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The relative abilities of C57BL and 101 marrow to repopulate the hematopoietic system of lethally x-irradiated mice was determined. The disproportionately greater ability of 101 than C57BL cells to repopulate (C57BL X 101) F/sub 1/ recipients is reported, and data are given on the relative ability of marrow from other strains of mice to repopulate lethally irradiated mice. Consistent with the observation that C57BL marrow is less efficient that 101 marrow is the finding that the number of C57BL cells required to produce functioral long-terra marrow grafts was much larger than the number of 101 cells. Factors that might influence the differential in the growth ability of the two types of marrow are discussed. (H.M.G.)

Hemoglobin (Hb) S Antilles is a naturally occurring form of sickling human Hb but causes a more severe phenotype than Hb S. Two homozygous viable Hb S Antilles transgene insertions from Tg58Ru and Tg98Ru mice were bred into MHOAH mice that express high oxygen affinity (P50 approximately 24.5 mm Hg) rather than normal (P50 approximately 40 mm Hg) mouse Hbs. The rationale was that the high oxygen affinity MHOAH Hb, the lower oxygen affinity of Hb S Antilles than Hb S (P50 approximately 40 v 26.5 mm Hg), and the lower solubility of deoxygenated Hb S Antilles than Hb S (approximately 11 v 18 g/dL) would favor deoxygenation and polymerization of human Hb S Antilles in MHOAH mouse red blood cells (RBCs). The Tg58 x Tg98 mice produced have a high and balanced expression (approximately 50% each) of h alpha and h beta(S Antilles) globins, 25% to 35% of their RBCs are misshapen in vivo, and in vitro deoxygenation of their blood induces 30% to 50% of the RBCs to form classical looking, elongated sickle cells with pointed ends. Tg58 x Tg98 mice exhibit reticulocytosis, an elevated white blood cell count and lung and kidney pathology commonly found in sickle cell patients, which should make these mice useful for experimental studies on possible therapeutic intervention of sickle cell disease.

Oxygen association-dissociation and hemoglobin stability analysis were performed on mouse hemoglobins with amino acid substitutions in an α-globin (α89, His to Leu) and a β-globin (β59, Lys to Ile). The variant α-globin, designated chain 5(m) in the Hba(g2) haplotype, had a high oxygen affinity and was stable. The variant β-globin, (β(s2)) of the Hbb(s2) haplotype, also had an elevated oxygen affinity and in addition was moderately unstable in 19% isopropanol. Hemoglobins from the expected nine (Hba(g2)/Hba(g2);Hbb(s)/Hbb(s) X Hba(a)/Hba(a);Hbb(s2)/Hbb(s2)) F(2) genotypes can be grouped into five classes of P(50) values characterized by strict additivity and dependency on mutant globin gene dosage; physiologically, both globin variants gave indistinguishable effects on oxygen affinity. The hemoglobin of normal mice (Hba(a)/Hba(a);Hbb(s)/Hbb(s)) had a P(50) = 40 mm Hg and the hemoglobin of Hba(g2)/Hba(g2);Hbb(s2)/Hbb(s2) F(2) mice had a P(50) = 25 mm Hg (human P(50) = 26 mm Hg). Peripheral blood from Hba(g2)/Hba(g2);Hbb(s)/Hbb(s), Hba(a)/Hba(a);Hbb(s2)/Hbb(s2) and Hba(g2)/Hba(g2);Hbb(s2)/Hbb(s2) mice exhibited normal hematological values except for a slightly higher hematocrit for Hba(g2)/Hba(g2);Hbb(s)/Hbb(s) and Hba(g2)/Hba(g2);Hbb(s2)/Hbb(s2) mice, slightly elevated red cell counts for mice of the three mutant genotypes, and significantly lower values for the mean corpuscular volume and mean corpuscular hemoglobin for Hba(g2)/Hba(g2);Hbb(s2)/Hbb(s2) mice.

