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Genomic structure and chromosomal mapping of the human and mouse hippocalcin genes1
Gene 225 (1998) 117–124
Genomic structure and chromosomal mapping of the human and mouse hippocalcin genes1 Tamotsu Masaki, Eiichi Sakai, Yoshitaka Furuta, Masaaki Kobayashi, Ken Takamatsu * Department of Physiology, Toho University School of Medicine, 5-21-16 Ohmori-nishi, Ohta-ku, Tokyo 143-8540, Japan Received 15 June 1998; received in revised form 29 September 1998; accepted 29 September 1998; Received by T. Sekiya
Abstract In an attempt to elucidate the possible relationship of hippocalcin to neurological disorders, we isolated and analyzed the human and mouse hippocalcin genes. The human and mouse hippocalcin genes contain three exons and two introns, and span approximately 7 and 8 kb, respectively. The exon/intron splice junctions of the human and mouse genes are all situated in exactly the same position and are not consistently placed with respect to the coding regions of the tandemly repeated EF-hand motifs. The amino acid sequences of human and mouse hippocalcins deduced from the genes are 100% identical. Within the 2-kb 3∞-flanking sequences of the human and mouse genes, one conserved polyadenylation signal was identified at positions 762 and 823 bp downstream from TAG, respectively. Within the 2.6-kb 5∞-flanking sequences of the human and mouse genes, neither a canonical ‘TATA’ box nor a ‘CAAT’ box was found. Southern blot analysis of the human and mouse genomic DNAs demonstrated that the positive bands coincide exactly with those expected from the sequence of the cloned genes, indicating that the human and mouse hippocalcin genes are present as a single-copy gene. Fluorescence in-situ hybridization revealed that the human hippocalcin gene is located at chromosome 1 p34.2–35 and the mouse hippocalcin gene at chromosome 4 D2–D3. © 1998 Elsevier Science B.V. All rights reserved. Keywords: Calcium-binding protein; Chromosomal mapping; EF-hand; FISH; Gene; Sequence
1. Introduction Hippocalcin is a 23-kDa calcium-binding protein with three EF-hand motifs, identified primarily in the rat hippocampus ( Kobayashi et al., 1992). In the last few years, a number of homologous proteins have been identified in the nervous system of vertebrate and invertebrate species (Polans et al., 1996). These form an EF-hand subfamily, designated as the neuron-specific * Corresponding author. Tel: +81 3 3762 4151; Fax: +81 3 3762 8225; e-mail: [email protected] 1 The sequence data in this article have been deposited in the DDBJ/EMBL/GenBank Data libraries under Accession Nos AB015201 and AB015102 for the human hippocalcin gene and AB015199 and AB015200 for the mouse hippocalcin gene. Abbreviations: aa, amino acid; bp, base pair(s); cAMP, cyclic adenosine 3∞, 5∞-monophosphate; cDNA, DNA complimentary to RNA; DAPI, 4,6-diamidino-2-phenylindole; FISH, fluorescence in-situ hybridization; kb, kilobase(s) or 1000 bp; NCBP, neuron-specific calcium-binding protein; RACE, rapid amplification of cDNA end; RT-PCR, reverse transcribed-polymerase chain reaction; SDS, sodium dodecyl sulfate.
calcium-binding protein (NCBP) family. The NCBP family has been rapidly expanding and comprises more than 20 members, including recoverin/visinin/S-modulin ( Yamagata et al., 1990; Dizhoor et al., 1991; Kawamura et al., 1993), guanylyl cyclase-activating proteins (GCAPs) (Subbaraya et al., 1994; Dizhoor et al., 1995), frequenin (Angaut-Petit et al., 1993) and visinin-like protein ( VILIP) (Lenz et al., 1992). Recoverin/visinin/Smodulin and GCAPs have been shown to be involved in photosignal transduction of photoreceptors ( Kawamura and Murakami, 1991; Gray-Keller et al., 1993; Kawamura et al., 1993; Gorczyca et al., 1995), frequenin in transmitter release from the motor nerve terminal (Rivosecchi et al., 1994), and VILIP in ligandactivated cAMP formation in transfected C6 cells (Braunewell et al., 1997). Thus, the members of the NCBP family are differentially distributed in the nervous system and are considered to be multifunctional calciumsensitive modulators. We previously isolated rat and human hippocalcin cDNAs and showed that their predicted amino acid sequences are identical ( Kobayashi et al., 1992;
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Takamatsu et al., 1994). The hippocalcin mRNA is expressed prominently in the hippocampus, but a considerable amount is also found in the cerebral cortex, cerebellum and caudate-putamen in the rat and human brains ( Kobayashi et al., 1992, 1997). Immunohistochemical analysis of the adult rat brain has shown that hippocalcin is abundantly expressed in the pyramidal cells of the hippocampus and weakly expressed in the pyramidal cells of the cerebral cortex and the Purkinje cells of the cerebellum (Saitoh et al., 1993). In these cells, hippocalcin is located in the cytoplasm and plasma membrane of the cell body and in dendrites. In in-vitro experiments using a rod outersegment membrane preparation, hippocalcin produced in E. coli inhibits rhodopsin phosphorylation at high calcium levels in a manner similar to recoverin/visinin/S-modulin ( Kawamura, 1994). Although the mechanism responsible for the modulation of phosphorylation in neurons is different from that in photoreceptors, hippocalcin might play an important role in neurons of the central nervous system in a number of species. The gene encoding rat hippocalcin has been cloned (Grant et al., 1996). In the present study, we report the primary structure and the chromosomal localization of the human and mouse hippocalcin genes. These data provide a basis for evolutionary comparisons of hippocalcin, and aid us in investigating its possible relationship to neurological disorders, in constructing and analyzing a mouse null mutant as a direct approach to understanding the biological function of hippocalcin, and in identifying the regulatory elements that specify tissue- and cell-specific expression.
