Intergenic DNA sequences flanking the pseudo alpha globin genes of human and chimpanzee.

We have determined the sequence of 2400 base pairs upstream from the human pseudo alpha globin (psi alpha) gene, and for comparison, 1100 base pairs of DNA within and upstream from the chimpanzee psi alpha gene. The region upstream from the promoter of the psi alpha gene shows no significant homology to the intergenic regions of the adult alpha 2 and alpha 1 globin genes. The chimpanzee gene has a coding defect in common with the human psi alpha gene, showing that the product of this gene, if any, was inactivated before the divergence of human and chimpanzee. However the chimpanzee gene contains a normal ATG initiation codon in contrast to the human gene which has GTG as the initiation codon. The psi alpha genes of both human and chimpanzee are flanked by the same Alu family member. The structure and position of this repeat have not been altered since the divergence of human and chimpanzee, and it is at least as well conserved as its immediate flanking sequence. Comparing human and chimpanzee, the 300 bp Alu repeat has accumulated only two base substitutions and one length mutation; the adjacent 300 bp flanking region has accumulated five base substitutions and twelve length mutations.     

Nucleotide sequence of the goat embryonic alpha globin gene (zeta) and linkage and evolutionary analysis of the complete alpha globin cluster

In previous studies we identified and sequenced clones containing two adult alpha globin genes of the goat. Additional studies have revealed the presence of an embryonic alpha globin gene termed zeta. Sequence analysis of the gene shows that it is the largest mammalian or avian globin gene cloned to date. Its unusual size is mainly due to a 14 base-pair tandem repeat sequence in its first intron. A similar sequence is also found in the first intron of the human zeta gene. The goat zeta coding sequence differs greatly from that of the adult alpha, particularly at amino acid position 38, where it codes for the amino acid replacement of Gln for Thr. This change may confer a higher intrinsic O2 affinity on the zeta globin protein, ensuring a sufficient O2 supply for the developing goat embryo. The cloning and sequencing of this gene completes the alpha globin locus of the goat, composed of three genes in the following order 5'-zeta-I alpha-II alpha-3'. Evolutionary comparisons of the goat alpha locus with other amphibian, avian and mammalian loci reveal several interesting features. Statistical analysis confirms the hypothesis that the embryonic alpha gene is much older (400 million years) than the embryonic beta gene (200 million years), and that it is descended from a primordial gene, whose present-day counterpart is the Xenopus larval alpha globin gene. Our results also suggest that after the divergence of the avian line, the alpha A gene converted the alpha D gene during the evolution of the pre-mammalian line. The alpha D globin gene remains unconverted in the avian line, potentially because of insertion/deletion sequences that may prevent any gene conversion event. The divergence rates of specific globin genes have been analyzed and found to form an essentially straight line, in agreement with the neutralist view of evolution.     

The organization of the tadpole and adult alpha globin genes of Xenopus laevis

Adult erythrocytes of X. laevis contain six electrophoretically resolvable globin polypeptides while tadpole erythrocytes contain four polypeptides, none of which comigrates with an adult protein. We show that three of the adult proteins are alpha globin polypeptides (alpha 1, alpha 2, alpha 3) and three are beta globin polypeptides (beta 1, beta 2, beta 3). We find that a tadpole alpha globin gene (alpha T1) is linked to the major adult locus in the sequence 5'-alpha T1-alpha 1-beta 1-3' with 5.2 kb separating alpha T1 from alpha 1. Another tadpole alpha globin gene (alpha T2) is linked to the minor adult locus in the sequence 5'-alpha T2-alpha 2-beta 2-3' with 10.7 kb separating alpha T2 from alpha 2. These linkage relationships are consistent with the major and minor loci having arisen by tetraploidization but the different separation of larval and adult globin genes at the two loci indicates the occurrence of some additional chromosomal rearrangement. Two alternative models are presented.     

