Proc. Nati. Acad. Sci. USA Vol. 88, pp. 3637-3641, May 1991
Neurobiology
Human monoamine oxidase A and B genes exhibit identical exon-intron organization (human MAOA aud MAOB genes/genomic organization)
JOSEPH GRIMSBY, KEVIN CHEN, Li-JIA WANG, NANCY C. LAN,
AND
JEAN C. SHIH*
Department of Molecular Pharmacology and Toxicology, School of Pharmacy, University of Southern California, 1985 Zonal Avenue, Los Angeles, CA 90033
Communicated by Norman Davidson, January 8, 1991 (received for review November 21, 1990)
ABSTRACT Monoamine oxidases A and B [MAOA and MAOB; amine:oxygen oxidoreductase (deaminating) (flavincontaning), EC 1.4.3.4] play important roles in the metabolism of neuroactive, vasoactive amines and the Parkinsonismproducing neurotoxin 1-methyl4-4phenyl-1,2,3,6-tetrahydropyridine (MPTP). Human MAOA and MAOB genes isolated from X chromosome-specific libraries span at least 60 kilobases, consist of 15 exons, and exhibit identical exon-intron organization. Exon 12 codes for the covalent FAD-binding-site and is the most conserved exon; the MAOA and MAOB exon 12 products share 93.9% peptide identity. These results suggest that MAOA and MAOB are derived from duplication of a common ancestral gene and provide insight on the structural/functional relationship of the enzyme products.
a MAOA mRNA species of 5 kilobases (kb) in most tissues except placenta and small intestine, which express an additional 2-kb mRNA species (11, 15). These two mRNA species arise in part from the use of alternative polyadenylylation sites (15). A 3-kb MAOB mRNA species is found in most tissues (15). These two genes are closely linked and located on the X chromosome, Xpll.23-Xp22.1. Both of them are deleted in some patients with Norrie disease (18), which is characterized by blindness, deafness, and mental retardation. However, the etiology of this disorder is still unknown. Abnormal levels of MAO activity have been reported in a number of mental disorders, such as depression (19), alcoholism (20), schizophrenia (21), and Alzheimer disease (22). Further, MAO inhibitors have been used as antidepressants (23). The structure of MAOA and MAOB genes will help us to understand the tissue-specific expression, the regulation, and the structural-functional relationship of their enzyme product and the possible role of these genes in mental disorders. In this report, we describe the partial structure of the MAOA and MAOB genes. These data show that both are comprised of 15 exons and 14 introns spanning at least 60 kb. The exon-intron organization is identical in both genes, which suggests that they are derived by duplication of a common ancestral gene.
Monoamine oxidases A and B [MAOA and MAOB; amine:oxygen oxidoreductase (deaminating) (flavin-containing), EC 1.4.3.4] catalyze the oxidative deamination of a number of neurotransmitters and dietary amines. Both forms are located in the outer mitochondrial membrane where they are used as marker enzymes (1). The two enzymes possess different substrate and inhibitor specificities (2-4), tissue and cell distribution (5), immunological properties (6), photodependent inactivation (7, 8), and amino acid sequences (9-11). Further, they are regulated differently by steroids (12). MAOA preferentially oxidizes the biogenic amines such as serotonin, norepinephrine, and epinephrine and is inactivated irreversibly by the acetylenic inhibitor clorgyline. MAOB preferentially oxidizes phenylethylamine and benzylamine and is inactivated by the irreversible inhibitors pargyline and deprenyl. Dopamine, tyramine, and tryptamine are the common substrates for both forms. Furthermore, the xenobiotic 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) can be oxidized by MAOB to form a neurotoxic species that can cause permanent Parkinsonian symptoms (13). Recently isolated cDNA clones that encode the human liver MAOA and MAOB showed that they share 70%o amino acid identity and may be derived from separate genes (9). Transient transfection of human liver MAOA or MAOB cDNAs in COS cells resulted in expression in these virally transformed African Green monkey kidney cells of functional enzymes that observe substrate and inhibitor preferences identical to those of the respective human enzyme (14). The tissue distribution of MAOA and MAOB mRNAs determined by RNA blot-hybridization (Northern) analysis (9, 15) is consistent with enzymatic studies (16). MAOA and MAOB are coexpressed in almost all human tissues except placenta, which predominantly expresses MAOA (11, 15, 16), and platelets and lymphocytes, which predominantly express MAOB. MAOA is expressed before MAOB in fetal brain (15, 17). Northern blot analysis reveals predominantly
