized the Lsamp gene on Chr 16B4. The exon/intron organization of the Lsamp gene The mouse Lsamp gene consists of 11 exons (Fig. 3). Five exons are used in alternative splicing events to generate three transcripts (Fig. 3C). Seven exons (1b – 6 and 9) encode the Lsamp cDNA. Two additional exons (7 and 8)

are included to generate the alternative spliced form with the 69-nt insertion described in Fig. 1. Two additional exons, 1a and 1aV, encode the alternative 5VUTR signal peptide (Fig. 5) of the mouse Lsamp-SP. To generate this transcript, an additional splice event takes place between exon 1aV and a putative acceptor splice site (score, 94.7) within exon 1b to include the sequences encoding the predicted cleavage site for the signal peptide and the N-terminus of LAMP (Figs. 3c and 5; Table 1). As noted in Fig. 5A, exon 1b contains the 5V UTR, the initiator ATG, and the nucleotide sequence encoding the signal peptide and N-terminus of LAMP. The DNA sequence encoding IgI is contained completely in exon 2, whereas the coding regions of IgII and IgIII are interrupted by an intron. Exon 1a contains the alternative 5V UTR with its initiator ATG and the sequence encoding the first 22 aa of the signal peptide. The remaining 20 aa of the signal peptide are encoded by exon 1aV. Exon 9 contains the nucleotide sequence encoding the C-terminus of LAMP and the 3VUTR which contains putative polyadenylation signals. Two very large introns, 799 and 838 kb, intercalate exons 1a, 1aV, and 1b (Fig. 3B; Table 1). Another large intron, 355 kb, is present between exons 1b and 2. Large 5V introns, particularly the first intron, are characteristic of members of the IgSF [20 – 26] and a number of other genes. In several instances, those large introns contain regulatory elements involved in silencing and spatial and temporal patterns of gene expression [27 –30]. All intron –exon boundary sequences conformed to consensus splice donor (GT) and acceptor (AG) sites. Junction scores (Table 1) were calculated using an algorithm based on Shapiro and Senapathy [31], publicly available at the Splice Site Finder program. All scores are in the normal range: scores for donor sites varied from 74.1 to 95.4 and those for acceptor sites from 79.4 to 96.1. The prediction program was basically used to validate the splice junctions and the highest scores were indeed attributed to the splice sites defined in alignments with genomic structures. False positives are predicted with this method but with lower scores. The 5V-noncoding regions Four genomic phage clones containing exon 1b were analyzed by restriction mapping and PCR amplification using exon-T3/T7 (vector) primers. Restriction fragments and PCR products were subcloned and sequenced to produce a 3058-nt sequence upstream of the initiator ATG. A putative Lsamp transcription initiation site at position 567 was determined by 5V RACE, defining a 5V UTR of 567 nt with an overall 58% G+C content and immediately upstream of the initiator ATG ( 1 to 127), a 75% G+C stretch. Genomic DNA fragments of 0.86, 2.0, and 2.5 kb, containing 5V sequences flanking exon 1b, were fused to the chloramphenicol acetyltransferase (cat) gene (Figs. 7A and 7B). In CAT reporter gene assays these genomic fragments exhibited general promoter activity in neuronal and nonneuronal cell lines (Fig. 7C), indicating the presence of a

A.F. Pimenta, P. Levitt / Genomics 83 (2004) 790–801

795

Fig. 5. Alternative use of exons generates isoforms of LAMP with distinct signal peptides. (A) Multiple sequence alignment of the deduced amino acid sequence encoded by Lsamp first exons. Alternative splice of exon 1a or 1a/1aVuses a putative splice site within exon 1b to include three amino acids (GLP), present in all isoforms, and the N-terminus of the mature polypeptide. Signal peptide sequences are underlined and the predicted putative cleavage sites for the signal peptides are denoted by an arrowhead. The first seven amino acids of the N-terminus are denoted (VRSVDFN/T). Rn indicates Rattus norvegicus; Mm, Mus musculus; Hs, Homo sapiens; Gg, Gallus gallus; and Ss, Sus scrofa. (Exon 1b) GenBank accession numbers for cDNA nucleotide sequences encoding exon 1b are as follows: rat (U31554), human (U41901) and chicken (Y08171, Z94719) Lsamp orthologs [6,7,18,51]; mouse (BY726997, BB645027, BB643056, BB618284, AI427225, BY135319) and pig (BI337752) ESTs. (Exon 1a) GenBank accession numbers for cDNA nucleotide sequences encoding exon 1a as follows are: chicken Lsamp ortholog [18], g9-isoform (Z94718); human (BI199955, BM696801) and pig (BI359722, BE012499) ESTs. All mouse ESTs nucleotide sequences from hippocampus (BB652926), visual cortex (BY237314), olfactory brain (BY004290) and 15 days embryo head (BB663069) library are generated by splicing of exon 1a and exon1aV. (B) Alignment of the Lsamp cDNA nucleotide sequences transcribed from exon 1b and exon 1a or 1a/ 1aV (mouse). The cDNA sequences encoding the signal peptides are shown, and the respective 5V UTRs are omitted. Alternative splicing of exon 1a and 1aV is used to generate the mouse transcript. Mouse genomic sequences encoding exon 1aVare aligned with an identical exon 1aVnucleotide sequence present in the rat genome, including the flanking conserved splice sites. In the human genome, a loss of the acceptor splice site (thick bar) prevents the inclusion of a mutated exon 1aV, containing at 5V an insertion of 2 nt that introduces a frame shift resulting in a termination codon indicated by an asterisk.

