scholarly journals The beta globin 3' enhancer element confers regulated expression on the human gamma globin gene in the human embryonic-fetal erythroleukemia cell line K562

Blood ◽  
1991 ◽  
Vol 77 (4) ◽  
pp. 855-860
Author(s):  
M Donovan-Peluso ◽  
S Acuto ◽  
D O'Neill ◽  
A Hom ◽  
A Maggio ◽  
...  
Keyword(s):  
Developmental Stage ◽  
Globin Gene ◽  
K562 Cells ◽  
Specific Gene ◽  
Fetal Life ◽  
Globin Genes ◽  
Beta Globin ◽  
Specific Expression ◽  
Enhancer Element ◽  
Gamma Globin

We have constructed fusion genes comprised of gamma and beta globin elements and globin sequences linked to neomycin resistance (neoR) genes to define the cis acting sequences responsible for developmental stage-specific expression and induction of fetal globin genes in embryonic-fetal erythroleukemia K562 cells. The results indicate that the gamma promoter is required for proper initiation of transcription. However, the accumulation of gamma globin transcripts in response to hemin induction requires the additional presence of either gamma intervening sequence 2 or the 3′ enhancer element of the beta globin gene. Thus, the gamma promoter may provide the elements for developmental stage-specific gene expression during fetal life. By contrast, the beta 3′ enhancer is erythroid-specific but not developmental stage- or gene-specific.

scholarly journals The beta globin 3' enhancer element confers regulated expression on the human gamma globin gene in the human embryonic-fetal erythroleukemia cell line K562

Blood ◽  
1991 ◽  
Vol 77 (4) ◽  
pp. 855-860 ◽  
Author(s):  
M Donovan-Peluso ◽  
S Acuto ◽  
D O'Neill ◽  
A Hom ◽  
A Maggio ◽  
...  
Keyword(s):  
Developmental Stage ◽  
Globin Gene ◽  
K562 Cells ◽  
Specific Gene ◽  
Fetal Life ◽  
Globin Genes ◽  
Beta Globin ◽  
Specific Expression ◽  
Enhancer Element ◽  
Gamma Globin

Abstract We have constructed fusion genes comprised of gamma and beta globin elements and globin sequences linked to neomycin resistance (neoR) genes to define the cis acting sequences responsible for developmental stage-specific expression and induction of fetal globin genes in embryonic-fetal erythroleukemia K562 cells. The results indicate that the gamma promoter is required for proper initiation of transcription. However, the accumulation of gamma globin transcripts in response to hemin induction requires the additional presence of either gamma intervening sequence 2 or the 3′ enhancer element of the beta globin gene. Thus, the gamma promoter may provide the elements for developmental stage-specific gene expression during fetal life. By contrast, the beta 3′ enhancer is erythroid-specific but not developmental stage- or gene-specific.

scholarly journals Increased expression of the G gamma and A gamma globin genes associated with a mutation in the A gamma enhancer

Blood ◽  
1994 ◽  
Vol 83 (12) ◽  
pp. 3727-3737 ◽  
Author(s):  
G Balta ◽  
HE Brickner ◽  
S Takegawa ◽  
HH Kazazian ◽  
T Papayannopoulou ◽  
...  
Keyword(s):  
Linkage Analysis ◽  
Globin Gene ◽  
Beta Thalassemia ◽  
Functional Assay ◽  
Globin Genes ◽  
Beta Globin ◽  
Enhancer Element ◽  
Beta Globin Gene ◽  
Gamma Globin ◽  
Common Polymorphism

