Raymond A. Popp

A mouse model for β-thalassemia

Abstract A mutation that produces an absolute deficiency of normal β-major globin polypeptides has been recovered from a DBA/2J male mouse. Most mice homozygous for the deficiency survived to adulthood and reproduced but were smaller at birth than their littermates and demonstrated a hypochromic, microcytic anemia with severe anisocytosis, poikilocytosis, and reticulocytosis and the presence of inclusion bodies in a high proportion of circulating erythrocytes. Mice heterozygous for the deficiency demonstrated a mild reticulocytosis but were not clinically anemic. Analysis of globin chain synthesis in vitro by 3 H-leucine incorporation revealed that β-globin synthesis was nearly normal (95%) in heterozygotes and about 75% of normal in deficiency homozygotes. Molecular characterization of the mutation by restriction analysis revealed a deletion of about 3.3 kb of DNA, including regulatory sequences and all coding blocks for β-major globin. Based on genetic and hematological criteria, mice homozygous for the mutant allele, designated Hbb th-1 , represent the first animal model of β-thalassemia (Cooleys anemia), a severe genetic disease of humans.

Hemoglobins of mice: Sequence and possible ambiguity at one position of the alpha chain☆

Abstract The sequences of amino acids in the α chains of hemoglobins from C57BL, BALB/c, and NB mice were studied. C57BL is a common strain of laboratory mice; therefore, it is used as the standard for comparison with others. The α chain of each has 141 amino acids and is homologous to that of man and other vertebrates. When these α chains were compared with that of normal adult human hemoglobin, amino acid exchanges were found at 16 positions. C57BL α chain differs from that of BALB/c at position 68 only, where C57BL consistently has asparagine while BALB/c apparently has two forms, differing only in whether there is serine or threonine inserted in the chain. The insertions of serine or threonine at position 68 occur at relatively equal frequencies. Several explanations for the production of two forms of α chains in BALB/c mice are presented. The α chain of NB hemoglobin shows three amino acid replacements when compared with that of C57BL: NB has valine rather than glycine at position 26, isoleucine rather than valine at position 62, and serine rather than asparagine at position 68.

Sequence of amino acids in the major and minor β chains of the diffuse hemoglobin from BALB/c mice

Abstract Hemoglobin from strain BALB/c mice contains two kinds of β-chain polypeptides. The relative amounts of the major and minor components are approximately 80 and 20%, respectively. These polypeptide chains differ from one another at six positions: the major chain has alanine, glycine, serine, alanine, aspartic acid and histidine and the minor chain has serine, alanine, proline, proline, glutamic acid and lysine at Residues 9, 16, 20, 58, 72, and 76, respectively. Both the major and minor β chains of BALB/c hemoglobin differ from the β chain of C57BL hemoglobin at three positions: C57BL β chain has glycine, alanine, and alanine and BALB/c β chains have cysteine, serine or proline, and threonine at Residues 13, 20, and 139, respectively. The two kinds of β chains in BALB/c mice appear to be controlled by two tightly-linked genes; one probably arose by gene duplication during the evolution of Rodentia .

ERYTHROCYTE REPOPULATION IN X-IRRADIATED RECIPIENTS OF NUCLEATED, PERIPHERAL BLOOD CELLS OF NORMAL MICE

Summary Strains of mice are being produced that are isogenic with inbred stocks except for the hemoglobin locus. With mice of such strains, experiments were carried out to ascertain whether peripheral blood of normal mice contain cells capable of repopulating the marrow of irradiated recipients. In 3 of 5 recipients injected intravenously with ~ 100 million nucleated peripheral blood cells after 600 r, increasing numbers of graft-derived erythrocytes were noted within 60 days after treatment, indicating the presence in peripheral blood of cells that are able to give rise to mature erythrocytes.

Sequence of amino acids in the β chain of single hemoglobins from C57BL, SWR, and NB mice

Abstract The sequences of amino acids in the β chains of hemoglobins from C57BL, SWR, and NB mice were studied. C57BL, a common strain of laboratory mice with an electrophoretically single hemoglobin, was used as the standard for comparison with others. Its β chain has 146 amino acid residues and is homologous in sequence to that of man and other vertebrates. There are a minimum of 27 amino acid exchanges found when the β-chain sequences of the hemoglobin from C57BL mice and of human A hemoglobin are compared. The β chains within the three mouse strains are identical except at Positions β72 and 73, where the C57BL β chain has Ser-Asp and SWR and NB have Asp-Ser, respectively.