2. Materials and methods 2.1. Isolation of the human and mouse hippocalcin genes A human placenta genomic pWE-15 cosmid library (Clontech, CA), approximately 5×105 clones, was screened with an EcoRI fragment (positions −9 to 825) of the human hippocalcin cDNA ( Takamatsu et al., 1994) as a probe. A mouse 129/SVJ liver genomic l FIX II phage library, approximately 1×106 clones, was screened with an EcoRI fragment (positions −174 to 1388) of the rat hippocalcin cDNA ( Kobayashi et al., 1992) as a probe. Each probe was labeled with a [a-32P]dATP random-primed labeling kit ( Takara, Kyoto, Japan). Hybridization was carried out in a buffer containing 4× SSC, 5× Denhardt’s solution, 0.5% SDS and 50 mg/ml of denatured salmon sperm DNA at 65°C for 12–16 h. The membranes were given a final wash in 0.5× SSC/0.1% SDS at 65°C for 30 min. Positive clones were purified by two or three rounds of plating and screening. Cosmid and l phage DNAs of the positive
clones were isolated according to standard procedures (Sambrook et al., 1989). 2.2. Restriction map construction and DNA sequence analysis Cosmid and l phage DNAs were digested with appropriate restriction enzymes and electrophoresed on 0.8% agarose gels, followed by hybridization with the human and rat hippocalcin cDNA probes, respectively. The hybridized fragments were subcloned into pBluescript II SK(−) or KS(+) (Stratagene, La Jolla, CA), and their detailed restriction maps were obtained. The restriction fragments containing exons were again subcloned into the same vectors. The clones were sequenced on both strands by the dideoxy chain termination method using fluorescent dye-primers in an automated DNA sequencer, model DSQ1000 (Shimadzu, Kyoto, Japan). The sequences were analyzed by DNASIS ( Hitachi, Tokyo, Japan) and GENETYX (Software Development, Tokyo, Japan) software packages. 2.3. Genomic Southern blots Human genomic DNA was isolated from whole blood, and mouse genomic DNA was from 129/SVJ mouse thymus as described previously (Blin and Stafford, 1976). Approximately 10 mg of genomic DNA were digested with appropriate restriction enzymes, electrophoresed on 0.8% agarose gels. The DNA fragments were transferred by capillary blotting on to Biodyne B nylon membranes (Pall BioSupport, NY ). The probe for human DNA was the EcoRI fragment (positions −9 to 825) of the human hippocalcin cDNA, and the probe for mouse DNA was the PCR-amplified mouse hippocaclin cDNA containing the entire coding region. Hybridization was carried out as described above. The membranes were given a final wash in 0.1× SSC/0.1% SDS at 65°C for 30 min and exposed to Kodak X-Omat films at −80°C for 3 days. 2.4. Determination of transcription start site Messenger RNA from human hippocampus (Clontech) and total RNAs from mouse and rat whole brains were used for primer extension and S1 mapping analyses. Primers were designed according to the untranslated leader sequences of the human (positions −91 to −70), mouse (positions −116 to −94) and rat (positions −116 to −94) hippocalcin cDNAs. For primer extension analysis, the 5∞ end of the primers was labeled with [c−32P]ATP using a T4 polynucleotide kinase. For S1 mapping analysis, the probes were extended by using the same primers and labeled with [a−32P]dCTP using a Klenow fragment. Primer exten-
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sion and S1 mapping analyses were carried out as described previously (Sambrook et al., 1989). 2.5. Chromosomal mapping by FISH A FISH analysis was performed as described previously (Heng et al., 1992; Heng and Tsui, 1993). Briefly, human lymphocytes were isolated from cord blood, and mouse lymphocytes were from C57BL/6J mouse spleen. The cultured lymphocytes were treated with BrdU to synchronize the cell population. Metaphase chromosome slides were made by standard procedures, including hypotonic treatment, fixation and air-drying. Plasmids containing a 10-kb XhoI fragment of the human hippocalcin gene and a 12-kb SmaI fragment of the mouse hippocalcin gene were labeled with biotinylated dATP using the BioNick labeling kit (BRL, CA) and used as a probe. Hybridization was carried out in a buffer containing 2× SSC, 50% formamide, 10% dextran sulfate, 2 mg/ml of BSA and 500 mg/ml of human or mouse cotI DNA at 37°C for 24 h. Human and mouse metaphase chromosomes were counter-stained with 4,6-diamidino-2-phenylindole (DAPI ). The assignment of the FISH mapping data with chromosomal bands was achieved by superimposing FISH signals with DAPI-banded chromosomes. A total of 100 typical R-banded metaphase spreads for human or mouse chromosomes were examined.