promoter region associated with exon 1b and therefore suggesting that a second promoter region must be associated with mRNA transcription from exon 1a. Indeed, additional in silico analysis corroborates this hypothesis: Exon 1b and

exon 1a are both immersed in predicted CpG islands (Fig. 3B). In addition, the 5V flanking regions of both exons were scanned for putative promoter and transcriptional regulatory elements. The 5V regions of both first exons lack the

796

A.F. Pimenta, P. Levitt / Genomics 83 (2004) 790–801

Discussion

Fig. 6. Northern blot analysis reveals a novel 3.3-kb Lsamp transcript expressed in neural tissue. Distribution of Lsamp transcripts in adult mice indicates the presence of three transcripts of 1.6, 3.3, and 8.0 kb in neural tissue (prefrontal cortex (lane 1) and cerebellum (lane 2)). No hybridization is detected in nonneural peripheral tissue (kidney (lane 3) and liver (lane 4)).

canonical TATA box and harbor GC-rich segments, which is a common feature of promoter regions of genes encoding cell adhesion molecules of the IgSF [20 –22,32,33]. The 5V region of exon 1a contains a predicted transcription start site at 298 preceded by two Sp1 sites. Indeed, four mouse ESTs contain 5V UTR extending to 290 and 294, the ESTs were derived, respectively, from hippocampus, P10 brain and visual cortex, and E15 embryo libraries (accession numbers are noted in Fig. 5). Both genomic regions were found to contain sequences for regulatory elements such as AP-1, SP1, CREB and CAAT. Rat and human LSAMP The comparison of the structure of the mouse Lsamp with the rat and human LSAMP genes reveals that the length and position of all exons are identical in the three orthologs, with the exception of exon 1aV. We identified, in the human genome, the nucleotide sequence of exon 1aV containing an insertion of one codon and two additional nucleotides that result in frame shift and introduction of a termination codon. In addition, the acceptor splice site in intron 1a is lost therefore preventing the splice of the mutated exon (Fig. 5B). All the introns are of similar size, including intron 1a, which, due to the lack of exon 1aV reflects, in human, the full-length genomic sequence that separates the two first exons encoding the signal peptide. The intron/exon structure is highly conserved among the species.

The sequencing of the human [34,35] and mouse [36] genome revealed a much smaller number of genes for both species than predicted from the expressed sequences, indicating that complex gene structure associated with intricate mechanisms of regulation would account for the diversity of gene expression through events such as differential splicing, RNA editing, and alternative 3V end polyadenylation. Here we report the genomic structure of the mouse Lsamp gene, revealing an unusually large gene that encodes three isoforms of LAMP generated through alternative splicing. The Lsamp gene extends over 2.2 mb and contains 11 exons that encode mRNAs of 1.6, 3.3, and 8 kb, the three transcripts expressed in the mouse CNS. In addition, the smallest transcript is of sufficient size to encode LAMP [6] and the isoform containing an insertion of 23 aa described here. LAMP is a 64- to 68-kDa glycoprotein, is heavily glycosylated, and appears as a broad band on Western blots [2,6]. Using monoclonal and polyclonal antibodies against LAMP, we have not detected bands of different molecular mass on Western blots of membrane proteins isolated from CNS, indicating that larger messages may contain long 5V and/or 3V UTR with regulatory functions. The unusual size of the Lsamp gene is primarily due to the presence of the separation of the two first exons (exon 1b and exon1a/1aV) by introns encompassing 1.6 Mb. Another large intron of 355 kb intercalates exons 1b and 2. The presence of large 5V introns has been reported and suggested to contain genomic information such as enhancer or silencer elements involved in the regulation of gene expression [37 – 41]. Indeed, in members of the IgSF, such as L1 and Ng-CAM, the first intron contains regulatory elements and silencers conferring tissue-specific expression [27 – 30]. Another potential role for very large introns may reside in the temporal regulation of transcription, which may be particularly important during development or pathophysiological processes. The presence of additional 5V or first exons driven by individual promoters has been reported for other genes [42 – 46]. Alternative promoter usage is believed to be an important evolutionary mechanism that provides flexibility to the transcriptional regulation of genes [47]. The unique feature of the two first introns of the Lsamp gene is the enormous distance that separates them, and the fact that they encode distinct signal peptides. Signal peptides are used by cells to target nascent membrane proteins to the ER and are cleaved in this compartment. Therefore, identical mature LAMP polypeptides attached to the membrane by a GPI anchor are generated whether transcription is driven by exon 1b or exon 1a promoters, suggesting that such use of alternative promoters/exon1 have regulatory implications. Besides contributing a mechanism for differential expression of genes at the transcriptional level, the use of more than one promoter generates mRNA isoforms containing alternative 5V UTRs for which translational efficiency may vary. The use of long

