- Email: [email protected]
Assignment of the gene for neuroendocrine protein 7B2 (SGNE1 locus) to mouse chromosome region 2[E3–F3] and to human chromosome region 15q11-q15
GENOMICS
6,436-440
(1990)
Assignment of the Gene for Neuroendocrine Protein 7B2 (SGNE1 Locus) to Mouse Chromosome Region 2[E3-F3] and to Human Chromosome Region 15ql l-q1 5 M.
GENEVIEVE MATTEI,* MAJAMBU MwAY,t BAKARY 5. SYLLA,* GILBERT J. FRANC06 MATTEL* NABIL G. SEIDAH,t AND MICHEL CHRETlENt
LEtvoiR,t*
l INSERM U.242, Centre de G&n&ique
Mhdicale, Hdpital d’Enfants de la Timone, F- 13385 Marseille Cedex 5, France; tlnstitut de Recherches Cliniques de Mont&al, Montrbal, Quhbec, Canada H2W 1R7; and SCentre International de Recherche sur le Cancer, 69372 Lyon Cedex 08, France Received
August
10, 1989;
16, 1989
MATERIALS
AND
METHODS
cDNA and Gene Cloning The cloning of alpartial cDNA for mouse pancreatic 7B2 has been described (Mbikay et al., 1989). A fulllength cDNA was obtained from a mouse pituitary cDNA library in pCDM8 expression vector (Seed, 1987) constructed with a kit from InVitrogen (San Diego, CA). Seven identical genomic clones containing SGNEl sequences were retrieved from a human genomic library in bacteriophage EMBL3, obtained from American Type Culture Collection (ATCC, Rockville, MD). Library screening, restriction enzyme analysis, and other DNA manipulations were conducted using standard protocols (Maniatis et al., 1982).
INTRODUCTION
Neuroendocrine protein 7B2 is an acidic protein of 186 amino acids, first isolated from human and porcine pituitaries (Hsi et al., 1982; Seidah et al., 1983). It was later detected at varying concentrations in the secretory granules of a wide variety of endocrine and neuronal cells (for reviews, see Marcinkiewicz et aZ., 1985; Mbikay et al., 1989). In the brain, it is most abundant in the supraoptic and paraventricular nuclei of the hypothalamus (Marcinkiewicz et aZ.,1985). Like chromogranins, synaptophysin, and neuron-specific enolase, it can be considered a marker of neurendocrine differentiation. Its role within the endocrine cell is still unknown, but the high conservation of its sequence through evolution could portend an important biological function (Martens, 1988; Brayton et al., 1988; Mbikay et al., 1989).
Probes A diagrammatic representation of the mouse pituitary 7B2 cDNA insert and a 12-kb fragment of its human gene is given in Fig. 1. The 1155bp-long cDNA sequence contains an open reading frame of 636 bp, preceded by a 5’ untranslated region of 78 bp and followed by a 3’ untranslated region of 441 bp (Fig. 1A). The complete sequence of the mouse 7B2 cDNA can be found in the EMBL/GenBank/DDBJ nucleotide sequence databases under Accession Number X15830.
436 Inc. reserved.
October
We plan to define the role of 7B2 by studying its molecular biology and its genetics in mouse and man. As part of this effort, the chromosomal localization of the 7B2 gene (hereafter designated SGNEl, for secretory granule, neuroendocrine protein 1) in both genomes was undertaken in hope of finding some known murine or human disorders mapping at or near the SGNEl locus.