3. Results and discussion 3.1. Structure of the human and mouse hippocalcin genes We isolated three overlapping human genomic clones that encompassed the hippocalcin gene. By comparing the genomic sequences to the human hippocalcin cDNA sequence, it was revealed that the human hippocalcin gene contained three exons and two introns, spanning approximately 7 kb ( Fig. 1A). The sequences of the exons and the exon/intron junctions are given in Fig. 1B. The sequence of the exons was consistent with the originally reported human cDNA sequence ( Takamatsu et al., 1994). The boundary sequence of GT and AG is highly conserved at each of the donor and acceptor splice sites. Exon 1 contains the N-terminus of the protein and the first two calcium-binding domains of the EF-hand motif. The splice junction between exon 2 and 3 is located within the region that encodes the third calcium-binding domain of the EF-hand motif. Exon 3 contains the stop codon of TAG and the putative polyadenylation signal of AATAAA at position 762 bp downstream from TAG. No other canonical polyadenylation signals were identified within the 2-kb 3∞-flanking sequence. The structure of the human hippocalcin gene is similar to that of the rat hippocalcin gene (Grant
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et al., 1996). The positions of intron interruptions are identical, even though the size of the introns differs somewhat. From the mouse genomic library, we isolated five overlapping clones. By comparing the genomic sequences to the rat cDNA sequence and the organization of the rat hippocalcin gene, putative exon/intron splice junctions were postulated for the mouse hippocalcin gene. The mouse hippocalcin gene contained three exons and two introns, spanning approximately 8 kb ( Fig. 1A). To confirm the deduced organization of the mouse hippocalcin gene and the nucleotide sequence of the mouse hippocalcin cDNA, RT-PCR was used to amplify a cDNA fragment spanning the entire coding region of mouse hippocalcin. The deduced amino acid sequence of mouse hippocalcin is 100% identical to those of rat and human hippocalcins. Furthermore, the mouse exon/intron splice junctions are all situated exactly as those in the human and rat hippocalcin genes. Within the 2-kb 3∞-flanking sequence, only one conserved polyadenylation signal was identified at position 823 bp downstream from TAG. The individual exons are more than 90% similar, whereas the analogous introns and flanking regions are less similar. Thus, the structure of the hippocalcin gene is highly conserved across species, and the intron positions are not consistently placed with respect to the coding regions of the tandemly repeated EF-hand motifs. We compared the structure of the hippocalcin gene to those of other NCBP gene. Genomic structures of the human recoverin and human and mouse GCAP1 and 2 genes have been analyzed. The human recoverin gene has a common gene structure with three exons and two introns. The protein-coding region of recoverin is divided by two introns at similar positions (Murakami et al., 1992). The GCAP1 and two genes have an additional intron between the first and second EF-hand motifs (Subbaraya et al., 1994; Surguchov et al., 1997). These four genes are believed to share a common ancestral gene, with GCAP genes possibly diverging in early evolutional stages. This idea is consistent with the fact that the amino acid sequence homology of GCAPs is the lowest among the NCBP family. Moreover, hippocalcin displays recoverin activity, a calcium-dependent inhibition of rhodopsin kinase ( Kawamura, 1994), but not GCAP activity, a calcium-dependent activation of guanylyl cyclase (unpublished data). 3.2. 5∞-flanking region of the human and mouse hippocalcin genes For 5∞-flanking regions of the human and mouse hippocalcin genes, over 2.6 kb of sequence upstream from ATG start codon were determined and compared with that of the rat gene. The 5∞-flanking regions of the hippocalcin gene from the three species were well con-
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Fig. 1. (A) Genomic clones, organizations and predicted protein of the human and mouse hippocalcin genes. Blackened boxes represent the exons containing the protein-coding region, and open boxes represent the exons containing the 5∞- and 3∞-untranslated regions. The positions of the restriction sites are indicated above the solid line (B, BamHI; H, HindIII; S, SacI; E, EcoRI; V, EcoRV; N, NcoI ). (B) Exon/intron junction sequences in the human and mouse hippocalcin genes. The first and last nine base sequences of each exon and intron are shown. Exonic sequences are shown in upper-case letters, and intronic sequences are shown in lower-case letters. The donor and acceptor sites are underlined. The sequence data of the human and mouse hippocalcin genes have been deposited in the DDBJ/EMBL/GenBank Data libraries under Accession Nos AB015201 and AB015202, and AB015199 and AB015200, respectively.
served as a whole; however, the regions between −500 and −900 differ somewhat. In particular, the mouse gene contained additional sequences of 170 bp at positions around −620 of the rat gene. The overall sequence identity of the human to mouse was 67%, the human to rat 69% and the mouse to rat 81%. In the rat hippocalcin gene, the multiple transcription initiation sites are assigned to positions between −639 and −547, and −265 and −136 by 5∞-RACE analysis (Grant et al., 1996). In our primer extension analysis, major extended products were revealed at positions −235, −206 and −153 in the human gene, at position −182 in the mouse gene, and at positions −229 and −179 in the rat gene. Moreover, S1 mapping analysis revealed only one major protected signal at position −182 in the mouse gene, and at position −229 in the rat gene (data not shown).