Adult alpha-globin in mice is synthesized in large amounts during development, first in the primitive, nucleated erythrocytes of yolk sac origin and later in the definitive, nonnucleated erythrocytes that differentiate in the fetal liver, spleen, and bone marrow. Isoelectric focusing analysis of hemoglobins of mice with theHba g2 andHba c haplotypes shows that the ratios of alpha chain 1 to chain 5m and alpha chain 1 to chain 4 in adult hemoglobins fromHba g2 andHba c mice, respectively, change between day 11.5 and day 16.5 of gestation in nucleated red cells, while no change occurs in nonnucleated red cells. The percentage ratios of the two different alpha-globin chains are different inHba g2 andHba c mice for EII, EIII, and adult hemoglobin. In nucleated red cells of yolk sac origin, differences and changes in alpha-globin ratios are a composite of changing globin gene transcription and posttranslational competitive affinities among globins to form embryonic and adult hemoglobin tetramers.

Two electrophoretic polymorphisms affecting lens crystallins, designated LEN-1 and LEN-2, have been discovered among inbred strains of mice. Analysis by isoelectric focusing demonstrated that both crystallins are monomeric proteins with isoelectric points at or above pH 7. Both proteins eluted in the low molecular weight (LM) fraction upon Sephadex G-200 gel filtration but LEN-2 was shown to be larger than LEN-1 by G75SF gel filtration and denaturing gel electrophoresis. Linkage analysis demonstrated that the genes encoding LEN-1 and LEN-2 assort independently. Amino acid analysis of the allelic products of the two genes revealed that genetic variants of each respective crystallin were very similar in amino acid compositions but that LEN-1 and LEN-2 were dissimilar crystallins.

The congenic pair of mice, C57BL/10 (B10) and C57BL/10.F (B10.F), differ at the H-2 locus and have mean ages at death of 706 and 456 days, respectively. B10.F also has reduced basal serum IgA levels compared with B10, 63 and 256 mg/dl, respectively. Controlled matings between the two strains of mice were used to identify genetic factors that govern longevity. F2 and backcross progeny from reciprocal F1 hybrids were classified for H-2 genotype and serum IgA levels and allowed to live out their lifespan. F2 and backcross progeny homozygous for the H-2 allele of B10.F had a mean age at death (602 days) significantly reduced from that of progeny homozygous for the H-2 allele of B10 (689 days). However, the greatest reduction of lifespan occurred among progeny of the (B10.F X B10)F1 mothers, 693 compared with 540 days. The strain of the maternal parent also has been shown to affect the segregation of IgA phenotypes. An increased incidence of low IgA phenotype associated with H-2 genotype was observed among progeny of (B10.F X B10)F1 mothers. Survival curves demonstrated a relationship between low serum IgA levels and shortened lifespan and no maternal effect was observed. The basis of the shortened lifespan among progeny of F1 hybrids in which the maternal parent was B10.F was the increased incidence of offspring with low IgA phenotypes. The apparent association of H-2 and shortened lifespan also was because the low IgA phenotype was more frequent among progeny that carried the H-2 allele of the B10.F strain. The B10.F mice spontaneously shed an endogenous ecotropic retrovirus which may be responsible for the maternal effect on immunoglobulin levels and lifespan.

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 at Adh-3 [encoding the stomach and ocular isozyme of alcohol dehydrogenase (ADH-C2)] on chromosome 3. Three chromosome 11 markers, hemoglobin alpha-chain (Hba), trembler (Tr), and rex (Re), were used in backcross analyses which established that Ahd-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.

A mutation that produces an absolute deficiency of normal beta-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 3H-leucine incorporation revealed that beta-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 beta-major globin. Based on genetic and hematological criteria, mice homozygous for the mutant allele, designated Hbbth-1, represent the first animal model of beta-thalassemia (Cooley's anemia), a severe genetic disease of humans.

