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.      

Rates of Synonymous Substitution and Base Composition of Nuclear Genes in Drosophila

We compared the rates of synonymous (silent) substitution among various genes in a number of species of Drosophila. First, we found that even for a particular gene, the rate of synonymous substitution varied considerably with Drosophila lineages. Second, we showed a large variation in synonymous substitution rates among nuclear genes in Drosophila. These rates of synonymous substitution were correlated negatively with C content and positively with A content at the third codon positions. Nucleotide sequences were also compared between pseudogenes and their functional homologs. The C content of the pseudogenes was lower than that of the functional genes and the A content of the former was higher than that of the latter. Because the synonymous substitution for functional genes and the nucleotide substitution for pseudogenes are exempted from any selective constraint at the protein level, these observations could be explained by a biased pattern of mutation in the Drosophila nuclear genome. Such a bias in the mutation pattern may affect the molecular clock (local clock) of each nuclear gene of each species. Finally, we obtained the average rates of synonymous substitution for three gene groups in Drosophila; 11.0 x 10(-9), 17.5 x 10(-9) and 27.1 x 10(-9)/site/year.     

Molecular evolution of mammalian lactate dehydrogenase-A genes and pseudogenes: association of a mouse processed pseudogene with a B1 repetitive sequence

A mouse genomic clone containing a lactate dehydrogenase-A (LDH-A) processed pseudogene and a B1 repetitive element was isolated, and a nucleotide sequence of approximately 3 kb was determined. The pseudogene and B1 element are flanked by perfect 13-bp repeats, and the B1 sequence starts at 14 nucleotides 3' to the presumptive polyadenylation signal of the pseudogene. The nucleotide sequences of the LDH-A genes and processed pseudogenes from mouse, rat, and human were compared, and a phylogenetic tree was constructed. The rate and pattern of nucleotide substitutions in the LDH-A pseudogenes are similar to previously reported results (Li et al. 1984). The average rate of nucleotide substitutions in the LDH-A pseudogenes is 4.3 X 10(- 9)/site/year. The substitutions of C----T and G----A are most frequent, and A----G substitutions are relatively high. The rate of synonymous substitutions in the LDH-A genes is 5.3 X 10(-9), which is not significantly higher than the average rate of 4.7 X 10(-9) for 35 mammalian genes. The rate of nonsynonymous substitutions in the LDH-A genes is 0.20 X 10(-9), which is considerably lower than the average rate of 0.88 X 10(-9) for 35 mammalian genes. Thus, the mammalian LDH-A gene appears to be highly conserved in evolution.      

Inference of selection from multiple species alignments

The selective pressure on a protein-coding gene can be measured by comparing silent (synonymous) and replacement (nonsynonymous) substitution rates. Higher replacement than silent rates provide unequivocal evidence for adaptive evolution driven by Darwinian selection. Previous employment of this criterion involved pairwise sequence comparison, averaging rates over time and sequences, resulting in virtually no power. Recent methods apply the criterion to particular lineages on a phylogeny or to individual sites in the gene and are much more powerful. Their application has led to detection of adaptive Darwinian selection in a number of genes and organisms.     

Comparative analysis of human masculinity

To study rapidly evolving male specific Y (MSY) genes we retrieved and analyzed nine such genes. VCY, HSFY and RBMY were found to have functional X gametologs, but the rest did not. Using chimpanzee orthologs for XKRY, CDY, HSFY, PRY, and TSPY, the average silent substitution is estimated as 0.017 +/- 0.006/site and the substitution rate is 1.42 x 10(-9)/site/year. Except for VCY, all other loci possess two or more pseudogenes on the Y chromosome. Sequence differences from functional genes show that BPY2, DAZ, XKRY, and RBMY each have one pseudogene for each one that is human specific, while others were generated well before the human-chimpanzee split, by means of duplication, retro-transposition or translocation. Some functional MSY gene duplication of VCY, CDY and HSFY, as well as X-linked VCX and HSFX duplication, occurred in the lineage leading to humans; these duplicates have accumulated nucleotide substitutions that permit their identification.     

