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.     

Amphi-panamic geminates of snook (Percoidei: Centropomidae) provide a calibration of the divergence rate in the mitochondrial DNA control region of fishes.

The mitochondrial DNA control region is one of the most frequently utilized sequences for both intra- and interspecific genetic studies of fishes, yet a tenable divergence rate specifically for fish control regions has not been established. We attempted to establish a rate through a comparative study of control region sequences and those of a protein-coding mitochondrial gene region from geminate species of snook (Centropomus) assumed to have been separated by the emergence of the Isthmus of Panama 3.5 million years ago. The divergence rate suggested from the control region alignments between the geminates was markedly higher than rates currently applied in many studies, as was the rate suggested from the ND 5/6 protein coding region alignments. However, the suggested ND 5/6 rate when applied to alignments with the outgroup species was not concordant with the scant centropomid fossil record and therefore the assumed separation time of 3.5 million years seemed implausible. An average control region divergence rate was then estimated based on separation times of snook species derived assuming a divergence rate of 1% per million years for transversion substitutions at third codon positions in the ND 5/6 region. Using these separation times, a tenable average divergence rate for fish control regions of approximately 3.6% per million years +/- 0.46% SE was calculated.     

Common 5' beta-globin RFLP haplotypes harbour a surprising level of ancestral sequence mosaicism

Blocks of linkage disequilibrium (LD) in the human genome represent segments of ancestral chromosomes. To investigate the relationship between LD and genealogy, we analysed diversity associated with restriction fragment length polymorphism (RFLP) haplotypes of the 5' beta-globin gene complex. Genealogical analyses were based on sequence alleles that spanned a 12.2-kb interval, covering 3.1 kb around the psibeta gene and 6.2 kb of the delta-globin gene and its 5' flanking sequence known as the R/T region. Diversity was sampled from a Kenyan Luo population where recent malarial selection has contributed to substantial LD. A single common sequence allele spanning the 12.2-kb interval exclusively identified the ancestral chromosome bearing the "Bantu" beta(s) (sickle-cell) RFLP haplotype. Other common 5' RFLP haplotypes comprised interspersed segments from multiple ancestral chromosomes. Nucleotide diversity was similar between psibeta and R/T-delta-globin but was non-uniformly distributed within the R/T-delta-globin region. High diversity associated with the 5' R/T identified two ancestral lineages that probably date back more than 2 million years. Within this genealogy, variation has been introduced into the 3' R/T by gene conversion from other ancestral chromosomes. Diversity in delta-globin was found to lead through parts of the main genealogy but to coalesce in a more recent ancestor. The well-known recombination hotspot is clearly restricted to the region 3' of delta-globin. Our analyses show that, whereas one common haplotype in a block of high LD represents a long segment from a single ancestral chromosome, others are mosaics of short segments from multiple ancestors related in genealogies of unsuspected complexity.     

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.     

Cloning and complete sequence of an HLA-A2 gene: analysis of two HLA-A alleles at the nucleotide level

The gene for the HLA-A2 antigen has been cloned from the human lymphoblastoid cell line 721. Comparison of this sequence with the published sequence for HLA-A3 permits the examination of two alleles at the extremely polymorphic HLA-A locus. A high degree of sequence conservation was seen in both coding and noncoding DNA, 97.2% and 94.5%, respectively. Interestingly, the 3' untranslated region was the most conserved, with 99.5% homology. The polymorphism of the HLA-A antigens results from a high proportion of amino acid substitutions relative to the total nucleotide changes in exons 2 and 3. Unlike the clustered differences seen in this region on comparison of two H-2K alleles of mouse, nucleotide substitutions between the HLA-A2 and A3 alleles are evenly distributed. Substitutions at silent sites and within introns were used to calculate an intra-allelic divergence time of at least 10 to 15 million years for these two HLA-A alleles.     

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.      

