RESUMEN
Divalent metal transporter 1 (DMT1; also called DCT1, Nramp2, or SLC11A2) has multiple isoforms that localize differently in many cell types. DMT1 +IRE species (encoded by mRNA with an iron-responsive element) are limited to the plasma membrane and cytosolic vesicles. In neural cells, -IRE isoforms of DMT1 (encoded by mRNA lacking an IRE) localize to the nucleus, plasma membrane, and cytosolic vesicles. In considering nuclear compartmentalization of -IRE isoforms, we hypothesized that the newly identified exon 1A in the N-terminus of this transporter might contain a nuclear localization signal. DNA constructs starting with exon 1A and ending with exons encoding alternative isoforms were made and transiently transfected into HEK293T and PC12 cells as well as rat sympathetic neurons. None of the constructs appeared in the nucleus despite the presence of exon 1A. Antibody specific for exon 1A was also used in both immunostaining and Western blots to investigate localization of exon 1A expressed both endogenously and ectopically in cells. Again, nuclear localization of DMT1 containing exon 1A was not observed. Our data suggest that exon 1A is neither sufficient nor necessary for DMT1 to appear in the nucleus.
Asunto(s)
Proteínas de Transporte de Catión/metabolismo , Núcleo Celular/metabolismo , Proteínas de Unión a Hierro/metabolismo , Neuronas/metabolismo , Señales de Localización Nuclear/metabolismo , Sistema Nervioso Simpático/citología , Animales , Animales Recién Nacidos , Western Blotting/métodos , Proteínas de Transporte de Catión/genética , Membrana Celular/metabolismo , Células Cultivadas , Citoplasma/metabolismo , Embrión de Mamíferos , Exones , Proteínas Fluorescentes Verdes , Humanos , Inmunohistoquímica/métodos , Proteínas de Unión a Hierro/genética , Riñón , Proteínas Luminiscentes/metabolismo , Neuronas/citología , Isoformas de Proteínas/metabolismo , Transporte de Proteínas , Proteínas/metabolismo , ARN Mensajero/biosíntesis , Ratas , Ratas Sprague-Dawley , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Transfección/métodosRESUMEN
Iron accumulation in the brain occurs in a number of neurodegenerative diseases. Two new iron transport proteins have been identified that may help elucidate the mechanism of abnormal iron accumulation. The Divalent Metal Transporter 1 (DMT1), is responsible for iron uptake from the gut and transport from endosomes. The Metal Transport Protein 1 (MTP1) promotes iron export. In this study we determined the cellular and regional expression of these two transporters in the brains of normal adult and Belgrade rats. Belgrade rats have a defect in DMT1 that is associated with lower levels of iron in the brain. In the normal rat, DMT1 expression is highest in neurons in the striatum, cerebellum, thalamus, ependymal cells lining the third ventricle, and vascular cells throughout the brain. The staining in the ependymal cells and endothelial cells suggests that DMT1 has an important role in iron transport into the brain. In Belgrade rats, there is generalized decrease in immunodetectable DMT1 compared to normal rats except in the ependymal cells. This decrease in immunoreactivity, however, was absent on immunoblots. The immunoblot analysis indicates that this protein did not upregulate to compensate for the chronic defect in iron transport. MTP1 staining is found in most brain regions. MTP1 expression in the brain is robust in pyramidal neurons of the cerebral cortex but is not detected in the vascular endothelial cells and ependymal cells. MTP1 staining in Belgrade rats was decreased compared to normal, but similar to DMT1 this decrease was not corroborated by immunoblotting. These results indicate that DMT1 and MTP1 are involved in brain iron transport and this involvement is regionally and cellularly specific.
