Genomic organization and localization of the gene encoding human preprogalanin

GENOMICS 18,473-477 (1993)

Genomic Organization and Localization of the Gene Encoding Human Preprogalanin HELEN EVANS, *,1 MARTINA BAUMGARTNER, t JOHN SHINE, * AND HERBERT HERZOG* *Garvan Institute of Medical Research, St. Vincent's Hospital, Darlinghurst, Sydney, New South Wales 2010, Australia; and tlnstitute far Biochemie, Universitat Innsbruck, Peter Mayr Strasse la, 6020 Innsbruck, Austria Received April 19, 1993; revised July 2, 1993

ingly, in some small-cell lung carcinoma cells galanin has also been recently shown to possess mitogenic activity (Sethi and Rozengurt, 1991). Human galanin is a peptide of 30 amino acids (Evans and Shine, 1991; Bersani et at., 1991) with a carboxy-terminal serine, in contrast to the equivalent 29-aminoacid C-terminally amidated peptide found in all other species examined so far. The C-terminal amidation appears to playa role in the stability of the peptide. Galanin is synthesized from a 123-amino-acid precursor, preprogalanin, which also contains a signal peptide and a carboxy-terminal 59-amino-acid galanin message-associated peptide (GMAP) (Kaplan et at., 1988) (Fig. 1). In the rat, GMAP contains an extra amino acid absent in other species (Kaplan et at., 1988). Galanin shows little similarity to other peptides but is well conserved across species (80-90% identity), with the first 15 N-terminal amino acids invariant. G MAP is less well conserved, particularly in the N-terminal portion of the peptide, although a region of 18 amino acids (amino acids 88-105) within the C-terminal portion of the peptide shows approximately 88-95% identity in amino acid sequence across species. Although the function of G MAP is not yet known, immunohistochemistry has shown that its distribution generally parallels that of galanin (H6kfelt et at., 1992). To investigate the regulation and molecular organization of the human preprogalanin gene, we isolated and characterized two genomic clones encoding galanin sequences. Analysis of the human gene reveale9 that it is composed of six exons spanning 6.5 kb. We also localized the position of the single human preprogalanin gene to chromosome llqI3.3-qI3.5 with high-resolution fluorescence in situ hybridization (FISH).

An approximately 35-kb region of genomic DNA encoding the human preprogalanin gene including 5' and 3' flanking sequences has been cloned and characterized. Exons and flanking introns were sequenced to determine the structural organization of the gene. The gene spans 6.5 kb, with the first exon encoding only the 5' untranslated sequence. The coding region of preprogalanin and the 3' untranslated sequence is divided into five exons. Using high-resolution fluorescence in situ hybridization, the position of the single human preprogalanin gene was localized to chromosome llq13.3q13.5. Several oncogenes have been mapped to this region, which is also the breakpoint for the translocation t(11;14)(q13;q32) in chronic lymphocytic leukemia and diffuse B-celllymphoma. © 1993 Academic Press, Inc.

INTRODUCTION

The neuropeptide galanin has diverse and often species-specific pharmacological actions. Although a precise physiological role for galanin has not yet been determined, the range of pharmacological actions and widespread distribution of galanin throughout the central and peripheral nervous system suggest a neuromodulatory as well as an endocrine role. The endocrine actions of galanin include inhibition of insulin release from the pancreas and stimulation of both feeding and the release of growth hormone, whereas its neuromodulatory actions include inhibition of both hippocampal acetylcholine release and firing of locus coeruleus cells (for review and references see Vrontakis et at., 1991; Bartfai et at., 1992). Considerable interest has been focused on the potential role of galanin in diabetes, appetite disorders, and Alzheimer's disease (Vrontakis et at., 1991). InterestThe nucleotide sequence reported in this paper has been deposited in the GenBank/EMBL Data Libraries under Accession No. L08285. 1 To whom correspondence should be addressed at Centre de Recherches Roussel Vclaf, Department Endocrinologie, 111 route de Noisy, 93230 Romainville, France. Telephone: 33 1 4991 4565. Fax: 33 1 4991 5257.

