Molecular evolution of the psi eta-globin gene locus: gibbon phylogeny and the hominoid slowdown

An 8.4-kb genomic region spanning both the psi eta-globin gene locus and flanking DNA was sequenced from the common gibbon (Hylobates lar). In addition, sequencing of the entire orthologous region from galago (Galago crassicaudatus) was completed. The gibbon and galago sequences, along with published orthologous sequences from 10 other species, were aligned. These noncoding nucleotide sequences represented four human alleles, four apes (chimpanzee, gorilla, organgutan, and gibbon), an Old World monkey (rhesus monkey), two New World monkeys (spider and owl monkeys), tarsier, two strepsirhines (galago and lemur), and goat. Divergence and maximum parsimony analyses of the psi eta genomic region first groups humans and chimpanzees and then, at progressively more ancient branch points, successively joins gorillas, orangutans, gibbons, Old World monkeys, New World monkeys, tarsiers, and strepsirhines (the lemuriform-lorisiform branch of primates). This cladistic pattern supports the taxonomic grouping of all extant hominoids into family Hominidae, the division of Hominidae into subfamilies Hylobatinae (gibbons) and Homininae, the division of Homininae into tribes Pongini (orangutans) and Hominini, and the division of Hominini into subtribes Gorillina (gorillas) and Hominina (chimpanzees and humans). The additional gibbon and galago sequence data provide further support for the occurrence of a graded evolutionary-rate slowdown in the descent of simian primates, with the slowing rate being more pronounced in the great-ape and human lineages than in the gibbon or monkey lineages. A comparison of global versus local molecular clocks reveals that local clock predictions, when focused on a specific number of species within a narrow time frame, provide a more accurate estimate of divergence dates than do those of global clocks.     

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.     

The phylogenetic history of New World monkey beta globin reveals a platyrrhine beta to delta gene conversion in the atelid ancestry

Orthologues of the beta globin gene locus from 10 New World monkey species were sequenced and aligned against available beta and delta globin sequences from rabbit and other primates. Where needed, additional primate sequencing was performed. Phylogenetic analysis identified a beta to delta conversion in the stem of the Anthropoidea, stretching from the 3' part of the proximal promotor to the 5' start of intron 2, consistent with earlier findings. No further conversion appeared to have occurred in the descent of the catarrhines. Within the New World monkey lineage that led to spider monkey and other atelids, another shorter gene conversion was found, spanning adjacent parts of exon 1 and intron 1. The analysis also confirmed that galago beta had replaced galago delta, that an earlier loriform-specific gene conversion extended over intron 2, and that gene conversion throughout the main gene conversion region occurred in the tarsiiform lineage. Platyrrhine phylogenetic relationships were investigated with beta sequences restricted to those that were not involved in gene conversions. This phylogeny generally agreed with results from other nuclear genes. The one exception was that the beta sequences did not place the callitrichine clade within the Cebidae but weakly joined the callitrichine and atelid clades.     

The beta globin gene cluster of the prosimian primate Galago crassicaudatus: nucleotide sequence determination of the 41-kb cluster and comparative sequence analyses.

The nucleotide sequence of the beta globin gene cluster of the prosimian Galago crassicaudatus has been determined. A total sequence spanning 41,101 bp contains and links together previously published sequences of the five galago beta-like globin genes (5'-epsilon-gamma-psi eta-delta-beta-3'). A computer-aided search for middle interspersed repetitive sequences identified 10 LINE (L1) elements, including a 5' truncated repeat that is orthologous to the full-length L1 element found in the human epsilon-gamma intergenic region. SINE elements that were identified included one Alu type I repeat, four Alu type II repeats, and two methionine tRNA-derived Monomer (type III) elements. Alu type II and Monomer sequences are unique to the galago genome. Structural analyses of the cluster sequence reveals that it is relatively A+T rich (about 62%) and regions with high G+C content are associated primarily with globin coding regions. Comparative analyses with the beta globin cluster sequences of human, rabbit, and mouse reveal extensive sequence homologies in their genic regions, but only human, galago, and rabbit sequences share extensive intergenic sequence homologies. Divergence analyses of aligned intergenic and flanking sequences from orthologous human, galago, and rabbit sequences show a gradation in the rate of nucleotide sequence evolution along the cluster where sequences 5' of the epsilon globin gene region show the least sequence divergence and sequences just 5' of the beta globin gene region show the greatest sequence divergence.     

