The rate and pattern of nucleotide substitution in Drosophila mitochondrial DNA.

The nucleotide sequences of a segment of mitochondrial DNA (mtDNA) have been determined for nine species or subspecies of the subgenus Drosophila of the genus Drosophila. This segment contains two complete protein-coding genes (i.e., NADH dehydrogenase subunit 1 and cytochrome b) and a transfer RNA gene (tRNA(ser)). The G+C content at third-codon positions for the two protein-coding genes was 1.5 times higher than that in the D. melanogaster species group, which belongs to the subgenus Sophophora. However, there was a substantial difference between the nucleotide frequencies of G and C. The number of nucleotide substitutions per silent site was more than three times higher than that for nuclear DNA, although it was only 60% of that for mammalian mtDNA. Both parametric and nonparametric analyses revealed a strong transition-transversion bias in nucleotide substitution, as was observed in mammalian mtDNA. Moreover, the rate of substitution of A and T for G and C is higher than that for the opposite direction. This bias seems to be responsible for the extremely A+T-rich base composition of Drosophila mtDNA. It is also noted that the rate of transitional change between A and G is higher than that between T and C.     

On the estimation of the rate of nucleotide substitution for the control region of human mitochondrial DNA

To apply molecular clock for studying human evolution, the pattern of nucleotide substitution for the control region of human mtDNA was analyzed in detail. It is well known that the rate of nucleotide substitution for the control region is much higher than that for any other part of mtDNA. In this study, the higher substitution rate was attributed to the higher rate of transition-type substitution between pyrimidines within the D-loop part, whereas the rates of other types of substitution were essentially the same over the entire mtDNA molecule. Even within the control region, the rate and pattern of nucleotide substitution were different between the D-loop part and the rest. The rate and pattern for the non-D-loop part were very similar to those for fourfold-degenerate sites in the protein-coding region. In contrast, the D-loop and non-D-loop parts showed similarities in the base composition, whereas the base composition of fourfold-degenerate sites slightly different from that of the both parts of the control region. It is concluded, therefore, that the nucleotide frequencies of the control region should be used to estimate the number of substitutions (d) between the control region sequences. However, a method to verify the accuracy of the estimation of d by means of the transition/transversion (s/v) ratio was theoretically studied. It was suggested that the s/v ratio becomes constant over a wide range of d values only when the estimation of d is unbiased. On the basis of this result, the estimates of d previously obtained between human sequences were evaluated.     

Transition and transversion rate in the evolution of animal mitochondrial DNA

We present a further application of the stochastic model previously described (Lanave et al., 1984, 1985) for measuring the nucleotide substitution rate in the mammalian evolution of the mitochondrial DNA (mtDNA). The applicability of this method depends on the validity of "stationarity conditions" (equal nucleotide frequencies at first, second and third silent codon positions in homologous protein coding genes). In the comparison of homologous sequences satisfying the stationarity condition at the silent sites, only the four codon families (quartets) for which both transitions and transversions are silent at the third position are considered here. This has allowed us to estimate the transition and transversion rates for any pair of species. We have analyzed the third silent codon position of the triplet rat-mouse-cow, of a series of slightly divergent primates and of two Drosophila species. In terms of two external dating input we have then determined the phylogenetic trees for rat, mouse, and cow as well as for a number of primates including man. The phylogenetic tree that we have derived for the triplet rat, mouse and cow agrees with that we had previously determined by analyzing the first, second and third silent codon positions (in both duets and quartets) of mt genes (Lanave et al., 1985). For primates our method leads to the following branching order from the oldest to the most recent: Gibbon, Orangutan, Gorilla, Chimpanzee and Man. In absolute time, fixing the distance Chimpanzee-Man as 5 million years (Myr) we estimate the dating of the divergence nodes as: Gorilla 7 Myr; Orangutan 16 Myr; Gibbon 20 Myr. In all cases analyzed, the transition rate has been found to be substantially higher than the transversion rate. Moreover we have found that the transition/transversion ratio is different in the various lineages. We suggest that this fact is probably related to the nucleotide frequencies at the third silent codon position.     