A.F. Pimenta, P. Levitt / Genomics 83 (2004) 790–801

797

Fig. 7. Nucleotide sequences upstream of Lsamp exon 1b show basic promoter activity in neuronal and nonneuronal cell lines. (A) Line diagram illustrating the Lsamp genomic region 5V of exon 1b. Size and position of the regions amplified and fused to the chloramphenicol acetyltransferase (cat) reporter gene are indicated by line bars. Selected restriction enzyme sites are H, HindIII, and P, PstI. (B) Schematic representation of the chimeric cat gene expression plasmids. Sizes of the genomic DNA fragments are indicated. Arrowhead indicates reverse orientation of the genomic DNA fragment. E, pCAT-Enhancer vector; SV, pCAT control vector; In, intronic sequence. (C) Thin-layer chromatograms of representative CAT assays illustrate the transcriptional activity of the genomic region associated with exon 1b. CAT activity was tested on extracts of CHO (lanes 1 – 7) and SN56 (lanes 8 – 10) cells transfected with Lsamp/cat reporter constructs as indicate in each lane. Transcriptional activity is indicated by the acetylated choramphenicol bands present in all lanes containing Lsamp sequences (constructs 1, 3 – 5) and in the control vector (SV40 promoter) (6). Vector alone (7) or containing sequences in the reverse orientation (2) show no transcriptional activity. (a) chloramphenicol, (b) acetylated chloramphenicol, (c) diacetylated chloramphenicol.

5V UTRs with secondary structures [48] or ATG codons upstream of the genuine initiation site [49] can impede/delay the scanning of the ribosome. Within the IgLON subfamily, an identical alternative use of two exons 1 encoding the signal peptide is evident from the alignment of the cDNAs encoding the isoforms of the rat OBCAM [50] and its chicken orthologs [51,52]. The entire sequences encoding the N-terminus and the Ig domains are identical. The two transcripts differ on the 5V UTRs and the sequence encoding the signal peptides. Two distinct exons 1 are found in the mouse and the human Opcml/OPCLM (opioid binding protein/cell adhesion molecule-like) genes. So far, one cDNA sequence has been described for Neurotrimin (Hnt) [53] and Kilon [54], two other members of the IgLON subfamily. In searches of the mouse EST database we found neurotrimin cDNAs encoding two alternative signal peptides and respective exons 1 are found in the mouse and human genome. We also describe here an isoform of LAMP generated by an insertion of 23 aa at the C-terminus between the third Ig domain and the GPI anchor. Two small exons (exon 7 and exon 8) encoding, respectively, 12 and 11 aa are included in this Lsamp mRNA. This alternative splice event is conserved in rat, mouse, and human and in the chicken

ortholog, where an exon homologous to the mammalian exon 7 is spliced at the same position [18]. Although the chicken genome is not sequenced and publicly available, the orthologs of Lsamp are highly conserved and thus are likely to exhibit a conserved genomic structure. An associated functional correlation with this insertion has not yet been established. The insertion of small stretches of amino acids in the IgSF members is a common event and ranges from micro splicing to include exons coding for one amino acid to the inclusion of several amino acids [55 – 60]. An insertion of 10 amino acids in the fourth Ig domain of NCAM (named VASE) [57] introduces a conformational change in the Ig domain structure and has functional implications for cell – cell interactions and neurite outgrowth [59 –63] and potential implication in schizophrenia [64]. Analyses of the genomic organization of IgSF members have shown that the Ig domains are encoded by two separated exons [21 – 26]. Each Ig domain therefore is encoded by a module containing two exons. Introns interrupting domain modules in general are in phase 1 (located after the first nucleotide of the codon) which would permit evolutionary movement of modules without disrupting the open reading frame of a gene. In contrast, the introns within the domain module are of any of the three possible phases.

