G2Cdb::Gene report

Gene id
Gene symbol
Ank1 (MGI)
Mus musculus
ankyrin 1, erythroid
G00001838 (Homo sapiens)

Databases (9)

ENSMUSG00000031543 (Ensembl mouse gene)
11733 (Entrez Gene)
991 (G2Cdb plasticity & disease)
Gene Expression
MGI:88024 (Allen Brain Atlas)
g00123 (BGEM)
ank1 (gensat)
182900 (OMIM)
Marker Symbol
MGI:88024 (MGI)
Protein Sequence
Q02357 (UniProt)

Synonyms (2)

  • Ank-1
  • pale

Literature (49)

Pubmed - other

  • A novel ENU-generated truncation mutation lacking the spectrin-binding and C-terminal regulatory domains of Ank1 models severe hemolytic hereditary spherocytosis.

    Hughes MR, Anderson N, Maltby S, Wong J, Berberovic Z, Birkenmeier CS, Haddon DJ, Garcha K, Flenniken A, Osborne LR, Adamson SL, Rossant J, Peters LL, Minden MD, Paulson RF, Wang C, Barber DL, McNagny KM and Stanford WL

    The Biomedical Research Centre, University of British Columbia, Vancouver, BC, Canada.

    Objective: Hereditary spherocytosis (HS) is a heterogeneous group of spontaneously arising and inherited red blood cell disorders ranging from very mild subclinical cases to severe and life-threatening cases, with symptoms linked directly to the severity of the mutation at the molecular level. We investigated a novel mouse model in which the heterozygotes present with the diagnostic hallmarks of mild HS and surviving homozygotes phenocopy severe hemolytic HS.

    We used N-ethyl-N-nitrosourea mutagenesis to generate random point mutations in the mouse genome and a dominant screen to identify mouse models of human hematopoietic disease. Gene mapping of the HS strain revealed a unique in-frame nonsense mutation arising from a single base transversion in exon 27 of Ank1 (strain designation: Ank1(E924X)). Employing conventional hematopoietic, pathological, biochemical, and cell biology assays, we characterized heterozygous and homozygous Ank1(E924X) mice at the biochemical, cellular, and pathophysiological levels.

    Results: Although Ank1(E924X/E924X) red blood cell ghosts lack abundant full-length ankyrin-1 isoforms, N-terminal epitope ankyrin-1 antibodies reveal a band consistent with the theoretical size of a truncated mutant ankyrin-1. Using domain-specific antibodies, we further show that this protein lacks both a spectrin-binding domain and a C-terminal regulatory domain. Finally, using antisera that detect C-terminal residues of the products of alternative Ank1 transcripts, we find unique immunoreactive bands not observed in red blood cell ghosts from wild-type or Ank1(E924X) heterozygous mice, including a band similar in size to full-length ankyrin-1.

    Conclusions: The Ank1(E924X) strain provides a novel tool to study Ank1 and model HS.

    Funded by: Canadian Institutes of Health Research: FRN 74611, MOP-93580; NHLBI NIH HHS: HL088468, R01 HL088468; NIDDK NIH HHS: R01 DK080040, R01 DK080040-01A2

    Experimental hematology 2011;39;3;305-20, 320.e1-2

  • Hereditary spherocytosis and hereditary elliptocytosis: aberrant protein sorting during erythroblast enucleation.

    Salomao M, Chen K, Villalobos J, Mohandas N, An X and Chasis JA

    The Red Cell Physiology Laboratory, The New York Blood Center, New York, NY, USA.

    During erythroblast enucleation, membrane proteins distribute between extruded nuclei and reticulocytes. In hereditary spherocytosis (HS) and hereditary elliptocytosis (HE), deficiencies of membrane proteins, in addition to those encoded by the mutant gene, occur. Elliptocytes, resulting from protein 4.1R gene mutations, lack not only 4.1R but also glycophorin C, which links the cytoskeleton and bilayer. In HS resulting from ankyrin-1 mutations, band 3, Rh-associated antigen, and glycophorin A are deficient. The current study was undertaken to explore whether aberrant protein sorting, during enucleation, creates these membrane-spanning protein deficiencies. We found that although glycophorin C sorts to reticulocytes normally, it distributes to nuclei in 4.1R-deficient HE cells. Further, glycophorin A and Rh-associated antigen, which normally partition predominantly to reticulocytes, distribute to both nuclei and reticulocytes in an ankyrin-1-deficient murine model of HS. We conclude that aberrant protein sorting is one mechanistic basis for protein deficiencies in HE and HS.

    Funded by: NHLBI NIH HHS: HL31579, R01 HL031579; NIDDK NIH HHS: DK26263, DK32094, DK56267, P01 DK032094, R01 DK026263, R01 DK056267, R37 DK026263

    Blood 2010;116;2;267-9

  • Regulation of PI3-kinase/Akt signaling by muscle-enriched microRNA-486.

    Small EM, O'Rourke JR, Moresi V, Sutherland LB, McAnally J, Gerard RD, Richardson JA and Olson EN

    Department of Molecular Biology, University of Texas Southwestern Medical Center, Dallas, TX 75390-9148, USA.

    microRNAs (miRNAs) play key roles in modulating a variety of cellular processes through repression of mRNA targets. In a screen for miRNAs regulated by myocardin-related transcription factor-A (MRTF-A), a coactivator of serum response factor (SRF), we discovered a muscle-enriched miRNA, miR-486, controlled by an alternative promoter within intron 40 of the Ankyrin-1 gene. Transcription of miR-486 is directly controlled by SRF and MRTF-A, as well as by MyoD. Among the most strongly predicted targets of miR-486 are phosphatase and tensin homolog (PTEN) and Foxo1a, which negatively affect phosphoinositide-3-kinase (PI3K)/Akt signaling. Accordingly, PTEN and Foxo1a protein levels are reduced by miR-486 overexpression, which, in turn, enhances PI3K/Akt signaling. Similarly, we show that MRTF-A promotes PI3K/Akt signaling by up-regulating miR-486 expression. Conversely, inhibition of miR-486 expression enhances the expression of PTEN and Foxo1a and dampens signaling through the PI3K/Akt-signaling pathway. Our findings implicate miR-486 as a downstream mediator of the actions of SRF/MRTF-A and MyoD in muscle cells and as a potential modulator of PI3K/Akt signaling.

    Funded by: NHLBI NIH HHS: R01 HL093039

    Proceedings of the National Academy of Sciences of the United States of America 2010;107;9;4218-23

  • A systems approach reveals that the myogenesis genome network is regulated by the transcriptional repressor RP58.

    Yokoyama S, Ito Y, Ueno-Kudoh H, Shimizu H, Uchibe K, Albini S, Mitsuoka K, Miyaki S, Kiso M, Nagai A, Hikata T, Osada T, Fukuda N, Yamashita S, Harada D, Mezzano V, Kasai M, Puri PL, Hayashizaki Y, Okado H, Hashimoto M and Asahara H

    Department of Systems BioMedicine, National Research Institute for Child Health and Development, Setagaya, Tokyo 157-8535, Japan.

    We created a whole-mount in situ hybridization (WISH) database, termed EMBRYS, containing expression data of 1520 transcription factors and cofactors expressed in E9.5, E10.5, and E11.5 mouse embryos--a highly dynamic stage of skeletal myogenesis. This approach implicated 43 genes in regulation of embryonic myogenesis, including a transcriptional repressor, the zinc-finger protein RP58 (also known as Zfp238). Knockout and knockdown approaches confirmed an essential role for RP58 in skeletal myogenesis. Cell-based high-throughput transfection screening revealed that RP58 is a direct MyoD target. Microarray analysis identified two inhibitors of skeletal myogenesis, Id2 and Id3, as targets for RP58-mediated repression. Consistently, MyoD-dependent activation of the myogenic program is impaired in RP58 null fibroblasts and downregulation of Id2 and Id3 rescues MyoD's ability to promote myogenesis in these cells. Our combined, multi-system approach reveals a MyoD-activated regulatory loop relying on RP58-mediated repression of muscle regulatory factor (MRF) inhibitors.

    Funded by: NIAMS NIH HHS: R01 AR052779, R01 AR052779-03, R01AR052779; Telethon: TCR05004

    Developmental cell 2009;17;6;836-48

  • Novel roles for erythroid Ankyrin-1 revealed through an ENU-induced null mouse mutant.

    Rank G, Sutton R, Marshall V, Lundie RJ, Caddy J, Romeo T, Fernandez K, McCormack MP, Cooke BM, Foote SJ, Crabb BS, Curtis DJ, Hilton DJ, Kile BT and Jane SM

    Rotary Bone Marrow Research Laboratory, Melbourne Health Research Directorate, Parkville, Australia.

    Insights into the role of ankyrin-1 (ANK-1) in the formation and stabilization of the red cell cytoskeleton have come from studies on the nb/nb mice, which carry hypomorphic alleles of Ank-1. Here, we revise several paradigms established in the nb/nb mice through analysis of an N-ethyl-N-nitrosourea (ENU)-induced Ank-1-null mouse. Mice homozygous for the Ank-1 mutation are profoundly anemic in utero and most die perinatally, indicating that Ank-1 plays a nonredundant role in erythroid development. The surviving pups exhibit features of severe hereditary spherocytosis (HS), with marked hemolysis, jaundice, compensatory extramedullary erythropoiesis, and tissue iron overload. Red cell membrane analysis reveals a complete loss of ANK-1 protein and a marked reduction in beta-spectrin. As a consequence, the red cells exhibit total disruption of cytoskeletal architecture and severely altered hemorheologic properties. Heterozygous mutant mice, which have wild-type levels of ANK-1 and spectrin in their RBC membranes and normal red cell survival and ultrastructure, exhibit profound resistance to malaria, which is not due to impaired parasite entry into RBC. These findings provide novel insights into the role of Ank-1, and define an ideal model for the study of HS and malarial resistance.

    Funded by: NHLBI NIH HHS: P01 HL053749, P01 HL53749-03

    Blood 2009;113;14;3352-62

  • Localization of ank1.5 in the sarcoplasmic reticulum precedes that of SERCA and RyR: relationship with the organization of obscurin in developing sarcomeres.