Upregulation of alpha globin promotes apoptotic cell death in the hematopoietic cell line FL5.12

The function of alpha globin in the context of oxygen transport in erythroid cells is well described. Recently the expression of alpha globin was shown to be upregulated upon specific apoptotic stimuli like cytokine deprivation or cisplatin treatment in the hematopoietic pro-B cell line, FL5.12. In contrast to alpha globin, beta globin or globin-like genes were expressed at a very low level or were not expressed at all. Further, we found that alpha globin was not associated with heme. Apoptotic cells neither produced hemoglobin nor displayed a phenotype of cells differentiating down the erythroid lineage. Also other cell lines of variable differentiation status (NIH3T3, HeLa, K562) upregulated alpha globin during treatment with apoptosis-inducing agents. Under IL-3-deprived conditions GFP-alpha globin accelerated the progression of apoptosis comparable to GFP-Bax. GFP-alpha globin was expressed at a low level and enrichment of FL5.12 cells expressing GFP-alpha globin was difficult even in the presence of IL-3. Caspase-8, -9 and -3 as well as the proapoptotic factor Bax and cytochrome c were activated. Antisense alpha globin downregulated the expression of endogenous alpha globin und reduced caspase activity. Taken together these data indicate that alpha globin is a new and crucial factor in apoptosis especially supporting the mitochondrial pathway.     

Human globin psi B2 is not a globin-related sequence

We have determined the complete nucleotide sequence of 3.4 kilobase pairs of DNA covering the region of the human beta globin gene cluster where a human globin-related sequence psi beta 2 was thought to lie (Fritsch, Lawn, and Maniatis (1980) Cell 19, 959-972). Analysis of the resulting data reveals no evidence for any globin-related sequences in this region. The region does, however, contain several stretches of poly (dA-dT). We have confirmed the observations of Fritsch et al. that DNA from the psi beta 2 region hybridizes to the poly (dA-dT)-tailed human fetal globin cDNA plasmid, pJW151 (Wilson et al., (1978) Nucl. Acids Res. 5, 563-581) under conditions of low stringency, but we find that this hybridization is abolished by the addition of poly(rA). We conclude that psi beta 2 is not a globin pseudogene, and that the earlier investigators were probably misled by hybridization between the poly (dA-dT) stretches within the psi beta 2 region and the tails used in constructing the cDNA plasmid.     

Independent recombination events between the duplicated human alpha globin genes; implications for their concerted evolution.

We have examined the molecular structure of the human alpha globin gene complex from individuals with a common form of alpha thalassaemia in which one of the duplicated pair of alpha genes (alpha alpha) has been deleted (-alpha 3-7). Restriction mapping and DNA sequence analysis of the mutants indicate that different -alpha 3.7 chromosomes are the result of at least three independent events. In each case the genetic crossover has occurred within a region of complete homology between the alpha 1 and alpha 2 genes. Since the -alpha chromosomes may reflect the processes of crossover fixation and gene conversion between the two genes, their structures may provide some insight into the mechanism by which the concerted evolution of the human alpha globin genes occurs.     

Molecular cloning and characterization of two sets of alpha-theta genes in the rat alpha-like globin gene cluster

The highly heterogeneous rat hemoglobin system was investigated at the gene level. Two regions of the alpha-like globin gene cluster from a Wistar rat were isolated. Four lambda Dash recombinant clones carrying rat alpha-like globin genes were localized on two distinct gene regions. A region of approximately 16kb was found to contain the 5'-IIalpha1-psi theta 1-3' loci, and another of approximately 24kb the 5'-IIalpha2-psi theta2-psiI alpha3-3' loci. Both IIalpha1 and IIalpha2 are considered to be active, coding the IIalpha-globin chain. The nt sequences of IIalpha1 and IIalpha2 are identical except for six nt in the non-coding region. The psiI alpha3 locus is a truncated pseudogene. The putative promoter region of an alpha-like globin gene is joined directly to the third exon, homologous to that of Ialpha-globin cDNA. psi theta1 and psi theta2 are also pseudogenes, as evidenced by several deletions located in the protein-coding regions of these loci. The psi theta1 and psi theta2 loci exhibit extensive homology, but the restriction maps of these genes and their flanking regions differ considerably. Genomic Southern blot analyses of the total liver DNA from six rats showed the existence of three theta-globin-related genes, including psi theta1 and psi theta2. These results indicate that the two gene regions investigated are not allelic variants, but may be generated by block duplication. This is the first report of the existence of rodent theta-globin genes.     

Genetic organization of the polymorphic equine alpha globin locus and sequence of the BII alpha 1 gene.

