Genes encoding the H,K-ATPase α and Na,K-ATPase α3 subunits are linked on mouse Chromosome 7 and human Chromosome 19

We have used linkage analysis and fluorescence in situ hybridization to determine the chromosomal organization and location of the mouse (Atp4a) and human (ATP4A) genes encoding the H,K-ATPase subunit. Linkage analysis in recombinant inbred (BXD) strains of mice localized Atp4a to mouse Chromosome (Chr) 7. Segregation of restriction fragment length polymorphisms in backcross progeny of Mus musculusxMus spretus mating confirmed this assignment and indicates that Atp4a and Atp1a3 (gene encoding the murine Na,K-ATPase 3 subunit) are linked and separated by a distance of 2 cM. Analysis of the segregation of simple sequence repeats suggested the gene order centromere-D7Mit21-D7Mit57/Atpla3-D7Mit72/Atp4a. A human Chr 19-enriched cosmid library was screened with both H,K-ATPase and Na,K-ATPase 3 subunit cDNA probes to isolate the corresponding human genes (ATP4A and ATP1A3, respectively). Fluorescence in situ hybridization with gene-specific cosmid clones localized ATP4A to the q13.1 region, and proximal to ATP1A3, which maps to the q13.2 region, of Chr 19. These results indicate that ATP4A and ATP1A3 are linked in both the mouse and human genomes.     

Alterations in the expression of the Atp7a gene in the early postnatal development of the mosaic mutant mice (Atp7amo-ms) – An animal model for Menkes disease

Copper is a trace element that is essential for the normal growth and development of all living organisms. In mammals, the ATP7A Cu-transporting ATPase is a key protein that is required for the maintenance of copper homeostasis. In both humans and mice, the ATP7A protein is coded by the X-linked ATP7A/Atp7a gene. Disturbances in copper metabolism caused by mutations in the ATP7A/Atp7a gene lead to severe metabolic syndromes Menkes disease in humans and the lethal mottled phenotype in mice. Mosaic is one of numerous mottled mutations and may serve as a model for a severe Menkes disease variant. In Menkes patients, mutations in the ATP7A gene often result in a decreased level of the normal ATP7A protein. The aim of this study was to analyse the expression of the Atp7a gene in mosaic mutants in early postnatal development, a critical period for starting copper supplementation therapy in both Menkes patients and mutant mice. Using real-time quantitative RT-PCR, we analysed the expression of the Atp7a gene in the brain, kidney and liver of newborn (P0.5) and suckling (P14) mice. Our results indicate that in mosaic P0.5 mutants, the Atp7a mRNA level is decreased in all analysed organs in comparison with wild-type animals. In two week-old mutants, a significant decrease was observed only in the kidney. In contrast, their hepatic level of Atp7a tended to be higher than in wild-type mice. We speculate that disturbance in the expression of the Atp7a gene and, consequently, change in the copper concentration of the organs, may contribute to the early fatal outcome of mosaic males.     

A mutation in the ATP7B copper transporter causes reduced dopamine beta-hydroxylase and norepinephrine in mouse adrenal

The copper-transporting ATPases Atp7A and Atp7B play a major role in controlling intracellular copper levels. In addition, they are believed to deliver copper to the copper-requiring proteins destined for the secretory vesicles. One cuproprotein, dopamine -hydroxylase (DBH) functions in the biosynthesis of norepinephrine and epinephrine, neurohormones in endocrine and nervous tissue. To evaluate the consequences of loss of Atp7B on the function of DBH, the level of proteins in adrenal gland were compared between normal mice and mice containing a null mutation in the ATP7B gene. The levels of DBH, as well as another vesicular protein, chromogranin A, are reduced in the ATP7B–/– mice. In addition to the lower level of enzyme, the products of DBH catalytic activity, norepinephrine and epinephrine, are also decreased. Although these changes are a consequence of ATP7B gene function, Atp7B mRNA is not normally expressed in the adrenal gland. Instead, Atp7A mRNA is present. The levels of copper and DBH RNA within adrenals of the ATP7B–/– mice are not different from the wild type. The results of these experiments suggest that copper-requiring enzymes are affected by a loss of ATP7B even in tissue not normally expressing this protein. Therefore the multisystemic effects observed in Wilson disease, the human disorder characterized by mutation in ATP7B, may be a secondary consequence of the major accumulation of copper in liver.     

