scholarly journals Molecular basis for alpha-thalassemia associated with the structural mutant hemoglobin Suan-Dok (alpha 2 109leu----arg) [published erratum appears in Blood 1991 Mar 15;77(6):1404]

Blood ◽  
1990 ◽  
Vol 76 (12) ◽  
pp. 2630-2636 ◽  
Author(s):  
I Weiss ◽  
FE Cash ◽  
MB Coleman ◽  
A Pressley ◽  
JG Adams ◽  
...  
Keyword(s):  
Messenger Rna ◽  
Globin Gene ◽  
Mrna Level ◽  
Early Time ◽  
In Vitro Translation ◽  
Globin Mrna ◽  
Time Points ◽  
Alpha Thalassemia ◽  
Alpha Globin

Hemoglobin (Hb) Suan-Dok (alpha 109Arg) is a rare alpha-globin structural mutation that is linked to an alpha-thalassemia (alpha-thal) determinant. When inherited in trans to an alpha-thal-1 mutation (-), it results in Hb H disease associated with low levels (9%) of the Suan- Dok Hb. The nature of the thalassemic defect associated with the alpha SD mutation has been investigated by structural and functional studies. Sequence analysis of the cloned Suan-Dok allele showed a missense mutation (T----G) at codon 109 in an otherwise normal alpha 2-globin gene. When the alpha 2SD-globin gene was introduced into mouse erythroleukemia cells, the steady state alpha-globin messenger RNA (mRNA) level was equivalent to the alpha A-globin gene control. Although in vitro translation of a synthetic alpha 2SD-globin mRNA generated levels of alpha globin equivalent to alpha 2A-globin mRNA at early time points, the ratio of alpha SD to alpha A globin decreased markedly at later time points. These data suggest that the thalassemic defect associated with the Suan-Dok mutation results from a significant instability of the alpha SD globin.

scholarly journals Molecular basis for alpha-thalassemia associated with the structural mutant hemoglobin Suan-Dok (alpha 2 109leu----arg) [published erratum appears in Blood 1991 Mar 15;77(6):1404]

Blood ◽  
1990 ◽  
Vol 76 (12) ◽  
pp. 2630-2636 ◽  
Author(s):  
I Weiss ◽  
FE Cash ◽  
MB Coleman ◽  
A Pressley ◽  
JG Adams ◽  
...  
Keyword(s):  
Messenger Rna ◽  
Globin Gene ◽  
Mrna Level ◽  
Early Time ◽  
In Vitro Translation ◽  
Globin Mrna ◽  
Time Points ◽  
Alpha Thalassemia ◽  
Alpha Globin

Abstract Hemoglobin (Hb) Suan-Dok (alpha 109Arg) is a rare alpha-globin structural mutation that is linked to an alpha-thalassemia (alpha-thal) determinant. When inherited in trans to an alpha-thal-1 mutation (-), it results in Hb H disease associated with low levels (9%) of the Suan- Dok Hb. The nature of the thalassemic defect associated with the alpha SD mutation has been investigated by structural and functional studies. Sequence analysis of the cloned Suan-Dok allele showed a missense mutation (T----G) at codon 109 in an otherwise normal alpha 2-globin gene. When the alpha 2SD-globin gene was introduced into mouse erythroleukemia cells, the steady state alpha-globin messenger RNA (mRNA) level was equivalent to the alpha A-globin gene control. Although in vitro translation of a synthetic alpha 2SD-globin mRNA generated levels of alpha globin equivalent to alpha 2A-globin mRNA at early time points, the ratio of alpha SD to alpha A globin decreased markedly at later time points. These data suggest that the thalassemic defect associated with the Suan-Dok mutation results from a significant instability of the alpha SD globin.

scholarly journals Locus assignment of two alpha-globin structural mutants from the Caribbean basin: alpha Fort de France (alpha 45 Arg) and alpha Spanish Town (alpha 27 Val)

Blood ◽  
1989 ◽  
Vol 74 (2) ◽  
pp. 833-835
Author(s):  
FE Cash ◽  
N Monplaisir ◽  
M Goossens ◽  
SA Liebhaber
Keyword(s):  
Dominant Role ◽  
Globin Gene ◽  
In Vitro Translation ◽  
Globin Mrna ◽  
Alpha Globin ◽  
Structural Mutations ◽  
The Caribbean ◽  
Spanish Town