Mammalian gene evolution: Nucleotide sequence divergence between mouse and rat

As a paradigm of mammalian gene evolution, the nature and extent of DNA sequence divergence between homologous protein-coding genes from mouse and rat have been investigated. The data set examined includes 363 genes totalling 411 kilobases, making this by far the largest comparison conducted between a single pair of species. Mouse and rat genes are on average 93.4% identical in nucleotide sequence and 93.9% identical in amino acid sequence. Individual genes vary substantially in the extent of nonsynonymous nucleotide substitution, as expected from protein evolution studies; here the variation is characterized. The extent of synonymous (or silent) substitution also varies considerably among genes, though the coefficient of variation is about four times smaller than for nonsynonymous substitutions. A small number of genes mapped to the X-chromosome have a slower rate of molecular evolution than average, as predicted if molecular evolution is male-driven. Base composition at silent sites varies from 33% to 95% G + C in different genes; mouse and rat homologues differ on average by only 1.7% in silent-site G + C, but it is shown that this is not necessarily due to any selective constraint on their base composition. Synonymous substitution rates and silent site base composition appear to be related (genes at intermediate G + C have on average higher rates), but the relationship is not as strong as in our earlier analyses. Rates of synonymous and nonsynonymous substitution are correlated, apparently because of an excess of substitutions involving adjacent pairs of nucleotides. Several factors suggest that synonymous codon usage in rodent genes is not subject to selection.     

High-resolution copy-number variation map reflects human olfactory receptor diversity and evolution

Olfactory receptors (ORs), which are involved in odorant recognition, form the largest mammalian protein superfamily. The genomic content of OR genes is considerably reduced in humans, as reflected by the relatively small repertoire size and the high fraction ( approximately 55%) of human pseudogenes. Since several recent low-resolution surveys suggested that OR genomic loci are frequently affected by copy-number variants (CNVs), we hypothesized that CNVs may play an important role in the evolution of the human olfactory repertoire. We used high-resolution oligonucleotide tiling microarrays to detect CNVs across 851 OR gene and pseudogene loci. Examining genomic DNA from 25 individuals with ancestry from three populations, we identified 93 OR gene loci and 151 pseudogene loci affected by CNVs, generating a mosaic of OR dosages across persons. Our data suggest that approximately 50% of the CNVs involve more than one OR, with the largest CNV spanning 11 loci. In contrast to earlier reports, we observe that CNVs are more frequent among OR pseudogenes than among intact genes, presumably due to both selective constraints and CNV formation biases. Furthermore, our results show an enrichment of CNVs among ORs with a close human paralog or lacking a one-to-one ortholog in chimpanzee. Interestingly, among the latter we observed an enrichment in CNV losses over gains, a finding potentially related to the known diminution of the human OR repertoire. Quantitative PCR experiments performed for 122 sampled ORs agreed well with the microarray results and uncovered 23 additional CNVs. Importantly, these experiments allowed us to uncover nine common deletion alleles that affect 15 OR genes and five pseudogenes. Comparison to the chimpanzee reference genome revealed that all of the deletion alleles are human derived, therefore indicating a profound effect of human-specific deletions on the individual OR gene content. Furthermore, these deletion alleles may be used in future genetic association studies of olfactory inter-individual differences.     

