The eta-globin gene. Its long evolutionary history in the beta-globin gene family of mammals

In phylogenetic reconstructions by the parsimony method, utilizing 62 sequenced globin genes and pseudogenes (including 34 of the beta-globin gene family from eutherian orders Primates, Lagomorpha, Artiodactyla and Rodentia), the branch of primate psi beta pseudogenes and the goat embryonically expressed epsilon II gene group monophyletically together as orthologues of a common ancestral gene (labelled eta) distinct from orthologues of epsilon, gamma, delta and beta. This primate psi eta-goat eta branch is cladistically closer to epsilon and gamma than to delta and beta branches. In each eutherian order gene conversions replaced portions of delta by beta sequences, whereas in descent of Primates epsilon, gamma and eta mostly retained their separate ancient identities predating the radiation of Eutheria in all their exons and non-coding regions. The loci of the ancestral beta-globin gene cluster in basal eutherians and proto-primates, as deduced from beta-clusters representing the four eutherian orders, were linked 5'-epsilon-gamma-eta-delta-beta-3' with epsilon, gamma and eta being embryonically expressed genes, and delta and beta ontogenetically later expressed genes. Through deletions gamma was lost in artiodactyl evolution, eta in lagomorph and rodent evolution, and all DNA between exon 2 3' boundaries of eta and delta in prosimian lemuriform evolution (lemur having the hybrid pseudogene psi eta delta). Simian primates retained intact the five loci of the ancestral cluster. Not only did eta, after it became a pseudogene in the basal primates, persist intact in descent to present-day simians but in the line to hominoids it evolved during the last 40 million years at the decelerated rate of 1 X 10(-9) substitutions/site per year which is one-fifth the expected neutral rate. The possibility is suggested that the psi eta locus situated between fetal and adult chromosomal domains of the simian beta-globin gene cluster might play some role in a mechanism for ontogenetic switches of globin gene expression. However, not enough sequence data on genes and intergenic regions in DNA of species of primates and other mammals as yet exist to know if the slow rate of 1 X 10(-9) reflects the rate of a conserved functional gene or primarily reflects a decelerated neutral rate of hominoid DNA evolution, conceivably from enhanced DNA repair and longer generation times in hominoids. The further possibility is raised that gene correction (repair of damaged DNA that prevents emergence of new alleles) and gene conversion both more often involve strand copying of conserved than of rapidly evolving DNA.     

Nucleotide sequence analysis of the lemur beta-globin gene family: evidence for major rate fluctuations in globin polypeptide evolution

Lemur beta-related globin genes have been isolated and sequenced. Orthology of prosimian and human epsilon-, gamma-, and beta-related globin genes was established by dot-matrix analysis. All of these lemur globin genes potentially encode functional beta-related globin polypeptides, though precisely when the gamma-globin gene is expressed remains unknown. The organization of the 18-kb brown lemur beta-globin gene cluster (5' epsilon-gamma-[psi eta-delta]-beta 3') is consistent with its evolution by contraction via unequal crossing-over from the putative ancestral mammalian beta-globin gene cluster (5' epsilon-gamma- eta-delta-beta 3'). The dwarf lemur nonadult globin genes are arranged as in the brown lemur. Similar levels of synonymous (silent) nucleotide substitutions and noncoding DNA sequence differences have accumulated between species in all of these genes, suggesting a uniform rate of noncoding DNA divergence throughout primate beta-globin gene clusters. These differences are comparable with those observed in the nonfunctional psi eta pseudogene and have therefore accumulated at the presumably maximal neutral rate. In contrast, nonsynonymous (replacement) nucleotide substitutions show a significant heterogeneity in distribution for both the same gene in different lineages and different genes in the same lineage. These major fluctuations in replacement but not silent substitution rates cannot be attributed to changes in mutation rate, suggesting that changes in the rate of globin polypeptide evolution in primates is not governed solely by variable mutation rates.      

