GENOMICS
10,569-575
(1991)
Chromosomal Mapping of Brain-Derived Neurotrophic and Neurotrophin-3 Genes in Man and Mouse TAYFUN &ELIK,*
ARNON
Factor
ROSENTHAL, t AND UTA FRANCKE*+
*Howard Hughes Medical Institute, *Departments of Genetics and Pediatrics, Stanford University Medical Center, Stanford, California 94305, and tDepartment of Developmental Biology, Genentech Inc., South San Francisco, California 94080 ReceivedJanuary30,1991
liver and skeletal muscle (Ernfors et al., 1990; Maisonpierre et aZ., 199Ob). It is likely to be important for the growth, differentiation, and survival of visceral and placode-derived sensory neurons that do not respond to NGF. The mature BDNF, NT-3, and NGF polypeptides share about 55% amino acid identity (Hohn et al., 1990; Maisonpierre et al., 1990a; Rosenthal et uZ., 1990; Jones and Reichardt, 1990). It has been proposed that neurotrophic molecules, such as NGF, may play an important therapeutic role in the treatment of neurodegenerative disorders including Alzheimer disease or Huntington disease (Ad Hoc Working Group on Nerve Growth Factor and Alzheimer’s Disease, 1989). The involvement of BDNF with the olfactory system and the basal forebrain cholinergic system makes it another potentially useful agent in the treatment of Alzheimer disease. The chromosomal mapping of the genes for neurotrophic factors in man and mouse is of interest for two reasons. First, inherited mutations of these genes may be the underlying mechanisms in the development of certain neurodegenerative disorders. By comparing the map positions of these genes with those of disease loci testable hypotheses can be derived for the involvement of these genes in inherited disorders. Second, determining the chromosomal locations of the member of a gene family contributes to our understanding of the patterns and mechanisms of genome evolution (Ohta et al., 1983). We have previously mapped the gene for P-NGF (NGFB) to the short arm of human chromosome 1, band p22 (Francke et al., 1983), and here we describe the locations of the genes for BDNF and NT-3 (assigned gene symbol NZ’F3) in man and mouse.
Brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NTF3) are two new members of the nerve growth factor gene family, which play important roles in the development and maintenance of the vertebrate nervous system. Here we describe the assignments of the BDNF and NTF3 gene loci to human and mouse chromosomes and discuss the evolutionary relationship of human chromosomes 11 and 12. BDNF has been mapped to human chromosome llp15.5-~11.2 and to mouse chromosome 2, and NTF3 to human chromosome 12p and mouse chromosome 6. 0 1991 Academic Press, Inc.
INTRODUCTION
A specific group of proteins regulate the development and maintenance of the vertebrate nervous system. Known as neurotrophic (or survival) factors, they are released to the projection fields of developing neurons by target cells and promote neuronal survival. Recently, the genes for two new neurotrophic factors named brain-derived neurotrophic factor (BDNF) (Leibrock et al., 1989) and neurotrophin-3 (NT-3) (Hohn et aZ., 1990; Maisonpierre et al., 1990a; Rosenthal et al., 1990; Jones and Reichardt, 1990) have been isolated. They both share extensive structural similarity with the /3 subunit of nerve growth factor (NGF) which is the prime example of these factors. BDNF is a 247-amino acid dimeric protein encoded by 1.45- and 3.5-kb messagesthat is widely distributed in the central nervous system (Ernfors et al., 1990; Maisonpierre et al., 1990b; Phillips et al., 1990) and is a survival factor for neural crest- and placodederived sensory neurons (Lindsay et aZ., 1985), CNSderived retinal neurons (Johnson et aZ., 1986), and rat septal cholinergic neurons (Alderson et aZ., 1990). NT-3 is a protein of 258 amino acids encoded by a 1.4-kb message that is present in cerebellum, CA2 cells of hippocampus, and peripheral tissues such as
MATERIALS
AND
METHODS
Hybrid Cell Lines The primary chromosomal assignments were made by using a human mapping panel of rodent X human 569 All
Copyright 0 1991 rights of reproduction
o&%-7543/91 $3.00 by Academic Press, Inc. in any form reserved.
570
GZCELIK, Rosmmfc,~~~
somatic cell hybrids from series XII, XIII, XV, XVII, XVIII, XXI, and 31, and a mouse mapping panel of Chinese hamster or rat X mouse hybrids from fusions I, EAS, EBS, and RTM. The derivation of hybrid cell lines has been summarized (Francke et al., 1986). The chromosome constitutions of hybrids were analyzed by trypsin-Giemsa banding when they were harvested for the preparation of high molecular weight DNA. Over many years, these hybrid cell lines were analysed for more than 100 genetic markers, thus their chromosomal content has been amply verified by biochemical and molecular genetic data. BDNF and NTF3 were regionally assigned to human chromosomes by analyzing six hamster or mouse X human hybrid cell lines carrying various portions of human chromosomes 11 and 12 in the absence of an intact copy of these chromosomes (Barton et al., 1988; Archer et al., 1990).
Hybridization
Probes
EcoRI-Hind111 fragments of human origin that contain the entire coding sequences of BDNF (1300 bp) (Rosenthal et al., 1991) and NT-3 (975 bp) (Rosenthal et al., 1990) were labeled by random priming and used as probes on Southern filters.
Southern
Hybridizations
Genomic DNA was extracted from human placental tissue, hybrid cell lines, and parental control cells by standard methods. Restriction enzyme digestion, electrophoresis, Southern transfer to Hybond nylon membranes (Amersham Corp.), and hybridizations to 32P-labeled probes were carried out as described (Barton et al., 1986). Filters were rinsed twice in 2~ SSC and washed twice in 1X SSC, 1% SDS at 55-65°C for lo-15 min. The filters were autoradiographed for 16 to 48 h at -70°C with Kodak X-Omat AR film and two intensifying screens.