Organization and chromosomal locations of Rap1a/Krev sequences in the mouse

The human RAP1A gene encodes a protein that apparently can antagonize the function of oncogenic ras genes in gene transfer experiments, but its normal function is unknown. To understand the function of this gene, we have undertaken a study of the mouse homolog, Rap1a. The complete coding sequence of a mouse Rap1a cDNA has been determined, and genomic clones representing three distinct Rap1a species were recovered. We find that Rap1a is located on distal mouse Chromosome (Chr) 3 near Nras, Ampd-1, Tshb, Ngfb, and Atp1a1. Two related sequences (Rap1a-rs1 and Rap1a-rs2) were also characterized. Rap1a-rs1, which was not localized, has a sequence very similar to the Rap1a cDNA, suggesting that it has been recently acquired by the mouse genome. Rap1a-rs2 is more distantly related to the gene sequence and is located on Chr 2 near Actc-1.     

Modeling human epilepsy by TALEN targeting of mouse sodium channel Scn8a

To evaluate the efficiency of TALEN technology for introducing mutations into the mouse genome we targeted Scn8a, a member of a multigene family with nine closely related paralogs. Our goal was to generate a model of early onset epileptic encephalopathy by introduction of the Scn8a missense mutation p.Asn1768Asp. We used a pair of TALENs that were highly active in transfected cells. The targeting template for homologous recombination contained a 4 kb genomic fragment. Microinjection of TALENs with the targeting construct into the pronucleus of 350 fertilized mouse eggs generated 67 live‐born potential founders, of which 5 were heterozygous for the pathogenic mutation, a yield of 7% correctly targeted mice. Twenty‐four mice carried one or two Scn8a indels, including 12 frameshift mutations and the novel amino acid deletion p.Asn1759del. Nine off‐site mutations in the paralogs sodium channel genes Scn5a and Scn4a were identified. The data demonstrate the feasibility and efficiency of targeting members of multigene families using TALENs. The Scn8a^tm1768DMm mouse model will be useful for investigation of the pathogenesis and therapy of early onset seizure disorders. genesis 52:141–148. © 2013 The Authors genesis Published by Wiley Periodicals, Inc.     

Hepatic copper-transporting ATPase ATP7B: function and inactivation at the molecular and cellular level

Copper-transporting ATPase ATP7B (Wilson disease protein) is a member of the P-type ATPase family with characteristic domain structure and distinct ATP-binding site. ATP7B plays a central role in the regulation of copper homeostasis in the liver by delivering copper to the secretory pathway and mediating export of excess copper into the bile. The dual function of ATP7B in hepatocytes is coupled with copper-dependent intracellular relocalization of the transporter. The final destination of ATP7B in hepatocytes during the copper-induced trafficking process is still under debate. We show the results of immunocytochemistry experiments in polarized HepG2 cells that support the model in which elevated copper induces trafficking of ATP7B to sub-apical vesicles, and transiently to the canalicular membrane. In Atp7b ^-/- mice, an animal model of Wilson disease, both copper delivery to the trans-Golgi network and copper export into the bile are disrupted despite large accumulation of copper in the cytosol. We review the biochemical and physiological changes associated with Atp7b inactivation in mouse liver and discuss the pleiotropic consequences of the common Wilson disease mutation, His1069Gln.     