Abstract We have previously described a unique type of delta beta-thalassemia in a Chinese family characterized by increased expression of the G gamma and A gamma fetal globin genes in the absence of a large deletion in the beta-globlin gene cluster. Our earlier study of the beta-globin gene on this delta beta-thalassemia chromosome showed a promoter mutation in the TATA box. In this report, we describe the results of our study of the fetal globin domain of this delta beta-thalassemia chromosome. We have cloned a 13-kb DNA fragment that includes the G gamma and the A gamma genes and the 3′ A gamma enhancer element of this delta beta-thalassemia chromosome. DNA sequence analysis of the G gamma and A gamma-globin genes including their promoters did not show any mutations, but analysis of the putative enhancer element downstream from the A gamma-globin gene showed a C to T substitution 2,401 nucleotides downstream from the A gamma cap site. We performed DNA linkage analysis to determine if this mutation is unique to this chromosome or represents a common polymorphism. Our linkage analysis showed that this mutation is not a common polymorphism and that it is also not an intrinsic part of the haplotype of the chromosome on which it was found. We also studied the interaction of nuclear proteins from erythroid and nonerythroid cells with the DNA sequences surrounding this mutation. We have shown by in vitro DNase I footprinting that this mutation falls within a region that is occupied by a novel DNA-binding protein that binds to this site in nuclear extracts from erythroid, but not nonerythroid cells. The binding of this nuclear protein to DNA appears to be dependent on GATA-1 binding to an adjacent GATA-1 site. We have also developed a new functional assay to compare the activity of the normal and mutant A gamma enhancer elements in erythroid cells. Analysis of the activity of the mutant enhancer shows that the mutation completely eliminates all enhancer activity in this assay. These findings suggest that this mutation of the A gamma enhancer on a chromosome that carries a partially inactivated beta-globin gene may be responsible for the increased expression of both gamma-globin genes seen in this condition.

scholarly journals Molecular characterization of a novel form of (A gamma delta beta)zero thalassemia deletion in a Chinese family

Blood ◽  
1993 ◽  
Vol 81 (6) ◽  
pp. 1624-1629
Author(s):  
JW Zhang ◽  
WF Song ◽  
YJ Zhao ◽  
GY Wu ◽  
ZM Qiu ◽  
...  
Keyword(s):  
Globin Gene ◽  
Fetal Hemoglobin ◽  
Chinese Family ◽  
Globin Genes ◽  
Beta Globin ◽  
Gamma Delta ◽  
Enhancer Element ◽  
Beta Globin Gene ◽  
Gamma Globin ◽  
Mild Anemia

We have identified and molecularly characterized a novel deletion in the beta-globin gene cluster that is associated with elevated fetal hemoglobin in the adult. The propositus is a homozygote from the Yunnan province of China. The deletion spans about 90 kb of DNA and removes the A gamma, delta, and beta-globin genes. The 5' breakpoint of the deletion is located about 0.13 kb upstream from the A gamma-globin gene, whereas the 3' breakpoint is located about 66 kb downstream from the beta-globin gene, about 13 kb upstream from the breakpoint of the Chinese (A gamma delta beta)zero-thalassemia. Heterozygotes for this Yunnanese form of (A gamma delta beta)zero-thalassemia express between 9% and 17% of fetal hemoglobin, whereas the homozygote present with a mild anemia (Hb = 10.7 g/dl). Comparison of the sites of 3' breakpoints of the Yunnanese and the Chinese (A gamma delta beta)zero-thalassemia mutants is compatible with the hypothesis that an enhancer element is located between the 3' breakpoints of these two mutants. Juxta-position to the G gamma gene of this element may be responsible for the efficient gamma-gene expression in the Yunnanese mutant.

scholarly journals Molecular characterization of a novel form of (A gamma delta beta)zero thalassemia deletion in a Chinese family

Blood ◽  
1993 ◽  
Vol 81 (6) ◽  
pp. 1624-1629 ◽  
Author(s):  
JW Zhang ◽  
WF Song ◽  
YJ Zhao ◽  
GY Wu ◽  
ZM Qiu ◽  
...  
Keyword(s):  
Globin Gene ◽  
Fetal Hemoglobin ◽  
Chinese Family ◽  
Globin Genes ◽  
Beta Globin ◽  
Gamma Delta ◽  
Enhancer Element ◽  
Beta Globin Gene ◽  
Gamma Globin ◽  
Mild Anemia