    Giacomello E and Sorrentino V

    Department of Neuroscience, Interuniversity Institute of Myology, University of Siena, Siena, Italy.

    Ank1.5 is a muscle-specific isoform of ankyrin1 localized on the sarcoplasmic reticulum (SR) membrane that has been shown to interact with obscurin, a sarcomeric protein. We report here studies on the localization of obscurin and ank1.5 in embryonic and postnatal rodent skeletal muscles. Using two antibodies against epitopes in the N- and C-terminus of obscurin, two distinct patterns of localization were observed. Before birth, the antibodies against the N- and the C-terminus of obscurin stained the Z-disk and M-band, respectively. At the same time, ank1.5 was detected at the Z-disk, rising the possibility that obscurin molecules at M-band may not be able to interact with ank1.5. Localization of ank1.5 at Z-disks in E14 muscle fibers revealed that ank1.5 is among the earliest SR proteins to assemble, since its organization preceded that of other SR proteins, like SERCA and RyR. After birth, the antibody against the N-terminus of obscurin stained the M-band while that against the C-terminus stained both M-bands and the Z-disks. Starting from postnatal day 1, ank1.5 was found at the level of both M-bands and Z-disks. Altogether, from these results we infer that exposure of some obscurin epitopes changes during skeletal muscle development, resulting in distinct, antibody-specific, localization pattern. Why this occurs is not clear, yet these data indicate that the organization of obscurin at different locations in the sarcomere changes during muscle development and that this might affect the interaction with ank1.5.

    Funded by: Telethon: GGP08153

    Histochemistry and cell biology 2009;131;3;371-82

  • Targeted deletion of alpha-adducin results in absent beta- and gamma-adducin, compensated hemolytic anemia, and lethal hydrocephalus in mice.

    Robledo RF, Ciciotte SL, Gwynn B, Sahr KE, Gilligan DM, Mohandas N and Peters LL

    The Jackson Laboratory, Bar Harbor, ME, USA.

    In the red blood cell (RBC), adducin is present primarily as tetramers of alpha- and beta-subunits at spectrin-actin junctions, or junctional complexes. Mouse RBCs also contain small amounts of gamma-adducin. Platelets contain alpha- and gamma-adducin only. Adducin functions as a barbed-end actin capping protein to regulate actin filament length and recruits spectrin to the ends of actin filaments. To further define adducin's role in vivo, we generated alpha-adducin knockout mice. alpha-Adducin is absent in all tissues examined in homozygous null mice. In RBCs, beta- and gamma-adducin are also absent, indicating that alpha-adducin is the limiting subunit in tetramer formation at the spectrin-actin junction. Similarly, gamma-adducin is absent in alpha-null platelets. alpha-Adducin-null mice display compensated hemolytic anemia with features characteristic of RBCs in hereditary spherocytosis (HS), including spherocytes with significant loss of surface area, decreased mean corpuscular volume (MCV), cell dehydration, and increased osmotic fragility. Platelets maintain their normal discoid shape, and bleeding times are normal. alpha-Adducin-null mice show growth retardation at birth and throughout adulthood. Approximately 50% develop lethal communicating hydrocephalus with striking dilation of the lateral, third, and fourth ventricles. These data indicate that adducin plays a role in RBC membrane stability and in cerebrospinal fluid homeostasis.

    Funded by: NCI NIH HHS: CA34196, P30 CA034196; NHLBI NIH HHS: HL075714, R01 HL075714

    Blood 2008;112;10;4298-307

  • Characterization of glycolytic enzyme interactions with murine erythrocyte membranes in wild-type and membrane protein knockout mice.

    Campanella ME, Chu H, Wandersee NJ, Peters LL, Mohandas N, Gilligan DM and Low PS

    Department of Chemistry, Purdue University, West Lafayette, IN 47907, USA.

    Previous research has shown that glycolytic enzymes (GEs) exist as multienzyme complexes on the inner surface of human erythrocyte membranes. Because GE binding sites have been mapped to sequences on the membrane protein, band 3, that are not conserved in other mammalian homologs, the question arose whether GEs can organize into complexes on other mammalian erythrocyte membranes. To address this, murine erythrocytes were stained with antibodies to glyceraldehyde-3-phosphate dehydrogenase, aldolase, phosphofructokinase, lactate dehydrogenase, and pyruvate kinase and analyzed by confocal microscopy. GEs were found to localize to the membrane in oxygenated erythrocytes but redistributed to the cytoplasm upon deoxygenation, as seen in human erythrocytes. To identify membrane proteins involved in GE assembly, erythrocytes from mice lacking each of the major erythrocyte membrane proteins were examined for GE localization. GEs from band 3 knockout mice were not membrane associated but distributed throughout the cytoplasm, regardless of erythrocyte oxygenation state. In contrast, erythrocytes from mice lacking alpha-spectrin, ankyrin, protein 4.2, protein 4.1, beta-adducin, or dematin headpiece exhibited GEs bound to the membrane. These data suggest that oxygenation-dependent assembly of GEs on the membrane could be a general phenomenon of mammalian erythrocytes and that stability of these interactions depends primarily on band 3.

    Funded by: NHLBI NIH HHS: HL075714, R01 HL075714; NIGMS NIH HHS: GM24417-29, R01 GM024417, R37 GM024417

    Blood 2008;112;9;3900-6

  • Molecular interactions with obscurin are involved in the localization of muscle-specific small ankyrin1 isoforms to subcompartments of the sarcoplasmic reticulum.

    Armani A, Galli S, Giacomello E, Bagnato P, Barone V, Rossi D and Sorrentino V

    Molecular Medicine Section, Department of Neuroscience, University of Siena, 53100 Siena, Italy.

    We report here on experiments aimed to characterise the molecular basis of the interactions between muscle-specific ankyrin1 isoforms localized on the sarcoplasmic reticulum and obscurin a protein associated with the contractile apparatus. A novel small muscle-specific ankyrin isoform, ank1.9 was identified that, similarly to the known ank1.5 isoform, was able to bind to obscurin in yeast two-hybrid assay and in pull-down experiments. Two distinct binding sites in the C-terminus of obscurin were found to mediate binding with ank1.5 and ank1.9. Interactions between ank1.5 and ank1.9 with recombinant proteins containing one or two of the binding sites of obscurin were confirmed by expressing recombinant proteins in NIH3T3 cells. In cultured myotubes, ank1.5 and ank1.9 colocalized with endogenous obscurin at the M-band region. In contrast with evidence of efficient binding between small ank1 isoforms and obscurin, in vitro interaction studies and transfection experiments in myotubes indicated that small ank1 isoforms do not efficiently interact with titin. Altogether, these results support a role of obscurin in mediating the subcellular localization of small ank1 isoforms in striated muscle cells. Given that the localization of small muscle-specific ank1 isoforms mirrors that of obscurin, we propose that obscurin and small ank1 isoforms may form stable interactions that may be relevant to connect the sarcoplasmic reticulum and the contractile apparatus in skeletal muscle cells.

    Funded by: Telethon: GGP02168

    Experimental cell research 2006;312;18;3546-58

  • Major erythrocyte membrane protein genes in EKLF-deficient mice.

    Nilson DG, Sabatino DE, Bodine DM and Gallagher PG

    Hematopoiesis Section, Genetics and Molecular Biology Branch, National Human Genome Research Institute, National Institutes of Health, Bethesda, MD, USA.

    Objectives: Mice deficient in the transcription factor erythroid Krüppel-like factor, KLF1 (EKLF) die approximately 14.5 days postcoitum of anemia, attributed to decreased expression of the beta-globin gene. The objectives of this study were to rescue EKLF-deficient embryos with mice expressing gamma-globin from beta-spectrin or ankyrin promoters and to characterize expression of the major erythrocyte membrane genes in EKLF-deficient cells.

    Methods: Transgenic beta-spectrin/gamma-globin or ankyrin/gamma-globin mice were bred onto EKLF-deficient and wild-type backgrounds. Animals were genotyped, gamma-globin mRNA levels measured, and hemoglobin electrophoresis performed. Steady-state mRNA levels and transcriptional rates of the major erythrocyte membrane protein genes were assayed.

    Results: beta-spectrin/gamma-globin or ankyrin/gamma-globin mice on EKLF-deficient and wild-type backgrounds had identical levels of gamma-globin mRNA, indicating EKLF-independence of these promoters. gamma-Globin expression improved globin chain imbalance, but hemolysis was not improved and no live-born EKLF-deficient/(A)gamma-globin mice were obtained. Circulating erythroid cells from EKLF-deficient/(A)gamma-globin embryos exhibited hemolysis reminiscent of that seen in patients with severe erythrocyte membrane defects. Levels of beta-spectrin, ankyrin, and band 3 mRNA, but not alpha-spectrin, were decreased in EKLF-deficient fetal liver RNA. In a run-on assay, levels of transcription of the ankyrin and band 3 genes were decreased in EKLF-deficient fetal liver nuclei.

    Conclusions: These results indicate that the EKLF-responsive regions of the ankyrin and beta-spectrin genes are outside their promoters and that EKLF is necessary for full transcriptional activity of the ankyrin and band 3 genes; the results also provide additional evidence that defects in addition to beta-globin deficiency, including an abnormal erythrocyte membrane, contribute to the anemia and embryonic lethality in EKLF-deficient mice.

    Funded by: NHLBI NIH HHS: HL65448; NIDDK NIH HHS: DK/HL62039

    Experimental hematology 2006;34;6;705-12

  • Comprehensive identification of phosphorylation sites in postsynaptic density preparations.

    Trinidad JC, Specht CG, Thalhammer A, Schoepfer R and Burlingame AL

    Mass Spectrometry Facility, Department of Pharmaceutical Chemistry, University of California, San Francisco, California 94143, USA.