It is possible that many transcripts with prematurely truncated 5∞-ends are produced when the mRNA is reverse-transcribed; however, the majority of mRNAs initiate at around −200 in the human, mouse and rat genes. Neither a canonical ‘TATA’ box nor a ‘CAAT’ box was found in the 5∞-flanking regions of the human and mouse genes. Functional promoter analysis of the rat hippocalcin gene using transgenic mice suggested that the −1.5 to −0.6-kb upstream region is the minimum sequence required to promote gene expression and that the −3.8 to −1.5-kb upstream region includes the elements for transcriptional enhancement and cell type-specific transcriptional regulation (Grant et al., 1996). An in-vitro reporter gene assay using cultured neuronal and nonneuronal cells demonstrates that the −2.4 to −2.1-kb
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upstream region has a strong transcriptional activity in NG108 cells but not in PC12, HEK and 3T3 cells (Grant and Wisden, 1997). The −2.4 to −2.1-kb upstream region of the three species was highly conserved and had a high GC content (78%) compared to other regions. There were two potential binding sites for activator protein-2 (AP-2) and one for both Sp1 and PEBP2 in this region of the three species ( Fig. 2). Conservation of this region might be requisite for binding some transcription factors for neuronal cell-specific transcriptional activation. On inspection of the sequence of other regions, two clusters of E-box consensus sites (Blackwell and Weintraub, 1990) were found at around −1.0 and −1.5 kb in the human, mouse and rat genes. An N-box consensus site ( Tietze et al., 1992) was also found in the proximity of the E-box cluster at around −1.0 kb in the three genes. An E-box sequence has been shown to be necessary for the cell type-specific expression of the tyrosine hydroxylase gene in cultured neuronal cells ( Wong et al., 1994). Transcription factors such as HES-1 and HES-5 bind the N-box consensus site to repress transcriptional activation from the adjacent E-box (Akazawa et al., 1992; Ishibashi et al., 1994, 1995). Though there is no experimental evidence, these regions
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might be important in determining the expression pattern. In addition, we also identified a number of potential binding sites for transcription factors in the hippocalcin gene, such as a TPA response element at position −1130 in mice and a cAMP response element at position −1080 in humans. Further investigation of the hippocalcin promoter region in vitro and in vivo is a prerequisite for understanding the molecular mechanism of region-specific expression. 3.3. Southern blot analysis of the human and mouse hippocalcin genes Southern blot analyses of human and mouse genomic DNAs with the human and mouse hippocalcin cDNAs as probes are shown in Fig. 3. In humans, one strong band was seen in a BamHI digest and in a HindIII digest and two strong bands in a SacI digest. In mice, one strong band was seen in an EcoRI digest and two strong bands in both an EcoRV digest and a NcoI digest. An EcoRI digest also showed one weak larger band corresponding to a partial digest in a size. These bands exactly coincided with those expected from the sequence of the genes, indicating that the human and mouse hippocalcin gene are present as a single copy gene.
Fig. 2. Nucleotide sequence of the 5∞-flanking regions of the human and mouse hippocalcin genes and sequence comparison of the corresponding regions (the position −2456 to −2101, EMBL Data Libraries Accession No. Y12514) of the rat hippocalcin gene. The first nucleotide upstream of the ATG start codon of the known cDNA sequences has been assigned as position −1. The asterisk indicates identical nucleotides. Putative transcription factor binding sites that were conserved in three species are boxed, and those that were found individually are underlined.
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Fig. 3. Genomic Southern blot analysis of the human and mouse hippocalcin genes. Human genomic DNA was digested with BamHI (B), HindIII (H ), and SacI (S) and mouse genomic DNA with EcoRI ( E ), EcoRV ( V ), and NcoI (N ). The blots were hybridized with a [32P]dATP-labeled human or mouse hippocalcin cDNA probe.
3.4. Chromosomal mapping of the human and mouse hippocalcin genes To localize the human and mouse hippocalcin genes, a FISH analysis was carried out. In humans, hybridization of the hippocalcin gene probe resulted in specific labeling only on chromosome 1 (Fig. 4A), consistent with a previous report on human-rodent somatic cell hybrid mapping ( Takamatsu et al., 1994). As a result, 32% exhibited complete twin spots on both homologs, and 67% had incomplete single and/or twin spots on one or both homologs. No twin spots were observed on other chromosomes. The detailed position was confirmed from a summary of 10 DAPI banding photos (Fig. 4B). The human hippocalcin gene is located at chromosome 1, region p34.2–35. In mice, hybridization of the hippocalcin gene probe resulted in specific labeling on chromosome 4. As a result, 29% exhibited complete twin spots on both homologs and 65% had incomplete single and/or twin
Fig. 4. Fluorescence in-situ hybridization analysis of the human (A and B) and mouse (C and D) hippocalcin genes. Biotin-labeled DNA from the human and mouse hippocalcin gene was hybridized to metaphase chromosomes from human cord lymphocytes and mouse spleen lymphocytes. The positions of the hybridization signals are indicated with arrows. The FISH signals (A and C ) were compared to the DAPI staining (B and D).
spots on one or both homologs. No twin spots were observed on other chromosomes. Further analysis confirmed that the mouse hippocalcin gene is located at chromosome 4, region D2–D3 ( Fig. 4C ). The existence of hybridization signals only on human chromosome 1 and mouse chromosome 4, consistent with the results of the Southern blot analyses, supports the view that only one copy of the hippocalcin gene is present in the genome. The human chromosome locus 1p34.2–35 has been reported to share a homology with the mouse chromosome 4D. In the human chromosome 1p34.2–35, no locus for hereditary neurological disorder has been identified. The mouse chromosome 4D includes two loci for hereditary neurological disorders, ecl and cla ( Taylor, 1976). A homozygote for two recessive genes, one ecl from C57L/J and one ecs from SWR mouse, shows epistatic circling behavior. The cla homozygote results in a fine whole-body tremor with clasping of both forefeet and hindfeet when held up by the tail. ecl is on chromosome 4D3-ter, and cla on 4C7-D1. These
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findings demonstrate the need for a further linkage analysis of the hippocalcin gene and these disorders, and for construction and analysis of a mouse null mutant as a direct approach to understanding the biological function of hippocalcin.
Acknowledgements This study was supported in part by the Science Research Promotion Fund from the Japan Private School Promotion Foundation (to K.T.), a Grant-inAid for Scientific Research from the Ministry of Education, Science and Culture of Japan (to K.T. and M.K.) and the Research Grant (6B-3) for Nervous and Mental Disorders from the Ministry of Health and Welfare of Japan (to K.T.).
