Production of mouse globin in heterokaryons of mouse erythroleukaemia cells and human fibroblasts

In an effort to activate the globin genes of non-erythroid cells, tetraploid murine erythroleukaemia cells (Friend cells) were fused with diploid human amniotic fibroblasts. When the Friend cells were pretreated with dimethylsulphoxide, an average of 27% heterokaryons was observed. These cells stained with benzidine, an indication that they contained haemoglobin. The cells incorporated radioactive amino acids into proteins. Electrophoresis of [3H]leucine-labelled lysates on SDS urea polyacrylamide gels indicated that up to 7% of the newly synthesized protein co-electrophoresed with globin. CM cellulose chromatography demonstrated the presence of mouse but not human globin chains. Hybridization analyses of cytoplasmic RNA also revealed only mouse globin mRNA in the heterokaryons. Although heterokaryons form readily between mouse erythroleukaemia cells and human fibroblasts, and globin synthesis does occur, only the erythroid partner in the fusion system employed here directs globin production.

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Effective erythropoiesis and HbF reactivation induced by kit ligand in β-thalassemia

Blood ◽

10.1182/blood-2007-06-097550 ◽

2008 ◽

Vol 111 (1) ◽

pp. 421-429 ◽

Cited By ~ 11

Author(s):

Marco Gabbianelli ◽

Ornella Morsilli ◽

Adriana Massa ◽

Luca Pasquini ◽

Paolo Cianciulli ◽

...

Keyword(s):

Fetal Hemoglobin ◽

Thalassemia Major ◽

Globin Genes ◽

Erythroid Progenitors ◽

Ineffective Erythropoiesis ◽

Kit Ligand ◽

Globin Chains ◽

In Vitro Response ◽

Globin Synthesis

In human β-thalassemia, the imbalance between α- and non–α-globin chains causes ineffective erythropoiesis, hemolysis, and anemia: this condition is effectively treated by an enhanced level of fetal hemoglobin (HbF). In spite of extensive studies on pharmacologic induction of HbF synthesis, clinical trials based on HbF reactivation in β-thalassemia produced inconsistent results. Here, we investigated the in vitro response of β-thalassemic erythroid progenitors to kit ligand (KL) in terms of HbF reactivation, stimulation of effective erythropoiesis, and inhibition of apoptosis. In unilineage erythroid cultures of 20 patients with intermedia or major β-thalassemia, addition of KL, alone or combined with dexamethasone (Dex), remarkably stimulated cell proliferation (3-4 logs more than control cultures), while decreasing the percentage of apoptotic and dyserythropoietic cells (<5%). More important, in both thalassemic groups, addition of KL or KL plus Dex induced a marked increase of γ-globin synthesis, thus reaching HbF levels 3-fold higher than in con-trol cultures (eg, from 27% to 75% or 81%, respectively, in β-thalassemia major). These studies indicate that in β-thalassemia, KL, alone or combined with Dex, induces an expansion of effective erythropoiesis and the reactivation of γ-globin genes up to fetal levels and may hence be considered as a potential therapeutic agent for this disease.

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Terminal stages of erythroid differentiation: persistent production of globin messenger RNA is necessary to sustain globin synthesis

Blood ◽

10.1182/blood.v52.1.171.bloodjournal521171 ◽

1978 ◽

Vol 52 (1) ◽

pp. 171-180

Author(s):

E Ullu ◽

MG Farace ◽

R Gambari ◽

P Orsi ◽

M Lunadei ◽

...

Keyword(s):

Messenger Rna ◽

Relative Proportion ◽

Terminal Differentiation ◽

Yolk Sac ◽

Globin Genes ◽

Erythroid Cells ◽

Globin Mrna ◽

Rna Molecules ◽

Cell Functions ◽

Globin Synthesis

The kinetic relationship between the globin mRNA accumulation and the rate of synthesis of globin chains was studied during the terminal stages of differentiation in erythroid cells derived from the yolk sac of mouse fetuses. RNA derived from the whole cells and from different cell compartments were hybridized to DNA complementary to embryonic globin mRNA. The relative proportion of embryonic globin RNA molecules and their absolute number per cell were estimated on the 11th, 12th, and 13th days of mouse fetal development. During erythroid terminal differentiation globin mRNA became progressively predominant on polyribosomes along with the progressive specialization of cell functions. The number of embryonic globin RNA molecules per cell remained constant while yolk sac erythroid cells underwent two rounds of cell division. These data indicate that the transcription of globin genes is operative throughout the last stages of terminal differentiation and that there is no detectable storage of globin RNA sequences in these cells. The rates of accumulation of mRNA molecules and of globin synthesis both seem correlated to the length of the cell cycle.