MATERIALS AND METHODS Genomic library screening A human lymphocyte genomic DNA library in the cosmid vector pWE 15 (Stratagene) was screened with a 32P-labeled 0.6-kb fragment (nucleotides -16 to 572) of the human preprogalanin cDNA (Evans and Shine, 1991). Cosmid DNA was

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0888-7543/93 $5.00 Copyright © 1993 by Academic Press, Inc. Al! rights of reproduction in any form reserved.

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EVANS ET AL. H

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FIG. 1. Organization of the human preprogalanin gene. (A) A schematic representation of the human preprogalanin gene and (B) the positions of the six exons with respect to the pituitary preprogalanin cDNA are shown. Numbers refer to the amino acid position ofpreprogalanin. The positions of the translation initiation site (ATG), the polyadenylation site, and the termination codon are indicated. The exact site of cleavage of the preprogalanin protein is unknown, but probably occurs at Ala 23, generating a propeptide that is then processed by a specific endopeptidase at the basic residues 31 and 32. Restriction sites indicated: E (EcoRI), H (HindIII), S (StuI), and P (PstI). GAL, galanin, SIG, signal peptide. transferred to Hybond N+ filters (Amersham) and hybridized with the probe in a solution containing 6X SSC, 5x Denhardt's solution, 0.1 % SDS, and 100 ILg/ml denatured and sheared salmon sperm DNA at 65°C for 16 h. Filters were washed twice for 15 min in 2X SSC/0.1% SDS at 65°C followed by a 15-min wash in O.lX SSC/0.1 % SDS and exposed to X-ray film (Kodak, X-Omat) using an intensifying screen at -70°C for 16 h. Positive colonies were purified and DNA was isolated using a standard alkaline lysis procedure (Sambrook et aI., 1989). The DNA was digested with PstI, Sac!, SaelI, and StuI and then subcloned into the Bluescript SK vector (Stratagene), generating clones covering most of the preprogalanin gene. Regions of the gene not included in the subclones were sequenced directly from the cosmid clone.

Nucleotide sequence determination. Supercoiled plasmid DNA was alkaline-denatured and sequenced by the dideoxy chain termination method using T7 polymerase (Promega) (Sambrook et aI., 1989). The oligonucleotide primers used initially were complementary to the flanking region of the vector or to the internal cDNA sequence. Additional primers based on the intron sequence obtained were also used to complete the sequence analysis. Chromosome preparation and R-banding. Metaphase chromosome spreads were prepared from lymphocyte cultures obtained from normal donors as previously described (Kasahara et al., 1977). 5-Bromo2' -deoxyuridine at a final concentration of 0.13 mM was applied after thymidine synchronization to visualize R-banding. After the chromosomes were spread, the slides were briefly rinsed in distilled water and stained with Hochst 33258 (0.1 ILg/lLl) for 25 min, washed with 2X SSC, and exposed to UV light (365 nm) for 25 min. The slides were dehydrated through an ethanol series, air-dried, and stored at -20°C until the day of hybridization. In situ hybridization. In situ hybridization was carried out as previously described (Baumgartner et ai., 1991) with some modifications. Slides were treated for 1 h with RNase (100 ILg/ml, 2X SSC) at 37°C, rinsed three times for 10 min in 2X SSC, and then dehydrated through an ethanol series. Chromosomes were denatured in 70% formamide, 2X SSC (pH 7) at 70°C for 2 min, rinsed at 4°C for 2 min in 2x SSC, and then dehydrated through an ethanol series. The DNA probe was nick-translated with biotin-ll-dUTP (Bio-Rad) according to GIBCO BRL protocols. To reduce background fluorescence, the biotinylated probe was first prehybridized with human Cot DNA and sonicated

salmon sperm DNA prior to hybridization with the chromosomes. For each slide, 50 ng biotinylated probe was combined with llLg human Cot DNA (GIBCO BRL) and 1.5ILg sonicated salmon sperm DNA in 10 ILl hybridization buffer (50% v/v deionized formamide, 10% (wt/vol) dextran sulfate, 2X SSC, 40 mM sodium phosphate, 0.1 % SDS, IX Denhardt's solution). The mixture was denatured at 95°C for 10 min and prehybridized at 37°C for 25 min. The slides were incubated overnight at 42°C with 10 ILl of the prehybridized probe solution.