Asunto(s)
Química Encefálica/genética , Encéfalo/metabolismo , Proteínas de Transporte de Catión/metabolismo , Proteínas de Unión a Hierro , Hierro/metabolismo , Enfermedades Neurodegenerativas/metabolismo , Neuronas/metabolismo , Animales , Astrocitos/citología , Astrocitos/metabolismo , Vasos Sanguíneos/citología , Vasos Sanguíneos/metabolismo , Barrera Hematoencefálica/fisiología , Encéfalo/citología , Modelos Animales de Enfermedad , Epéndimo/citología , Epéndimo/metabolismo , Heterocigoto , Homocigoto , Inmunohistoquímica , Microcirculación/citología , Microcirculación/metabolismo , Enfermedades Neurodegenerativas/fisiopatología , Neuronas/citología , Oligodendroglía/citología , Oligodendroglía/metabolismo , Ratas , Ratas Mutantes , Valores de ReferenciaRESUMEN
Two isoforms of divalent metal transporter 1 (DMT1) (Nramp2 and DCT1) are encoded by two mRNA species, one of which contains an iron response element (IRE) motif in the 3'-noncoding region. The subcellular distribution of the two isoforms of DMT1 is distinct, and the -IRE species accumulates in the nucleus of neuronal or neuronal-like cells. Reverse transcription-PCR and Western blot analysis of PC12 cells reveals that these cells express both forms of DMT1. Immunofluorescence and immunoblotting studies, using immunospecific antibodies to the -IRE form of DMT1, demonstrate that this form of the transporter, in PC12 cells, is predominantly localized in the nucleus, cell membrane, and neurites with only weak staining of the cell body. Studies using antibodies to the +IRE form indicate that this species of DMT1 is distributed within vesicles in the cell body and neurite projections, with minimal nuclear staining. Similar staining patterns are observed for the two forms of DMT1 in cultures of sympathetic ganglion neurons isolated from perinatal rat pups. To determine whether nuclear localization of the -IRE form of DMT1 is constrained to neuronal or neuronal-like cells, immunocytochemical studies were performed with human embryonic kidney 293T (HEK293T), HEP2G hepatoma and medulloblastoma, and rat Schwann cells. The -IRE-specific antibodies stained nuclei from medulloblastoma, whereas little nuclear staining was observed with HEK293T, hepatoma, or Schwann cells. The unexpected finding that the -IRE species of DMT1 selectively accumulates in the nucleus of neuronal and neuronal-like cells leads us to postulate that the two proteins may have different functions in vivo.
Asunto(s)
Proteínas Portadoras/metabolismo , Proteínas de Transporte de Catión , Proteínas de Unión a Hierro , Proteínas de la Membrana/metabolismo , Células PC12/metabolismo , Sistema Nervioso Simpático/metabolismo , Animales , Sitios de Unión/genética , Transporte Biológico , Proteínas Portadoras/genética , Membrana Celular/metabolismo , Núcleo Celular/metabolismo , Células Cultivadas , Técnica del Anticuerpo Fluorescente , Humanos , Immunoblotting , Inmunohistoquímica , Hierro/metabolismo , Proteínas de la Membrana/genética , Neuritas/metabolismo , Células PC12/citología , Isoformas de Proteínas/metabolismo , ARN Mensajero/metabolismo , Ratas , Receptores de Transferrina/metabolismo , Sistema Nervioso Simpático/citologíaRESUMEN
Separate pathways for transport of nontransferrin ferric and ferrous iron into tissue cultured cells were demonstrated. Neither the ferric nor ferrous pathway was shared with either zinc or copper. Manganese shared the ferrous pathway but had no effect on cellular uptake of ferric iron. We postulate that ferric iron was transported into cells via beta(3)-integrin and mobilferrin (IMP), whereas ferrous iron uptake was facilitated by divalent metal transporter-1 (DMT-1; Nramp-2). These conclusions were documented by competitive inhibition studies, utilization of a beta(3)-integrin antibody that blocked uptake of ferric but not ferrous iron, development of an anti-DMT-1 antibody that blocked ferrous iron and manganese uptake but not ferric iron, transfection of DMT-1 DNA into tissue culture cells that showed enhanced uptake of ferrous iron and manganese but neither ferric iron nor zinc, hepatic metal concentrations in mk mice showing decreased iron and manganese but not zinc or copper, and data showing that the addition of reducing agents to tissue culture media altered iron binding to proteins of the IMP and DMT-1 pathways. Although these experiments show ferric and ferrous iron can enter cells via different pathways, they do not indicate which pathway is dominant in humans.