RESULTS Mapping
of Human
BDNF
The location of the human BDNF gene was determined with a panel of 16 well-characterized rodent X human hybrid cell lines carrying different subsets of human chromosomes. Hybridization of human probe to BamHI-digested genomic DNA revealed strongly hybridizing single fragments in each species:
FRANCKE 7-kb human, 5-kb Chinese hamster, and 9-kb mouse (Fig. 1A). The 7-kb human fragment was concordant with human chromosome 11. All other chromosomes were excluded by at least four discordant hybrids (Table 1). Regional localization of BDNF was carried out with four hybrids from series VII, XV, and XXI fusions that contain defined regions of chromosome 11 in the absence of an intact 11. Southern analysis of these hybrid cell lines assigned the BDNF locus to the short arm region llpll.Z-~15.5 (Fig. 2).
Mapping
of Mouse Bdnf
The murine brain derived neurotrophic factor gene (symbol Bdnf ) was mapped by using the same 1300bp human probe and a panel of Chinese hamster or rat X mouse somatic cell hybrids that carry different subsets of mouse chromosomes. Single 4.3-kb Chinese hamster (Fig. lB, lane l), 11-kb mouse (lane Z), and 7.6-kb rat (lane 3) fragments were detected in control genomic DNA samples digested with EcoRI. The llkb mouse fragment was present in hybrids that had retained mouse chromosome 2 (Fig. lB, lanes 5 and 6) and absent in hybrids that are lacking this chromosome (lanes 4 and 7). One hybrid had mouse chromosome 2 as the only mouse chromosome and was positive for the mouse Bdnf fragment. In addition, all other mouse chromosomes were excluded as possible sites for Bdnf by at least 20% discordant hybrids (Table 2). These results place Bdnf on mouse chromosome 2.
Mapping
of Human
NTF3
The human neurotrophin-3 gene (symbol NTF3) was mapped with a BglII-digested mapping panel of 13 Chinese hamster X human somatic cell hybrids having reduced numbers of human chromosomes. The 900-bp human probe recognized a 7.6-kb human and a 4-kb Chinese hamster fragment (Fig. 3A). The 7.6-kb BgZII human fragment was concordant with human chromosome 12. All other chromosomes were excluded by at least two discordant hybrids (Table 1). Two hybrid lines from fusions XII and XXI that carry defined regions of human chromosome 12 in the absence of an intact copy of this chromosome were used for regional localization of NTF3. Hybrid cell line XII-2A retaining the region lZpter-q21 was positive (Fig. 3A, lane 5) and hybrid cell line XXI-63A containing the region 12cen-qter was negative (Fig. 3A, lane 4) for human neurotrophin-3 sequences. These results place the NTF3 locus on the short arm of chromosome 12 (Fig. 4).
MAPPING
1
2
3
4
OF
5
6
BDNF
7
AND
8
NTF3
IN
MAN
AND
9 10 11 12
571
MOUSE
1234567 -
kb 11
- 1.6
Human
Mouse
BDNF FIG. 1. Chromosomal mapping of the human (A) and mouse (B) BDNF gene. 32P-labeled BDNF probe was hybridized to Southern blots of BarnHI-digested DNA from human X rodent and EcoRI-digested DNA from mouse X Chinese hamster or rat hybrid cell lines and controls. A: lane 1, human placental tissue; lane 2, Chinese hamster cell line V79/380-6; lane 3, mouse 3T3 cell line; lanes 4-9 and 12, human X Chinese hamster cell hybrids of which lanes 4,6,7,8, and 12 contain the human specific 7-kb fragment; lane 10, human X mouse hybrid with region A (Fig. 2) positive for the human specific fragment; lane 11, mouse X human hybrid negative for the human signal B: lane 1, V79/380-6: lane 2,3T3; lane 3, rat hepatoma cell line 7777-14b aza; lanes 4,5, and 6, Chinese hamster X mouse cell hybrids of which lanes 5 and 6 are positive for the 11-kb mouse fragment; lane ‘7, rat X mouse cell hybrid negative for the mouse fragment.
TaqI, and Hind111 in five inbred mouse strains, DBA/ 25, AKR/J, C3H/HeJ, C57BL/6J, and C57L/J.
Mapping of Mouse Ntf-3 In rodent genomic DNA samples digested with PstI the human NT-3 probe hybridized strongly with single fragments of 1.9 kb for Chinese hamster (Fig. 3B, lane l), 3 kb for mouse (lane 2), and 2.7 kb for rat (lane 3). The 3-kb mouse fragment was present in all hybrid cell lines that had retained mouse chromosome 6. All other mouse chromosomes were excluded aspossible sites for the gene by at least two discordant hybrids (Table 2). Thus, the mouse neurotrophin-3 gene (gene symbol Ntf-3) is assigned to chromosome 6.
DISCUSSION
The human brain derived neurotrophic factor gene (BDNF) has been mapped to the short arm of human chromosome 11 (HSA ll), bands p11.2-~15.5, by Southern blot analysis of DNA from rodent X human hybrid lines carrying different parts of HSA 11. With a panel of Chinese hamster or rat X mouse hybrid lines the murine B&f gene has been mapped to mouse chromosome 2 (MMU 2). Four other loci, CAT (catalase), FSHB (follicle-stimulating hormone, /3polypeptide), AN2 (aniridia, small eye), and ACP2 (acid phosphatase-2, lysosomal) have also been assigned to HSA 11 and MMU 2 (for review, see Searle et al., 1989). The conserved region spans the proximal short arm of HSA 11, bands pll-~13, and region D-E4 in
Polymorphism Search with BDNF and NTF3 Probes No restriction fragment length polymorphism was found after digestion with P&I, TuqI, BamHI, HinfI, NsiI, HincII, StyI, Eco0109, AZuI, P&I, and MspI in DNA from 8 to 10 unrelated Caucasians. Likewise, no strain differences were detected with PstI, BamHI,
TABLE Presence
1
of Human BDNF and NTF3 Sequences and Human Cell Hybrids in Rodent X Human Somatic Human
BDNF discordant hybrids Informative hybrids” Percentage discordant NTF3 discordant hybrids Informative hybrids” Percentage discordant a Chromosomes
with
Chromotimes
chromosome
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
X
11 17 65 4 16 25
11 18 61 5 17 29
9 17 53 7 15 47
9 14 64 5 15 33
8 18 44 8 18 44
9 17 53 8 16 50
11 17 65 8 16 50
8 18 44 6 17 35
14 18 78 7 16 44
12 18 67 8 18 44
0 16 0 4 16 25
7 18 39 0 18 0
12 17 ‘71 11 18 61
11 16 69 10 17 59
8 18 44 7 18 39
9 17 53 5 16 31
10 18 56 10 18 56
10 18 56 9 18 50
9 18 50 6 17 35
4 18 22 5 18 28
7 17 41 6 17 35
8 16 50 7 17 41
4 6 67 3 7 43
rearrangements
or present
at a frequency
of 0.1 or less were excluded.
