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Ahch, the mouse homologue of DAX1: cloning, characterization and synteny with GyK, the glycerol kinase locus
GENE AN I N T E R N A T I O N A L * J O U R N A L ON GENES AND GENOMES
ELSEVIER
Gene 178 (1996) 31-34
Ahch, the mouse homologue of DAXI" cloning, characterization and synteny with GyK, the glycerol kinase locus Weiwen Guo a, Rhonda S. Lovell b Yao-Hua Zhang a, Bing-Ling Huang William J. Craigen b, Edward R.B. McCabe a.,
a,
Thomas P. Burris ~,
a Department of Pediatrics, UCLA School of Medicine, 10833 Le Conte Avenue, Los Angeles, CA 90024, USA b Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX 77030, USA
Received 5 December 1995; revised 2 February 1996; accepted 6 February 1996
Abstract
We cloned the murine full-length cDNA encoding Ahch, the mouse homologue of DAX1 (DSS-AHC Region on Human X Chromosome, Genel) which is the gene responsible for human X-linked adrenal hypoplasia congenita (AHC) and hypogonadotropic hypogonadism (HH). Sequence analysis revealed that the murine and human cDNAs have 65% aa identity and 75% aa similarity overall. The cysteine residues in the putative DNA binding domain, which may interact with Zn 2÷ ions to form zinc fingers, are 100% conserved between the two species, indicating that the novel zinc-finger structures in DAX1 may be functional. In addition, mouse interspecific backcrosses show that the Ahch gene is closely linked to the glycerol kinase locus, GyK, on the mouse X chromosome, indicating that the order of the loci is conserved in this syntenic region between mouse and human. Keywords: Adrenal hypoplasia congenita; Hypogonadotropic hypogonadism; Duchenne muscular dystrophy; Steroidogenic factor
1 response element; Dosage sensitive sex reversal locus; Zinc-finger domain
I. Introduction
D A X I is the gene responsible for X-linked adrenal hypoplasia congenita (AHC) and hypogonadotropic hypogonadism (HH) in the human (Muscatelli et al., 1994; Zanaria et al., 1994; Guo et al., 1995a). The DAX1 gene maps to the human X chromosome in Xp21, telomeric to the glycerol kinase (GK) gene, and is frequently deleted in patients with a contiguous gene syndrome that involves the loss of the AHC locus along with the GK deficiency or G K D and Duchenne muscular dystrophy (DMD) loci (Walker et al., 1992; Worley et al., 1993).
*Corresponding author. Tel.: (+1-310) 825-5095; Fax: (+1-310) 206-4584; e-mail: [email protected] Abbreviations: aa, amino acid; AHC, adrenal hypoplasia congenita; Ahch, mouse locus for adrenal hypoplasia congenita in the human; Amel, amelogenin; bp, base pair(s); cDNA; DNA complementary to RNA; DAX1, DSS-AHC Region on Human X Chromosome, Genel; DMD, Duchenne muscular dystrophy; DSS; dosage sensitive sex reversal locus; Flnl, filamin 1, actin binding protein 280; GKD, glycerol kinase deficiency; GyK, mouse glycerol kinase locus; hDAXI, human DAXI; kb, kilobase(s) or 1000 bp; SF-1, steroidogenic factor-1. 0378-1119/96/$15.00 © 1996 Elsevier Science B.V. All rights reserved PII S0378-1119(96)00320-4
The human DAX1 gene was identified by positional cloning methods, and the cDNA sequence from both testis and fetal adrenal gland revealed it tO be a member of the nuclear hormone receptor superfamily based on the presence of a conserved C-terminal ligand binding domain (Zanaria et al., 1994; Guo et al., 1995a). The amino terminal portion of DAX1, which is composed of 3.5 repeats of a 65-67-aa motif, contains two putative zinc fingers that may define a novel DNA binding domain (Guo et al., 1995a). The genomic sequence in the promoter region of DAX1 revealed a putative steroidogenic factor-1 (SF-1) response element (Guo et al., 1996). Recently, we showed that SF-1 binds in vitro to the putative SF-1 response element found in the D A X I promoter, indicating that SF-1 may directly regulate the expression of DAX-1 (Burris et al., 1995). Northern blot and RT-PCR studies show that DAX1 is expressed in adrenal gland, testis, ovary, hypothalamus and pituitary gland, suggesting the involvement of DAX-1 in the development and regulation of the hypothalamic-pituitary-gonadal axis (Guo et al., 1995b). To further study the function of DAX1, it will be necessary to utilize an animal model that is well-characterized genetically and developmentally, such as the
32
W. Guo et al./Gene 178 (1996) 31-34
mouse. In addition, cloning the homologues of DAX1 from different species will help us to study the evolution of this gene and to define its functional domains, such as the potential zinc-finger structure in the putative D N A binding domain. In this report, we describe the cloning of the murine homologue of DAX1, Ahch, from a mouse testis c D N A library.