MATERIALS AND METHODS Isolation of MAOA and MAOB Genes. Genes encoding human MAOA and MAOB were isolated by screening four A bacteriophage libraries with human liver MAOA and MAOB cDNA clones (9). These libraries were screened independently with MAOA or MAOB cDNA subfragments 32Plabeled by the random priming method (24, 25). The subfragments used included the 5' end (A8:nucleotides 1-552), the middle region (A9:nucleotides 553-1200), and the 3' end (A1O:nucleotides 1201-1958) of the MAOA cDNA and the 5' end (B1:nucleotides 1-602), the middle region (B2:nucleotides 603-1434), and the 3' end (B3:nucleotides 14352498) of the MAOB cDNA (18). Hybridization was carried out in 50% (vol/vol) formamide in 5x SSC (1x SSC = 0.15 M NaCl/0.015 M sodium citrate) at 370C overnight and washed at high stringency [0.1x SSC/0.1% sodium dodecyl sulfate (SDS) at 600C]. Twenty-one MAOA' positive clones were isolated from a partially digested X chromosomespecific library cloned in phage A EMBL3 (a gift from L. Hood) and A Charon 35 (American Type Culture Collection). Forty-three MAOB' clones were isolated from the A Charon 35 library and from a completely HindIll-digested (a gift from S. A. Latt) or EcoRI-digested (American Type Culture Collection) X chromosome-specific library cloned in A Charon 21. Southern blot analysis of phage DNA isolated from 30 A bacteriophage clones identified exon-containing restriction
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Abbreviations: MAO, monoamine oxidase; MAOA and MAOB, MAOs A and B. *To whom reprint requests should be addressed. 3637
Neurobiology: Grimsby et al.
3638
Proc. Natl. Acad. Sci. USA 88 (1991)
MAO A
r-p35-A8a--l
p35-A8b
EH 1.,
E
I
E E I .1 2
i
p35-A8c-1 np35-A8dl1
r
HE
IL
H
5
1.1.1
HH Il.. 7 89 >
HH
E
EHEH H
H
.1
pEMBL-Al1O---
p35-A9
L..1
4 5
->->
+
6 4-
>
*
Neurobiology
Human monoamine oxidase A and B genes exhibit identical exon-intron organization (human MAOA aud MAOB genes/genomic organization)
JOSEPH GRIMSBY, KEVIN CHEN, Li-JIA WANG, NANCY C. LAN,
AND
JEAN C. SHIH*
Department of Molecular Pharmacology and Toxicology, School of Pharmacy, University of Southern California, 1985 Zonal Avenue, Los Angeles, CA 90033
Communicated by Norman Davidson, January 8, 1991 (received for review November 21, 1990)
ABSTRACT Monoamine oxidases A and B [MAOA and MAOB; amine:oxygen oxidoreductase (deaminating) (flavincontaning), EC 1.4.3.4] play important roles in the metabolism of neuroactive, vasoactive amines and the Parkinsonismproducing neurotoxin 1-methyl4-4phenyl-1,2,3,6-tetrahydropyridine (MPTP). Human MAOA and MAOB genes isolated from X chromosome-specific libraries span at least 60 kilobases, consist of 15 exons, and exhibit identical exon-intron organization. Exon 12 codes for the covalent FAD-binding-site and is the most conserved exon; the MAOA and MAOB exon 12 products share 93.9% peptide identity. These results suggest that MAOA and MAOB are derived from duplication of a common ancestral gene and provide insight on the structural/functional relationship of the enzyme products.
a MAOA mRNA species of 5 kilobases (kb) in most tissues except placenta and small intestine, which express an additional 2-kb mRNA species (11, 15). These two mRNA species arise in part from the use of alternative polyadenylylation sites (15). A 3-kb MAOB mRNA species is found in most tissues (15). These two genes are closely linked and located on the X chromosome, Xpll.23-Xp22.1. Both of them are deleted in some patients with Norrie disease (18), which is characterized by blindness, deafness, and mental retardation. However, the etiology of this disorder is still unknown. Abnormal levels of MAO activity have been reported in a number of mental disorders, such as depression (19), alcoholism (20), schizophrenia (21), and Alzheimer disease (22). Further, MAO inhibitors have been used as antidepressants (23). The structure of MAOA and MAOB genes will help us to understand the tissue-specific expression, the regulation, and the structural-functional relationship of their enzyme product and the possible role of these genes in mental disorders. In this report, we describe the partial structure of the MAOA and MAOB genes. These data show that both are comprised of 15 exons and 14 introns spanning at least 60 kb. The exon-intron organization is identical in both genes, which suggests that they are derived by duplication of a common ancestral gene.