572
GZCELIK,
ROSENTHAL,
logical mutations in this region that could involve Bndf, the best candidate is am (anorexia) a recessive lethal mutation of abnormal behavior and neurological dysfunction that leads to starvation due to insufficient food intake (Maltais et al., 1984). On the composite map of mouse chromosome 2, anx is close to Sey (Searle et aE., 1989). The NTF3 gene has been localized to HSA 12p and MMU 6. By using the same panels of somatic cell hybrids, we have recently mapped a synaptic vesicle protein gene, synaptobrevin 1 (SYBI) to the same chromosome regions (Archer et aZ., 1990). In fact, as summarized in Searle et al. (1989), all genes known to be on HSA 12p, including CD4 (L3T4 T cell differentiation antigen), GAPD (glyceraldehyde-3-phosphate dehydrogenase), PRH (proline-rich salivary protein), TPIl (triose phosphate isomerase l), LDHB (lactate dehydrogenase B), PTHLH (parathyroid hormonelike hormone), and KRAS2 (Kirsten rus oncogene 2), have their homologues on the distal third of MMU 6. The orientation of this conserved syntenic region seemsto be inverted with respect to the centromere. More proximal parts of MMU 6 contain loci homologous to genes on human chromosomes 2, 3, and 7. Thus, interspecies comparative mapping allows one to predict the regional localization of genes in conserved chromosome region. However, independent proof by higher resolution mapping methods is still necessary, as disruption of conserved syntenic regions are frequent in mouse and are also seen on human chromosomes (Barton et al., 1988). Our data mapping BDNF and NTF3 to human chromosomes llp and 12p, respectively, add to the list of structurally related loci on these two human chromosomes. It has been proposed that a tetraploidization event during evolution doubled the chromosome number of mammals (Ohno, 1967; Ohno et aZ., 1968). HSA 11 and 12 are good candidates for a pair of
1
BDNF
J
FIG. 2. Ideogram of G-banding patterns of human chromosome 11 with regional mapping data using somatic cell hybrids VII-3 HAT (A), XV-N4 (B), XXI-23A-2c (C), and XXI-54B-1J (D). The vertical bars illustrate different portions of chromosome 11 present in these hybrids. + is hybrid positive for human BDNF sequence; - is hybrid negative. The bracket indicates the location of the BDNF gene.
the middle of MMU 2. All genes from 11~14 to the telomere are homologous to loci on MMU 7. Therefore, it is likely that BDNF is located within the conserved region llp11.2-p13 (Fig. 5). This region also includes the genes for Wilms tumor and aniridia. The latter is thought to be homologous to the Sey locus on MMU 2 (Searle et aZ., 1989). In terms of murine neuro-
TABLE Presence
AND FRANCKE
2
of Mouse Bdnf and Ntf-3 Sequences and Mouse Chromosomes Somatic Cell Hybrids in Mouse X Chinese Hamster Mouse chromosome
discordant hybrids Informative hybrids” Percentage discordant Ntf-3 discordant hybrids Informative hybrids” Percentage discordant Bdnf
1
2
3
4
5
6
‘7
8
9
10
11
12
13
14
15
16
17
18
19
X
4 10 40 3 11 27
0 11 0 6 13 46
4 11 36 4 13 31
3 10 30 5 12 42
4 9 44 4 11 36
5 10 50 0 12 0
3 10 30 5 12 42
3 11 27 4 13 31
8 11 73 3 13 23
7 11 64 5 12 42
9 11 82 7 13 54
7 11 64 5 13 38
7 10 70 5 12 42
4 11 36 5 12 42
5 11 45 3 13 23
6 10 60 4 11 36
3 11 27 3 13 23
4 11 36 5 12 42
2 10 20 3 12 25
5 10 50 3 12 25
o Chromosomes with rearrangements
or present at a frequency of 0.1 or less were excluded.
MAPPING
OF
BDNF
AND
IN
MAN
AND
573
MOUSE
B
A
1 2
123456
kb
NTF3
3
4
5
6 7
8
9
10
kb -3 - 2.7
7.6-
-
Human
1.9
Mouse
NTF 3 FIG. 3. Chromosomal mapping of the human (A) and mouse (B) NTF-3. 32P-Labsled NTF3 probe was hybridized to Southern blots of BglII-digested DNA from human X rodent and PstI-digested DNA from mouse X Chinese hamster or rat hybrid cell lines and controls. A: lane 1, human placental tissue; lane 2, Chinese hamster cell line V79/380-6; lanes 3-6, human X Chinese hamster hybrid cell lines. Lanes 3 and 6 are positive for the human 7.6-kb fragment, and lanes 4 and 6 are negative. B: lane 1, V79/380-6; lane 2,3T3; lane 3, rat hepatoma cell line 7777-14b aza; lanes 4-9 Chinese hamster X mouse cell hybrids of which lanes 4, 7, and 8 are positive for the 3-kb mouse specific fragment; lane 10, mouse X rat hybrid negative for the mouse fragment.
tetraploidization derivatives because of their similar banding patterns and content of structurally related loci. As seen in Fig. 5, the genes for HRASl (Harvey rat sarcoma oncogene), PTH (parathyroid hormone), and LDHA (lactate dehydrogenase A) are on the short arm of HSA 11, and related genes for KRAS2
TF3
FIG. 4. Ideogram of G banding patterns of human chromosome 12 with regional mapping data using Chinese hamster x human somatic cell hybrids XII-2A (A) and XXI-63A (B). The vertical bars indicate portions of chromosome 12 present in these cell hybrids. + is hybrid positive for human NTF3 sequences; - is hybrid negative. The bracket indicates the location of the NTF3 gene.
