Assay of Thalassaemic Messenger RNA in the Wheat Germ System

Messenger RNA(mRNA) has been prepared from reticulocytes obtained from patients with different types of thalassaemia and assayed in the wheat germ system. Since normal human reticulocyte mRNA directs the synthesis of equal numbers of alpha- and beta-globin chains in this system it offers a rapid and simple technique for assaying mRNA in the thalassaemic disorders. In mRNA from beta+ thalassaemics the deficiency of beta-globin synthesis mirrored that observed in intact reticulocytes while that prepared from patients wiht haemoglobin H disease gave alpha/beta globin chain production ratios which showed consistently greater imbalance than was found in reticulocytes. Messenger RNA prepared from haemoglobin E-beta0 thalassaemics from Thailand directed no detectable beta-chain synthesis while that prepared from a betaO thalassaemic from Ferrara synthesized a fraction with the chromatographic characteristics of beta-globin chains.     

Further evidence of a quantitative deficiency of chain-specific globin mRNA in the thalassemia syndromes.

Formamide gel electrophoresis separates the mRNA fraction from reticulocyte polyribosomes of adult humans into two major RNA species with migratory rates identical to those of the alpha- and beta-globin mRNAs of the rabbit. That these two RNAs of human origin are the globin mRNAs is further supported by the deficiency of the presumed beta mRNA in reticulocyte polyribosomes of fetuses and premature infants, whose cells make gamma chains in preference to beta chains. The globin mRNAs of reticulocyte polyribosomes from patients with hematological disorders were estimated by scanning the stained formamide gels. In contrast to individuals with either hemolytic anemia without hemoglobinopathy or sickle cell anemia who had beta mRNA to alpha mRNA ratios of approximately one, a patient with Hb S-beta-thalassemia had a ratio of beta mRNA to alpha mRNA of 0.75 while two subjects with homozygous beta-thalassemia had severe deficiencies of beta mRNA. Conversely, a patient with alpha-thalassemia (Hb H disease) had a ratio of beta mRNA to alpha mRNA on reticulocyte polyribosomes of 6. These data provide further evidence of a quantitative deficiency of chain-specific globin mRNA in patients with the thalassemia syndromes.     

Regulation of hemoglobin beta-chain synthesis in bone marrow erythroid cells by alpha chains

Synthesis of alpha and beta chains of hemoglobin was studied in vitro in intact reticulocytes and bone marrow cells. The cells were from rabbits having a variant form of hemoglobin in which L-isoleucine is in the alpha but not in the beta chains. This characteristic permitted a selective inhibition of alpha-chain synthesis to be produced by addition to the incubation medium of L-O-methylthreonine, an inhibitor of protein synthesis that is a specific antagonist of L-isoleucine.In studies with reticulocytes, 25 mM L-O-methylthreonine produced a 60-70% inhibition of alpha-chain synthesis, but beta-chain synthesis was unaffected even after incubation times for 4 hr. Because reticulocytes contain a pool of uncombined alpha chains which might have obscured the demonstration of an alpha chain-dependent mechanism for beta-chain synthesis, subsequent studies were done with bone marrow cells. The latter had little or no detectable alpha-chain pool. A substantial inhibition of alpha-chain synthesis by the bone marrow cells was produced by the isoleucine antagonist but was also accompanied by a significantly decreased rate of beta-chain synthesis.These findings suggest that the coordinated synthesis of the complementary alpha- and beta-globin chains of hemoglobin may reflect in part a modifying effect of alpha-chain synthesis on the synthesis of beta chains.     

Cell-free hemoglobin synthesis in beta-thalassemia

Human ribosomes obtained from the reticulocytes of patients having either homozygous beta-thalassemia (thalassemia ribosomes) or a hematological disorder unrelated to thalassemia („normal” ribosomes) have been utilized in a cell-free system highly active in the synthesis of intact human globin chains. This system is dependent on the addition of a ribosomal wash fraction from reticulocytes that contains factors necessary for chain initiation. In response to the ribosomal wash fraction, isolated from either thalassemia, normal human, or rabbit reticulocytes, normal human ribosomes synthesize equal amounts of alpha and beta chains. In contrast, in response to all three types of ribosomal wash fractions, thalassemia ribosomes synthesize 8-times more α than β chains, a ratio similar to that produced in the intact cells of these patients. The molecular defect in beta-thalassemia, therefore, does not appear to be associated with initiation factors.     