Abstract Two alpha-globin structural mutants were mapped to their encoding loci by in vitro translation of hybrid-selected alpha 1- and alpha 2-globin mRNA. The more highly expressed mutant, alpha Spanish Town (alpha 27Val), is encoded at the alpha 2 locus and the less expressed mutant, alpha Fort de France (alpha 45Arg), is encoded at the alpha 1 locus. These results further define the distribution of alpha-globin structural mutations within the alpha-globin gene cluster and substantiate the dominant role of the alpha 2-globin locus in alpha- globin expression.

scholarly journals Locus assignment of two alpha-globin structural mutants from the Caribbean basin: alpha Fort de France (alpha 45 Arg) and alpha Spanish Town (alpha 27 Val)

Blood ◽  
1989 ◽  
Vol 74 (2) ◽  
pp. 833-835
Author(s):  
FE Cash ◽  
N Monplaisir ◽  
M Goossens ◽  
SA Liebhaber
Keyword(s):  
Dominant Role ◽  
Globin Gene ◽  
In Vitro Translation ◽  
Globin Mrna ◽  
Alpha Globin ◽  
Structural Mutations ◽  
The Caribbean ◽  
Spanish Town

Two alpha-globin structural mutants were mapped to their encoding loci by in vitro translation of hybrid-selected alpha 1- and alpha 2-globin mRNA. The more highly expressed mutant, alpha Spanish Town (alpha 27Val), is encoded at the alpha 2 locus and the less expressed mutant, alpha Fort de France (alpha 45Arg), is encoded at the alpha 1 locus. These results further define the distribution of alpha-globin structural mutations within the alpha-globin gene cluster and substantiate the dominant role of the alpha 2-globin locus in alpha- globin expression.

scholarly journals Developmental switching of messenger RNA expression from the human alpha-globin cluster: fetal/adult pattern of theta-globin gene expression

Blood ◽  
1992 ◽  
Vol 80 (6) ◽  
pp. 1586-1591 ◽  
Author(s):  
M Albitar ◽  
A Care ◽  
C Peschle ◽  
SA Liebhaber
Keyword(s):  
Steady State ◽  
Gene Cluster ◽  
Messenger Rna ◽  
Globin Gene ◽  
Mrna Level ◽  
Globin Genes ◽  
Globin Mrna ◽  
Globin Gene Cluster ◽  
Adult Pattern ◽  
Alpha Globin

Abstract The alpha-globin gene cluster contains four functional globin genes, zeta, alpha 2, alpha 1, and theta. The developmental regulation of the embryonic zeta and fetal/adult alpha 2- and alpha 1-globin genes is well characterized at the level of protein synthesis. The developmental pattern of the theta-globin gene is not well characterized due to the inability to detect its encoded protein. Direct analysis of the globin switching at the steady-state messenger RNA (mRNA) level has been hampered by the difficulty in obtaining quantities of embryonic and early fetal mRNA sufficient for analysis. We analyzed the relative levels of the steady-state zeta-, alpha-, and theta-globin mRNAs in yolk sac in 5-, 6-, 7-, and 8-week postconception embryonic liver, and in cord and adult blood reticulocytes. We show that the switch in the alpha-globin gene cluster from the embryonic to fetal/adult pattern of expression begins at 5 to 6 weeks of gestation. Both the theta- and alpha-globin genes show similar patterns of developmental control that are reciprocal to zeta. alpha-globin RNA is barely detectable or undetectable at 5 weeks, and increases in the 6- to 8-week period, while theta-globin mRNA shows a parallel increase at 5 to 8 weeks postconception and is expressed in cord blood and adult reticulocytes. These data show that the theta-globin gene represents a fetal/adult gene, albeit expressed at a low level.

scholarly journals Developmental switching of messenger RNA expression from the human alpha-globin cluster: fetal/adult pattern of theta-globin gene expression

Blood ◽  
1992 ◽  
Vol 80 (6) ◽  
pp. 1586-1591
Author(s):  
M Albitar ◽  
A Care ◽  
C Peschle ◽  
SA Liebhaber
Keyword(s):  
Steady State ◽  
Gene Cluster ◽  
Messenger Rna ◽  
Globin Gene ◽  
Mrna Level ◽  
Globin Genes ◽  
Globin Mrna ◽  
Globin Gene Cluster ◽  
Adult Pattern ◽  
Alpha Globin