Rabbit globin pseudogene psi beta 2 is a hybrid of delta- and beta-globin gene sequences

The evolutionary history of the rabbit globin pseudogene psi beta 2 was studied by completing its nucleotide sequence and aligning the sequence with that of the rabbit adult globin gene beta 1 and the human minor adult globin gene delta. The 5' flanking region and exon 1 of psi beta 2 were most similar to rabbit beta 1, but the large intervening sequence and the 3' untranslated region were most similar to human delta. Intron 1 and exon 2 were equally similar to both delta and beta 1. This pattern indicates that psi beta 2 was originally a delta-like gene that acquired the 5' portion of gene beta 1 by intrachromosomal gene conversion. The presence of a delta-globin gene sequence in both rabbits and humans shows that it is an ancient gene, predating the mammalian radiation that occurred over 85 Myr ago. Delta has shown a pronounced tendency to be altered in its 5' end during the course of mammalian evolution. Quantitative divergence analysis shows that the ancestor to rabbit psi beta 2 was active until 20-30 Myr ago, during which time the lagomorph beta-globin gene family apparently functioned without a pseudogene.      

Expression of chimeric human beta- and delta-globin genes during erythroid differentiation

To determine whether sequences contained within the small intervening sequence (IVS 1) or large intervening sequence (IVS 2) are involved in the regulated expression of the human beta-globin gene, chimeric genes containing portions of the human beta- and delta-globin genes were stably transfected into mouse erythroleukemia (MEL) cells. Since MEL cells can be induced to differentiate in culture, the expression of the chimeric genes was compared to the expression of beta and delta both before and after the induction of erythroid differentiation. The expression of beta delta 1, a beta-globin gene containing delta IVS 1 in place of beta IVS 1, was comparable to the expression of a beta-globin gene both before and after erythroid differentiation. However, the base-line expression of human beta-globin genes containing delta IVS 2 in place of beta IVS 2 was dramatically decreased. Furthermore, the substitution of delta IVS 2 for beta IVS 2 prevented the regulated increase in expression of the beta-globin gene upon induction. The results also indicate that sequences present in beta IVS 2 are not sufficient for this induced increase in expression since the substitution of beta IVS 2 for delta IVS 2 in a delta gene does not increase the regulated expression of delta during differentiation. These experiments suggest that either the presence of delta IVS 2 in a beta gene interrupts sequences required for the induced expression of beta-globin or that sequences in beta IVS 2 act in concert with other beta globin sequences not present in the delta-globin gene to permit optimal expression.     

Isolation and sequence analysis of a hybrid delta-globin pseudogene from the brown lemur

The β-globin gene cluster of the brown lemur, a prosimian, is very short and contains a single ?-, γ- and β-globin gene, with an additional β-related gene sequence between the γ- and β-globin genes. Brown lemur DNA was cloned into the bacteriophage vector λL47.1 and a recombinant was isolated which contained an 11 × 10³ base insert including the β-globin gene and the additional putative β-globin pseudogene. The nucleotide sequence of this β-related gene was completely determined. A complete gene sequence was found, containing four frameshift mutations sufficient to establish its pseudogene status. The gene was interrupted by two intervening sequences with sizes and locations typical of mammalian β-related globin genes. The pseudogene sequence was compared in detail with human ?-, γ-, δ- and β-globin genes. The beginning of the pseudogene, from the 5′ flanking region to the second exon, was homologous to the corresponding regions of the human ?- and γ-globin genes. In contrast, the second intron, third exon and 3′ flanking region showed a remarkably close homology to the δ-globin, but not β-globin, gene of man. This suggests that the δ-globin gene is not the product of a recent gene duplication, but instead is present in most or all primates. This gene has been silenced on at least two separate occasions in primate evolution (in lemurs and in old world monkeys). In addition, the 5′ end of the lemur ψδ gene appears to have exchanged sequences with an ?- or γ-globin gene, and an analogous exchange with the β-globin gene seems to have occurred recently in the human δ-globin gene. The evolution and function of the δ-globin gene are discussed.     