Immunofluorescence detection. The slides were rinsed twice in 50% formamide/2X SSC, three times for 2 min in 2X SSC at 42°C and 5 min in modified bicarbonate buffer (BN: 0.1 MNa 2 C0 3 , pH 8, 0.05% Nonidet P40, 5% fat-free milk powder). Immunofluorescence was detected by fluorescein isothiocyanate-conjugated avidin according to the protocol of Pinkel et al. (1986). Chromosomes were counterstained with propidium iodide at a final concentration of 0.5-1ILg/ml for 3 min and briefly rinsed with PBS. Slides were mounted with p-phenyldiamine and observed and photographed with a Zeiss Photo microscope III using the filter combination 487 709 and Kodak T-Max 400 for fluorescent signals and 487 715 and Hford PAN F for R-banding.

RESULTS

Isolation of the Human Preprogalanin Gene A human genomic DNA library was screened with a 32P-labeled preprogalanin cDNA clone (nucleotides -16 to 572) isolated from a human pituitary cDNA library (Evans and Shine, 1991). Two positive clones were obtained from 7.5 X 105 colonies. Clone 1 contained an insert of approximately 35 kb. The restriction map for clone 1 is shown in Fig. 1. The insert of this clone was digested with different enzymes and the fragments were subcloned into the Bluescript SK vector for sequencing. The exon sequence of the human preprogalanin gene is identical to that of the human neuroblastoma and pitu-

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GENOMIC ORGANIZATION OF HUMAN PREPROGALANIN

TABLE 1 ExonJIntron Boundaries of the Human Preprogalanin Gene 5' Intron boundary

GCAGCTCAAGlqtactactgg

Intron number and size (bpj Intron I 190 bp

3' Intron boundary +1 · ................................ tcccttccag/ATGGCCCGAG

MAR +81 CTGGTCGCCGlqtaagtgcgg

W S

+l38

G

+225

+303

L

Intron III 2.3 kb

K

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E

+139 · ................................ ctgcaaacag/ATGCCGTTGG

H A

P

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H

A

P

ATGAAACCAGlqtgagaggac

M K

+82 ................................. tcccttcaaqlGCCAAGGAAA

P

CTGGGCCCAClqtaaggtact

L

Intron II 586 bp

V

G

+226 ................................. tctcttctaqlGAAGCTTTGA G S F D

+304 · ................................ ccctttgcag/AGGCCGGTGC

K

E

A

G

A

Note. Exon and intron sequences are shown in upper- and lowercase letters, respectively. Nucleotides are numbered according to the cDNA sequence. The nucleotide consensus sequences of the introns adjoining the splice junctions are shown in boldface.

itary cDNA previously reported (Evans and Shine, 1991). The human preprogalanin gene consists of six exons spanning 6.5 kb from the start of exon 1 to the site of polyadenylation (Fig. 1). The first exon (approximately 190 bp) encodes only the 5' untranslated region of the preprogalanin mRNA. This region of the human preprogalanin gene shows a high degree of nucleotide identity with the 5' untranslated sequence of the bovine (approximately 73% over 173 nucleotides) (R6kaeus and Carlquist, 1988) and the rat (approximately 55% over 167 nucleotides) (Kaplan et al., 1988) preprogalanin cDNA sequences, suggesting a important role in the transcriptional and/or translational regulation of the gene. The second exon starts with the methionine codon of the signal peptide and terminates just before the tryptic cleavage site preceding galanin. Following a 586-bp intron, the tryptic cleavage site and the first 13 amino acids of galanin are encoded on the third exon. Separated by a 2.3-kb intron, the remaining portion of galanin and the first 10 amino acids of G MAP form the fourth exon. The fifth exon encoding the next 26 amino acids ofGMAP follows after an 800-bp intron. An intron of 1.9 kb separates the sixth exon containing the remaining portion of GMAP and the 3' untranslated region (Table 1). The nucleotide sequences of the introns adjoining the splice junctions (Table 1) are consistent with the recognized consensus sequence GT / AG (Breathnach and Chambon, 1981).