798

A.F. Pimenta, P. Levitt / Genomics 83 (2004) 790–801

In the Lsamp gene as reported here, the first Ig domain is encoded entirely by one exon, whereas Ig2 and Ig3 are each encoded by a module of two exons. Introns interrupting the modules encoding Ig2 and Ig3, typical C2 type Ig domains, are in phase one. However the module encoding Ig1 is interrupted 5V by an intron in phase 2 and 3V by an intron in phase 1, implying that evolutionary shuffling of this domain would not be favorable. We also noted the unusual feature of a very large intron, 179 kb, disrupting the sequence encoding Ig2, whereas a much smaller intron of 0.5 kb interrupts the sequence encoding Ig3. The Lsamp gene exhibits a highly conserved structure, but with potentially complex regulation and splicing. While exonic sequence and gene structure are conserved, expression patterns in the nervous system of LAMP orthologs in nonmammalian vertebrates are different. For example, the LAMP ortholog in chick is expressed in the retina and sensory ganglia, two structures that do not contain Lsamp transcripts or protein in mammals [18,52]. In mammals, the expression patterns become more refined and specific to limbic system structures in gyrencephalic species such as monkey and human [10 –13]. Thus, region-specific regulation of Lsamp, and its alternatively spliced forms, will become important to investigate in the context of human neuropsychiatric and neurological disorders, where LAMP expression may be altered.

Materials and methods Genomic library screening A 129/ReJ mouse genomic library constructed in the EFIX II vector (Stratagene), containing inserts of 15 –25 kb (gift from Dr. J. Pintar, Robert Wood Johnson Medical School), was screened with 32P random primed labeled probes derived from the rat Lsamp cDNA. Six hundred thousand plaques were screened. Eighteen positive clones were submitted to restriction map and Southern blot analysis. Initial orientation and alignment of the clones were done by restriction mapping and PCR amplification using exon – T3/T7 (vector) or exon– exon primers. PCR products of interest were subcloned into pGEM-T (Promega) and sequenced. Molecular biology techniques Unless otherwise indicated, all standard molecular biology techniques were performed essentially as described by Sambrook et al. [65] and Ausubel et al. [66]. cDNA library screening and RT-PCR analysis of splice variants An adult rat hippocampus cDNA library constructed in the Egt11 expression vector (BD Biosciences) was screened

with 32P end labeled Lsamp oligonucleotide probes, and plaque purified positive clones were subcloned and sequenced as described previously [6]. To identify the human LSAMP cDNA sequence containing the 69-nt insertion, human cerebral cortex cDNA (BD Biosciences) was amplified by PCR using Lsamp oligonucleotide primers complementary to the rat Lsamp nucleotide sequence, as described previously [7]. The sequence of the mouse Lsamp cDNA was obtained by amplification of mouse cerebral cortex cDNA (BD Biosciences) using Lsamp oligonucleotide primers complementary to the rat Lsamp nucleotide sequence, as described previously [7]. RT-PCR analysis of the Lsamp 69-nt insertion Total cellular RNA from dissected areas of the rat brain was isolated using the TRIzol reagent (Invitrogen) following manufacturer’s protocol. The integrity of the isolated RNA was verified on formaldehyde – agarose gel. Reverse transcription (RT) and PCR amplification were performed with the Superscript II one-step RT-PCR system (Stratagene) following manufacturer’s protocol. Forward (6c-F35) and reverse (6c-R47) primers used for amplification of the 69-nt insertion were designed to produce a 237 – bp cDNA fragment. 5V-RACE 5V-RACE was carried out with the 5V- RACE system for rapid amplification of cDNA ends, Version 2.0 (Invitrogen), using mouse total RNA as template for RT. Total RNA was isolated using the TRIzol reagent (Invitrogen) according to the manufacturer’s protocol. Primers AP1 and AP2 were supplied with the RACE system. The first round of PCR amplification was performed using oligo Race L-1 and AP1. The nested round of amplification was carried out using oligo Race L-2 and AP2. The resulting RACE products were cloned into the pGEM-T vector (Promega) and sequenced using Race L-3, SP6, and T7 oligonucleotides. Synthetic oligonucleotides The oligonucleotides designed and used in these studies are the following: 6c-F35 5V-CGGGATGACACCAGGATAAACAG-3V 6c-R47 5V-CGGGTCCTTTTTGCTTGAAGTG-3V Race L-1 5V-GTCTCAGTAGGACCAGCGGCAACTGTTTCC-3V Race L-2 5V-GCTCTTTCCCTCGCTTAGTC-3V Race L-3 5V-GCTTAGTCTCTTTTCCCTCTG-3V Northern blot analysis Total cellular RNA was isolated from adult C57Bl/6J mice using the TRIzol reagent (Invitrogen) following manufacturer’s protocol. The poly(A)+ RNA fraction was puri-