Retrotransposed genes such as Frat3 in the mouse Chromosome 7C Prader-Willi syndrome region acquire the imprinted status of their insertion site

Prader-Willi syndrome (PWS) results from loss of function of a 1.0- to 1.5-Mb domain of imprinted, paternally expressed genes in human Chromosome (Chr) 15q11-q13. The loss of imprinted gene expression in the homologous region in mouse Chr 7C leads to a similar neonatal PWS phenotype. Several protein-coding genes in the human PWS region are intronless, possibly arising by retrotransposition. Here we present evidence for continued acquisition of genes by the mouse PWS region during evolution. Bioinformatic analyses identified a BAC containing four genes, Mkrn3, Magel2, Ndn, Frat3, and the Atp5l-ps1 pseudogene, the latter two genes derived from recent L1-mediated retrotransposition. Analyses of eight overlapping BACs indicate that these genes are clustered within 120 kb in two inbred strains, in the order tel–Atp5l-ps1–Frat3–Mkrn3–Magel2–Ndn–cen. Imprinting analyses show that Frat3 is differentially methylated and expressed solely from the paternal allele in a transgenic mouse model of Angelman syndrome, with no expression from the maternal allele in a mouse model of PWS. Loss of Frat3 expression may, therefore, contribute to the phenotype of mouse models of PWS. The identification of five intronless genes in a small genomic interval suggests that this region is prone to retroposition in germ cells or their zygotic and embryonic cell precursors, and that it allows the subsequent functional expression of these foreign sequences. The recent evolutionary acquisition of genes that adopt the same imprint as older, flanking genes indicates that the newly acquired genes become `innocent bystanders' of a primary epigenetic signal causing imprinting in the PWS domain. Received: 22 May, 2001 / Accepted: 16 July 2001     

Linkage of a 7S RNA sequence and kappa light chain genes in the mouse

A mouse 7S RNA cDNA plasmid clone was employed to identify and map DNA restriction fragment variants using recombinant inbred (RI) and congenic mouse strains. More than a dozen such restriction variants were identified and mapped to different regions of the mouse genome. One such variant, designated Rn7s-6, showed close linkage to the Ly-2,3-Igk-V (T lymphocyte antigens 2 and 3, kappa immunoglobulin variable region) cluster of markers on chromosome 6. No recombinants were detected among three of these markers in 59 RI strains. On the basis of these data, the Rn7s-6 sequence may be placed within 1.3 centimorgans of Ly-3 and one of the Igk-V-region markers, Igk-Efl. Two mouse stocks with previously identified crossovers within the Ly2,3-Igk-V region were used to sublocalize Rn7s-6. The results are consistent with the gene order (Ly-2, Ly-3)-(Rn7s-6, Igk-Efl)-Igk-Ef2. Several mouse plasmacytomas, known to have various parts of the kappa chain complex deleted, retain the Rn7s-6 sequence. The Rn7s-6 variant is a plus/minus variant; no sequence allelic to Rn7s-6 is found in inbred strains that share the Ly-3 ^a-Igk-Efl^a haplotype.     

<Emphasis Type="Italic">In vivo</Emphasis> expression of copper-transporting proteins in rat brain regions

Expression of two copper-transporting P1-type ATPases (ATP7A and ATP7B), the CTR1 protein, a high-affinity copper transporter, and ceruloplasmin (Cp), a copper-containing ferroxidase was studied. The level of mRNA of these proteins was determined by RT-PCR analysis, the distribution of polypeptides encoded by these genes was determined by immunoblotting, and the type of cells expressing these genes was identified immunohistochemically. It was found that the major product of Cp gene in the brain is the cell membrane-bound Cp. Secretory Cp, whose molecule contains the greatest number of weakly associated copper atoms, is synthesized in the choroid plexus. CTR1 mRNA is evenly distributed in the brain; however, its content is twice higher in the vascular plexus. The Atp7a gene is active in all brain regions, whereas the Atp7b gene is active only in the hypothalamus. The membrane-bound Cp is expressed in glial cells of all types and in ependyma cells. ATP7B and ATP7A are expressed predominantly in ependymyocytes and neurons, respectively. The organization of copper transport in mammalian brain is discussed.Translated from Izvestiya Akademii Nauk, Seriya Biologicheskaya, No. 2, 2005, pp. 141–154.Original Russian Text Copyright © 2005 by Platonova, Barabanova, Povalikhin, Tsymbalenko, Danilovskii, Voronina, Dorokhova, Puchkova.     