The gene for 7B2, a protein found in the secretory granules of neural and endocrine cells (gene symbol SGNEl) was localized to the E3-F3 region of mouse chromosome 2 and to the qll-ql6 region of human chromosome 16. This was determined by in situ hybridization, using a mouse 7B2 cDNA and an intronic fragment of the corresponding human gene as probes. The respective locations of SGNEl in the two species correlate with the conservation of loci between these subregions of mouse chromosome 2 and human chromosome 16. Clinically, the human SGNEl DNA fragment may serve as a molecular probe of this locus in both the Prader-Willi and the Angelman syndromes, which are often accompanied by submicroscopic chromosomal deletions in the 16ql l-16q13 region. o leso Academic Press,Inc.
osss-7543/90 $3.00 Copyright 0 1990 by Academic Press, AII rights of reproduction in any form
revised
7B2
GENE
IS ON
MOUSE
CHROMOSOME
2 AND
ON
HUMAN
CHROMOSOME
437
15
A cc
KKRAKRRKK
I I 702
A
KR
I
l 186
(b) mRNA
ORF
3’ UT
636bp
441bp
B ~
(01 GENE FRAGMENT
II \
(b) PROTElN
-. \
I -.
II
--
‘\
Ilrl
-.
I
I
V -. \ \
--
G’y49
--
\
-.
-- \ I AsplOl
-\
-.
I
-.
1Kb
I
,
II / -2 ’ / / / / / / / / I/ Asn 138
I
I
J
%55
FIG. 1. Diagrammatic representation of 7B2 macromolecules. (A) (a) Structure of mouse pre-7B2: a signal peptide (SP) of 26 amino acids precedes a mature protein (7B2) of 166 residues containing 2 Cys (C) and pairs of basic residues, Arg (R) or Lys (K), clustered in the carboxyl-terminal portion of the protein. (b) cDNA-derived structure of mouse 7B2 mRNA: the positions of hexameric restriction sites as well as the sizes of the open-reading frame (ORF) and the untranslated (UT) regions are indicated. (B) Partial map of a 12-kb human SGNEl fragment: the splicing of the 3 exons (vertical black bars) produce a sequence encoding residues 49 to 155 of 7B2 (open box). The horizontal black bars in A and B indicate the segments used as probes for in situ hybridization.
A partial restriction map of the human SGNEl fragment is shown in Fig. 1B. The three exons of the genomic insert are interrupted by two introns of 4.2 and 6.4 kb, respectively. These exons were fully sequenced and their junctions to introns were determined. Altogether they comprise 317 nucleotides of the 7B2 mRNA encoding amino acids +49 to +155 of the mature 7B2 protein. By Southern blot analysis, it is estimated that the SGNEl locus extends over 25 kb. Two additional screenings of the ATCC human genomic library have failed to provide the missing sequences. A 0.9-kb fragment of the mouse cDNA and a 1.3-kb intronic fragment of the human gene (horizontal black boxes in Fig. 1) were subcloned into Bluescript plasmid and used as probes for chromosomal localization. Chromosome Mapping by in Situ Hybridization In situ hybridization experiments were carried out on metaphase spreads of phytohemaglutinin-stimu-
lated human lymphocytes or of concanavalin A-stimulated lymphocytes from a male mouse of the WMP/ Pas inbred strain in which all the autosomes except 19 were in the form of metacentric Robertsonian translocations (a gift from J. L. Guenet, Pasteur Institute, Paris). The lymphocytes were cultured at 37°C for 72 h, with 5-bromodeoxyuridine added for the final 7 h of culture (60 pg/ml) to ensure a posthybridization chromosomal banding of good quality. Recombinant Bluescript plasmids containing a 7B2 DNA insert were labeled with tritium by nick-translation to a specific activity of 1.3 X lo8 dpm/pg. The radioactive probes were hybridized to the metaphase spreads at a final concentration of 15 rig/ml of hybridization solution. After a coating with nuclear track emulsion (Kodak NTB2), the slides were exposed for 8 days at 4°C and then developed. To avoid the slipping of silver grains during the banding procedure, chromosome spreads were first stained with buffered Giemsa solution before
MATTE1
438
the metaphases were photographed. R-banding was then performed by the fluorochrome-photolysisGiemsa (FPG) method and metaphases were photographed before analysis. This protocol for gene mapping has been previously described (Mattei et al., 1985). RESULTS
The Sgne-1 Locus in Mouse One hundred metaphase cells were examined after in situ hybridization with the mouse cDNA probe. Only metaphase cells with good chromosome morphology and at least one silver grain located on a chromosome or touching a chromatid were scored. There was a total of 207 silver grains associated with chromosomes; 46 of these (22%) were located on chromosome 2. The distribution of the 46 grains on this chromosome was not random: 35 of them (76%) mapped to the E3-F3 region of the standard chromosome 2 idiogram (Nesbitt and Francke, 1973), with a maximum on the E5 band. A typical illustration of these results is given in Fig. 2,
ET
AL.