The alpha-globin gene cluster contains four functional globin genes, zeta, alpha 2, alpha 1, and theta. The developmental regulation of the embryonic zeta and fetal/adult alpha 2- and alpha 1-globin genes is well characterized at the level of protein synthesis. The developmental pattern of the theta-globin gene is not well characterized due to the inability to detect its encoded protein. Direct analysis of the globin switching at the steady-state messenger RNA (mRNA) level has been hampered by the difficulty in obtaining quantities of embryonic and early fetal mRNA sufficient for analysis. We analyzed the relative levels of the steady-state zeta-, alpha-, and theta-globin mRNAs in yolk sac in 5-, 6-, 7-, and 8-week postconception embryonic liver, and in cord and adult blood reticulocytes. We show that the switch in the alpha-globin gene cluster from the embryonic to fetal/adult pattern of expression begins at 5 to 6 weeks of gestation. Both the theta- and alpha-globin genes show similar patterns of developmental control that are reciprocal to zeta. alpha-globin RNA is barely detectable or undetectable at 5 weeks, and increases in the 6- to 8-week period, while theta-globin mRNA shows a parallel increase at 5 to 8 weeks postconception and is expressed in cord blood and adult reticulocytes. These data show that the theta-globin gene represents a fetal/adult gene, albeit expressed at a low level.

The switch from larval to adult globin gene expression in Xenopus laevis is mediated by erythroid cells from distinct compartments

Development ◽  
1991 ◽  
Vol 112 (4) ◽  
pp. 1021-1029
Author(s):  
R. Weber ◽  
B. Blum ◽  
P.R. Muller
Keyword(s):  
Xenopus Laevis ◽  
Globin Gene ◽  
Northern Blotting ◽  
Precursor Cells ◽  
Globin Mrna ◽  
Erythroid Precursor ◽  
Main Site ◽  
Alpha Globin ◽  
Successive Generations

The transition of hemoglobins during metamorphosis of Xenopus laevis involves replacement of the larval erythrocytes by adult ones, suggesting that the developmental control of this event depends upon the growth characteristics of the precursor cells. To identify the erythroid precursor cells and to investigate their developmental fate, we analyzed the distribution of stage-specific globin mRNAs by northern blotting in dorsal and ventral fragments of stage 32 embryos after in vitro culture as well as presumptive erythropoietic tissues of tadpoles during metamorphosis. The histological analysis shows that erythrocytes differentiate only in ventral fragments, suggesting that the ventral blood islands and most likely also the dorsolateral mesoderm are the primary sites of erythropoiesis. We also demonstrate that the first generations of erythrocytes, already express the predominating larval-specific alpha-globin mRNAs. The globin mRNA patterns obtained from presumptive erythropoietic tissues suggest an important role of circulating precursor cells in larval erythropoiesis, whereas the liver appears to be the main site of formation and maturation of the adult erythrocytes. Tentatively we propose that anuran erythropoiesis is dependent upon a self-perpetuating stem-cell line and that the larval and the adult erythrocytes are derived from successive generations of erythroid precursors, whose commitment may be imposed by the erythropoietic sites.

Alpha Hemoglobin Stabilizing Protein (AHSP) Optimizes Hemoglobin A Synthesis by Maintaining a Pool of Viable Alpha Globin Subunits.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 650-650
Author(s):  
Xiang Yu ◽  
Yi Kong ◽  
Louis C. Dore ◽  
Anne M. Katein ◽  
John K. Choi ◽  
...  
Keyword(s):  
In Vitro Translation ◽  
Beta Thalassemia ◽  
Beta Globin ◽  
Fully Depleted ◽  
Alpha Thalassemia ◽  
Gene Ablation ◽  
Mutant Strains ◽  
Alpha Globin

Abstract AHSP binds alpha hemoglobin (Hb) to maintain its structure and limit its prooxidant activities. In addition, AHSP binds and stabilizes apo-alpha globin, which lacks heme. Previously, we demonstrated that Ahsp−/− mice exhibit hemolytic anemia with Hb precipitation in erythroid cells. Through interbreeding of mutant strains, we also showed that loss of AHSP exacerbates beta thalassemia. Together, these studies indicate that AHSP participates in Hb homeostasis and may act to neutralize potentially toxic excess alpha globin that is known to accumulate in normal erythroid precursors, and to a greater extent, in beta thalassemic ones. However, additional functions for AHSP may exist. In particular, AHSP-alpha globin complexes may also promote HbA synthesis. To test this, we depleted the pool of excess alpha globin in Ahsp−/− mice by interbreeding with alpha thalassemic ones. Compared to mice with either mutation alone, compound mutants missing both AHSP and one alpha globin allele (genotype Ahsp−/− //alpha globin*α/αα) exhibited more severe erythroid defects, including worsened anemia, hypochromia, Hb instability and ineffective erythropoiesis. Pulse-labeling of double-mutant reticulocytes showed that alpha to beta globin synthetic ratios were unaffected by loss of AHSP, but precipitation of both alpha and beta nascent chains into cell membranes was strongly enhanced. These data indicate that AHSP is important for erythropoiesis and Hb production even when alpha globin is not produced in excess. In vitro translation studies using wheat germ extracts showed that recombinant AHSP present during the synthesis of alpha globin improved its ability to become incorporated into HbA. Moreover, AHSP conferred protease resistance to nascent alpha globin, suggesting enhanced folding into the native state. Further supporting this interpretation, circular dichroism studies showed that AHSP accelerated refolding of purified denatured alpha Hb. Finally, through pulse labeling of reticulocytes followed by isoelectric focusing of soluble cytosolic fractions, we identified transient pools of free alpha Hb and AHSP-alpha Hb in vivo and showed that Ahsp gene ablation fully depleted both pools. Together, our studies indicate that AHSP acts as a molecular chaperone to promote alpha globin folding and stability prior to its incorporation into HbA. In addition, it is possible that alterations in AHSP gene function or expression could modulate alpha thalassemia severity in patients.