Chromosomal assignment of 11 loci in the rat by mouse-rat somatic hybrids and linkage

Eleven rat genes have been assigned to rat chromosomes by use of mouse × rat somatic hybrids and/or use of linkage to known chromosome markers. Among them, the genes for the inducible nitric oxide synthase (Nos2) and for a vasoactive intestinal peptide receptor (Vipr) are potential candidates for genetic regulation of blood pressure and were localized to rat Chromosomes (Chrs) 10 and 8 respectively. Genes for gastric H,K-ATPase alpha subunit (Atp4a). Class I alcohol dehydrogenase (Adh), and aldolase C (Aldoc) were localized to Chrs 1, 2, and 10 respectively, and thus provide more DNA markers for genetic mapping of quantitative trait loci for blood pressure on those chromosomes. Genes for alkaline phosphatase (Alp1) and cardiac AE-3 Cl^-/HCO₃ ^- exchanger (Ae3) were both localized to Chr 9. Genes for glutamate dehydrogenase (Glud) and gastric H,K-ATPase beta subunit (Atp4b) were localized to Chr 16. The ornithine decarboxylase (Odc) gene and ornithine decarboxylase pseudogene (Odcp) were localized to Chrs 6 and 11 respectively.     

Atp1a3‐deficient heterozygous mice show lower rank in the hierarchy and altered social behavior

Atp1a3 is the Na‐pump alpha3 subunit gene expressed mainly in neurons of the brain. Atp1a3‐deficient heterozygous mice (Atp1a3^+/?) show altered neurotransmission and deficits of motor function after stress loading. To understand the function of Atp1a3 in a social hierarchy, we evaluated social behaviors (social interaction, aggression, social approach and social dominance) of Atp1a3^+/? and compared the rank and hierarchy structure between Atp1a3^+/? and wild‐type mice within a housing cage using the round‐robin tube test and barbering observations. Formation of a hierarchy decreases social conflict and promote social stability within the group. The hierarchical rank is a reflection of social dominance within a cage, which is heritable and can be regulated by specific genes in mice. Here we report: (1) The degree of social interaction but not aggression was lower in Atp1a3^+/? than wild‐type mice, and Atp1a3^+/? approached Atp1a3^+/? mice more frequently than wild type. (2) The frequency of barbering was lower in the Atp1a3^+/? group than in the wild‐type group, while no difference was observed in the mixed‐genotype housing condition. (3) Hierarchy formation was not different between Atp1a3^+/? and wild type. (4) Atp1a3^+/? showed a lower rank in the mixed‐genotype housing condition than that in the wild type, indicating that Atp1a3 regulates social dominance. In sum, Atp1a3^+/? showed unique social behavior characteristics of lower social interaction and preference to approach the same genotype mice and a lower ranking in the hierarchy.     

Exome sequencing identifies a nonsense mutation in <Emphasis Type="Italic">Fam46a</Emphasis> associated with bone abnormalities in a new mouse model for skeletal dysplasia

We performed exome sequencing for mutation discovery of an ENU (N-ethyl-N-nitrosourea)-derived mouse model characterized by significant elevated plasma alkaline phosphatase (ALP) activities in female and male mutant mice, originally named BAP014 (bone screen alkaline phosphatase #14). We identified a novel loss-of-function mutation within the Fam46a (family with sequence similarity 46, member A) gene (NM_001160378.1:c.469G>T, NP_001153850.1:p.Glu157*). Heterozygous mice of this mouse line (renamed Fam46a ^E157*Mhda) had significantly high ALP activities and apparently no other differences in morphology compared to wild-type mice. In contrast, homozygous Fam46a ^E157*Mhda mice showed severe morphological and skeletal abnormalities including short stature along with limb, rib, pelvis, and skull deformities with minimal trabecular bone and reduced cortical bone thickness in long bones. ALP activities of homozygous mutants were almost two-fold higher than in heterozygous mice. Fam46a is weakly expressed in most adult and embryonic tissues with a strong expression in mineralized tissues as calvaria and femur. The FAM46A protein is computationally predicted as a new member of the superfamily of nucleotidyltransferase fold proteins, but little is known about its function. Fam46a ^E157*Mhda mice are the first mouse model for a mutation within the Fam46a gene.     