Replicating animal mitochondrial DNA

The field of mitochondrial DNA (mtDNA) replication has been experiencing incredible progress in recent years, and yet little is certain about the mechanism(s) used by animal cells to replicate this plasmid-like genome. The long-standing strand-displacement model of mammalian mtDNA replication (for which single-stranded DNA intermediates are a hallmark) has been intensively challenged by a new set of data, which suggests that replication proceeds via coupled leading- and lagging-strand synthesis (resembling bacterial genome replication) and/or via long stretches of RNA intermediates laid on the mtDNA lagging-strand (the so called RITOLS). The set of proteins required for mtDNA replication is small and includes the catalytic and accessory subunits of DNA polymerase γ, the mtDNA helicase Twinkle, the mitochondrial single-stranded DNA-binding protein, and the mitochondrial RNA polymerase (which most likely functions as the mtDNA primase). Mutations in the genes coding for the first three proteins are associated with human diseases and premature aging, justifying the research interest in the genetic, biochemical and structural properties of the mtDNA replication machinery. Here we summarize these properties and discuss the current models of mtDNA replication in animal cells.     

Site-by-site estimation of the rate of substitution and the correlation of rates in mitochondrial DNA

A maximum likelihood method for independently estimating the relative rate of substitution at different nucleotide sites is presented. With this method, the evolution of DNA sequences can be analyzed without assuming a specific distribution of rates among sites. To investigate the pattern of correlation of rates among sites, the method was applied to a data set consisting of the protein-coding regions of the mitochondrial genome from 10 vertebrate species. Rates appear to be strongly correlated at distances up to 40 codons apart. Furthermore, there appears to be some higher order correlation of sites approximately 75 codons apart. The method of site-by-site estimation of the rate of substitution may also be applied to examine other aspects of rate variation along a DNA sequence and to assess the difference in the support of a tree along the sequence.     

Analysis of directional mutation pressure and nucleotide content in mitochondrial cytochrome b genes

We present a new approach for analyzing directional mutation pressure and nucleotide content in protein-coding genes. Directional mutation pressure, the heterogeneity in the likelihood of different nucleotide substitutions, is used to explain the increasing or decreasing guanine-cytosine content (GC%) in DNA and is represented by µ_D, in agreement with Sueoka (1962, Proc Natl Acad Sci USA 48:582–592). The new method uses simulation to facilitate identification of significant A + T or G + C pressure as well as the comparison of directional mutation pressure among genes, even when they are translated by different genetic codes. We use the method to analyze the evolution of directional mutation pressure and nucleotide content of mitochondrial cytochrome b genes. Results from a survey of 110 taxa indicate that the cytochrome b genes of most taxa are subjected to significant directional mutation pressure and that the gene is subject to A + T pressure in most cases. Only in the anseriform bird Cairina moschata is the cytochrome b gene subject to significant G + C pressure. The GC% at nonsynonymous codon sites decreases proportionately with increasing A + T pressure, and with a slope less than one, indicating a presence of selective constraints. The cytochrome b genes of insects, nematodes, and eumycotes are subject to extreme A + T pressures (µ_D = 0.123, 0.224, and 0.130) and, in parallel, the GC% of the nonsynonymous codon sites has decreased from about 0.44 in organisms that are not subjected to A + T or G + C pressure to about 0.332, 0.323, and 0.367, respectively. The distribution of taxa according to the GC% at nonsynonymous codon sites and directional mutation pressure supports the notion that variation in these parameters is a phylogenetic component.     

Structure of animal mitochondrial DNA: nucleotide composition, pyrimidine clusters, and methylation character.

