Direct selection of mutations in the human mitochondrial tRNAThr gene: reversion of an 'uncloneable' phenotype

Several regions of the human mitochondrial genome are refractory to cloning in plasmid and bacteriophage DNA vectors. For example, recovery of recombinant M13 clones containing a 462 basepair MboI-Kpn I restriction fragment that spans nucleotide positions 15591 to 16053 of HeLa cell mitochondrial DNA was as much as 100-fold lower than the recovery of M13 clones containing other regions of the human mitochondrial genome. All of 50 recombinant M13 clones containing this 'uncloneable' fragment had one or more changes in nucleotide sequence. Each clone contained at least one alteration in two nucleotide positions within the tRNAThr gene that encode portions of the anticodon loop and D-stem of the HeLa mitochondrial tRNAThr. These results imply that the HeLa mitochondrial tRNAThr gene is responsible for the 'uncloneable' phenotype of this region of human mitochondrial (mt) DNA. A total of 61 nucleotide sequence alterations were identified in 50 independent clones containing the HeLa mt tRNAThr gene. 56 mutations were single-base substitutions; 5 were deletions. Approximately 80% of the base substitution mutations were A:T----G:C transitions. A preference for A:T----G:C transition mutations also characterizes polymorphic base substitution variants in the mitochondrial DNA of unrelated individuals. This similarity suggests that human mitochondrial DNA sequence variation within and between individuals may have a common origin.     

Nucleotide sequence and evolution of the rat mitochondrial cytochrome b gene containing the ochre termination codon

The nucleotide sequences of the genes for cytochrome b and three potential transfer RNAs (tRNAPro, tRNAThr and tRNAGlu) in cloned rat mitochondrial DNA were determined. The derived amino acid sequence of the cytochrome b protein from the light strand indicated that the C-terminal amino acid is asparagine and the ochre termination codon is encoded in the DNA, in contrast to the the lack of termination codon in the reading frame of human [Anderson et al., Nature 290 (1981) 457] or mouse [Bibb et al., Cell 26 (1981) 167] mitochondrial DNA. The first ATG codon of the cytochrome b gene was spaced five nucleotides from the 5'-end of the tRNAGlu gene on the heavy strand. There was a single nucleotide spacing between the termination codon of the cytochrome b gene and the 5' end of the tRNAThr gene in the light strand. There was also a single nucleotide spacing between the 3'-end of the tRNAThr gene and the 3'-end of the tRNAPro gene on the heavy strand. The amino acid and nucleotide sequences of the cytochrome b genes of mammals and yeast [Nobrega and Tzagoloff, J. Biol. Chem. 255 (1980) 9828] were compared to reveal structural differences in two very different species. At the same time, amino acid substitutions in particular regions of the mammalian gene corresponding to the exon-intron boundaries in the yeast gene were noted. These genetic features are discussed in relation to the extreme compression of genetic information in the mammalian mitochondrial genome as related to the evolution of the gene organization and its sequence.     

Characterization of a yeast nuclear gene (MST1) coding for the mitochondrial threonyl-tRNA1 synthetase

The wild-type yeast nuclear gene MST1 complements mutants defective in mitochondrial protein synthesis. The gene has been sequenced and shown to code for a protein of 54,030 kDa. The predicted product of MST1 is 36% identical over its 462 residues to the Escherichia coli threonyl-tRNA synthetase. Amino-acylation of wild-type mitochondrial tRNAs with a mitochondrial extract from mst1 mutants fail to acylate tRNAThr1 (anticodon: 3'-GAU-5') but show normal acylation of tRNAThr2 (anticodon: 3'-UGU-5'). These data suggest the presence of two separate threonyl-tRNA synthetases in yeast mitochondria. Antibodies were prepared against a trpE/MST1 fusion protein containing the 321 residues from the amino-terminal region of the E. coli anthranilate synthetase and 118 residues of the mitochondrial threonyl-tRNA synthetase. Antibodies to the fusion protein detect a 50-55-kDa protein in wild type yeast mitochondria but not in mitochondria of a strain in which the chromosomal MST1 gene was replaced by a copy of the same gene disrupted by insertion of the yeast LEU2 gene. The ability of the mutant with the inactive MST1 gene to charge tRNAThr2 argues strongly for the existence of a second threonyl-tRNA synthetase gene.     