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Genomic structure and chromosomal mapping of the human and mouse hippocalcin genes1 Tamotsu Masaki, Eiichi Sakai, Yoshitaka Furuta, Masaaki Kobayashi, Ken Takamatsu * Department of Physiology, Toho University School of Medicine, 5-21-16 Ohmori-nishi, Ohta-ku, Tokyo 143-8540, Japan Received 15 June 1998; received in revised form 29 September 1998; accepted 29 September 1998; Received by T. Sekiya
Abstract In an attempt to elucidate the possible relationship of hippocalcin to neurological disorders, we isolated and analyzed the human and mouse hippocalcin genes. The human and mouse hippocalcin genes contain three exons and two introns, and span approximately 7 and 8 kb, respectively. The exon/intron splice junctions of the human and mouse genes are all situated in exactly the same position and are not consistently placed with respect to the coding regions of the tandemly repeated EF-hand motifs. The amino acid sequences of human and mouse hippocalcins deduced from the genes are 100% identical. Within the 2-kb 3∞-flanking sequences of the human and mouse genes, one conserved polyadenylation signal was identified at positions 762 and 823 bp downstream from TAG, respectively. Within the 2.6-kb 5∞-flanking sequences of the human and mouse genes, neither a canonical ‘TATA’ box nor a ‘CAAT’ box was found. Southern blot analysis of the human and mouse genomic DNAs demonstrated that the positive bands coincide exactly with those expected from the sequence of the cloned genes, indicating that the human and mouse hippocalcin genes are present as a single-copy gene. Fluorescence in-situ hybridization revealed that the human hippocalcin gene is located at chromosome 1 p34.2–35 and the mouse hippocalcin gene at chromosome 4 D2–D3. © 1998 Elsevier Science B.V. All rights reserved. Keywords: Calcium-binding protein; Chromosomal mapping; EF-hand; FISH; Gene; Sequence
1. Introduction Hippocalcin is a 23-kDa calcium-binding protein with three EF-hand motifs, identified primarily in the rat hippocampus ( Kobayashi et al., 1992). In the last few years, a number of homologous proteins have been identified in the nervous system of vertebrate and invertebrate species (Polans et al., 1996). These form an EF-hand subfamily, designated as the neuron-specific * Corresponding author. Tel: +81 3 3762 4151; Fax: +81 3 3762 8225; e-mail: [email protected] 1 The sequence data in this article have been deposited in the DDBJ/EMBL/GenBank Data libraries under Accession Nos AB015201 and AB015102 for the human hippocalcin gene and AB015199 and AB015200 for the mouse hippocalcin gene. Abbreviations: aa, amino acid; bp, base pair(s); cAMP, cyclic adenosine 3∞, 5∞-monophosphate; cDNA, DNA complimentary to RNA; DAPI, 4,6-diamidino-2-phenylindole; FISH, fluorescence in-situ hybridization; kb, kilobase(s) or 1000 bp; NCBP, neuron-specific calcium-binding protein; RACE, rapid amplification of cDNA end; RT-PCR, reverse transcribed-polymerase chain reaction; SDS, sodium dodecyl sulfate.
calcium-binding protein (NCBP) family. The NCBP family has been rapidly expanding and comprises more than 20 members, including recoverin/visinin/S-modulin ( Yamagata et al., 1990; Dizhoor et al., 1991; Kawamura et al., 1993), guanylyl cyclase-activating proteins (GCAPs) (Subbaraya et al., 1994; Dizhoor et al., 1995), frequenin (Angaut-Petit et al., 1993) and visinin-like protein ( VILIP) (Lenz et al., 1992). Recoverin/visinin/Smodulin and GCAPs have been shown to be involved in photosignal transduction of photoreceptors ( Kawamura and Murakami, 1991; Gray-Keller et al., 1993; Kawamura et al., 1993; Gorczyca et al., 1995), frequenin in transmitter release from the motor nerve terminal (Rivosecchi et al., 1994), and VILIP in ligandactivated cAMP formation in transfected C6 cells (Braunewell et al., 1997). Thus, the members of the NCBP family are differentially distributed in the nervous system and are considered to be multifunctional calciumsensitive modulators. We previously isolated rat and human hippocalcin cDNAs and showed that their predicted amino acid sequences are identical ( Kobayashi et al., 1992;
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Takamatsu et al., 1994). The hippocalcin mRNA is expressed prominently in the hippocampus, but a considerable amount is also found in the cerebral cortex, cerebellum and caudate-putamen in the rat and human brains ( Kobayashi et al., 1992, 1997). Immunohistochemical analysis of the adult rat brain has shown that hippocalcin is abundantly expressed in the pyramidal cells of the hippocampus and weakly expressed in the pyramidal cells of the cerebral cortex and the Purkinje cells of the cerebellum (Saitoh et al., 1993). In these cells, hippocalcin is located in the cytoplasm and plasma membrane of the cell body and in dendrites. In in-vitro experiments using a rod outersegment membrane preparation, hippocalcin produced in E. coli inhibits rhodopsin phosphorylation at high calcium levels in a manner similar to recoverin/visinin/S-modulin ( Kawamura, 1994). Although the mechanism responsible for the modulation of phosphorylation in neurons is different from that in photoreceptors, hippocalcin might play an important role in neurons of the central nervous system in a number of species. The gene encoding rat hippocalcin has been cloned (Grant et al., 1996). In the present study, we report the primary structure and the chromosomal localization of the human and mouse hippocalcin genes. These data provide a basis for evolutionary comparisons of hippocalcin, and aid us in investigating its possible relationship to neurological disorders, in constructing and analyzing a mouse null mutant as a direct approach to understanding the biological function of hippocalcin, and in identifying the regulatory elements that specify tissue- and cell-specific expression.
