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.     

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 cloned delta-globin genes from a normal subject and a patient with delta-thalassemia; sequence polymorphisms found in the delta-globin gene region of Japanese individuals.

The delta-globin genes of a normal Japanese and a Japanese patient with homozygous delta-thalassemia were cloned, and the nucleotide sequence of a region including the gene was determined. Comparison of the nucleotide sequences of these two individuals with that of pH delta 1, delta-globin clone from the gene library constructed by Maniatis et al., showed differences in the large intervening sequence (IVS 2), at positions 137, 151, 186, 188, 291, 292 and 540 as one base substitutions, at 339 and 823 as one base additions, at 548 as a one base deletion, and a 9 bp duplication between positions 651 and 659, and differences in the 3'-flanking sequence at 51 and 98 nucleotides 3' to the AATAAA sequence. However, in the region studied, no differences was observed in the nucleotide sequences of the normal subject and the patient with delta-thalassemia. Therefore, these differences may represent polymorphisms of the delta-globin gene present in Japanese individuals. These data suggest that IVS 2 is more divergent than other regions, and that a DNA region(s) other than the globin gene may affect expression of the gene.     

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.      

Evolutionary rate of immunoglobulin alpha noncoding region is greater in hominoids than in Old World monkeys.

Recent studies on the molecular evolution of primates show that the evolutionary rate among hominoids is considerably slower than that among nonhominoid primates. However, this observation at the nucleotide-sequence level is restricted to the beta-globin family region. In this study, we sequenced orthologous immunoglobulin alpha (C alpha) genes of chimpanzee, gorilla, orangutan, and crab-eating macaque (an Old World monkey) and compared them with that of the human by using noncoding regions for analysis. Since significant differences in rates among hominoids were not found by using the relative rate test, we evaluated the ratio (R) of the evolutionary distance between Old World monkey and human to the distance between orangutan and human. The R value (1.12) for the C alpha gene was much smaller than the expected value (1.38-2.33), showing that the nucleotide substitution rate (= mutation rate per year under selective neutrality) of the C alpha gene is greater in the human lineage than in the Old World monkey lineage. We also did a similar analysis for the gamma 1-, gamma 2-, psi eta-, and delta-globin genes and found a considerable heterogeneity (1.12-2.37) among the R values, including that for the C alpha gene. This indicates that the hominoid slowdown of the evolutionary rate is not a universal phenomenon in primate evolution.     

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.].     

Mitochondrial-nuclear interactions and accelerated compensatory evolution: evidence from the primate cytochrome C oxidase complex

Accelerated rates of mitochondrial protein evolution have been proposed to reflect Darwinian coadaptation for efficient energy production for mammalian flight and brain activity. However, several features of mammalian mtDNA (absence of recombination, small effective population size, and high mutation rate) promote genome degradation through the accumulation of weakly deleterious mutations. Here, we present evidence for "compensatory" adaptive substitutions in nuclear DNA- (nDNA) encoded mitochondrial proteins to prevent fitness decline in primate mitochondrial protein complexes. We show that high mutation rate and small effective population size, key features of primate mitochondrial genomes, can accelerate compensatory adaptive evolution in nDNA-encoded genes. We combine phylogenetic information and the 3D structure of the cytochrome c oxidase (COX) complex to test for accelerated compensatory changes among interacting sites. Physical interactions among mtDNA- and nDNA-encoded components are critical in COX evolution; amino acids in close physical proximity in the 3D structure show a strong tendency for correlated evolution among lineages. Only nuclear-encoded components of COX show evidence for positive selection and adaptive nDNA-encoded changes tend to follow mtDNA-encoded amino acid changes at nearby sites in the 3D structure. This bias in the temporal order of substitutions supports compensatory weak selection as a major factor in accelerated primate COX evolution.     