Comparison of the beta-like globin gene families of rabbits and humans indicates that the gene cluster 5''-epsilon-gamma-delta-beta-3'' predates the mammalian radiation

The members of the rabbit and human beta-like globin gene families have been compared both by a computer-generated dot matrix graphical analysis of each entire gene and by calculating divergences in the coding regions. The rabbit-human gene pairs beta 4-epsilon, beta 3- gamma, psi beta 2-delta, and beta 1-beta were identified as orthologous on the basis of sequence similarities found in flanking and intervening sequences as well as by quantitative divergence calculations. The orthologous genes are in the same order on the chromosome in each species, which suggests that an ancestral family with the arrangement 5'-epsilon-gamma-delta-beta-3' preceded the mammalian radiation. Descendants of ancestral epsilon have diverged more slowly than other beta-like genes and are expressed only in embryonic life. Descendants of ancestral gamma and beta diverged at a higher rate and are expressed at wider range of developmental times. Descendants of delta have undergone nonreciprocal recombination at a high frequency and are often pseudogenes. Paralogous comparisons among the rabbit beta-like globin genes show that the beta 4-beta 3 and psi beta 2-beta 1 pairs are most similar and that beta 4 and beta 3 are more closely related to beta 1 than to psi beta 2. This fits with a branching pattern where the primordial beta split into ancestral epsilon/gamma and delta/beta genes, which later split into epsilon and gamma or delta and beta, respectively. Rabbit genes beta 4 and beta 1 acquired similar 3' untranslated regions after the epsilon/gamma split but prior to the mammalian radiation, presumably via a gene conversion event. The 5' end of beta 2 apparently converted with beta 1 after the radiation, and afterward it became a pseudogene.      

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.     

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.     

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.      

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.     

Primate evolution of the alpha-globin gene cluster and its Alu-like repeats

The arrangement of alpha-globin genes in Old World and New World monkeys and a prosimian, galago, has been determined by restriction mapping. Recombinant DNAs containing galago and Old World monkey alpha-globin genes have been isolated and subjected to a partial sequence determination for comparison to alpha-globin genes in human, chimpanzee and non-primate mammals. The results of this extensive structural analysis are relevant to several topics concerning the evolution of primate alpha-globin genes and Alu family repeats. All orders of higher primates (i.e. Old and New World monkeys, chimpanzee and human) have the same arrangement of alpha-globin genes. In contrast, the arrangement and correction of galago alpha-globin genes differ from those of higher primates, but are similar to those of non-primate mammals. The 5' and 3'-flanking regions of the human alpha 1 gene are orthologous to the corresponding region in galago, identifying the human alpha 2 gene as the more recently duplicated gene. The human psi alpha 1 gene is found to be inactivated after divergence of the human and galago lineages but prior to the divergence of human and monkey. Orthologous Alu family members in human and monkey DNAs indicate that the dispersion of some Alu repeats occurred prior to the divergence of these lineages. However, the Alu-like repeats of prosimian and higher primates result from entirely independent events giving rise to different repeat elements inserted at distinct genomic positions.     

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.     

Genetic diversity at class II DRB loci of the primate MHC

The evolution of polymorphism at loci encoding the beta-chains of the MHC class II DR Ag was studied in primates by DNA amplification (polymerase chain reaction). Phylogenetic analysis of 63 DRB sequences from the polymorphic second exon (first domain) of nonhuman primates and 53 human sequences indicates the presence of five DRB loci in primates, derived from a DRB1-like ancestral locus over 20 million yr ago. Many of the allelic types at the DRB1 locus predate the divergence of hominoids (5 million yr ago) and some (DR4, DR3, 5, 6) predate the divergence of Old world monkeys and hominoids (20 million yr ago). The DRB3 locus appears to have arisen before the divergence of hominoids on an ancestral DRB1 lineage. The DRB2 and DRB5 loci were generated more than 20 million yr ago and the DRB4 locus more than 5 million yr ago. The DRB2 locus, a pseudogene in humans, is polymorphic in the nonhuman primates.     

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.     

