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.     

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.     

The primate psi beta 1 gene. An ancient beta-globin pseudogene

The human beta-globin gene cluster contains five functional genes plus a single pseudogene termed psi beta 1. Hybridization and comparative sequence analysis show that this pseudogene is not the product of a recent gene duplication, but is ancient and has been maintained in all major primate groups ranging from prosimians to anthropoids, at the same position as in man, between gamma- and delta-globin genes. In the lemur, a prosimian, the central exons of the psi beta 1 and delta-globin genes have undergone an unequal exchange, which has resulted in a contraction of the beta-globin gene cluster and the formation of a Lepore-type psi beta 1-delta globin pseudogene. Comparisons of defects shared by prosimian, New World monkey and human psi beta 1 sequences suggest that the ancestral primate gene was probably a pseudogene with an abnormal initiation codon but few if any additional defects, and that most contemporary pseudogene defects were accumulated relatively recently by slow neutral drift. We suggest that psi beta 1 arose early in primate evolution by silencing of a pre-existing discrete functional gene, and show that psi beta 1-related sequences are also present in other mammalian orders. In view of the antiquity of psi beta 1-related sequences, we propose that this gene be renamed the eta-globin gene.     

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.     

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.      

Molecular evolution of intergenic DNA in higher primates: pattern of DNA changes, molecular clock, and evolution of repetitive sequences

A 3.1-kb intergenic DNA fragment located between the psi beta-globin and delta-globin genes in the beta-globin gene cluster was cloned from gorilla, orangutan, rhesus monkey, and spider monkey, and the nucleotide sequence of each fragment was determined. The phylogeny of these four sequences, together with two previously published allelic sequences from humans and one from chimpanzee, was constructed, and the accumulation of mutations in the region was analyzed. The sites of base substitutions are not evenly distributed within the region: two Alu repeats have accumulated 0.21 + 0.02 substitutions/site with 0.15 + 0.008 substitutions/site in the remainder of the fragment. The occurrence of substitutions at neighboring sites is more frequent than would be expected if they were independent. The observed excesses disappear when ancestral -CG- dinucleotide sites are excluded. The phylogenetic relationships of the sequences indicate that the human sequence shares a most recent coancestor with the chimpanzee sequence. The data also show that great apes have accumulated fewer mutations in this part of the genome than has the rhesus monkey. The relative rates of accumulation of 12 kinds of nucleotide substitution in the region during primate evolution are asymmetric in the DNA strands. From these rates of accumulation, the origin of a simple stretch of sequence near the 3' end of the 3.1-kb fragment was deduced to be a sequence comprising 50% T and 50% C on one strand. The two oppositely oriented Alu sequences in the 3.1-kb region were inserted at their present positions before the divergence of the New-World monkeys from other lineages. Our analysis shows that the nucleotide sequences of the two Alu repeats in spider monkey are unexpectedly similar both to each other and to the deduced ancestral sequence of Alu repeats. The data suggest that there has been some type of recombinational event between the spider monkey Alu repeats but that it was not a simple gene conversion.      

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.     

A silent deletion in the beta-globin gene cluster.

A survey of the gamma-globin gene region of over 1000 normal individuals revealed a novel 2.5 kb deletion which removes the 5' end of the A gamma-globin gene. Unusually, this deletion in the beta-globin gene cluster is not associated with increased fetal haemoglobin production. Sequence analysis of the deletion endpoints revealed no significant homology at the breakpoint and failed to support a role for a proposed recombination hotspot in IVS-2 in the generation of this illegitimate recombination event. The existence of small "silent" deletions in the beta-globin gene cluster emphasizes the importance of deletion size in altering expression of the fetal globin genes.     

A molecular and evolutionary study of the beta-globin gene family of the Australian marsupial Sminthopsis crassicaudata

Beta-globin gene families in eutherians (placental mammals) consist of a set of four or more developmentally regulated genes which are closely linked and, in general, arranged in the order 5'-embryonic/fetal genes- adult genes-3'. This cluster of genes is proposed to have arisen by tandem duplication of ancestral beta-globin genes, with the first duplication occurring 200 to 155 MYBP just prior to a period in mammalian evolution when eutherians and marsupials diverged from a common ancestor. In this paper we trace the evolutionary history of the beta-globin gene family back to the origins of these mammals by molecular characterization of the beta-globin gene family of the Australian marsupial Sminthopsis crassicaudata. Using Southern and restriction analysis of total genomic DNA and bacteriophage clones of beta-like globin genes, we provide evidence that just two functional beta-like globin genes exist in this marsupial, including one embryonic- expressed gene (S.c-epsilon) and one adult-expressed gene (S.c-beta), linked in the order 5'-epsilon-beta-3'. The entire DNA sequence of the adult beta-globin gene is reported and shown to be orthologous to the adult beta-globin genes of the North American marsupial Didelphis virginiana and eutherian mammals. These results, together with results from a phylogenetic analysis of mammalian beta-like globin genes, confirm the hypothesis that a two-gene cluster, containing an embryonic- and an adult-expressed beta-like globin gene, existed in the most recent common ancester of marsupials and eutherians. Northern analysis of total RNA isolated from embryos and neonatals indicates that a switch from embryonic to adult gene expression occurs at the time of birth, coinciding with the transfer of the marsupial from a uterus to a pouch environment.      