    In the mammalian central nervous system, the structure known as the postsynaptic density (PSD) is a dense complex of proteins whose function is to detect and respond to neurotransmitter released from presynaptic axon terminals. Regulation of protein phosphorylation in this molecular machinery is critical to the activity of its components, which include neurotransmitter receptors, kinases/phosphatases, scaffolding molecules, and proteins regulating cytoskeletal structure. To characterize the phosphorylation state of proteins in PSD samples, we combined strong cation exchange (SCX) chromatography with IMAC. Initially, tryptic peptides were separated by cation exchange and analyzed by reverse phase chromatography coupled to tandem mass spectrometry, which led to the identification of phosphopeptides in most SCX fractions. Because each of these individual fractions was too complex to characterize completely in single LC-MS/MS runs, we enriched for phosphopeptides by performing IMAC on each SCX fraction, yielding at least a 3-fold increase in identified phosphopeptides relative to either approach alone (SCX or IMAC). This enabled us to identify at least one site of phosphorylation on 23% (287 of 1,264) of all proteins found to be present in the postsynaptic density preparation. In total, we identified 998 unique phosphorylated peptides, mapping to 723 unique sites of phosphorylation. At least one exact site of phosphorylation was determined on 62% (621 of 998) of all phosphopeptides, and approximately 80% of identified phosphorylation sites are novel.

    Funded by: NCRR NIH HHS: RR14606; Wellcome Trust

    Molecular & cellular proteomics : MCP 2006;5;5;914-22

  • BGEM: an in situ hybridization database of gene expression in the embryonic and adult mouse nervous system.

    Magdaleno S, Jensen P, Brumwell CL, Seal A, Lehman K, Asbury A, Cheung T, Cornelius T, Batten DM, Eden C, Norland SM, Rice DS, Dosooye N, Shakya S, Mehta P and Curran T

    Department of Developmental Neurobiology, St. Jude Children's Research Hospital, Memphis, Tennessee, United States.

    Funded by: NINDS NIH HHS: 5R37NS036558, N01-NS-0-2331, R37 NS036558

    PLoS biology 2006;4;4;e86

  • The status, quality, and expansion of the NIH full-length cDNA project: the Mammalian Gene Collection (MGC).

    Gerhard DS, Wagner L, Feingold EA, Shenmen CM, Grouse LH, Schuler G, Klein SL, Old S, Rasooly R, Good P, Guyer M, Peck AM, Derge JG, Lipman D, Collins FS, Jang W, Sherry S, Feolo M, Misquitta L, Lee E, Rotmistrovsky K, Greenhut SF, Schaefer CF, Buetow K, Bonner TI, Haussler D, Kent J, Kiekhaus M, Furey T, Brent M, Prange C, Schreiber K, Shapiro N, Bhat NK, Hopkins RF, Hsie F, Driscoll T, Soares MB, Casavant TL, Scheetz TE, Brown-stein MJ, Usdin TB, Toshiyuki S, Carninci P, Piao Y, Dudekula DB, Ko MS, Kawakami K, Suzuki Y, Sugano S, Gruber CE, Smith MR, Simmons B, Moore T, Waterman R, Johnson SL, Ruan Y, Wei CL, Mathavan S, Gunaratne PH, Wu J, Garcia AM, Hulyk SW, Fuh E, Yuan Y, Sneed A, Kowis C, Hodgson A, Muzny DM, McPherson J, Gibbs RA, Fahey J, Helton E, Ketteman M, Madan A, Rodrigues S, Sanchez A, Whiting M, Madari A, Young AC, Wetherby KD, Granite SJ, Kwong PN, Brinkley CP, Pearson RL, Bouffard GG, Blakesly RW, Green ED, Dickson MC, Rodriguez AC, Grimwood J, Schmutz J, Myers RM, Butterfield YS, Griffith M, Griffith OL, Krzywinski MI, Liao N, Morin R, Morrin R, Palmquist D, Petrescu AS, Skalska U, Smailus DE, Stott JM, Schnerch A, Schein JE, Jones SJ, Holt RA, Baross A, Marra MA, Clifton S, Makowski KA, Bosak S, Malek J and MGC Project Team

    The National Institutes of Health's Mammalian Gene Collection (MGC) project was designed to generate and sequence a publicly accessible cDNA resource containing a complete open reading frame (ORF) for every human and mouse gene. The project initially used a random strategy to select clones from a large number of cDNA libraries from diverse tissues. Candidate clones were chosen based on 5'-EST sequences, and then fully sequenced to high accuracy and analyzed by algorithms developed for this project. Currently, more than 11,000 human and 10,000 mouse genes are represented in MGC by at least one clone with a full ORF. The random selection approach is now reaching a saturation point, and a transition to protocols targeted at the missing transcripts is now required to complete the mouse and human collections. Comparison of the sequence of the MGC clones to reference genome sequences reveals that most cDNA clones are of very high sequence quality, although it is likely that some cDNAs may carry missense variants as a consequence of experimental artifact, such as PCR, cloning, or reverse transcriptase errors. Recently, a rat cDNA component was added to the project, and ongoing frog (Xenopus) and zebrafish (Danio) cDNA projects were expanded to take advantage of the high-throughput MGC pipeline.

    Funded by: PHS HHS: N01-C0-12400

    Genome research 2004;14;10B;2121-7

  • Rh-RhAG/ankyrin-R, a new interaction site between the membrane bilayer and the red cell skeleton, is impaired by Rh(null)-associated mutation.

    Nicolas V, Le Van Kim C, Gane P, Birkenmeier C, Cartron JP, Colin Y and Mouro-Chanteloup I

    INSERM U76, Institut National de la Transfusion Sanguine, 6 Rue Alexandre Cabanel, 75015 Paris, France.

    Several studies suggest that the Rh complex represents a major interaction site between the membrane lipid bilayer and the red cell skeleton, but little is known about the molecular basis of this interaction. We report here that ankyrin-R is capable of interacting directly with the C-terminal cytoplasmic domain of Rh and RhAG polypeptides. We first show that the primary defect of ankyrin-R in normoblastosis (nb/nb) spherocytosis mutant mice is associated with a sharp reduction of RhAG and Rh polypeptides. Secondly, our flow cytometric analysis of the Triton X-100 extractability of recombinant fusion proteins expressed in erythroleukemic cell lines suggests that the C-terminal cytoplasmic domains of Rh and RhAG are sufficient to mediate interaction with the erythroid membrane skeleton. Using the yeast two-hybrid system, we demonstrate a direct interaction between the cytoplasmic tails of Rh and RhAG and the second repeat domain (D2) of ankyrin-R. This finding is supported by the demonstration that the substitution of Asp-399 in the cytoplasmic tail of RhAG, a mutation associated with the deficiency of the Rh complex in one Rhnull patient, totally impaired interaction with domain D2 of ankyrin-R. These results identify the Rh/RhAG-ankyrin complex as a new interaction site between the red cell membrane and the spectrin-based skeleton, the disruption of which might result in the stomato-spherocytosis typical of Rhnull red cells.

    The Journal of biological chemistry 2003;278;28;25526-33

  • Normoblastosis, a murine model for ankyrin-deficient hemolytic anemia, is caused by a hypomorphic mutation in the erythroid ankyrin gene Ank1.

    Birkenmeier CS, Gifford EJ and Barker JE

    The Jackson Laboratory, Bar Harbor, ME 04609, USA. cb@jax.org

    Ankyrin deficiency is one of the most common causes of hereditary spherocytosis in humans. A spontaneous mutation, normoblastosis (Ank1nb), discovered in 1969 in a mouse stock maintained at the Jackson Laboratory, provides an important animal model for these human ankyrin-deficient anemias. Study of this model has led to the finding of multiple isoforms of Ank1 as well as Ank1nb-related pathology in nonerythroid tissues. To enhance the usefulness of this model, we have identified the Ank1nb mutation as the deletion of a guanosine residue in exon 36 of the erythroid ankyrin gene (Ank1). This results in a frame shift that introduces a stop 13 codons downstream and predicts a 157 kDa nb-ankyrin lacking the regulatory domain but including intact membrane- and spectrin-binding domains. By epitope scanning on immunoblots, we show that a previously reported protein (p150) found in nb reticulocytes is the predicted nb-ankyrin. Existing evidence indicates that this protein is functional, making the normoblastosis mutation a hypomorph rather than a null as originally thought. The nb-ankyrin provides an explanation for the milder phenotype displayed by nb/nb animals relative to the murine spectrin-deficient anemias, spherocytosis (Spna1(sph), Spna1(sph-1J), Spna1(sph-2BC), Spna1(sph-DEM)) and jaundiced (Spnb1(ja)), and suggests that truncated ankyrins could be useful in gene replacement therapy.

    The hematology journal : the official journal of the European Haematology Association 2003;4;6;445-9

  • A minimal ankyrin promoter linked to a human gamma-globin gene demonstrates erythroid specific copy number dependent expression with minimal position or enhancer dependence in transgenic mice.

    Sabatino DE, Wong C, Cline AP, Pyle L, Garrett LJ, Gallagher PG and Bodine DM

    Hematopoiesis Section, Genetics and Molecular Biology Branch, NHGRI, National Institutes of Health, Bethesda, Maryland 20892, USA.

    In red blood cells ankyrin (ANK-1) provides the primary linkage between the erythrocyte membrane skeleton and the plasma membrane. We have previously demonstrated that a 271-bp 5'-flanking region of the ANK-1 gene has promoter activity in erythroid, but not non-erythroid, cell lines. To determine whether the ankyrin promoter could direct erythroid-specific expression in vivo, we analyzed transgenic mice containing the ankyrin promoter fused to the human (A)gamma-globin gene. Sixteen of 17 lines expressed the transgene in erythroid cells indicating nearly position-independent expression. We also observed a significant correlation between the level of Ank/(A)gamma-globin mRNA and transgene copy number. The level of Ank/(A)gamma mRNA averaged 11% of mouse alpha-globin mRNA per gene copy at all developmental stages. The addition of the HS2 enhancer from the beta-globin locus control region to the Ank/(A)gamma-globin transgene resulted in Ank/(A)gamma-globin mRNA expression in embryonic and fetal erythroid cells in six of eight lines but resulted in absent or dramatically reduced levels of Ank/(A)gamma-globin mRNA in adult erythroid cells in eight of eight transgenic lines. These data indicate that the minimal ankyrin promoter contains all sequences necessary and sufficient for erythroid-specific, copy number-dependent, position-independent expression of the human (A)gamma-globin gene.

    The Journal of biological chemistry 2000;275;37;28549-54

  • Gene transfer to ankyrin-deficient bone marrow corrects spherocytosis in vitro.

    Dooner GJ, Barker JE, Gallagher PG, Debatis ME, Brown AH, Forget BG and Becker PS

    Cancer Center and Gene Therapy Link Laboratory, University of Massachusetts Medical School, Worcester, USA.

