Beta-globin gene haplotypes in Polynesians are predominantly southern Chinese in type

beta-Globin gene haplotypes obtained in Polynesian Samoans were similar to those described in Southern Chinese. An atypical HindIII restriction fragment length polymorphism detected with pRK29, a 3' beta-globin gene probe, was present at a gene frequency of 7% in Samoans. Haplotype patterns suggest that this polymorphism may have arisen by 1 or 2 mutational events. DNA haplotypes derived from the beta-globin gene cluster confirm nuclear and mitochondrial DNA data that Polynesian precursor populations were East Asian in origin.     

Molecular characterization of seven beta-thalassemia mutations in Asian Indians

To characterize systematically the mutations which produce beta-thalassemia in Asian Indians, we first determined the DNA polymorphism haplotype in the beta-globin gene cluster of 44 beta-thalassemia chromosomes in the ethnic group. Nine different haplotypes were observed. Upon molecular cloning and partial DNA sequencing of one beta-gene from each of eight haplotypes and two from the ninth, seven different mutations were found. None of these have been identified in Mediterranean patients, even among the five haplotypes which appeared identical in the two groups. Asian Indian mutations included one nonsense and three frameshift mutations, one deletion affecting an acceptor splice site, and two mutations affecting a donor splice site. The correlation of a specific mutation with a specific haplotype was high but not invariant. Two mutations were associated with more than one haplotype but, in each instance, the mutation spread to a new haplotype could be explained most simply by recombination 5' to the beta-globin gene. In addition, four mutations, one reported here and three others previously reported, have been observed on two chromosome backgrounds that are identical except for the status of a polymorphic HinfI site 5' to the beta gene. This HinfI site does not show significant linkage disequilibrium with markers both 5' and 3' to it, suggesting that it lies within a region of relative sequence randomization.     

DNA sequence variants in the G gamma-, A gamma-, delta- and beta-globin genes of man

DNA prepared from 60 unrelated individuals was cleaved with one of eight different restriction endonucleases and the resulting DNA fragments were separated by agarose gel electrophoresis. DNA fragments containing G gamma-, A gamma-, delta- or beta-globin genes were detected by Southern blot hybridization, using as probe either a 32P-labeled cloned DNA copy of rabbit beta-globin messenger RNA or labeled human beta- and G gamma- globin cDNA plasmids. Three types of variant restriction enzyme patterns of globin DNA fragments were detected in otherwise normal individuals. One variant pattern, found in only one person, was caused by an additional restriction endonuclease Pst I cleavage site in the center of the delta- globin gene intervening sequence; the subject was heterozygous for the presence of this cleavage site and was shown to have inherited it from her mother. Another variant pattern resulted from the appearance of an endonuclease Hind III cleavage site in the intervening sequence of the A gamma-globin gene; this variant is polymorphic, with a gene frequency for the presence of the intragenic Hind III site of 0.23. This Hind III cleavage site polymorphism is also found in the G gamma-globin gene intervening sequence and thus the polymorphism itself appears to be duplicated over the pair of gamma-globin loci. These variants can be used to derive an approximate estimate of the total number of different DNA sequence variants in man.     

The DNA sequence of the 5' flanking region of the human beta-globin gene: evolutionary conservation and polymorphic differences.

We have determined the DNA sequence of a 1464 bp segment immediately flanking the 5' side of the human beta-globin gene. The sequence shows little similarity to the corresponding regions of the epsilon- or gamma-globin genes. There is about 75% homology, however, between the 5' extragenic regions of the beta-globin genes of man, goat and rabbit respectively. The mouse beta minor globin gene, but not the mouse beta major globin gene, also shares this extensive homology. A short segment of simple sequence DNA is found from about 1418 to 1388 bp upstream from the human beta-globin gene which consists of repeats of the sequence (TTTTA). Similar DNA sequences are also found at several sites in the large intron of the beta-globin gene. We have compared the DNA sequence of the 5' extragenic region of the normal beta-globin gene with the same segment of the beta-globin gene of a patient with beta thalassaemia. Of the two nucleotide differences observed, one generates a polymorphic HinfI site present 990 bp upstream from the beta-globin gene in the thalassaemic beta-globin and absent in the normal gene. A second beta thalassemic beta-globin gene which has the same molecular defect as the above mentioned case, however, lacks this HinfI site. It is therefore not yet clear whether this HinfI site will have any value in prenatal diagnosis of beta thalassaemia.     

A family of long reiterated DNA sequences, one copy of which is next to the human beta globin gene.

