scholarly journals A directly repeated sequence in the beta-globin promoter regulates transcription in murine erythroleukemia cells.

1990 ◽  
Vol 10 (3) ◽  
pp. 972-981 ◽  
Author(s):  
L L Stuve ◽  
R M Myers
Keyword(s):  
Direct Repeat ◽  
Repeated Sequence ◽  
Beta Globin ◽  
Transcript Levels ◽  
Cis Acting ◽  
Promoter Sequences ◽  
Erythroleukemia Cells ◽  
Pair Sequence ◽  
Globin Promoter ◽  
Murine Erythroleukemia

We have identified a previously undetected cis-acting element in the mouse beta-major globin promoter region that is necessary for maximal transcription levels of the gene in the inducible preerythroid murine erythroleukemia (MEL) cell line. This element, termed the beta-globin direct-repeat element (beta DRE), consists of a directly repeated 10-base-pair sequence, 5'-AGGGCAG(G)AGC-3', that lies just upstream from the TATA box of the promoter. The beta DRE motif is highly conserved in all adult mammalian beta-globin promoter sequences known. Mutation of either single repeat alone caused less than a twofold decrease in transcript levels. However, simultaneous mutation of both repeated regions resulted in a ninefold decrease in accumulated transcripts when the gene was transiently transfected into MEL cells. Attachment of the beta DRE to a heterologous promoter had little effect on levels of accumulated transcripts initiated from the promoter in undifferentiated MEL cells but resulted in a threefold increase in transcript levels in induced (differentiated) MEL cells. Similarly, a comparison of the relative effects of mutations in the beta DRE in uninduced and induced MEL cells indicated that the element was more active in induced cells. The increase in beta DRE activity upon MEL cell differentiation and the more pronounced effects of mutations in both repeats of the beta DRE have implications for the mechanism of action of the element in regulating beta-globin transcription and for mutational studies of other repetitive or redundant transcription elements.

A directly repeated sequence in the beta-globin promoter regulates transcription in murine erythroleukemia cells

1990 ◽  
Vol 10 (3) ◽  
pp. 972-981
Author(s):  
L L Stuve ◽  
R M Myers
Keyword(s):  
Direct Repeat ◽  
Repeated Sequence ◽  
Beta Globin ◽  
Transcript Levels ◽  
Cis Acting ◽  
Promoter Sequences ◽  
Erythroleukemia Cells ◽  
Pair Sequence ◽  
Globin Promoter ◽  
Murine Erythroleukemia

We have identified a previously undetected cis-acting element in the mouse beta-major globin promoter region that is necessary for maximal transcription levels of the gene in the inducible preerythroid murine erythroleukemia (MEL) cell line. This element, termed the beta-globin direct-repeat element (beta DRE), consists of a directly repeated 10-base-pair sequence, 5'-AGGGCAG(G)AGC-3', that lies just upstream from the TATA box of the promoter. The beta DRE motif is highly conserved in all adult mammalian beta-globin promoter sequences known. Mutation of either single repeat alone caused less than a twofold decrease in transcript levels. However, simultaneous mutation of both repeated regions resulted in a ninefold decrease in accumulated transcripts when the gene was transiently transfected into MEL cells. Attachment of the beta DRE to a heterologous promoter had little effect on levels of accumulated transcripts initiated from the promoter in undifferentiated MEL cells but resulted in a threefold increase in transcript levels in induced (differentiated) MEL cells. Similarly, a comparison of the relative effects of mutations in the beta DRE in uninduced and induced MEL cells indicated that the element was more active in induced cells. The increase in beta DRE activity upon MEL cell differentiation and the more pronounced effects of mutations in both repeats of the beta DRE have implications for the mechanism of action of the element in regulating beta-globin transcription and for mutational studies of other repetitive or redundant transcription elements.

scholarly journals Identification and characterization of a beta-globin promoter-binding factor from murine erythroleukemia cells.