Mice of the mutant haplotype ( Hbb s2 ) produce a variant β-s globin (β-s2major) which can be distinguished from β-smajor and β-sminor by cellulose acetate electrophoresis and ion exchange chromatography. Mice homozygous for this mutation were used to study the relative quantities of the mutant β-s2major and normal β-sminor globins specified by the two adult β-globin genes of the Hbb s2 haplotype during development. At 11.5 days of gestation, β-s2major comprises under 20% and β-sminor over 80% of the adult β-globin. The relative level of β-sminor decreases through fetal development; at birth β-sminor represents 33.7% of the β-globin. The adult value of 71.0% β-s2major and 29.0% β-sminor globin is expressed in mice 6 days after birth. In mildly anemia α-thalassemic heterozygotes ( Hba b2(th) Hba b ; Hbb s2 Hbb s2 ) the level of β-sminor globin increases from 29.0 to 37.9%, but β-sminor is elevated only slightly (29.0 to 33.9%) in asymptomatic β-thalassemic heterozygotes ( Hba b Hba b ; Hbb c3(th) Hbb s2 ). The relative quantity of β-sminor is increased significantly (29.0 to 41.4%) in doubly heterozygous α-thalassemic, β-thalassemic mice ( Hba b2(th) Hba b ; Hbb d3(th) Hbb s2 ). The relative levels of expression of the β1 s2major and β2 sminor globin genes of Hbb s2 Hbb s2 mice correlates well with the expression of the β1 dmajor and β2 dminor globin genes of Hbb d Hbb d mice during development and in response to hematological stress caused by thalassemia. Expression of the β1 sminor globin gene should not have been affected by the ENU-induced base substitution in the β1 smajor gene. Therefore, we propose that the β1 sminor gene is also expressed in mice of the Hbb s haplotype. The results also indicated that the two adult β-globin genes of the Hbb s2 and, presumably, of the Hbb s haplotypes are regulated independently as are the β1 dmajor and β2 dminor genes of the Hbb d and Hbb p haplotypes.

To explore the pathogenesis of nontransfusional iron overload in iron- loading anemia, we examined features of external iron exchange, internal iron kinetics, and tissue iron burden in adult mice with inherited gene-deletion beta-thalassemia. Mice homozygous for beta- thalassemia display moderate anemia, reticulocytosis, and shortened red cell survival, whereas heterozygous carriers appear hematologically normal. Quantitative iron determination revealed that iron content and concentration in liver, spleen, and kidney, but not heart, were far higher (P less than .01) in 15-to 35-week old homozygous thalassemic mice than in age-matched normal and heterozygous controls; of these tissues, iron content increased with age only in kidneys (P = .01) of homozygous affected mice. Although plasma iron levels were only minimally elevated in homozygotes, plasma iron turnover was threefold greater (P less than .001) than that seen in heterozygote controls. Nevertheless hyperabsorption of enteric radioiron, discernible among homozygous thalassemic mice as late as 6 to 8 weeks after birth, was not observed in older mice, additionally, thalassemic and control mice at 18 to 34 weeks showed comparable iron excretion after intravenous radioiron. We conclude that adult mice with beta-thalassemia regain balanced external iron exchange, despite substantial tissue iron excess and accelerated internal iron transit.

The breakpoints of the deletion responsible for the Hbb(th-1) mouse model of beta-thalassemia have been isolated. A 3709 (+/- 2)-base pair (bp) region, including the entire beta major globin gene and 2 kilobases of 5' flanking region, is deleted. A novel 66 (+/- 2)-bp sequence, ending in a stretch of 25 dA:dT base pairs, was found to bridge the deletion. A region of the normal murine genome, containing the first 43 bp of the deletion-associated insert (DAI), but lacking the 25-bp dA:dT sequence, was isolated. All normal mice tested contain this DAI-like element and several inbred strains contain an additional DAI-like element. The sequence spanning the Hbb(th-1) deletion may be a reverse transcript of this region.

The tRNAs that are bound to the genomic RNAs of several murine, feline, and primate retroviruses have been identified. Transfer RNAs were divided into those loosely bound and those tightly bound by stepwise thermal dissociation of the 70 S RNA. They were then identified and semiquantitated by amino-acylation. Proline tRNA is the most tenaciously bound tRNA in several strains of murine leukemia virus, two strains of feline leukemia virus, and the primate viruses simian sarcoma, baboon endogenous, and gibbon ape lymphoma. In the feline xenotropic virus, RD-114, tRNAGly is enriched in the most tightly bound fraction. In Mason-Pfizer monkey virus, as in the murine mammary tumor virus, tRNALys is the tRNA most tenaciously bound to its genomic RNA. Besides the most tightly associated tRNA, one or more different tRNAs are found in relatively large amounts in association with the 70 S RNA. (For convenience, we refer to the largest RNA complex (50–70 S) isolated from any of the retroviruses studied as ‘70 S’ RNA.) These tRNAs can be distinguished from the most tightly bound tRNA by the fact that they can be dissociated at lower temperatures. However, they occur in the same relative abundance as the tightly bound tRNA.