(Kirsten rat sarcoma oncogene-2), PTHLH (parathyroid hormone like hormone), and LDHB (lactate dehydrogenase B) are on the short arm of HSA 12 (Searle et al., 1989). On the other hand, although the genes for IGF2 (insulin like growth factor 2), INS (insulin), and TH (thyrosine hydroxylase) are also on llp, related genes for IGFl (insulin like growth factor 1) and PAH (phenylalanine hydroxylase) are on 12q. In order to explain this difference, it has been postulated that intrachromosomal rearrangements have altered the organization of genes on HSA 11 and 12 (Brissenden et al, 1984; Barton et al., 1988). Relationships with regions on other chromosomes are suggested by the observation that NGFB, a member of the nerve growth factor/insulin/relaxin family of small peptides, is on human chromosome 1~22.1 (neuroblastoma oncogene), a along with NRAS member of the rm family of oncogenes (Mtinke et al., 1984) and NRASL, an NRAS-like gene is on 9p toand RLN2 gether with the two relaxin loci RLNl (Smith and Simpson, 1989). If the structurally related genes mapped to human chromosomes 11 and 12 support the hypothesis of evolutionary kinship between these two chromosomes, the map positions of the same genes in the mouse genome should serve to identify the mouse chromosomes that are evolutionarily related to each other. However, this is not straightforward. First, a comparison of the banding patterns of the mouse chromosomes involved does not lead to an obvious alignment of chromosome pairs. Second, as illustrated in Fig. 5, genes on HSA 11 and 12 have homologs on eight different mouse chromosomes. This observation suggests that mouse chromosomes have undergone more rearrangements since separation from a
574 IGF2
3”HARSl MMU7 12.3 12.2 12.1
P
MMU
6
MMU
15
MMU
5
19
7
MMU
9
FIG. 5. Comparative maps of human chromosomes 11 and 12. The locations of structurally explanation of gene symbols, see text. Vertical bars indicate proposed regions of homology with on gene mapping data summarized in Searle et al. (Ref. (26) and Kuo et al. (Ref. (13)).
common ancestor which is consistent of mice.
than have human chromosomes, with the shorter generation time
7.
ACKNOWLEDGMENTS This work was supported by Research Grant and by Genentech Inc. U.F. is an investigator ciate of the Howard Hughes Medical Institute.
6.
HG00298 and T.O.
(to U.F.) an asso-
2.
3.
4.
5.
Ad Hoc Working Group on Nerve Growth Factor and Alzheimer’s Disease (National Institute of Aging) (1989). Potential use of nerve growth factor to treat Alzheimer’s disease. Science 243: 11. ALDERSON, R. F., ALTERMAN, A. L., BARDE, Y. A., AND LINDSAY, R. M. (1990). Brain-derived neurotrophic factor increases survival and differentiated functions of rat septal cholinergic neurons in culture. Neuron 5: 297-306. ARCHER, B. T., III, GZCELIK, T., JAHN, R., FRANCKE, U., AND S~DHOF, T. C. (1990). Structures and chromosomal localizations of two human genes encoding synaptobrevins 1 and 2. J. Biol. Chem. 265: 17,267-17,273. BARTON, D. E., YANG-FENG, T. L., AND FRANCKE, U. (1986). The human tyrosine aminotransferase mapped to the long arm of chromosome 16 (region 16q22-q24) by somatic cell hybrid analysis and in situ hybridization. Hum. Genet. 72: 221-224. BARTON, D. E., KWON, B. S., AND FRANC+ U. (1988). Human tyrosinase gene, mapped to chromosome 11 (q14-q21), defines second region of homology with mouse chromosome 7. Genomics 3: 17-24.
BRISSENDEN, J. E., ULLRICH, A., AND FRANC-, U. (1984). Human chromosomal mapping of genes for insulin-like growth factors I and II and epidermal growth factor. Nature 310: 781-784. ENFORS, P., WETMORE, C., OLSON, L., AND &EN, P. (1990). Identification of cells in rat brain and peripheral tissues expressing mRNA for members of the nerve growth factor family. Neuron 5: 511-526.
8.
FRANC=, U., DE MARTINVILLE, B., COUSSENS, L., AND ULLRICH, A. (1983). The human gene for the fi subunit of nerve growth factor is located on the proximal short arm of chromosome 1. Science 222: 1248-1251.
9.
FRANCKE, ULLRICH, in growth 855-866.
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related genes are shown in brackets. For various mouse (MMU) chromosomes based
U., YANG-FENG, T. L., BFUSSENDEN, J. E., AND A. (1986). Chromosomal mapping of genes involved control. Cold Spring Harbor Symp. Quad. Biol. 51:
10.
HOHN, A., LEIBROCK, J., BAILEY, K., (1990). Identification and characterization of the nerve growth factor/brain-derived family. Nature 344: 339-341.
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JOHNSON, J. E., BARDE, Y.-A., SCHWAB, M., AND THOENEN, H. (1986). Brain-derived neurotrophic factor supports the survival of cultured rat retinal ganglion cells. J. Neurosci. 6: 3031-3038.
12.
JONES, K. R., AND REICHARDT, L. F. (1990). Molecular ing of a gene that is a member of the nerve growth family. Proc. Natl. Acad. Sci. USA 87: 8060-8064.
13.