indicating the mapping of the Sgne-1 locus to the E3F3 region of mouse chromosome 2. The SGNEl Locus in Man Similarly, 100 human metaphase cells were examined after in situ hybridization with the 1.3-kb human gene probe. There was a total of 198 grains associated with chromosomes; 38 of these (19%) were located on chromosome 15. Of these 38 grains, 33 (87%) mapped to the 15qll-q15 region, with a maximum on the 15q13 band. This is illustrated in Fig. 3. The 15qll-q15 region is therefore the most probable SGNEl locus in man. DISCUSSION
The location of Sgne-1 on the 2 [E3-F3] region in mouse and of SGNEl on the 15qll-ql5 region in man is in keeping with the known conservation of these chromosomal segments between the two species.Thus, the genes coding for sorbitol dehydrogenase (human SORD and mouse Sdh-I), &-microglobulin (human
A
B -A
FIG. 2. Localization of Sgne-I on chromosome. Top: Arrowheads silver grains subsequently identified sites.
16
to mouse chromosome 2. (A) Two partial WMP mouse metaphases showing the specific site of hybridization indicate silver grains on Giemsa-stained chromosomes after autoradiography. Bottom: Chromosomes with by R-banding. (B) Idiogram of WMP mouse Rb (2;16) chromosome, indicating the distribution of labeled
7B2
GENE
IS ON
MOUSE
CHROMOSOME
2 AND
ON
HUMAN
CHROMOSOME
15
439
B
.P q ::-
FIG. 3. Localization of SGNEl to human chromosome 15. (A) Two partial human to chromosome 15. Top: Arrowheads indicate grains on Giemsa-stained chromosomes silver grains subsequently identified by R-banding. (B) Idiogram of the human G-banded sites for the SGNEl probe.
B2it4 and mouse B2m), and cardiac actin (human ACTC and mouse Actc) have already been assigned to the proximal long arm of human chromosome 15 (Ropers et al., 1987) and to the E-F region of mouse chromosome 2 (Lyon, 1988; Crosby et al., 1989). Analysis of mouse recombinant inbreds, outcrosses, and interspecies backcrosses is needed to determine whether Sgne-1 belongs to this linkage group. Our review of the literature on mouse mutants and variants has failed to show any neuroendocrine pathology linked to the E3-F3 region of chromosome 2. However, it is interesting to note that the SGNEl locus in man is in the region of chromosome 15 that is affected by deletions, translocations, and other rearrangements in nearly half of the patients suffering from the Prader-Willi syndrome (PWS) (Mattei et al., 1984). This congenital syndrome is characterized by hypotonia in infancy, hypogonadism, obesity, and mental deficiency. A hypothalamic dysfunction has been suggested as its most probable cause (Tze et al., 1979). Several DNA fragments of unknown sequence have been mapped to this region of chromosome 15 and have
metaphases showing the specific site of hybridization after autoradiography. Bottom: Chromosomes with chromosome 15 illustrating the distribution of labeled
been used as molecular probes to detect genetic deletions in the PWS (Donlon et aZ., 1986; Donlon, 1988; Tasset et aZ., 1988). Donlon (1988) has recently described chromosomal deletions in the Angelman syndrome involving the same DNA probes as those in the PWS. If alterations of SGNEl can also be observed in the PWS, the 7B2 protein, to our knowledge, will be the first neuroendocrine gene product to be associated with the altered locus in this apparently neuroendocrine pathology. The association would be further strengthened by our previous finding that 7B2 neuronal cell bodies are predominantly localized in the hypothalamus (Marcinkiewicz et al., 1985). In addition, the SGNEl probes could be useful tools for the molecular comparison of the two pathologies. ACKNOWLEDGMENTS We thank Haidy Tadros, Francine Sirois, and Edith Passage for their technical assistance and Sylvie Emond for her secretarial help. This research is funded by grants from the Medical Research Council of Canada and from La Succession J. A. de S&e.