Hemoglobins of mouse embryos at 11.5 through 16.5 days of gestation were separated by electrophoresis on cellulose acetate and quantitated by a scanning densitometer to study the effects of two radiation-induced mutations on the expression of embryonic hemoglobin genes in mice. Normal mice produce three kinds of embryonic hemoglobins. In heterozygous α-thalassemic embryos, expression of EI (x2y2) and EII (α2y2) is deficient because the x- and α-globin genes of one of the allelic pairs of Hba on chromosome 11 was deleted or otherwise inactivated by X irradiation. Simultaneous inactivation of the x- and α-globin genes indicates that these genes must be closely linked. Reduced x- and α-chain synthesis results in an excess of y chains that associate as homotetramers. This unique y4 hemoglobin also appears in β-duplication embryos where excess y chains are produced by the presence of three rather than two functional alleles of y- and β-globin genes. In double heterozygotes, which have a single functional allele of x- and α-globin genes and three functional alleles of y- and β-globin genes, synthesis of α and non-α chains is severely imbalanced and half of the total hemoglobin is y4. Mouse y4 has a high affinity for oxygen, P50 of less than 10 mm Hg, but it lacks cooperativity so is inefficient for oxygen transport. The death of double heterozygotes in late fetal or neonatal life may be due in large part to oxygen deprivation to the tissues.

The primary structures of the alpha globins from CE/J, DBA/2J, and a stock of Potter's 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 alpha 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 alpha globin called chain 5. It differs from the single kind of alpha globin (chain 1) in C57BL/6 by having alanine rather than glycine at position 78. DBA/2J hemoglobin has two kinds of alpha globins: one half is like chain 5 and the other half is like chain 1. The hemoglobin from Potter's 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 a alpha 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 alpha chains.

The results of a study of the expression of embryonic hemoglobin genes in mice which show an imbalance of alpha and non-alpha chain synthesis are reported. (ACR)

Three induced, heritable mutations in the mouse cause alpha-thalassemias. The adult alpha-globin genes on each mutant chromosome are no longer expressed. Embryos heterozygous for one normal and any of the three mutant chromosomes also seem to be deficient in embryonic alpha-globin-like x-globin, suggesting that the x-globin gene is nearby and also inactivated. A normal genetic polymorphism for a specific EcoRI site in or around the mouse alpha-globin gene complex has been used here to show that each of the three mutated chromosomes has a deletion that includes the segment of a 12-kilobase EcoRI band which normally carries one of the two adult alpha-globin genes. The deletion of the comparable part of the second alpha-globin gene site is also inferred. Nonetheless, a 4.7-kilobase EcoRI segment which carries a characterized alpha-globin-like pseudogene is still present in each mutant. These mutations were recovered after triethylenemelamine or x-ray treatments.

Dot blot hybridization was used to determine the relative amounts of the beta-major and beta-minor globin RNAs present in reticulocytes of mice at 14.5 and 17.5 days of gestation, newborns, and adults of the Hbab/Hbab;Hbbs2/Hbbs2 globin genotype. RNAs isolated from embryonic yolk sac, fetal liver, and adult reticulocytes were hybridized with the following labeled DNA probes: alpha-1, beta-minor specific, and beta-major specific. The level of beta-sminor RNA in reticulocytes at 14.5 and 17.5 days of gestation is nearly the same as in induced reticulocytes of adult mice. In contrast, the level of beta-s2major RNA in reticulocytes at 14.5 days of gestation is 0.23 X and at 17.5 days of gestation is 0.66 X the amount found in reticulocytes of adult mice. These results correlate well with earlier observations that the beta-sminor globin gene approaches its normal adult level of expression by 14.5 days of gestation, whereas the beta-s2major globin gene expression increases between 14.5 days of gestation and 6 days postnatally. They indicate that the differential expression of beta-sminor and beta-s2major globins during development is regulated at the level of transcription. Expression of the beta-minor globin gene in reticulocytes of adult normal mice is not maximal, however, because the levels of the beta-minor globin and its RNA are increased further in reticulocytes of thalassemic mice.

The amino acid sequences of the beta major and beta minor globin chains from DBA/2 mice have been determined. This information is of interest because DBA/2 mice are the strain of origin for most murine erythroleukemia lines. The primary structure of DBA/2 beta globins agrees completely with that predicted from the coding properties of BALB/c beta globin genes. This identity does not support a rapid evolutionary divergence in inbred mouse strains, at least at these loci in these strains.