2. Experimental and discussion 2.1. Characterization of Ahch cDNA Four positive c D N A clones were identified from a mouse testis c D N A library that was cloned into the Uni-ZAPXR polylinker with EcoRI and XhoI adapters on the cDNAs, and all four were sequenced. One of the clones, MC4, contained 1806 bp of murine c D N A sequence in addition to 12 bp of adapter sequence and included the entire coding region (Fig. 1) (GenBank accession No. U41568). The complete coding sequence of the mouse Ahch c D N A was 1419 bp in length from the start codon (ATG) through the stop codon (TGA). The MC4 clone contained 1 9 b p of 5'-untranslated region and 368 bp of 3'-untranslated region, including an AATAAA polyadenylation signal that was followed by polyadenosine residues.
2.2. Sequence analysis The Ahch c D N A predicts a 472-aa protein which is two aa longer than h u m a n DAX1. The mouse Ahch deduced protein sequence shows highly significant similarity to hDAX1, with 65% identity and 75% similarity overall, and identity that is even higher in the ligand binding domain: 93% in region II and 96% in region III (Fig. 2). The cysteine residues (aa positions 38, 41, 43, 66, 67, 69, 105, 108, 110, 134, 135 and 137) in the putative D N A binding domain, which we have predicted to interact with Zn 2+ ions to form zinc fingers (Guo et al., 1995a), are 100% conserved between the mouse and h u m a n proteins, but aa around the cysteine residues are less conserved (Fig. 2). The deduced aa sequence for mouse Ahch was used as a query to search the nonredundant protein database of the National Center for Biological Information (NCBI) using the BLAST network server (Altschul et al., 1990). Ahch showed a highly significant similarity between its carboxy terminus and the ligand binding domain of members of the nuclear hormone receptor gene superfamily, as had been observed previously for hDAX1 (Zanaria et al., 1994; Guo et al., 1995a). The N-terminal portion of the Ahch gene was rich in small aa (glycine and alanine) and consisted of three and a half repeats of a 65-67-aa motif, like hDAX1 (Zanaria et al., 1994; G u o et al., 1996).
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Fig. l. Complete nucleotide and predicted aa sequence of mouse Ahch cDNA clone MC4. The cloning adapter sequences for the EcoRI and Xhol sites are underlined. The potential polyadenylation signal identified in the 3' noncoding region is underlined. The deduced aa sequence of the Ahch protein is given in one-letter symbols. Two putative zinc fingers are bolded, italicized and underlined. Methods: Cloning and sequencing. The 3.5-kb EcoRI restriction fragment containing the first exon of the human DAX1 gene (Guo et al., 1995a) was used to screen a mouse testis cDNA library in the Uni-ZAPXR vector (Cat. No. 937308, Stratagene, La Jolla, CA, USA). The positive clones that were identified were sequenced in both directions by a combination of manual dideoxy sequencing (Sequenase, US Biochemical, Cleveland, OH, USA) and automated cyclesequencing (ABI-377 DNA Sequencer, Applied Biosystems, Foster City, CA, USA). The sequencing began with the universal and reverse primers, followed by walking primers. The Gel programs of the GCG (Version 8.0; Madison, WI, USA) were used for sequence assembly.Evaluation of the nucleotide and deduced aa sequences relied on the GCG programs (Version 8.0). Nucleotide sequence analysisof the cloned cDNA utilized the Blast Serverat NCBI (Altschul et al., 1990).
2.3. Mapping Ahch to the X chromosome and linkage with GyK Interspecific backcrosses of C57BL/6J and Mus spretus showed that there was no recombination between the GyK locus and the Ahch locus, indicating tight linkage between GyK and Aheh (Fig. 3). DXMit7, a
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Fig. 2. Amino-acid sequence comparison between Ahch and hDAX1. The Gap program of the GCG (Version 8.0) was used for sequence comparison. The top portion represents the mouse Ahch protein and the bottom portion represents the hDAX1 protein. Vertical lines indicate that the aa are identical. Double dots indicate aa with similar chemical characteristics. Singledots indicate aa with less similar chemical characteristics and spaces indicate aa with no chemical similarities at all. The cysteine residues that may interact with Znz+ to form zinc fingers are bolded and italicized. The conserved regions in the putative ligand binding domain are italicized and underlined.