Kuo, C. J., CO-Y, P. B., HSIEH, C.-L., FRANCKE, U., AND CRABTREE, G. R. (1990). Molecular cloning, expression, and chromosomal localization of mouse hepatocyte nuclear factor 1. Proc. Natl. Acad. Sci. USA 67: 9838-9842. LEIBROCK, J., LO?TSPEXCH, F., HOHN, A., HOFER, M., HENGERER, B., MASIAKOWSKI, P., THOENIZN, H., AND BARDE,
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AND BARDE, Y. A. of a novel member neurotrophic factor
clonfactor
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OF
Y. A. (1989). Molecular cloning and expression rived neurotrophic factor. Nature 341: 149-152. 15.
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LINDSAY, R. M., THOENEN, H., AND BARDE, Y. A. (1985). Placode and neural crest-derived sensory neurons are responsive at early developmental stages to brain-derived neurotrophic factor. Deu. Biol. 112: 319-328.
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OHNO, S., WOLF, U., AND ATKIN, N. B. (1968). Evolution from fish to mammals by gene duplication. Hereditas 69: 169-187.
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OHTA, T., AND DOVER, G. A. (1983). Population genetics of multigene families that are dispersed into two or more chromosomes. Proc. Natl. Acad. Sci. USA 80: 4079-4083.
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MAISONPIERRE, P. C., BELLUSCIO, L., SQUINTO, S., IP, N. Y., FURTH, M. E., LINDSAY, R. M., AND Y~~co~ou~os, G. D. (1990a). Neurotrophin-3: A neurotrophic factor related to NGF and BDNF. Science 247: 1446-1451.
23.
PHILLIPS, H. S., HAINS, J. M., LARAMEE, G. R., ROSENTHAL, A., AND WINSLOW, J. W. (1990). Widespread expression of BDNF but not NT3 by target areas of basal forebrain cholinergic neurons. Science 260: 290-294.
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MAISONPIERRE, P. C., BELLUSCIO, L., FRIEDMAN, B., ALDERSON, R. F., WIEGAND, S. J., FURTH, M. E., LINDSAY, R. M., AND YANCOPOULOS, G. D. (1990b). NT-3, BDNF, and NGF in the developing rat nervous system: Parallel as well as reciprocal patterns of expression. Neuron 6: 501-509.
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ROSENTHAL, A., GOEDDEL, D. V., NGUYEN, T., LEWIS, M., SHIH, A., LAF~AMEE, G. R., NIKOLICS, K., AND WINSLOW, J. W. (1990). Primary structure and biological activity of a novel human neurotrophic factor. Neuron 4: 767-773.
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MONKE, M., LINDGREN, V., DE MARTINVILLE, B., AND FRANCKE, U. (1984). Comparative analysis of mouse-human hybrids with rearranged chromosomes 1 by in situ hybridization and Southern blotting: High-resolution mapping of NRAS, NGFB, and AMY on human chromosome 1. Somatic Cell Mol. Genet. 10: 589-599.
ROSENTHAL, A., GOEDDEL, D. V., NGUYEN, T., MARTIN, E., BURTON, L. E., SHIH, A., LARAMEE, G. R., NIKOLICS, K., AND WINSLOW, J. W. (1991). Primary structure and biological activity of human brain-derived neurotrophic factor. (submitted).
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SMITH, M., AND SIMPSON, N. E. (1989). Report of the committee on the genetic constitution of chromosomes 9 and 10. Cytogenet. Cell Genet. 51: 202-225.
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OHNO, S. (1967). Springer-Verlag,
“Sex New
Chromosomes York.
and Sex-linked
Genes,”
10,569-575
(1991)
Chromosomal Mapping of Brain-Derived Neurotrophic and Neurotrophin-3 Genes in Man and Mouse TAYFUN &ELIK,*
ARNON
Factor
ROSENTHAL, t AND UTA FRANCKE*+
*Howard Hughes Medical Institute, *Departments of Genetics and Pediatrics, Stanford University Medical Center, Stanford, California 94305, and tDepartment of Developmental Biology, Genentech Inc., South San Francisco, California 94080 ReceivedJanuary30,1991
liver and skeletal muscle (Ernfors et al., 1990; Maisonpierre et aZ., 199Ob). It is likely to be important for the growth, differentiation, and survival of visceral and placode-derived sensory neurons that do not respond to NGF. The mature BDNF, NT-3, and NGF polypeptides share about 55% amino acid identity (Hohn et al., 1990; Maisonpierre et al., 1990a; Rosenthal et uZ., 1990; Jones and Reichardt, 1990). It has been proposed that neurotrophic molecules, such as NGF, may play an important therapeutic role in the treatment of neurodegenerative disorders including Alzheimer disease or Huntington disease (Ad Hoc Working Group on Nerve Growth Factor and Alzheimer’s Disease, 1989). The involvement of BDNF with the olfactory system and the basal forebrain cholinergic system makes it another potentially useful agent in the treatment of Alzheimer disease. The chromosomal mapping of the genes for neurotrophic factors in man and mouse is of interest for two reasons. First, inherited mutations of these genes may be the underlying mechanisms in the development of certain neurodegenerative disorders. By comparing the map positions of these genes with those of disease loci testable hypotheses can be derived for the involvement of these genes in inherited disorders. Second, determining the chromosomal locations of the member of a gene family contributes to our understanding of the patterns and mechanisms of genome evolution (Ohta et al., 1983). We have previously mapped the gene for P-NGF (NGFB) to the short arm of human chromosome 1, band p22 (Francke et al., 1983), and here we describe the locations of the genes for BDNF and NT-3 (assigned gene symbol NZ’F3) in man and mouse.
Brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NTF3) are two new members of the nerve growth factor gene family, which play important roles in the development and maintenance of the vertebrate nervous system. Here we describe the assignments of the BDNF and NTF3 gene loci to human and mouse chromosomes and discuss the evolutionary relationship of human chromosomes 11 and 12. BDNF has been mapped to human chromosome llp15.5-~11.2 and to mouse chromosome 2, and NTF3 to human chromosome 12p and mouse chromosome 6. 0 1991 Academic Press, Inc.