Molecular Nature of Spontaneous Mutations in Mouse Lactate Dehydrogenase-a Processed Pseudogenes

The presence of at least ten mouse LDH-A pseudogenes was demonstrated in the genomic blot analysis, and four different processed pseudogenes have thus far been isolated and characterized. In this report, the nucleotide sequences to two different mouse lactate dehydrogenase-A processed pseudogenes, M11 and M14, were determined and compared with the protein-coding sequences of the mouse and rat LDH-A functional genes. In the pseudogene M11, the sequence of 64 nucleotides from codon no. 257 to 278 was tandemly duplicated. In the pseudogene M14, the sequence of 22 nucleotides from codon no. 68 to 75 was replaced by an inserted repetitive sequence of 242 nucleotides homologous to a mouse truncated R element. The pattern of nucleotide substitutions accumulated in mouse LDH-A pseudogenes M11 and M14, as well as that of pseudogene M10 identified previously, was analyzed, and the substitution frequencies of the C or G at the CG dinucleotide were found to be high.     

A simple method for estimating the intensity of purifying selection in protein-coding genes.

We propose a method by which the intensity of purifying selection on a functional protein-coding gene is estimated by using three aligned homologous sequences: a processed pseudogene (psi), a functional paralog from the same species (g), and a functional ortholog from a different species (o). For each such trio, we calculate the numbers of nucleotide substitutions along the branches leading to psi and g, i.e., K psi and K(g). If we assume that the mutation rates are the same in the genes and the pseudogenes and that mutations occurring in a pseudogene do not affect the fitness of the organism, we can show that the fraction of mutations that are selectively neutral, fg, is equal to the ratio K(g)/K psi. Since advantageous mutations occur only very rarely, such that they do not contribute significantly to the rate of molecular evolution, the fraction of deleterious mutations that are subject to purifying selection is 1-fg. Therefore, the K(g)/K psi ratio can be used directly to estimate the intensity of purifying selection, thereby isolating its effects on the rate of evolution from those of mutation. We compared the selection intensities of 12 orthologous protein-coding pairs from humans and murids. As expected, the fraction of mutations that are subject to purifying selection is strongest in the second codon position and weakest in the third. Interestingly, the mean fractions of effectively neutral mutations in the third codon position were only 41% and 42% for murids and humans, respectively, indicating that many synonymous mutations are subject to selective constraint. In several orthologous genes, we found that the intensity of purifying selection is very different between murid and human orthologous genes. There was no statistically significant difference in overall intensity of purifying selection between humans and murids. Thus, purifying selection does not seem to be an important factor contributing to the observed differences in the rates of evolution between these two taxa.     

Evolution in bacteria: Evidence for a universal substitution rate in cellular genomes

Summary This paper constructs a temporal scale for bacterial evolution by tying ecological events that took place at known times in the geological past to specific branch points in the genealogical tree relating the 16S ribosomal RNAs of eubacteria, mitochondria, and chloroplasts. One thus obtains a relationship between time and bacterial RNA divergence which can be used to estimate times of divergence between other branches in the bacterial tree. According to this approach,Salmonella typhimurium andEscherichia coli diverged between 120 and 160 million years (Myr) ago, a date which fits with evidence that the chief habitats occupied now by these two enteric species became available that long ago.The median extent of divergence betweenS. typhimurium andE. coli at synonymous sites for 21 kilobases of protein-coding DNA is 100%. This implies a silent substitution rate of 0.7–0.8%/Myr—a rate remarkably similar to that observed in the nuclear genes of mammals, invertebrates, and flowering plants. Similarities in the substitution rates of eucaryotes and procaryotes are not limited to silent substitutions in protein-coding regions. The average substitution rate for 16S rRNA in eubacteria is about 1%/50 Myr, similar to the average rate for 18S rRNA in vertebrates and flowering plants. Likewise, we estimate a mean rate of roughly 1%/25 Myr for 5S rRNA in both eubacteria and eucaryotes.For a few protein-coding genes of these enteric bacteria, the extent of silent substitution since the divergence ofS. typhimurium andE. coli is much lower than 100%, owing to extreme bias in the usage of synonymous codons. Furthermore, in these bacteria, rates of amino acid replacement were about 20 times lower, on average, than the silent rate. By contranst, for the mammalian genes studied to date, the average replacement rate is only four to five times lower than the rate of silent substitution.     