microsatellite D N A for a p o l y m o r p h i s m m a r k e r on the m o u s e X c h r o m o s o m e was also linked to the G y K locus (Fig. 3). Since D X M i t 7 was s h o w n to be a b o u t 3 cM from the m u r i n e d y s t r o p h i n locus (Dmd) ( H e r m a n et al., 1994) then the G y K also was linked to the Dmd locus (Blair et al., 1994, 1995; H e r m a n et al., 1994). These results indicated that the Ahch, G y K a n d Dmd loci were in the same region of the m o u s e X c h r o m o s o m e a n d in the same order as in the h u m a n , and, therefore, this region was syntenic with the h o m o l o g o u s region of the h u m a n X chromosome.
3. Conclusions The putative Ahch p r o t e i n derived from m o u s e c D N A clones is two aa longer t h a n the h D A X 1 protein, with 75% similarity a n d 65% identity to the h u m a n D A X I p r o t e i n sequence overall, i n d i c a t i n g that the gene responsible for A H C has been highly conserved d u r i n g the 100 million years of e v o l u t i o n a r y time that separate the two species (Searle et al., 1989). The more striking feature of
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Fig. 3. Haplotype analysis of the BSS interspecificbackcross panel for Ahch. Each column represents a chromosomal haplotype, with the number of offspring possessing the haplotype indicated at the bottom of the column. Relevant typed loci are listed on the left side of the fgure, while the black boxes indicate the C57BL/6J allele and the white boxes the M. spretus allele. On the right is the recombination frequency between the listed loci. Methods: Genetic mapping. Ahch was mapped by an interspecific backcross panel. To identify species-specific DNA polymorphisms, a genomic Southern blot was prepared using matched DNA samples of C57BL/6J and Mus spretus (obtained from the Jackson Laboratory, Bar Harbor, ME, USA) that were digested with nine different restriction enzymes.The entire Ahch cDNA was used as a radiolabeled probe. A number of polymorphic fragments were identified with various restriction enzymes (data not shown). PstI was chosen since it gave 4.8-kb C57BL/6J-specificand 5.7-kb Mus spretusspecific bands that were easily resolved by Southern blotting. Three micrograms per lane of DNA from the 93 interspecific backcross progeny from the Jackson Laboratory BSS backcross panel [(C57BL/6J x Mus spretus) F1 x Mus spretus] (Rowe et al., 1994) were digested in a 96-wellmicrotiter dish using PstI and blotted to Hybond N+ nylon membranes (Dupont, Boston, MA, USA) following the manufacturer's recommendations. The membranes were hybridized under standard conditions to the same entire Ahch cDNA probe used to identify the polymorphism, and washed twice in 0.5 x standard saline citrate/0.01% SDS for 15 min at 65°C. The blots were then scored for the presence of the C57BL/6J allele and the data were sent to the Jackson Laboratory for chromosomal assignment. The GyK locus was analyzed using an intronic fragment from the murine GyK locus as a probe (Huq et al., 1996). The Fin1 locus, which mapped to the Drnd region, was analyzed as described previously (Gariboldi et al., 1994). All other markers were analyzed and scored as previously described (Herman et al., 1994)
the Ahch p r o t e i n is that the cysteine residues that m a y interact with zinc ions to form the two zinc fingers in the putative D N A b i n d i n g d o m a i n of the h D A X 1 protein ( G u o et al., 1995a) are 100% conserved, a l t h o u g h the aa residues a r o u n d the cysteines are less conserved, suggesting the functional i m p o r t a n c e of the cysteine residues. Regions II a n d I I I in the ligand b i n d i n g d o m a i n also show a very high degree of sequence identity, with 93% of the aa identical between mouse Ahch a n d h D A X 1 in region II a n d 96% in region III, i n d i c a t i n g the importance of these d o m a i n s as well for the function of the protein. These observations also m a y explain why missense m u t a t i o n s in regions II cause A H C a n d H H (Muscatelli et al., 1994). S o u t h e r n blots of EcoRI-digested m o u s e genomic D N A show that the m o u s e Ahch gene has a very simple g e n o m i c structure, since the entire gene is located on
34
W. Guo et al./Gene 178 (1996) 31-34
one 6-kb EcoRI restriction fragment (data not shown). This is similar to the human DAX1 in which the entire gene has only two exons, separated by a single intron, and is contained on three EcoRI restriction fragments totaling about 9 kb (Guo et al., 1995a). The simplicity of genomic structure of the AHC gene together with its unusual DNA binding domain suggest that it may be one of the most primitive receptors in the superfamily (Guo et al., 1996; Burris et al., 1996). Since Dmd, GyK and Ahch loci in the mouse lie in a syntenic block of DNA on the X chromosome, we are afforded the opportunity to study mouse models for human genetic disease in this region other than AHC and HH. Bardoni et al. (1994) demonstrated that the dosage sensitive sex reversal (DSS) locus maps to Xp21, and is within a 160-kb region that includes the AHC locus as well as additional telomeric DNA. The evidence presented here supports speculation that the DSS locus in the mouse may be located in the same subchromosoreal block of DNA as Dmd, GyK and Ahch loci. By generating transgenic mice with an additional copy of the genomic fragment from this region, one may be able to demonstrate whether an extra copy of Ahch or another gene in this region will affect gonadal development and cause phenotypically sex-reversed females from genotypic male mice, eventually identifying the gene responsible for the phenotype.
