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H E Brickner

Publications and source records attributed to H E Brickner.

8 recordsLinked to original sources
PubMed1994

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

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.

Base Sequence↗
PubMed1991

Regulated expression of p18, a major phosphoprotein of leukemic cells.

p18 is a phosphoprotein that is present in great abundance in acute leukemia blasts and in less abundance in proliferating lymphocytes. This protein undergoes major changes in its state of phosphorylation upon induction of differentiation of leukemic cells in culture. The same protein appears to be involved in a variety of other cellular processes that include regulation of hormone secretion, T cell activation, muscle differentiation, and brain development. In this report, we describe our studies of the regulation of expression of this gene in leukemic cells. We show that the expression of this gene is markedly reduced upon induction of differentiation of a variety of leukemic cells in culture. We use a cDNA clone that we constructed earlier which encodes this protein as a probe to isolate the human chromosomal p18 gene. We characterize the 5' end of this gene in detail and identify its promoter element. We also identify a regulatory element in the first intervening sequence (IVS-1) of this gene which loses its DNase I hypersensitivity upon induction of differentiation of leukemic cells in culture. Our DNase I footprinting experiments demonstrate nuclear protein binding to multiple sequence motifs within its promoter element and its IVS-1 regulatory element. Functional studies using a transient expression system show that deletion of these sequence motifs has profound effects on the expression of this gene. These studies begin to shed some light on the mechanism of regulation of a gene that may be involved in control of cell growth and differentiation and in a variety of other vital cellular processes.

Base Sequence↗
PubMed1991

A novel regulatory element of the human alpha-globin gene responsible for its constitutive expression.

The alpha-globin gene is expressed at a constitutively high level upon gene transfer into both erythroid and nonerythroid cells. The beta-globin gene, on the other hand, is dependent on the presence of a linked viral enhancer for its efficient expression upon transfer into heterologous cells. In this report, we describe a novel regulatory element within the structural alpha-globin gene which can activate its own promoter to result in a high level of expression in both erythroid and non-erythroid cells. This regulatory element does not appear to have the properties of a classical enhancer. While this element exerts a positive effect on its own promoter, we have demonstrated in a previous study that the same element exerts a negative effect on heterologous genes such as the beta- and gamma-globin genes. In this study, we localize this element to a 259 nucleotide fragment immediately downstream from the translation initiation codon which is partially overlapped by a DNase I hypersensitive domain only in erythroid cells. We propose that this element may activate the alpha-globin gene promoter in all cell types in vivo as it does in vitro. The specificity of erythroid expression of the alpha-globin gene in vivo is probably determined by a "permissive" chromatin configuration in erythroid cells and a "nonpermissive" configuration in non-erythroid cells.

Blotting, Southern↗
PubMed1989

Abnormal processing of beta-Malay globin RNA.

Hemoglobin Malay (alpha 2 beta 2 19 Asn----Ser) has been observed in a few Malaysian patients with thalassemia intermedia. The beta Malay substitution increases the homology of the cryptic splice site at codons 17/18/19 of the beta-globin gene to the donor consensus splice sequence, suggesting that the beta-thalassemia associated with this mutation may be due to the generation of a new splice site. To test this hypothesis, we constructed a hybrid gene where we replaced part of a normal beta-globin gene with a PCR amplified region of the beta Malay gene. The expression of this mutant gene was studied in a heterologous transient expression system. The data show that nearly 25% of globin mRNA produced by this gene is abnormally spliced at the new splice site, providing a molecular mechanism for the beta-thalassemia associated with the mutation.

Base Sequence↗
PubMed1988

A silencer element from the alpha-globin gene inhibits expression of beta-like genes.

We have studied the cis and trans interactions of the alpha- and beta-globin genes in a transient expression system. We found that the alpha-globin gene inhibited beta-globin expression in cis but not in trans. The silencer element responsible for this inhibition was localized to a 259-base-pair fragment at the 5' end of the alpha-globin gene.

Enhancer Elements, Genetic↗
PubMed1988

New amber mutation in a beta-thalassemic gene with nonmeasurable levels of mutant messenger RNA in vivo.

We have identified a beta-thalassemia gene that carries a novel nonsense mutation in a Chinese patient. This mutation, a G to T substitution at the first position of codon 43, changes the glutamic acid coding triplet (GAG) to a terminator codon (TAG). Based on oligonucleotide hybridization studies of 78 Chinese and Southeast Asian beta-thalassemia chromosomes, we estimate that this mutation accounts for a small minority of the beta-thalassemia mutations in that population. Study of the expression of this cloned gene in a transient expression system demonstrated a 65% decrease in levels of normally spliced mutant beta-globin mRNA. However, the study of reticulocyte RNA isolated from an individual heterozygous for this mutation demonstrated a total absence of this mutant mRNA in vivo. The basis for this big discrepancy between the level of accumulated mRNA in vivo and in vitro is probably the result of differences in the stabilities of the mutant mRNA in erythroid cells.

Amino Acid Sequence↗
PubMed1987

The beta-globin gene on the Chinese delta beta-thalassemia chromosome carries a promoter mutation.

A new type of delta beta-thalassemia characterized by decreased expression of the beta-globin gene and increased expression of both G gamma and A gamma globin gene in the absence of a detectable deletion has recently been described in the Chinese population. In this study we characterize the mutant beta-globin gene from this delta beta-thalassemia chromosome. An A to G transversion is identified in the "ATA" sequence of the promoter region that leads to decreased expression of the beta-globin gene in vivo and in vitro. We also demonstrate the presence of this mutation in every individual with a high fetal hemoglobin phenotype in this family and its absence in every individual with a normal hemoglobin phenotype. This same promoter mutation has recently been detected in Chinese beta-thalassemia genes where it is present on chromosomes of the same haplotype as that of the delta beta-thalassemia chromosome we are studying. These data support the hypothesis that an as yet unidentified mutation occurred on the ancestral chromosome carrying the promoter mutation and subsequently gave rise to the delta beta-thalassemia phenotype.

China↗