The equine alpha globin gene complex comprises two functional alpha genes and an alpha-like pseudogene arranged in the order 5'-alpha 2-(5kb)-alpha 1-(3kb)-psi alpha-3'. A single (embryonic) zeta-like sequence lies within a 12 kb region 5' to the alpha 2 gene. We have determined the sequence of the alpha 1 gene of the BII haplotype, one of two most common haplotypes (the other being BI) which encode alpha globins with either Tyr (BI) or Phe (BII) at codon 24 in both linked alpha genes. In BI and BII the non-allelic alpha 2 and alpha 1 genes respectively code for Gln or Lys at codon 60, thus accounting for the 4 alpha globin types seen in BI/BII heterozygotes. Genomic restriction enzyme maps of the BII alpha complex (24Phe/60Lys,Gln) and the allelic BI (24Tyr/60Lys,Gln) are identical to each other, and to those of a rarer normal haplotype, A, which encodes only alpha 24Tyr/60Gln globin, and a low expression mutant of BII which encodes only 24Phe/60Lys globin. These two latter haplotypes must therefore have a linked pair of alpha genes, as in BI and BII, but with identical coding properties, and it is suggested that this has arisen by gene conversion.     

Analysis of the human alpha globin upstream regulatory element (HS-40) in transgenic mice.

We have analysed the effect of a 1.4 kb segment of DNA containing the upstream alpha globin regulatory element (HS-40) on human alpha globin gene expression in fetal mice and lines of transgenic mice. High levels of tissue-specific, human alpha mRNA expression were seen in all transgenic animals and in this sense expression was position independent. However, the level of human alpha mRNA expression per integrated gene copy decreased during development and was inversely related to copy number. The limitation in expression with increasing gene copy number was shown to be in cis since homozygotes for the transgene produced twice as much human alpha mRNA as hemizygotes. In many respects HS -40 appears similar to single elements within the previously described beta globin locus control region and in cross breeding experiments we have shown that HS -40 behaves in a similar manner to such elements in transgenic mice.     

Highly variable regions of DNA flank the human alpha globin genes.

A series of restriction fragment length polymorphisms which are due to DNA rearrangements have been identified within two highly variable regions flanking the human alpha globin genes. The existence of such highly polymorphic areas provides a large number of individual genetic markers for the alpha globin gene cluster on chromosome 16. If, as seems likely, such regions occur frequently throughout the human genome they should be of considerable value in the antenatal diagnosis of genetic disease.     

Cloning of two adult hamster globin cDNAs (alpha and beta major).

A cDNA library was prepared from poly(A) mRNA extracted from adult anemic hamster spleen erythroid cells. cDNA clones containing inserts coding for adult alpha and beta major globin chains were isolated. Their identity was confirmed by (a) translation of hybrid selected mRNA and (b) nucleotide sequence analysis of the inserts and comparison to the adult globin cDNAs of mouse, rabbit and human. Availability of sequences for embryonic (Li et al. (1992) Biochim. Biophys. Acta 1130, 218-220) and adult globin cDNAs (this report) will aid in investigations of the molecular mechanisms involved in the globin ontogeny of hamsters.     

Developmental regulation of the human zeta globin gene in transgenic mice.

We have characterized the expression of the human zeta (zeta) gene, which encodes an embryonic alpha-like globin, in transgenic mice. We find that a 777 base pair fragment spanning erythroid specific hypersensitive site II (HSII) from the distal 5. region of the human beta globin gene cluster potentiates expression of the zeta globin gene. In the absence of the HSII fragment, no zeta expression is observed. Expression of the human zeta gene in mice parallels expression of a murine embryonic alpha-like globin gene (x). Thus, expression of the human zeta gene in mice requires linkage to an erythroid-specific enhancer sequence, but the presence of the enhancer does not affect the developmental regulation of the transgene. Our results indicate that the factors involved in switching from embryonic to adult alpha globin gene expression during development are evolutionarily conserved, and suggest that the transgenic mouse is an in vivo system in which the requirements for the developmental switch in alpha globin gene expression can be analyzed in detail.     

Structure and evolution of the horse zeta globin locus

The equine zeta globin gene locus consists of an intact 5' gene and a truncated 3' pseudogene (psi zeta) that has only 5' control sequences and a first exon and intron. Nevertheless, the psi zeta gene has retained almost perfect homology with its neighbour, presumably by gene conversion. The first introns of both zeta and psi zeta genes contain a number of degenerate tandem repeats of a 14 base-pair sequence that has been found in the zeta genes of goats and humans and that is related to a family of human minisatellite sequences. Comparisons of sequences flanking the zeta and psi zeta genes reveal areas of considerable interspecies homology, which can be explained by a zeta gene duplication that pre-dated the mammalian radiation.