Asunto(s)
Proteínas Portadoras/metabolismo , Proteínas de Transporte de Catión , Compuestos Férricos/farmacocinética , Compuestos Ferrosos/farmacocinética , Proteínas de Unión a Hierro , Sustitución de Aminoácidos , Animales , Antígenos CD/metabolismo , Transporte Biológico , Cationes/metabolismo , Cationes Bivalentes/metabolismo , Línea Celular , Cloruros/farmacocinética , Humanos , Integrina beta3 , Células K562 , Riñón , Compuestos de Manganeso/farmacocinética , Ratones , Glicoproteínas de Membrana Plaquetaria/metabolismo , Ratas , Proteínas Recombinantes/metabolismo , Transfección , Compuestos de Zinc/farmacocinéticaRESUMEN
In this study, we investigated the cellular distribution of iron in the brain of Belgrade rats. These rats have a mutation in Divalent Metal Transporter 1, which has been implicated in iron transport from endosomes. The Belgrade rats have iron-positive pyramidal neurons, but these are fewer in number and less intensely stained than in controls. In the white matter, iron is normally present in patches of intensely iron-stained oligodendrocytes and myelin, but there is dramatically less iron staining in the Belgrade rat. Those oligodendrocytes that stained for iron did so strongly and were associated with blood vessels. Astrocytic iron staining was seen in the cerebral cortex for both normal rats and Belgrade rats, but the iron-stained astrocytes were less numerous in the mutants. Iron staining in tanycytes, modified astrocytes coursing from the third ventricle to the hypothalamus, was not affected in the Belgrade rat, but was affected by diet. The results of this study indicate that Divalent Metal Transporter 1 is important to iron transport in the brain. Iron is essential in the brain for basic metabolic processes such as heme formation, neurotransmitter production and ATP synthesis. Excess brain iron is associated with a number of common neurodegenerative diseases. Consequently, elucidating the mechanisms of brain iron delivery is critical for understanding the role of iron in pathological conditions.
Asunto(s)
Anemia Hipocrómica/metabolismo , Química Encefálica , Proteínas Portadoras/metabolismo , Proteínas de Transporte de Catión , Proteínas de Unión a Hierro , Hierro/análisis , Proteínas de la Membrana/deficiencia , Proteínas de la Membrana/metabolismo , Proteínas del Tejido Nervioso/deficiencia , Ratas Mutantes/metabolismo , Sustitución de Aminoácidos , Anemia Hipocrómica/genética , Animales , Astrocitos/química , Proteínas Portadoras/genética , Cruzamientos Genéticos , Dieta , Endosomas/metabolismo , Femenino , Masculino , Proteínas de la Membrana/genética , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Neuronas/metabolismo , Oligodendroglía/química , Oligodendroglía/patología , Especificidad de Órganos , Mutación Puntual , Ratas , Ratas Endogámicas F344 , Ratas WistarRESUMEN
The Belgrade rat has a microcytic, hypochromic anemia inherited as an autosomal recessive trait (gene symbol b). Transferrin-dependent iron uptake is defective because of a mutation in Nramp2 (now DMT1, also called DCT1), the protein responsible for endosomal iron efflux. Hence, Belgrade reticulocytes are iron deficient. We show that a chromatographic method is able to measure the amount of 'free' heme in reticulocytes. Most of the 'free' heme is the result of biosynthesis. Succinylacetone, an inhibitor of heme synthesis, decreases the level of 'free' heme and cycloheximide, an inhibitor of globin synthesis, increases the 'free' heme level. In a pulse-chase experiment with 59Fe-transferrin, the 'free' heme pool behaves as an intermediate, with a half-life of just over 2 h. Belgrade reticulocytes contain about 40% as much 'free' heme as do heterozygous or homozygous reticulocytes. This deficiency of 'free' heme slows initiation of translation in Belgrade reticulocytes by increasing the level of an inhibitor of initiation. Thus the Belgrade rat makes a whole animal model available with chronic heme deficiency.
Asunto(s)
Anemia Hipocrómica/genética , Hemo/deficiencia , Reticulocitos/metabolismo , Anemia Hipocrómica/sangre , Animales , Sistema Libre de Células , Cicloheximida/farmacología , Modelos Animales de Enfermedad , Hemo/biosíntesis , Heptanoatos/farmacología , Hierro/metabolismo , Ratas , Reticulocitos/efectos de los fármacosRESUMEN
Genomic imprinting is an epigenetic modification that can lead to parental-specific monoallelic expression of specific autosomal genes. While methylation of CpG dinucleotides is thought to be a strong candidate for this epigenetic modification, little is known about the establishment or maintenance of parental origin-specific methylation patterns. We have recently identified a portion of mouse chromosome 9 containing a paternally methylated region associated with a paternally expressed imprinted gene, Ras protein-specific guanine nucleotide-releasing factor 1 (Rasgrf1). This area of chromosome 9 also contains a short, direct tandem repeat in close proximity to a paternally methylated NotI site 30 kb upstream of Rasgrf1. Short, direct tandem repeats have been found associated with other imprinted genes and may act as important regulatory structures. Here we demonstrate that two rodent species (Mus and Rattus) contain a similar direct repeat structure associated with a region of paternal-specific methylation. In both species, the Rasgrf1 gene shows paternal-specific monoallelic expression in neonatal brain. A more divergent rodent species (Peromyscus) appears to lack a similar repeat structure based on Southern Blot analysis. Peromyscus animals show biallelic expression of Rasgrf1 in neonatal brain. These results suggest that direct repeat elements may play an important role in the imprinting process.