    Objective: The goal of this study was to transfer by retroviral vector the cDNA for ankyrin to progenitors from normal bone marrow and from the nb/nb spherocytosis mutant deficient in expression of full-length ankyrin to achieve erythroid expression of functional ankyrin protein.

    A minigene composed of the human ankyrin promoter, murine ankyrin cDNA, and the 3' human domain corresponding to the ankyrin 2.2 isoform was assembled in the retroviral vector, pG1. Murine erythroleukemia (MEL) cells, normal murine bone marrow cells, 3T3 fibroblasts, and nb/nb mutant bone marrow and spleen cells were transduced with the retroviral supernatant. Transduced mutant cells were induced to differentiate in liquid culture. Gene transfer was assessed by colony polymerase chain reaction (PCR) and reverse transcriptase (RT)-PCR, immunofluorescence, and Southern, Northern, and Western blot analysis.

    Results: MEL cells, normal bone marrow progenitors, and nb/nb cells were all successfully transduced and expressed ankyrin by RT-PCR and Western blot. Transduced murine 3T3 fibroblasts and MEL cells exhibited cell membrane staining by immunofluorescence. Colony RT-PCR demonstrated dependence of expression on erythropoietin. In vitro, the transduced nb/nb cells matured to polychromatophils, whereas nontransduced nb/nb cells matured to microspherocytes.

    Conclusion: Retroviral transfer of ankyrin corrected the defect leading to formation of microspherocytes in erythroid differentiation cultures from the nb/nb mutant. The human ankyrin promoter conferred erythropoietin-dependent expression in normal and mutant erythroid progenitors, which could have implications for the gene therapy of human hemolytic anemias.

    Funded by: NHLBI NIH HHS: R01 HL29305, R01 HL63184; NIDDK NIH HHS: R01 DK27726; ...

    Experimental hematology 2000;28;7;765-74

  • Erythroid phosphatidyl serine exposure is not predictive of thrombotic risk in mice with hemolytic anemia.

    Wandersee NJ, Tait JF and Barker JE

    The Jackson Laboratory, Bar Harbor, Maine 04609, USA.

    Thrombosis is a major complication of human hemolytic anemias such as sickle cell disease, thalassemia, and severe hereditary spherocytosis (HS). Mice with severe HS and severe hereditary elliptocytosis (HE) also suffer from thrombosis, with incidences ranging from 15 and 22% in beta-spectrin- and ankyrin-deficient mice, respectively, to 85 to 100% in alpha-spectrin-deficient and band 3 knockout mice. A contributing factor to thrombosis could be loss of phospholipid asymmetry of the mutant red blood cells (RBCs), with concomitant exposure of the aminophospholipid phosphatidylserine (PS). Increased PS exposure occurs in RBCs from sickle cell and thalassemia patients and in RBCs from band 3-deficient mice. To determine if increased PS exposure correlates with thrombotic risk in HS and HE mice with ankyrin, beta-spectrin, and alpha-spectrin deficiencies, measurements of FITC-labeled annexin V binding to externalized PS on RBCs were performed. PS exposure is elevated in all mice with HS and HE, but the percentage of RBCs with exposed PS does not correlate with thrombotic risk in these mice.

    Funded by: NCI NIH HHS: CA34193; NHLBI NIH HHS: R01 HL29305; NIDDK NIH HHS: F32 DK09482

    Blood cells, molecules & diseases 2000;26;1;75-83

  • The modular nature of apoptotic signaling proteins.

    Hofmann K

    MEMOREC Stoffel GmbH, Köln, Germany. Kay.Hofmann@memorec.com

    Apoptosis, initiated by a variety of stimuli, is a physiological process that engages a well-ordered signaling cascade, eventually leading to the controlled death of the cell. The most extensively studied apoptotic stimulus is the binding of death receptors related to CD95 (Fas/Apo1) by their respective ligands. During the last years, a considerable number of proteins have been identified which act together in the receptor-proximal part of the signaling pathway. Based on localized regions of sequence similarity, it has been predicted that these proteins consist of several independently folding domains. In several cases these predictions have been confirmed by structural studies; in other cases they are at least supported by experimental data. This review focuses on the three most widespread domain families found in the apoptotic signaling proteins: the death domain, the death effector domain and the caspase recruitment domain. The recently discovered analogies between these domains, both in structure and in function, have shed some light on the overall architecture of the pathway leading from death receptor ligation to the activation of caspases and eventually to the apoptotic phenotype.

    Cellular and molecular life sciences : CMLS 1999;55;8-9;1113-28

  • An alternative first exon in the distal end of the erythroid ankyrin gene leads to production of a small isoform containing an NH2-terminal membrane anchor.

    Birkenmeier CS, Sharp JJ, Gifford EJ, Deveau SA and Barker JE

    The Jackson Laboratory, Bar Harbor, Maine 04609, USA. cb@aretha.jax.org

    Mouse erythroid ankyrin is encoded by the Ank1 gene on Chromosome 8. The best studied isoform is 210 kDa and contains three large functional domains. We have recently reported a small Ank1 isoform (relative mobility 25 kDa) that localizes to the M and Z lines in skeletal muscle. Analyses of cDNA and genomic clones show that three transcripts of 3.5, 2.0, and 1.6 kb code for this protein. The different transcript sizes are due to their 3'-untranslated regions. They are encoded by a new first exon located in intron 39 of the Ank1 gene and three previously described Ank1 exons (40, 41, and 42). The 5'-flanking region contains a putative muscle-specific promoter. The sequence of the first 72 amino acids is novel and is predicted to form a transmembrane helix at the NH2-terminus. Functional testing of the putative transmembrane segment indicates that it acts as a membrane anchor, suggesting that the new Ank1 isoform may play an important role in organizing the contractile apparatus within the cell.

    Funded by: NCI NIH HHS: CA34196; NHLBI NIH HHS: HL29305; NIDDK NIH HHS: DK27726

    Genomics 1998;50;1;79-88

  • A high-resolution genetic map of the nervous locus on mouse chromosome 8.

    De Jager PL, Harvey D, Polydorides AD, Zuo J and Heintz N

    Howard Hughes Medical Institute, Laboratory of Molecular Biology, Rockefeller University, New York, New York 10021, USA.

    The nervous (nr) mutant mouse displays two gross recessive traits: both an exaggeration of juvenile hyperactivity and a pronounced ataxia become apparent during the third and fourth postnatal weeks. Using an intersubspecific intercross, we have established a high-resolution map of a segment of mouse chromosome 8 that places the nr locus in a genomic segment defined by D8Rck1 on the centromeric end and D8Mit3 on the telomeric end. This map position places the nr locus within the BALB/cGr congenic region of the C3HeB/ FeJ-nr strain, confirming the accuracy of our study. We used this map position to identify and evaluate three genes-ankyrin 1, cortexin, and farnesyltransferase-as candidates for the nr gene. These three genes were eliminated from consideration but allowed us to establish the conservation of synteny between the region containing the nr locus and a segment of the short arm of human chromosome 8 (8p21-p11.2). Finally, the incomplete penetrance of the nr phenotype led us to perform a screen for modifier loci, and we present evidence that such a nervous modifier locus may exist on mouse chromosome 5.

    Funded by: NIGMS NIH HHS: GM07739

    Genomics 1998;48;3;346-53

  • Red cell membranes of ankyrin-deficient nb/nb mice lack band 3 tetramers but contain normal membrane skeletons.

    Yi SJ, Liu SC, Derick LH, Murray J, Barker JE, Cho MR, Palek J and Golan DE

    Department of Biomedical Research, St. Elizabeth's Medical Center of Boston, Tufts University School of Medicine, Boston, Massachusetts, USA.

    The role of ankyrin in the formation and stabilization of the spectrin-based skeletal meshwork and of band 3 oligomers was studied by characterizing, in nb/nb mouse red cells, the effect of ankyrin deficiency on skeletal ultrastructure, band 3-skeleton associations, and band 3 oligomeric states. Despite severe ankyrin deficiency, nb/nb mouse red cell skeletal components formed a relatively uniform two-dimensional hexagonal array of junctional complexes cross-linked by spectrin tetramers. Treatment of nb/nb ghosts with the nonionic detergent C12E8 (octaethylene glycol n-dodecyl monoether) resulted in nearly complete extraction of band 3. The extracted band 3 was present exclusively as band 3 dimers. Fluorescence photobleaching recovery and polarized fluorescence depletion measurements showed increases in the laterally (33% vs 10%) and rotationally (90% vs 76%) mobile fractions of band 3 in intact nb/nb compared to control red cells. The rotational correlation time of the major fraction of band 3 molecules was 10-fold shorter in nb/nb compared to control red cells, indicating a significant relaxation of rotational constraints in nb/nb cells. These data suggest that, although ankyrin plays a major role in strengthening the attachment of the skeleton to the membrane bilayer, ankyrin is not required for the formation of a stable two-dimensional spectrin-based skeleton. The absence of band 3 tetramers in the membrane of ankyrin-deficient red cells suggests that ankyrin is required for the formation of stable band 3 tetramers.

    Funded by: NHLBI NIH HHS: HL 37462, HL27215, HL32854; ...

    Biochemistry 1997;36;31;9596-604

  • Small, membrane-bound, alternatively spliced forms of ankyrin 1 associated with the sarcoplasmic reticulum of mammalian skeletal muscle.

    Zhou D, Birkenmeier CS, Williams MW, Sharp JJ, Barker JE and Bloch RJ

    Department of Physiology, University of Maryland School of Medicine, Baltimore 21201, USA.

