Chromosomal arrangement of the duck alpha-globin genes and primary structure of the embryonic alpha-globin gene pi

A recombinant lambda Charon 4A bacteriophage, D alpha G-1, carrying the genes coding for the duck embryonic (pi') and adult (alpha A, alpha D) alpha-like globins was isolated from a previously constructed duck DNA recombinant library. The three globin genes are transcribed from the same DNA strand and are arranged in the order of their expression during development: 5'-pi'-alpha D-alpha A-3'. We have determined the complete nucleotide sequence of the duck pi'-globin gene, including the flanking regions. Due to the unusual length of intron 1 (963 bp) and intron 2 (568 bp) the 2167-bp duck pi'-globin gene is by far the largest among all known mammalian or avian alpha- and beta-globin genes. For instance, the duck pi'-globin gene introns are almost twice as long as those of the chicken pi'-globin genes. A surprisingly high degree of nucleotide sequence homology (88%) has been found for the 5' flanking region (positions -1 to -223) of the duck and chicken pi'-globin gene.     

Identification and characterization of a chicken alpha-globin enhancer.

We identify and describe the properties of an enhancer within the chicken alpha-globin gene cluster. This cluster consists of one gene (pi) expressed only in primitive erythrocytes and two (alpha A and alpha D) expressed in both primitive and definitive cell lineages. The genes are linked together in the order 5'-pi-alpha D-alpha A-3' and occupy a region about 10 kilobase pairs long. The enhancer is located at the 3' end of the cluster, about 750 base pairs 3' to the alpha A translation stop site. When assayed by transfection into either primitive or definitive primary chicken erythrocytes, this element stimulated expression from plasmids containing the alpha D- or alpha A-globulin gene promoters. Except for sites in the alpha-globin promoters, no other stimulatory activity was observed in DNA taken from other regions of the alpha-globin locus. Moderate resolution DNase I hypersensitivity studies as well as DNase I footprinting revealed three regions of protein binding, each containing a similar core DNA sequence within the enhancer element. Gel mobility shift studies demonstrated that all three regions bind the recently identified erythrocyte-specific factor, EryfI, which has binding sites in the regulatory regions of all chicken globin genes. Our data suggest that the enhancer we have identified may act in vivo only on the alpha A gene; expression of the alpha D gene is affected by another EryfI site located in the alpha D promoter. Such a mechanism would be consistent with the observed relative abundances of alpha A- and alpha D-globin in vivo. The simplicity of these regulatory elements may reflect the limited repertoire of expression of these genes during development.     

Conserved sequences and cell-specific DNase I hypersensitive sites upstream from the co-ordinately expressed alpha I- and alpha II-globin genes of Xenopus laevis

The globin gene family of Xenopus laevis comprises pairs of closely related genes that are arranged in two clusters, each pair of genes being co-ordinately and stage-specifically expressed. To get information on putative regulatory elements, we compared the DNA sequences and the chromatin conformation 5' to the co-ordinately expressed adult alpha-globin genes. Sequence analysis revealed a relatively conserved region from the cap site up to position -289, and further upstream seven distinct boxes of homology, separated by more diverged sequences or deletions/insertions. The homology boxes comprise 22 to 194 base-pairs showing 78 to 95% homology. Analysis of chromatin conformation showed that DNase I preferentially cuts the upstream region of both genes at similar positions, 5' to the T-A-T-A and the C-C-A-A-T boxes, only in chromatin of adult erythroblasts and erythrocytes, where adult globin genes are expressed, but not in chromatin of adult liver cells or larval erythrocytes, where these genes are silent. This suggests that cell- and stage-specific activation of these genes coincides with specific changes in chromatin conformation within the proximal upstream region. No difference was found in the nucleotide sequence within the DNase I hypersensitive region proximal to the adult alpha 1-globin gene in DNA from embryonic cells, in which this gene is inactive, and adult erythrocytes, expressing this gene.     