The identification of the ceruloplasmin region interacting with the copper transferring menkes ATPase

The interaction was studied of ceruloplasmin (Cp, EC 1.16.3.1), a copper-containing plasma protein, with two synthetic peptides P15 and P16 whose structures correlate with those of the noncytosolic regions of the copper transfer P1 type ATPase (ATP7A), apparently encoded by the Menkes disease gene (Atp7a). Pentadecapeptide P15 and hexadecapeptide P16 were synthesized using the solid phase method. They correspond to fragments of two extracellular loops ATP7A, of which one loop is apparently involved in the copper ion transfer (P16) whereas the otheris not (P15). The protein footprinting showed that P16 binds to a fragment of the ceruloplasmin domain 6. Kinetics of the ceruloplasmin-P16 binding was studied by affinity chromatography on P16 immobilized on a macroporous disk, and theK _d value (1.5￠10⁻⁶ M) of this interaction was determined. The ATP7A involvement in the copper ion transfer to nonhepatocyte cells is discussed. Deceased.     

Three ENU-induced neurological mutations in the pore loopof sodium channel <Emphasis Type="Italic">Scn8a</Emphasis> (Na<Subscript>v</Subscript>1.6) and a genetically linkedretinal mutation, <Emphasis Type="Italic">rd13</Emphasis>

The goal of The Jackson Laboratory Neuroscience Mutagenesis Facility is to generate mouse models of human neurological disease. We describe three new models obtained from a three-generation screen for recessive mutations. Homozygous mutant mice from lines nmf2 and nmf5 exhibit hind limb paralysis and juvenile lethality. Homozygous nmf58 mice exhibit a less severe movement disorder that includes sustained dystonic postures. The mutations were mapped to the distal region of mouse Chromosome (Chr) 15. Failure to complement a mutant allele of a positional candidate gene, Scn8a, demonstrated that the mutations are new alleles of Scn8a. Missense mutations of evolutionarily conserved residues of the sodium channel were identified in the three lines, with the predicted amino acid substitutions N1370T, I1392F, and L1404H. These residues are located within the pore loop of domain 3 of sodium channel Na_v1.6. The lethal phenotypes suggest that the new alleles encode proteins with partial or complete loss of function. Several human disorders are caused by mutation in the pore loop of domain 3 of paralogous sodium channel genes. Line nmf5 contains a second, independent mutation in the rd13 locus that causes a reduction in cell number in the outer nuclear layer of the retina. rd13 was mapped to the distal 4 Mb of Chr 15. No coding or splice site mutations were detected in Pde1b, a candidate gene for rd13. The generation of three independent Scn8a mutations among 1100 tested G3 families demonstrates that the Scn8a locus is highly susceptible to ENU mutagenesis. The new alleles of Scn8a will be valuable for analysis of sodium channel physiology and disease.(David A. Buchner and Kevin L. Seburn) These authors contributed equally.     

The mouse homolog of FRG1, a candidate gene for FSHD, maps proximal to the myodystrophy mutation on chromosome 8

The human autosomal dominant neuromuscular disorder facioscapulohumeral muscular dystrophy (FSHD) is associated with deletions within a complex tandem DNA repeat (D4Z4) on Chromosome (Chr) 4q35. The molecular mechanism underlying this association of FSHD with DNA rearrangements is unknown, and, thus far, no gene has been identified within the repeat. We isolated a gene mapping 100 kb proximal to D4Z4 (FSHD Region Gene 1:FRG1), but were unable to detect any alterations in total or allele-specific mRNA levels of FRG1 in FSHD patients. Human Chr 4q35 exhibits synteny homology with the region of mouse Chr 8 containing the gene for the myodystrophy mutation (myd), a possible mouse homolog of FSHD. We report the cloning of the mouse gene (Frg1) and show that it maps to mouse Chr 8. Using a cross segregating the myd mutation and the European Collaborative Interspecific Backcross, we showed that Frg1 maps proximal to the myd locus and to the Clc3 and Ant1 genes. Received: 24 September 1996 / Accepted: 7 February 1997