MATTE1
440 REFERENCES
1. BRAYTON, K. A., AIMI, J., QUI, H., YAZDANPARAST, R., GHATEI, M. A., POLAK, J. M., BLOOM, S. R., AND DIXON, J. E. (1988). Cloning, characterization, and the sequence of a porcine cDNA encoding a secreted neuronal and endocrine protein. DNA 7: 713-719. 2. CROSBY,J. L., PHILLIPS, S. J., AND NADEAU, J. H. (1989). The cardiac actin locus (Actc-1) is not on mouse chromosome 17 but is linked to &-microglobulin on chromosome 2. Genomics 6: 19-23. 3. DONLON, T. A. (1988). Similar molecular deletions on chromosome 15q11.2 are encountered in both the Prader-Willi and Angelman syndrome. Hum. Genet. 46: 322-328. 4. DONLON, T. A., LALANDE, M., WYMAN, A., BRUNS, G., AND LA’IT, S. A. (1986). Isolation of molecular probes associated with the chromosome 15 instability in the Prader-Willi syndrome. Proc. Natl. Acad. 5%. USA 83: 4408-4412. 5. HSI, K. L., SEIDAH, N. G., DE SERRES, G., AND CHRETIEN, M. (1982). Isolation and NHr-terminal sequence of a novel porcine anterior pituitary polypeptide. FEBS Z&t. 147: 261-266. 6. LYON, M. F. (1988). Mouse chromosome atlas. Mouse News Z&t.
81:
20-41.
MANIATIS, T., FRITSCH, E. F., AND SAMBROOK, J. (1982). “Molecular Cloning: A Laboratory Manual,” Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. 8. MARCINKIEWICZ, M., BENJANNET, S., SEIDAH, N. G., CANTIN, M., AND CHRETIEN, M. (1985). Immunological localization of a novel pituitary protein (7B2) within the rat brain and hypophysis. J. Histochem. Cytochem. 33: 1219-1226. 9. MARTENS, G. J. M. (1988). Cloning and sequence analysis of human pituitary cDNA encoding the novel polypeptide 7B2. FEBS. L&t. 234: 160-164. IMA~EI, M. G., SOUIAH, N., AND MAT’I’EI, J. F. (1984). Chro10. mosome 15 anomalies and the Prader-Willi syndrome: Cytogenetic analysis. Hum. Genet. 66: 313-334. 7.
ET AL. 11. MATTEI, M. G., PHILIP, N., PASSAGE, E., MOISAN, J. P., AND MATTEI, J. F. (1985). DNA probe localization at 18~11.3 band by in situ hybridization and identification of a small supernumerary chromosome. Hum. Genet. 69: 268-271. 12. MBIKAY, M., GRANT, S. G. N., SIROIS, F., TADROS, H., SKOWRONSKI, J., LAZURE, C., SEIDAH, N. G., HANAHAN, D., AND CHRETIEN, M. (1989). cDNA sequence of neuroendocrine protein 7B2 expressed in beta cell tumors of transgenic mice. Znt. J. Peptide
Protein
Res. 33:
39-45.