Structure of the goat psi beta y beta-globin pseudogene. Analysis of goat pseudogene evolutionary patterns

The 12-member beta-globin gene locus of the goat contains three beta(adult)-type pseudogenes, one in each of three four-gene subsets of the locus. We have determined the complete nucleotide sequence of psi beta y, the pseudogene present in the most downstream four-gene subset, which also contains the functional fetal gene, beta F. psi beta y contains, throughout its length, numerous incapacitating mutations in common with the previously sequenced goat psi beta x and psi beta z pseudogenes consistent with the model that all were descended from a common pseudogene ancestor which became defective prior to the expansion of the beta-globin locus in the goat lineage. Evolutionary analysis of the psi beta y sequence in comparison to psi beta x and psi beta z provides evidence that nucleotide substitutions were fixed in a random manner within these pseudogenes with respect to polarity, coding versus non-coding regions, and replacement sites versus silent sites. However, substitutions appear to have accumulated asymmetrically between different pseudogenes in a manner that provides evidence for partial gene conversion. Moreover, the presence of deletions in goat psi beta y, which are also observed in the cow pseudogene psi 2, but not in the cow psi 1 pseudogene, indicate that goat psi beta y and cow psi 2 are orthologous but cow psi 1 actually arose prior to the goat/cow divergence. The authentic goat orthologue to cow psi 1 temporarily existed in the goat lineage but was deleted, probably prior to the divergence of goats and sheep.     

Isolation and nucleotide sequence analysis of the beta-type globin pseudogene from human, gorilla and chimpanzee

The beta-globin gene cluster of human, gorilla and chimpanzee contain the same number and organization of beta-type globin genes: 5'-epsilon (embryonic)-G gamma and A gamma (fetal)-psi beta (inactive)-delta and beta (adult)-3'. We have isolated the psi beta-globin gene regions from the three species and determined their nucleotide sequences. These three pseudogenes each share the same substitutions in the initiator codon (ATG----GTA), a substitution in codon 15 which generates a termination signal TGG----TGA, nucleotide deletion in codon 20 and the resulting frame shift which yields many termination signals in exons 2 and 3. The basic structure of these psi beta-globin genes, however, remains consistent with that found for functional beta-globin genes: their coding regions are split by two introns, IVS 1 (which splits codon 30, 121 base-pairs in length) and IVS 2 (which splits codon 104, 840 to 844 base-pairs in length). These introns retain the normal splice junctions found in other eukaryotic split genes. The three hominoid psi beta-globin genes show a high degree of sequence correspondence, with the number of differences found among them being only about one-third of that predicted for DNA sites evolving at the neutral rate (i.e. for sites evolving in the absence of purifying selection). Thus, there appears to be a deceleration in the rate of evolution of the psi beta-globin locus in higher primates.     

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.     

Analysis of the beta-delta-globin gene loci in normal and Hb Lepore DNA: direct determination of gene linkage and intergene distance

Total human DNA was cleaved with a variety of restriction enzymes, and the fragments were fractionated by gel electrophoresis and transferred to nitrocellulose filter strips. The restricted DNA was then hybridized to nick-translated radioactive recombinant plasmid DNA containing sequences derived from human beta-globin messenger RNA. Under suitable conditions, this probe hybridizes with both the beta--and delta-globin genes. Using this probe, a restriction map of the human beta--and delta-globin genes and the surrounding genomic DNA regions has been constructed. The beta-globin gene contains a nonglobin DNA insert approximately 899-1000 base pairs in length, present within the sequence coding for amino acids 101-120 of the 146 amino acid long globin polypeptide. A similar sequence may be present within the same sequence of the delta-globin gene. The distance between the beta--and delta-globin genes is approximately 7000 nucleotide pairs, and the delta-globin gene is to the 5' side of the beta-globin gene, as predicted by genetic evidence. Both genes are transcribed from the same DNA strand. The structure of the Hb Lepore gene is shown to be a fused delta--and beta-globin gene, and to be completely consistent with the derived map of normal beta--and delta-globin genes. [Restriction enzyme nomenclature follows that of Smith and Nathans (1973) and Roberts (1976). A genomic DNA restriction fragment containing part or all of one globin gene will be designated by that globin chain--for instance, the Pst I fragment containing the beta-globin gene sequence will be designated Pst I beta. A similar convention will be used for double digests. Throughout this paper, when reference is made to the 5' or 3' side or fragment of a gene, this refers to the 5' or 3' side of the mRNA coded by that sequence. Thus the 5' side (N terminal) of the beta-globulin gene is the sequence to the 5' side of the anti-sense strand.].     