Origin and Evolution of a New Gene Descended from Alcohol Dehydrogenase in Drosophila

Drosophila alcohol dehydrogenase (Adh) is highly conserved in size, organization, and amino acid sequence. Adh-ψ was hypothesized to be a pseudogene derived from an Adh duplication in the repleta group of Drosophila; however, several results from molecular analyses of this gene conflict with currently held notions of molecular evolution. Perhaps the most difficult observations to reconcile with the pseudogene hypothesis are that the hypothetical replacement sites of Adh-ψ evolve only slightly more quickly than replacement sites of closely related, functional Adh genes, and that the replacement sites of the pseudogenes evolve considerably more slowly than neighboring silent sites. The data have been presented as a paradox that challenges our understanding of the mechanisms underlying DNA sequence divergence. Here I show that Adh-ψ is actually a new, functional gene recently descended from an Adh duplication. This descendant recruited ~60 new N-terminal amino acids, is considerably more basic than ADH, and is evolving at a faster rate than Adh. Furthermore, though the descendant is clearly functional, as inferred from molecular evolution and population genetic data, it retains no obvious ADH activity. This probably reflects functional divergence from its Adh ancestor.     

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.     

Pseudogene evolution and natural selection for a compact genome

Pseudogenes are nonfunctional copies of protein-coding genes that are presumed to evolve without selective constraints on their coding function. They are of considerable utility in evolutionary genetics because, in the absence of selection, different types of mutations in pseudogenes should have equal probabilities of fixation. This theoretical inference justifies the estimation of patterns of spontaneous mutation from the analysis of patterns of substitutions in pseudogenes. Although it is possible to test whether pseudogene sequences evolve without constraints for their protein-coding function, it is much more difficult to ascertain whether pseudogenes may affect fitness in ways unrelated to their nucleotide sequence. Consider the possibility that a pseudogene affects fitness merely by increasing genome size. If a larger genome is deleterious--for example, because of increased energetic costs associated with genome replication and maintenance--then deletions, which decrease the length of a pseudogene, should be selectively advantageous relative to insertions or nucleotide substitutions. In this article we examine the implications of selection for genome size relative to small (1-400 bp) deletions, in light of empirical evidence pertaining to the size distribution of deletions observed in Drosophila and mammalian pseudogenes. There is a large difference in the deletion spectra between these organisms. We argue that this difference cannot easily be attributed to selection for overall genome size, since the magnitude of selection is unlikely to be strong enough to significantly affect the probability of fixation of small deletions in Drosophila.     

Human glyceraldehyde-3-phosphate dehydrogenase pseudogenes: Molecular evolution and a possible mechanism for amplification

We screened two human genomic libraries and isolated 14 different clones, designated λG1 and EG1-EG13, homologous to human glyceraldehyde-3-phosphate dehydrogenase (GAPD) cDNA. Subcloning and sequencing these recombinant phages led us to classify them as five different pseudogenes (ψG1–ψG5). All these sequences show such features typical of processed pseudogenes as numerous mutations, insertions, and deletions. The identity of numerous mutated sites among these pseudogenes and the presence of two Alu sequences flanking both ends of ψG1 suggest that GAPD pseudogenes originated from a unique reverse transcribed mRNA followed by gene duplication. The rate of nucleotide substitutions per site per year for known GAPD functional genes is low both for the synonymous substitutions (1.87×10⁻⁹) and for the nonsynonymous substitutions (0.12￠10⁻⁹) and indicates that the GAPD cDNA sequence is well conserved not only at the amino acid level, but also at the nucleotide level. The rate of nucleotide substitutions per site per year for GAPD pseudogenes shows a higher value (5.9×10⁻⁹) and suggests that these pseudogenes do not have any functional role. This work was supported by grants from the Consiglio Nazionale delle Ricerche and the Ministero Pubblica Istruzione (Rome, Italy). Special acknowledgment is given to the “Progetto Finalizzato Ingegneria Genetica e Basi Molecolari delle Malattie Ereditarie.”     

