Heterogeneity in the substitution process of amino acid sites of proteins coded for by mitochondrial DNA

Summary Several forms of maximum likelihood models are applied to aligned amino acid sequence data coded for in the mitochondrial DNA of six species (chicken, frog, human, bovine, mouse, and rat). These models range in form from relatively simple models of the type currently used for inferring phylogenetic tree structure to models more complex than those that have been used previously. No major discrepancies between the optimal trees inferred by any of these methods are found, but there are huge differences in adequacy of fit. A very significant finding is that the fit of any of these models is vastly improved by allowing a certain proportion of the amino acid sites to be invariant. An even more important, although disquieting, finding is that none of these models fits well, as judged by standard statistical criteria. The primary reason for this is that amino acid sites undergo substitution according to a process that is very heterogeneous. Because most phylogenetic inference is accomplished by choosing the optimal tree under the assumption that a homogeneous process is acting on the sites, the potential invalidity of some such conclusions is raised by this article's results. The seriousness of this problem depends upon the robustness of the phylogenetic inferential procedure to departures from the underlying model.     

Amino acid specificity of the transfer RNA species coded for by HeLa cell mitochondrial DNA

The amino acid specificity of the tRNA species coded for by HeLa cell mitochondrial DNA has been investigated by carrying out hybridizations between amino acid-tRNA complexes labeled in the amino acid and separated mitochondrial DNA strands.The results indicate that there are in HeLa cell mitochondria at least 17 distinct tRNA species hybridizable with mitochondrial DNA, which are specific for 16 amino acids. For 14 of the 16 amino acids, amino-acyl-tRNA synthetase activities distinct from the cytoplasmic ones have been detected in mitochondria. The remaining four amino acids (asparagine, glutamine, histidine and proline) have consistently failed to charge to any detectable extent mitochondrial tRNA species hybridizable with mitochondrial DNA.No obvious relationship appears to exist between the amino acids incorporated into tRNAs hybridizable to mitochondrial DNA and the previously observed pattern of chloramphenicol-sensitive amino acid incorporation by HeLa cell mitochondria.     

Model of amino acid substitution in proteins encoded by mitochondrial DNA

Mitochondrial DNA (mtDNA) sequences are widely used for inferring the phylogenetic relationships among species. Clearly, the assumed model of nucleotide or amino acid substitution used should be as realistic as possible. Dependence among neighboring nucleotides in a codon complicates modeling of nucleotide substitutions in protein-encoding genes. It seems preferable to model amino acid substitution rather than nucleotide substitution. Therefore, we present a transition probability matrix of the general reversible Markov model of amino acid substitution for mtDNA-encoded proteins. The matrix is estimated by the maximum likelihood (ML) method from the complete sequence data of mtDNA from 20 vertebrate species. This matrix represents the substitution pattern of the mtDNA-encoded proteins and shows some differences from the matrix estimated from the nuclear-encoded proteins. The use of this matrix would be recommended in inferring trees from mtDNA-encoded protein sequences by the ML method. Received: 3 May 1995 / Accepted: 31 October 1995     

The preference of the mitochondrial endonuclease for a conserved sequence block in mitochondrial DNA is highly conserved during mammalian evolution.

Endonuclease activity identified in crude preparations of rat and human heart mitochondria has each been partially purified and characterized. Both the rat and human activities purify as a single enzyme that closely resembles the endonuclease of bovine-heart mitochondria (Cummings, O.W. et. al. (1987) J. Biol. Chem. 262:2005-2015). All three enzymes, for example elute similarly during gel filtration and DNA-cellulose chromatography, and exhibit similar enzymatic properties. Although the nucleotide sequences of the mtDNAs indicate that there has occurred an unusual degree of divergence in the displacement-loop region during mammalian evolution, the nucleotide specificities of the mt endonucleases appear highly conserved and show a striking preference for an evolutionarily-conserved sequence tract that is located upstream from the heavy (H)-strand origin of DNA replication (OriH).     

Amino acid preferences of small proteins. Implications for protein stability and evolution.

The dependence of amino acid frequency on sequence length has been examined for the 20 natural amino acids using a set of 2275 protein sequences with little sequence identity. As expected, the frequency of cysteine increases dramatically for sequences shorter than 100 amino acids with a length-dependence that corresponds to an average of two Cys per sequence independent of length. Surprisingly dramatic changes were also observed for the frequencies of arginine, lysine, aspartic acid, and glutamic acid: Arg and Lys frequencies increase for short sequences whereas Asp and Glu frequencies decrease. These changes do not appear to be due to an over-abundance of DNA- and membrane-binding proteins in the database and may, therefore, be related to protein stability. Possible stabilizing mechanisms include increased hydrogen bonding by Arg and increased hydrophobic stabilization due to the amphiphilic character of Arg and Lys. These observations suggest that amino acid composition played an important role in the evolution of small proteins.     