Differences in human alpha-, beta- and delta-globin gene expression in monkey kidney cells

We have compared the function of the human alpha-, beta- and delta-globin genes using various plasmid expression vectors derived from pBR322. Amplification of recombinants occurred after their introduction, by calcium-phosphate-mediated DNA transfer, into monkey kidney cells that constitutively produce T antigen (COS cells). The human alpha-globin gene promoter functioned independently, but the beta-globin gene promoter was nearly totally dependent on the enhancing activity of the 72 bp direct repeats from the SV40 genome. Furthermore, when the human alpha- and beta-globin genes were linked in the same vector, the alpha promoter was active but the beta promoter was not. Function of the delta-globin gene promoter also depended on the enhancer element. In vectors containing the 72 bp repeats and the beta- or delta-globin gene, the activity of the beta-globin gene was approximately 50 times greater than that of the delta-globin gene, approximating the ratio of beta and delta mRNA observed in normal human bone marrow cells.     

Structure and organization of the bovine beta-globin genes

Genomic clones spanning the entire cow beta-globin gene locus have been isolated and characterized. These clones demonstrate that the linkage of embryonic-like (epsilon) genes and pseudogenes (psi) to the previously described fetal (gamma) and adult (beta) genes is as follows: 5'-epsilon 3-epsilon 4-psi 3-beta-epsilon 1-epsilon 2-psi 1- psi 2-gamma-3'. Present data indicate that, like that of the goat, the fetal and adult genes arose via block duplication of an ancestral four- gene set: epsilon-epsilon-psi-beta. This duplication event preceded the divergence of cows and goats, which occurred greater than or equal to 18-20 Myr ago. However, cows do not have the additional four-gene block containing a preadult/stress globin gene (beta C). Furthermore, the cow fetal cluster contains an extra beta-like pseudogene, which apparently arose by a small-scale duplication. The fixation of this duplication may indicate a possible evolutionary role for pseudogenes.      

New genes originated via multiple recombinational pathways in the beta-globin gene family of rodents

Species differences in the size or membership composition of multigene families can be attributed to lineage-specific additions of new genes via duplication, losses of genes via deletion or inactivation, and the creation of chimeric genes via domain shuffling or gene fusion. In principle, it should be possible to infer the recombinational pathways responsible for each of these different types of genomic change by conducting detailed comparative analyses of genomic sequence data. Here, we report an attempt to unravel the complex evolutionary history of the beta-globin gene family in a taxonomically diverse set of rodent species. The main objectives were: 1) to characterize the genomic structure of the beta-globin gene cluster of rodents; 2) to assign orthologous and paralogous relationships among duplicate copies of beta-like globin genes; and 3) to infer the specific recombinational pathways responsible for gene duplications, gene deletions, and the creation of chimeric fusion genes. Results of our comparative genomic analyses revealed that variation in gene family size among rodent species is mainly attributable to the differential gain and loss of later expressed beta-globin genes via unequal crossing-over. However, two distinct recombinational mechanisms were implicated in the creation of chimeric fusion genes. In muroid rodents, a chimeric gamma/epsilon fusion gene was created by unequal crossing-over between the embryonic epsilon- and gamma-globin genes. Interestingly, this gamma/epsilon fusion gene was generated in the same fashion as the "anti-Lepore" 5'-delta-(beta/delta)-beta-3' duplication mutant in humans (the reciprocal exchange product of the pathological hemoglobin Lepore deletion mutant). By contrast, in the house mouse, Mus musculus, a chimeric beta/delta fusion pseudogene was created by a beta-globin --> delta-globin gene conversion event. Although the gamma/epsilon and beta/delta fusion genes share a similar chimeric gene structure, they originated via completely different recombinational pathways.     

Physical mapping of the globin gene deletion in (delta beta (0)) -thalassaemia.

We have constructed a physical map of restriction endonuclease cleavage sites in the (delta (+) beta)-globin gene region in the DNA of patients with (delta beta(0))-thalassaemia. This map shows that a 10 kb deletion has occured in (delta beta (0))-thalassaemia to remove the entire beta-globin gene and the 3' portion of the delta-globin gene. The 5' terminus of the deletion is in the large intron of the delta-globin gene and the 3' terminus 1.8 kb to the 3'-side of the beta-globin gene. A similar deletion of about 7 kb has been described previously in the DNA of patients with Hb Lepore; the 5' terminus of the deletion is also in the delta-globin gene but the 3' terminus is in the beta-globin gene. Comparison of the foetal (gamma) globin gene expression in adults with (delta beta(0))-thalassaemia and Hba Lepore suggests that the 3' extragenic regions of the beta-globin gene contain DNA sequences involved in the regulation of gamma-globulin gene expression.     