Asunto(s)
Impresión Genómica , Proteínas/genética , Secuencias Repetitivas de Ácidos Nucleicos/genética , Animales , Secuencia de Bases , Southern Blotting , Encéfalo/embriología , Encéfalo/metabolismo , Mapeo Cromosómico , Regulación del Desarrollo de la Expresión Génica , Factores de Intercambio de Guanina Nucleótido , Pulmón/embriología , Pulmón/metabolismo , Ratones , Datos de Secuencia Molecular , Muridae , Peromyscus , Reacción en Cadena de la Polimerasa , Polimorfismo Conformacional Retorcido-Simple , Ratas , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Especificidad de la Especie , Factores de Intercambio de Guanina Nucleótido ras , Proteínas rasRESUMEN
Belgrade (b) rats have an autosomal recessive, microcytic, hypochromic anemia. Transferrin (Tf)-dependent iron uptake is defective because of a mutation in DMT1 (Nramp2), blocking endosomal iron efflux. This experiment of nature permits the present study to address whether the mutation also affects non-Tf-bound iron (NTBI) uptake and to use NTBI uptake compared to Tf-Fe utilization to increase understanding of the phenotype of the b mutation. The distribution of 59Fe2+ into intact erythroid cells and cytosolic, stromal, heme, and nonheme fractions was different after NTBI uptake vs. Tf-Fe uptake, with the former exhibiting less iron into heme but more into stromal and nonheme fractions. Both reticulocytes and erythrocytes exhibit NTBI uptake. Only reticulocytes had heme incorporation after NTBI uptake. Properly normalized, incorporation into b/b heme was approximately 20% of +/b, a decrease similar to that for Tf-Fe utilization. NTBI uptake into heme was inhibited by bafilomycin A1, concanamycin, NH4Cl, or chloroquine, consistent with the endosomal location of the transporter; cellular uptake was uninhibited. NTBI uptake was unaffected after removal of Tf receptors by Pronase or depletion of endogenous Tf. Concentration dependence revealed that NTBI uptake into cells, cytosol, stroma, and the nonheme fraction had an apparent low affinity for iron; heme incorporation behaved like a high-affinity process, as did an expression assay for DMT1. DMT1 serves in both apparent high-affinity NTBI membrane transport and the exit of iron from the endosome during Tf delivery of iron in rat reticulocytes; the low-affinity membrane transporter, however, exhibits little dependence on DMT1.
Asunto(s)
Anemia Hipocrómica/sangre , Proteínas Portadoras/metabolismo , Proteínas de Transporte de Catión , Proteínas de Unión a Hierro , Hierro/sangre , Proteínas de la Membrana/metabolismo , Reticulocitos/metabolismo , Transferrina/metabolismo , Anemia Hipocrómica/genética , Animales , Transporte Biológico , Línea Celular , Endosomas/metabolismo , Heterocigoto , Humanos , Técnicas In Vitro , Hierro/metabolismo , Cinética , Mutación Puntual , Ratas , Ratas Mutantes , Proteínas Recombinantes/metabolismo , Valores de Referencia , Transfección , Transferrina/genéticaRESUMEN
The Belgrade (b) rat has an autosomal recessively inherited, microcytic, hypochromic anemia associated with abnormal reticulocyte iron uptake and gastrointestinal iron absorption. The b reticulocyte defect appears to be failure of iron transport out of endosomes within the transferrin cycle. Aspects of this phenotype are similar to those reported for the microcytic anemia (mk) mutation in the mouse. Recently, mk has been attributed to a missense mutation in the gene encoding the putative iron transporter protein Nramp2. To investigate the possibility that Nramp2 was also mutated in the b rat, we established linkage of the phenotype to the centromeric portion of rat chromosome 7. This region exhibits synteny to the chromosomal location of Nramp2 in the mouse. A polymorphism within the rat Nramp2 gene cosegregated with the b phenotype. A glycine-to-arginine missense mutation (G185R) was present in the b Nramp2 gene, but not in the normal allele. Strikingly, this amino acid alteration is the same as that seen in the mk mouse. Functional studies of the protein encoded by the b allele of rat Nramp2 demonstrated that the mutation disrupted iron transport. These results confirm the hypothesis that Nramp2 is the protein defective in the Belgrade rat and raise the possibility that the phenotype shared by mk and b animals is unique to the G185R mutation. Furthermore, the phenotypic characteristics of these animals indicate that Nramp2 is essential both for normal intestinal iron absorption and for transport of iron out of the transferrin cycle endosome.