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IN SITU LOCALIZATION OF GLOBIN MESSENGER RNA FORMATION

The Journal of Cell Biology ◽

10.1083/jcb.63.2.414 ◽

1974 ◽

Vol 63 (2) ◽

pp. 414-419 ◽

Cited By ~ 32

Author(s):

D. Conkie ◽

N. Affara ◽

P. R. Harrison ◽

J. Paul ◽

K. Jones

Keyword(s):

Messenger Rna ◽

Mrna Levels ◽

Globin Mrna ◽

Mrna Accumulation ◽

True Solution ◽

Cytoplasmic Rna ◽

Friend Cells ◽

Experimental Approaches ◽

Mouse Cells

Globin messenger RNA (mRNA) levels in Friend virus-transformed mouse cells have been estimated by in situ hybridization of DNA copy (cDNA) to fixed preparations of cells and by hybridization of cDNA to extracted cytoplasmic RNA in true solution. The results obtained by both methods agree in showing that a low level of globin mRNA can be detected in untreated Friend cells. The levels of hemoglobin and globin mRNA have also been correlated after treatment of Friend cells with dimethyl sulfoxide (DMSO). The results obtained by both experimental approaches show that there is a minimum period of treatment with DMSO required in order that Friend cells may become hemoglobinized, and that this period coincides with the time when globin mRNA accumulates. Moreover, bromodeoxyuridine prevents both hemoglobin and globin mRNA accumulation.

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Terminal stages of erythroid differentiation: persistent production of globin messenger RNA is necessary to sustain globin synthesis

Blood ◽

10.1182/blood.v52.1.171.171 ◽

1978 ◽

Vol 52 (1) ◽

pp. 171-180 ◽

Cited By ~ 2

Author(s):

E Ullu ◽

MG Farace ◽

R Gambari ◽

P Orsi ◽

M Lunadei ◽

...

Keyword(s):

Messenger Rna ◽

Relative Proportion ◽

Terminal Differentiation ◽

Yolk Sac ◽

Globin Genes ◽

Erythroid Cells ◽

Globin Mrna ◽

Rna Molecules ◽

Cell Functions ◽

Globin Synthesis

Abstract The kinetic relationship between the globin mRNA accumulation and the rate of synthesis of globin chains was studied during the terminal stages of differentiation in erythroid cells derived from the yolk sac of mouse fetuses. RNA derived from the whole cells and from different cell compartments were hybridized to DNA complementary to embryonic globin mRNA. The relative proportion of embryonic globin RNA molecules and their absolute number per cell were estimated on the 11th, 12th, and 13th days of mouse fetal development. During erythroid terminal differentiation globin mRNA became progressively predominant on polyribosomes along with the progressive specialization of cell functions. The number of embryonic globin RNA molecules per cell remained constant while yolk sac erythroid cells underwent two rounds of cell division. These data indicate that the transcription of globin genes is operative throughout the last stages of terminal differentiation and that there is no detectable storage of globin RNA sequences in these cells. The rates of accumulation of mRNA molecules and of globin synthesis both seem correlated to the length of the cell cycle.