1993 ◽  
Vol 13 (7) ◽  
pp. 4311-4322 ◽  
Author(s):  
L L Stuvé ◽  
R M Myers
Keyword(s):  
Binding Affinity ◽  
Regulatory Element ◽  
Direct Repeat ◽  
Binding Activity ◽  
Beta Globin ◽  
Recognition Sequence ◽  
Dna Binding Activity ◽  
Globin Promoter ◽  
Murine Erythroleukemia

We have identified a DNA-binding activity with specificity for the beta DRE, an evolutionarily conserved transcriptional regulatory element in mammalian adult beta-globin promoters. This binding activity, which we term beta DRf, for beta-globin direct repeat factor, was detected in fractionated nuclear extracts from the murine erythroleukemia cell line and has been partially purified from undifferentiated cells. beta DRf makes symmetric contacts on the two copies of its recognition sequence on both strands and introduces a bend into the DNA helix upon binding. While the factor displays a low binding affinity for the beta DRE in isolation, it binds to the intact beta-globin promoter and DNA fragments containing multiple beta DRE-binding sites with high affinity. A correlation between beta DRf binding affinity and transcriptional activity of beta DRE mutant promoters suggests that this factor stimulates transcription of the beta-globin promoter in vivo.

Identification and characterization of a beta-globin promoter-binding factor from murine erythroleukemia cells

1993 ◽  
Vol 13 (7) ◽  
pp. 4311-4322
Author(s):  
L L Stuvé ◽  
R M Myers
Keyword(s):  
Binding Affinity ◽  
Regulatory Element ◽  
Direct Repeat ◽  
Binding Activity ◽  
Beta Globin ◽  
Recognition Sequence ◽  
Dna Binding Activity ◽  
Globin Promoter ◽  
Murine Erythroleukemia

We have identified a DNA-binding activity with specificity for the beta DRE, an evolutionarily conserved transcriptional regulatory element in mammalian adult beta-globin promoters. This binding activity, which we term beta DRf, for beta-globin direct repeat factor, was detected in fractionated nuclear extracts from the murine erythroleukemia cell line and has been partially purified from undifferentiated cells. beta DRf makes symmetric contacts on the two copies of its recognition sequence on both strands and introduces a bend into the DNA helix upon binding. While the factor displays a low binding affinity for the beta DRE in isolation, it binds to the intact beta-globin promoter and DNA fragments containing multiple beta DRE-binding sites with high affinity. A correlation between beta DRf binding affinity and transcriptional activity of beta DRE mutant promoters suggests that this factor stimulates transcription of the beta-globin promoter in vivo.

Human globin gene promoter sequences are sufficient for specific expression of a hybrid gene transfected into tissue culture cells

1987 ◽  
Vol 7 (1) ◽  
pp. 398-402
Author(s):  
T Rutherford ◽  
A W Nienhuis
Keyword(s):  
Gene Expression ◽  
Globin Gene ◽  
Gene Promoter ◽  
Gene Promoters ◽  
Globin Genes ◽  
Specific Expression ◽  
Tissue Specific ◽  
Promoter Sequences ◽  
Erythroleukemia Cells ◽  
Murine Erythroleukemia

The contribution of the human globin gene promoters to tissue-specific transcription was studied by using globin promoters to transcribe the neo (G418 resistance) gene. After transfection into different cell types, neo gene expression was assayed by scoring colony formation in the presence of G418. In K562 human erythroleukemia cells, which express fetal and embryonic globin genes but not the adult beta-globin gene, the neo gene was expressed strongly from a fetal gamma- or embryonic zeta-globin gene promoter but only weakly from the beta promoter. In murine erythroleukemia cells which express the endogenous mouse beta genes, the neo gene was strongly expressed from both beta and gamma promoters. In two nonerythroid cell lines, human HeLa cells and mouse 3T3 fibroblasts, the globin gene promoters did not allow neo gene expression. Globin-neo genes were integrated in the erythroleukemia cell genomes mostly as a single copy per cell and were transcribed from the appropriate globin gene cap site. We conclude that globin gene promoter sequences extending from -373 to +48 base pairs (bp) (relative to the cap site) for the beta gene, -385 to +34 bp for the gamma gene, and -555 to +38 bp for the zeta gene are sufficient for tissue-specific and perhaps developmentally specific transcription.

scholarly journals Stability of alpha and beta globin messenger RNA during induced differentiation of mouse erythroleukemia cells

Blood ◽  
1979 ◽  
Vol 54 (4) ◽  
pp. 933-939
Author(s):  
R Gambari ◽  
RA Rifkind ◽  
PA Marks
Keyword(s):  
Messenger Rna ◽  
Erythroid Differentiation ◽  
Beta Globin ◽  
Globin Mrna ◽  
Induced Differentiation ◽  
Hexamethylene Bisacetamide ◽  
Erythroleukemia Cells ◽  
Mrna Content ◽  
Murine Erythroleukemia ◽  
Murine Erythroleukemia Cells