Acknowledgement This work was supported by a grant to ERBM from the NIH (RO1 HD22563), a March of Dimes Basil O'Connor Scholar Award #0095 for W.J.C., and an ACS postdoctoral grant (TPB; PF-4074).
References Altschul, S.F., Gish, W., Miller, W., Myers, E.W. and Lipman, D.J. (1990) Basic local alignment search tool. J. Mol. Biol. 215, 403-410. Bardoni, B., Zanaria, E., Guioli, S., Floridia, G., Worley, K., Tonini, G., Ferrante, E., Chiumello, G., McCabe, E.R.B., Fraccaro, M., Zuffardi, O. and Camcrino, G. (1994) X-linked sex reversal due to a dosage sensitive gene which interferes testis formation. Nature Genet. 7, 497-501. Blair, H.J., Reed, V., Laval, S.H. and Boyd, Y. (1994) New insights into the man-mouse comparative map of the X chromosome. Genomics 19, 212-220. Blair, H.J., Ho, M., Monaco, A.P., Fisher, S., Craig, I.W. and Boyd, Y. (1995) High-resolution comparative mapping of the proximal region of the mouse X chromosome. Genomics 28, 305-310. Burris, T.P., Guo, W. and McCabe, E.R.B. (1995) Identification of a
putative steroidogenic factor-1 response element in the DAX-1 promoter. Biochem. Biophys. Res. Commun. 214, 576-581. Burris, T.P., Guo, W. and McCabe, E.R.B. (1996) The gene responsible for adrenal hypoplasia congenita, DAXI, encodes a nuclear hormone receptor that defines a new class within the superfamily. Rec. Prog. Hormone Res. 51,241-260. Gariboldi, M., Maestrini, E., Canzian, F., Manenti, G., De Gregorio, L., Rivella, S., Chatterjee, A., Herman, G.E., Archidiacono, N., Antonacci, R., Pierotti, M.A., Dragani, T.A. and Toniolo, D. (1994) Comparative mapping of the actin-binding protein 280 genes in human and mouse. Genomics 21, 428-430. Guo, W., Mason, J., Charles, S.G., Stacy, M.A., Muda, S., Baldini, A., Lindsay, E.A., Biesecker, L.G., Copelland, K., Horlick, M.N.B., Pettigrew, A., Zanaria, E. and McCabe, E.R.B. (1995a) Diagnosis of X-linked adrenal hypoplasia congenita by mutation analysis of the DAX1 gene. J. Am. Med. Ass. 274, 324-330. Guo, W., Burris, T.P. and McCabe, E.R.B. (1995b) Expression of DAX1, the gene responsible for x-linked adrenal hypoplasia congenita and hypogonadotropic hypogonadism, in the hypothalamicpituitary-gonadal axis. Biochem. Mol. Med. 56, 8-13. Guo, W., Burris, T.P., Zhang, Y.H., Huang, B.L., Mason, J., Copeland, K.C., Kupfer, S.R., Pagon, R.A. and McCabe, E.R.B. (1996) The genomic sequence of the DAX1 gene: an orphan nuclear receptor responsible for X-linked adrenal hypoplasia congenita and hypogonadotropic hypogonadism. J. Clin. Endocrinol. Metab. 81, 2481-2486. Herman, G.E., Boyd, Y., Chapman, V., Chatterjee, A. and Brown, S.D. (1994) Mouse X chromosome. Mammal. Genome 5, $276 $288. Huq, A.H.M.M., Lovell, R.S., Sampson, M.J., Decker, W.K., Dinulos, M.B., Disteche, C.M. and Craigen, W.J. (1996) Isolation, mapping and functional expression of the mouse X chromosome glycerol kinase gene. Genomics, in press. Muscatelli, F., Strom, T.M., Walker, A.P., Zanaria, E., Recan, D., Meindl, A., Bardoni, B., Guioli, S., Zehetner, G., Rabl, W., Schwarz, H.P., Kaplan, J.C., Camerino, G., Meitinger, T. and Monaco, A.P. (1994) Mutation in the DAX1 gene gives rise to both X-linked adrenal hypoplasia congenita and hypogonadotropic hypogonadism. Nature 372, 672-676. Rowe, L.B., Nadeau, J.H., Turner, R., Frankel, W.N., Letts, V.A., Eppig, J.T., Ko, M.S., Thurston, S.J. and Birkenmeier, E.H. (1994) Maps from two interspecific backcross DNA panels available as a community genetic mapping resource. Mammal. Genome 5, 253-274. Searle, A.G., Peters, J., Lyon, M.F., Hall, J.G., Evans, E.P., Edwards, J.H. and Buckle, V.J. (1989) Chromosome maps of man and mouse. IV. Ann. Hum. Genet. 