13. NESBITT, M. N., AND FRANCKE, U. (1973). A system of nomenclature for band patterns of mouse chromosomes. ChromOsoma 41: 145-158. 14. PEMBREY, M., FENNEL, S. J., VAN DEN BERGHE, J., FITCHETT, M., SUMMERS, D., BUTLER, L., CLARKE, C., GRIFFITHS, M., THOMPSON, E., SUPER, M., AND BARAITSER, M. (1989). The association of Angelman’s syndrome with deletion within 15qll-13. J. Med. Genet. 26: 73-77. 15. ROPERS, H. H., GEDDE-DAHL, T., AND Cox, D. W. (1987). Report of the committee on the genetic constitution of chromosomes 13,14,15 and 16. Cytogenet. Cell Genet. 46: 213-241. 16. SEED, B. (1987). An LFA-3 cDNA encodes a phospholipid-linked membrane protein homologous to its receptor CD2. Nature (London)
329:
840-842.
17. SEIDAH, N. G., HSI, K. L., DE SERRES, G., ROCHEMONT, J., HAMELIN, J., ANTAKLY, T., CANTIN, M., AND CHRETIEN, M. (1983). Isolation and NH,-terminal sequence of a highly conserved human and porcine pituitary protein belonging to a new superfamily. Arch. Biochem. Biophys. 225: 525-534. 18. TASSET, D. M., HARZT, J. A., AND KAo, F-T. (1988). Isolation and analysis of DNA markers specific to human chromosome 15. Amer. J. Hum. Genet. 42: 854-866. 19. TZE, W. J., DUNN, H. G., AND ROTHSTEIN, R. L. (1979). Endocrine and metabolic aspects of Prader-Willi syndrome. In “The Prader-Willi Syndrome” (V. A. Holm, P. L. Pipes, and M. J. St&es, Eds.), pp. 281-290, University Park Press, Baltimore.
6,436-440
(1990)
Assignment of the Gene for Neuroendocrine Protein 7B2 (SGNE1 Locus) to Mouse Chromosome Region 2[E3-F3] and to Human Chromosome Region 15ql l-q1 5 M.
GENEVIEVE MATTEI,* MAJAMBU MwAY,t BAKARY 5. SYLLA,* GILBERT J. FRANC06 MATTEL* NABIL G. SEIDAH,t AND MICHEL CHRETlENt
LEtvoiR,t*
l INSERM U.242, Centre de G&n&ique
Mhdicale, Hdpital d’Enfants de la Timone, F- 13385 Marseille Cedex 5, France; tlnstitut de Recherches Cliniques de Mont&al, Montrbal, Quhbec, Canada H2W 1R7; and SCentre International de Recherche sur le Cancer, 69372 Lyon Cedex 08, France Received
August
10, 1989;
16, 1989
MATERIALS
AND
METHODS
cDNA and Gene Cloning The cloning of alpartial cDNA for mouse pancreatic 7B2 has been described (Mbikay et al., 1989). A fulllength cDNA was obtained from a mouse pituitary cDNA library in pCDM8 expression vector (Seed, 1987) constructed with a kit from InVitrogen (San Diego, CA). Seven identical genomic clones containing SGNEl sequences were retrieved from a human genomic library in bacteriophage EMBL3, obtained from American Type Culture Collection (ATCC, Rockville, MD). Library screening, restriction enzyme analysis, and other DNA manipulations were conducted using standard protocols (Maniatis et al., 1982).
INTRODUCTION
Neuroendocrine protein 7B2 is an acidic protein of 186 amino acids, first isolated from human and porcine pituitaries (Hsi et al., 1982; Seidah et al., 1983). It was later detected at varying concentrations in the secretory granules of a wide variety of endocrine and neuronal cells (for reviews, see Marcinkiewicz et aZ., 1985; Mbikay et al., 1989). In the brain, it is most abundant in the supraoptic and paraventricular nuclei of the hypothalamus (Marcinkiewicz et aZ.,1985). Like chromogranins, synaptophysin, and neuron-specific enolase, it can be considered a marker of neurendocrine differentiation. Its role within the endocrine cell is still unknown, but the high conservation of its sequence through evolution could portend an important biological function (Martens, 1988; Brayton et al., 1988; Mbikay et al., 1989).