An unusually long repeated DNA sequence was identified in cloned DNA, three kb 3' to the human beta-globin gene. Other members of this repeated sequence family were isolated from a human genomic DNA library and characterized by Southern blotting techniques, electron microscopy, and solution hybridization. The copy located next to the beta-globin gene was found to be 6.4 +/- 0.2 kb long and continuous over that length. This repeated sequence family comprises about 1% of the human genome and contains 3000-4800 copies of moderate sequence divergence which are interspersed with other less-highly repeated DNA. The 6.4 kb repeated unit does not appear to be composed of any smaller tandemly repeated subunits, nor is it expressed at a high level in bone marrow cell RNA.     

Restriction fragment length polymorphism of the human beta-globin gene cluster: analysis of a beta-thalassemic family in Taiwan

We have analysed seven polymorphic restriction sites of the human beta-globin gene cluster of six members of a Chinese family with a beta +-thalassemic sibling. The seven polymorphic sites analysed are the HincII site at the 5'-end of the epsilon-globin gene, the HindIII sites in the two gamma-globin genes, two HincII sites within and at the 3'-end of the psi beta 1 pseudogene, the AvaII site in the beta-globin gene and the BamHI site located at the 3' side of the beta-globin gene. The beta thal chromosome has been identified to have a haplotype of +----++ with respect to these seven polymorphic sites. This is also the most predominant haplotype associated with beta +-thalassemia in Mediterranean and Chinese populations (Chen et al., 1984; Orkin et al., 1982). Of the seven sites analysed in this family, four will be useful in prenatal diagnosis of beta-thalassemia in subsequent pregnancies in the family.     

LDL受体基因内含子15结构分析及其在家族性高胆固醇血症基因诊断中的应用

为了解人类ＬＤＬ受体基因内含子１５的遗传背景,利用长链ＰＣＲ和锚定ＰＣＲ分离了ＬＤＬ受体基因外显子１５－内含子１５－外显子１６和内含子１５的３‘末端片段。利用Ｄｙｎａｌｂｅａｄｓ固相单链分离ＰＣＲ产物直接测序法测定了内含子１５ ３’末端１２２２个碱基序列。序列显示：３‘末端含有由１６个碱基组成的典型３’末端剪接位点;３‘端上游第３１个碱基处含有经典分支位点,除了经典分支位点外,在３’末端上游第２０     

Structure and function of the murine beta-globin locus control region 5' HS-3.

We previously identified the murine homologue of the human beta-globin Locus Control Region (LCR) 5' HS-2. The lambda clone containing murine 5' HS-2 extends approximately 12 kb upstream from this site; here, we report the sequence of this entire upstream region. The murine homologue of 5' HS-3 is located approximately 16.0 kb upstream from the mouse epsilon y-globin gene, but no region homologous to human 5' HS-4 was present in our clone. Using a reporter system consisting of a human gamma-globin promoter driving the neomycin phosphotransferase gene (gamma-neo), we tested murine LCR fragments extending from -21 to -9 kb (with respect to the epsilon y-globin gene cap site) for activity in classical enhancer and integration site assays in K562 and MEL cells. 5' HS-2 behaved as a powerful enhancer and increased the number of productive integration events (as measured by a colony assay) in both K562 and MEL cells. 5' HS-3 had no activity in K562 cells or in transiently transfected MEL cells, but was nearly as active as 5' HS-2 in the MEL cell colony assay. Two additional tests confirmed the identification of murine 5' HS-3: first, a DNA fragment containing 5' HS-3 confers copy number-dependent, integration-site independent inducibility on a linked beta-globin gene in the MEL cell environment. Secondly, a strong DNAseI hypersensitive site maps to the location of the 5' HS-3 functional core in chromatin derived from MEL cells. Collectively, these data suggest that we have identified the murine homologue of human 5' HS-3, and that this site is functional when integrated into the chromatin of MEL cells but not K562 cells. 5' HS-3 may therefore contain information that contributes to the development-specific expression of the beta-like globin genes.     

High-mobility group protein 2 may be involved in the locus control region regulation of the beta-globin gene cluster.

Expression regulation of the beta-globin gene cluster is a result of synergistic interactions between cis-elements and trans-acting factors. Previous studies usually concentrated on the core sequence of each hypersensitive site in the locus control region of the beta-globin gene cluster. But more and more evidence illustrates that the flanking regions are indispensable also. Using electrophoretic mobility shift assay and solid-phase DNase I footprinting methods, we identified a small nuclear protein from K562 cells that binds specifically to the first AT-rich region flanking the hypersensitive site 2 core sequence of the human beta-globin gene locus control region. N-terminal sequencing of the enriched protein proved that it is a member of the high-mobility group protein 2 family. This indicates that the AT-rich region in human hypersensitive site 2 may take part in the regulation of the beta-globin gene cluster by facilitating DNA bending, which is a prerequisite for the looping mechanism in this region.     

An intron nucleotide sequence variant in a cloned beta +-thalassaemia globin gene.