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.      

Concerted evolution led to high expression of a prosimian primate delta globin gene locus

The delta globin gene in simian primates is either weakly expressed (in hominoids and New World monkeys) or silent (in Old World monkeys). In prosimian primates, however, an unequal homologous crossover between the psi eta and delta loci of lemurs produced a hybrid psi eta delta pseudogene locus, whereas in tarsier the delta locus encodes a beta-type chain found in 18% of adult tarsier hemoglobin molecules. In the present study, the nucleotide and amino acid sequences of the galago delta and beta globin genes and their encoded peptides were determined, and evidence is provided showing that the galago delta locus encodes a beta-type chain (beta 2) found in 40% of the galago fetal and postnatal hemoglobin molecules, whereas the beta locus encodes the remaining 60% of the beta-type chain (beta 1). Galago beta 1 and beta 2 chains differ from each other by only one amino acid residue. The homology between the galago delta and beta loci extends from 800 base pairs 5' of the proximal CCAAT element to near the end of exon 3 as a result of a recombination event in which beta sequence replaced delta sequence. After this initial recombination event, concerted evolution between the loci continued over their conserved coding, intron 1, and promoter regions but failed to occur between the two loci in their intron 2 and distal 5'-flanking sequences where the two loci have now diverged by 20%. Calculations based on this divergence value and on a rate of noncoding sequence evolution of 4.2 x 10(-9) to 5.5 x 10(-9) substitutions/site/year for the lorisiform lineage to galago yielded a date of 18-24 million years ago for the initial recombination event. The fact that the promoter sequences of the galago delta locus are the same as that of the galago beta locus may account for the high level of expression of the galago delta gene.     

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.      

An intron nucleotide sequence variant in a cloned beta +-thalassaemia globin gene.

A 7.5 kb Hsu I restriction fragment of genomic DNA containing a beta-globin gene has been isolated from a patient doubly heterozygous for beta + thalassaemia and a delta beta (Lepore globin fusion gene. This fragment must be derived from the chromosome carrying the beta +-thalassaemia determinant. The gross structure of the cloned gene plus flanking sequences is indistinguishable from that of a normal beta-globin gene. Within in 1606 base-pair transcribed region of the gene there is only one nucleotide difference from the normal beta-globin gene sequence. This is a G leads to A replacement 21 nucleotides upstream from the 3' terminus of the small intron. This nucleotide lies within a 10 base-pair sequence repeated in an inverted configuration near the 5' terminus of the small intron. The nucleotide replacement may result in a precursor mRNA less amenable to RNA splicing than its normal counterpart.     

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.     

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.      

Silent nucleotide substitutions and G+C content of some mitochondrial and bacterial genes

Summary The G+C content of DNA varies widely in different organisms, especially microorganisms. This variation is accompanied by changes in the nucleotide composition of silent positions in codons. (Silent positions are defined and explained in the text.) These changes are mostly neutral or near neutral, and appear to result from mutation pressure in the direction of increasing either A+T (AT pressure) or G+C(GC pressure) content. Variations in G+C content are also accompanied by substitutions at replacement positions in codons. These substituions produce changes in the amino acid content of homologous proteins. The examples studied were genes for 13 mitochondrial proteins in five species, and A and B genes for bacterial tryptophan synthase in four species.In microorganisms, varying AT and GC mutational pressures, presumably resulting from shifts in the DNA polymerase system, exert strong effects on molecular evolution by changing the G+C content of DNA. These effects may be greater than those of random drift. The effects of GC pressure on silent substitutions in the systems examined are several times as great as the effects on replacement substitutions.GC pressure is exerted on noncoding as well as coding regions in mitochondrial DNA. This is shown by the close correlation (correlation coefficient, 0.99) of the G+C content of the noncoding D loop of mitochondria with the G+C content of silent positions in the corresponding mitochondrial genes.     

Neutral evolution of the sex-determining gene transformer in Drosophila

The amino acid sequence of the transformer (tra) gene exhibits an extremely rapid rate of evolution among Drosophila species, although the gene performs a critical step in sex determination. These changes in amino acid sequence are the result of either natural selection or neutral evolution. To differentiate between selective and neutral causes of this evolutionary change, analyses of both intraspecific and interspecific patterns of molecular evolution of tra gene sequences are presented. Sequences of 31 tra alleles were obtained from Drosophila americana. Many replacement and silent nucleotide variants are present among the alleles; however, the distribution of this sequence variation is consistent with neutral evolution. Sequence evolution was also examined among six species representative of the genus Drosophila. For most lineages and most regions of the gene, both silent and replacement substitutions have accumulated in a constant, clock-like manner. In exon 3 of D. virilis and D. americana we find evidence for an elevated rate of nonsynonymous substitution, but no statistical support for a greater rate of nonsynonymous relative to synonymous substitutions. Both levels of analysis of the tra sequence suggest that, although the gene is evolving at a rapid pace, these changes are neutral in function.