Asunto(s)
Anemia Hipocrómica/genética , Proteínas Portadoras/genética , Proteínas Portadoras/fisiología , Proteínas de Transporte de Catión , Endosomas/metabolismo , Proteínas de Unión a Hierro , Hierro/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/fisiología , Mutación , Alelos , Secuencia de Aminoácidos , Anemia Hipocrómica/metabolismo , Animales , Arginina/genética , Arginina/metabolismo , Transporte Biológico , Mapeo Cromosómico , Codón , Ligamiento Genético , Genotipo , Glicina/genética , Glicina/metabolismo , Ratones , Datos de Secuencia Molecular , Ratas , Alineación de Secuencia , Transferrina/metabolismoRESUMEN
Belgrade rats have an autosomal recessive anemia with hypochromia and microcytosis. Iron uptake into reticulocytes is approximately 20% of normal, but transferrin uptake is unimpaired. We have systematically compared the transferrin cycle in Belgrade versus normal reticulocytes to locate the defect more precisely. Belgrade transferrin was functionally normal as purified transferrin or whole plasma. Transferrin affinity of Belgrade receptors was indistinguishable from normal, but Belgrade reticulocytes had twice as many receptors. Belgrade transferrin endocytosis was 1.5 times faster than normal, whereas exocytosis is about twice as fast. Initially Belgrade reticulocytes internalize iron at an unimpaired rate, but they lag behind normal by 5 min. During reincubation, they release 25-33% of iron taken up during a 30-min preincubation, whereas normal cells do not lose a detectable fraction. Unexpectedly, transferrin cycle time was unchanged. Hence another kinetic step of the cycle is slower, compensating for increases in Belgrade endocytosis and exocytosis. After one cycle, Belgrade reticulocytes retain only half of the iron that entered, but over 90% of iron entering normal cells remains within. Iron unloading is ineffective inside the Belgrade vesicle; 85% of iron that entered on transferrin returned to the medium after exocytosis, whereas only 45% of iron entering normal reticulocytes exits. Ineffective utilization of iron in or near Belgrade endosomes accounts for the Belgrade defect.
Asunto(s)
Reticulocitos/metabolismo , Transferrina/metabolismo , Anemia/genética , Anemia/metabolismo , Animales , Células Cultivadas , Medios de Cultivo , Endocitosis , Exocitosis , Hierro/metabolismo , Cinética , Ratas , Ratas MutantesRESUMEN
We have used succinylacetone (4,6-dioxoheptanoic acid), a specific inhibitor of delta-aminolevulinic acid dehydrase, to gain insight into the defect in iron metabolism in the Belgrade anemia. The Belgrade rat has an inherited microcytic, hypochromic anemia associated with poor iron uptake into developing erythroid cells. Succinylacetone inhibits heme synthesis, leading to nonheme iron accumulation in mitochondria and cytosol of normal reticulocytes. When succinylacetone is used to inhibit Belgrade heme synthesis, iron from diferric transferrin does not accumulate in the stromal fraction that contains mitochondria, nor does 59Fe accumulate in the nonheme cytosolic fraction. Hence, the defect in the Belgrade rat reticulocyte occurs in the endocytic vesicle or in a step subsequent to iron transit from the vesicle but before the nonheme cytosolic or mitochondrial iron fractions. Therefore, the mutation affects either the release of iron from transferrin or iron transport from the vesicle to the mitochondrion.
