Beta globin gene transcripts originating in the promoter region during early hexamethylene bisacetamide and dimethylsulfoxide induction of Friend erythroleukemia cells

1989 ◽  
Vol 90 (2) ◽  
Author(s):  
DouglasA. Weidner ◽  
Ilga Winicov
Keyword(s):  
Promoter Region ◽  
Globin Gene ◽  
Beta Globin ◽  
Hexamethylene Bisacetamide ◽  
Beta Globin Gene ◽  
Erythroleukemia Cells ◽  
Gene Transcripts ◽  
Friend Erythroleukemia Cells

scholarly journals A human beta-globin gene fused to the human beta-globin locus control region is expressed at high levels in erythroid cells of mice engrafted with retrovirus-transduced hematopoietic stem cells

Blood ◽  
1993 ◽  
Vol 81 (5) ◽  
pp. 1384-1392 ◽  
Author(s):  
I Plavec ◽  
T Papayannopoulou ◽  
C Maury ◽  
F Meyer
Keyword(s):  
Bone Marrow Cells ◽  
Globin Gene ◽  
Beta Globin ◽  
Erythroid Cells ◽  
Hematopoietic Stem ◽  
Beta Globin Gene ◽  
Erythroleukemia Cells ◽  
Murine Erythroleukemia ◽  
In Erythroid Cells

Abstract Retroviral-mediated gene transfer of human beta-globin provides a model system for the development of somatic gene therapy for hemoglobinopathies. Previous work has shown that mice receiving a transplant of bone marrow cells infected with a retroviral vector containing the human beta-globin gene can express human beta-globin specifically in erythroid cells; however, the level of expression of the transduced globin gene was low (1% to 2% per gene copy as compared with that of the endogenous mouse beta-globin gene). We report here the construction of a recombinant retrovirus vector encoding a human beta- globin gene fused to the 4 major regulatory elements of the human beta- globin locus control region (LCR). The LCR cassette increases the level of expression of the globin gene in murine erythroleukemia cells by 10- fold. To study the level of expression in vivo, mouse bone marrow cells were infected with virus-producing cells and the transduced cells were injected into lethally irradiated recipients. In the majority of provirus-containing mice (up to 75%), expression of human beta-globin in peripheral blood was detected at least 3 to 6 months after transplantation. Twelve animals representative of the level of expression of the transduced gene in blood (0.04% to 3.2% of the endogenous mouse beta-globin RNA) were selected for further analysis. A range of 0.4% to 12% of circulating erythrocytes stained positive for human beta-globin protein. Based on these values, the level of expression of the transduced gene per cell was estimated to be 10% to 39% of the endogenous mouse beta-globin gene. These data demonstrate that fusion of the LCR to the beta-globin gene in a retroviral vector increases the level of beta-globin expression in murine erythroleukemia cells and suggest that high-level expression can be obtained in erythroid cells in vivo after transduction into hematopoietic stem cells.

scholarly journals An approximately 300 bp deletion involving part of the 5' beta-globin gene region is observed in members of a Turkish family with beta- thalassemia

Blood ◽  
1987 ◽  
Vol 70 (2) ◽  
pp. 583-586 ◽  
Author(s):  
JC Diaz-Chico ◽  
KG Yang ◽  
A Kutlar ◽  
AL Reese ◽  
M Aksoy ◽  
...  
Keyword(s):  
Promoter Region ◽  
Genomic Dna ◽  
Restriction Site ◽  
Globin Gene ◽  
Beta Thalassemia ◽  
Beta Globin ◽  
Promoter Sequences ◽  
Beta Globin Gene ◽  
Turkish Family ◽  
Thalassemia Trait

Abstract Detailed gene mapping analyses of genomic DNA from two Turkish subjects with a beta-thalassemia trait demonstrated an approximately 300 bp deletion, which is located between the Rsa I restriction site 128 bp 5′ to the Cap site and the Acc I restriction site 284 bp 3′ to the same Cap site; it includes the 5′ beta promoter region, the first exon, and (part of) the IVS-I. Heterozygotes for this and two other beta- thalassemia types, which are also caused by deletions involving 5′ beta promoter sequences, appear to have higher hemoglobin (Hb) A2 levels, perhaps because the loss of this promoter results in an increased transcription of the delta globin gene, as delta and beta promoters may be influenced by the same enhancing sequences 3′ to the beta globin gene.

