Inter-ethnic polymorphism of the β-globin gene locus control region (LCR) in sickle-cell anemia patients

Sequence polymorphisms within the 5HS2 segment of human locus control region is described among sickle cell anemia patients. Distinct polymorphic patterns of a simple sequence repeat are observed in strong linkage disequilibrium with each of the five major ^S haplotypes. Potential functional relevance of this polymorphic region in globin gene expression is discussed.     

Insulin analogues with modifications in the β-turn of the B-chain

The β-turn formed by the amino acid residues 20–23 of the B-chain of insulin has been implicated as an important structural feature of the molecule. In other biologically active peptides, stabilization of β-turns has resulted in increases in activity. We have synthesized three insulin analogues containing modifications which would be expected to increase the stability of the β-turn. In two analogues, we have substituted α-aminoisobutyric acid (Aib) for the Glu residue normally present in position B21 or for the Arg residue normally present in position B22; in a third compound, we have replaced the Glu residue with its D-isomer. Biological evaluation of these compounds showed that [B21 Aib]insulin displays a potencyca. one-fourth that of natural insulin, while [B22 Aib]insulin is less than 10% as potent. In contrast, [B21 D-Glu]insulin is equipotent with natural insulin. We conclude that the β-turn region of the insulin molecule normally possesses considerable flexibility, which may be necessary for it to assume a conformation commensurate with high biological activity. If this is the case, [B21 D-Glu]insulin may exhibit a stabilized geometry similar to that of natural insulin when bound to the insulin receptor.     

Evolution of the β-globin gene cluster in man and the primates

The arrangement of primate β-related globin genes has been determined by restriction endonuclease mapping of genomic DNA from species ranging from prosimians to man. The arrangement of the entire ?γγδβ-globin gene cluster in the gorilla and the yellow baboon is indistinguishable from that of man. Restriction site differences between these species are consistent with a surprisingly low overall rate of intergenic DNA sequence divergence of approximately 1% in 5 million years. A new world monkey (owl monkey) has a single γ-globin gene, suggesting that the ^Gγ-^Aγ-globin gene duplication in man is ancient, and occurred about 20 to 40 million years ago. The β-globin gene cluster in the brown lemur, a prosimian, is remarkably short (about 20,000 base-pairs) and contains single ?-, γ- and β-globin genes. The γ- and β-globin genes in this animal are separated by a curious gene containing the 3′ end of a β-globin gene preceded by sequences related to the 5′ end of the ?-globin gene.     

Gene evolution in the chicken β-globin cluster

We have determined the cDNA sequence of the chicken embryonic β-like ?-globin gene. Comparison with the sequences of the chicken ρ-globin and β-globin genes reveals the presence of two regions that are identical or nearly identical in ? and ρ. The first contains the 5′ untranslated sequence and exon 1, while the second region includes the second half of axon 2. Outside these regions ρ and ? are less homologous to each other than to the adult β-globin gene. The embryonic ρ and ? genes are located at opposite ends of the β-globin-gene cluster, not contiguously as are all other known pairs of simultaneously expressed globin genes. We suggest a role for gene conversion in the synchronization of expression of two highly diverged genes.     

The interaction between the human β-globin locus control region and nuclear matrix

Our previous study showed that hydroxyurea (Hu) could induce HEL cells to express humanβ-globin gene. However the molecular mechanisms by which the expression of β-globin gene is activated and regulated are poorly understood. Here we show that the binding patterns between the core DNA sequences (HS2 core sequence -10681- -10971 bp , HS3 core sequence -14991- -14716 bp and HS4 core sequence -18586- -18306 bp) of DNase I hypersensitive sites in the human β-globin LCR and nuclear matrix proteins isolated from Hu induced and uninduced HEL cells are quite different. Results demonstrated that nuclear matrix proteins might play important roles in regulating the expression of humanβ-like globin genes through their interaction with HSs (HS2,HS3 and HS4 core sequences) in the LCR. Moreover, the results obtained from the in vitro DNA-matrix binding assay showed that the core DNA sequences of DNase I hypersensitive sites (HS2, HS3 and HS4) were unable to bind to the nuclear matrix isolated from uninduced HEL cel     

High-resolution analysis of cis-acting regulatory networks at the α-globin locus

We have combined the circular chromosome conformation capture protocol with high-throughput, genome-wide sequence analysis to characterize the cis-acting regulatory network at a single locus. In contrast to methods which identify large interacting regions (10–1000 kb), the 4C approach provides a comprehensive, high-resolution analysis of a specific locus with the aim of defining, in detail, the cis-regulatory elements controlling a single gene or gene cluster. Using the human α-globin locus as a model, we detected all known local and long-range interactions with this gene cluster. In addition, we identified two interactions with genes located 300 kb (NME4) and 625 kb (FAM173a) from the α-globin cluster.     

Restriction fragment length polymorphism in the 3′ flanking region of the rabbit β1-globin gene

By Southern blot analysis, a restriction fragment length polymorphism in the 3' flanking region of the rabbit beta 1-globin gene was detected. Two alleles, characterized by 9.7- and 12.4-kb BamHI fragments and by 15.3- and 18.0-kb HindIII fragments, have been detected in a small population of White New Zealand rabbits. The long allele is the most frequent (about 70%). The simultaneous changes in the restriction patterns of the two endonucleases and the constant distance between BamHI and HindIII sites in short and long fragments suggest the possibility that the two alleles arise from a rearrangement phenomenon involving a DNA segment 2.7 kb long. In addition, the presence of the two alleles in individuals genetically unrelated to the White New Zealand breed suggests that this polymorphism is widespread.     

DNA methylation in the human γδβ-globin locus in erythroid and nonerythroid tissues

We have analyzed DNA modification in the human γδβ-globin gene region at 17 cleavage sites of restriction endonucleases which are unable to cleave DNA if 5-methylcytosine is present at certain positions in their respective cleavage sites. Using this criterion, all sites tested in the globin gene region are fully modified in the germ line (sperm) DNA. In somatic tissues, however, methyl groups are absent at specific sites in the globin gene region. In tissues not expressing the genes, these losses range from one of these cleavage sites in lymphocyte DNA to essentially all of these sites in the entire region in placental DNA. In the DNA of tissues expressing the globin genes, the region surrounding and including the genes expressed shows a low level of modification, whereas the neighboring DNA regions have a high level of modification. The data suggest that a low level of DNA methylation may be a necessary, but not a sufficient, condition for gene expression in higher eucaryotes.     

γ-H2AX and other histone post-translational modifications in the clinic

Chromatin is a dynamic complex of DNA and proteins that regulates the flow of information from genome to end product. The efficient recognition and faithful repair of DNA damage, particularly double-strand damage, is essential for genomic stability and cellular homeostasis. Imperfect repair of DNA double-strand breaks (DSBs) can lead to oncogenesis. The efficient repair of DSBs relies in part on the rapid formation of foci of phosphorylated histone H2AX (γ-H2AX) at each break site, and the subsequent recruitment of repair factors. These foci can be visualized with appropriate antibodies, enabling low levels of DSB damage to be measured in samples obtained from patients. Such measurements are proving useful to optimize treatments involving ionizing radiation, to assay in vivo the efficiency of various drugs to induce DNA damage, and to help diagnose patients with a variety of syndromes involving elevated levels of γ-H2AX. We will survey the state of the art of utilizing γ-H2AX in clinical settings. We will also discuss possibilities with other histone post-translational modifications. The ability to measure in vivo the responses of individual patients to particular drugs and/or radiation may help optimize treatments and improve patient care. This article is part of a Special Issue entitled: Chromatin in time and space.