Decreased alpha globine mRNA in nucleated red cell precursors in alpha thalassemia.

The alpha thalassemias are associated with a decrease in alpha chain synthesis. Hemoglobin H (HbH) disease is a moderately severe form of alpha thalassemia characterized by the production of 5%--20% of HbH, while alpha thalassemia trait is a milder form of alpha thalassemia. In two patients with HbH disease, the ratio of alpha chain synthesis to beta chain synthesis (alpha/beta ratio) was decreased in both bone marrow cells and reticulocytes. When isolated mRNA from bone marrow cells and reticulocytes was translated in a heterologous cell-free system, the alpha/beta ratios were lower than the intact cell ratios. These findings were confirmed by hybridization of the mRNA of both marrow cells and reticulocytes using purified alpha and beta cDNA probes. In the intact cells of two patients with alpha thalassemia trait, the alpha/beta ratios were also decreased and were similar in marrow cells and reticulocytes. Cell-free studies of translatable mRNA also demonstrated decreased alpha/beta ratios, but, unlike the HbH studies, the cell-free alpha/beta ratios were similar to the intact cell ratios. One hybridization study utilizing peripheral blood mRNA had an alpha/beta ratio consistent with the cell-free ratios. These results indicated that, in both HbH disease and alpha thalassemia trait, there was decreased alpha globin mRNA present in both nucleated red cell precursors and reticulocytes. In addition, the data suggested that there may be translational mechanisms that operate in intact HbH cells which attempt to balance globin chain production. In alpha thalassemia trait cells, no such controls appeared to be active and globin chain synthesis was directly proportional to the amount of alpha and beta globin mRNA in the cells.     

Beta Thalassemia and Translation of Globin Messenger RNA

To define the quality and relative quantity of beta and alpha messenger RNA in human nonthalassemic and thalassemic reticulocytes, intact cells were incubated with [(35)S]methionine. The relative amounts of beta- and alpha-nascent chains on polysomes of different sizes were measured by tryptic digestion of pooled polysomes and by determination of the specific activities of beta and alpha peptides that contain methionine. Betachain synthesis predominated on heavy polysomes in nonthalassemic, as well as in thalassemic cells. Since beta chains in thalassemia are made on normal-size polyribosomes, we conclude that the defect in thalassemia does not involve reduction in the rate of initiation of translation due to the production of an abnormal beta message. Such would lead to beta-chain synthesis on very small polysomes. We therefore suggest that the decreased production of beta-globin chains results from a decreased amount of functional beta-globin messenger RNA.     

betaMinor-globin messenger RNA accumulation in reticulocytes governs improved erythropoiesis in beta thalassemic mice after erythropoietin complementary DNA electrotransfer in muscles

Mechanisms governing the induction of effective erythropoiesis in response to erythropoietin (Epo) oversecretion have been investigated in beta thalassemic C57Bl/6(Hbbth) mice. Naked DNA encoding an expression vector for mouse Epo was introduced into skeletal muscles by electrotransfer. A transient increase of serum Epo concentrations with a proportional augmentation of hematocrit values was observed. Various parameters relevant to beta thalassemia were surveyed in blood samples taken before treatment, at the peak of Epo secretion, and when the phenotype reverted to anemia. We measured globin messenger RNA (mRNA) levels in reticulocytes by real-time quantitative polymerase chain reaction, globin chain synthesis levels, and several indicators of erythrocyte membrane quality, including bound alpha chains, bound immunoglobulins, main protein components, and iron compartmentalization. Data indicated that high serum Epo levels primarily affect betaminor-globin mRNA accumulation in reticulocytes. Other changes subsequent to intense Epo stimulation, like increased betaminor/alpha-globin chain synthesis ratio, reduced levels of alpha chains and immunoglobulins bound to membranes, improved spectrin/band 3 ratio, increased red blood cell survival, and improved erythropoiesis appeared as consequences of increased betaminor-globin mRNA levels. This conclusion is consistent with models postulating that intense Epo stimulation induces the expansion and differentiation of erythroid progenitors committed to fetal erythropoiesis. Although phenotypic correction was partial in mice, and comparable achievements will probably be more difficult to obtain in humans, naked DNA electrotransfer may provide a safe and low-cost method for reassessing the potentials of Epo as an inducer of fetal erythropoiesis reactivation in patients with beta thalassemia.     