The nucleotide composition, relative concentration of pyrimidine clusters, and the degree of methylation of the mitochondrial and nuclear DNA's of various vertebrates and the protozoan Crithidia oncopelti have been studied. With respect to the relative concentration of GC pairs, the mtDNA of animals (bull, rat) does not differ from the corresponding nDNA. The relative concentration of GC pairs in the mtDNA of certain fish and birds is 1.5-2.5 mole% higher than in the respective nDNA. The kinetoplast DNA of the protozoan C. oncopelti (where the relative concentration of the GC pairs is 42.9 mole %) differs very sharply in composition from the nDNA (where the relative concentration of GC pairs is 51.3 mole %). The mtDNA's and kDNA's studied are distinguished from the respective nDNA'S by a lower degree of clustering of pyrimidine nucleotides. The proportion of mono- and dipyrimidine fragments in the mtDNA and kDNA is 30 mole %, while in the nDNA it does not exceed 23 mole %. The relative concentration of long pyrimidine clusters (hexapyrimidine clusters of larger) in the mtDNA is smaller than in the nDNA by a factor of 2-5. The low degree of clustering of the pyrimidine nucleotides is apparently characteristic of all the known mtDNA's and may support the fact that they have a single type of organization and are of a single origin. All the vertebrate mtDNA's studied contain 5-methylcytosine as a minor base (1.5-3.15 mole %), and their level of methylation is 1.5-2 times greater than that in the respective nDNA's. It has been shown that animals display species specificity with respect to the 5-methylcytosine content in the mtDNA. Its distribution among the pyrimidine clusters in the bovine heart mtDNA differs substantially from that in the nDNA. This suggests that the methylation specificities of nuclear and mitochondrial DNA are different. A DNA methylase, which effects the in vitro methylation of cytosine residues both in the homologous mtDNA and in different heterologous DNA's, has been found in rat liver and bovine heart mitochondria. The specificity of the in vitro methylation of the cytosine residues in the same heterologous Escherichia coli B DNA by the nuclear and mitochondrial enzymes is different: The mitochondrial enzyme methylates predominantly in monopyrimidine fragments, and the nuclear enzyme methylates mostly in di- and tripyrimidine fragments. They, therefore, recognize different nucleotide sequences.     

Rapid concerted evolution in animal mitochondrial DNA

Recombinational genetic processes are thought to be rare in the uniparentally inherited mitochondrial (mt) DNA molecules of vertebrates and other animals. Here, however, we document extremely rapid concerted microevolution, probably mediated by frequent gene conversion events, of duplicated sequences in the mtDNA control region of mangrove killifishes (Kryptolebias marmoratus). In local populations, genetic distances between paralogous loci within an individual were typically smaller (and often zero) than those between orthologous loci in different specimens. These findings call for the recognition of concerted evolution as a microevolutionary process and gene conversion as a likely recombinational force in animal mtDNA. The previously unsuspected power of these molecular phenomena could greatly impact mtDNA dynamics within germ cell lineages and in local animal populations.     

Evolution of hominoid mitochondrial DNA with special reference to the silent substitution rate over the genome

Summary Focusing on the synonymous substitution rate, we carried out detailed sequence analyses of hominoid mitochondrial (mt) DNAs of ca. 5-kb length. Owing to the outnumbered transitions and strong biases in the base compositions, synonymous substitutions in mtDNA reach rapidly a rather low saturation level. The extent of the compositional biases differs from gene to gene. Such changes in base compositions, even if small, can bring about considerable variation in observed synonymous differences and may result in the region-dependent estimate of the synonymous substitution rate. We demonstrate that such a region dependency is due to a failure to take proper account of heterogeneous compositional biases from gene to gene but that the actual synonymous substitution rate is rather uniform. The synonymous substitution rate thus estimated is 2.37 ± 0.11 × 10^–8 per site per year and comparable to the overall rate for the noncoding region. On the other hand, the rate of nonsynonymous substitutions differs considerably from gene to gene, as expected under the neutral theory of molecular evolution. The lowest rate is 0.8 × 10^–9 per site per year forCOI and the highest rate is 4.5 × 10^–9 forATPase 8, the degree of functional constraints (measured by the ratio of the nonsynonymous to the synonymous substitution rate) being 0.03 and 0.19, respectively. Transfer RNA (tRNA) genes also show variability in the base contents and thus in the nucleotide differences. The average rate for 11 tRNAs contained in the 5-kb region is 3.9 × 10^–9 per site per year. The nucleotide substitutions in the genome suggest that the transition rate is about 17 times faster than the transversion rate.     