Probes A diagrammatic representation of the mouse pituitary 7B2 cDNA insert and a 12-kb fragment of its human gene is given in Fig. 1. The 1155bp-long cDNA sequence contains an open reading frame of 636 bp, preceded by a 5’ untranslated region of 78 bp and followed by a 3’ untranslated region of 441 bp (Fig. 1A). The complete sequence of the mouse 7B2 cDNA can be found in the EMBL/GenBank/DDBJ nucleotide sequence databases under Accession Number X15830.
436 Inc. reserved.
October
We plan to define the role of 7B2 by studying its molecular biology and its genetics in mouse and man. As part of this effort, the chromosomal localization of the 7B2 gene (hereafter designated SGNEl, for secretory granule, neuroendocrine protein 1) in both genomes was undertaken in hope of finding some known murine or human disorders mapping at or near the SGNEl locus.
The gene for 7B2, a protein found in the secretory granules of neural and endocrine cells (gene symbol SGNEl) was localized to the E3-F3 region of mouse chromosome 2 and to the qll-ql6 region of human chromosome 16. This was determined by in situ hybridization, using a mouse 7B2 cDNA and an intronic fragment of the corresponding human gene as probes. The respective locations of SGNEl in the two species correlate with the conservation of loci between these subregions of mouse chromosome 2 and human chromosome 16. Clinically, the human SGNEl DNA fragment may serve as a molecular probe of this locus in both the Prader-Willi and the Angelman syndromes, which are often accompanied by submicroscopic chromosomal deletions in the 16ql l-16q13 region. o leso Academic Press,Inc.
osss-7543/90 $3.00 Copyright 0 1990 by Academic Press, AII rights of reproduction in any form
revised
7B2
GENE
IS ON
MOUSE
CHROMOSOME
2 AND
ON
HUMAN
CHROMOSOME
437
15
A cc
KKRAKRRKK
I I 702
A
KR
I
l 186
(b) mRNA
ORF
3’ UT
636bp
441bp
B ~
(01 GENE FRAGMENT
II \
(b) PROTElN
-. \
I -.
II
--
‘\
Ilrl
-.
I
I
V -. \ \
--
G’y49
--
\
-.
-- \ I AsplOl
-\
-.
I
-.
1Kb
I
,
II / -2 ’ / / / / / / / / I/ Asn 138
I
I
J
%55
FIG. 1. Diagrammatic representation of 7B2 macromolecules. (A) (a) Structure of mouse pre-7B2: a signal peptide (SP) of 26 amino acids precedes a mature protein (7B2) of 166 residues containing 2 Cys (C) and pairs of basic residues, Arg (R) or Lys (K), clustered in the carboxyl-terminal portion of the protein. (b) cDNA-derived structure of mouse 7B2 mRNA: the positions of hexameric restriction sites as well as the sizes of the open-reading frame (ORF) and the untranslated (UT) regions are indicated. (B) Partial map of a 12-kb human SGNEl fragment: the splicing of the 3 exons (vertical black bars) produce a sequence encoding residues 49 to 155 of 7B2 (open box). The horizontal black bars in A and B indicate the segments used as probes for in situ hybridization.