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Potential new approaches to the management of the Hb Bart’s hydrops fetalis syndrome: the most severe form of α-thalassemia

Hematology ◽

10.1182/asheducation-2018.1.353 ◽

2018 ◽

Vol 2018 (1) ◽

pp. 353-360 ◽

Cited By ~ 4

Author(s):

Andrew J. King ◽

Douglas R. Higgs

Keyword(s):

Globin Gene ◽

Perinatal Care ◽

Severe Form ◽

Hydrops Fetalis ◽

Globin Genes ◽

Globin Chains ◽

Intrauterine Transfusion ◽

Globin Synthesis ◽

Primitive Erythropoiesis ◽

Hb Bart's

Abstract The α-thalassemia trait, associated with deletions removing both α-globin genes from 1 chromosome (genotype ζ αα/ζ--), is common throughout Southeast Asia. Consequently, many pregnancies in couples of Southeast Asian origin carry a 1 in 4 risk of producing a fetus inheriting no functional α-globin genes (ζ--/ζ--), leading to hemoglobin (Hb) Bart’s hydrops fetalis syndrome (BHFS). Expression of the embryonic α-globin genes (ζ-globin) is normally limited to the early stages of primitive erythropoiesis, and so when the ζ-globin genes are silenced, at ∼6 weeks of gestation, there should be no α-like globin chains to pair with the fetal γ-globin chains of Hb, which consequently form nonfunctional tetramers (γ4) known as Hb Bart’s. When deletions leave the ζ-globin gene intact, a low level of ζ-globin gene expression continues in definitive erythroid cells, producing small amounts of Hb Portland (ζ2γ2), a functional form of Hb that allows the fetus to survive up to the second or third trimester. Untreated, all affected individuals die at these stages of development. Prevention is therefore of paramount importance. With improvements in early diagnosis, intrauterine transfusion, and advanced perinatal care, there are now a small number of individuals with BHFS who have survived, with variable outcomes. A deeper understanding of the mechanism underlying the switch from ζ- to α-globin expression could enable persistence or reactivation of embryonic globin synthesis in definitive cells, thereby providing new therapeutic options for such patients.

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The transport of neutral amino acids in cultured human fibroblasts.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(19)86122-7 ◽

1980 ◽

Vol 255 (3) ◽

pp. 929-936 ◽

Cited By ~ 2

Author(s):

G.C. Gazzola ◽

V. Dall'Asta ◽

G.G. Guidotti

Keyword(s):

Amino Acids ◽

Human Fibroblasts ◽

Neutral Amino Acids ◽

Cultured Human Fibroblasts

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Calcium-binding proteins in a vorticellid contractile organelle

Journal of Cell Science ◽

10.1242/jcs.19.1.203 ◽

1975 ◽

Vol 19 (1) ◽

pp. 203-213

Author(s):

W.B. Amos ◽

L.M. Routledge ◽

F.F. Yew

Keyword(s):

Amino Acids ◽

Molecular Weight ◽

Calcium Binding ◽

Binding Proteins ◽

Sodium Dodecyl ◽

Aromatic Amino Acids ◽

Calcium Binding Proteins ◽

Polyacrylamide Gels ◽

Protein Species ◽

Low Concentrations

The proteins of the contractile spasmoneme of Zoothamnium have been examined for comparison with other motile systems. Though capable of calcium-induced contraction, glycerinated preparations of the spasmoneme contain neither actin nor tubulin at levels that can be detected in polyacrylamide gels. Sixty per cent of the protein in sodium dodecyl sulphate gels migrates in a band at a molecular weight of approximately 20,000, consisting largely of 2 similar protein species which are here given the name of spasmins. The amino acid composition of 2 spasmin fractions has been determined by a fluorimetric method. They are rich in Asx, Glx and serine, but have few aromatic amino acids and no cystine or methionine. In calcium-buffered polyacrylamide gels, it was observed that a reduction in the electrophoretic mobility of the spasmins was induced specifically by calcium (but not magnesium) at the same low concentrations as induce contraction. This indicates that the spasmins are calcium-binding proteins which may be involved directly in the calcium-induced contraction of the spasmoneme.