Murine erythroleukemia cells (MELC) are induced to express erythroid differentiation when cultured with hexamethylene bisacetamide (HMBA). Newly synthesized alpha and beta globin mRNA are both relatively stable, half-life (t1/2) greater than 50 hr, early in the course of induced differentiation. In fully induced cells there is a decrease in stability of both newly synthesized alpha and beta globin mRNA. The decay of alpha mRNA is faster, (t 1/2, 10--12 hr) than beta globin mRNA (t1/2, 20--22 hr). Thus, differences in stability of alpha and beta globin mRNA plays a role in determining the ratio of alpha to beta mRNA content in differentiated erythroid 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.

DNA sequences involved in transcriptional regulation of the mouse beta-globin promoter in murine erythroleukemia cells

1988 ◽  
Vol 8 (8) ◽  
pp. 3122-3128
Author(s):  
A Cowie ◽  
R M Myers
Keyword(s):  
Transcriptional Regulation ◽  
Dimethyl Sulfoxide ◽  
Dna Sequences ◽  
Mutational Analysis ◽  
Point Mutations ◽  
Cis Acting ◽  
Sequence Elements ◽  
Induction Of Differentiation ◽  
Globin Promoter ◽  
Murine Erythroleukemia

We have developed a transient assay in murine erythroleukemia (MEL) cells to analyze the cis-acting sequence requirements for transcriptional regulation of the mouse beta-major-globin promoter. From deletion analysis, a fragment of the promoter region, from -106 to +26 relative to the RNA cap site, was found to be sufficient for regulated transcription in MEL cells following induction of differentiation by dimethyl sulfoxide. Single-base mutational analysis of this 132-base-pair promoter fragment identified three sequence elements required for transcription in MEL cells. These are the ATATAA sequence at -31 to -26, the CCAATC sequence between -77 and -72, and the GCCACACCC sequence between -95 and -87. In addition, we found a requirement for sequences adjacent to the CCAAT and ATATAA consensus motifs. Point mutations within the promoter did not abolish transcriptional regulation following induction of differentiation by dimethyl sulfoxide. However, mutations that resulted in reduced transcription levels in uninduced MEL cells gave similarly decreased levels in induced MEL cells.

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 Expression of transfected vimentin genes in differentiating murine erythroleukemia cells reveals divergent cis-acting regulation of avian and mammalian vimentin sequences.

1987 ◽  
Vol 7 (11) ◽  
pp. 3955-3970 ◽  
Author(s):  
J Ngai ◽  
V C Bond ◽  
B J Wold ◽  
E Lazarides
Keyword(s):  
Posttranscriptional Regulation ◽  
Mrna Levels ◽  
Vimentin Expression ◽  
Cis Acting ◽  
Erythroleukemia Cells ◽  
The Difference ◽  
Murine Erythroleukemia ◽  
Vimentin Filaments

We studied the expression of transfected chicken and hamster vimentin genes in murine erythroleukemia (MEL) cells. MEL cells normally repress the levels of endogenous mouse vimentin mRNA during inducermediated differentiation, resulting in a subsequent loss of vimentin filaments. Expression of vimentin in differentiating MEL cells reflects the disappearance of vimentin filaments during mammalian erythropoiesis in vivo. In contrast, chicken erythroid cells express high levels of vimentin mRNA and vimentin filaments during terminal differentiation. We demonstrate here that chicken vimentin mRNA levels increase significantly in differentiating transfected MEL cells, whereas similarly transfected hamster vimentin genes are negatively regulated. In conjunction with in vitro nuclear run-on transcription experiments, these results suggest that the difference in vimentin expression in avian and mammalian erythropoiesis is due to a divergence of cis-linked vimentin sequences that are responsible for transcriptional and posttranscriptional regulation of vimentin gene expression. Transfected chicken vimentin genes produce functional vimentin protein and stable vimentin filaments during MEL cell differentiation, further demonstrating that the accumulation of vimentin filaments is determined by the abundance of newly synthesized vimentin.

Sign in / Sign up

Export Citation Format

Share Document