The structure and evolution of the spider monkey delta-globin gene

We have isolated the delta-globin gene of the New-World spider monkey, Ateles geoffroyi, and compared its nucleotide sequence with those of other primate delta- and beta-globin genes. Among primate delta-globin genes, the rate of nonsynonymous substitutions is much less than the rate of synonymous substitutions. This suggests that primate delta- globin genes may remain under evolutionary conservation, perhaps because hemoglobin A2 has an as yet unknown physiological importance.      

Haplotype analysis of the human beta-globin gene complex using multiple locus specific oligonucleotide probes

Three oligonucleotide probes complementary to specific DNA sequences of the six human globin genes (epsilon, G gamma, A gamma, psi beta, delta, beta) were synthesized. The oligonucleotides were used either singly or in combination as hybridization probes to determine the haplotype of the human beta-globin gene cluster employing the four conventionally used restriction endonucleases HincII, HindIII, AvaII, and BamHI, in addition to HpaI. Polymorphism in the epsilon- and psi beta-genes (HincII) can be simultaneously determined with a single probe mixture. One of the probes complementary to both the psi beta- and gamma-genes is useful for determining both HindIII and HincII polymorphisms. The advantages of these probes relative to conventional cDNA probes are discussed.     

BP1, a homeodomain-containing isoform of DLX4, represses the beta-globin gene

In earlier studies we identified a putative repressor of the human beta-globin gene, termed beta protein 1 (BP1), which binds to two silencer DNA sequences upstream of the adult human beta-globin gene and to a negative control region upstream of the adult delta-globin gene. Further studies demonstrated an inverse correlation between the binding affinity of the BP1 protein for the distal beta-globin silencer sequence and the severity of sickle cell anemia, suggesting a possible role for BP1 in determining the production of hemoglobin S. We have now cloned a cDNA expressing the BP1 protein. Sequencing revealed that BP1 is a member of the homeobox gene family and belongs to the subfamily called Distal-less (DLX), genes important in early development. Further analysis showed that BP1 is an isoform of DLX4. BP1 protein has repressor function towards the beta-globin promoter, acting through the two beta-globin DNA silencers, demonstrated in transient transfection assays. Strong BP1 expression is restricted to placenta and kidney tissue, with no expression in 48 other human tissues. BP1 exhibits regulated expression in the human erythroid cell line MB-02, where its expression decreases upon induction of the beta-globin gene. BP1 is thus the first member of the DLX family with known DNA binding sites and a function in globin gene regulation.     

Tarsius delta- and beta-globin genes: conversions, evolution, and systematic implications

Comparisons between duplicated genes have shown that gene conversions play an important role in the evolution of multigene families. Previous comparisons have documented in the recently duplicated gamma-fetal globin genes of catarrhine primates, over 15 separate conversions affecting extensive stretches of coding and noncoding sequences. In the present study, delta- and beta- globin genes from a lower primate Tarsius syrichta, and the delta-globin gene of the Asian great ape, Pongo pygmaeus, have been isolated and sequenced. Comparisons of these sequences with other primate delta and beta sequences confirmed a previously reported conversion in an anthropoid ancestor and revealed additional conversions in basal primate, stem haplorhine, tarsier, and early lemur lineages. Conversions found between primate delta- and beta-globin genes contrast with those found in the gamma-genes in that delta-beta conversions appear much less frequently and are more restricted to regions conserved by selection (i.e. coding and 5'-regulatory sequences). These differences indicate that soon after a duplication occurs, conversions can be quite frequent and encompass extensive portions of the duplicated region. With time, sequence differences accumulate, particularly in noncoding regions, and limit both the frequency and size of the conversions. Sequences conserved by selection accumulate differences more slowly and are therefore subject to gene conversions for a longer period of time. Both unconverted and converted sequences were consistent in supporting the placement of tarsier with anthropoids.