Proliferation is required for induction of terminal differentiation of Friend erythroleukemia cells

1992 ◽  
Vol 70 (7) ◽  
pp. 555-564 ◽  
Author(s):  
Louaine L. Spriggs ◽  
Steven M. Hill ◽  
James R. Jeter Jr.
Keyword(s):  
Cell Proliferation ◽  
Cell Division ◽  
Cell Growth ◽  
Structural Changes ◽  
Globin Gene ◽  
Inhibition Of Proliferation ◽  
Hexamethylene Bisacetamide ◽  
Erythroleukemia Cells ◽  
Friend Erythroleukemia Cells ◽  
The Relationship

The relationship between cell proliferation and differentiation has long been a source of controversy. Stimulation of normal erythroid maturation results in a finite number of cell divisions accompanied by a concomitant accumulation of hemoglobin. Friend erythroleukemia cells treated with hexamethylene bisacetamide differentiate in a similar manner, while agents such as hemin apparently induce differentiation without limiting cell proliferation. Aphidicolin, an inhibitor of DNA synthesis, has been reported to induce differentiation in the absence of cell proliferation. Using these three chemicals we have investigated the relationship between cell proliferation and erythrocytic maturation by exposing Friend erythroleukemia cells to either hexamethylene bisacetamide (5 mM), hemin (100 μM), or aphidicolin 1.2 μM) and examining the effects on cell growth, morphology, and hemoglobin production. Proliferation in the presence of hexamethylene bisacetamide is limited to four to five rounds of cell division, while hemin has no inhibitory effect. Hexamethylene bisacetamide initiates the complete erythrocytic maturation program, including cellular structural changes and hemoglobin synthesis. Hemin stimulates only globin gene transcription, not differentiation. Aphidicolin inhibits cell growth within 24 h, but does not induce differentiation. Furthermore, inhibition of proliferation by aphidicolin prevents subsequent hexamethylene bisacetamide induced differentiation. These results indicate that at least one round of cell division is required for initiation of erythrocytic differentiation.Key words: proliferation, erythrocytic differentiation, hexamethylene bisacetamide, hemin, aphidicolin.

Expression of the human beta-globin gene after retroviral transfer into murine erythroleukemia cells and human BFU-E cells

1988 ◽  
Vol 8 (4) ◽  
pp. 1725-1735
Author(s):  
M A Bender ◽  
A D Miller ◽  
R E Gelinas
Keyword(s):  
Globin Gene ◽  
Globin Genes ◽  
Beta Globin ◽  
Globin Mrna ◽  
Beta Globin Gene ◽  
Erythroleukemia Cells ◽  
New Gene ◽  
Normal Human ◽  
Murine Erythroleukemia ◽  
Murine Erythroleukemia Cells

Replication-defective amphotropic retrovirus vectors containing either the human beta-globin gene with introns or an intronless beta-globin minigene were constructed and used to study beta-globin expression following gene transfer into hematopoietic cells. The beta-globin genes were marked by introducing a 6-base-pair insertion into the region corresponding to the 5' untranslated region of the beta-globin mRNA to allow detection of RNA encoded by the new gene in human cells expressing normal human beta-globin RNA. Introduction of a virus containing the beta-globin gene with introns into murine erythroleukemia cells resulted in inducible expression of human beta-globin RNA and protein, while the viruses containing the minigene were inactive. The introduced human beta-globin gene was 6 to 110% as active as the endogenous mouse beta maj-globin genes in six randomly chosen cell clones. Introduction of the viruses into human BFU-E cells, followed by analysis of marked and unmarked globin RNAs in differentiated erythroid colonies, revealed that the introduced beta-globin gene was about 5% as active as the endogenous genes in these normal human erythroid cells and that again the minigene was inactive. These data are discussed in terms of the potential treatment of genetic disease by gene therapy.