Human alpha-globin gene expression. The dominant role of the alpha 2-locus in mRNA and protein synthesis

The two human alpha-globin genes, alpha 1 and alpha 2, are coexpressed in normal erythroid cells and encode identical alpha-globin protein products. Based upon genetic studies, it has been assumed that these two adjacent and highly homologous genes are equally expressed. In previous studies we have, however, demonstrated that the alpha 2 gene encodes a 2-3-fold higher steady state level of mRNA than the alpha 1 gene. In the present study, we monitor the relative levels of protein production from these two loci by quantitating the synthesis of specific alpha-globin structural mutants encoded by each alpha-globin gene. These values are then used to infer the relative contributions of the normal alpha 1 and alpha 2 loci to total alpha-globin production. The results of eight separate studies, each based upon a different alpha-globin structural mutant mapped to either the alpha 1 or the alpha 2 locus, are internally consistent. The data demonstrate that the alpha 2 gene encodes 2-3-fold more protein than the alpha 1 gene. These results suggest that the human alpha-globin gene cluster contains a major and a minor locus. The dominant expression of the alpha 2 gene predicts a greater impact of mutations at this locus, in comparison to mutations at the alpha 1 locus, in the generation of the alpha-thalassemia phenotype.     

Analysis of a 70 kb segment of DNA containing the human zeta and alpha-globin genes linked to their regulatory element (HS-40) in transgenic mice.

We have ligated two cosmids through an oligonucleotide linker to produce a single fragment spanning 70 kb of the human alpha-globin cluster, in which the alpha-like globin genes (zeta 2, alpha 2 and alpha 1), their regulatory element (HS-40) and erythroid-specific DNase I hypersensitive sites accurately retain their normal genomic organization. The zeta (embryonic) and alpha (embryonic, fetal and adult) globin genes were expressed in all 17 transgenic embryos. Similarly, all fetal and adult mice from seven transgenic lines that contained one or more copies of the fragment, produced up to 66% of the level of endogenous mouse alpha-globin mRNA. However, as for smaller constructs containing these elements, human alpha-globin expression was not copy number dependent and decreased by 1.5-9.0 fold during development. These findings suggest that either it is not possible to obtain full regulation of human alpha-globin expression in transgenic mice or, more likely, that additional alpha-globin regulatory elements lie beyond the 70 kb segment of DNA analysed.     

The nucleotide sequence of the adult chicken alpha-globin genes

The complete nucleotide sequence is reported of the two adult chicken alpha-globin genes, alpha A and alpha D. These two genes, expressed in a 3:1 ratio, respectively, in adult red cells, are widely divergent, suggesting that they have evolved separately for several hundred million years. Although the genes are closely linked in the chicken chromosome, the nucleotide sequences determined clearly rule out any recent gene conversion events. As expected, both genes contain two relatively short intervening sequences. The 3' intron of the alpha D gene begins with the dinucleotide GC rather than the typical GT. Extensive flanking sequences are reported for both genes. The chromosomal sequences of the two genes are compared to each other and to sequenced mammalian alpha-globin genes.     

Adult chicken alpha-globin gene expression in transfected QT6 quail cells: evidence for a negative regulatory element in the alpha D gene region.

The chicken adult alpha-globin genes, alpha A and alpha D, are closely linked in chromosomal DNA and are coordinately expressed in vivo in an approximate 3:1 ratio, respectively. When subcloned DNAs containing one or the other gene are stably transfected into QT6 quail fibroblasts, the alpha A-globin gene is expressed at measurable RNA levels, but the alpha D gene is not. The alpha A gene expression can be considerably increased by the presence of a linked Rous sarcoma virus long terminal repeat enhancer, but that of the alpha D gene remains undetectable. Transfection with subclones containing both genes, either in cis or in trans, leads to considerably reduced alpha A RNA levels and still no observable alpha D gene expression. Transfection with deleted subclones suggests that maximal expression levels in this system require the alpha A-globin gene promoter, as opposed to that of the alpha D gene, but that such expression is greatly reduced by one or more DNA sequences which lie approximately 2,000 base pairs upstream of the alpha A gene, within the body of the alpha D gene.     

Variation in the number of alpha-globin loci in sheep

Southern blot analysis was used to compare sheep and goat restriction- endonuclease maps of the DNA region containing the alpha-globin genes. The identical digestion patterns observed in both species with three endonucleases (BamHI, BstEII, and PstI) show that in sheep a single chromosome normally bears two nonallelic alpha-globin genes positioned at the same distance as in goat. Variant digestion patterns with enzymes that cleave outside (BamHI and HindIII) and within (EcoRI) the alpha-globin loci allowed us to infer that chromosomes with different numbers of alpha-globin loci are also present in sheep. In particular, in the 60 sheep considered, four individuals were heterozygous (alpha alpha/alpha alpha alpha) and one was homozygous (alpha alpha alpha/alpha alpha alpha) for chromosomes with three loci and one individual was heterozygous for a chromosome with four loci (alpha alpha/alpha alpha alpha alpha). This variation in the number of copies of alpha-globin loci can be explained by means of unequal crossovers.      