INTRODUCTION
A specific group of proteins regulate the development and maintenance of the vertebrate nervous system. Known as neurotrophic (or survival) factors, they are released to the projection fields of developing neurons by target cells and promote neuronal survival. Recently, the genes for two new neurotrophic factors named brain-derived neurotrophic factor (BDNF) (Leibrock et al., 1989) and neurotrophin-3 (NT-3) (Hohn et aZ., 1990; Maisonpierre et al., 1990a; Rosenthal et al., 1990; Jones and Reichardt, 1990) have been isolated. They both share extensive structural similarity with the /3 subunit of nerve growth factor (NGF) which is the prime example of these factors. BDNF is a 247-amino acid dimeric protein encoded by 1.45- and 3.5-kb messagesthat is widely distributed in the central nervous system (Ernfors et al., 1990; Maisonpierre et al., 1990b; Phillips et al., 1990) and is a survival factor for neural crest- and placodederived sensory neurons (Lindsay et aZ., 1985), CNSderived retinal neurons (Johnson et aZ., 1986), and rat septal cholinergic neurons (Alderson et aZ., 1990). NT-3 is a protein of 258 amino acids encoded by a 1.4-kb message that is present in cerebellum, CA2 cells of hippocampus, and peripheral tissues such as
MATERIALS
AND
METHODS
Hybrid Cell Lines The primary chromosomal assignments were made by using a human mapping panel of rodent X human 569 All
Copyright 0 1991 rights of reproduction
o&%-7543/91 $3.00 by Academic Press, Inc. in any form reserved.
570
GZCELIK, Rosmmfc,~~~
somatic cell hybrids from series XII, XIII, XV, XVII, XVIII, XXI, and 31, and a mouse mapping panel of Chinese hamster or rat X mouse hybrids from fusions I, EAS, EBS, and RTM. The derivation of hybrid cell lines has been summarized (Francke et al., 1986). The chromosome constitutions of hybrids were analyzed by trypsin-Giemsa banding when they were harvested for the preparation of high molecular weight DNA. Over many years, these hybrid cell lines were analysed for more than 100 genetic markers, thus their chromosomal content has been amply verified by biochemical and molecular genetic data. BDNF and NTF3 were regionally assigned to human chromosomes by analyzing six hamster or mouse X human hybrid cell lines carrying various portions of human chromosomes 11 and 12 in the absence of an intact copy of these chromosomes (Barton et al., 1988; Archer et al., 1990).
Hybridization
Probes
EcoRI-Hind111 fragments of human origin that contain the entire coding sequences of BDNF (1300 bp) (Rosenthal et al., 1991) and NT-3 (975 bp) (Rosenthal et al., 1990) were labeled by random priming and used as probes on Southern filters.
Southern
Hybridizations
Genomic DNA was extracted from human placental tissue, hybrid cell lines, and parental control cells by standard methods. Restriction enzyme digestion, electrophoresis, Southern transfer to Hybond nylon membranes (Amersham Corp.), and hybridizations to 32P-labeled probes were carried out as described (Barton et al., 1986). Filters were rinsed twice in 2~ SSC and washed twice in 1X SSC, 1% SDS at 55-65°C for lo-15 min. The filters were autoradiographed for 16 to 48 h at -70°C with Kodak X-Omat AR film and two intensifying screens.
RESULTS Mapping
of Human
BDNF
The location of the human BDNF gene was determined with a panel of 16 well-characterized rodent X human hybrid cell lines carrying different subsets of human chromosomes. Hybridization of human probe to BamHI-digested genomic DNA revealed strongly hybridizing single fragments in each species:
FRANCKE 7-kb human, 5-kb Chinese hamster, and 9-kb mouse (Fig. 1A). The 7-kb human fragment was concordant with human chromosome 11. All other chromosomes were excluded by at least four discordant hybrids (Table 1). Regional localization of BDNF was carried out with four hybrids from series VII, XV, and XXI fusions that contain defined regions of chromosome 11 in the absence of an intact 11. Southern analysis of these hybrid cell lines assigned the BDNF locus to the short arm region llpll.Z-~15.5 (Fig. 2).
Mapping
of Mouse Bdnf
The murine brain derived neurotrophic factor gene (symbol Bdnf ) was mapped by using the same 1300bp human probe and a panel of Chinese hamster or rat X mouse somatic cell hybrids that carry different subsets of mouse chromosomes. Single 4.3-kb Chinese hamster (Fig. lB, lane l), 11-kb mouse (lane Z), and 7.6-kb rat (lane 3) fragments were detected in control genomic DNA samples digested with EcoRI. The llkb mouse fragment was present in hybrids that had retained mouse chromosome 2 (Fig. lB, lanes 5 and 6) and absent in hybrids that are lacking this chromosome (lanes 4 and 7). One hybrid had mouse chromosome 2 as the only mouse chromosome and was positive for the mouse Bdnf fragment. In addition, all other mouse chromosomes were excluded as possible sites for Bdnf by at least 20% discordant hybrids (Table 2). These results place Bdnf on mouse chromosome 2.
Mapping
of Human
NTF3
The human neurotrophin-3 gene (symbol NTF3) was mapped with a BglII-digested mapping panel of 13 Chinese hamster X human somatic cell hybrids having reduced numbers of human chromosomes. The 900-bp human probe recognized a 7.6-kb human and a 4-kb Chinese hamster fragment (Fig. 3A). The 7.6-kb BgZII human fragment was concordant with human chromosome 12. All other chromosomes were excluded by at least two discordant hybrids (Table 1). Two hybrid lines from fusions XII and XXI that carry defined regions of human chromosome 12 in the absence of an intact copy of this chromosome were used for regional localization of NTF3. Hybrid cell line XII-2A retaining the region lZpter-q21 was positive (Fig. 3A, lane 5) and hybrid cell line XXI-63A containing the region 12cen-qter was negative (Fig. 3A, lane 4) for human neurotrophin-3 sequences. These results place the NTF3 locus on the short arm of chromosome 12 (Fig. 4).
