Alpha-globin Synthesis Research Articles

Heat shock-induced translational control of HSP70 and globin synthesis in chicken reticulocytes.

Incubation of chicken reticulocytes at elevated temperatures (43 to 45 degrees C) resulted in a rapid change in the pattern of protein synthesis, characterized by the decreased synthesis of normal proteins, e.g., alpha and beta globin, and the preferential and increased synthesis of only one heat shock protein, HSP70. The repression of globin synthesis was not due to modifications of globin mRNA because the level of globin mRNA and its ability to be translated in vitro were unaffected. The HSP70 gene in reticulocytes was transcribed in non-heat-shocked cells, yet HSP70 was not efficiently translated until the cells had been heat shocked. In non-heat-shocked reticulocytes, HSP70 mRNA was a moderately abundant mRNA present at 1 to 2% of the level of globin mRNA. The rapid 20-fold increase in the synthesis of HSP70 after heat shock was not accompanied by a corresponding increase in the rate of transcription of the HSP70 gene or accumulation of HSP70 mRNA. These results suggest that the elevated synthesis of HSP70 is due to the preferential utilization of HSP70 mRNA in the heat-shocked reticulocyte. The heat shock-induced alterations in the reticulocyte protein-synthetic apparatus were not reversible. Upon return to control temperatures (37 degrees C), heat-shocked reticulocytes continued to synthesize HSP70 at elevated levels whereas globin synthesis continued to be repressed. Despite the presence of HSP70 mRNA in non-heat-shocked reticulocytes, we found that continued transcription was necessary for the preferential translation of HSP70 in heat-shocked cells. Preincubation of reticulocytes with the transcription inhibitor actinomycin D or 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole blocked the heat shock-induced synthesis of HSP70. Because the level of HSP70 mRNA was only slightly diminished in cells treated with actinomycin D, we suggest two possible mechanisms for the preferential translation of HSP70 mRNA: the translation of only newly transcribed HSP70 mRNA or the requirement of a newly transcribed RNA-containing factor.

Coordinate regulation of polypeptide chain initiation and elongation in rabbit reticulocytes.

Under normal conditions, reticulocytes synthesize alpha- and beta-globin polypeptides at equal rates. Incubation in the absence of hemin or under anoxia or hypertonic stress (100 mM excess NaCl) reduces the rate of protein synthesis to 30-50% of control levels. However, only hemin deprivation causes a reduction in polyribosome size and preferential inhibition of alpha-globin synthesis consistent with specific reduction in the rate of polypeptide chain initiation. Polyribosomal profiles are unaffected by anoxic or hypertonic stress and the ratio of alpha:beta globin synthesis remains close to unity. Measurement of ribosome transit time indicates that anoxic or hypertonic stress causes a decrease in the rate of polypeptide chain elongation that varies with the degree of inhibition of protein synthesis. Ribosomes isolated from stressed cells exhibit a reduced ability to bind 35S-met-tRNAf, suggesting that the ability to form initiation complexes is also impaired. These results suggest that reticulocytes, unlike nucleated cell lines, can coordinately reduce rates of initiation and elongation in response to certain physiological stresses.

Hemin Preferentially Stimulates Synthesis of Alpha-Globin in K562 Human Erythroleukemia Cells

K562 human erythroleukemia cells are an established cell line derived from an adult with chronic myelogenous leukemia. Hemin stimulates their synthesis of embryonic and fetal hemoglobins. We have found that their globin synthetic pattern depends on the concentration of added hemin. Clone RA6 was cultured with 0--100 microM hemin and the globin synthetic pattern determined by 3H-leucine incorporation and analysis of 3H-globins by polyacrylamide gel electrophoresis in Triton X acid urea followed by fluorography and densitometry. The higher the hemin concentration, the greater the synthetic rate of each type of globin. However, the relative increase was greatest for alpha-globin. We propose that the differential dependence of alpha synthesis on added hemin is a reflection of translational inefficiency of alpha messenger RNA and that this property is exposed when the translational capacity of the cell is limited by hemin deficiency. We suggest that the differential dependence of alpha-chain synthesis on added hemin in clone RA6 is evidence of an intrinsic deficiency in heme synthesis.