    We have recently found that the erythroid ankyrin gene, Ank1, expresses isoforms in mouse skeletal muscle, several of which share COOH-terminal sequence with previously known Ank1 isoforms but have a novel, highly hydrophobic 72-amino acid segment at their NH2 termini. Here, through the use of domain-specific peptide antibodies, we report the presence of the small ankyrins in rat and rabbit skeletal muscle and demonstrate their selective association with the sarcoplasmic reticulum. In frozen sections of rat skeletal muscle, antibodies to the spectrin-binding domain (anti-p65) react only with a 210-kD Ank1 and label the sarcolemma and nuclei, while antibodies to the COOH terminus of the small ankyrin (anti-p6) react with peptides of 20 to 26 kD on immunoblots and decorate the myoplasm in a reticular pattern. Mice homozygous for the normoblastosis mutation (gene symbol nb) are deficient in the 210-kD ankyrin but contain normal levels of the small ankyrins in the myoplasm. In nb/nb skeletal muscle, anti-p65 label is absent from the sarcolemma, whereas anti-p6 label shows the same distribution as in control skeletal muscle. In normal skeletal muscle of the rat, anti-p6 decorates Z lines, as defined by antidesmin distribution, and is also present at M lines where it surrounds the thick myosin filaments. Immunoblots of the proteins isolated with rabbit sarcoplasmic reticulum indicate that the small ankyrins are highly enriched in this fraction. When expressed in transfected HEK 293 cells, the small ankyrins are distributed in a reticular pattern resembling the ER if the NH2-terminal hydrophobic domain is present, but they are uniformly distributed in the cytosol if this domain is absent. These results suggest that the small ankyrins are integral membrane proteins of the sarcoplasmic reticulum. We propose that, unlike the 210-kD form of Ank1, previously localized to the sarcolemma and believed to be a part of the supporting cytoskeleton, the small Ank1 isoforms may stabilize the sarcoplasmic reticulum by linking it to the contractile apparatus.

    Funded by: NHLBI NIH HHS: HL29305; NICHD NIH HHS: HD16596; NINDS NIH HHS: NS17282

    The Journal of cell biology 1997;136;3;621-31

  • Ankyrin-1 mutations are a major cause of dominant and recessive hereditary spherocytosis.

    Eber SW, Gonzalez JM, Lux ML, Scarpa AL, Tse WT, Dornwell M, Herbers J, Kugler W, Ozcan R, Pekrun A, Gallagher PG, Schröter W, Forget BG and Lux SE

    Division of Hematology/Oncology, Children's Hospital, Boston, Massachusetts 02115, USA.

    Hereditary spherocytosis (HS) is the most common inherited haemolytic anaemia in Northern Europeans. The primary molecular defects reside in the red blood cell (RBC) membrane, particularly in proteins that link the membrane skeleton to the overlying lipid bilayer and its integral membrane constituents. Ankyrin-1 is the predominant linker molecule. It attaches spectrin, the major skeletal protein, to the cytoplasmic domain of band 3, the RBC anion exchanger. Two-thirds of patients with HS have combined spectrin and ankyrin-1 deficiency; deficiency of band 3 occurs in about 15 to 20% (ref.1). These data suggest that ankyrin-1 or band 3 defects may be common in HS. To test this we screened all 42 coding exons plus the 5' untranslated/promoter region of ankyrin-1 and the 19 coding exons of band 3 in 46 HS families. Twelve ankyrin-1 mutations and five band 3 mutations were identified. Missense mutations and a mutation in the putative ankyrin-1 promoter were common in recessive HS. In contrast, ankyrin-1 and band 3 frameshift and nonsense null mutations prevailed in dominant HS. Increased accumulation of the normal protein product partially compensated for the ankyrin-1 or band 3 defects in some of these null mutations. Our findings indicate that ankyrin-1 mutations are a major cause of dominant and recessive HS (approximately 35 to 65%), that band 3 mutations are less common (approximately 15 to 25%), and that the severity of HS is modified by factors other than the primary gene defect.

    Funded by: NHLBI NIH HHS: HL15157, HL32262; NIDDK NIH HHS: DK34083; ...

    Nature genetics 1996;13;2;214-8

  • Increased cation permeability in mutant mouse red blood cells with defective membrane skeletons.

    Joiner CH, Franco RS, Jiang M, Franco MS, Barker JE and Lux SE

    Department of Pediatrics, University of Cincinnati College of Medicine, OH, USA.

    Cellular cation homeostasis in mouse erythrocytes with defective membrane skeletons was examined in three mouse mutants, hemolytic anemia (sphha/sphha), spherocytosis (sph/sph), and normoblastosis (nb/nb), and compared with reticulocytes produced by repetitive bleeding of congenic normal mice. To assess reticulocyte maturity, nucleic acid and transferrin receptor contents were measured by fluorescence flow cytometry; mutant cells were somewhat more mature than normal reticulocytes by these criteria. Red blood cell (RBC) sodium contents (Nac+) in homozygous sphha/sphha, sph/sph, and nb/nb animals were 30.1 +/- 0.9, 28.9 +/- 0.3, and 26.9 +/- 1.5 mmol/L cell, respectively, whereas cellular potassium (Kc+) was 102 +/- 2.6, 101 +/- 7.8, and 97.4 +/- 3.0. Nac+ and Kc+ in normal reticulocyte preparations were 11.3 +/- 0.7 and 123 +/- 10, respectively. Net Na+ and K+ fluxes in the presence of ouabain were markedly increased in mutant RBCs. Sodium uptake was 14.8 +/- 1.6, 15.4 +/- 3.3, and 14.7 +/- 3.1 mmol/L cell/h in sphha/sphha, sph/sph, and nb/nb mutants, respectively, whereas K+ loss was 17.0 +/- 4.0, 15.0 +/- 3.8, and 14.1 +/- 2.6. Normal mouse reticulocytes gained Na+ at a rate of 3.9 +/- 1.0 mmol/L cell/h and lost K+ at 6.0 +/- 2.1, rates indistinguishable from those in mature mouse RBCs. Potassium loss from sphha/sphha and nb/nb cells was not dependent on the presence of a Na+ gradient, and net cation movements were insensitive to bumetanide (sphha/sphha and nb/nb RBCs) and to chloride replacement with sulfamate (nb/nb cells). We conclude that mutant mouse RBCs with dysfunctional membrane skeletons have increased passive permeability to monovalent cations. These findings support a role of the membrane skeleton in the maintenance of the membrane permeability barrier and suggest that the abnormal permeability associated with human hereditary spherocytosis and elliptocytosis may be a consequence of the membrane skeleton defects reported in these disorders.

    Funded by: NHLBI NIH HHS: HL 29305, HL 37515, HL 51174; ...

    Blood 1995;86;11;4307-14

  • Analysis of the role of membrane polarity in polycystic kidney disease of transgenic SBM mice.

    Barisoni L, Trudel M, Chrétien N, Ward L, van Adelsberg J and D'Agati V

    Department of Pathology, Columbia University, New York, New York 10032, USA.

    Altered membrane polarity has been proposed as an important pathogenetic factor in the development of renal cysts in polycystic kidney disease. To determine whether this alteration in epithelial phenotype is a primary or secondary phenomenon, we examined the epithelial membrane polarity of SBM transgenic mice, in which epithelial proliferation mediated by the c-myc oncogene is an established primary event. Kidneys from 32 transgenic mice and 10 age-matched controls from fetal to adult age were immunostained with antibodies to Na,K-ATPase, fodrin, ankyrin, E-cadherin, and tubule segment-specific lectins. In normal control mice, Na,K-ATPase localization was apical in fetal kidneys but became translocated to the basolateral membrane at maturity. Early microcysts in fetal transgenic kidneys displayed similar (95 to 100%) apical Na,K-ATPase. In young and newborn transgenic mice (1 to 8 days of age), Na,K-ATPase localization was extremely heterogeneous. Noncystic tubules demonstrated either apical (mean 23 to 28%), basolateral (mean 48 to 58%), mixed (mean 4 to 15%), or absent (mean 10 to 13%) staining for Na,K-ATPase. Apical Na,K-ATPase was more frequently observed in early cysts (mean 55%) in young transgenic mice but became less prevalent in adult mice (mean 22%), where 30% of cysts had basolateral staining, 39% mixed patterns, and 9% absent staining. Macrocysts typically lost all Na,K-ATPase reactivity. At all ages, Na,K-ATPase colocalized well with cytoskeletal proteins ankyrin and fodrin. These heterogeneous patterns of Na,K-ATPase staining indicate that although altered cell polarity is frequent in early cystic epithelium of SBM mice, it is not a prerequisite to cystogenesis or progressive cyst enlargement. In conclusion, our results support the view that altered cystic membrane polarity is not a primary process, but represents the persistence of an immature epithelial phenotype characteristic of proliferative polycystic kidney disease epithelia.

    Funded by: NIDDK NIH HHS: DK44864

    The American journal of pathology 1995;147;6;1728-35

  • Decreased content of protein 4.2 in ankyrin-deficient normoblastosis (nb/nb) mouse red blood cells: evidence for ankyrin enhancement of protein 4.2 membrane binding.

    Rybicki AC, Musto S and Schwartz RS

    Albert Einstein College of Medicine-Montefiore Medical Center, Division of Hematology, Bronx, NY 10467, USA.

    Homozygous normoblastosis (nb/nb) mice, whose red blood cell (RBC) membranes are nearly completely deficient in full-length 210-kD ankyrin, were used to study interactions between ankyrin and protein 4.2 (P4.2). Although it is unclear whether or not these proteins interact in the membrane, both ankyrin and P4.2 bind to the cytoplasmic domain of band 3 (cdb3). In addition to the complete deficiency of full-length ankyrin, nb/nb RBC membranes are also partially spectrin deficient, resulting in morphologically spherocytic and mechanically fragile cells. A new finding was that nb/nb RBC membranes are severely (approximately 73%) P4.2 deficient compared with wild type (+/+) or high reticulocyte mouse RBC membranes. Metabolic labeling of nb/nb reticulocytes showed active P4.2 synthesis at levels comparable with high reticulocyte controls suggesting that the nb/nb P4.2 deficiency was not the result of defective P4.2 synthesis. Reconstitution of nb/nb inside-out vesicles (IOVs) with human RBC ankyrin restored ankyrin levels to approximately 80% of +/+ IOV levels and increased binding of exogenously added human RBC P4.2 by approximately 60%. These results suggest that ankyrin is required for normal associations of P4.2 with the RBC membrane.

    Funded by: NHLBI NIH HHS: HL29305, HL38655, HL41382

    Blood 1995;86;9;3583-9

  • Ank3 (epithelial ankyrin), a widely distributed new member of the ankyrin gene family and the major ankyrin in kidney, is expressed in alternatively spliced forms, including forms that lack the repeat domain.