Increased genetic diversity in mitochondrial genes is correlated with the evolution of parasitism in the Hymenoptera

A higher AT content and rate of mtDNA sequence divergence was found in parasitic wasps (Apocrita) compared with nonparasitic wasps (Symphyta). The compositional bias was reflected in extreme codon bias for a cytochrome oxidase I protein coding gene fragment as well as in the types of amino acid substitutions that have occurred during the evolution of this gene fragment. In some instances, compositional bias influenced the definition of a conservative amino acid change. The increased rate of mtDNA sequence evolution probably arose during the early Jurassic, coincident with the first appearance of parasitic wasps in the fossil record. Our results suggest a causal link between the rate of sequence divergence and the parasitic lifestyle.Abbreviations AT adenosine-thymine - CO-1 cytochrome oxidase 1 - mtDNA mitochondrial DNA - Myr million years Correspondence to: M. Dowton     

The sequence of the gorilla fetal globin genes: evidence for multiple gene conversions in human evolution

Two fetal globin genes (G gamma and A gamma) from one chromosome of a lowland gorilla (Gorilla gorilla gorilla) have been sequenced and compared to three human loci (a G gamma-gene and two A gamma-alleles). A comparison of regions of local homology among these five sequences indicates that long after the duplication that produced the two nonallelic gamma-globin loci of catarrhine primates, about 35 million years (Myr) ago, at least one gene conversion event occurred between these loci. This conversion occurred not long before the ancestral divergence (about 6 Myr ago) of Homo and Gorilla. After this ancestral divergence, a minimum of three more gene conversion events occurred in the human lineage. Each human A gamma-allele shares specific sequence features with the gorilla A gamma-gene; one such distinctive allelic feature involves the simple repeated sequence in IVS 2. This suggests that early in the human lineage the A gamma-genes may have undergone a crossing-over event mediated by this simple repeated sequence. The DNA sequences from coding regions of both G gamma- and A gamma-loci, a comparison of 292 codons in the corresponding gorilla and human genes, show an unusually low evolutionary rate, with only two nonsilent differences and, surprisingly, not even one silent substitution. The two nonsynonymous substitutions observed predict a glycine at codon 73 and an arginine at codon 104 in the gorilla A gamma-sequence rather than aspartic acid and lysine, respectively, in human A gamma. Because only arginine has been found at position 104 in gamma-chains of Old World monkeys, it may represent the ancestral residue lost in gorilla and human G gamma-chains and in the human A gamma-chain. Possibly the arginine codon (AGG) was replaced by the lysine codon (AAG) in the G gamma-gene of a common ancestor of Homo and Gorilla and then was transferred to the A gamma-gene by subsequent conversions in the human lineage. DNA sequence conversions, similar to that attributed to the fetal gamma-globin genes, appear to be relatively frequent phenomena and, if widespread throughout the genome, may have profound evolutionary consequences.      

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.     

Evolution of a multigene family of V kappa germ line genes

We have isolated a series of related V kappa germ line genes from a BALB/c sperm DNA library. DNA sequence analysis of four members of this V kappa 24 multigene family implies that three V kappa genes are functional whereas the fourth one (psi V kappa 24) is a pseudogene. The prototype gene (V kappa 24) encodes the variable region gene segment expressed in an immune response against phosphorylcholine. The other two functional genes (V kappa 24A and V kappa 24B) may be expressed against streptococcal group A carbohydrate. The time of divergence of the four genes was estimated by the rate of synonymous nucleotide changes. This implies that an ancestral gene has duplicated approximately 33-35 million years ago and a subsequent gene duplication event has occurred approximately 23 million years ago.     

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.      

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.].     

New dates and new rates for divergence across the Isthmus of Panama

Sister species separated by the Isthmus of Panama have been widely used to estimate rates of molecular evolution. These estimates are based on the assumption that geographic isolation occurred nearly simultaneously for most taxa, when connections between the Caribbean and eastern Pacific closed approximately three million years ago. Here we show that this assumption is invalid for the only genus for which many taxa and multiple genetic markers have been analysed. Patterns of divergence exhibited by allozymes and the mitochondrial COI gene are highly concordant for 15 pairs of snapping shrimp in the genus Alpheus, indicating that they provide a reasonable basis for estimating time since cessation of gene flow. The extent of genetic divergence between pairs of sister species varied over fourfold. Sister species from mangrove environments showed the least divergence, as would be expected if these were among the last habitats to be divided. Using this pair yields a rate of sequence divergence of 1.4% per one million years, with implied times of separation for the 15 pairs of 3 to 18 million years ago. Many past studies may have overestimated rates of molecular evolution because they sampled pairs that were separated well before final closure of the Isthmus.     