C>A substitution in NT 46 of the 3’ UTR region (the α complex protected region) of the alpha-1 globin gene: a non-deletional mutation or polymorphism?

2019 ◽  
Vol 73 (1) ◽  
pp. 14-16
Author(s):  
Paloma Ropero ◽  
Fernando Ataúlfo González ◽  
Jorge M Nieto ◽  
Ana Villegas ◽  
Julian Sevilla ◽  
...  
Keyword(s):  
Mrna Stability ◽  
Messenger Rna ◽  
Globin Gene ◽  
Regulation Of Gene Expression ◽  
Erythroid Cell ◽  
Subcellular Localisation ◽  
Translation Efficiency ◽  
Globin Genes ◽  
Globin Mrna ◽  
Alpha Globin

AimsUntranslated regions (UTRs) play an important role in post-transcriptional regulation of gene expression, including by modulating messenger RNA (mRNA) transport out of the nucleus, translation efficiency, subcellular localisation and stability. Any mutation in this region could alter the stability of mRNA and thereby affect protein synthesis. We analysed if a mutation located in the α complex protected region of the α1 globin gene could cause non-deletional α-thalassaemia by affecting post-transcriptional stability (mRNA stability).MethodsA total of 14 patients without anaemia, normal or slight microcytosis and hypochromia (medium concentration haemoglobin [MCH] <27 pg) were studied. Haemoglobin subtypes were screened using capillary zone electrophoresis and ion-exchange high-performance liquid chromatography (VARIANT II β-Thalassaemia Short Program). The most common α-globin mutations were identified by multiplex PCR (Alpha-Globin StripAssay kit) and the molecular characterisation by automatic sequencing of alpha globin genes.ResultsAll of them shown a novel transversion mutation in nt 778 (C>A), which is located in the 3' UTR in the α complex protected region [HBA1: c.*+46C>A].ConclusionsThis mutation is in the αRNAmin binding site, so a single nucleotide substitution in this region can decrease mRNA stability by potentially compromising the binding of α-complex protein to αRNAmin, favouring the decay of α-globin mRNA via erythroid cell-enriched endoribonuclease cleavage. In this case, it is a non-deletional α-thalassaemia. However, in silico and empirical studies predicted that it could be a silent polymorphism. Functional studies should be carried out to confirm whether it is a pathological mutation or a silent polymorphism.

Preparation of a cell-free translation system from a wild-type strain of Neurospora crassa and translation of pyruvate kinase messenger RNA

1984 ◽  
Vol 4 (11) ◽  
pp. 979-986 ◽  
Author(s):  
M. Devchand ◽  
M. Kapoor
Keyword(s):  
Neurospora Crassa ◽  
Messenger Rna ◽  
In Vitro Translation ◽  
Translation System ◽  
Wild Type ◽  
Post Translational Modification ◽  
Globin Mrna ◽  
Free Translation ◽  
A Cell

A cell-free in vitro translation system exhibiting high activity has been developed from wild-type Neurospora crassa mycelium. The isolation is simple and fast, and the homogenization does not appear to affect the activity of mycelial proteases and nucleases. This system is capable of supporting efficient translation of exogenously added homologous RNA as demonstrated by the experiments with PK-specific mRNA. In addition, it translates heterologous RNA efficiently, shown by the translation of globin mRNA. We did not examine the Neurospora lysate for post-translational modification activity. The procedure used for the preparation of Neurospora cell-free extracts should be readily applicable to the other filamentous fungi.

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