Matings were made between (1) normal mice, (2) heterozygous alpha thalassemic females and normal males, and (3) heterozygous alpha thalassemic mice to produce litters of (1) normal mice, (2) normal and heterozygous alpha thalassemic mice, and (3) normal, heterozygous, and homozygous alpha thalassemic mice, respectively, in order to determine what effects the heterozygous and homozygous forms of alpha thalassemia might have on mouse development. Genetic tests showed that the viable alpha thalassemic progeny of matings between heterozygous alpha thalassemic mice were heterozygotes which suggests that the homozygotes die. Examination of preimplantation embryos from the uterus of normal and heterozygous alpha thalassemic females at 86 h after mating with normal males showed that these embryos had reached the blastocyst stage of development. When heterozygous alpha thalassemic females were mated with heterozygous alpha thalassemic males, however, only three-fourths of the embryos had reached the early blastocyst stage of development at 86 h, while one-fourth of them (presumed homozygous alpha thalassemic embryos) were still at the morula stage of development. At 11.5 though 15.5 days of gestation, about one-fourth of the implantation sites did not contain live fetuses, which suggests that death had occurred earlier. Histologic examination of embryos at 5.5 though 8.5 days of gestation showed that the homozygous alpha thalassemic embryos implanted and developed to the late blastocyst stage, at which time they became necrotic. Microscopic examination of blood films from fetuses at 11.5 through 15.5 days of development showed that the yolk sac-derived erythrocytes of alpha thalassemic heterozygotes were morphologically normal, but the fetal liver-derived erythrocytes contained eosinophilic inclusions. Electrophoresis of blood from the fetuses showed that alpha thalassemic heterozygotes contained a unique embryonic hemoglobin (y4), which is homologous to Bart's γ4 in humans.

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 for newly developed iron chelators.

Genetically different mouse hemoglobins differ in solubility and crystal formation. Differences in hemoglobin solubility and crystal formation can therefore be used to identify homologous erythrocytes in irradiated mouse chinreras if the hemoglobin types of donor and recipient mice are distinguishable by these properties to begin with. (auth)

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.

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 .

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.

A study was made of the effect of S, 2-aminoethylisothiuronium bromide hydrobromide (AET) on the changes in sodium and potassium concentrations in the walls and contents of the gastrointestinal tracts of mice exposed to 1500 r. Mice were given AET and then irradiated. At intervals thereafter, portions of the gastrointestinal tract and its contents were analyzed for sodium and potassiura in a flame spectrophotometer. In mice given AET before exposure to 1500 r, the following radiation-induced abnormalities were completely or partially corrected: decrease in potassium and increase in sodium in the wall of the small intestine; decrease in potassium in the colon wall; and increase in sodium and potassium in the contents of the stomach, small intestine and colon. Extracts from the walls of the small intestines of AET-treated or nontreated, irradiated mice were analyzed chemically for esterase activity. In AET-treated, irradiated mice, complete recovery of esterase activity was observed by day 7 after 900 r; and partial recovery occurred after 1500 r. Since AET reduces radiation damage and thereby allows recovery, the beneficongestion; in irradiated (400 r) mice, it was slightly shrunk, and considerably shrurk after combined irradiation. It was concluded that the effect of internal (topical) irradiation onmore » tumor cells and on blood vessels was considerably increased by combination with external (systemic) irradiation or vice versa. (auth)« less

The two adult beta-globin genes (beta 1s2major and beta 2sminor) of the Hbbs2 haplotype are differentially expressed during development. Centrifugal elutriation was used to separate the two populations of erythrocytes present in developing fetuses. Hemoglobin analysis showed that the larger, nucleated erythrocytes (yolk sac-derived) have relatively larger amounts of beta-sminor hemoglobin than do smaller, nonnucleated cells (fetal liver-, spleen-, and bone marrow-derived) at the same stage of development.