A partial restriction map of the human SGNEl fragment is shown in Fig. 1B. The three exons of the genomic insert are interrupted by two introns of 4.2 and 6.4 kb, respectively. These exons were fully sequenced and their junctions to introns were determined. Altogether they comprise 317 nucleotides of the 7B2 mRNA encoding amino acids +49 to +155 of the mature 7B2 protein. By Southern blot analysis, it is estimated that the SGNEl locus extends over 25 kb. Two additional screenings of the ATCC human genomic library have failed to provide the missing sequences. A 0.9-kb fragment of the mouse cDNA and a 1.3-kb intronic fragment of the human gene (horizontal black boxes in Fig. 1) were subcloned into Bluescript plasmid and used as probes for chromosomal localization. Chromosome Mapping by in Situ Hybridization In situ hybridization experiments were carried out on metaphase spreads of phytohemaglutinin-stimu-
lated human lymphocytes or of concanavalin A-stimulated lymphocytes from a male mouse of the WMP/ Pas inbred strain in which all the autosomes except 19 were in the form of metacentric Robertsonian translocations (a gift from J. L. Guenet, Pasteur Institute, Paris). The lymphocytes were cultured at 37°C for 72 h, with 5-bromodeoxyuridine added for the final 7 h of culture (60 pg/ml) to ensure a posthybridization chromosomal banding of good quality. Recombinant Bluescript plasmids containing a 7B2 DNA insert were labeled with tritium by nick-translation to a specific activity of 1.3 X lo8 dpm/pg. The radioactive probes were hybridized to the metaphase spreads at a final concentration of 15 rig/ml of hybridization solution. After a coating with nuclear track emulsion (Kodak NTB2), the slides were exposed for 8 days at 4°C and then developed. To avoid the slipping of silver grains during the banding procedure, chromosome spreads were first stained with buffered Giemsa solution before
MATTE1
438
the metaphases were photographed. R-banding was then performed by the fluorochrome-photolysisGiemsa (FPG) method and metaphases were photographed before analysis. This protocol for gene mapping has been previously described (Mattei et al., 1985). RESULTS
The Sgne-1 Locus in Mouse One hundred metaphase cells were examined after in situ hybridization with the mouse cDNA probe. Only metaphase cells with good chromosome morphology and at least one silver grain located on a chromosome or touching a chromatid were scored. There was a total of 207 silver grains associated with chromosomes; 46 of these (22%) were located on chromosome 2. The distribution of the 46 grains on this chromosome was not random: 35 of them (76%) mapped to the E3-F3 region of the standard chromosome 2 idiogram (Nesbitt and Francke, 1973), with a maximum on the E5 band. A typical illustration of these results is given in Fig. 2,
ET
AL.
indicating the mapping of the Sgne-1 locus to the E3F3 region of mouse chromosome 2. The SGNEl Locus in Man Similarly, 100 human metaphase cells were examined after in situ hybridization with the 1.3-kb human gene probe. There was a total of 198 grains associated with chromosomes; 38 of these (19%) were located on chromosome 15. Of these 38 grains, 33 (87%) mapped to the 15qll-q15 region, with a maximum on the 15q13 band. This is illustrated in Fig. 3. The 15qll-q15 region is therefore the most probable SGNEl locus in man. DISCUSSION
The location of Sgne-1 on the 2 [E3-F3] region in mouse and of SGNEl on the 15qll-ql5 region in man is in keeping with the known conservation of these chromosomal segments between the two species.Thus, the genes coding for sorbitol dehydrogenase (human SORD and mouse Sdh-I), &-microglobulin (human
A
B -A
FIG. 2. Localization of Sgne-I on chromosome. Top: Arrowheads silver grains subsequently identified sites.
16
to mouse chromosome 2. (A) Two partial WMP mouse metaphases showing the specific site of hybridization indicate silver grains on Giemsa-stained chromosomes after autoradiography. Bottom: Chromosomes with by R-banding. (B) Idiogram of WMP mouse Rb (2;16) chromosome, indicating the distribution of labeled
7B2
GENE
IS ON
MOUSE
CHROMOSOME
2 AND
ON
HUMAN
CHROMOSOME
15
439
B
.P q ::-
FIG. 3. Localization of SGNEl to human chromosome 15. (A) Two partial human to chromosome 15. Top: Arrowheads indicate grains on Giemsa-stained chromosomes silver grains subsequently identified by R-banding. (B) Idiogram of the human G-banded sites for the SGNEl probe.