Asunto(s)
Anemia Hipocrómica/sangre , Hemo/biosíntesis , Heptanoatos/farmacología , Hierro/sangre , Reticulocitos/metabolismo , Anemia Hipocrómica/genética , Animales , Citosol/metabolismo , Radioisótopos de Hierro , Mitocondrias/metabolismo , Mutación , Porfobilinógeno Sintasa/antagonistas & inhibidores , Ratas , Ratas Mutantes , Reticulocitos/ultraestructura , Transferrina/metabolismoRESUMEN
The Belgrade rat has a hypochromic, microcytic anemia inherited as an autosomal recessive mutation. Although transferrin binds normally to reticulocytes and internalizes normally, iron accumulation into cells and heme is much slower than normal. We have investigated the role of the transferrin cycle in this mutant by bypassing transferrin iron delivery with the iron chelate ferric salicylaldehyde isonicotinoyl hydrazone (Fe-SIH). Fe-SIH increases iron uptake into heme by Belgrade reticulocytes, restoring it almost to normal levels. This increase indicates that Fe-SIH delivers iron to a step in iron utilization that is after the Belgrade defect. Depleting reticulocytes of transferrin did not alter these observations. Failure to achieve above normal rates of iron incorporation could indicate damage due to chronic intracellular iron deficiency. Also, iron delivery by Fe-SIH restored globin synthesis to near-normal levels in Belgrade reticulocytes. The rates of glycine incorporation into porphyrin and heme in Belgrade reticulocytes incubated with Fe2-transferrin or Fe-SIH paralleled the rates of iron incorporation into heme. These data are consistent with the concept that iron availability limits protoporphyrin formation in rat reticulocytes. The protoporphyrin used for heme synthesis is provided by de novo synthesis and not by a pool of pre-existing protoporphyrin. The Belgrade defect occurs in the movement of iron from transferrin to a step prior to the ferrous state and insertion into heme. This defect diminishes the synthesis of heme and, consequently, that of protoporphyrin and globin.
Asunto(s)
Quelantes del Hierro/farmacología , Hierro/metabolismo , Isoniazida/análogos & derivados , Reticulocitos/metabolismo , Animales , Células Cultivadas , Globinas/metabolismo , Glicina/metabolismo , Hemo/metabolismo , Isoniazida/farmacología , Porfirinas/metabolismo , Ratas , Transferrina/metabolismo , Transferrina/fisiologíaRESUMEN
Homozygous beta-thalassemic mice show many of the features seen in human beta-thalassemia, such as decreased hemoglobin, hematocrit, and red blood cell count as well as increased reticulocyte count. They also exhibit splenomegaly and a decrease in osmotic fragility of red cells. beta-thalassemic mice were examined for spontaneous iron overload at ages ranging from 20 to 595 days. Accumulation of iron was shown to occur in the spleen, liver, and kidneys but not in the heart. Sections of spleen, liver, kidney, and heart were stained for iron and subjectively scored. Image analysis microscopy was used to examine sections of spleen and liver. Nonheme iron in the four tissues was quantitated using the bathophenanthroline sulfonate colorimetric assay. An increase in tissue iron occurred primarily in the spleen, even before weaning, despite the low iron content of milk. Iron accumulation in the absence of blood transfusion is of interest because iron overload is the major cause of death in human beta-thalassemia.
Asunto(s)
Hierro/farmacocinética , Talasemia/metabolismo , Envejecimiento/metabolismo , Animales , Peso Corporal , Riñón/metabolismo , Hígado/metabolismo , Ratones , Microscopía/métodos , Tamaño de los Órganos , Bazo/metabolismoRESUMEN
We compared the transcription and translation of globin genes in three mouse erythroleukemia cell lines under different conditions in which there is differential expression of beta-major (beta ma) and beta-minor (beta mi). Transcription was measured by pulse labeling of whole cell RNA with [3H]uridine, and translation by labeling whole cell protein synthesis with [3H]leucine. Induction with dimethyl sulfoxide led to increased transcription of alpha, beta ma, and beta mi genes, and the proportion of beta mi RNA synthesized was similar to the proportion of beta mi globin protein produced, whether the cells produced 30-40% beta mi (lines 745 and 9M) or greater than 80% beta mi (clone 25-66). Induction with hemin did not lead to increased globin gene transcription in any line, although globin protein synthesis did increase. Thus, the mechanism of beta globin chain induction depends on the inducing agent, and not on the cell line or the type of beta globin gene product. The relative proportion of beta mi and beta ma globin chain proteins reflects the relative transcription of the two beta genes, whether or not transcription increased following induction.