A mouse with a variant hemoglobin was discovered during electrophoretic screening of (C57BL/6J X DBA/2J)F1 progeny of females treated with ethylnitrosourea. The variant trait was transmitted as a simple Mendelian alternate at the Hbb locus in all crosses except those involving the original carrier of the mutation. The proband mouse which received the mutation directly from the mutagen-treated parent was a germinal mosaic for the mutant and normal Hbbs alleles. The mutant allele was designated Hbbs2. The mutant haplotype specifies both an electrophoretically fast hemoglobin band and a hemoglobin band in the normal beta single hemoglobin position. Thus, the mutation has altered one of the tandemly duplicated genes at the Hbbs locus. A comparison of the relative concentrations of the two hemoglobins in Hbbs2 mice demonstrates preferential expression of the mutant gene, possibly analogous to the enhanced expression of Hbbdmaj in the Hbbd haplotype. Analysis of the amino acid sequence of the variant beta-globin revealed that the valine at position 60 was changed to glutamic acid. The simplest mutation mechanism for such an alteration is an A X T----T X A transversion.

Both hemoglobin type and red cell serotype seem to be autonomously controlled by the genotype of the nucleated cell from which the erythrocyte is derived. Thus, genetic differences in hemoglobin can be used as markers for bone marrow transplantation in irradiated mice. Hemoglobin typing may be particularly useful where the H-2 markers cannot be used. (auth)

C57BL mouse spleen cells incubated with (C57BL x 101) F/sub 1/ liver homogenate killed only 17% of sublethally irradiated (C57BL x 1O1)F/sub 1/ recipients, whereas when incubated with C57BL liver homogenate or injected without incubation such cells killed 100% of sublethally irradiated (C57BL x 1O1)F/sub 1/ recipients within 35 days. Although the immunologically inactivated spleen cells showed a decreased ability to kill, circulating donor-type erythrocytes were found in surviving recipients, indicating repopulation of the bone marrow by surviving graft-derived stem cells. From this, it is inferred that immunologic inactivation did not result from nonspecific destruction of all types of implanted cells.

Strains of mice are being prcduced that are isogenic with inbred stocks except for the hemoglobin locus. With mice of such strains, experiments were carried out to ascertain whether peripheral blcod of normal mice contains cells capable of repopulating the marrow of irradiated recipients. In 3 of 5 recipients injected intravenously with approximately 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. (auth)

Three types of mice with globin gene mutations, called 352HB, 27HB, and Hbath-J, appear to be true animal models of human thalassemia. Expression of the alpha-globin genes in three stocks of mice, each one heterozygous for one of the alpha-globin mutations, was examined at the polypeptide, RNA, and DNA levels. alpha-Globin polypeptide chains, relative to beta-globin chains in heterozygous thalassemic mice, are present at approximately 80% of normal. The ratios of alpha-globin to beta-globin RNA sequences are also 75-80% of normal, exactly reflecting the alpha-globin to beta-globin chain ratios. In the case of mutant 352HB, at least one alpha-globin gene is deleted. Thalassemic mouse erythroid cells appear to compensate partially for the loss of half of their alpha-globin genes.

The primary structures of the alpha chains in hemoglobins from three stocks of mice with the Hbaw2, Hbaw3, and Hbaw4 haplotypes were determined to establish whether the tentative alpha-chain assignments based on the results of isoelectric focusing patterns were correct. These Hba haplotypes were identified in laboratory descendants of feral mice captured in different parts of the world. Hemoglobin from "Centreville", Maryland, Mus musculus domesticus (Hbaw2) contains equal amounts of alpha chains 1 and 3. Hemoglobin from "Czech" Mus musculus musculus (Hbaw4) contains equal amounts of alpha chains 3 and 4. Amino acid analysis of the alpha-globins of "Skive" Danish Mus musculus musculus (Hbaw3) establishes that its hemoglobin is comprised of about one-third alpha chain 2 as expected plus a greater amount of a unique alpha chain that has not been described previously. This unique alpha chain has glycine at position 25, isoleucine at position 62, and serine at position 68; it is called chain 7. It may represent an intermediate in the evolution of genes that code for chain 2 (which has glycine, valine, and serine at positions 25, 62, and 68, respectively) and chain 4 (which has valine, isoleucine, and serine at positions 25, 62, and 62, respectively).

Hemopoietic tissue is vulnerable to perturbations, and data show that it is an appropriate tissue in which to look for age-associated alterations. This tissue has a high regenerative capacity, is composed of a heterogeneous population of stem cells that are capable of self renewal or differentiation, or both, and is sustained by a pool of resting cells. The heterogeneity of bone marrow has made characterization of the cellular elements difficult. Techniques commonly used to identify and quantify the various maturation levels of hemopoietic stem cells and the limitations of these techniques are discussed. Most techniques used to assay age-associated changes in bone marrow have not differentiated between specific cellular alterations or shifts in the distribution of the cellular elements. In particular, it has been difficult to determine the stability of the non-dividing stem cell because of the low incidence of this cell (6 per 1000) and the lack of a specific assay for this important cell type. The use of cell cycle-specific drugs has provided quantitative information on specific subpopulations of hemopoietic stem cells and seems to be the most promising approach towards determining qualitative and quantitative differences in the hemopoietic stem cells of young and old individuals.