    Peters LL, John KM, Lu FM, Eicher EM, Higgins A, Yialamas M, Turtzo LC, Otsuka AJ and Lux SE

    Division of Hematology/Oncology, Children's Hospital, Boston, Massachusetts 02115, USA.

    We cloned a novel ankyrin, Ank3, from mouse kidney cDNA. The full-length transcript is predicted to encode a 214-kD protein containing an 89 kD, NH2 terminal "repeat" domain; a 65 kD, central "spectrin-binding" domain; and a 56 kD, COOH-terminal "regulatory" domain. The Ank3 gene maps to mouse Chromosome 10, approximately 36 cM from the centromere, a locus distinct from Ank1 and Ank2. Ank3 is the major kidney ankyrin. Multiple transcripts of approximately 7.5, 6.9, 6.3, 5.7, 5.1, and 4.6 kb are highly expressed in kidney where Ank1 and Ank2 mRNAs are barely detectable. The smaller mRNAs (< or = 6.3 kb) lack the entire repeat domain. These transcripts have a unique 5'untranslated region and NH2-terminal sequence and encode a predicted protein of 121 kD. Two small sequences of 21 and 18 amino acids are alternatively spliced at the junction of the repeat and spectrin-binding domains in the larger (> or = 6.9 kb) RNAs. Alternative splicing of a 588 bp sequence (corresponding to a 21.5-kD acidic amino acid sequence) within the regulatory domain also occurs. Ank3 is much more widely expressed than previously described ankyrins. By Northern hybridization or immunocytochemistry, it is present in most epithelial cells, in neuronal axons, in muscle cells, and in megakaryocytes/platelets, macrophages, and the interstitial cells of Leydig (testis). On immunoblots, an antibody raised to a unique regions of the regulatory domain detects multiple Ank3 isoforms in the kidney (215, 200, 170, 120, 105 kD) and in other tissues. The 215/200 kD and 120/105-kD kidney proteins are close to the sizes predicted for the 7.5/6.9- and 6.3/5.7-kb RNAs (with/without the 588-bp acidic insert). Interestingly, it appears that Ank3 exhibits a polarized distribution only in tissues that express the approximately 7.0-kb isoforms, the only isoforms in the kidney that contain the repeat domain. In tissues where smaller transcripts (< or = 6.3 kb) are expressed. Ank3 is diffusely distributed in some or all cells and may be associated with cytoplasmic structures. We conclude that Ank3 is a broadly distributed epithelial ankyrin and is the major ankyrin in the kidney and other tissues, where it plays an important role in the polarized distribution of many integral membrane proteins.

    Funded by: NCRR NIH HHS: RR01183; NHLBI NIH HHS: HL32262; NIDDK NIH HHS: DK34083

    The Journal of cell biology 1995;130;2;313-30

  • Homologs of genes and anonymous loci on human chromosome 13 map to mouse chromosomes 8 and 14.

    Koizumi T, Hendel E, Lalley PA, Tchetgen MB and Nadeau JH

    Laboratory Animal Center, Fukui Medical School, Japan.

    To enhance the comparative map for human Chromosome (Chr) 13, we identified clones for human genes and anonymous loci that cross-hybridized with their mouse homologs and then used linkage crosses for mapping. Of the clones for four genes and twelve anonymous loci tested, cross-hybridization was found for six, COL4A1, COL4A2, D13S26, D13S35, F10, and PCCA. Strong evidence for homology was found for COL4A1, COL4A2, D13S26, D13S35, and F10, but only circumstantial homology evidence was obtained for PCCA. To genetically map these mouse homologs (Cf10, Col4a1, Col4a2, D14H13S26, D8H13S35, and Pcca-rs), we used interspecific and intersubspecific mapping panels. D14H13S26 and Pcca-rs were located on the distal portion of mouse Chr 14 extending by approximately 30 cM the conserved linkage between human Chr 13 and mouse Chr 14, assuming that Pcca-rs is the mouse homolog of PCCA. By contrast, Cf10, Col4a1, Col4a2, and D8H13S35 mapped near the centromere of mouse Chr 8, defining a new conserved linkage. Finally, we identified either a closely linked sequence related to Col4a2, or a recombination hot-spot between Col4a1 and Col4a2 that has been conserved in humans and mice.

    Funded by: NHGRI NIH HHS: HG00189

    Mammalian genome : official journal of the International Mammalian Genome Society 1995;6;4;263-8

  • Novel inheritance of the murine severe combined anemia and thrombocytopenia (Scat) phenotype.

    Peters LL and Barker JE

    Jackson Laboratory, Bar Harbor, Maine 04609.

    The phenotype of the autosomal recessive mutation scat includes severe intermittent bleeding, depletion of platelets, and circulating anti-platelet antibodies. In this study, we have mapped the scat mutation to mouse chromosome 8 and shown that the immune component is a secondary consequence of the gene defect. Surprisingly, the phenotype of the scat/scat pups depends on the genotype of the mother. Maternal homozygosity prevents disease transmission; crosses between scat homozygotes produce few affected young, while the expected frequency is generated from normal (+/+) mice bearing scat/scat ovaries. The results suggest a novel method of maternal-fetal interaction that relies neither on transfer of maternal mitochondria nor on parental imprinting. We conclude that contribution from the maternal wild-type allele is required for expression of the scat phenotype in homozygotes.

    Funded by: NHLBI NIH HHS: HL29305, HL49761; NIDDK NIH HHS: DK27726

    Cell 1993;74;1;135-42

  • Complex patterns of sequence variation and multiple 5' and 3' ends are found among transcripts of the erythroid ankyrin gene.

    Birkenmeier CS, White RA, Peters LL, Hall EJ, Lux SE and Barker JE

    Jackson Laboratory, Bar Harbor, Maine 04609.

    The structural protein ankyrin functions in red blood cells to link the spectrin-based membrane skeleton to the plasma membrane. Ankyrin proteins are now known to occur in most cell types, and two distinct ankyrin genes have been identified (erythroid (Ank-1) and brain (Ank-2)). We have characterized transcripts of the mouse erythroid ankyrin gene by cDNA cloning and DNA sequencing. Ank-1 transcripts of 7.5 and 9.0 kilobases are found in erythroid tissues, and a 9.0-kilobase transcript is found in cerebellum. RNA hybridization blot analysis of 13 additional mouse tissues has detected four novel Ank-1 transcripts (5.0, 3.5, 2.0, and 1.6 kilobases in size). Sequencing of Ank-1 cDNA clones isolated from mouse reticulocyte, spleen, and cerebellar libraries has identified (i) multiple 5' ends that indicate possible multiple promoters; (ii) alternative polyadenylation sites that probably account for the 7.5- and 9.0-kilobase size difference; (iii) a variety of small insertions and deletions that could produce transcripts (and ultimately proteins) of nearly identical size, but different functions; and (iv) clones with large deletions of coding sequence that account for the smaller transcripts seen in spleen, skeletal muscle, and heart.

    Funded by: NHLBI NIH HHS: HL15157, HL29305; NIDDK NIH HHS: DK27726; ...

    The Journal of biological chemistry 1993;268;13;9533-40

  • Distinct fetal Ank-1 and Ank-2 related proteins and mRNAs in normal and nb/nb mice.

    Peters LL, Turtzo LC, Birkenmeier CS and Barker JE

    Jackson Laboratory, Bar Harbor, ME 04609.

    Mice homozygous for the mutation normoblastosis (gene symbol nb on chromosome 8) are deficient in erythroid ankyrin (ANK-1) and have a severe hemolytic anemia throughout life. Characteristic of the disease is a dramatic decrease in the level of expression of the Ank-1 gene (chromosome 8). The other major ankyrin transcript, brain ankyrin (Ank-2 on chromosome 3) is expressed at normal levels in nb/nb mice. Surprisingly, nb/nb fetuses have normal erythrocyte counts despite the decreased levels of Ank-1 transcripts. We previously hypothesized that fetal-specific ankyrin-related proteins could exist in nb/nb fetuses to account for the lack of detrimental effects of ANK-1 deficiency. In the present report, Western and Northern blot analyses were performed on hematopoietic cells isolated from nb/nb and +/+ fetuses. An ANK-1-related protein (165 Kd) in fetal reticulocytes persisted in adult nb/nb but not in +/+ reticulocytes. An Ank-1-related transcript of 5.5 kb was found in fetal reticulocytes. This transcript appeared to be upregulated in nb/nb but not in +/+ adult reticulocytes. A fetal-specific ANK-2-related protein (155 Kd) was present in nb/nb and in +/+ fetal reticulocytes. Ank-2-related fetal liver mRNAs were present during the time the liver was actively generating erythrocytes. Neither the Ank-2-related transcripts nor the 155-Kd ANK-2-related protein were found in +/+ or mutant adult reticulocytes. The data indicate that (1) unique ankyrin-related proteins and mRNAs present in fetal erythrocytes may stabilize the ankyrin-deficient nb/nb erythrocytes and (2) adult nb/nb mice may upregulate fetal gene transcripts to compensate for the ANK-1 deficiency.

    Funded by: NHLBI NIH HHS: HL 29305; NICHD NIH HHS: HD 07065

    Blood 1993;81;8;2144-9

  • Fetal compensation of the hemolytic anemia in mice homozygous for the normoblastosis (nb) mutation.

    Peters LL, Birkenmeier CS and Barker JE

    Jackson Laboratory, Bar Harbor, ME 04609.

    The mouse autosomal recessive mutation nb causes a deficiency of erythroid ankyrin and generates a life-threatening hemolytic anemia in adult mice; however, at birth, nb/nb mice appear to be robust and show no pallor. In our study, the time of disease onset was sought by comparison of nb/nb and +/? mice both in utero and postnatally. Erythroid ankyrin messenger RNA (mRNA) is expressed in fetal erythroid progenitors from normal mice, but is reduced to 10% of normal levels in mutant fetuses. Despite the deficiency of erythroid ankyrin mRNA, 16 and 18 day nb/nb fetuses have normal levels of red blood cells (RBCs) and the RBCs are morphologically normal by scanning electron microscopy. The earliest signs of any clinical anomaly are an increase in the number of circulating reticulocytes and the deposition of minor amounts of iron just before birth in the 18 day fetal nb/nb liver, suggesting that RBCs are being destroyed. Within 24 hours after birth, nb/nb neonates have a slight but significant decrease of their RBC counts. During the next 5 days, the nb/nb RBC counts decrease markedly, the reticulocyte counts assume the mutant adult levels of 60%, the erythrocytes become microcytic and fragmented, and iron deposits accumulate in the liver. The rapid onset of clinical disease postnatally, coupled with our findings that the erythroid ankyrin gene is transcribed in fetal erythroid cell precursors from normal mice, suggest that mechanisms exist in the nb/nb fetus to compensate for the erythroid ankyrin deficiency.