Identification of a heat-shock pseudogene from Caenorhabditis elegans

While characterizing the hsp70 gene family from Caenorhabditis elegans we encountered an unusual member of this family. Sequence data reveal that the hsp-2ps gene is a pseudogene of the constitutively expressed, heat-inducible hsp-1 gene. Two stop codons generated near the 5' end of the sequence as well as several frameshift mutations and a large internal deletion confirm the identification of hsp-2ps as a pseudogene. The nucleotide substitution rate of the third codon position was twice that of the first and second codon positions, suggesting that the hsp-2ps gene was nonfunctional since the time of the duplication event. The hsp-2ps gene duplicates a region of the hsp-1 gene that lies exclusively within the transcribed region and retains the introns. We feel that the hsp-2ps gene was produced by a transpositional duplication event, which occurred approximately 8.5 million years ago.     

The mitochondrial genome of spotted green pufferfish Tetraodon nigroviridis (Teleostei: Tetraodontiformes) and divergence time estimation among model organisms in fishes

We determined the whole mitochondrial genome sequence for spotted green pufferfish, Tetraodon nigroviridis (Teleostei: Tetraodontiformes). The genome (16,488 bp) contained 37 genes (two ribosomal RNA genes, 22 transfer RNA genes, and 13 protein-coding genes) plus control region as found in other vertebrates, with the gene order identical to that of typical vertebrates. The sequence was used to estimate phylogenetic relationships and divergence times among major lineages of fishes, including representative model organisms in fishes. We employed partitioned Bayesian approaches for these two analyses using two datasets that comprised concatenated amino acid sequences from 12 protein-coding genes (excluding the ND6 gene) and concatenated nucleotide sequences from the 12 protein-coding genes (without 3rd codon positions), 22 transfer RNA genes, and two ribosomal RNA genes. The resultant trees from the two datasets were well resolved and largely congruent with those from previous studies, with spotted green pufferfish being placed in a reasonable phylogenetic position. The approximate divergence times between spotted green pufferfish and model organisms in fishes were 85 million years ago (MYA) vs. torafugu, 183 MYA vs. three-spined stickleback, 191 MYA vs. medaka, and 324 MYA vs. zebrafish, all of which were about twice as old as the divergence times estimated by their earliest occurrences in fossil records.     

Pervasive purifying selection characterizes the evolution of I2 homologs

We sampled 384 sequences related to the Solanum pimpinellifolium (=Lycopersicon pimpinellifolium) disease resistance (R) gene 12 from six species, potato, S. demissum, tomato, eggplant, pepper, and tobacco. These species represent increasing phylogenetic distance from potato to tobacco, within the family Solanaceae. Using sequence data from the nucleotide binding site (NBS) region of this gene, we tested models of gene family evolution and inferred patterns of selection acting on the NBS gene region and I2 gene family. We find that the I2 family has diversified within the family Solanaceae for at least 14 million years and evolves through a slow birth-and-death process requiring approximately 12 million years to homogenize gene copies within a species. Analyses of selection resolved a general pattern of strong purifying selection acting on individual codon positions within the NBS and on NBS lineages through time. Surprisingly, we find nine codon positions strongly affected by positive selection and six pairs of codon positions demonstrating correlated amino acid substitutions. Evolutionary analyses serve as bioinformatic tools with which to sort through the vast R gene diversity in plants and find candidates for new resistance specificities or to identify specific amino acid positions important for biochemical function. The slow birth-and-death evolution of I2 genes suggests that some NBS-leucine rich repeat-mediated resistances may not be overcome rapidly by virulence evolution and that the natural diversity of R genes is a potentially valuable source for durable resistance.