A.F. Pimenta, P. Levitt / Genomics 83 (2004) 790–801

fied using the Oligotex mRNA isolation system (Qiagen) and 1 Al of each sample was analyzed on an Agilent 2100 bioanalyzer (Agilent Technologies) for quantification and integrity of the mRNA. Poly(A)+ RNA (1 Ag) was separated on 1.2% agarose –formaldehyde gel, transferred to a nylon membrane (Nytran SuperCharge, Schleicher and Schuel), UV cross-linked, and hybridized overnight under stringent conditions with 32P-labeled RNA probes. Antisense probes were transcribed in vitro using T7 RNA polymerase from rat Lsamp cDNA template linearized with MscI (nt 464 – 1238), while control sense probes were prepared with SP6 RNA polymerase from rat Lsamp cDNA digested with NarI (nt 55 to 471). Reporter plasmid constructs and CAT assays Fragments of genomic DNA were amplified by PCR and cloned into the pCAT-Basic and pCAT-Enhancer promoterless vectors (Promega). The size, position, and orientation of DNA fragments in each construct are indicated in Fig. 7. For CAT assays, plasmids were introduced into CHO and SN56 [67] cell lines using calcium phosphate-mediated transfection [66] and cell extracts were prepared 48 h posttransfection. CAT assays were performed as described by Kumar et al. [68]. Thin-layer chromatography using silica gel (Chromagram, Kodak) was performed using standard protocol [66]. Chromatograms were exposed for autoradiography and scanned on a phosphor imager. Bioinformatics Alternative Lsamp transcripts were confirmed or identified by searching the expression sequence tags (EST divisions) and the nonredundant (nr) GenBank databases (http:// www.ncbi.nlm.nih.gov/) using the rat, mouse, and human Lsamp cDNAs. Genomic sequences were oriented and assembled in contigs using the GCG package and BLAST2 program. For examining genomic organization, BLAST searches were performed using the annotated human and mouse genome GenBank databases (http://www.ncbi.nlm. nih.gov/) and Celera Discovery System (http://www.celera. com). Junction scores were calculated using an algorithm based on Shapiro and Senapathy [31], publicly available at the Splice Site Finder program (http://www.genet.sickkids. on.ca/~ali/splicesitefinder.html). Putative transcription factor binding sites were predicted using TESS (http://www. cbil.upenn.edu/tess). The accession number for the Lsamp-6c isoform is AY326256.

Acknowledgments We thank Mrs. Pamela K. Cornuet for excellent technical assistance. This work was supported by NIMH Grant MH45507 and NICHD Core Grant P30 HD15052.

799

References [1] P. Levitt, A monoclonal antibody to limbic system neurons, Science 223 (1984) 299 – 301. [2] A. Zacco, V. Cooper, P.D. Chantler, S. Fisher-Hyland, H.L. Horton, P. Levitt, Isolation, biochemical characterization and ultrastructural analysis of the limbic system associated membrane protein (LAMP), a protein expressed by neurons comprising functional neural circuits, J. Neurosci. 10 (1990) 73 – 90. [3] C.E. Byrum, J.E. Thompson, E.R. Heinz, K.R. Krishnan, R.D. Tien, Limbic circuits and neuropsychiatric disorders. Functional anatomy and neuroimaging findings, Neuroimaging Clin. N. Am. 7 (1997) 79 – 99. [4] A. Kumar, I.A. Cook, White matter injury, neural connectivity and the pathophysiology of psychiatric disorders, Dev. Neurosci. 24 (2002) 255 – 261. [5] A.R. Hariri, D.R. Weinberger, Imaging genomics, Br. Med. Bull. 65 (2003) 259 – 270. [6] A.F. Pimenta, V. Zhukareva, M.F. Barbe, B.S. Reinoso, C. Grimley, W. Henzel, I. Fischer, P. Levitt, The limbic system-associated membrane protein is an Ig superfamily member that mediates selective neuronal growth and axon targeting, Neuron 15 (1995) 287 – 297. [7] A.F. Pimenta, I. Fischer, P. Levitt, cDNA cloning and structural analysis of the human limbic-system-associated membrane protein (LAMP), Gene 170 (1996) 189 – 195. [8] A.F. Pimenta, B.S. Reinoso, P. Levitt, Expression of the mRNAs encoding the limbic system-associated membrane protein (LAMP). II. Fetal rat brain, J. Comp. Neurol. 375 (1996) 289 – 302. [9] B.S. Reinoso, A.F. Pimenta, P. Levitt, Expression of the mRNAs encoding the limbic system-associated membrane protein (LAMP). I. Adult rat brain, J. Comp. Neurol. 375 (1996) 274 – 288. [10] L. Prensa, S. Richard, A. Parent, Chemical anatomy of the human ventral striatum and adjacent basal forebrain structures, J. Comp. Neurol. 460 (2003) 345 – 367. [11] L. Prensa, J.M. Gimenez-Amaya, A. Parent, Chemical heterogeneity of the striosomal compartment in the human striatum, J. Comp. Neurol. 413 (1999) 603 – 618. [12] P.-Y. Cote, P. Levitt, A. Parent, Distribution of limbic system-associated membrane protein immunoreactivity in primate basal ganglia, Neuroscience 69 (1995) 71 – 81. [13] P.Y. Cote, P. Levitt, A. Parent, Limbic system-associated membrane protein (LAMP) in primate amygdala and hippocampus, Hippocampus 6 (1996) 483 – 494. [14] V. Zhukareva, P. Levitt, The limbic system-associated membrane protein (LAMP) selectively mediates interactions with specific central neuron populations, Development 121 (1995) 1161 – 1172. [15] F. Mann, V. Zhukareva, A. Pimenta, P. Levitt, J. Bolz, Membraneassociated molecules guide limbic and non-limbic thalamocortical projections, J. Neurosci. 18 (1998) 9409 – 9419. [16] F. Keller, K. Rimvall, M.F. Barbe, P. Levitt, A membrane glycoprotein associated with the limbic system mediates the formation of the septohippocampal pathway in vitro, Neuron 3 (1989) 551 – 561. [17] O.D. Gil, L. Zhang, S. Chen, Y.Q. Ren, A. Pimenta, G. Zanazzi, D. Hillman, P. Levitt, J.L. Salzer, Complementary expression and heterophilic interactions between IgLON family members neurotrimin and LAMP, J. Neurobiol. 51 (2002) 190 – 204. [18] T. Brummendorf, F. Spaltmann, U. Treubert, Cloning and characterization of a neural cell recognition molecule on axons of the retinotectal system and spinal cord, Eur. J. Neurosci. 9 (1997) 1105 – 1116. [19] A.F. Pimenta, L.C. Tsui, H.H. Heng, P. Levitt, Assignment of the gene encoding the limbic system-associated membrane protein (LAMP) to mouse chromosome 16B5 and human chromosome 3q13.2 – q21, Genomics 49 (1998) 472 – 474. [20] G. Colwell, B. Li, D. Forrest, R. Brackenbury, Conserved regulatory elements in the promoter region of the N-CAM gene, Genomics 14 (1992) 875 – 882.