Distribution of nucleotide substitutions in human mitochondrial DNA genes

To analyze the distribution pattern of nucleotide substitutions in human mitochondrial DNA (mtDNA), mutational spectra of the mitochondrial genes were reconstructed. The reconstruction procedure is based on the mutation distribution data for 47 monophyletic mtDNA clusters, to which 794 examined mtDNA sequences encoding for tRNAs, rRNAs, and mitochondrial proteins are attributed. One of specific features of mitochondrial mutational spectra revealed was homoplasy of the mutations (the mean mutation number per variable nucleotide site in the coding region varied from 1.09 to 1.43). It was established that in the mtDNA genes maximum mutational constraint fell onto the guanine bases, albeit the content of these bases in the mtDNA L-chains was minimal. Maximal bias towards parallel G to A transitions was observed for rRNA genes, with the protein- and tRNA-encoding genes ranking next. Despite the fact that the differences in the average G-nucleotides content and variability between the genes of two mtDNA segments located between the OriH and OriL were statistically significant, the results did not provide the conclusion that the G-nucleotide instability observed in the mtDNA L-spectra was determined by the mechanism of asynchronous mtDNA replication, along with the deamination of cytosines in the H-chain regions, which remained single-stranded during replication.     

Distribution of nucleotide substitutions in human mitochondrial DNA genes

To analyze the distribution pattern of nucleotide substitutions in human mitochondrial DNA (mtDNA), mutational spectra of the mitochondrial genes were reconstructed. The reconstruction procedure is based on the mutation distribution data for 47 monophyletic mtDNA clusters, to which 794 examined mtDNA sequences encoding for tRNAs, rRNAs, and mitochondrial proteins are attributed. One of specific features of mitochondrial mutational spectra revealed was homoplasy of the mutations (the mean mutation number per variable nucleotide site in the coding region varied from 1.09 to 1.43). It was established that in the mtDNA genes maximum mutational constraint fell onto the guanine bases, albeit the content of these bases in the mtDNA L-chains was minimal. Maximal bias towards parallel G to A transitions was observed for rRNA genes, with the protein-and tRNA-encoding genes ranking next. Despite the fact that the differences in the average G-nucleotides content and variability between the genes of two mtDNA segments located between the OriH and OriL were statistically significant, the results did not provide the conclusion that the G-nucleotide instability observed in the mtDNA L-spectra was determined by the mechanism of asynchronous mtDNA replication, along with the deamination of cytosines in the H-chain regions, which remained single-stranded during replication.Translated from Genetika, Vol. 41, No. 1, 2005, pp. 93–99.Original Russian Text Copyright © 2005 by Malyarchuk.     

Estimation of average number of nucleotide substitutions when the rate of substitution varies with nucleotide

Summary A formal mathematical analysis of Kimura's (1981) six-parameter model of nucleotide substitution for the case of unequal substitution rates among different pairs of nucleotides is conducted, and new formulae for estimating the number of nucleotide substitutions and its standard error are obtained. By using computer simulation, the validities and utilities of Jukes and Cantor's (1969) one-parameter formula, Takahata and Kimura's (1981) four-parameter formula, and our sixparameter formula for estimating the number of nucleotide substitutions are examined under three different schemes of nucleotide substitution. It is shown that the one-parameter and four-parameter formulae often give underestimates when the number of nucleotide substitutions is large, whereas the six-parameter formula generally gives a good estimate for all the three substitution schemes examined. However, when the number of nucleotide substitutions is large, the six-parameter and four-parameter formulae are often inapplicable unless the number of nucleotides compared is extremely large. It is also shown that as long as the mean number of nucleotide substitutions is smaller than one per nucleotide site the three formulae give more or less the same estimate regardless of the substitution scheme used.On leave of absence from the Department of Biology, Faculty of Science, Kyushu University 33, Fukuoka 812, Japan     