53 (Pt2), 89-140. Walker, A.P., Chelly, J., Love, D.R., Brush, Y.I., Recan, D., Chaussain, J.L., Oley, C.A., Connor, J.M., Yates, J., Price, D.A., Super, M., Bottani, A., Steinman, B., Kaplan, J.C., Davies, K.E. and Monaco, A.P. (1992) A YAC contig in Xp21 containing the adrenal hypoplasia congentia and glycerol kinase deficiency genes. Hum. Mol. Genet. 1, 579 585. Worley, K.C., Ellison, K.A., Zhang, Y.H., Wang, D.F., Mason, J., Roth, E.J., Adams, V., Fogt, D.D., Zhu, X.M., Tobin, J.A., Chinault, A.C., Zoghbi, H. and McCabe, R.E.B. (1993) Yeast artifical chromosome cloning in the glycerol kinase and adrenal hypoplasia congenita region of Xp21. Genomics 16, 407-416. Zanaria, E., Muscatelli, F., Bardoni, B., Strom, T.M., Guioli, S., Guo, W., Lalli, E., Moser, C., Walker, A.P., McCabe, E.R.B., Meitinger, T., Monaco, A.P., Sassone-Corsi, P. and Camerino, G. (1994) An unusual member of the nuclear hormone receptor superfamily responsible for X-linked adrenal hypoplasia congenita. Nature 372, 635-641.
ELSEVIER
Gene 178 (1996) 31-34
Ahch, the mouse homologue of DAXI" cloning, characterization and synteny with GyK, the glycerol kinase locus Weiwen Guo a, Rhonda S. Lovell b Yao-Hua Zhang a, Bing-Ling Huang William J. Craigen b, Edward R.B. McCabe a.,
a,
Thomas P. Burris ~,
a Department of Pediatrics, UCLA School of Medicine, 10833 Le Conte Avenue, Los Angeles, CA 90024, USA b Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX 77030, USA
Received 5 December 1995; revised 2 February 1996; accepted 6 February 1996
Abstract
We cloned the murine full-length cDNA encoding Ahch, the mouse homologue of DAX1 (DSS-AHC Region on Human X Chromosome, Genel) which is the gene responsible for human X-linked adrenal hypoplasia congenita (AHC) and hypogonadotropic hypogonadism (HH). Sequence analysis revealed that the murine and human cDNAs have 65% aa identity and 75% aa similarity overall. The cysteine residues in the putative DNA binding domain, which may interact with Zn 2÷ ions to form zinc fingers, are 100% conserved between the two species, indicating that the novel zinc-finger structures in DAX1 may be functional. In addition, mouse interspecific backcrosses show that the Ahch gene is closely linked to the glycerol kinase locus, GyK, on the mouse X chromosome, indicating that the order of the loci is conserved in this syntenic region between mouse and human. Keywords: Adrenal hypoplasia congenita; Hypogonadotropic hypogonadism; Duchenne muscular dystrophy; Steroidogenic factor
1 response element; Dosage sensitive sex reversal locus; Zinc-finger domain
I. Introduction
D A X I is the gene responsible for X-linked adrenal hypoplasia congenita (AHC) and hypogonadotropic hypogonadism (HH) in the human (Muscatelli et al., 1994; Zanaria et al., 1994; Guo et al., 1995a). The DAX1 gene maps to the human X chromosome in Xp21, telomeric to the glycerol kinase (GK) gene, and is frequently deleted in patients with a contiguous gene syndrome that involves the loss of the AHC locus along with the GK deficiency or G K D and Duchenne muscular dystrophy (DMD) loci (Walker et al., 1992; Worley et al., 1993).