Monoamine oxidases A and B [MAOA and MAOB; amine:oxygen oxidoreductase (deaminating) (flavin-containing), EC 1.4.3.4] catalyze the oxidative deamination of a number of neurotransmitters and dietary amines. Both forms are located in the outer mitochondrial membrane where they are used as marker enzymes (1). The two enzymes possess different substrate and inhibitor specificities (2-4), tissue and cell distribution (5), immunological properties (6), photodependent inactivation (7, 8), and amino acid sequences (9-11). Further, they are regulated differently by steroids (12). MAOA preferentially oxidizes the biogenic amines such as serotonin, norepinephrine, and epinephrine and is inactivated irreversibly by the acetylenic inhibitor clorgyline. MAOB preferentially oxidizes phenylethylamine and benzylamine and is inactivated by the irreversible inhibitors pargyline and deprenyl. Dopamine, tyramine, and tryptamine are the common substrates for both forms. Furthermore, the xenobiotic 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) can be oxidized by MAOB to form a neurotoxic species that can cause permanent Parkinsonian symptoms (13). Recently isolated cDNA clones that encode the human liver MAOA and MAOB showed that they share 70%o amino acid identity and may be derived from separate genes (9). Transient transfection of human liver MAOA or MAOB cDNAs in COS cells resulted in expression in these virally transformed African Green monkey kidney cells of functional enzymes that observe substrate and inhibitor preferences identical to those of the respective human enzyme (14). The tissue distribution of MAOA and MAOB mRNAs determined by RNA blot-hybridization (Northern) analysis (9, 15) is consistent with enzymatic studies (16). MAOA and MAOB are coexpressed in almost all human tissues except placenta, which predominantly expresses MAOA (11, 15, 16), and platelets and lymphocytes, which predominantly express MAOB. MAOA is expressed before MAOB in fetal brain (15, 17). Northern blot analysis reveals predominantly
MATERIALS AND METHODS Isolation of MAOA and MAOB Genes. Genes encoding human MAOA and MAOB were isolated by screening four A bacteriophage libraries with human liver MAOA and MAOB cDNA clones (9). These libraries were screened independently with MAOA or MAOB cDNA subfragments 32Plabeled by the random priming method (24, 25). The subfragments used included the 5' end (A8:nucleotides 1-552), the middle region (A9:nucleotides 553-1200), and the 3' end (A1O:nucleotides 1201-1958) of the MAOA cDNA and the 5' end (B1:nucleotides 1-602), the middle region (B2:nucleotides 603-1434), and the 3' end (B3:nucleotides 14352498) of the MAOB cDNA (18). Hybridization was carried out in 50% (vol/vol) formamide in 5x SSC (1x SSC = 0.15 M NaCl/0.015 M sodium citrate) at 370C overnight and washed at high stringency [0.1x SSC/0.1% sodium dodecyl sulfate (SDS) at 600C]. Twenty-one MAOA' positive clones were isolated from a partially digested X chromosomespecific library cloned in phage A EMBL3 (a gift from L. Hood) and A Charon 35 (American Type Culture Collection). Forty-three MAOB' clones were isolated from the A Charon 35 library and from a completely HindIll-digested (a gift from S. A. Latt) or EcoRI-digested (American Type Culture Collection) X chromosome-specific library cloned in A Charon 21. Southern blot analysis of phage DNA isolated from 30 A bacteriophage clones identified exon-containing restriction
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Abbreviations: MAO, monoamine oxidase; MAOA and MAOB, MAOs A and B. *To whom reprint requests should be addressed. 3637
Neurobiology: Grimsby et al.
3638
Proc. Natl. Acad. Sci. USA 88 (1991)
MAO A
r-p35-A8a--l
p35-A8b
EH 1.,
E
I
E E I .1 2
i
p35-A8c-1 np35-A8dl1
r
HE
IL
H
5
1.1.1
HH Il.. 7 89 >
HH
E
EHEH H
H
.1
pEMBL-Al1O---
p35-A9
L..1
4 5
->->
+
6 4-
>
*
