Assignment of orthologous relationships among mammalian alpha-globin genes by examining flanking regions reveals a rapid rate of evolution

In order to study the relationships among mammalian alpha-globin genes, we have determined the sequence of the 3' flanking region of the human alpha 1 globin gene and have made pairwise comparisons between sequenced alpha-globin genes. The flanking regions were examined in detail because sequence matches in these regions could be interpreted with the least complication from the gene duplications and conversions that have occurred frequently in mammalian alpha-like globin gene clusters. We found good matches between the flanking regions of human alpha 1 and rabbit alpha 1, human psi alpha 1 and goat I alpha, human alpha 2 and goat II alpha, and horse alpha 1 and goat II alpha. These matches were used to align the alpha-globin genes in gene clusters from different mammals. This alignment shows that genes at equivalent positions in the gene clusters of different mammals can be functional or nonfunctional, depending on whether they corrected against a functional alpha-globin gene in recent evolutionary history. The number of alpha-globin genes (including pseudogenes) appears to differ among species, although highly divergent pseudogenes may not have been detected in all species examined. Although matching sequences could be found in interspecies comparisons of the flanking regions of alpha- globin genes, these matches are not as extensive as those found in the flanking regions of mammalian beta-like globin genes. This observation suggests that the noncoding sequences in the mammalian alpha-globin gene clusters are evolving at a faster rate than those in the beta-like globin gene clusters. The proposed faster rate of evolution fits with the poor conservation of the genetic linkage map around alpha-globin gene clusters when compared to that of the beta-like globin gene clusters. Analysis of the 3' flanking regions of alpha-globin genes has revealed a conserved sequence approximately 100-150 bp 3' to the polyadenylation site; this sequence may be involved in the expression or regulation of alpha-globin genes.      

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.     

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.     

Manipulating the mouse genome to engineer precise functional syntenic replacements with human sequence

We have devised a strategy (called recombinase-mediated genomic replacement, RMGR) to allow the replacement of large segments (>100 kb) of the mouse genome with the equivalent human syntenic region. The technique involves modifying a mouse ES cell chromosome and a human BAC by inserting heterotypic lox sites to flank the proposed exchange interval and then using Cre recombinase to achieve segmental exchange. We have demonstrated the feasibility of this approach by replacing the mouse alpha globin regulatory domain with the human syntenic region and generating homozygous mice that produce only human alpha globin chains. Furthermore, modified ES cells can be used iteratively for functional studies, and here, as an example, we have used RMGR to produce an accurate mouse model of human alpha thalassemia. RMGR has general applicability and will overcome limitations inherent in current transgenic technology when studying the expression of human genes and modeling human genetic diseases.     

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.     

Genetic modifiers in hemoglobinopathies

Hereditary anemias show considerable variation in their clinical presentation. In some cases, the causes of these variations are easily apparent. In thalassemia (or in HbE/thalassemia), genetic variation is primarily caused by the severity of the thalassemia mutation. However, not uncommonly, there is variation unexplained by the globin gene mutations themselves, which may be caused by genetic modifiers. In sickle cell disease, the primary mutation is the same in all patients. Therefore, variations in disease severity generally are due to genetic modifiers. In most genetic diseases involving beta globin, the most clearcut influence on phenotype results from elevated fetal hemoglobin levels. In addition, alpha globin gene number can influence disease phenotype. In thalassemia major or intermedia, reduction in the number of alpha globin genes can ameliorate the disease phenotype; conversely, excess alpha globin genes can convert beta thalassemia trait to a clinical picture of thalassemia intermedia. In sickle cell disease, the number of alpha globin genes has both ameliorating and exacerbating effects, depending on which disease manifestation is being examined. Unlinked genetic factors have substantial effects on the phenotype of hereditary anemias, both on the anemia and other disease manifestations. Recently, studies using genome-wide techniques, particularly studying QTLs causing elevated HbF, or affecting HbE/thalassemia, have revealed other genetic elements whose mechanisms are under study. The elucidation of genetic modifiers will hopefully lead to more rational and effective management of these diseases.     

The primary structure of the hemoglobin of the Cormorant (Phalacrocorax carbo, Pelecaniformes)

The erythrocytes of the adult Cormorant contain two hemoglobin components in a ratio of 83% Hb A to 17% Hb D. The primary structures of the alpha A-, alpha D- and beta-chains are presented. The globin chains were separated by high-performance liquid chromatography and cleaved enzymatically and/or chemically. The native chains and their fragments were sequenced using liquid- or gas-phase sequencers, and the peptides aligned using the homology to human and to avian hemoglobin sequences. Compared to human hemoglobin, there are 46 amino-acid replacements in the alpha A-chains (67.4% homology), 65 replacements in the alpha D-chains (53.9% homology) and 45 replacements in the beta-chains (69.2% homology). In the functionally important regions, the percentage of amino-acid substitutions, as compared to human hemoglobin, is 13.2% in the alpha A-, 19.0% in the alpha D - and 16.0% in the beta-chains. The importance of the replacement beta 135 arginine (other birds)----glycine (Cormorant) in the phosphate-binding pocket and its effect on phosphate binding will be discussed.     