Abstract We have identified and molecularly characterized a novel deletion in the beta-globin gene cluster that is associated with elevated fetal hemoglobin in the adult. The propositus is a homozygote from the Yunnan province of China. The deletion spans about 90 kb of DNA and removes the A gamma, delta, and beta-globin genes. The 5' breakpoint of the deletion is located about 0.13 kb upstream from the A gamma-globin gene, whereas the 3' breakpoint is located about 66 kb downstream from the beta-globin gene, about 13 kb upstream from the breakpoint of the Chinese (A gamma delta beta)zero-thalassemia. Heterozygotes for this Yunnanese form of (A gamma delta beta)zero-thalassemia express between 9% and 17% of fetal hemoglobin, whereas the homozygote present with a mild anemia (Hb = 10.7 g/dl). Comparison of the sites of 3' breakpoints of the Yunnanese and the Chinese (A gamma delta beta)zero-thalassemia mutants is compatible with the hypothesis that an enhancer element is located between the 3' breakpoints of these two mutants. Juxta-position to the G gamma gene of this element may be responsible for the efficient gamma-gene expression in the Yunnanese mutant.

scholarly journals Increased expression of the G gamma and A gamma globin genes associated with a mutation in the A gamma enhancer

Blood ◽  
1994 ◽  
Vol 83 (12) ◽  
pp. 3727-3737
Author(s):  
G Balta ◽  
HE Brickner ◽  
S Takegawa ◽  
HH Kazazian ◽  
T Papayannopoulou ◽  
...  
Keyword(s):  
Linkage Analysis ◽  
Globin Gene ◽  
Beta Thalassemia ◽  
Functional Assay ◽  
Globin Genes ◽  
Beta Globin ◽  
Enhancer Element ◽  
Beta Globin Gene ◽  
Gamma Globin ◽  
Common Polymorphism

We have previously described a unique type of delta beta-thalassemia in a Chinese family characterized by increased expression of the G gamma and A gamma fetal globin genes in the absence of a large deletion in the beta-globlin gene cluster. Our earlier study of the beta-globin gene on this delta beta-thalassemia chromosome showed a promoter mutation in the TATA box. In this report, we describe the results of our study of the fetal globin domain of this delta beta-thalassemia chromosome. We have cloned a 13-kb DNA fragment that includes the G gamma and the A gamma genes and the 3′ A gamma enhancer element of this delta beta-thalassemia chromosome. DNA sequence analysis of the G gamma and A gamma-globin genes including their promoters did not show any mutations, but analysis of the putative enhancer element downstream from the A gamma-globin gene showed a C to T substitution 2,401 nucleotides downstream from the A gamma cap site. We performed DNA linkage analysis to determine if this mutation is unique to this chromosome or represents a common polymorphism. Our linkage analysis showed that this mutation is not a common polymorphism and that it is also not an intrinsic part of the haplotype of the chromosome on which it was found. We also studied the interaction of nuclear proteins from erythroid and nonerythroid cells with the DNA sequences surrounding this mutation. We have shown by in vitro DNase I footprinting that this mutation falls within a region that is occupied by a novel DNA-binding protein that binds to this site in nuclear extracts from erythroid, but not nonerythroid cells. The binding of this nuclear protein to DNA appears to be dependent on GATA-1 binding to an adjacent GATA-1 site. We have also developed a new functional assay to compare the activity of the normal and mutant A gamma enhancer elements in erythroid cells. Analysis of the activity of the mutant enhancer shows that the mutation completely eliminates all enhancer activity in this assay. These findings suggest that this mutation of the A gamma enhancer on a chromosome that carries a partially inactivated beta-globin gene may be responsible for the increased expression of both gamma-globin genes seen in this condition.