800

A.F. Pimenta, P. Levitt / Genomics 83 (2004) 790–801

[21] S.V. Kozlov, R.J. Giger, T. Hasler, E. Korvatska, D.F. Schorderet, P. Sonderegger, The human TAX1 gene encoding the axon-associated cell adhesion molecule TAG-1/axonin-1: genomic structure and basic promoter, Genomics 30 (1995) 141 – 148. [22] R.J. Giger, L. Vogt, R.A. Zuellig, C. Rader, A. Henehan-Beatty, D.P. Wolfer, P. Sonderegger, The gene of chicken axonin-1: complete structure and analysis of the promoter, Eur. J. Biochem. 227 (1995) 617 – 628. [23] B. Hassel, F.G. Rathjen, H. Volkmer, Organization of the neurofascin gene and analysis of developmentally regulated alternative splicing, J. Biol. Chem. 272 (1997) 28742 – 28749. [24] A. Plagge, T. Brummendorf, The gene of the neural cell recognition molecule F11: conserved exon – intron arrangement in genes of neural members of the immunoglobulin superfamily, Gene 192 (1997) 215 – 225. [25] O. Coutelle, G. Nyakatura, S. Taudien, G. Elgar, S. Brenner, M. Platzer, B. Drescher, M. Jouet, S. Kenwrick, A. Rosenthal, The neural cell adhesion molecule L1: genomic organisation and differential splicing is conserved between man and the pufferfish Fugu, Gene 208 (1998) 7 – 15. [26] K.R. Cho, J.D. Oliner, J.W. Simons, L. Hedrick, E.R. Fearon, A.C. Preisinger, P. Hedge, G.A. Silverman, B. Vogelstein, The DCC gene: structural analysis and mutations in colorectal carcinomas, Genomics 19 (1994) 525 – 531. [27] P. Kallunki, S. Jenkinson, G.M. Edelman, F.S. Jones, R.H. Waterston, K. Lindblad-Toh, E. Birney, Silencer elements modulate the expression of the gene for the neuron – glia – cell adhesion molecule NgCAM, J. Biol. Chem. 270 (1995) 21291 – 21298. [28] P. Kallunki, G.M. Edelman, F.S. Jones, Tissue-specific expression of the L1 cell adhesion molecule is modulated by the neural restrictive silencer element, J. Cell. Biol. 138 (1997) 1343 – 1354. [29] P. Kallunki, G.M. Edelman, F.S. Jones, The neural restrictive silencer element can act as both a repressor and enhancer of L1 cell adhesion molecule gene expression during postnatal development, Proc. Natl. Acad. Sci. USA 95 (1998) 3233 – 3238. [30] G.M. Edelman, F.S. Jones, Gene regulation of cell adhesion: a key step in neural morphogenesis, Brain Res. Rev. 26 (1998) 337 – 352. [31] M.B. Shapiro, P. Senapathy, RNA splice junctions of different classes of eukaryotes: sequence statistics and functional implications in gene expression, Nucleic Acids Res. 15 (1987) 7155 – 7174. [32] M.R. Hirsch, L. Gaugler, H. Deagostini-Bazin, L. Bally-Cuif, C. Goridis, Identification of positive and negative regulatory elements governing cell-type-specific expression of the neural cell adhesion molecule gene, Mol. Cell Biol. 10 (1990) 1959 – 1968. [33] M. Buttiglione, G. Cangiano, G. Goridis, G. Gennarini, Characterization of the 5V and promoter regions of the gene encoding the mouse neuronal cell adhesion molecule F3, Brain Res. Mol. Brain. Res. 29 (1995) 297 – 309. [34] J.C. Venter, M.D. Adams, E.W. Myers, et al., The sequence of the human genome, Science 291 (2001) 1304 – 1351. [35] E.S. Lander, L.M. Linton, B. Birren, et al., Initial sequencing and analysis of the human genome. International Human Genome Sequencing Consortium, Nature 409 (2001) 860 – 921. [36] R.H. Waterston, K. Lindblad-Toh, E. Birney, et al., Initial sequencing and comparative analysis of the mouse genome. Mouse Genome Sequence Consortium, Nature 420 (2002) 520 – 562. [37] J. Lazar, B. Desvergne, E.C. Zimmerman, D.B. Zimmer, M.A. Magnuson, V.M. Nikodem, A role for intronic sequences on expression of thyroid hormone receptor alpha gene, J. Biol. Chem. 269 (1994) 20352 – 20359. [38] C.J. Storbeck, L.A. Sabourin, J.D. Waring, R.G. Korneluk, Definition of regulatory sequence elements in the promoter region and the first intron of the myotonic dystrophy protein kinase gene, J. Biol. Chem. 273 (1998) 9139 – 9147. [39] P. Bornstein, Regulation of expression of the alpha 1 (I) collagen gene: a critical appraisal of the role of the first intron, Matrix Biol. 15 (1996) 3 – 10.