Models of amino acid substitution and applications to mitochondrial protein evolution

Models of amino acid substitution were developed and compared using maximum likelihood. Two kinds of models are considered. "Empirical" models do not explicitly consider factors that shape protein evolution, but attempt to summarize the substitution pattern from large quantities of real data. "Mechanistic" models are formulated at the codon level and separate mutational biases at the nucleotide level from selective constraints at the amino acid level. They account for features of sequence evolution, such as transition-transversion bias and base or codon frequency biases, and make use of physicochemical distances between amino acids to specify nonsynonymous substitution rates. A general approach is presented that transforms a Markov model of codon substitution into a model of amino acid replacement. Protein sequences from the entire mitochondrial genomes of 20 mammalian species were analyzed using different models. The mechanistic models were found to fit the data better than empirical models derived from large databases. Both the mutational distance between amino acids (determined by the genetic code and mutational biases such as the transition-transversion bias) and the physicochemical distance are found to have strong effects on amino acid substitution rates. A significant proportion of amino acid substitutions appeared to have involved more than one codon position, indicating that nucleotide substitutions at neighboring sites may be correlated. Rates of amino acid substitution were found to be highly variable among sites.      

An analysis of the dynamics of mammalian mitochondrial DNA sequence evolution

The dynamics of the substitution process for mammalian mitochondrial DNA have been modeled. The temporal behavior of several quantities has been studied and the model's predictions have been compared with estimates obtained from recent mtDNA sequence data for an increasingly divergent series of primates, the mouse and the cow (Anderson et al. 1981, 1982; Bibb et al. 1981; Brown et al. 1982). The results are consistent with the hypothesis that the decrease in the proportion of transitions observed as divergence increases is a consequence of the highly biased substitution process. In addition, the results support the hypothesis that, although a portion of the mtDNA molecule evolves at an extremely rapid rate, a significant portion of the molecule is under strong selective constraints.      

Amino acid sequence homology of mammalian type C RNA virus major internal proteins.

The NH2-terminal amino acid sequence of the major group-specific antigen, the major internal virion protein (p30; approximate molecular weight 30,000) of several mammalian type C RNA viruses was determined by the Edman degradation procedure using an automated protein sequenator. All of the proteins analyzed show a high degree of over-all sequence homology and also contain specific regions or single residues. All p30s begin with the sequence prolyl-leucylarginyl (Pro-Leu-Arg) and have an invariant, conserved region from residues 11 to 24. In this region only a single amino acid difference appears between the cat and mouse p30s. At position 17 alanine is found in the cat, and serine in all the mouse proteins. This homologous region starts at position 10 for RD-114 and baboon virus p30s, and at position 18 in the protein of the virus isolated from gibbon ape. The region extending from residue 4 to 10 shows considerable variability between p30s isolated from different mammalian species. Out of 24 residues compared, only a single amino acid difference was found between six different mouse p30s. At position 4, three have leucine, two have alanine, and one has serine. The comparative sequence data demonstrate that the viral p30s are products of related genes in the viruses from various mammalian species.     

Amino acid substitution in the C-terminal arm domain of HU-2 results in an enhanced affinity for DNA.

Three mutants of the Escherichia coli hupA gene, encoding the HU-2 protein, were constructed by synthetic oligodeoxyribonucleotide-directed, site-specific mutagenesis on M13mp18 vectors. The resulting HupAN10, HupAN11 and HupAN12 proteins contained Thr59-->Lys, Gln64-->Lys and Asn53-->Arg substitutions, respectively. These amino acid (aa) changes increased the positive charge of the N-terminal half of the two-strand, antiparallel beta-ribbon of the arm structure, which is believed to be a domain for DNA binding. The three mutant proteins bound to DNA more tightly than wild-type HU-2, and their affinities for DNA increased in the order of HupAN10, HupAN11, HupAN12. The mutant proteins showed a slightly increased HU activity for supporting Mu phage development. A mutant HU-2 protein with increased basicity, but with an altered aa sequence in the arm region due to a frameshift mutation, was also constructed. This mutant protein showed a reduced affinity to DNA and was unable to support Mu growth, suggesting that a unique aa sequence of the arm domain, rather than mere basicity of this domain, is required for efficient binding to DNA.     

Heterogeneity of tempo and mode of mitochondrial DNA evolution among mammalian orders

A statistical analysis of complete DNA sequence data of mitochondrial genomes from human, bovine, and mouse (and partial sequence of rat) indicates that the transversion rate is lower in bovine than in human and murids, and that it is probably lower in human than in murids. However, it is unknown whether the transition rate is also lower in bovine. It is shown that the L-strand of human mitochondrial DNA has significantly higher C content and lower T and A contents than those of bovine and murids. This suggests that a directional mutation pressure which tends to change base composition has been operating in the human lineage.     