scholarly journals Linker scanning mutagenesis of the 5'-flanking region of the mouse beta-major-globin gene: sequence requirements for transcription in erythroid and nonerythroid cells.

1985 ◽  
Vol 5 (6) ◽  
pp. 1498-1511 ◽  
Author(s):  
P Charnay ◽  
P Mellon ◽  
T Maniatis
Keyword(s):  
Hela Cell ◽  
Gene Transcription ◽  
Hela Cells ◽  
Globin Gene ◽  
Beta Globin ◽  
Sequence Element ◽  
Beta Globin Gene ◽  
Erythroleukemia Cells ◽  
Flanking Region ◽  
Mouse Erythroleukemia

We analyzed the sequences required for transcription of the mouse beta-major-globin gene by introducing deletion and linker scanning mutations into the 5'-flanking region and then studying the effects of these mutations on beta-globin gene transcription in a HeLa cell transient expression assay or after stable introduction into mouse erythroleukemia cells. Consistent with earlier studies, we found that three distinct regions upstream from the RNA capping site are required for efficient beta-globin gene transcription in HeLa cells: the ATA box located 30 base pairs upstream from the mRNA capping site (-30), the CCAAT box located at -75, and the distal sequence element CCACACCC located at -90. In the ATA and CAAT box regions, the sequences necessary for efficient transcription extend beyond the limits of the canonical sequences. Mutations in the sequences located between the three transcriptional control elements do not significantly affect transcription in HeLa cells. Although the promoter defined in HeLa cell transfection experiments is also required for efficient transcription in mouse erythroleukemia cells, none of the mutations tested affects the regulation of beta-globin gene transcription during mouse erythroleukemia cell differentiation. Thus, DNA sequences downstream from the mRNA cap site appear to be sufficient for the regulation of beta-globin gene expression during the differentiation of mouse erythroleukemia cells in culture.

scholarly journals Expression of the human beta-globin gene after retroviral transfer into murine erythroleukemia cells and human BFU-E cells.

1988 ◽  
Vol 8 (4) ◽  
pp. 1725-1735 ◽  
Author(s):  
M A Bender ◽  
A D Miller ◽  
R E Gelinas
Keyword(s):  
Globin Gene ◽  
Globin Genes ◽  
Beta Globin ◽  
Globin Mrna ◽  
Beta Globin Gene ◽  
Erythroleukemia Cells ◽  
New Gene ◽  
Normal Human ◽  
Murine Erythroleukemia ◽  
Murine Erythroleukemia Cells

Replication-defective amphotropic retrovirus vectors containing either the human beta-globin gene with introns or an intronless beta-globin minigene were constructed and used to study beta-globin expression following gene transfer into hematopoietic cells. The beta-globin genes were marked by introducing a 6-base-pair insertion into the region corresponding to the 5' untranslated region of the beta-globin mRNA to allow detection of RNA encoded by the new gene in human cells expressing normal human beta-globin RNA. Introduction of a virus containing the beta-globin gene with introns into murine erythroleukemia cells resulted in inducible expression of human beta-globin RNA and protein, while the viruses containing the minigene were inactive. The introduced human beta-globin gene was 6 to 110% as active as the endogenous mouse beta maj-globin genes in six randomly chosen cell clones. Introduction of the viruses into human BFU-E cells, followed by analysis of marked and unmarked globin RNAs in differentiated erythroid colonies, revealed that the introduced beta-globin gene was about 5% as active as the endogenous genes in these normal human erythroid cells and that again the minigene was inactive. These data are discussed in terms of the potential treatment of genetic disease by gene therapy.