[40] A. Wutz, D.P. Barlow, Imprinting of the mouse Igf2r gene depends on an intronic CpG island, Mol. Cell. Endocrinol. 140 (1998) 9 – 14. [41] S. Finkbeiner, New roles for introns: sites of combinatorial regulation of Ca2+- and cyclic AMP-dependent gene transcription, Sci. STKE 94 (2001) PE1. [42] I. Bar, F. Tissir, C. Lambert de Rouvroit, O. De Backer, A.M. Goffinet, The gene encoding disabled-1 (DAB1), the intracellular adaptor of the Reelin pathway, reveals unusual complexity in human and mouse, J. Biol. Chem. 278 (2003) 5802 – 5812. [43] C. Corti, R.W. Clarkson, L. Crepaldi, C.F. Sala, J.H. Xuereb, F. Ferraguti, Gene structure of the human metabotropic glutamate receptor 5 and functional analysis of its multiple promoters in neuroblastoma and astroglioma cells, J. Biol. Chem. 278 (2003) 33105 – 33119. [44] J. Mankertz, J.S. Waller, B. Hillenbrand, S. Tavalali, P. Florian, T. Schoneberg, M. Fromm, J.D. Schulzke, Gene expression of the tight junction protein occludin includes differential splicing and alternative promoter usage, Biochem. Biophys. Res. Commun. 298 (2002) 657 – 666. [45] J.R. Landry, D.L. Mager, Widely spaced alternative promoters, conserved between human and rodent, control expression of the Opitz syndrome gene MID1, Genomics 80 (2002) 499 – 508. [46] J.L. Ko, H.C. Chen, H.H. Loh, Differential promoter usage of mouse mu-opioid receptor gene during development, Brain Res. Mol. Brain Res. 104 (2002) 184 – 193. [47] T.A. Ayoubi, W.J. Van De Ven, Regulation of gene expression by alternative promoters, FASEB J. 10 (1996) 453 – 460. [48] M. Kozak, An analysis of vertebrate mRNA sequences: intimations of translational control, J. Cell Biol. 115 (1991) 887 – 903. [49] M. Kozak, Adherence to the first-AUG rule when a second AUG codon follows closely upon the first, Proc. Natl. Acad. Sci. USA 92 (1995) 2662 – 2666. [50] D.A. Lippman, N.M. Lee, H.H. Loh, Opioid-binding cell adhesion molecule (OBCAM)-related clones from a rat brain cDNA library, Gene 117 (1992) 249 – 254. [51] D.J. Wilson, D.S. Kim, G.A. Clarke, S. Marshall-Clarke, D.J. Moss, A family of glycoproteins (GP55), which inhibit neurite outgrowth, are members of the Ig superfamily and are related to OBCAM, neurotrimin, LAMP and CEPU-1, J. Cell Sci. 109 (1996) 3129 – 3138. [52] A.P. Lodge, M.R. Howard, C.J. McNamee, D.J. Moss, Co-localisation, heterophilic interactions and regulated expression of IgLON family proteins in the chick nervous system, Brain Res. Mol. Brain Res. 82 (2000) 84 – 94. [53] A.F. Struyk, P.D. Canoll, M.J. Wolfgang, C.L. Rosen, P. D’Eustachio, J.L. Salzer, Cloning of neurotrimin defines a new subfamily of differentially expressed neural cell adhesion molecules, J. Neurosci. 15 (1995) 2141 – 2156. [54] N. Funatsu, S. Miyata, H. Kumanogoh, M. Shigeta, K. Hamada, Y. Endo, Y. Sokawa, S. Maekawa, Characterization of a novel rat brain glycosylphosphatidylinositol-anchored protein (Kilon), a member of the IgLON cell adhesion molecule family, J. Biol. Chem. 274 (1999) 8224 – 8230. [55] A.A. Reyes, S.J. Small, R. Akeson, At least 27 alternatively spliced forms of the neural cell adhesion molecule mRNA are expressed during rat heart development, Mol. Cell. Biol. 11 (1991) 1654 – 1661. [56] A.A. Reyes, S.V. Schulte, S. Small, R. Akeson, Distinct NCAM splicing events are differentially regulated during rat brain development, Brain Res. Mol. Brain Res. 17 (1993) 201 – 211. [57] S.J. Small, S.L. Haines, R.A. Akeson, Polypeptide variation in an NCAM extracellular immunoglobulin-like fold is developmentally regulated through alternative splicing, Neuron 1 (1988) 1007 – 1017. [58] B. Hassel, F.G. Rathjen, H. Volkmer, Organization of the neurofascin gene and analysis of developmentally regulated alternative splicing, J. Biol. Chem. 272 (1997) 28742 – 28749. [59] E. De Angelis, T. Brummendorf, L. Cheng, V. Lemmon, S. Kenwrick, Alternative use of a mini exon of the L1 gene affects L1 binding to neural ligands, J. Biol. Chem. 276 (2001) 32738 – 32742.