A 7.5 kb Hsu I restriction fragment of genomic DNA containing a beta-globin gene has been isolated from a patient doubly heterozygous for beta + thalassaemia and a delta beta (Lepore globin fusion gene. This fragment must be derived from the chromosome carrying the beta +-thalassaemia determinant. The gross structure of the cloned gene plus flanking sequences is indistinguishable from that of a normal beta-globin gene. Within in 1606 base-pair transcribed region of the gene there is only one nucleotide difference from the normal beta-globin gene sequence. This is a G leads to A replacement 21 nucleotides upstream from the 3' terminus of the small intron. This nucleotide lies within a 10 base-pair sequence repeated in an inverted configuration near the 5' terminus of the small intron. The nucleotide replacement may result in a precursor mRNA less amenable to RNA splicing than its normal counterpart.     

DNA length polymorphism located 5'' to the human myelin basic protein gene.

A region of DNA 5' to the human myelin basic protein (MBP) gene, located on the long arm of chromosome 18, is a site of restriction-fragment-length polymorphism (RFLP) showing 37% heterozygosity in 40 subjects studied. Southern transfer analysis using a 0.9-kb genomic fragment encompassing the first exon of the human MBP gene reveals this polymorphism with at least nine restriction enzymes, indicating that insertion, deletion, or both is the basis for the DNA length variation. Double restriction-enzyme digest analysis suggests that this polymorphism is within the region 0.5-2.0 kb upstream of the coding region of the first exon of the human MBP gene. Eleven different allelic RFLPs were identified, differing in size by as many as 450 bp. The distribution of insertion/deletion-size variants from this region is bimodal, with most restriction fragments varying in size over a 0.1-kb range. Pedigree analysis of polymorphism at this site in one three-generation family shows Mendelian assortment of parental haplotypes. The form and frequency of polymorphism generated by this site is similar to that reported for human DNA regions comprised of homologous short tandem repeats.     

Characterization of a spontaneous mutation to a beta-thalassemia allele.

We have studied a nuclear family containing a single child with severe beta-thalassemia intermedia, a Greek-Cypriot mother with hematological findings of beta-thalassemia trait, and a Polish father who is hematologically normal. Since both the child and her father were heterozygous for a DNA polymorphism within the beta-globin gene, it was possible to clone and sequence the beta-globin gene identical by descent from both the child and her father. A nonsense mutation in codon 121 (GAA----TAA) was found in the beta-globin gene of the child, while the same gene from her father lacked this mutation and was normal. This mutation has not been previously observed among over 200 beta-thalassemia genes characterized in Caucasians. Since the mutation eliminates an EcoRI site in the beta-globin gene, we could show that the mutation is not present in genomic DNA of the father. To rule out germinal mosaicism, sperm DNA of the father was also digested with EcoRI, and the mutant EcoRI fragment was not observed under conditions that would detect the mutation if it were present in at least 2% of sperm cells. Routine HLA and blood group testing supported stated paternity. In addition, studies with 17 DNA probes that detect multiple allele polymorphisms increased the probability of stated paternity to at least 10(8):1. These data provide evidence that the G----T change in codon 121 of the beta-globin gene in the child is the result of a spontaneous mutation that occurred during spermatogenesis in a paternal germ cell.     

A molecular and evolutionary study of the beta-globin gene family of the Australian marsupial Sminthopsis crassicaudata

Beta-globin gene families in eutherians (placental mammals) consist of a set of four or more developmentally regulated genes which are closely linked and, in general, arranged in the order 5'-embryonic/fetal genes- adult genes-3'. This cluster of genes is proposed to have arisen by tandem duplication of ancestral beta-globin genes, with the first duplication occurring 200 to 155 MYBP just prior to a period in mammalian evolution when eutherians and marsupials diverged from a common ancestor. In this paper we trace the evolutionary history of the beta-globin gene family back to the origins of these mammals by molecular characterization of the beta-globin gene family of the Australian marsupial Sminthopsis crassicaudata. Using Southern and restriction analysis of total genomic DNA and bacteriophage clones of beta-like globin genes, we provide evidence that just two functional beta-like globin genes exist in this marsupial, including one embryonic- expressed gene (S.c-epsilon) and one adult-expressed gene (S.c-beta), linked in the order 5'-epsilon-beta-3'. The entire DNA sequence of the adult beta-globin gene is reported and shown to be orthologous to the adult beta-globin genes of the North American marsupial Didelphis virginiana and eutherian mammals. These results, together with results from a phylogenetic analysis of mammalian beta-like globin genes, confirm the hypothesis that a two-gene cluster, containing an embryonic- and an adult-expressed beta-like globin gene, existed in the most recent common ancester of marsupials and eutherians. Northern analysis of total RNA isolated from embryos and neonatals indicates that a switch from embryonic to adult gene expression occurs at the time of birth, coinciding with the transfer of the marsupial from a uterus to a pouch environment.      