The coordinate replication of the human beta-globin gene domain reflects its transcriptional activity and nuclease hypersensitivity

1988 ◽  
Vol 8 (11) ◽  
pp. 4958-4965
Author(s):  
V Dhar ◽  
D Mager ◽  
A Iqbal ◽  
C L Schildkraut
Keyword(s):  
Cell Lines ◽  
Dna Sequences ◽  
Globin Gene ◽  
K562 Cells ◽  
Globin Genes ◽  
Beta Globin ◽  
Beta Globin Gene ◽  
Hypersensitive Sites ◽  
Flanking Sequences ◽  
Globin Gene Domain

The temporal order of replication of DNA sequences in the chromosomal domain containing the human beta-globin gene cluster and its flanking sequences (140 kilobases) was measured and compared in two different human cell lines. In human erythroleukemia (K562) cells, in which embryonic and fetal globin genes are transcribed, all of the sequences we examined from the beta-globin domain replicated early during S phase, while in HeLa cells, in which globin genes are transcriptionally silent, these sequences replicated late during S. Potential sites of initiation of DNA replication within this domain were identified. The beta-globin gene domain was also found to differ with respect to the nuclease sensitivity of the chromatin in these two cell lines. In K562 cells, hypersensitive sites for endogenous nucleases and DNase I were present in the chromatin near the earliest-replicating segments in the beta-globin domain.

scholarly journals Molecular analysis of Japanese delta beta-thalassemia

Blood ◽  
1988 ◽  
Vol 72 (5) ◽  
pp. 1771-1776
Author(s):  
S Shiokawa ◽  
H Yamada ◽  
Y Takihara ◽  
E Matsunaga ◽  
Y Ohba ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Globin Gene ◽  
Fetal Hemoglobin ◽  
Beta Thalassemia ◽  
Globin Genes ◽  
Beta Globin ◽  
Beta Globin Gene ◽  
Gamma Globin ◽  
Large Deletions ◽  
Deletion Junction

A DNA fragment containing the deletion junction region from a Japanese individual with homozygous delta beta-thalassemia has been cloned. A clone containing the normal DNA surrounding the 3′ breakpoint of this deletion and a clone carrying the G gamma- and A gamma-globin genes of this patient were also isolated. Sequences of the deletion junction and both gamma-globin genes were determined. A comparison of these sequences with previously determined sequences of the normal counterparts revealed that the 5′ breakpoint is located between 2,134 and 2,137 base pairs (bp) 3′ to the polyA site of the A gamma-globin gene, the 5′ breakpoint is located just downstream of the 3′ border of the fetal gamma-globin duplication unit, and no molecular defects are evident within the gamma-globin gene region. A comparison between the sequences of the normal DNA surrounding the 3′ breakpoint and the normal DNA surrounding the 5′ breakpoint shows that deletion is the result of a nonhomologous recombination event. There are A+T-rich stretches near the 5′ and 3′ breakpoints in the normal DNA, and a portion of an Aly repeat is located in the region 3′ to the 3′ breakpoint. Southern blot analysis using probes 3′ to the beta-globin gene showed that the deletion extends in the 3′ direction further than any other deletions associated with delta beta-thalassemia and hereditary persistence of fetal hemoglobin (HPFH) heretofore reported. These results are discussed in terms of the mechanism generating large deletions in mammalian cells and three models for the regulation of gamma-globin and beta-globin gene expression in humans.

Upstream G gamma-globin and downstream beta-globin sequences required for stage-specific expression in transgenic mice

1987 ◽  
Vol 7 (11) ◽  
pp. 4024-4029
Author(s):  
M Trudel ◽  
J Magram ◽  
L Bruckner ◽  
F Costantini
Keyword(s):  
Transgenic Mice ◽  
Fusion Gene ◽  
Globin Gene ◽  
Regulatory Elements ◽  
Globin Genes ◽  
Beta Globin ◽  
Erythroid Cells ◽  
Base Pairs ◽  
Beta Globin Gene ◽  
Gamma Globin