Absence of functional messenger RNA activity for beta globin chain synthesis in beta 0-thalassemia

Functional human globin messenger RNA was isolated from reticulocytes of two patients with homozygous beta 0-thalassemia, three patients with sickle cell beta 0-thalassemia, and one patient doubly heterozygous for beta 0-thalassemia and hemoglobin Lepore. When incubated in the Krebs type II mouse ascites tumor-cell-free system, messenger RNA from these patients actively directed the synthesis of human beta s and/or alpha- and gamma-globin chains but failed to stimulate the synthesis of any beta A-chains, even though nonthalassemic human globin mRNA preparations consistently stimulated two to four times as much beta A- or beta S-globin chain synthesis as alpha-chain synthesis when incubated in the same system under the same conditions. These results strongly suggest that functional beta A-chain-specific globin mRNA is absent in beta 0-thalassemia.     

Increased Efficiency of Exogenous Messenger RNA Translation in a Krebs Ascites Cell Lysate

Addition of a 0.5 M KCl wash fraction from rabbit reticulocyte ribosomes causes a 3- to 10-fold increase in the extent of translation of natural mRNAs by Krebs-cell lysates. In the presence of the wash fraction, 1 pmol of rabbit or mouse 10S RNA directs the incorporation of 80 pmol of leucine into rabbit globin. The addition of human 10S RNA results in the synthesis of equal amounts of human alpha and beta chains, identified by column chromatography. The stimulation by the wash fraction is almost completely dependent on added mammalian tRNA. In contrast to the wash fraction from rabbit reticulocytes, the wash fraction isolated from Krebs-cell ribosomes is inhibitory to both endogenous and exogenous mRNA translation. The stimulation by the wash fraction from rabbit ribosomes is not specific for globin mRNAs, but also increases endogenous, phage Qbeta, and viral RNA-directed protein synthesis.     

A Limited Number of Globin Genes in Human DNA

The number of globin genes in human cells was determined by hybridizing DNA from human spleens to (3)H-labeled DNA complementary to human globin mRNA. Assuming the rates of reannealing of complementary DNA and cellular DNA are similar, the extent of hybridization of complementary DNA at various ratios of cellular DNA to complementary DNA indicate that there are fewer than 10 globin gene copies per haploid human genome. An alternative analysis of the data, which introduces no assumptions concerning the relative rates of reaction of complementary DNA and cellular DNA, indicates fewer than 20 globin gene copies are present. DNA isolated from the spleen of a patient with beta(+) thalassemia contained a number of globin gene copies similar to that of normal DNA.     

Autonomous synthesis of alpha and beta hemoglobin chains in rabbit erythroid cells

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.     

Isolation of distinct cDNA clones encoding HLA-DR beta chains by use of an expression assay.

cDNA clones encoding different human Ia antigen beta chains were isolated by use of a complementation-expression assay in Xenopus oocytes. The assay was based on two previous findings. First, oocytes injected with mRNA from a human B-cell line express HLA-DR antigen. The three intracellular DR chains are assembled in oocytes and can be immunoprecipitated with anti-DR monoclonal antibodies. Second, we have isolated cDNA clones encoding DR alpha and intermediate chains. In order to identify beta-chain cDNA clones, mRNA was hybrid-selected with pools of cDNA clones, mixed with mRNA for the alpha and intermediate chains, and injected into oocytes. We isolated two distinct clones that could select DR beta-chain mRNA as demonstrated by assembly of the translation product with DR alpha chains and immunoprecipitation with DR-specific monoclonal antibodies. One clone is specific for a beta chain of the DR locus. The other clone, much weaker in its ability to select DR mRNA, encodes another Ia-like beta chain. Full-length cDNA clones corresponding to the DR and Ia-like beta chains were isolated and compared. Cross-hybridization was detectable in the coding regions but not in the 3' untranslated regions. Distinct RNAs homologous to the DR and the Ia-like beta-chain clones were present in B cells but were undetectable in three T-cell lines.     