Detection of a high frequency RsaI polymorphism in the human pro alpha 2(I) collagen gene which is linked to an autosomal dominant form of osteogenesis imperfecta.

Screening of the pro alpha 2(I) collagen genes of Southern African populations for restriction fragment length polymorphisms (RFLPs) has revealed a locus polymorphic for the restriction enzyme RsaI. The frequency of the RFLP was 0.38 in Afrikaners, but much lower in indigenous Southern African populations, which suggests that it is of European origin. The polymorphism was used to study 19 affected and non-affected individuals in a four generation family with the autosomal dominant disorder, osteogenesis imperfecta (OI) type I. Co-inheritance of the loss of the RsaI site and the OI phenotype was observed with a lod score of 3.91 at a recombination fraction (theta) of zero, indicating strong linkage. This suggests that the defect in this family is caused by a structural mutation within or close to the pro alpha 2(I) collagen gene. The use of this high frequency RFLP together with other recently described polymorphisms at this locus will facilitate the analysis of the role of this gene in OI and other inherited disorders of connective tissue.     

Transcriptional control of the mouse prealbumin (transthyretin) gene: both promoter sequences and a distinct enhancer are cell specific.

The mouse genomic clone for the prealbumin (transthyretin) gene was cloned, and its upstream regulatory regions were analyzed. The 200 nucleotides 5' to the cap site when placed within a recombinant plasmid were sufficient to direct transient expression in HepG2 (human hepatoma) cells, but this DNA region did not support expression in HeLa cells. The sequence of the 200-nucleotide region is highly conserved between mouse and human DNA and can be considered a cell-specific promoter. Deletions of this promoter region identified a crucial element for cell-specific expression between 151 and 110 nucleotides 5' to the RNA start site. A region situated at about 1.6 to 2.15 kilobases upstream of the RNA start site was found to stimulate expression 10-fold in HepG2 cells but not in HeLa cells. This far upstream element was invertible and increased expression from the beta-globin promoter in HepG2 cells. Unlike the simian virus 40 enhancer, the prealbumin enhancer would not stimulate beta-globin synthesis in HeLa cells, and even the simian virus 40 enhancer did not stimulate the prealbumin promoter in HeLa cells. Thus, we identified in the prealbumin gene two DNA elements that respond in a cell-specific manner: a proximal promoter including a crucial sequence between -108 and -151 nucleotides and a distant enhancer element located between 1.6 and 2.15 kilobases upstream.     

Nuclease hypersensitivity in the beta-globin gene region of K562 cells

We have investigated chromatin structure in the beta-globin gene region of the K562 human erythroleukemic cell line by using S1 and DNase I nuclease sensitivity assays. Despite the lack of beta-globin gene expression in these cells, we find nuclease-hypersensitive sites to these enzymes in its 5' and 3' flanking regions in K562 chromatin. This result is in contrast to previous reports in which no hypersensitive sites were found in the immediate vicinity of this gene. In the 3' region, one major hypersensitive site at 0.9 kpb 3' and three minor hypersensitive sites at 0.7 kbp, 0.5 kbp 3' and 0.2 kbp 5' of the polyadenylation site were observed; these sites are very similar to those found in fetal liver and adult bone marrow cells in which the beta-globin gene is expressed. We find hypersensitive sites to both enzymes in the 5' region of the beta-globin gene: a major site 0.8 kbp 5' to the cap site, and two minor sites 1.2 and 1.5 kbp 5' to the cap site. The -0.8 kbp site is also present in plasmids containing the beta-globin gene. Our results suggest that the lack of beta-globin gene expression may be related to the lack of hypersensitivity sites in the immediate (150 bp) 5' flanking region of the beta-globin gene, as occurs in other active globin genes.     

An insulator element and condensed chromatin region separate the chicken beta-globin locus from an independently regulated erythroid-specific folate receptor gene.

We have identified a folate receptor gene upstream of the chicken beta-globin locus and separated from it by a 16 kbp region of silent chromatin. We find that this receptor is expressed only at a stage of erythroid differentiation (CFU-E) preceding the activation of beta-globin genes, consistent with the role of folate receptors in proliferation. This discovery raises the question of how these two loci are regulated during erythropoiesis. Our data suggest that the folate receptor gene and the beta-globin locus are regulated independently. We show that a 3.3 kbp DNA region upstream of the folate receptor gene is sufficient to induce strong expression of a transgene in CFU-E stage cells. We also find that the region between the beta-globin locus and the folate receptor gene is fully methylated and condensed at this stage of differentiation. Its 3' boundary coincides with the 5' beta-globin insulator. We speculate that the 5' beta-globin boundary element might be important for the proper regulation of two adjacent domains activated at two different stages during differentiation.