MAPPING
1
2
3
4
OF
5
6
BDNF
7
AND
8
NTF3
IN
MAN
AND
9 10 11 12
571
MOUSE
1234567 -
kb 11
- 1.6
Human
Mouse
BDNF FIG. 1. Chromosomal mapping of the human (A) and mouse (B) BDNF gene. 32P-labeled BDNF probe was hybridized to Southern blots of BarnHI-digested DNA from human X rodent and EcoRI-digested DNA from mouse X Chinese hamster or rat hybrid cell lines and controls. A: lane 1, human placental tissue; lane 2, Chinese hamster cell line V79/380-6; lane 3, mouse 3T3 cell line; lanes 4-9 and 12, human X Chinese hamster cell hybrids of which lanes 4,6,7,8, and 12 contain the human specific 7-kb fragment; lane 10, human X mouse hybrid with region A (Fig. 2) positive for the human specific fragment; lane 11, mouse X human hybrid negative for the human signal B: lane 1, V79/380-6: lane 2,3T3; lane 3, rat hepatoma cell line 7777-14b aza; lanes 4,5, and 6, Chinese hamster X mouse cell hybrids of which lanes 5 and 6 are positive for the 11-kb mouse fragment; lane ‘7, rat X mouse cell hybrid negative for the mouse fragment.
TaqI, and Hind111 in five inbred mouse strains, DBA/ 25, AKR/J, C3H/HeJ, C57BL/6J, and C57L/J.
Mapping of Mouse Ntf-3 In rodent genomic DNA samples digested with PstI the human NT-3 probe hybridized strongly with single fragments of 1.9 kb for Chinese hamster (Fig. 3B, lane l), 3 kb for mouse (lane 2), and 2.7 kb for rat (lane 3). The 3-kb mouse fragment was present in all hybrid cell lines that had retained mouse chromosome 6. All other mouse chromosomes were excluded aspossible sites for the gene by at least two discordant hybrids (Table 2). Thus, the mouse neurotrophin-3 gene (gene symbol Ntf-3) is assigned to chromosome 6.
DISCUSSION
The human brain derived neurotrophic factor gene (BDNF) has been mapped to the short arm of human chromosome 11 (HSA ll), bands p11.2-~15.5, by Southern blot analysis of DNA from rodent X human hybrid lines carrying different parts of HSA 11. With a panel of Chinese hamster or rat X mouse hybrid lines the murine B&f gene has been mapped to mouse chromosome 2 (MMU 2). Four other loci, CAT (catalase), FSHB (follicle-stimulating hormone, /3polypeptide), AN2 (aniridia, small eye), and ACP2 (acid phosphatase-2, lysosomal) have also been assigned to HSA 11 and MMU 2 (for review, see Searle et al., 1989). The conserved region spans the proximal short arm of HSA 11, bands pll-~13, and region D-E4 in
Polymorphism Search with BDNF and NTF3 Probes No restriction fragment length polymorphism was found after digestion with P&I, TuqI, BamHI, HinfI, NsiI, HincII, StyI, Eco0109, AZuI, P&I, and MspI in DNA from 8 to 10 unrelated Caucasians. Likewise, no strain differences were detected with PstI, BamHI,
TABLE Presence
1
of Human BDNF and NTF3 Sequences and Human Cell Hybrids in Rodent X Human Somatic Human
BDNF discordant hybrids Informative hybrids” Percentage discordant NTF3 discordant hybrids Informative hybrids” Percentage discordant a Chromosomes
with
Chromotimes
chromosome
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
X
11 17 65 4 16 25
11 18 61 5 17 29
9 17 53 7 15 47
9 14 64 5 15 33
8 18 44 8 18 44
9 17 53 8 16 50
11 17 65 8 16 50
8 18 44 6 17 35
14 18 78 7 16 44
12 18 67 8 18 44
0 16 0 4 16 25
7 18 39 0 18 0
12 17 ‘71 11 18 61
11 16 69 10 17 59
8 18 44 7 18 39
9 17 53 5 16 31
10 18 56 10 18 56
10 18 56 9 18 50
9 18 50 6 17 35
4 18 22 5 18 28
7 17 41 6 17 35
8 16 50 7 17 41
4 6 67 3 7 43
rearrangements
or present
at a frequency
of 0.1 or less were excluded.
572
GZCELIK,
ROSENTHAL,
logical mutations in this region that could involve Bndf, the best candidate is am (anorexia) a recessive lethal mutation of abnormal behavior and neurological dysfunction that leads to starvation due to insufficient food intake (Maltais et al., 1984). On the composite map of mouse chromosome 2, anx is close to Sey (Searle et aE., 1989). The NTF3 gene has been localized to HSA 12p and MMU 6. By using the same panels of somatic cell hybrids, we have recently mapped a synaptic vesicle protein gene, synaptobrevin 1 (SYBI) to the same chromosome regions (Archer et aZ., 1990). In fact, as summarized in Searle et al. (1989), all genes known to be on HSA 12p, including CD4 (L3T4 T cell differentiation antigen), GAPD (glyceraldehyde-3-phosphate dehydrogenase), PRH (proline-rich salivary protein), TPIl (triose phosphate isomerase l), LDHB (lactate dehydrogenase B), PTHLH (parathyroid hormonelike hormone), and KRAS2 (Kirsten rus oncogene 2), have their homologues on the distal third of MMU 6. The orientation of this conserved syntenic region seemsto be inverted with respect to the centromere. More proximal parts of MMU 6 contain loci homologous to genes on human chromosomes 2, 3, and 7. Thus, interspecies comparative mapping allows one to predict the regional localization of genes in conserved chromosome region. However, independent proof by higher resolution mapping methods is still necessary, as disruption of conserved syntenic regions are frequent in mouse and are also seen on human chromosomes (Barton et al., 1988). Our data mapping BDNF and NTF3 to human chromosomes llp and 12p, respectively, add to the list of structurally related loci on these two human chromosomes. It has been proposed that a tetraploidization event during evolution doubled the chromosome number of mammals (Ohno, 1967; Ohno et aZ., 1968). HSA 11 and 12 are good candidates for a pair of
1
BDNF
J
FIG. 2. Ideogram of G-banding patterns of human chromosome 11 with regional mapping data using somatic cell hybrids VII-3 HAT (A), XV-N4 (B), XXI-23A-2c (C), and XXI-54B-1J (D). The vertical bars illustrate different portions of chromosome 11 present in these hybrids. + is hybrid positive for human BDNF sequence; - is hybrid negative. The bracket indicates the location of the BDNF gene.