A codon-based model of nucleotide substitution for protein-coding DNA sequences

A codon-based model for the evolution of protein-coding DNA sequences is presented for use in phylogenetic estimation. A Markov process is used to describe substitutions between codons. Transition/transversion rate bias and codon usage bias are allowed in the model, and selective restraints at the protein level are accommodated using physicochemical distances between the amino acids coded for by the codons. Analyses of two data sets suggest that the new codon-based model can provide a better fit to data than can nucleotide-based models and can produce more reliable estimates of certain biologically important measures such as the transition/transversion rate ratio and the synonymous/nonsynonymous substitution rate ratio.      

The effect of nucleotide substitution on DNA denaturation profiles.

The melting profiles were obtained for DNA restriction fragments of approx. 1150 bp with deletion of one, five or six base pairs making them different from each other. In all cases the deletions caused a shift of one melting peak without affecting the positions of the other three peaks. The effect amounted to 0.28 +/- 0.03C upon the deletion of one GC pair. The melting of DNA fragments was also studied by electrophoresis in denaturing gradient gels. The deletion of one GC pair was shown to cause an appreciable shift of the electrophoretic denaturation profile.     

Using the nucleotide substitution rate matrix to detect horizontal gene transfer

Background  

Horizontal gene transfer (HGT) has allowed bacteria to evolve many new capabilities. Because transferred genes perform many medically important functions, such as conferring antibiotic resistance, improved detection of horizontally transferred genes from sequence data would be an important advance. Existing sequence-based methods for detecting HGT focus on changes in nucleotide composition or on differences between gene and genome phylogenies; these methods have high error rates.     

Rates of gene rearrangement and nucleotide substitution are correlated in the mitochondrial genomes of insects

A number of studies indicated that lineages of animals with high rates of mitochondrial (mt) gene rearrangement might have high rates of mt nucleotide substitution. We chose the hemipteroid assemblage and the Insecta to test the idea that rates of mt gene rearrangement and mt nucleotide substitution are correlated. For this purpose, we sequenced the mt genome of a lepidopsocid from the Psocoptera, the only order of hemipteroid insects for which an entire mtDNA sequence is not available. The mt genome of this lepidopsocid is circular, 16,924 bp long, and contains 37 genes and a putative control region; seven tRNA genes and a protein-coding gene in this genome have changed positions relative to the ancestral arrangement of mt genes of insects. We then compared the relative rates of nucleotide substitution among species from each of the four orders of hemipteroid insects and among the 20 insects whose mt genomes have been sequenced entirely. All comparisons among the hemipteroid insects showed that species with higher rates of gene rearrangement also had significantly higher rates of nucleotide substitution statistically than did species with lower rates of gene rearrangement. In comparisons among the 20 insects, where the mt genomes of the two species differed by more than five breakpoints, the more rearranged species always had a significantly higher rate of nucleotide substitution than the less rearranged species. However, in comparisons where the mt genomes of two species differed by five or less breakpoints, the more rearranged species did not always have a significantly higher rate of nucleotide substitution than the less rearranged species. We tested the statistical significance of the correlation between the rates of mt gene rearrangement and mt nucleotide substitution with nine pairs of insects that were phylogenetically independent from one another. We found that the correlation was positive and statistically significant (R2 = 0.73, P = 0.01; Rs = 0.67, P < 0.05). We propose that increased rates of nucleotide substitution may lead to increased rates of gene rearrangement in the mt genomes of insects.