The human G gamma-globin and beta-globin genes are expressed in erythroid cells at different stages of human development, and previous studies have shown that the two cloned genes are also expressed in a differential stage-specific manner in transgenic mice. The G gamma-globin gene is expressed only in murine embryonic erythroid cells, while the beta-globin gene is active only at the fetal and adult stages. In this study, we analyzed transgenic mice carrying a series of hybrid genes in which different upstream, intragenic, or downstream sequences were contributed by the beta-globin or G gamma-globin gene. We found that hybrid 5'G gamma/3'beta globin genes containing G gamma-globin sequences upstream from the initiation codon were expressed in embryonic erythroid cells at levels similar to those of an intact G gamma-globin transgene. In contrast, beta-globin upstream sequences were insufficient for expression of 5'beta/3'G gamma hybrid globin genes or a beta-globin-metallothionein fusion gene in adult erythroid cells. However, beta-globin downstream sequences, including 212 base pairs of exon III and 1,900 base pairs of 3'-flanking DNA, were able to activate a 5'G gamma/3'beta hybrid globin gene in fetal and adult erythroid cells. These experiments suggest that positive regulatory elements upstream from the G gamma-globin and downstream from the beta-globin gene are involved in the differential expression of the two genes during development.

scholarly journals Expression of the chicken beta-globin gene cluster in mice: correct developmental expression and distributed control.

1995 ◽  
Vol 15 (1) ◽  
pp. 407-414 ◽  
Author(s):  
M M Mason ◽  
E Lee ◽  
H Westphal ◽  
M Reitman
Keyword(s):  
Developmental Stage ◽  
Globin Gene ◽  
Stage Iv ◽  
Gene Clusters ◽  
Developmental Expression ◽  
Single Element ◽  
Globin Genes ◽  
Beta Globin ◽  
Variable Expression ◽  
Hypersensitive Sites

To investigate the regulation of gene clusters, we introduced the entire chicken beta-globin cluster into mice. This 35-kb region includes the four globin genes (rho-beta H-beta A-epsilon), the four upstream hypersensitive sites, and the intergenic beta A/epsilon enhancer. The chicken globins are not arranged in order of developmental expression, which is unlike the case for the human beta-globin cluster, in which gene order plays a role in the regulation of globin expression. Mice carrying the chicken cluster expressed the transgenes with the same developmental patterns as seen in the chicken. Therefore, stage-specific erythroid transcriptional milieus existed before the divergence of birds and mammals and have been conserved since then. Mice bearing the complete cluster except for a deletion removing the beta A/epsilon enhancer displayed markedly reduced expression of the beta H, beta A, and epsilon genes with efficient (but variable) rho expression. Mice carrying the four genes and beta A/epsilon enhancer but without the upstream hypersensitive sites showed reduced expression of rho, beta H, and beta A, with variable expression of epsilon. We conclude that (i) all of the genes (except possibly rho) are under the control of both the upstream hypersensitive sites and the enhancer, (ii) the influence of the control elements can extend beyond the nearest active gene, (iii) a single element (the enhancer) can influence more than one gene in a single developmental stage, (iv) the enhancer can work bidirectionally, and (v) neither the upstream sites (as a group) nor the enhancer showed developmental stage specificity. Thus, the regulation of this cluster is achieved by interaction of two distinct control regions with each of the globin genes.

scholarly journals The detection and use of hemoglobin mutants in the direct analysis of human globin genes

Blood ◽  
1980 ◽  
Vol 55 (6) ◽  
pp. 1060-1062 ◽  
Author(s):  
PF Little ◽  
E Whitelaw ◽  
G Annison ◽  
R Williamson ◽  
JM Kooter ◽  
...  
Keyword(s):  
Restriction Site ◽  
Globin Gene ◽  
Direct Analysis ◽  
Globin Genes ◽  
Beta Globin ◽  
Gene Sequences ◽  
Gamma Globin ◽  
Alpha Globin ◽  
Specific Restriction ◽  
Alpha Beta

Abstract Many human globin-chain mutants contain amino acid replacements that result from single base changes in the corresponding globin gene. Using recombinants, the coding sequences of each of the alpha-, beta-, Ggamma- , and Agamma-globin genes have now been determined. Those sequences of DNA that are cleaved by a number of specific restriction endonucleases have been identified and accurately positioned. Mutations at these sequences abolish the restriction site, and therefore, the pattern of DNA fragments containing hybridizing globin-gene sequences is altered compared to DNA from normal persons. This allows the identification of one of a pair of cross-hybridizing human globin-gene sequences, as is shown here for the two alpha-globin, the two gamma-globin, and the delta- and beta-globin genes.

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