Structure,molecular evolution,and gene expression of primate superoxide dismutases

Mn- and Cu,Zn-superoxide dismutase (SOD) cDNAs of eight primate species, Pan troglodytes, Pongo pygmaeus, Hylobates lar, Macaca fuscata, Macaca fascicularis, Macaca mulatta, Cebus apella, and Callithrix jacchus, were cloned. The whole protein-coding sequences were covered, comparing 198 and 153 (or 154) amino acids, for Mn- and Cu,Zn-SODs, respectively. Residues forming metal ligands were completely conserved in the two primate SODs and nucleotide/amino acid substitutions were more frequent in Cu,Zn-SODs than in Mn-SODs. Molecular evolutionary analyses showed Mn-SOD to have evolved at a constant rate and its phylogenetic tree well reflected primate phylogeny. Cu,Zn-SOD was shown to have evolved differently between primate lineages. The significant high ratio of a non-synonymous/synonymous rate was found in the lineage leading to great apes and humans, showing that this lineage underwent positive Darwinian selection. Southern hybridization suggested that the genes for primate Mn- and Cu,Zn-SOD exist as single copies. Northern analysis in various Japanese monkey tissues showed Mn- and Cu,Zn-SOD expression to be high in the liver, kidneys, and adrenal glands.     

Origin heterogeneity of Hb Lepore-Boston gene in Italy

Forty-three hybrid delta-beta-globin genes were characterized by DNA sequence analysis and associated RFLP haplotypes in 40 families from Abruzzo and Campania, which are on the east and west coast of Italy, respectively. All the genes had the delta-globin sequence up to the exon 2 codon 87 and had the beta-globin sequence from IVS-2-8; between these two ends, they had 58 bp in common with the delta- and beta-globin genes. Thus, they were all of the Lepore-Boston type. A chromosomal background heterogeneity was present among the mutant genes. In fact, they were all associated with (+ - - - -) 5' subhaplotype, but 23/31 from Campania were associated with (+ +) 3' subhaplotype, whereas 12/12 genes from Abruzzo and 8/31 from Campania were associated with (+ -). DNA sequencing of homozygous subjects showed that (+ +) 3' subhaplotype was associated, at IVS-2-74, with G, while (+ -) was associated with T; that is they were associated with the beta-globin gene sequence of frameworks 1 and 2, respectively. The molecular characteristics of this heterogeneity, as well as its geographical patterns in the eastern and western regions of Italy, represent strong evidence for the recurrent and multicentric origins of the mutation.     

Recent gene conversion between genes encoding human red and green visual pigments

Nucleotide sequences of the human X-linked red and green pigment genes were compared, and the number of silent substitutions per site (KSc) between these genes was analysed in comparison with the corresponding values of primate genes. Taking the retarded mutation rate of X-linked genes into consideration (Miyata et al., 1987), the red and green pigment genes were shown to have undergone gene conversion at around the time of separation of African apes and orangutan. Thus the recent gene conversion and retarded mutation rate in these X-linked genes are probably responsible for the strong sequence similarity between these genes, which is likely to facilitate the occurrence of red-green color blindness in the human population. It was also shown that the red pigment gene evolved about five times more rapidly than the green pigment gene since the latest gene conversion.     

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.     

Molecular Evolution of Cytochrome c Oxidase Subunit IV: Evidence for Positive Selection in Simian Primates

Cytochrome c oxidase (COX) is a multi-subunit enzyme complex that catalyzes the final step of electron transfer through the respiratory chain on the mitochondrial inner membrane. Up to 13 subunits encoded by both the mitochondrial (subunits I, II, and III) and nuclear genomes occur in eukaryotic organisms ranging from yeast to human. Previously, we observed a high number of amino acid replacements in the human COX IV subunit compared to mouse, rat, and cow orthologues. Here we examined COX IV evolution in the two groups of anthropoid primates, the catarrhines (hominoids, cercopithecoids) and platyrrhines (ceboids), as well as one prosimian primate (lorisiform), by sequencing PCR-amplified portions of functional COX4 genes from genomic DNAs. Phylogenetic analysis of the COX4 sequence data revealed that accelerated nonsynonymous substitution rates were evident in the early evolution of both catarrhines and, to a lesser extent, platyrrhines. These accelerated rates were followed later by decelerated rates, suggesting that positive selection for adaptive amino acid replacement became purifying selection, preserving replacements that had occurred. The evidence for positive selection was especially pronounced along the catarrhine lineage to hominoids in which the nonsynonymous rate was first faster than the synonymous rate, then later much slower. The rates of three types of ``neutral DNA' nucleotide substitutions (synonymous substitutions, pseudogene nucleotide substitutions, and intron nucleotide substitutions) are similar and are consistent with previous observations of a slower rate of such substitutions in the nuclear genomes of hominoids than in the nuclear genomes of other primate and mammalian lineages. Received: 22 May 1996 / Accepted: 24 November 1996     