Nucleotide substitution pattern in rice paralogues: implication for negative correlation between the synonymous substitution rate and codon usage bias

Understanding the correlation between synonymous substitution rate and GC content is essential to decipher the gene evolution. However, it has been controversial on their relationship. We analyzed the GC content and synonymous substitution rate in 1092 paralogues produced by two large-scale duplication events in the rice genome. According to the GC content at the third codon sites (GC3), the paralogues were classified into GC3-rich and GC3-poor genes. By referring to their outgroup sequences, we inferred the last common ancestor of sister paralogues and, consequently, calculated the average synonymous substitution rate for two gene classes. The results suggest that average synonymous substitution rate is lower in GC3-rich genes than that in GC3-poor genes, indicating that the synonymous substitution rate is negatively correlated with GC content in the rice genome. Through characterizing the synonymous nucleotide substitution pattern, we found a strong synonymous nucleotide substitution frequency bias from AT to GC in GC3-rich genes. This indicates possible limitations of commonly used methods developed to estimate the synonymous substitution rate. Their estimates might produce misleading results on correlation between the synonymous substitution rate and GC content.     

Adaptive and Slightly Deleterious Evolution in a Conifer, <Emphasis Type="Italic">Cryptomeria japonica</Emphasis>

In order to evaluate effects of the population structure and natural selection on organisms having long generation times, we surveyed DNA polymorphisms at five loci encoding 9-cis-epoxycarotenoid dioxygenase (NCED), ammonium transporter, calmodulin, aquaporin, and the second major allergen with polymethylgalacturonase enzyme activity in the pollen (Cryj2) in a conifer, Cryptomeria japonica. The average nucleotide diversity at silent sites across 12 loci including the previously analyzed seven loci was 0.0044. The population recombination rate (4Nr, where N and r are the effective population size and recombination rate per base per generation, respectively) was estimated as 0.00046 and a slow reduction in the population size was indicated, according to the maximum likelihood method implemented in LAMARC. At NCED, the McDonald-Kreitman (MK) test revealed an excess of replacement polymorphisms, suggesting contributions of slightly deleterious mutations. In contrast, the MK test revealed an excess of replacement divergence at Cryj2 and a maximum likelihood approach using the PAML package revealed that certain amino acid sites had a nonsynonymous/synonymous substitution rate ratio (omega) > 4.0, indicating adaptive evolution at this locus. The overall analysis of the 12 loci suggested that adaptive, neutral, and slightly deleterious evolution played important roles in the evolution of C. japonica.     

Mitochondrial genome sequence evolution in Chlamydomonas

The mitochondrial genomes of the Chlorophyta exhibit significant diversity with respect to gene content and genome compactness; however, quantitative data on the rates of nucleotide substitution in mitochondrial DNA, which might help explain the origin of this diversity, are lacking. To gain insight into the evolutionary forces responsible for mitochondrial genome diversification, we sequenced to near completion the mitochondrial genome of the chlorophyte Chlamydomonas incerta, estimated the evolutionary divergence between Chlamydomonas reinhardtii and C. incerta mitochondrial protein-coding genes and rRNA-coding regions, and compared the relative evolutionary rates in mitochondrial and nuclear genes. Synonymous and nonsynonymous substitution rates do not differ significantly between the mitochondrial and nuclear protein-coding genes. The mitochondrial rRNA-coding regions, however, are evolving much faster than their nuclear counterparts, and this difference might be explained by relaxed functional constraints on the mitochondrial translational apparatus due to the small number of proteins synthesized in Chlamydomonas mitochondria. Substitution rates at synonymous sites in a nonstandard mitochondrial gene (rtl) and at intronic and synonymous sites in nuclear genes expressed at low levels suggest that the mutation rate is similar in these two genetic compartments. Potential evolutionary forces shaping mitochondrial genome evolution in Chlamydomonas are discussed.