2. Materials and methods 2.1. Isolation of the human and mouse hippocalcin genes A human placenta genomic pWE-15 cosmid library (Clontech, CA), approximately 5×105 clones, was screened with an EcoRI fragment (positions −9 to 825) of the human hippocalcin cDNA ( Takamatsu et al., 1994) as a probe. A mouse 129/SVJ liver genomic l FIX II phage library, approximately 1×106 clones, was screened with an EcoRI fragment (positions −174 to 1388) of the rat hippocalcin cDNA ( Kobayashi et al., 1992) as a probe. Each probe was labeled with a [a-32P]dATP random-primed labeling kit ( Takara, Kyoto, Japan). Hybridization was carried out in a buffer containing 4× SSC, 5× Denhardt’s solution, 0.5% SDS and 50 mg/ml of denatured salmon sperm DNA at 65°C for 12–16 h. The membranes were given a final wash in 0.5× SSC/0.1% SDS at 65°C for 30 min. Positive clones were purified by two or three rounds of plating and screening. Cosmid and l phage DNAs of the positive
clones were isolated according to standard procedures (Sambrook et al., 1989). 2.2. Restriction map construction and DNA sequence analysis Cosmid and l phage DNAs were digested with appropriate restriction enzymes and electrophoresed on 0.8% agarose gels, followed by hybridization with the human and rat hippocalcin cDNA probes, respectively. The hybridized fragments were subcloned into pBluescript II SK(−) or KS(+) (Stratagene, La Jolla, CA), and their detailed restriction maps were obtained. The restriction fragments containing exons were again subcloned into the same vectors. The clones were sequenced on both strands by the dideoxy chain termination method using fluorescent dye-primers in an automated DNA sequencer, model DSQ1000 (Shimadzu, Kyoto, Japan). The sequences were analyzed by DNASIS ( Hitachi, Tokyo, Japan) and GENETYX (Software Development, Tokyo, Japan) software packages. 2.3. Genomic Southern blots Human genomic DNA was isolated from whole blood, and mouse genomic DNA was from 129/SVJ mouse thymus as described previously (Blin and Stafford, 1976). Approximately 10 mg of genomic DNA were digested with appropriate restriction enzymes, electrophoresed on 0.8% agarose gels. The DNA fragments were transferred by capillary blotting on to Biodyne B nylon membranes (Pall BioSupport, NY ). The probe for human DNA was the EcoRI fragment (positions −9 to 825) of the human hippocalcin cDNA, and the probe for mouse DNA was the PCR-amplified mouse hippocaclin cDNA containing the entire coding region. Hybridization was carried out as described above. The membranes were given a final wash in 0.1× SSC/0.1% SDS at 65°C for 30 min and exposed to Kodak X-Omat films at −80°C for 3 days. 2.4. Determination of transcription start site Messenger RNA from human hippocampus (Clontech) and total RNAs from mouse and rat whole brains were used for primer extension and S1 mapping analyses. Primers were designed according to the untranslated leader sequences of the human (positions −91 to −70), mouse (positions −116 to −94) and rat (positions −116 to −94) hippocalcin cDNAs. For primer extension analysis, the 5∞ end of the primers was labeled with [c−32P]ATP using a T4 polynucleotide kinase. For S1 mapping analysis, the probes were extended by using the same primers and labeled with [a−32P]dCTP using a Klenow fragment. Primer exten-
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sion and S1 mapping analyses were carried out as described previously (Sambrook et al., 1989). 2.5. Chromosomal mapping by FISH A FISH analysis was performed as described previously (Heng et al., 1992; Heng and Tsui, 1993). Briefly, human lymphocytes were isolated from cord blood, and mouse lymphocytes were from C57BL/6J mouse spleen. The cultured lymphocytes were treated with BrdU to synchronize the cell population. Metaphase chromosome slides were made by standard procedures, including hypotonic treatment, fixation and air-drying. Plasmids containing a 10-kb XhoI fragment of the human hippocalcin gene and a 12-kb SmaI fragment of the mouse hippocalcin gene were labeled with biotinylated dATP using the BioNick labeling kit (BRL, CA) and used as a probe. Hybridization was carried out in a buffer containing 2× SSC, 50% formamide, 10% dextran sulfate, 2 mg/ml of BSA and 500 mg/ml of human or mouse cotI DNA at 37°C for 24 h. Human and mouse metaphase chromosomes were counter-stained with 4,6-diamidino-2-phenylindole (DAPI ). The assignment of the FISH mapping data with chromosomal bands was achieved by superimposing FISH signals with DAPI-banded chromosomes. A total of 100 typical R-banded metaphase spreads for human or mouse chromosomes were examined.