B2it4 and mouse B2m), and cardiac actin (human ACTC and mouse Actc) have already been assigned to the proximal long arm of human chromosome 15 (Ropers et al., 1987) and to the E-F region of mouse chromosome 2 (Lyon, 1988; Crosby et al., 1989). Analysis of mouse recombinant inbreds, outcrosses, and interspecies backcrosses is needed to determine whether Sgne-1 belongs to this linkage group. Our review of the literature on mouse mutants and variants has failed to show any neuroendocrine pathology linked to the E3-F3 region of chromosome 2. However, it is interesting to note that the SGNEl locus in man is in the region of chromosome 15 that is affected by deletions, translocations, and other rearrangements in nearly half of the patients suffering from the Prader-Willi syndrome (PWS) (Mattei et al., 1984). This congenital syndrome is characterized by hypotonia in infancy, hypogonadism, obesity, and mental deficiency. A hypothalamic dysfunction has been suggested as its most probable cause (Tze et al., 1979). Several DNA fragments of unknown sequence have been mapped to this region of chromosome 15 and have
metaphases showing the specific site of hybridization after autoradiography. Bottom: Chromosomes with chromosome 15 illustrating the distribution of labeled
been used as molecular probes to detect genetic deletions in the PWS (Donlon et aZ., 1986; Donlon, 1988; Tasset et aZ., 1988). Donlon (1988) has recently described chromosomal deletions in the Angelman syndrome involving the same DNA probes as those in the PWS. If alterations of SGNEl can also be observed in the PWS, the 7B2 protein, to our knowledge, will be the first neuroendocrine gene product to be associated with the altered locus in this apparently neuroendocrine pathology. The association would be further strengthened by our previous finding that 7B2 neuronal cell bodies are predominantly localized in the hypothalamus (Marcinkiewicz et al., 1985). In addition, the SGNEl probes could be useful tools for the molecular comparison of the two pathologies. ACKNOWLEDGMENTS We thank Haidy Tadros, Francine Sirois, and Edith Passage for their technical assistance and Sylvie Emond for her secretarial help. This research is funded by grants from the Medical Research Council of Canada and from La Succession J. A. de S&e.
MATTE1
440 REFERENCES
1. BRAYTON, K. A., AIMI, J., QUI, H., YAZDANPARAST, R., GHATEI, M. A., POLAK, J. M., BLOOM, S. R., AND DIXON, J. E. (1988). Cloning, characterization, and the sequence of a porcine cDNA encoding a secreted neuronal and endocrine protein. DNA 7: 713-719. 2. CROSBY,J. L., PHILLIPS, S. J., AND NADEAU, J. H. (1989). The cardiac actin locus (Actc-1) is not on mouse chromosome 17 but is linked to &-microglobulin on chromosome 2. Genomics 6: 19-23. 3. DONLON, T. A. (1988). Similar molecular deletions on chromosome 15q11.2 are encountered in both the Prader-Willi and Angelman syndrome. Hum. Genet. 46: 322-328. 4. DONLON, T. A., LALANDE, M., WYMAN, A., BRUNS, G., AND LA’IT, S. A. (1986). Isolation of molecular probes associated with the chromosome 15 instability in the Prader-Willi syndrome. Proc. Natl. Acad. 5%. USA 83: 4408-4412. 5. HSI, K. L., SEIDAH, N. G., DE SERRES, G., AND CHRETIEN, M. (1982). Isolation and NHr-terminal sequence of a novel porcine anterior pituitary polypeptide. FEBS Z&t. 147: 261-266. 6. LYON, M. F. (1988). Mouse chromosome atlas. Mouse News Z&t.
81:
20-41.
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