Chromosomal Localization High-resolution fluorescence in situ hybridization was used to determine the chromosomal location of the hu-

man preprogalanin gene. The genomic clone 1 was labeled by standard nick-translation with biotin -11dUTP and hybridized to human metaphase spreads. Analysis of 59 metaphase spreads revealed a total of 310 fluorescent signals (5.2/metaphase). Ofthe total fluorescent signals recorded, 47 (15.2%) were located on the distal part of band qI3.3-qI3.5 of chromosome 11. The other signals were distributed randomly over all chromosomes (Fig. 2). In 53% of these metaphase spreads, there was at least one signal on chromosome 11qI3, whereas 10 of the metaphase spreads showed double signals on chromosomes 11, suggesting that the probe hybridized with complementary sequences on each of the chromatids. Localization to chromosome 11 was confirmed with PCR analysis of somatic cell hybrid DNA using oligonucleotides complementary to preprogalanin genomic sequence (data not shown). These results demonstrate that there is a single gene for human prep rogalanin, which is located on chromosome 11qI3.3-qI3.5.

DISCUSSION

Sequence and restriction analyses revealed that the human preprogalanin gene consists of six exons spanning 6.5 kb from the start of exon 1 to the site ofpolyadenylation. In the context of a comparison of the exon/intron structure of the gene to the function of the corresponding regions of prep rogal an in, it is noteworthy that the first 13 amino acids of galanin are encoded on a separate exon. Structure-activity studies of galanin from several species, and of galanin fragments and analogues, have shown that it is this highly conserved N-terminal portion of galanin that is necessary and sufficient for

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FIG. 2. Fluorescence in situ hybridization of the human preprogalanin gene. Hybridization of the human preprogalanin clone to human chromosome llq13.3-q13.5: (A) Fluorescent signals (indicated by arrows) observed with the filter combination 487 709; (B) R-banding with the filter 487 715; the corresponding sites of fluorescence are indicated by arrows; (C) histogram of the distribution of the 310 fluorescent signals analyzed in 59 metaphase spreads.

recognition by the galanin receptor in both exocrine and nervous tissue (for review and references see Bartfai et al., 1992). GMAP is coded over three exons, but the intron/exon boundaries do not correspond to regions of the peptide showing the highest sequence conservation across species. The high level of conservation between the human preprogalanin gene 5' untranslated sequence and the equivalent region in rat and bovine cDNAs suggests that the functional role of this region is conserved across the species. Although the exon/intron boundaries of the rat preprogalanin gene have not yet been reported, a preliminary analysis has shown that this gene is encoded by the same number of exons as the human gene (Vrontakis et al., 1991). Our results have demonstrated that there is a single gene for human preprogalanin located in the region qI3.3-qI3.5 of chromosome 11. This region is of interest since it is the breakpoint region for the translocation t(1l;14)(qI3;q32), an important abnormality associated with chronic lymphocytic leukemia and diffuse B-cell lymphoma (Croce, 1986). It is accepted that chromosomal trans locations disrupt proto-oncogenes involved in the pathogenesis of these malignancies. A number of candidate oncogenes have been mapped to this locus, including the genes for cyclin Dl (PRADl) (Schuuring et al., 1992; Withers et at., 1991), the mUltiple endocrine neoplasia type 1 disease (MENl), EMSI (Schuuring et al., 1992) (function unknown), and the two mitogenic fibroblast growth factor genes HSTFI and INT2 (Szepetowski et al., 1991; Richard et al., 1991) found to be amplified in some solid tumors. The localization of the preprogalanin gene to this locus is particular interesting given the observation that gal an in acts as a mitogen in small-cell lung carcinoma cells (Sethi and Rozengurt, 1991). In summary, this study describes the first characterization of a preprogalanin gene and its localization to human chromosome llqI3.3-qI3.5. This information provides a basis for future studies on the neuroendocrine

role of galanin and its possible involvement in a variety of metabolic and behavioral disorders.

ACKNOWLEDGMENTS We gratefully acknowledge the assistance of M. Liu with DNA sequencing. Helen Evans holds a postgraduate research scholarship from the National Health and Medical Research Council (Australia). Herbert Herzog is a fellow of the E. Schrodinger Foundation (Austria).