Asunto(s)
Globinas/biosíntesis , Leucemia Eritroblástica Aguda/metabolismo , Transcripción Genética , Animales , ADN/metabolismo , ADN Recombinante/metabolismo , Dimetilsulfóxido/farmacología , Virus de la Leucemia Murina de Friend , Globinas/genética , Hemina/farmacología , Leucina/metabolismo , Ratones , Hibridación de Ácido Nucleico , Biosíntesis de Proteínas/efectos de los fármacos , Transcripción Genética/efectos de los fármacos , Células Tumorales Cultivadas , Uridina/metabolismoRESUMEN
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.
Asunto(s)
Globinas/genética , Ratones Endogámicos DBA/genética , Secuencia de Aminoácidos , Aminoácidos/análisis , Animales , Evolución Biológica , Ratones , Ratones Endogámicos BALB C/genéticaRESUMEN
Rat hemoglobins are unusually heterogeneous among mammals. This heterogeneity provides multiple opportunities for asynchronies in synthesis and degradation. We have examined rat hemoglobin turnover after an intravenous injection of 2-14C-glycine. After analyzing incorporation into the seven nonallelic globin chains of adult rats, we found the percentage of the major beta chain decreases over the red cell lifespan while the percentage of one of the minor beta chains increases. This suggests that a post-synthetic modification event converts a portion of the latter into the former.
Asunto(s)
Hemoglobinas/metabolismo , Procesamiento Proteico-Postraduccional , Animales , Femenino , Globinas/metabolismo , Hemo/metabolismo , RatasAsunto(s)
Clorometilcetonas de Aminoácidos/farmacología , Globinas/biosíntesis , Biosíntesis de Proteínas/efectos de los fármacos , Clorometilcetona Tosilisina/farmacología , Animales , Cromatografía por Intercambio Iónico , Técnicas In Vitro , Metionina/análisis , Conejos , Reticulocitos/efectos de los fármacos , Reticulocitos/metabolismoRESUMEN
Hemoglobin beta-chain synthesis by rabbit erythroid cells was tested for dependence on availability of complementary alpha-chains. Reticulocytes and bone marrow cells were obtained from variant rabbits that have hemoglobin with isoleucine in alpha-chains but not in beta-chains. This characteristic permits the use of L-O-methylthreonine, a specific isoleucine antagonist, to inhibit selectively the synthesis of hemoglobin alpha-chains without directly affecting that of beta-chains. Study of hemoglobin synthesis by bone marrow cells presents two problems that require careful management: (A) the fragility of the globin-synthesizing apparatus and (B) the isolation of globin from the various proteins made by the mixture of nucleated cells. Disruption of synthetic activity was minimized by collecting the bone marrow in autologous plasma then removing fat and connective tissue while the cells were suspended in this medium. Purification involved gel filtration of hemoglobin and globin then CM-cellulose chromatography of globin chains. Absence of radioactive isoleucine in beta-chains demonstrated the efficacy of this scheme in removing isoleucine-containing proteins that otherwise elute with beta-chains on CM-cellulose columns. In reticulocytes, when synthesis of alpha-chains is inhibited by 30%--80%, that of beta-chains is stimulated by 20%--60%, but in marrow cells, incorporation into beta-chains stays at control level when alpha incorporation is inhibited. The data indicate that beta-chain synthesis is independent of the availability of complementary alpha-chains.
Asunto(s)
Eritrocitos/análisis , Hemoglobinas/biosíntesis , Animales , Proteínas Sanguíneas , Células de la Médula Ósea , Separación Celular , Globinas/aislamiento & purificación , Hemoglobinas/metabolismo , Conejos , Reticulocitos/análisis , Treonina/análogos & derivados , Treonina/farmacologíaRESUMEN
Tosyl-L-lysyl chloromethyl ketone (TLCK) inhibits protein synthesis in intact cells and lysates. Its presence leads to polyribosome disaggregation. This inhibition is probably at the level of chain initiation because it does not slow poly(U)-dependent elongation of poly(F). This drug activates an inhibitor similar to the heme-controlled repressor in the postribosomal supernatant. High concentrations of ATP and GTP affect inhibitions of translation by the heme-controlled repressor and TLCK in a similar fashion. The latter also interferes with formation of the 40S initiation complex. Its action on translation can be prevented by preliminary incubation with thiols.