Hemin preferentially stimulates synthesis of alpha-globin in K562 human erythroleukemia cells

K562 human erythroleukemia cells are an established cell line derived from an adult with chronic myelogenous leukemia. Hemin stimulates their synthesis of embryonic and fetal hemoglobins. We have found that their globin synthetic pattern depends on the concentration of added hemin. Clone RA6 was cultured with 0--100 microM hemin and the globin synthetic pattern determined by 3H-leucine incorporation and analysis of 3H-globins by polyacrylamide gel electrophoresis in Triton X acid urea followed by fluorography and densitometry. The higher the hemin concentration, the greater the synthetic rate of each type of globin. However, the relative increase was greatest for alpha-globin. We propose that the differential dependence of alpha synthesis on added hemin is a reflection of translational inefficiency of alpha messenger RNA and that this property is exposed when the translational capacity of the cell is limited by hemin deficiency. We suggest that the differential dependence of alpha-chain synthesis on added hemin in clone RA6 is evidence of an intrinsic deficiency in heme synthesis.

Molecular characteristics of a non-deletion alpha-thalassaemia of the Po River Delta.

The form of alpha-thalassaemia of the Po river delta presents haematological and globin biosynthetic characteristics similar to alpha-thalassaemia-1 but never gives rise to HB H disease nor to hydrops foetalis. In alpha-thalassaemic subjects originally from this region globin mRNA translation and alpha-globin gene arrangement have been investigated. The data obtained indicate that alpha-globin synthesis and reticulocyte alpha-globin mRNA are reduced by one fourth; in addition, since the defect in alpha-globin synthesis does not change with cell ageing, a possible instability of alpha-globin mRNA is excluded. Restriction enzyme analysis of the DNA shows a normal alpha-globin gene organization. This form of alpha-thalassaemia is therefore of the non-deletion type; its molecular lesion is either at the level of alpha-globin mRNA transcription or processing. The fact that this, as well as other forms of non-deletion alpha-thalassaemia, have a phenotypic expression similar to alpha-thalassaemia 1 is discussed.

Translational stability of native and deadenylylated rabbit globin mRNA injected into HeLa cells.

HeLa human cells were injected with a natural mixture of rabbit alpha and beta globin mRNA. They were incubated for 6 hr with [35S]methionine either immediately after injection or 20 hr later. The labeled proteins in the injected cells were analyzed by fluorography of two-dimensional electrophoresis gels. By using this procedure, it was possible to show that, during the first few hours after injection, both alpha and beta globin molecules are synthesized with an alpha to beta ratio approximately equal to 0.6. The rate of synthesis of alpha globin decreased significantly faster than that of beta globin over a 26-hr period after injection of the two mRNAs. It thus seems that two messenger RNAs coding for closely related polypeptides possess a markedly different translational stability. When deadenylylated rabbit globin mRNAs were injected into HeLa cells, no globin synthesis could be detected by the techniques used. We conclude that the translational half-life of mRNAs lacking poly(A) is very short in these cells. It is thus clear that the poly(A) segment is required to ensure stability to globin mRNA in somatic cells as in Xenopus oocytes.

Unequal alpha and beta globin mRNA in reticulocytes of normal and mutant f/f fetal mice.

Messenger RNA (mRNA) was isolated from adult as well as normal and mutant f/f fetal mouse reticulocytes by poly-U affinity chromatography. mRNA from normal adult reticulocytes directed equal synthesis of alpha and beta globin chains in wheat germ cell free translational system. mRNA from either normal or mutant f/f fetal reticulocytes directed 40% more alpha globin synthesis than beta globin synthesis. These observations are suggestive that there is significantly more alpha mRNA than beta mRNA in normal and mutant f/f fetal mouse reticulocytes.

Free cytoplasmic messenger ribonucleoprotein complexes from rabbit reticulocytes.

Free cytoplasmic globin mRNA containing mRNP-particles were isolated from rabbit reticulocytes by zonal sucrose gradient centrifugation and their properties were compared with mRNP particles isolated in the same way from EDTA-dissociated reticulocyte polyribosomes. The average poly(A)-length of 9S mRNA from free cytoplasmic mRNP was 17-20 nucleotides being about two times shorter than the average poly(A)-length of polysomal 9S mRNA. The protein composition of the free cytoplasmic mRNP particles disclosed the absence of the 76,000 dalton protein which is associated with the 3'poly(A)-segment of polysomal globin mRNA. It was concluded that free cytoplasmic mRNP-particles from rabbit reticulocytes can be classified as "old" mRNP in a post-translational phase. Free cytoplasmic mRNPs were translated in heterologous cell-free systems as well as in Xenopus laevis oocytes. Addition of hemin stimulated the synthesis of alpha-globin in all systems, while the presence of the cap analogue m7G(5')p inhibited translation of free cytoplasmic mRNA completely. The latter finding suggested that free cytoplasmic mRNA has a 5' terminal "cap". Shortening of the poly(A)-segment with concomitant loss of the 76,000 dalton protein may lead to less efficient translation of free cytoplasmic mRNP.