scholarly journals A human beta-globin gene fused to the human beta-globin locus control region is expressed at high levels in erythroid cells of mice engrafted with retrovirus-transduced hematopoietic stem cells

Blood ◽  
1993 ◽  
Vol 81 (5) ◽  
pp. 1384-1392
Author(s):  
I Plavec ◽  
T Papayannopoulou ◽  
C Maury ◽  
F Meyer
Keyword(s):  
Bone Marrow Cells ◽  
Globin Gene ◽  
Beta Globin ◽  
Erythroid Cells ◽  
Hematopoietic Stem ◽  
Beta Globin Gene ◽  
Erythroleukemia Cells ◽  
Murine Erythroleukemia ◽  
In Erythroid Cells

Retroviral-mediated gene transfer of human beta-globin provides a model system for the development of somatic gene therapy for hemoglobinopathies. Previous work has shown that mice receiving a transplant of bone marrow cells infected with a retroviral vector containing the human beta-globin gene can express human beta-globin specifically in erythroid cells; however, the level of expression of the transduced globin gene was low (1% to 2% per gene copy as compared with that of the endogenous mouse beta-globin gene). We report here the construction of a recombinant retrovirus vector encoding a human beta- globin gene fused to the 4 major regulatory elements of the human beta- globin locus control region (LCR). The LCR cassette increases the level of expression of the globin gene in murine erythroleukemia cells by 10- fold. To study the level of expression in vivo, mouse bone marrow cells were infected with virus-producing cells and the transduced cells were injected into lethally irradiated recipients. In the majority of provirus-containing mice (up to 75%), expression of human beta-globin in peripheral blood was detected at least 3 to 6 months after transplantation. Twelve animals representative of the level of expression of the transduced gene in blood (0.04% to 3.2% of the endogenous mouse beta-globin RNA) were selected for further analysis. A range of 0.4% to 12% of circulating erythrocytes stained positive for human beta-globin protein. Based on these values, the level of expression of the transduced gene per cell was estimated to be 10% to 39% of the endogenous mouse beta-globin gene. These data demonstrate that fusion of the LCR to the beta-globin gene in a retroviral vector increases the level of beta-globin expression in murine erythroleukemia cells and suggest that high-level expression can be obtained in erythroid cells in vivo after transduction into hematopoietic stem cells.

scholarly journals An approximately 300 bp deletion involving part of the 5' beta-globin gene region is observed in members of a Turkish family with beta- thalassemia

Blood ◽  
1987 ◽  
Vol 70 (2) ◽  
pp. 583-586
Author(s):  
JC Diaz-Chico ◽  
KG Yang ◽  
A Kutlar ◽  
AL Reese ◽  
M Aksoy ◽  
...  
Keyword(s):  
Promoter Region ◽  
Genomic Dna ◽  
Restriction Site ◽  
Globin Gene ◽  
Beta Thalassemia ◽  
Beta Globin ◽  
Promoter Sequences ◽  
Beta Globin Gene ◽  
Turkish Family ◽  
Thalassemia Trait

Detailed gene mapping analyses of genomic DNA from two Turkish subjects with a beta-thalassemia trait demonstrated an approximately 300 bp deletion, which is located between the Rsa I restriction site 128 bp 5′ to the Cap site and the Acc I restriction site 284 bp 3′ to the same Cap site; it includes the 5′ beta promoter region, the first exon, and (part of) the IVS-I. Heterozygotes for this and two other beta- thalassemia types, which are also caused by deletions involving 5′ beta promoter sequences, appear to have higher hemoglobin (Hb) A2 levels, perhaps because the loss of this promoter results in an increased transcription of the delta globin gene, as delta and beta promoters may be influenced by the same enhancing sequences 3′ to the beta globin gene.

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