    Funded by: NHLBI NIH HHS: HL 29305; NICHD NIH HHS: HD 07065

    Blood 1992;80;8;2122-7

  • Changing patterns in cytoskeletal mRNA expression and protein synthesis during murine erythropoiesis in vivo.

    Peters LL, White RA, Birkenmeier CS, Bloom ML, Lux SE and Barker JE

    Jackson Laboratory, Bar Harbor, ME 04609.

    The major cytoskeletal proteins alpha-spectrin, beta-spectrin, and ankyrin are synthesized and assembled into a supportive membrane skeleton during erythroid differentiation. Information on the temporal appearance of mRNA and protein species is essential for understanding both the cytoskeletal assembly process and the function of various isoforms. We have isolated highly enriched populations of fetal erythroid cells at various stages of maturation. mRNAs for erythroid ankyrin, alpha-spectrin, and beta-spectrin were expressed at all stages but there were differences in transcript types and levels. The ratio of 9-kilobase (kb) to 7.5-kb erythroid ankyrin transcripts decreased markedly during differentiation, but there was no change in the ratio of the 10.1-kb and 9.3-kb erythroid beta-spectrin transcripts. The relative amounts of ankyrin, alpha-spectrin, and beta-spectrin mRNA increased during yolk sac cell differentiation, whereas only alpha-spectrin mRNA increased during differentiation of the fetal liver cells. The amounts of beta-spectrin mRNA exceeded the amounts of alpha-spectrin mRNA in the early precursors from both yolk sac and fetal liver; protein synthetic levels showed the same pattern. The 16-day fetal peripheral reticulocytes, on the other hand, had the adult mRNA and protein synthetic ratios with alpha/beta greater than 1. The data indicate that at least two mechanisms exist to meet changing erythroid membrane cytoskeletal requirements during development in utero: (i) stage-specific processing of the mRNA for the major cytoskeletal linker protein ankyrin and (ii) developmentally regulated alpha/beta-spectrin protein synthetic rates.

    Funded by: NHLBI NIH HHS: HL29305

    Proceedings of the National Academy of Sciences of the United States of America 1992;89;13;5749-53

  • Mapping of the motor neuron degeneration (Mnd) gene, a mouse model of amyotrophic lateral sclerosis (ALS).

    Messer A, Plummer J, Maskin P, Coffin JM and Frankel WN

    Wadsworth Center for Laboratories and Research, New York State Department of Health.

    The motor neuron degeneration mutation (Mnd) causes a late-onset, progressive degeneration of upper and lower motor neurons in mice. After establishing genetic and environmental conditions that distinguish the phenotypes of Mnd/Mnd from +/Mnd mice, Mnd was mapped to proximal Chr 8, using endogenous retroviruses as markers. The map location was confirmed with additional linked polymorphic markers. The outcross/intercross matings to the strain AKR/J, which were used to follow the segregation of the retroviral markers with respect to Mnd, also revealed the existence of a timing effect. Approximately one-fourth of the affected Mnd/Mnd F2 progeny showed accelerated disease. The Mnd mouse model should allow study of mechanisms affecting onset and progression of specific neuronal degeneration in both animal and human neurological disease.

    Funded by: NCI NIH HHS: R35-CA44385

    Genomics 1992;13;3;797-802

  • Molecular pathology of inherited erythrocyte membrane disorders: hereditary spherocytosis and elliptocytosis.

    Iolascon A, Miraglia del Giudice E and Camaschella C

    Dipartimento di Pediatria, Clinica Pediatrica Ia, Università, Napoli, Italy.

    Hereditary spherocytosis and elliptocytosis are common genetic defects of the red blood cell membrane skeleton. In recent years rapid advances have been made in the knowledge of the protein structure and assembly of the cytoskeleton. Thanks to the wide use of protein analysis methods several alterations have been discovered in functionally important domains of the different cytoskeletal proteins in these diseases. The cloning of cDNA for the majority of the cytoskeletal proteins allows us to begin elucidating some of these defects at the DNA level. This paper will review the effects of recent advances upon: cytoskeleton structure and assembly; molecular pathology of spherocytosis, elliptocytosis and pyropoikilocytosis.

    Haematologica 1992;77;1;60-72

  • Murine erythrocyte ankyrin cDNA: highly conserved regions of the regulatory domain.

    White RA, Birkenmeier CS, Peters LL, Barker JE and Lux SE

    Hematology/Oncology Division, Children's Hospital, Boston, Massachusetts.

    Ankyrin is an essential link between cytoskeletal proteins, such as spectrin, and membrane bound proteins, such as protein 3, the erythrocyte anion exchanger. Although the amino acid structure of human ankyrin is known, the functional regions have been only partially defined. Sequence comparisons between mouse and human ankyrin offer one mechanism of identifying highly conserved regions that probably have functional significance. We report the isolation and sequencing of a series of overlapping murine erythroid ankyrin (Ank-1) cDNAs from spleen and reticulocyte libraries (total span 6238 bp) and identify potentially important regions of murine-human reticulocyte ankyrin homology. Comparison of the predicted peptide sequences of mouse and human erythroid ankyrins shows that these ankyrins are highly conserved in both the N-terminal, protein 3 binding domain (96% amino acid identity) and in the central spectrin-binding domain (97% identity), but differ in the C-terminal regulatory domain (79% identity). However, the C-terminal regulatory domain contains two regions of peptide sequence that are perfectly conserved. We postulate these regions are important in the regulatory functions of this domain.

    Funded by: NHLBI NIH HHS: HL15157, HL32262; NIDDK NIH HHS: DK34083; ...

    Mammalian genome : official journal of the International Mammalian Genome Society 1992;3;5;281-5

  • Distinct ankyrin isoforms at neuron cell bodies and nodes of Ranvier resolved using erythrocyte ankyrin-deficient mice.

    Kordeli E and Bennett V

    Howard Hughes Medical Institute, Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27710.

    Isoforms of ankyrin (ankyrinsR) immunologically related to erythrocyte ankyrin (ankyrinRo) are associated with distinct neuronal plasma membrane domains of functional importance, such as cell bodies and dendrites, axonal hillock and initial segments, and nodes of Ranvier. AnkyrinRo is expressed in brain, and accounts for at least one of the ankyrinR isoforms. Another ankyrin isoform of brain, ankyrinB, is encoded by a distinct gene and is immunologically distinct from ankyrinsR. Mutant mice with normoblastosis (nb/nb) constitute the first described genetic model of ankyrin deficiency: they display a severe hemolytic anemia due to a significantly reduced expression of the ankyrinRo gene in reticulocytes as well as brain (Peters L. L., C. S. Birkenmeier, R. T. Bronson, R. A. White, S. E. Lux, E. Otto, V. Bennett, A. Higgins, and J. E. Barker. 1991. J. Cell Biol. 114:1233-1241). In the present report, we distinguish between ankyrinRo and other ankyrinR isoforms using immunoblot analysis and immunofluorescence localization of ankyrinsR throughout the nervous system (forebrain, cerebellum, brain stem, spinal cord, and sciatic nerve) of nb/nb and normal mice. This is the first immunocytochemical characterization of the neurological component of the nb mutation and shows the following. (a) The isoform of ankyrin at the nodes of Ranvier and initial axonal segments is present in the nb/nb mice and does not cross-react with an ankyrinRo-specific antibody; this isoform, therefore, is distinct from both ankyrin isoforms identified in brain, ankyrinRo and ankyrinB, and is probably the product of a distinct gene and a unique component of the specialized membrane skeleton associated with nodes of Ranvier. (b) AnkyrinRo missing from nb/nb mice is selectively associated with neuronal cell bodies and dendrites, excluded from myelinated axons, and displays a selective pattern of expression in the nervous system whereby expression is almost ubiquitous in neurons of the cerebellum (Purkinje and granule cells) and spinal cord, and restricted to a very minor subset of neurons in hippocampus and neocortex of forebrain.

    The Journal of cell biology 1991;114;6;1243-59

  • Purkinje cell degeneration associated with erythroid ankyrin deficiency in nb/nb mice.

    Peters LL, Birkenmeier CS, Bronson RT, White RA, Lux SE, Otto E, Bennett V, Higgins A and Barker JE

    Jackson Laboratory, Bar Harbor, Maine 04609.

    Mice homozygous for the nb mutation (Chromosome 8) have a severe hemolytic anemia and develop a psychomotor disorder at 6 mo of age. The nb/nb mice are deficient in erythroid ankyrin (Ank-1) but, until the present study, the role of Ank-1 and of Ank-2 (brain ankyrin) in disease genesis was unknown. In normal erythroid tissues, we show that two major transcripts are expressed from Ank-1, and one of these is also present at high levels in the cerebellum. By in situ hybridization and immunocytochemistry, Ank-1 localizes to the cerebellar Purkinje cells and, to a lesser extent, the granule cells. In nb/nb mice, Ank-1 transcripts are markedly reduced in both erythroid and neural tissue, and nb/nb Purkinje cells and granule cells are nearly devoid of Ank-1. The neurological syndrome appears concurrently with a dramatic loss of Purkinje cells. Ank-2 maps to Chromosome 3 and its expression is unaffected by the nb mutation. We conclude that Ank-1 is specifically required for Purkinje cell stability and, in its absence, Purkinje cell loss and neurological symptoms appear.

    The Journal of cell biology 1991;114;6;1233-41

  • Resistance to malaria in ankyrin and spectrin deficient mice.

    Shear HL, Roth EF, Ng C and Nagel RL

    Department of Medical and Molecular Parasitology, New York University School of Medicine, N.Y.