3. Results and discussion 3.1. Structure of the human and mouse hippocalcin genes We isolated three overlapping human genomic clones that encompassed the hippocalcin gene. By comparing the genomic sequences to the human hippocalcin cDNA sequence, it was revealed that the human hippocalcin gene contained three exons and two introns, spanning approximately 7 kb ( Fig. 1A). The sequences of the exons and the exon/intron junctions are given in Fig. 1B. The sequence of the exons was consistent with the originally reported human cDNA sequence ( Takamatsu et al., 1994). The boundary sequence of GT and AG is highly conserved at each of the donor and acceptor splice sites. Exon 1 contains the N-terminus of the protein and the first two calcium-binding domains of the EF-hand motif. The splice junction between exon 2 and 3 is located within the region that encodes the third calcium-binding domain of the EF-hand motif. Exon 3 contains the stop codon of TAG and the putative polyadenylation signal of AATAAA at position 762 bp downstream from TAG. No other canonical polyadenylation signals were identified within the 2-kb 3∞-flanking sequence. The structure of the human hippocalcin gene is similar to that of the rat hippocalcin gene (Grant
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et al., 1996). The positions of intron interruptions are identical, even though the size of the introns differs somewhat. From the mouse genomic library, we isolated five overlapping clones. By comparing the genomic sequences to the rat cDNA sequence and the organization of the rat hippocalcin gene, putative exon/intron splice junctions were postulated for the mouse hippocalcin gene. The mouse hippocalcin gene contained three exons and two introns, spanning approximately 8 kb ( Fig. 1A). To confirm the deduced organization of the mouse hippocalcin gene and the nucleotide sequence of the mouse hippocalcin cDNA, RT-PCR was used to amplify a cDNA fragment spanning the entire coding region of mouse hippocalcin. The deduced amino acid sequence of mouse hippocalcin is 100% identical to those of rat and human hippocalcins. Furthermore, the mouse exon/intron splice junctions are all situated exactly as those in the human and rat hippocalcin genes. Within the 2-kb 3∞-flanking sequence, only one conserved polyadenylation signal was identified at position 823 bp downstream from TAG. The individual exons are more than 90% similar, whereas the analogous introns and flanking regions are less similar. Thus, the structure of the hippocalcin gene is highly conserved across species, and the intron positions are not consistently placed with respect to the coding regions of the tandemly repeated EF-hand motifs. We compared the structure of the hippocalcin gene to those of other NCBP gene. Genomic structures of the human recoverin and human and mouse GCAP1 and 2 genes have been analyzed. The human recoverin gene has a common gene structure with three exons and two introns. The protein-coding region of recoverin is divided by two introns at similar positions (Murakami et al., 1992). The GCAP1 and two genes have an additional intron between the first and second EF-hand motifs (Subbaraya et al., 1994; Surguchov et al., 1997). These four genes are believed to share a common ancestral gene, with GCAP genes possibly diverging in early evolutional stages. This idea is consistent with the fact that the amino acid sequence homology of GCAPs is the lowest among the NCBP family. Moreover, hippocalcin displays recoverin activity, a calcium-dependent inhibition of rhodopsin kinase ( Kawamura, 1994), but not GCAP activity, a calcium-dependent activation of guanylyl cyclase (unpublished data). 3.2. 5∞-flanking region of the human and mouse hippocalcin genes For 5∞-flanking regions of the human and mouse hippocalcin genes, over 2.6 kb of sequence upstream from ATG start codon were determined and compared with that of the rat gene. The 5∞-flanking regions of the hippocalcin gene from the three species were well con-
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Fig. 1. (A) Genomic clones, organizations and predicted protein of the human and mouse hippocalcin genes. Blackened boxes represent the exons containing the protein-coding region, and open boxes represent the exons containing the 5∞- and 3∞-untranslated regions. The positions of the restriction sites are indicated above the solid line (B, BamHI; H, HindIII; S, SacI; E, EcoRI; V, EcoRV; N, NcoI ). (B) Exon/intron junction sequences in the human and mouse hippocalcin genes. The first and last nine base sequences of each exon and intron are shown. Exonic sequences are shown in upper-case letters, and intronic sequences are shown in lower-case letters. The donor and acceptor sites are underlined. The sequence data of the human and mouse hippocalcin genes have been deposited in the DDBJ/EMBL/GenBank Data libraries under Accession Nos AB015201 and AB015202, and AB015199 and AB015200, respectively.
served as a whole; however, the regions between −500 and −900 differ somewhat. In particular, the mouse gene contained additional sequences of 170 bp at positions around −620 of the rat gene. The overall sequence identity of the human to mouse was 67%, the human to rat 69% and the mouse to rat 81%. In the rat hippocalcin gene, the multiple transcription initiation sites are assigned to positions between −639 and −547, and −265 and −136 by 5∞-RACE analysis (Grant et al., 1996). In our primer extension analysis, major extended products were revealed at positions −235, −206 and −153 in the human gene, at position −182 in the mouse gene, and at positions −229 and −179 in the rat gene. Moreover, S1 mapping analysis revealed only one major protected signal at position −182 in the mouse gene, and at position −229 in the rat gene (data not shown).
