Molecular Cloning of the Human α-Globin Gene Family

During human development a series of α-like and β-like subunits of hemoglobin are produced. Five β-like polypeptides, embryonic (Ɛ), fetal (Gγ, Aγ) and adult (δ, β), and two α-like polypeptides, embryonic (z;) and adult (α), have been identified. A structural analysis of the gene family encoding the human α-like globins is described. An improved gene isolation procedure was developed. This procedure makes it possible to isolate genes and their flanking sequences, and thus to directly determine the linkage relationship between genes, without requiring partial purification of genes. Large random genomic DNA fragments resulting from physical shear of DNA were joined to phage lambda vectors using synthetic DNA linkers. The resulting recombinant DNA molecules were packaged in vitro into viable phage to yield a collection of cloned overlapping DNA fragments which were then amplified to produce a permanent library of genomic DNA sequences. This library can be screened repeatedly using nucleic acid probes and an in situ plaque hybridization procedure in order to isolate many different genes. Clones containing the duplicated human α-globin genes (a1 and a2) were isolated from a library of human DNA. Also present on these clones are an α-like pseudogene (ψα1) and an embryonic α-like gene (ς1) which were identified by blot hybridization experiments and DNA sequence analysis. Genomic blotting using the ς1 coding sequence as probe identified a second embryonic gene (ς2). The ς2 gene was isolated by cloning a DNA fragment which overlaps the clones containing the other α-like genes. All five genes are transcribed from the same DNA strand and are arranged in the order 5'-ς2-ς1ψα1-α2-α1-3'. Comparison of α1 and α2 by restriction mapping and heteroduplex analysis of DNA fragments containing al or α2 plus 5' flanking sequences demonstrated that each gene is located within an approximately 4 kb region of homology interrupted by two short regions of nonhomology. The association of these large blocks of homology with genes which are thought to have duplicated long ago suggests the existence of a mechanism for sequence matching. Two types of deletions invariably occur during propagation of clones containing α1 and α2. The breakpoints of these two types of deletions are located within the two blocks of al-a2 homology. The positions and the precise lengths of these deletions indicate that deletion occurs by homologous but unequal crossing-over between corresponding regions of al and a2. The lengths and positions of these deletions are indistinguishable from those of the two types of deletions which are associated with α-thalassemia 2, suggesting that this common genetic disease results from homologous but unequal crossing-over between regions within and/or surrounding the adult α-globin genes.