Testing with puromycin for amino acyl trnas that limit the rate of peptide chain extension

With puromycin one can recognize when the synthesis of a given protein is dependent on amino acyl tRNA that is present in rate limiting amount. We demonstrate this use of puromycin by its interaction with another inhibitor, L-o-methylthreonine. L-o-methylthreonine lowers the Ile-tRNA concentration in the cell, thereby inhibiting synthesis of proteins containing isoleucine. In certain rabbits, the alpha hemoglobin chain has three isoleucyl residues and the beta chain none. L-o-methylthreonine thus inhibits alpha globin synthesis in intact reticulocytes from these rabbits. When puromycin and L-o-methylthreonine are used together, the two inhibitors synergize in inhibiting alpha globin synthesis. Hence, puromycin is a more effective inhibitor when the Ile-tRNA concentration is lowered. Cycloheximide and sodium fluoride have different modes of action from puromycin. Neither synergizes with L-o-methylthreonine; instead, the interaction is less than additive. We have found that beta chain synthesis in rabbit reticulocytes is more sensitive than alpha to inhibition by puromycin. This difference could reflect either differences in amino acid sequence or tRNA dependent limitations of beta chain elongation. The switch from fetal to adult hemoglobin in humans does not involve changes in limiting amino acyl tRNA because, for cord blood from infants of different developmental ages, the puromycin sensitivity of incorporation into gamma and beta chains remains constant.

Maintenance of the ratio of alpha and beta globin synthesis in rabbit reticulocytes.

The synthesis of rabbit alpha and beta globins under various conditions was studied using intact reticulocytes and reticulocyte cell-free systems. Raising salt concentration of media in which reticulocytes were incubated with radioactive amino acids reduced the total protein synthesis but did not affect the ratio of alpha to beta globins produced. Using a reticulocyte lysate which had been incubated with micrococcal nuclease to remove the endogenous globin messenger RNA activity, it was found that unlike the intact cell or the untreated lysate very little alpha globin was synthesised on adding purified globin mRNA. These results are discussed in terms of their compatibility with some proposed models of coordination of alpha and beta globin production.

Unequal accumulation of alpha- and beta-globin mRNA in erythropoietic mouse spleen.

Relative amounts and rates of synthesis of alpha- and beta-globin mRNAs were determined during splenic erythropoiesis in mice. At times after injection of mice with phenylhydrazine, alpha- and beta-globin mRNAs were separated by gel electrophoresis and quantitated by densitometric scanning of stained gels. At 66 hr after injection, the ratio of beta to alpha mRNA is about 1.2. By 138 hr, total globin mRNA is 5-fold greater in spleen cells, and the beta to alpha mRNA ratio approaches 2. This ratio remains around 1 in reticulocytes throughout this period. Analyses of globin products directed by these mRNAs from spleen cells and reticulocytes in the ascites cell-free system reflect the beta to alpha mRNA ratio observed by electrophoresis. Relative rates of synthesis of globin mRNAs were estimated after incubation of spleen cells with either [3H] uridine or [3H] adenosine. Although synthesis of both mRNAs is maximal at 114 hr and then declines sharply, beta mRNA is synthesized at a greater rate than alpha mRNA at every developmental stage. In contrast to the excess accumulation of beta mRNA in spleen cells, synthesis of alpha- and beta-globin chains remains balanced throughout erythroid development. These data suggest that during erythropoiesis in this system, equal synthesis of alpha and beta globin involves regulation at both transcriptional and post-transcriptional levels.