A.F. Pimenta, P. Levitt / Genomics 83 (2004) 790–801 [60] S.J. Small, R. Akeson, Expression of the unique NCAM VASE exon is independently regulated in distinct tissues during development, J. Cell Biol. 111 (1990) 2089 – 2096. [61] A. Chen, S. Haines, K. Maxson, R.A. Akeson, VASE exon expression alters NCAM-mediated cell – cell interactions, J. Neurosci. Res. 38 (1994) 483 – 492. [62] J.L. Saffell, F.S. Walsh, P. Doherty, Expression of NCAM containing VASE in neurons can account for a developmental loss in their neurite outgrowth response to NCAM in a cellular substratum, J. Cell Biol. 125 (1994) 427 – 436. [63] F. Lahrtz, R. Horstkorte, H. Cremer, M. Schachner, D. Montag, VASE-encoded peptide modifies NCAM- and L1-mediated neurite outgrowth, J. Neurosci. Res. 50 (1997) 62 – 68. [64] M.P. Vawter, M.A. Frye, J.J. Hemperly, D.M. VanderPutten, N. Usen, P. Doherty, J.L. Saffell, F. Issa, R.M. Post, R.J. Wyatt, W.J. Freed,

[65]

[66]

[67]

[68]

801

Elevated concentration of N-CAM VASE isoforms in schizophrenia, J. Psychiatr. Res. 34 (2000) 25 – 34. J. Sambrook, E.F. Fritsch, T. Maniatis, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989. F.M. Ausubel, R. Brent, R.E. Kingston, D.D. Moore, J.G. Seidman, J.A. Smith, K. Struhl, Current Protocols in Molecular Biology, Wiley & Sons, New York, 1998. H.J. Lee, D.N. Hammond, T.H. Large, B.H. Wainer, Immortalized young adult neurons from the septal region: generation and characterization, Brain Res. Dev. Brain Res. 52 (1990) 219 – 228. S. Kumar, A.B. Rabson, C. Gelinas, The RxxRxRxxC motif conserved in all Rel/kappa B proteins is essential for the DNA-binding activity and redox regulation of the v-Rel oncoprotein, Mol. Cell. Biol. 12 (1992) 3094 – 3106.