It is possible that many transcripts with prematurely truncated 5∞-ends are produced when the mRNA is reverse-transcribed; however, the majority of mRNAs initiate at around −200 in the human, mouse and rat genes. Neither a canonical ‘TATA’ box nor a ‘CAAT’ box was found in the 5∞-flanking regions of the human and mouse genes. Functional promoter analysis of the rat hippocalcin gene using transgenic mice suggested that the −1.5 to −0.6-kb upstream region is the minimum sequence required to promote gene expression and that the −3.8 to −1.5-kb upstream region includes the elements for transcriptional enhancement and cell type-specific transcriptional regulation (Grant et al., 1996). An in-vitro reporter gene assay using cultured neuronal and nonneuronal cells demonstrates that the −2.4 to −2.1-kb
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upstream region has a strong transcriptional activity in NG108 cells but not in PC12, HEK and 3T3 cells (Grant and Wisden, 1997). The −2.4 to −2.1-kb upstream region of the three species was highly conserved and had a high GC content (78%) compared to other regions. There were two potential binding sites for activator protein-2 (AP-2) and one for both Sp1 and PEBP2 in this region of the three species ( Fig. 2). Conservation of this region might be requisite for binding some transcription factors for neuronal cell-specific transcriptional activation. On inspection of the sequence of other regions, two clusters of E-box consensus sites (Blackwell and Weintraub, 1990) were found at around −1.0 and −1.5 kb in the human, mouse and rat genes. An N-box consensus site ( Tietze et al., 1992) was also found in the proximity of the E-box cluster at around −1.0 kb in the three genes. An E-box sequence has been shown to be necessary for the cell type-specific expression of the tyrosine hydroxylase gene in cultured neuronal cells ( Wong et al., 1994). Transcription factors such as HES-1 and HES-5 bind the N-box consensus site to repress transcriptional activation from the adjacent E-box (Akazawa et al., 1992; Ishibashi et al., 1994, 1995). Though there is no experimental evidence, these regions
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might be important in determining the expression pattern. In addition, we also identified a number of potential binding sites for transcription factors in the hippocalcin gene, such as a TPA response element at position −1130 in mice and a cAMP response element at position −1080 in humans. Further investigation of the hippocalcin promoter region in vitro and in vivo is a prerequisite for understanding the molecular mechanism of region-specific expression. 3.3. Southern blot analysis of the human and mouse hippocalcin genes Southern blot analyses of human and mouse genomic DNAs with the human and mouse hippocalcin cDNAs as probes are shown in Fig. 3. In humans, one strong band was seen in a BamHI digest and in a HindIII digest and two strong bands in a SacI digest. In mice, one strong band was seen in an EcoRI digest and two strong bands in both an EcoRV digest and a NcoI digest. An EcoRI digest also showed one weak larger band corresponding to a partial digest in a size. These bands exactly coincided with those expected from the sequence of the genes, indicating that the human and mouse hippocalcin gene are present as a single copy gene.
Fig. 2. Nucleotide sequence of the 5∞-flanking regions of the human and mouse hippocalcin genes and sequence comparison of the corresponding regions (the position −2456 to −2101, EMBL Data Libraries Accession No. Y12514) of the rat hippocalcin gene. The first nucleotide upstream of the ATG start codon of the known cDNA sequences has been assigned as position −1. The asterisk indicates identical nucleotides. Putative transcription factor binding sites that were conserved in three species are boxed, and those that were found individually are underlined.
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Fig. 3. Genomic Southern blot analysis of the human and mouse hippocalcin genes. Human genomic DNA was digested with BamHI (B), HindIII (H ), and SacI (S) and mouse genomic DNA with EcoRI ( E ), EcoRV ( V ), and NcoI (N ). The blots were hybridized with a [32P]dATP-labeled human or mouse hippocalcin cDNA probe.
3.4. Chromosomal mapping of the human and mouse hippocalcin genes To localize the human and mouse hippocalcin genes, a FISH analysis was carried out. In humans, hybridization of the hippocalcin gene probe resulted in specific labeling only on chromosome 1 (Fig. 4A), consistent with a previous report on human-rodent somatic cell hybrid mapping ( Takamatsu et al., 1994). As a result, 32% exhibited complete twin spots on both homologs, and 67% had incomplete single and/or twin spots on one or both homologs. No twin spots were observed on other chromosomes. The detailed position was confirmed from a summary of 10 DAPI banding photos (Fig. 4B). The human hippocalcin gene is located at chromosome 1, region p34.2–35. In mice, hybridization of the hippocalcin gene probe resulted in specific labeling on chromosome 4. As a result, 29% exhibited complete twin spots on both homologs and 65% had incomplete single and/or twin
Fig. 4. Fluorescence in-situ hybridization analysis of the human (A and B) and mouse (C and D) hippocalcin genes. Biotin-labeled DNA from the human and mouse hippocalcin gene was hybridized to metaphase chromosomes from human cord lymphocytes and mouse spleen lymphocytes. The positions of the hybridization signals are indicated with arrows. The FISH signals (A and C ) were compared to the DAPI staining (B and D).
spots on one or both homologs. No twin spots were observed on other chromosomes. Further analysis confirmed that the mouse hippocalcin gene is located at chromosome 4, region D2–D3 ( Fig. 4C ). The existence of hybridization signals only on human chromosome 1 and mouse chromosome 4, consistent with the results of the Southern blot analyses, supports the view that only one copy of the hippocalcin gene is present in the genome. The human chromosome locus 1p34.2–35 has been reported to share a homology with the mouse chromosome 4D. In the human chromosome 1p34.2–35, no locus for hereditary neurological disorder has been identified. The mouse chromosome 4D includes two loci for hereditary neurological disorders, ecl and cla ( Taylor, 1976). A homozygote for two recessive genes, one ecl from C57L/J and one ecs from SWR mouse, shows epistatic circling behavior. The cla homozygote results in a fine whole-body tremor with clasping of both forefeet and hindfeet when held up by the tail. ecl is on chromosome 4D3-ter, and cla on 4C7-D1. These
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findings demonstrate the need for a further linkage analysis of the hippocalcin gene and these disorders, and for construction and analysis of a mouse null mutant as a direct approach to understanding the biological function of hippocalcin.
Acknowledgements This study was supported in part by the Science Research Promotion Fund from the Japan Private School Promotion Foundation (to K.T.), a Grant-inAid for Scientific Research from the Ministry of Education, Science and Culture of Japan (to K.T. and M.K.) and the Research Grant (6B-3) for Nervous and Mental Disorders from the Ministry of Health and Welfare of Japan (to K.T.).
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