    Inbred mice carrying mutations in ankyrin and/or spectrin synthesis and assembly were studied for their ability to support the growth of the rodent malarias, Plasmodium chabaudi adami and P. berghei, in vivo. Mice carrying the nb/nb (normoblastosis) mutation which do not synthesize ankyrin and therefore also have a deficiency in membrane-bound spectrin, were refractory to P. chabaudi adami, which invades mature erythrocytes and to P. berghei, which invades reticulocytes. Similarly, sph/sph mice which do not synthesize the alpha chain of spectrin but do synthesize ankyrin, were also resistant to both parasites. The heterozygote for the nb defect (nb/+) exhibited a diminution of parasitaemia. We conclude that the host cell spectrin may be necessary for the invasion and/or growth of rodent malarial parasites.

    Funded by: NHLBI NIH HHS: HL21016, HL38655; NIAID NIH HHS: AI15235

    British journal of haematology 1991;78;4;555-60

  • Zinc finger protein gene complexes on mouse chromosomes 8 and 11.

    Nadeau JH, Birkenmeier CS, Chowdhury K, Crosby JL and Lalley PA

    Jackson Laboratory, Bar Harbor, Maine 04609.

    Two murine homologs of the Drosophila Krüppel gene, a member of the gap class of developmental control genes that encode a protein with zinc fingers, were mapped to mouse chromosomes 8 and 11 by using somatic cell hybrids and an interspecific backcross. Surprisingly, both genes were closely linked to two previously mapped, Krüppel-related zinc finger protein genes, suggesting that they are part of gene complexes.

    Funded by: NHGRI NIH HHS: HG00189; NHLBI NIH HHS: HL29305

    Genomics 1990;8;3;469-76

  • Ankyrin and the hemolytic anemia mutation, nb, map to mouse chromosome 8: presence of the nb allele is associated with a truncated erythrocyte ankyrin.

    White RA, Birkenmeier CS, Lux SE and Barker JE

    Jackson Laboratory, Bar Harbor, ME 04609.

    Mice with normoblastosis, nb/nb, have a severe hemolytic anemia. The extreme fragility and shortened lifespan of the mutant erythrocytes result from a defective membrane skeleton. Previous studies in our laboratory indicated a 50% deficiency of spectrin and an absence of normal ankyrin in erythrocyte membranes of nb/nb mice. We now report genetic mapping data that localize both the nb and erythroid ankyrin (Ank-1) loci to the centromeric end of mouse chromosome 8. Using immunological and biochemical methods, we have further characterized the nature of the ankyrin defect in mutant erythrocytes. We do not detect normal sized (210 kDa) erythroid ankyrin by immunoblot analysis in nb/nb reticulocytes. However, nb/nb reticulocytes do contain a 150-kDa ankyrin immunoreactive protein. The 150-kDa protein is present with normal-sized ankyrin in nb/+ reticulocytes but is not found in +/+ reticulocytes. Our genetic and biochemical data indicate that the nb mutation results from a defect in the erythroid ankyrin gene. A human hereditary spherocytosis putatively resulting from an ankyrin defect maps to a segment of human chromosome 8 that is homologous to the nb-ankyrin region of mouse chromosome 8. The linkage data suggest that the mouse and human diseases result from mutations in homologous loci.

    Funded by: NHLBI NIH HHS: HL 29305

    Proceedings of the National Academy of Sciences of the United States of America 1990;87;8;3117-21

  • Spectrin deficient inherited hemolytic anemias in the mouse: characterization by spectrin synthesis and mRNA activity in reticulocytes.

    Bodine DM, Birkenmeier CS and Barker JE

    We have investigated spectrin synthesis and mRNA activity in mice homozygous and heterozygous for six mutations occurring at three distinct loci (nb, ja, sph). When homozygous, these mutations cause severe hemolytic anemias that are characterized by specific spectrin deficiencies. Our results indicate that the primary effect of the nb mutation is a deficiency of another erythrocyte membrane skeletal protein, ankyrin. The severe deficiency of spectrin in the red blood cells of ja/ja mice is the result of a beta spectrin defect. Analysis of spectrin synthesis in mice homozygous and heterozygous for several alleles of sph indicates that the sph locus is the structural gene locus for alpha spectrin. We have mapped the sph locus to mouse Chromosome 1.

    Funded by: NCI NIH HHS: T32-CA09217-04; NHLBI NIH HHS: HL29305; NIADDK NIH HHS: AM27726

    Cell 1984;37;3;721-9

  • Studies on the pathogenesis of pigment gallstones in hemolytic anemia: description and characteristics of a mouse model.

    Trotman BW, Bernstein SE, Bove KE and Wirt GD

    The pathogenesis of hemolysis-induced gallstones was studied in mice with a hereditary hemolytic disease called normoblastic anemia (genotype nb/nb) and in their normal controls (genotype +/+). Infrared spectroscopy demonstrated that spontaneously formed gallstones from nb/nb mice were nearly identical to stones from patients with chronic hemolysis as the result of sickle cell disease, and both mouse and human stones strikingly resembled synthetic calcium bilirubinate. 57% of 115 nb/nb mice, but none of 109 control mice, developed calcium bilirubinate pigment gallstones (P < 0.001). The incidence of luminal gallstones in nb/nb mice was both sex and age dependent. Female nb/nb mice formed stones twice as frequently as male nb/nb mice (P < 0.001). Before 6 mo of age neither sex developed stones, but thereafter the incidence of stones increased with age. Neither hematocrit, reticulocyte count, nor total plasma bilirubin values, were useful in distinguishing between nb/nb mice with or without gallstones. In gallbladder bile, nb/nb mice with gallstones had higher concentrations of hydrogen ion, total bilirubin, calcium, and bile acids than nb/nb mice without stones. Although total unconjugated bilirubin was similar in both nb/nb groups, the ionized fraction of unconjugated bilirubin was higher in bile from nb/nb mice without stones than those with stones. In nb/nb mice, neutral mucin plugs and pigment concentrations were observed histologically in the glandular crypts of the gallbladder in 33% of nb/nb mice without stones and in 80% of nb/nb mice with luminal stones. This suggested that luminal pigment stone disease in mice with hemolysis may be preceded by microscopic precipitation of calcium bilirubinate in the glandular crypts of the gallbladder. These precipitates may then migrate into the lumen and grow by accretion.

    Funded by: NCRR NIH HHS: RR 05415-16; NIADDK NIH HHS: AM20361; NICHD NIH HHS: HD00254

    The Journal of clinical investigation 1980;65;6;1301-8

  • Inherited hemolytic disease in mice: a review and update.

    Bernstein SE

    There are four overt hemolytic diseases in the mouse which have almost identical clinical syndromes but are genetically distinct. These include hemolytic anemia (gene symble ha), jaundice (ja), normoblastic anemia (nb) and spherocytosis (sph). Each of the hemolytic disorders arises from an intrinsic defect in erythrocyte membrane proteins. Each of the responsible genes is involved, in some fashion, in the common process of membrane development and the maintenance of its integrity. Because each gene has its own special function, the gross phenotypic manifestations, although similar, must have a different functional basis.

    Funded by: NCI NIH HHS: CA01074; NIADDK NIH HHS: AM25305; NICHD NIH HHS: HD00254

    Laboratory animal science 1980;30;2 Pt 1;197-205

  • Studies of erythrocyte protoporphyrin in anemic mutant mice: use of a modified hematofluorometer for the detection of heterozygotes for hemolytic disease.

    Sassa S and Bernstein SE

    Erythrocyte protoporphyrin concentration was determined on a single drop of blood from normal and anemic mutant mice using a modified hematofluorometer. Mice showed age- and sex-dependent changes in protoporphyrin concentrations. Mice with hypoplastic anemias had a slight elevation and mice with iron deficiency anemia had a moderate increase in red cell protoporphyrin. Those with inherited hemolytic anemias had a marked elevation of erythrocyte protoporphyrin. Mice heterozygous for hemolytic anemias had no overt hemolysis and reticulocytosis, yet their erythrocyte protoporphyrin concentrations were distinctly higher than their littermate controls. Previously these heterozygotes were differentiated only by laborious and expensive progeny testing. This paper is the first demonstration of a quick and practical way of detecting mice heterozygous for hemolytic anemias by erythrocyte protoporphyrin determination. The data of this study demonstrate not only the usefulness of the hematofluorometer for protoporphyrin determination in the diagnosis of mouse anemias, but also the significance of elevated erythrocyte protoporphyrin as a very sensitive indicator of increased erythropoiesis, or of iron deficiency.

    Experimental hematology 1978;6;5;479-87

  • Hereditary disorders of the red cell in animals.

    Bannerman RM, Edwards JA and Pinkerton PH

    Progress in hematology 1973;8;131-79

  • Pyrrole pigments in normal and congenitally anaemic mice (+:+, W-W v , ha-ha, nb-nb, mk-mk, f-f and sla-Y).

    Kreimer-Birnbaum M, Bannerman RM, Russell ES and Bernstein SE

    Comparative biochemistry and physiology. A, Comparative physiology 1972;43;1;21-30

  • Kinetic aspects of endogenous carbon monoxide production in experimental animals.

    Landaw SA

    Annals of the New York Academy of Sciences 1970;174;1;32-48

Gene lists (6)

Gene List Source Species Name Description Gene count
L00000001 G2C Mus musculus Mouse PSD Mouse PSD adapted from Collins et al (2006) 1080
L00000008 G2C Mus musculus Mouse PSP Mouse PSP adapted from Collins et al (2006) 1121
L00000060 G2C Mus musculus BAYES-COLLINS-HUMAN-PSD-CONSENSUS Human cortex PSD consensus (ortho) 748
L00000062 G2C Mus musculus BAYES-COLLINS-MOUSE-PSD-CONSENSUS Mouse cortex PSD consensus 984
L00000070 G2C Mus musculus BAYES-COLLINS-HUMAN-PSD-FULL Human cortex biopsy PSD full list (ortho) 1461
L00000072 G2C Mus musculus BAYES-COLLINS-MOUSE-PSD-FULL Mouse cortex PSD full list 1556
© G2C 2014. The Genes to Cognition Programme received funding from The Wellcome Trust and the EU FP7 Framework Programmes:
EUROSPIN (FP7-HEALTH-241498), SynSys (FP7-HEALTH-242167) and GENCODYS (FP7-HEALTH-241995).

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