Conversion and compensatory evolution of the gamma-crystallin genes and identification of a cataractogenic mutation that reverses the sequence of the human CRYGD gene to an ancestral state

We identified a mutation in the CRYGD gene (P23S) of the gamma-crystallin gene cluster that is associated with a polymorphic congenital cataract that occurs with frequency of approximately 0.3% in a human population. To gain insight into the molecular mechanism of the pathogenesis of gamma-crystallin isoforms, we undertook an evolutionary analysis of the available mammalian and newly obtained primate sequences of the gamma-crystallin genes. The cataract-associated serine at site 23 corresponds to the ancestral state, since it was found in CRYGD of a lower primate and all the surveyed nonprimate mammals. Crystallin proteins include two structurally similar domains, and substitutions in mammalian CRYGD protein at site 23 of the first domain were always associated with substitutions in the structurally reciprocal sites 109 and 136 of the second domain. These data suggest that the cataractogenic effect of serine at site 23 in the N-terminal domain of CRYGD may be compensated indirectly by amino acid changes in a distal domain. We also found that gene conversion was a factor in the evolution of the gamma-crystallin gene cluster throughout different mammalian clades. The high rate of gene conversion observed between the functional CRYGD gene and two primate gamma-crystallin pseudogenes (CRYGEP1 and CRYGFP1) coupled with a surprising finding of apparent negative selection in primate pseudogenes suggest a deleterious impact of recently derived pseudogenes involved in gene conversion in the gamma-crystallin gene cluster.     

Recent insertion of an alu sequence in the beta-globin gene cluster of the gorilla

We present the nucleotide sequence of a new Alu family member that lies between the delta- and beta-globin genes in gorilla DNA. The sequence exhibits 91% similarity with a consensus sequence of the Alu family. It is flanked by a perfect repetition of a 16-nucleotide target sequence and terminates with 24 adenylic residues. As this sequence is absent at this locus in other primate DNAs, its insertion occurred less than 8 million years ago, thus supporting the idea that Alu sequences are still mobile elements in the hominoid genome.     

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.      

Molecular evolution of mRNA: A method for estimating evolutionary rates of synonymous and amino acid substitutions from homologous nucleotide sequences and its application

Summary A method for estimating the evolutionary rates of synonymous and amino acid substitutions from homologous nucleotide sequences is presented. This method is applied to genes of øX174 and G4 genomes, histone genes and-globin genes, for which homologous nucleotide sequences are available for comparison to be made. It is shown that the rates of synonymous substitutions are quite uniform among the non-overlapping genes of øX174 and G4 and among histone genes H4, H2B, H3 and H2A. A comparison between øX174 and G4 reveals that, in the overlapping segments of the A-gene, the rate of synonymous substitution is reduced more significantly than the rate of amino acid substitution relative to the corresponding rate in the nonoverlapping segment. It is also suggested that, in the coding regions surrounding the splicing points of intervening sequences of-globin genes, there exist rigid secondary structures. It is in only these regions that the-globin genes show the slowing down of evolutionary rates of both synonymous and amino acid substitutions in the primate line.     

Intra- and interspecies analyses of the carcinoembryonic antigen (CEA) gene family reveal independent evolution in primates and rodents

Summary Various rodent and primate DNAs exhibit a stronger intra- than interspecies cross-hybridization with probes derived from the N-terminal domain exons of human and rat carcinoembryonic antigen (CEA)-like genes. Southern analyses also reveal that the human and rat CEA gene families are of similar complexity. We counted at least 10 different genes per human haploid genome. In the rat, approximately seven to nine different N-terminal domain exons that presumably represent different genes appear to be present. We were able to assign the corresponding genomic restriction endonuclease fragments to already isolated CEA gene family members of both human and rat. Highly similar subgroups, as found within the human CEA gene family, seem to be absent from the rat genome. Hybridization with an intron probe from the human nonspecific cross-reacting antigen (NCA) gene and analysis of DNA sequence data indicate the conservation of noncoding regions among CEA-like genes within primates, implicating that whole gene units may have been duplicated. With the help of a computer program and by calculating the rate of synonymous substitutions, evolutionary trees have been derived. From this, we propose that an independent parallel evolution, leading to different CEA gene families, must have taken place in, at least, the primate and rodent orders.