*Corresponding author. Tel.: (+1-310) 825-5095; Fax: (+1-310) 206-4584; e-mail: [email protected] Abbreviations: aa, amino acid; AHC, adrenal hypoplasia congenita; Ahch, mouse locus for adrenal hypoplasia congenita in the human; Amel, amelogenin; bp, base pair(s); cDNA; DNA complementary to RNA; DAX1, DSS-AHC Region on Human X Chromosome, Genel; DMD, Duchenne muscular dystrophy; DSS; dosage sensitive sex reversal locus; Flnl, filamin 1, actin binding protein 280; GKD, glycerol kinase deficiency; GyK, mouse glycerol kinase locus; hDAXI, human DAXI; kb, kilobase(s) or 1000 bp; SF-1, steroidogenic factor-1. 0378-1119/96/$15.00 © 1996 Elsevier Science B.V. All rights reserved PII S0378-1119(96)00320-4
The human DAX1 gene was identified by positional cloning methods, and the cDNA sequence from both testis and fetal adrenal gland revealed it tO be a member of the nuclear hormone receptor superfamily based on the presence of a conserved C-terminal ligand binding domain (Zanaria et al., 1994; Guo et al., 1995a). The amino terminal portion of DAX1, which is composed of 3.5 repeats of a 65-67-aa motif, contains two putative zinc fingers that may define a novel DNA binding domain (Guo et al., 1995a). The genomic sequence in the promoter region of DAX1 revealed a putative steroidogenic factor-1 (SF-1) response element (Guo et al., 1996). Recently, we showed that SF-1 binds in vitro to the putative SF-1 response element found in the D A X I promoter, indicating that SF-1 may directly regulate the expression of DAX-1 (Burris et al., 1995). Northern blot and RT-PCR studies show that DAX1 is expressed in adrenal gland, testis, ovary, hypothalamus and pituitary gland, suggesting the involvement of DAX-1 in the development and regulation of the hypothalamic-pituitary-gonadal axis (Guo et al., 1995b). To further study the function of DAX1, it will be necessary to utilize an animal model that is well-characterized genetically and developmentally, such as the
32
W. Guo et al./Gene 178 (1996) 31-34
mouse. In addition, cloning the homologues of DAX1 from different species will help us to study the evolution of this gene and to define its functional domains, such as the potential zinc-finger structure in the putative D N A binding domain. In this report, we describe the cloning of the murine homologue of DAX1, Ahch, from a mouse testis c D N A library.
2. Experimental and discussion 2.1. Characterization of Ahch cDNA Four positive c D N A clones were identified from a mouse testis c D N A library that was cloned into the Uni-ZAPXR polylinker with EcoRI and XhoI adapters on the cDNAs, and all four were sequenced. One of the clones, MC4, contained 1806 bp of murine c D N A sequence in addition to 12 bp of adapter sequence and included the entire coding region (Fig. 1) (GenBank accession No. U41568). The complete coding sequence of the mouse Ahch c D N A was 1419 bp in length from the start codon (ATG) through the stop codon (TGA). The MC4 clone contained 1 9 b p of 5'-untranslated region and 368 bp of 3'-untranslated region, including an AATAAA polyadenylation signal that was followed by polyadenosine residues.
2.2. Sequence analysis The Ahch c D N A predicts a 472-aa protein which is two aa longer than h u m a n DAX1. The mouse Ahch deduced protein sequence shows highly significant similarity to hDAX1, with 65% identity and 75% similarity overall, and identity that is even higher in the ligand binding domain: 93% in region II and 96% in region III (Fig. 2). The cysteine residues (aa positions 38, 41, 43, 66, 67, 69, 105, 108, 110, 134, 135 and 137) in the putative D N A binding domain, which we have predicted to interact with Zn 2+ ions to form zinc fingers (Guo et al., 1995a), are 100% conserved between the mouse and h u m a n proteins, but aa around the cysteine residues are less conserved (Fig. 2). The deduced aa sequence for mouse Ahch was used as a query to search the nonredundant protein database of the National Center for Biological Information (NCBI) using the BLAST network server (Altschul et al., 1990). Ahch showed a highly significant similarity between its carboxy terminus and the ligand binding domain of members of the nuclear hormone receptor gene superfamily, as had been observed previously for hDAX1 (Zanaria et al., 1994; Guo et al., 1995a). The N-terminal portion of the Ahch gene was rich in small aa (glycine and alanine) and consisted of three and a half repeats of a 65-67-aa motif, like hDAX1 (Zanaria et al., 1994; G u o et al., 1996).