Translation of human globin mRNA: globin synthesis in cells containing Hb Leiden.

Most structurally abnormal hemoglobins are present in smaller amounts than HbA in the erythrocytes of heterozygous subjects. In the presence of a hemoglobinopathy, alpha and beta globin synthesis remains balanced with equal production of the two types of chains. In reticulocytes of subjects with Hb Leiden (beta 6 or 7 glu leads to 0) there is greater production of alpha than beta globin in vitro (beta/alpha = 0.67), and slightly more beta A is synthesized than beta Leiden (beta A/beta Leiden = 1.28). Differences in specific mRNA content, rates of initiation of chain synthesis, or rates of chain elongation could be responsible for such differential polypeptide synthesis. In the present study, the ribosomal assembly of beta A, beta Leiden, and alpha globin chains was examined in peripheral blood. The translation times of the three chains did not differ significantly (average times: beta A = 65.4 sec, beta Leiden = 70.8 sec, alpha = 53.5 sec). These results indicated that an altered rate of translation was not the source of the anomalous globin synthesis observed in vitro in cells containing Hb Leiden. The experiments suggested that the observed imbalance in alpha/beta production was due to either differential rates of initiation of globin chain synthesis or quantitative differences in the amounts of the specific mRNAs present in the cells.     

Gene encoding the human beta-hexosaminidase beta chain: extensive homology of intron placement in the alpha- and beta-chain genes.

Lysosomal beta-hexosaminidase (EC 3.2.1.52) is composed of two structurally similar chains, alpha and beta, that are the products of different genes. Mutations in either gene causing beta-hexosaminidase deficiency result in the lysosomal storage disease GM2-gangliosidosis. To enable the investigation of the molecular lesions in this disorder and to study the evolutionary relationship between the alpha and beta chains, the beta-chain gene was isolated, and its organization was characterized. The beta-chain coding region is divided into 14 exons distributed over approximately 40 kilobases of DNA. Comparison with the alpha-chain gene revealed that 12 of the 13 introns interrupt the coding regions at homologous positions. This extensive sharing of intron placement demonstrates that the alpha and beta chains evolved by way of the duplication of a common ancestor.     

Proteolytic activity in erythrocyte precursors

Thalassemia is characterized by unequal rates of synthesis of the alpha and beta globin chains that are part of the hemoglobin tetramer. In the type of thalassemia due to a defect in beta-chain synthesis (beta-thalassemia), this imbalance results in a relative exoess of alpha-chains. We have studied the susceptibility of excess free alpha-chains to proteolysis. Incubation of isotopically labeled peripheral blood lysates from individuals with beta-thalassemia trait in the presence of bone marrow or normoblast lysates from thalassemic or hematologically normal individuals resulted in a decrease in the alpha/beta ratio and a loss of free alpha-chain radioactivity. Neither contamination with leukocytes nor higher ATP contents in young erythrocytes appeared to be responsible for this activity in normoblasts and bone marrow. We propose that erythroid precursor cells possess proteolytic activity that is markedly diminished in mature cells. This activity serves an important control function in the regulation of hemoglobin synthesis. It accounts at least in part for the more balanced synthesis of alpha- and beta-chains observed in bone marrow than in peripheral blood in heterozygous beta-thalassemia. It also plays a fine-tuning role in maintaining balanced synthesis in non-thalassemic erythrocytes.     

Acquired hemoglobin H disease in idiopathic myelofibrosis.

A 68-year-old male, diagnosed 1 year previously as having myelofibrosis, developed hemolysis, red cell inclusions, and 37% Hb H. The alpha/beta globin synthetic ratio for circulating reticulocytes, determined by 3H-leucine incorporation and globin chain separation by carboxymethylcellulose chromatography in urea, was 0.049. When total RNA was purified from peripheral blood cells and translated in a wheat germ cell-free translation system, the alpha/beta ratio of the translation products was 0.26, indicating mRNA as a major cause of the globin synthetic imbalance. This study demonstrates that myelofibrosis is one setting in which acquired Hb H disease occurs; that the synthetic imbalance may be extreme; and that it can be associated with an imbalance in the activities of specific globin mRNAs.