the middle of MMU 2. All genes from 11~14 to the telomere are homologous to loci on MMU 7. Therefore, it is likely that BDNF is located within the conserved region llp11.2-p13 (Fig. 5). This region also includes the genes for Wilms tumor and aniridia. The latter is thought to be homologous to the Sey locus on MMU 2 (Searle et aZ., 1989). In terms of murine neuro-
TABLE Presence
AND FRANCKE
2
of Mouse Bdnf and Ntf-3 Sequences and Mouse Chromosomes Somatic Cell Hybrids in Mouse X Chinese Hamster Mouse chromosome
discordant hybrids Informative hybrids” Percentage discordant Ntf-3 discordant hybrids Informative hybrids” Percentage discordant Bdnf
1
2
3
4
5
6
‘7
8
9
10
11
12
13
14
15
16
17
18
19
X
4 10 40 3 11 27
0 11 0 6 13 46
4 11 36 4 13 31
3 10 30 5 12 42
4 9 44 4 11 36
5 10 50 0 12 0
3 10 30 5 12 42
3 11 27 4 13 31
8 11 73 3 13 23
7 11 64 5 12 42
9 11 82 7 13 54
7 11 64 5 13 38
7 10 70 5 12 42
4 11 36 5 12 42
5 11 45 3 13 23
6 10 60 4 11 36
3 11 27 3 13 23
4 11 36 5 12 42
2 10 20 3 12 25
5 10 50 3 12 25
o Chromosomes with rearrangements
or present at a frequency of 0.1 or less were excluded.
MAPPING
OF
BDNF
AND
IN
MAN
AND
573
MOUSE
B
A
1 2
123456
kb
NTF3
3
4
5
6 7
8
9
10
kb -3 - 2.7
7.6-
-
Human
1.9
Mouse
NTF 3 FIG. 3. Chromosomal mapping of the human (A) and mouse (B) NTF-3. 32P-Labsled NTF3 probe was hybridized to Southern blots of BglII-digested DNA from human X rodent and PstI-digested DNA from mouse X Chinese hamster or rat hybrid cell lines and controls. A: lane 1, human placental tissue; lane 2, Chinese hamster cell line V79/380-6; lanes 3-6, human X Chinese hamster hybrid cell lines. Lanes 3 and 6 are positive for the human 7.6-kb fragment, and lanes 4 and 6 are negative. B: lane 1, V79/380-6; lane 2,3T3; lane 3, rat hepatoma cell line 7777-14b aza; lanes 4-9 Chinese hamster X mouse cell hybrids of which lanes 4, 7, and 8 are positive for the 3-kb mouse specific fragment; lane 10, mouse X rat hybrid negative for the mouse fragment.
tetraploidization derivatives because of their similar banding patterns and content of structurally related loci. As seen in Fig. 5, the genes for HRASl (Harvey rat sarcoma oncogene), PTH (parathyroid hormone), and LDHA (lactate dehydrogenase A) are on the short arm of HSA 11, and related genes for KRAS2
TF3
FIG. 4. Ideogram of G banding patterns of human chromosome 12 with regional mapping data using Chinese hamster x human somatic cell hybrids XII-2A (A) and XXI-63A (B). The vertical bars indicate portions of chromosome 12 present in these cell hybrids. + is hybrid positive for human NTF3 sequences; - is hybrid negative. The bracket indicates the location of the NTF3 gene.
(Kirsten rat sarcoma oncogene-2), PTHLH (parathyroid hormone like hormone), and LDHB (lactate dehydrogenase B) are on the short arm of HSA 12 (Searle et al., 1989). On the other hand, although the genes for IGF2 (insulin like growth factor 2), INS (insulin), and TH (thyrosine hydroxylase) are also on llp, related genes for IGFl (insulin like growth factor 1) and PAH (phenylalanine hydroxylase) are on 12q. In order to explain this difference, it has been postulated that intrachromosomal rearrangements have altered the organization of genes on HSA 11 and 12 (Brissenden et al, 1984; Barton et al., 1988). Relationships with regions on other chromosomes are suggested by the observation that NGFB, a member of the nerve growth factor/insulin/relaxin family of small peptides, is on human chromosome 1~22.1 (neuroblastoma oncogene), a along with NRAS member of the rm family of oncogenes (Mtinke et al., 1984) and NRASL, an NRAS-like gene is on 9p toand RLN2 gether with the two relaxin loci RLNl (Smith and Simpson, 1989). If the structurally related genes mapped to human chromosomes 11 and 12 support the hypothesis of evolutionary kinship between these two chromosomes, the map positions of the same genes in the mouse genome should serve to identify the mouse chromosomes that are evolutionarily related to each other. However, this is not straightforward. First, a comparison of the banding patterns of the mouse chromosomes involved does not lead to an obvious alignment of chromosome pairs. Second, as illustrated in Fig. 5, genes on HSA 11 and 12 have homologs on eight different mouse chromosomes. This observation suggests that mouse chromosomes have undergone more rearrangements since separation from a
574 IGF2
3”HARSl MMU7 12.3 12.2 12.1
P
MMU
6
MMU
15
MMU
5
19
7
MMU
9
FIG. 5. Comparative maps of human chromosomes 11 and 12. The locations of structurally explanation of gene symbols, see text. Vertical bars indicate proposed regions of homology with on gene mapping data summarized in Searle et al. (Ref. (26) and Kuo et al. (Ref. (13)).
common ancestor which is consistent of mice.
than have human chromosomes, with the shorter generation time
7.
ACKNOWLEDGMENTS This work was supported by Research Grant and by Genentech Inc. U.F. is an investigator ciate of the Howard Hughes Medical Institute.
6.
HG00298 and T.O.
(to U.F.) an asso-
2.
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“Sex New
Chromosomes York.
and Sex-linked
Genes,”