Isolation and In Vitro Differentiation of Human Erythroid Precursor Cells

There is decreased beta-globin production in beta-thalassemic reticulocytes and nucleated erythroid cells. In this study, we have examined whether unbalanced globin synthesis is expressed at all stages of human erythroid cell maturation. In order to determine the pattern of globin synthesis in early erythroid cells during erythroid cell maturation, an in vitro culture system using human bone marrow erythroid precursor cells has been developed. Early erythroid precursor cells (proerythroblasts and basophilic erythroblasts) have been isolated from nonthalassemic and thalassemic human bone marrows by lysing more mature erythroid cells, using complement and a rabbit antiserum prepared against normal human red cells. In the presence of erythropoietin, differentiation and proliferation of erythroid cells in demonstrable in liquid suspension culture for 24-48 hr, as determined by morphological criteria and by an increase in globin synthesis. The ratio of alpha- to beta-globin chain synthesis in nonthalassemic cells in approximately 1 at all stages of erythroid cell differentiation during culture. In cells from four patients with homozygous beta- thalassemia there is decreased beta-globin synthesis compared to alpha-globin synthesis, both in early erythroid precursor cells and during their maturation in culture. These findings indicate that unbalanced globin chain synthesis is expressed at all stages of red cell maturation in homozygous beta-thalassemia.

Free alpha-globin pool in human bone marrow.

A pool of free alpha-globin chains was found in the bone marrow samples from three controls, two patients with beta-thalassemia trait, three with sickle beta-thalassemia, three with hemoglobin (Hb) Lepore trait, one with alphabeta-thalassemia, four with homozygous beta-thalassemia, and one doubly heterozygous for Hb Lepore and beta-thalassemia. The average percentage of newly synthesized alpha-chains found in the free alpha-globin pool was 6.2% in the controls and 33.0% in the patients heterozygous for thalassemia or Hb Lepore. These controls and patients had balanced beta- and alpha-globin synthesis in the bone marrow. In the homozygous patients and in the one patient doubly heterozygous for thalassemia and Hb Lepore, there was a marked deficit of beta-chain synthesis in the bone marrow and also a large pool of newly synthesized free alpha-chains. The function of this pool of free alpha-chains is not known, but it may be involved in the regulation of globin chain synthesis in normal patients and in the compensatory synthesis of beta-chains that occurs in the bone marrow of patients heterozygous for thalassemia or for Hb Lepore.

Excess of alpha-globin synthesis in homozygous beta-thalassemia. Its cytoplasmic molecular forms.

The cytoplasmic distribution of the excess newly synthesized α-globin, present in the red cell lysate of β-thalassemic subjects has been investigated. The labelled globins present in the cytoplasm, as well as in purified Hb A and Hb F, were separated by CM-cellulose chromatography, and the ratios 3Hα/3Hβ and 3Hα/3Hγ were determined. The results obtained show that an intracellular exchange occurs between non radioactive α-chains of existing hemoglobins and the newly synthesized ones. Since the amount of labelled α-globin chains which is found in combination with Hb A or Hb F does not account for all the newly synthesized α-globin, it is concluded that part of this α-globin must exist in a free form. A series of experimental data support the conclusion that this “free fraction” is probably composed of non heminated α-globin molecules.

Excess of alpha-globin synthesis in homozygous beta-thalassemia and its removal from the red blood cell cytoplasm.

The intracellular behaviour of the excess newly synthesized α‐globin has been studied by incubating peripheral blood and bone marrow samples from 9 homozygous β‐thalassemic subjects with labeled amino acids. The globin chains present in the cytoplasm and in association with the stromal material have been separated by column chromatography, and the incorporation of radioactivity has been determined for each globin at various incubation times.The results obtained show that the excess of α‐globin is removed from the cell cytoplasm and becomes associated with the stromal material.

Absence of β Globin Synthesis and Excess of α Globin Synthesis in Homozygous β‐Thalassemia

Globin chain synthesis has been studied in β‐thalassemia by incubating red blood cells of 10 homozygous thalassemic patients with [3H] amino acids, and by measuring the incorporation of radioactivity into the globin chains, separated by column chromatography.Five of the ten patients were never transfused, five were polytransfused.The following results were obtained: (a) absence of β chain synthesis in both polytransfused and never trasfused patients; (b) excess of α chain synthesis in the ten cases examined (ratio α synthesis/(γ+δ) synthesis = 3.0).In three heterozygous subjects β globin synthesis and an excess of α chain synthesis was observed (ratio α/(β+γ+δ) = 1.6). The possible interpretations of these results are presented in the discussion.