ECORI
25 G~TTCGGACGA~AGCCTCAGGCC 1 AT~CGGGTGAGGACCACCCGTGGCAG~CAGCATCCTCTAC~TCTACTGATGAGCGCG M A G E D H P W Q G S I L Y N L L M S A 61 ~GCAG~GCACGCGTCTCAGG~GAGCGAGAGGTGCGCTTGGGGGCTCAG~CTGGGGT K Q K H A S Q E E R E V R L G A Q ~ w G 121 TGCGCCTGCGGTOCTCAGCCCGTCCTGGGTGGGGAGAGACTGTCCGGCGG~GCCAGG ~ C G A O P V L G G E R L S G q Q A R 181 T C C C T C T ~ T A C C G C T G C ~ C T T T T G T G G ~ A G ~ T C A C C C G C G C C A G G G T G G C A T C C T C ~
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Fig. l. Complete nucleotide and predicted aa sequence of mouse Ahch cDNA clone MC4. The cloning adapter sequences for the EcoRI and Xhol sites are underlined. The potential polyadenylation signal identified in the 3' noncoding region is underlined. The deduced aa sequence of the Ahch protein is given in one-letter symbols. Two putative zinc fingers are bolded, italicized and underlined. Methods: Cloning and sequencing. The 3.5-kb EcoRI restriction fragment containing the first exon of the human DAX1 gene (Guo et al., 1995a) was used to screen a mouse testis cDNA library in the Uni-ZAPXR vector (Cat. No. 937308, Stratagene, La Jolla, CA, USA). The positive clones that were identified were sequenced in both directions by a combination of manual dideoxy sequencing (Sequenase, US Biochemical, Cleveland, OH, USA) and automated cyclesequencing (ABI-377 DNA Sequencer, Applied Biosystems, Foster City, CA, USA). The sequencing began with the universal and reverse primers, followed by walking primers. The Gel programs of the GCG (Version 8.0; Madison, WI, USA) were used for sequence assembly.Evaluation of the nucleotide and deduced aa sequences relied on the GCG programs (Version 8.0). Nucleotide sequence analysisof the cloned cDNA utilized the Blast Serverat NCBI (Altschul et al., 1990).
2.3. Mapping Ahch to the X chromosome and linkage with GyK Interspecific backcrosses of C57BL/6J and Mus spretus showed that there was no recombination between the GyK locus and the Ahch locus, indicating tight linkage between GyK and Aheh (Fig. 3). DXMit7, a
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Fig. 2. Amino-acid sequence comparison between Ahch and hDAX1. The Gap program of the GCG (Version 8.0) was used for sequence comparison. The top portion represents the mouse Ahch protein and the bottom portion represents the hDAX1 protein. Vertical lines indicate that the aa are identical. Double dots indicate aa with similar chemical characteristics. Singledots indicate aa with less similar chemical characteristics and spaces indicate aa with no chemical similarities at all. The cysteine residues that may interact with Znz+ to form zinc fingers are bolded and italicized. The conserved regions in the putative ligand binding domain are italicized and underlined.
microsatellite D N A for a p o l y m o r p h i s m m a r k e r on the m o u s e X c h r o m o s o m e was also linked to the G y K locus (Fig. 3). Since D X M i t 7 was s h o w n to be a b o u t 3 cM from the m u r i n e d y s t r o p h i n locus (Dmd) ( H e r m a n et al., 1994) then the G y K also was linked to the Dmd locus (Blair et al., 1994, 1995; H e r m a n et al., 1994). These results indicated that the Ahch, G y K a n d Dmd loci were in the same region of the m o u s e X c h r o m o s o m e a n d in the same order as in the h u m a n , and, therefore, this region was syntenic with the h o m o l o g o u s region of the h u m a n X chromosome.