Aminoacyl-tRNA(anticodon): codon adaptation in human and rabbit reticulocytes

A highly significant correlation was observed between the relative abundances of isoacceptor aminoacyl-tRNAs in human and rabbit reticulocytes and the frequency of their cognate codons in alpha and beta globin mRNAs from the respective tissue. The correlation coefficient (r value) for human reticulocytes was 0.78 and for rabbit reticulocytes was 0.88. These results provide strong evidence that the amounts of most isoacceptors in human and rabbit reticulocytes are adapted to requirements for translating their cognate codons in globin mRNA, i.e., there is an aa-tRNA(anticodon): codon adaptation in these tissues.     

3' non-translated region of human alpha-globin messenger RNA does not affect translation efficiency

The relative translation efficiency of three synthetic alpha-globin mRNAs differing by their 3' non-translated end was measured in vitro in a rabbit reticulocyte lysate. Results showed that substituting the 3' non-translated end of human alpha 2 globin mRNA by the 3' non-translated end of chimpanzee alpha 1 or alpha 2 mRNAs has no effect on translation efficiency. In contrast, the introduction of the alpha-Quong-Sze mutation (alpha 125, Leu----Pro) in human alpha 2 mRNA led to a 50% apparent reduction in globin synthesis due to the instability of the alpha-Quong-Sze globin chain. We conclude that human alpha 1 and alpha 2 globin mRNAs have the same translation efficiency, and that the reduction, previously reported, in the kinetics of alpha-globin synthesis by alpha 2 mRNA carrying the alpha-Quong-Sze mutation is due to the instability of the alpha-Quong-Sze globin chain only.     

Gene mapping by fluorescent in situ hybridization

We describe a new method for the mapping of mammalian genes, utilizing in situ hybridization of mRNA to DNA of chromosomes. It involves the hydrogen bonding of the polyadenylic acid at the 3' end of hybridized mRNA to the polyuridylic acid tail of a highly fluorescent latex microsphere. The resultant double hybrid can be visualized by fluorescence microscopy. The chromosomal localization of human alpha + beta globin genes has been explored by this method. Our data point ot the long arms of chromosomes 4 and 5 as the loci for the human globin genes.     

Sequence and comparative analysis of the rabbit alpha-like globin gene cluster reveals a rapid mode of evolution in a G + C-rich region of mammalian genomes

A sequence of 10,621 base-pairs from the alpha-like globin gene cluster of rabbit has been determined. It includes the sequence of gene zeta 1 (a pseudogene for the rabbit embryonic zeta-globin), the functional rabbit alpha-globin gene, and the theta 1 pseudogene, along with the sequences of eight C repeats (short interspersed repeats in rabbit) and a J sequence implicated in recombination. The region is quite G + C-rich (62%) and contains two CpG islands. As expected for a very G + C-rich region, it has an abundance of open reading frames, but few of the long open reading frames are associated with the coding regions of genes. Alignments between the sequences of the rabbit and human alpha-like globin gene clusters reveal matches primarily in the immediate vicinity of genes and CpG islands, while the intergenic regions of these gene clusters have many fewer matches than are seen between the beta-like globin gene clusters of these two species. Furthermore, the non-coding sequences in this portion of the rabbit alpha-like globin gene cluster are shorter than in human, indicating a strong tendency either for sequence contraction in the rabbit gene cluster or for expansion in the human gene cluster. Thus, the intergenic regions of the alpha-like globin gene clusters have evolved in a relatively fast mode since the mammalian radiation, but not exclusively by nucleotide substitution. Despite this rapid mode of evolution, some strong matches are found 5' to the start sites of the human and rabbit alpha genes, perhaps indicating conservation of a regulatory element. The rabbit J sequence is over 1000 base-pairs long; it contains a C repeat at its 5' end and an internal region of homology to the 3'-untranslated region of the alpha-globin gene. Part of the rabbit J sequence matches with sequences within the X homology block in human. Both of these regions have been implicated as hot-spots for recombination, hence the matching sequences are good candidates for such a function. All the interspersed repeats within both gene clusters are retroposon SINEs that appear to have inserted independently in the rabbit and human lineages.     

Contrasting effects of alpha and beta globin regulatory elements on chromatin structure may be related to their different chromosomal environments.

Expression of the human alpha and beta globin gene clusters is regulated by remote sequences, referred to as HS -40 and the beta-locus control region (beta-LCR) that lie 5-40 kb upstream of the genes they activate. Because of their common ancestry, similar organization and coordinate expression it has often been assumed that regulation of the globin gene clusters by HS -40 and the beta-LCR occurs via similar mechanisms. Using interspecific hybrids containing chromosomes with naturally occurring deletions of HS -40 we have shown that, in contrast to the beta-LCR, this element exerts no discernible effect on long-range chromatin structure and in addition does not influence formation of DNase I hypersensitive sites at the alpha globin promoters. These differences in the behaviour of HS -40 and the beta-LCR may reflect their contrasting influence on gene expression in transgenic mice and may result from the differing requirements of these elements in their radically different, natural chromosomal environments; the alpha cluster lying within a region of constitutively 'open' chromatin and the beta cluster in a segment of chromatin which opens in a tissue-specific manner. Differences in the hierarchical control of the alpha and beta globin clusters may exemplify more general differences in the regulation of eukaryotic genes which lie in similar open or closed chromosomal regions.