Design and Development of Reverse Slot Blot for the Simultaneous Detection of Rare and Regional Specific Mutations in the Beta Globin Gene in Khuzestan Province of Iran

Abstract We report the characterization of a beta zero-thalassemia in an American Black with unusually high HbA2 and HbF levels. Genomic southern analysis indicated that the individual was heterozygous for a deletion that began within the second intervening sequence of the beta- globin gene and extended approximately 1.4 kb in the 5′ direction. A clone spanning the breakpoint on the abnormal chromosome was isolated and further mapped, and the deletion joint was sequenced. Comparison of the normal beta-globin gene and its 5′ flanking region with the deletion joint sequence indicated that the 5′ breakpoint for this deletion was 484 base pairs (bp) 5′ to the transcriptional start site for the beta-globin gene and the 3′ breakpoint was 908 bp into the beta- globin gene; the deletion removed a total of 1,393 bp. Comparison of the normal 5′ and 3′ breakpoint sequences indicated that this deletion was the result of a “clean” nonhomologous breakage and reunion event; ie, no spurious bases were added during the recombinational event. Analysis of the breakpoints of this deletion together with the breakpoints of two other small deletions involving the beta-globin gene suggests that the breakpoints may occur at DNA polymerase alpha pause sites.

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Serum factors can modulate the developmental clock of gamma- to beta-globin gene switching in somatic cell hybrids.

Molecular and Cellular Biology ◽

10.1128/mcb.13.8.4844 ◽

1993 ◽

Vol 13 (8) ◽

pp. 4844-4851 ◽

Cited By ~ 4

Author(s):

G Zitnik ◽

Q Li ◽

G Stamatoyannopoulos ◽

T Papayannopoulou

Keyword(s):

Globin Gene ◽

Cumulative Number ◽

Beta Globin ◽

Serum Factors ◽

Beta Globin Gene ◽

Cell Divisions ◽

Growth Promoting ◽

Gene Switching ◽

Mouse Erythroleukemia

The fusion of human fetal erythroid (HFE) cells with mouse erythroleukemia (MEL) cells produces stable synkaryons (HFE x MEL) which can be monitored for extended periods of time in culture. Initially these hybrids express a human fetal globin program (gamma >> beta), but after weeks or months in culture, they switch to an adult pattern of globin expression (beta >> gamma). The rate at which hybrids switch to the adult phenotype is roughly dependent on the gestational age of the fetal erythroid cells used in the fusion, suggesting that the rate of switching in vitro may be determined by a developmental clock type of mechanism, possibly involving the cumulative number of divisions experienced by the human fetal cells. To investigate whether the number or rate of cell divisions postfusion can influence the rate of switching, we monitored the rate of switching in hybrids from independent fusions under growth-promoting (serum-replete) and growth-suppressing (serum-deprived) conditions. We found that hybrids grown under serum-deprived or serumless conditions switched more rapidly to adult globin expression than did their counterparts in serum-replete conditions. Neither the number of cumulative cell divisions nor time in culture per se predicted the rate of switching in vitro. Our data suggest that factors present in serum either retard switching of hybrids by their presence or promote switching by their absence, indicating that globin switching in vitro can be modulated by the environment; however, once switching in HFE x MEL hybrids is complete, serum factors cannot reverse this process.

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Role for DNA replication in beta-globin gene activation.

Molecular and Cellular Biology ◽

10.1128/mcb.8.3.1301 ◽

1988 ◽

Vol 8 (3) ◽

pp. 1301-1308 ◽

Cited By ~ 14

Author(s):

T Enver ◽

A C Brewer ◽

R K Patient

Keyword(s):

Dna Replication ◽

Transcriptional Activation ◽

Globin Gene ◽

Gene Activation ◽

Simian Virus 40 ◽

Simian Virus ◽

Covalent Modification ◽

Beta Globin ◽

Beta Globin Gene ◽

Template Copy

Transcriptional activation of the Xenopus laevis beta-globin gene requires the synergistic action of the simian virus 40 enhancer and DNA replication in DEAE-dextran-mediated HeLa cell transfections. Replication does not act through covalent modification of the template, since its requirement was not obviated by the prior replication of the transfected DNA in eucaryotic cells. Transfection of DNA over a 100-fold range demonstrates that replication does not contribute to gene activation simply increasing template copy number. Furthermore, in cotransfections of replicating and nonreplicating constructs, only replicating templates were transcribed. Replication is not simply a requirement of chromatin assembly, since even unreplicated templates generated nucleosomal ladders. Stimulation of beta-globin transcription by DNA replication, though less marked, was also observed in calcium phosphate transfections. We interpret these results as revealing a dynamic role for replication in gene activation.

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Beta-Globin Gene Cluster Haplotypes of Amerindian Populations from the Brazilian Amazon Region

Human Heredity ◽

10.1159/000154206 ◽

1994 ◽

Vol 44 (3) ◽

pp. 142-149 ◽

Cited By ~ 15

Author(s):

João Farias Guerreiro ◽

Mauro Silvério Figueiredo ◽

Marco Antonio Zago

Keyword(s):

Gene Cluster ◽

Globin Gene ◽

Brazilian Amazon ◽

Amazon Region ◽

Beta Globin ◽

Beta Globin Gene ◽

Globin Gene Cluster ◽

Brazilian Amazon Region

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5'-flanking sequences mediate butyrate stimulation of embryonic globin gene expression in adult erythroid cells.

Molecular and Cellular Biology ◽

10.1128/mcb.11.9.4690 ◽

1991 ◽

Vol 11 (9) ◽

pp. 4690-4697 ◽

Cited By ~ 74

Author(s):

J G Glauber ◽

N J Wandersee ◽

J A Little ◽

G D Ginder

Keyword(s):

Gene Expression ◽

Cell Differentiation ◽

Globin Gene ◽

Beta Globin ◽

Erythroid Cells ◽

Adult Chicken ◽

Beta Globin Gene ◽

Flanking Sequences ◽

Murine Erythroleukemia ◽

Globin Gene Expression

A stable transfection assay was used to test the mechanism by which embryonic globin gene transcription is stimulated in adult erythroid cells exposed to butyric acid and its analogs. To test the appropriate expression and inducibility of chicken globin genes in murine erythroleukemia (MEL) cells, an adult chicken beta-globin gene construct was stably transfected. The chicken beta-globin gene was found to be coregulated with the endogenous adult mouse alpha-globin gene following induction of erythroid differentiation of the transfected MEL cells by incubation with either 2% dimethyl sulfoxide (DMSO) or 1 mM sodium butyrate (NaB). In contrast, a stably transfected embryonic chicken beta-type globin gene, rho, was downregulated during DMSO-induced MEL cell differentiation. However, incubation with NaB, which induces MEL cell differentiation, or alpha-amino butyrate, which does not induce differentiation of MEL cells, resulted in markedly increased levels of transcription from the stably transfected rho gene. Analysis of histone modification showed that induction of rho gene expression was not correlated with increased bulk histone acetylation. A region of 5'-flanking sequence extending from -569 to -725 bp upstream of the rho gene cap site was found to be required for both downregulation of rho gene expression during DMSO-induced differentiation and upregulation by treatment with NaB or alpha-amino butyrate. These data are support for a novel mechanism by which butyrate compounds can alter cellular gene expression through specific DNA sequences. The results reported here are also evidence that 5'-flanking sequences are involved in the suppression of embryonic globin gene expression in terminally differentiated adult erythroid cells.

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