Genetics of ocular NAD+-dependent alcohol dehydrogenase and aldehyde dehydrogenase in the mouse: Evidence for genetic identity with stomach isozymes and localization ofAhd-4 on chromosome 11 near trembler

Electrophoretic and activity variation of the stomach and ocular isozyme of aldehyde dehydrogenase (designated AHD-4) was observed between C57BL/6J and SWR/J inbred strains of mice. The phenotypes were inherited in a normal mendelian fashion, with two alleles at a single locus (Ahd-4) showing codominant expression. The alleles assorted independently of those atAdh-3 [encoding the stomach and ocular isozyme of alcohol dehydrogenase (ADH-C2)] on chromosome 3. Three chromosome 11 markers, hemoglobin α-chain (Hba), trembler (Tr), and rex (Re), were used in backcross analyses which established thatAhd-4 is closely linked to trembler. The distribution patterns for stomach and ocular AHD-4 phenotypes were examined among SWXL recombinant inbred mice, and those for stomach and ocular ADH-C2 among BXD recombinant inbred strains. The data provided evidence for the genetic identity of stomach and ocular ADH-C2 and of stomach and ocular AHD-4.

Some physical and chemical properties of albumin esterase and albumin from mouse serum

Abstract 1. 1. A pH and ionic strength gradient over DEAE-cellulose is described for the separation of the principal arylesterase (aryl-ester hydrolase, EC 3.1.1.2) (albumin esterase) from mouse serum. Rechromatography over Sephadex G-200 was done to remove some small polypeptides and macromolecules which eluted coincidently with albumin esterase on DEAE-cellulose. 2. 2. Microdensitometer tracings of acrylamide gels showed that such preparations contained less than 5% other serum proteins. At pH 8.9 the electrophoretic mobility of albumin esterase in acrylamide gel is slightly greater than that of mouse albumin. 3. 3. The s 20 value of albumin esterase is approx. 4.4. S. This data, as well as the chromatographic property of albumin esterase in Sephadex, suggested that the molecular weight of albumin esterase is approx. 70 000. 4. 4. The amino acid compositions of mouse albumin and albumin esterase were determined. They differ significantly in their content of cysteine, glycine, alanine, methionine, and isoleucine.

The primary structure of genetic variants of mouse hemoglobin

The primary structures of the α globins from CE/J, DBA/2J, and a stock of Potters mice were determined to identify the amino acid substitutions associated with the unique isoelectric focusing patterns of these hemoglobins. In addition, the primary structures of the α globins from MOL III and PERU mice were studied in search of amino acid substitutions that may not be detected by isoelectric focusing. CE/J hemoglobin contains a unique kind of α globin called chain 5. It differs from the single kind of α globin (chain 1) in C57BL/6 by having alanine rather than glycine at position 78. DAB/2J hemoglobin has two kinds of α globins: one half is like chain 5 and the other half is like chain 1. The hemoglobin from Potters stock of Mus musculus molossinus also contains chains 1 and 5, but they are expressed at different levels, i.e., 80% chain 1 and 20% chain 5. MOL III hemoglobin has a single kind of α globin identical to that in C57BL/6, and PERU hemoglobin contains approximately 40% chain 1 and 60% chain 4. Chains 1 and 4 have different amino acids at positions 25, 62, and 68. These studies confirm that mouse hemoglobins separable by isoelectric focusing, but not by other means of electrophoresis, have substitutions of neutrally charged amino acids in their α chains.

Hematology of a murine beta-thalassemia: a longitudinal study.

Mice homozygous for a spontaneous mutation, in which the beta-major globin gene is deleted, have clinical symptoms of beta-thalassemia. These mice have a hypocellular, hypochromic, microcytic anemia that becomes more severe with increasing age. The defective red cell morphology, decreased osmotic fragility of erythrocytes and shortened red cell life span found in beta-thalassemic mice are similar to those observed in human beta-thalassemia. Synthesis of beta-globin is depressed but not as much as might be expected because the expression of the beta-minor globin gene is enhanced to encode two to three times more globin than in normal mice. Splenomegaly, an enlarged pool of stem cells for erythropoiesis, and iron overloading occur in older mice. The fact that these mice remain moderately healthy makes them a very suitable animal model in which to develop and test alternative techniques of gene therapy that could be successfully applied to the treatment of human thalassemia. Homozygous beta-thalassemic mice have large deposits of iron in their tissues, which might make these mice also useful for in vivo tests of the effectiveness and possible long-term side effects of newly developed iron chelators.

REPOPULATION OF THYMUS BY IMMUNOLOGICALLY COMPETENT CELLS DERIVED FROM DONOR MARROW

Summary Many of the sublethally irradiated BlF1 and B6D2F1 mice injected intraperitoneally with thymus cells from parental strain donors died within 90 days, owing presumably to an immunological reaction by the implanted cells against the recipient. Likewise, regenerated thymus tissue of lethally irradiated BIF1 mice injected with C57BL bone marrow cells induced a fatal wasting disease on implantation in sublethally irradiated B6D2F1 mice. It was concluded, therefore, that regenerated thymus tissue in lethally irradiated BlF1 mice injected with C57BL bone marrow was repopulated by immunologically competent cells derived from the donor marrow.