Birth of a gene: locus of neuronal BC200 snmRNA in three prosimians and human BC200 pseudogenes as archives of change in the Anthropoidea lineage

The gene encoding brain-specific dendritic BC200 small non-messenger RNA is limited to the primate order and arose from a monomeric Alu element. It is present and neuronally expressed in all Anthropoidea examined. By comparing the human sequence of about 13.2 kb with each of the prosimian (lemur 14.6 kb, galago 12 kb, and tarsier 13.8 kb) orthologous loci, we could establish that the BC200 RNA gene is absent from the prosimian lineages. In Strepsirhini (lemurs and lorises), a dimeric AluJ-like element integrated very close to the BC200 insertion point, while the corresponding tarsier region is devoid of any repetitive element. Consequently, insertion of the Alu monomer that gave rise to the BC200 RNA gene must have occurred after the anthropoid lineage diverged from the prosimian lineage(s). Shared insertions of other repetitive elements favor proximity of simians and tarsiers in support of their grouping into Haplorhini and the omomyid hypothesis. On the other hand, the nucleotide sequences in the segment that is available for comparison in all four species reveal less exchanges between Strepsirhini (lemur and galago) and human than between tarsier and human. Our data imply that the early activity of dimeric Alu sequences must have been concurrent with the activity of monomeric Alu elements that persisted longer than is usually thought. As BC200 RNA gave rise to more than 200 pseudogenes, we used their consensus sequence variations as a molecular archive recording the BC200 RNA sequence changes in the anthropoid lineage leading to Homo sapiens and timed these alterations over the past 35-55 million years.     

Primate evolution at the DNA level and a classification of hominoids

Summary The genetic distances among primate lineages estimated from orthologous noncoding nucleotide sequences of -type globin loci and their flanking and intergenic DNA agree closely with the distances (delta T₅₀H values) estimated by cross hybridization of total genomic single-copy DNAs. These DNA distances and the maximum parsimony tree constructed for the nucleotide sequence orthologues depict a branching pattern of primate lineages that is essentially congruent with the picture from phylogenetic analyses of morphological characters. The molecular evidence, however, resolves ambiguities in the morphological picture and provides an objective view of the cladistic position of humans among the primates. The molecular data group humans with chimpanzees in subtribe Hominina, with gorillas in tribe Hominini, orangutans in subfamily Homininae, gibbons in family Hominidae, Old World monkeys in infraorder Catarrhini, New World monkeys in semisuborder Anthropoidea, tarsiers in suborder Haplorhini, and strepsirhines (lemuriforms and lorisiforms) in order Primates. A seeming incongruency between organismal and molecular levels of evolution, namely that morphological evolution appears to have speeded up in higher primates, especially in the lineage to humans, while molecular evolution has slowed down, may have the trivial explanation that relatively small genetic changes may sometimes result in marked phenotypic changes.     

A delta T-cell receptor deleting element transgenic reporter construct is rearranged in alpha beta but not gamma delta T-cell lineages.

T cells can be divided into two groups on the basis of the expression of either alpha beta or gamma delta T-cell receptors (TCRs). Because the TCR delta chain locus lies within the larger TCR alpha chain locus, control of the utilization of these two receptors is important in T-cell development, specifically for determination of T-cell type: rearrangement of the alpha locus results in deletion of the delta coding segments and commitment to the alpha beta lineage. In the developing thymus, a relative site-specific recombination occurs by which the TCR delta chain gene segments are deleted. This deletion removes all D delta, J delta, and C delta genes and occurs on both alleles. This delta deletional mechanism is evolutionarily conserved between mice and humans. Transgenic mice which contain the human delta deleting elements and as much internal TCR delta chain coding sequence as possible without allowing the formation of a complete delta chain gene were developed. Several transgenic lines showing recombinations between deleting elements within the transgene were developed. These lines demonstrate that utilization of the delta deleting elements occurs in alpha beta T cells of the spleen and thymus. These recombinations are rare in the gamma delta population, indicating that the machinery for utilization of delta deleting elements is functional in alpha beta T cells but absent in gamma delta T cells. Furthermore, a discrete population of early thymocytes containing delta deleting element recombinations but not V alpha-to-J alpha rearrangements has been identified. These data are consistent with a model in which delta deletion contributes to the implementation of a signal by which the TCR alpha chain locus is rearranged and expressed and thus becomes an alpha beta T cell.