Chemical analyses were done on the abnormal hemoglobins of five (101 × SEC)F1 offspring of X-irradiated adult SEC mice to determine which hemoglobin genes were expressed in each hemoglobin variant. Three offspring of irradiated SEC males did not express either of the two kinds of α-chains normally found in all SEC mice. The deficient α-chain synthesis caused these mice to exhibit an α-thalassemia similar to human α-thalassemia. Scanning electron microscopy was used to show that many erythrocytes of mice with α-thalassemia have bizarre shapes; e.g. many erthrocytes appeared flattened or had thorny projections (acanthocytes). One mutant with a tandem duplication of a segment of chromosome 7 (site of locus determining β-chain structure) produced twice as much SEC as 101 β-chain polypeptides. One mutant that probably arose by non-disjunction of chromosome 7's in its unirradiated 101 mother and loss of chromosome 7 from the gamete of its irradiated SEC father did not express the SEC β-chain gene.

The field of biochemical genetics relies heavily upon the detection by electrophoresis of genetically determined variants of proteins. Most of these variants differ by substitutions that involve charged amino acids. Genetic variants of another large class, ones that involve substitutions among neutral amino acids, are not easily detected and are often ignored. Ampholyte isoelectric focusing in some cases can separate proteins indistinguishable by standard electrophoresis, including genetic variants of mouse hemoglobins that differ only by neutral amino acid substitutions. A revolutionary variation of isoelectric focusing, in which gradients covering a small pH range are fixed into place in a polyacrylamide gel, provides greater resolution of these nearly identical proteins. Mouse hemoglobin tetramers that differ only by the substitution of alanine for glycine in the alpha-globin chains are resolved by several millimeters with the new technique; by comparison, these tetramers are imperfectly resolved on a standard pH 7-9 isoelectric focusing gel. This improved technique of isoelectric focusing was used to identify a variety of previously unreported genetic variants of mouse hemoglobin alpha chains. Immobilized gradients tailored to the requirements of the proteins being analyzed will extend greatly the ranges of protein variations that can be easily recognized for diverse applications, including genetic quality-control analyses and in studies of genetics, mutagenesis, and evolution.

Mice homozygous for a deletion of the beta-dmajor globin gene exhibit clinical symptoms of human beta-thalassemia and are good experimental animals for investigating the regulation of globin gene expression, perturbation of hematopoiesis, and potential methods for treating patients with beta-thalassemia. Homozygous beta-thalassemic mice have a microcytic anemia, their red blood cells display anisocytosis, poikilocytosis and a shortened life span, and iron overloading occurs in several tissues in response to increased erythropoiesis2,3. Mice heterozygous for the beta-thalassemia1,2 mutation are clinically normal.

Two loci which influence the structure of the alpha and beta chains of hemoglobin have been identified and are designated Sol and Hb. At least four alleles at Sol and two at Hb have been distinguished among inbred strains.

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.

A comparative study was made of the proteins in various tissues of Sprague-Dawley rats and (101 x C3H)F/sub 1/ mice. The results were used as a basis for studying changes in the protein patterns in tissues of rat-mouse chimeras: i.e., (101 x C3H)F/sub 1/ mice were given 950 r of x rays and then received an injection of bone marrow from Sprague-Dawley rats. Electrophoretic mobilities of rat proteins extracted from the bone marrow, spleen, lymph node, and thymus of chimeras and rats were similar. The quantity of rat proteins and the rate and time at which they appeared in different hematopoietic organs were quite different. Some mouse-type proteins not found in chimera tissues examined 30 days after treatment were frequently present on the 50th day or later, which suggests regression of the graft. The results are consistent with the idea that rat-type proteins synthesized in the cytoplasm of transplanted rat cells are controlled by the genotype of these cells, even in a mouse environment. They also indicate that there are organ-specific differences, in the rate and degree of replacement of transplanted cells, that require further investigation. (auth)