3. Conclusions The putative Ahch p r o t e i n derived from m o u s e c D N A clones is two aa longer t h a n the h D A X 1 protein, with 75% similarity a n d 65% identity to the h u m a n D A X I p r o t e i n sequence overall, i n d i c a t i n g that the gene responsible for A H C has been highly conserved d u r i n g the 100 million years of e v o l u t i o n a r y time that separate the two species (Searle et al., 1989). The more striking feature of
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o.oo 3.,9 20.2, 15.96
1
Fig. 3. Haplotype analysis of the BSS interspecificbackcross panel for Ahch. Each column represents a chromosomal haplotype, with the number of offspring possessing the haplotype indicated at the bottom of the column. Relevant typed loci are listed on the left side of the fgure, while the black boxes indicate the C57BL/6J allele and the white boxes the M. spretus allele. On the right is the recombination frequency between the listed loci. Methods: Genetic mapping. Ahch was mapped by an interspecific backcross panel. To identify species-specific DNA polymorphisms, a genomic Southern blot was prepared using matched DNA samples of C57BL/6J and Mus spretus (obtained from the Jackson Laboratory, Bar Harbor, ME, USA) that were digested with nine different restriction enzymes.The entire Ahch cDNA was used as a radiolabeled probe. A number of polymorphic fragments were identified with various restriction enzymes (data not shown). PstI was chosen since it gave 4.8-kb C57BL/6J-specificand 5.7-kb Mus spretusspecific bands that were easily resolved by Southern blotting. Three micrograms per lane of DNA from the 93 interspecific backcross progeny from the Jackson Laboratory BSS backcross panel [(C57BL/6J x Mus spretus) F1 x Mus spretus] (Rowe et al., 1994) were digested in a 96-wellmicrotiter dish using PstI and blotted to Hybond N+ nylon membranes (Dupont, Boston, MA, USA) following the manufacturer's recommendations. The membranes were hybridized under standard conditions to the same entire Ahch cDNA probe used to identify the polymorphism, and washed twice in 0.5 x standard saline citrate/0.01% SDS for 15 min at 65°C. The blots were then scored for the presence of the C57BL/6J allele and the data were sent to the Jackson Laboratory for chromosomal assignment. The GyK locus was analyzed using an intronic fragment from the murine GyK locus as a probe (Huq et al., 1996). The Fin1 locus, which mapped to the Drnd region, was analyzed as described previously (Gariboldi et al., 1994). All other markers were analyzed and scored as previously described (Herman et al., 1994)
the Ahch p r o t e i n is that the cysteine residues that m a y interact with zinc ions to form the two zinc fingers in the putative D N A b i n d i n g d o m a i n of the h D A X 1 protein ( G u o et al., 1995a) are 100% conserved, a l t h o u g h the aa residues a r o u n d the cysteines are less conserved, suggesting the functional i m p o r t a n c e of the cysteine residues. Regions II a n d I I I in the ligand b i n d i n g d o m a i n also show a very high degree of sequence identity, with 93% of the aa identical between mouse Ahch a n d h D A X 1 in region II a n d 96% in region III, i n d i c a t i n g the importance of these d o m a i n s as well for the function of the protein. These observations also m a y explain why missense m u t a t i o n s in regions II cause A H C a n d H H (Muscatelli et al., 1994). S o u t h e r n blots of EcoRI-digested m o u s e genomic D N A show that the m o u s e Ahch gene has a very simple g e n o m i c structure, since the entire gene is located on
34
W. Guo et al./Gene 178 (1996) 31-34
one 6-kb EcoRI restriction fragment (data not shown). This is similar to the human DAX1 in which the entire gene has only two exons, separated by a single intron, and is contained on three EcoRI restriction fragments totaling about 9 kb (Guo et al., 1995a). The simplicity of genomic structure of the AHC gene together with its unusual DNA binding domain suggest that it may be one of the most primitive receptors in the superfamily (Guo et al., 1996; Burris et al., 1996). Since Dmd, GyK and Ahch loci in the mouse lie in a syntenic block of DNA on the X chromosome, we are afforded the opportunity to study mouse models for human genetic disease in this region other than AHC and HH. Bardoni et al. (1994) demonstrated that the dosage sensitive sex reversal (DSS) locus maps to Xp21, and is within a 160-kb region that includes the AHC locus as well as additional telomeric DNA. The evidence presented here supports speculation that the DSS locus in the mouse may be located in the same subchromosoreal block of DNA as Dmd, GyK and Ahch loci. By generating transgenic mice with an additional copy of the genomic fragment from this region, one may be able to demonstrate whether an extra copy of Ahch or another gene in this region will affect gonadal development and cause phenotypically sex-reversed females from genotypic male mice, eventually identifying the gene responsible for the phenotype.
Acknowledgement This work was supported by a grant to ERBM from the NIH (RO1 HD22563), a March of Dimes Basil O'Connor Scholar Award #0095 for W.J.C., and an ACS postdoctoral grant (TPB; PF-4074).
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