REFERENCES Bartfai, T., Fisone, G., and Langel, D. (1992). Galanin and galanin antagonists: Molecular and biochemical perspectives. Trends Phys. Sci. 13: 313-317. Baumgartner, M., Dutrillax, B., Lemieux, N., Lilienbaum, A., Paulien, D., and Viegas-Pequignot (1991). Genes occupy a fixed and symmetrical position on sister chromatids. Cell 64: 761-766. Bersani, M., Johnsen, A. H., Hl'ljrup, P., Dunning, B. E., Andreasen, J. J., and Holst, J. J. (1991). Human galanin: Primary structure and identification of two molecular forms. FEBS Lett. 283: 189-194. Breathnach, R., and Chambon, P. (1981). Organisation and expression of eucaryotic split genes coding for proteins. Annu. Rev. Biochem. 50: 349-383. Croce, C. M. (1986). Chromosome translocation and human cancer. Cancer Res. 46: 6019-6023. Evans, H. F., and Shine, J. (1991). Human galanin: Molecular cloning reveals a unique structure. Endocrinology 129: 1682-1684. Hokfelt, H., Aman, K., Arvidsson, D., Bedecs, K., Ceccatelli, S., Hulting, A., Langel, D., Meister, B., Pieribone, V., and Bartfai, T. (1992). Galanin message-associated peptide (GMAP)- and galanin-like immunoreactivities: Overlapping and differential distributions in the rat. Neurosci. Lett. 142: 139-142. Kaplan, L. M., Spindel, E. R., Isselbacher, K. J., and Chin, W. W. (1988). Galanin is an estrogen-inducible, secretory product of the rat anterior pituitary. Proc. Natl. A cad. Sci. USA 85: 1065-1069. Kasahara, S., Viegas-Pequignot, E., and Frota-Pessoa, O. (1977). A search on karyotypic mosaicism in mongoloid patients and their parents. Rev. Bras. Pesqui. Med. Biol. 10: 225-235. Pinkel, D., Straume, T., and Gray, J. W. (1986). Cytogenic analysis using quantitative, high-sensitivity fluorescence hybridisation. Proc. Natl. Acad. Sci. USA 83: 2934-2938.

GENOMIC ORGANIZATION OF HUMAN PREPROGALANIN Richard, C. W., Withers, D. A., Meeker, T. C., and Myers, R. M. (1991). Cytogen. Cell Genet. 58: 1970. Rokaeus, A., and Carlquist, M. (1988). Nucleotide sequence analysis of cDNAs encoding a bovine galanin precursor protein in the adrenal medulla and chemical isolation of bovine gut galanin. FEBS Lett. 234: 400-406. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989). "Molecular Cloning: A Laboratory Manual," Cold Spring Harbor Laboratory Press, New York. Schuuring, E., Verhoeven, E., Mooi, W. J., and Michalides, J. A. M. (1992). Identification and cloning of two overexpressed genes, U21B31/PRAD1 and EMS1, within the amplified chromosome llq13 region in human carcinomas. Oncogene 7: 355-361. Sethi, T., and Rozengurt, E. (1991). Multiple neuropeptides stimulate clonal growth of small cell lung cancer: Effects of bradykinin, vaso-

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pressin, cholecystokinin, galanin, and neurotensin. Cancer Res. 51: 1674-1679. Szepetowski, P., Nguyen, C., Percucca-Lostanlen, D., Carle, G. F., Tsujimoto, Y., Birnbaum, D., Theillet, C., and Gaudray, P. (1991). DllS146 and BCL1 are physically linked but can be discriminated by their amplification status in human breast cancer. Genomics 10: 410-416. Vrontakis, M. E., Torsello, A., and Friesen, H. G. (1991). Galanin. J. Endocrinol. Invest. 14: 785-794. Withers, D. A., Harvey, R. C., Faust, J. B., Melnyk, 0., Carey, K., and Meeker, C. (1991). Characterization of a candidate bcl-1 gene. Mol. Cell. BioI. 11: 4846-4853. Woll, P. J., and Rozengurt, E. (1989). Multiple neuropeptides mobilise calcium in small cell lung cancer: Effects of vasopressin, bradykinin, cholecystokinin, galanin and neurotensin. Biochem. Biophys. Res. Commun. 164: 66-73.