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The identification of globin messenger ribonucleic acid in newt erythropoietic cells

Biochemical Journal ◽

10.1042/bj1830105 ◽

1979 ◽

Vol 183 (1) ◽

pp. 105-114

Author(s):

J A Grasso ◽

G P Casale

Keyword(s):

Ionic Strength ◽

Mrna Translation ◽

High Ionic Strength ◽

Globin Mrna ◽

Triturus Cristatus ◽

Messenger Ribonucleic Acid ◽

Reticulocyte Lysate ◽

Globin Synthesis ◽

26S Rrna ◽

Mrna Precursor

Polyadenylated [poly(A)+]-RNA isolated from newt (Triturus cristatus) erythropoietic cells contained two main species sedimenting at 9S and 25S, and minor amounts of a 15-20S component. The 9S poly(A)+-RNA fraction induced synthesis of newt haemoglobin and globins in frog oocytes and in an mRNA-dependent rabbit reticulocyte lysate, confirming its identity as newt globin mRNA. Translation of 9S globin mRNA in reticulocyte lysate was concentration-dependent, the patterns of globin synthesis suggesting both preferential utilization and unequal amounts of the different globin mRNA subspecies. Globin mRNA activity was also evident in the 25S poly(A)+-RNA fraction whose localization in polyribosomes excluded its function as a nuclear globin mRNA precursor. Denaturation in formamide and estimation of its relative methyl content indicated that the 25S poly(A)+-RNA fraction contained equimolar amounts of 9S globin mRNA and 26S rRNA. Translation of the 25S fraction in reticulocyte lysate was less efficient than that of comparable amounts of 9S globin mRNA and induced a pattern of globin synthesis similar to that obtained with subsaturating amounts of 9S mRNA. The 25S mRNA-rRNA complex was considered to be a non-physiological aggregate generated by extraction of RNA in the presence of buffers of moderate to high ionic strength.

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Globin synthesis in fibroblasts fused with erythroblasts. II. Human fibroblasts

Somatic Cell Genetics ◽

10.1007/bf01538664 ◽

1975 ◽

Vol 1 (4) ◽

pp. 323-333

Author(s):

Elena Citkowitz ◽

Michael G. Riggs ◽

Vernon M. Ingram

Keyword(s):

Human Fibroblasts ◽

Globin Synthesis

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Globin synthesis in hybrid cells constructed by transplantation of dormant avian erythrocyte nuclei into enucleated fibroblasts

Molecular and Cellular Biology ◽

10.1128/mcb.1.12.1163-1176.1981 ◽

1981 ◽

Vol 1 (12) ◽

pp. 1163-1176

Author(s):

J Bruno ◽

N Reich ◽

J J Lucas

Keyword(s):

Ribonucleic Acid ◽

Hybrid Cell ◽

Globin Gene ◽

Cell System ◽

Nuclear Transplantation ◽

Hybrid Cells ◽

Two Dimensional Gel Electrophoresis ◽

Messenger Ribonucleic Acid ◽

Globin Chains ◽

Globin Synthesis

The polypeptides synthesized by mature embryonic erythrocytes prepared from the peripheral blood of 14- to 15-day-old chicken embryos were analyzed by two-dimensional gel electrophoresis. Fewer than 200 species of polypeptides were detected; the major polypeptides made at this time were identified as the alpha A-, alpha D-, and beta-globin chains. The dormant erythrocyte nuclei were next reactivated to transcriptional competence by transplantation into enucleated mouse or chicken embryo fibroblasts, with frequencies of cytoplast renucleation of about 50 and 90%, respectively. Since large numbers of hybrid cells could be constructed, a biochemical analysis was possible. Electrophoretic analysis of the [35S]methionine-labeled polypeptides made in the hybrid cell types showed that polypeptides having the mobilities of only two (alpha A and alpha D) of the three major adult globin chains were made as major constituents of the hybrid cells. However, analysis of 14C-amino acid-labeled polypeptides revealed that a beta-like polypeptide that lacked methionine was also synthesized in large amounts. This polypeptide was tentatively identified as the early embryonic globin species rho. Globin synthesis was detected as early as 3 h after nuclear transplantation and as late as 18 h, the last time measured in these experiments. It appeared that globin polypeptides made at very early times were translated at least partially from chicken messenger ribonucleic acid introduced into the hybrid cells during fusion, whereas those made at later times were translated primarily from newly synthesized globin messenger ribonucleic acid. The potential usefulness of this hybrid cell system in analyzing mechanisms regulating globin gene expression is discussed.

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