Amino acid transport in mammalian oocytes

Uptake of ¹⁴C-labelled amino acids into single oocytes was determined using ³H-labelled choline to correct for extracellular space. Cycloleucine, a non-metabolisable amino acid sharing an entry mechanism with methionine and phenylalanine, was transported in accord with Michaelis-Menten kinetics. At extracellular levels below 8 mM, cycloleucine was concentrated within the oocyte. The proportion of sheep oocytes having a functional amino acid transport system (i.e. cycloleucine flux > 1 nmole cm⁻² h⁻¹) was highest in pre-ovulatory follicles (97%), and lowest in atretic follicles (59%). Amino acid fluxes in functional germinal vesicle oocytes were similar at all stages of development studied. An increase in V_max but not K_m during meiotic maturation resulted in a doubling of amino acid uptake in metaphase II oocytes. These increased fluxes were under gonadotropic regulation and were independent of nuclear maturation. Amino acid uptake by mouse oocytes was approximately half that measured in sheep oocytes.     

Amino acid changes coded by bacteriophage T4 DNA polymerase mutator mutants. Relating structure to function

Previous studies on the selection of bacteriophage T4 mutator mutants have been extended and a method to regulate the mutator activity of DNA polymerase mutator strains has been developed. The nucleotide changes of 17 bacteriophage T4 DNA polymerase mutations that confer a mutator phenotype and the nucleotide substitutions of several other T4 DNA polymerase mutations have been determined. The most striking observation is that the distribution of DNA polymerase mutator mutations is not random; almost all mutator mutations are located in the N-terminal half of the DNA polymerase. It has been shown that the T4 DNA polymerase shares several regions of homology at the protein sequence level with DNA polymerases of herpes, adeno and pox viruses. From studies of bacteriophage T4 and herpes DNA polymerase mutants, and from analyses of similar protein sequences from several organisms, we conclude that DNA polymerase synthetic activities are located in the C-terminal half of the DNA polymerase and that exonucleolytic activity is located nearer the N terminus.     

Template-directed arrest of mammalian mitochondrial DNA synthesis.

Mammalian mitochondrial DNA often contains a short DNA displacement loop at the heavy-strand origin of replication. This short nascent DNA molecule has been used to study site-specific termination of mitochondrial DNA synthesis in human and mouse cells. We examined D-loop strand termination in two distantly related artiodactyls, the pig and the cow. Porcine mitochondrial DNA was unique among mammals in that it contained only a single species of D-loop single-stranded DNA. Its 3' end mapped to a site 187 nucleotides from the 5' end of the proline tRNA gene. This site was 21 and 47 nucleotides 5' to two very similar sequences (5' ACATATPyATTAT 3') which are closely related to the human and mouse termination-associated sequences noted by Doda et al. (J. N. Doda, D. T. Wright, and D. A. Clayton, Proc. Nat. Acad. Sci. USA 78:616-6120, 1981). Bovine mitochondrial DNA contained three major D-loop DNA species whose 3' ends mapped to three different sites. These sites were not found in the porcine sequence. However, the bovine termination sites were located 60 to 64 base pairs 5' from sequences which were also very similar to the termination-associated sequences present in pigs and other mammals. These results firmly establish the concept that arrest of heavy-strand DNA synthesis is an event determined, at least in part, by template sequence. They also suggest that arrest is determined by sequences which are a considerable physical distance away from the actual termination site.     

DNA asymmetric strand bias affects the amino acid composition of mitochondrial proteins.

Variations in GC content between genomes have been extensively documented. Genomes with comparable GC contents can, however, still differ in the apportionment of the G and C nucleotides between the two DNA strands. This asymmetric strand bias is known as GC skew. Here, we have investigated the impact of differences in nucleotide skew on the amino acid composition of the encoded proteins. We compared orthologous genes between animal mitochondrial genomes that show large differences in GC and AT skews. Specifically, we compared the mitochondrial genomes of mammals, which are characterized by a negative GC skew and a positive AT skew, to those of flatworms, which show the opposite skews for both GC and AT base pairs. We found that the mammalian proteins are highly enriched in amino acids encoded by CA-rich codons (as predicted by their negative GC and positive AT skews), whereas their flatworm orthologs were enriched in amino acids encoded by GT-rich codons (also as predicted from their skews). We found that these differences in mitochondrial strand asymmetry (measured as GC and AT skews) can have very large, predictable effects on the composition of the encoded proteins.