Demonstration of an enhancer activity in a fragment of DNA located at the 3' of the adult alpha globin gene in the duck

We found an enhancer element placed at the 3' side of the adult duck alpha A globin gene. The duck alpha globin gene cluster contains three genes from the 5' to 3' side: the pi embryonic gene, the alpha D minor adult gene and the alpha A adult major gene. We analyzed a 16 kb genomic domain extending from 2 kb upstream of the pi gene to 5 kb downstream of the alpha A gene. This enhancer is active in AEV transformed chicken erythroblasts. Its is inactive both in HeLa cells and in the human erythroid cells K562 which express only embryonic genes. These findings are discussed in relation to previous results concerning the duck beta globin enhancer located at the 3' side of the beta A globin gene.     

Alpha-globin gene markers identify genetic differences between Australian aborigines and Melanesians.

Australian aborigines exhibit a number of alpha-globin cluster rearrangements involving both alpha- and zeta-globin genes. alpha+-Thalassemia (-alpha/) in this population is heterogeneous and includes the 3.7 types I, II, and III gene deletions. The alpha alpha alpha/ and zeta zeta zeta/ rearrangements are each found in association with two haplotypes, indicating origins from at least two separate DNA crossover events. Differences in alpha-globin cluster rearrangements and in haplotypes between Australian aborigines, Papua New Guinea highlanders and island Melanesians, are consistent with multiple colonizing events into Australia.     

Conservation and variation in the large scale organisation of the globin gene domains of duck and chicken

Summary The genomic DNA of cloned recombinants containing the duck globin genes was compared to that of the analogous domains of the chicken. A 36 kb insert including the three alpha-type globin genes was isolated from a newly prepared duck genomic library in the cosmid PJB8; another recombinant contained a 45 kb insert with the four beta globin genes. In the alpha globin gene domain, the relative positions of genes, of repetitive sequences, and of the A+T-rich segments (AT-rich linkers, ATRLs) which frame the gene cluster (Moreau et al. 1982), were found to be closely maintained between duck and chicken. Although ATRLs and repetitive sequences also frame the gene cluster in the beta globin domains of duck and chicken, there is more genetic drift in their relative positions than in the alpha domain. It is of interest that several repetitive DNA segments were detected in the chicken beta globin domain which do not exist in corresponding positions in the duck. In view of the strict conservation in both species of genes and their relative positions in the cluster, this observation seems to exclude a simple function of repetitive sequences in the control of individual genes. The data are discussed with regard to the possible significance of repetitive and AT-rich DNA segments in genome organisation and function.     

Initiation codon mutation as a cause of alpha thalassemia

Cloning and sequence analysis of the alpha-globin genes from a Sardinian patient with the nondeletion type of hemoglobin-H disease revealed a new type of thalassemia lesion. A mutation in the alpha 2-globin gene changes the initiation codon ATG to ACG and abolishes the function of this gene. Globin mRNA output from the affected alpha 2 locus is decreased relative to the alpha 1 locus. The mutation is detectable in genomic DNA by restriction analysis with the enzyme NcoI. Of the seven Sardinian patients with nondeletion alpha thalassemia screened with this enzyme, six had the initiation codon lesion.     

Superhelicity induces hypersensitivity of a human polypyrimidine . polypurine DNA sequence in the human alpha 2-alpha 1 globin intergenic region to S1 nuclease digestion--high resolution mapping of the clustered cleavage sites.

Supercoiled recombinant DNAs containing the human adult alpha-globin gene region have been probed with nuclease S1 in vitro. While agarose gel electrophoresis showed only one predominant, double-stranded cleavage generated by S1 within 6 kb of human DNA and 4 kb of pBR322 sequence, a high resolution gel analysis reveals that the unique S1-hypersensitive locus in the human adult alpha-globin gene region actually contains more than 15 authentic S1 cleavage sites closely spaced together. The mapping approach used here locates the specific S1 cleavage sites on both DNA strands at the nucleotide sequence level. Interestingly, most of these sites are mapped within a 90 bp stretch of GC-rich (66%) polypyrimidine . polypurine DNA that is located 1060 to 1150 bp upstream from alpha 1-globin gene. These results provide the first high resolution map of double-stranded S1-cleavage sites induced within a specific DNA sequence under supercoil strain. The distribution and relative cutting frequencies of these sites mapped are consistent with a slippage mechanism in which the simple repeating sequences are organized into base-mismatched duplex on supercoiled DNA.