Organization of the Mitochondrial Genome of a Deep-Sea Fish, Gonostoma gracile (Teleostei: Stomiiformes): First Example of Transfer RNA Gene Rearrangements in Bony Fishes

We determined the complete nucleotide sequence of the mitochondrial genome (except for a portion of the putative control region) for a deep-sea fish, Gonostoma gracile. The entire mitochondrial genome was purified by gene amplification using long polymerase chain reaction (long PCR), and the products were subsequently used as templates for PCR with 30 sets of newly designed, fish-universal primers that amplify contiguous, overlapping segments of the entire genome. Direct sequencing of the PCR products showed that the genome contained the same 37 mitochondrial structural genes as found in other vertebrates (two ribosomal RNA, 22 transfer RNA, and 13 protein-coding genes), with the order of all rRNA and protein-coding genes, and 19 tRNA genes being identical to that in typical vertebrates. The gene order of the three tRNAs (tRNA^Glu, tRNA^Thr, and tRNA^Pro) relative to cytochrome b, however, differed from that determined in other vertebrates. Two steps of tandem duplication of gene regions, each followed by deletions of genes, can be invoked as mechanisms generating such rearrangements of tRNAs. This is the first example of tRNA gene rearrangements in a bony fish mitochondrial genome. Received August 5, 1998; accepted February 19, 1999.     

The complete nucleotide sequence and gene organization of carp (Cyprinus carpio) mitochondrial genome

The complete sequence of the carp mitochondrial genome of 16,575 base pairs has been determined. The carp mitochondrial genome encodes the same set of genes (13 proteins, 2 rRNAs, and 22 tRNAs) as do other vertebrate mitochondrial DNAs. Comparison of this teleostean mitochondrial genome with those of other vertebrates reveals a similar gene order and compact genomic organization. The codon usage of proteins of carp mitochondrial genome is similar to that of other vertebrates. The phylogenetic relationship for mitochondrial protein genes is more apparent than that for the mitochondrial tRNA and rRNA genes.Correspondence to: F. Huang     

Complete mitochondrial genome of the longtooth grouper Epinephelus bruneus (Perciformes, Serranidae)

We determined the complete nucleotide sequence of the mitochondrial (mt) genome for the longtooth grouper, Epinephelus bruneus (Perciformes, Serranidae). This mt genome, consisting of 16,686 base pairs (bp), encoded genes for 13 protein-coding genes, 2 ribosomal RNAs, 22 transfer RNAs, and a noncoding control region as those found in other vertebrates, with the gene order identical to that of typical vertebrates. A major noncoding region between the trnP and trnF genes (991 bp) was considered to be the control region (D-loop). Within this sequence, 22 copies of a 17-bp tandem repeat element, 5'-TGATATTACATATATGC-3', were identified in the control region unlike previous reported Epinephelus species.     

Complete mitochondrial DNA sequence of Aulopus japonicus (Teleostei: Aulopiformes), a basal Eurypterygii: longer DNA sequences and higher-level relationships

For the first step toward resolution of the higher-level relationships of the order Aulopiformes (Teleostei: Eurypterygii) using longer DNA sequences, we determined the complete mitochondrial DNA sequence for Aulopus japonicus (Aulopodidae). The entire genome was purified by gene amplification using a long PCR technique, and the products were subsequently used as templates for PCR with 63 fish-versatile and 3 species-specific primers that amplify contiguous, overlapping segments of the entire genome. Direct sequencing of the PCR products demonstrated that the genome (16 653 base pairs [bp]) contained the same 37 mitochondrial genes (2 ribosomal RNA, 22 transfer RNA, and 13 protein-coding genes) as found in other vertebrates, with the gene order identical to that in typical vertebrates. Maximum-parsimony analysis using nucleotide sequences from the concatenated 12 protein-coding genes (no third codon positions and excluding the ND6 gene) plus 22 tRNA genes (stem regions only) from eight teleosts placed A. japonicus in a reasonable phylogenetic position; those from individual protein-coding genes and the concatenated 22 tRNA genes alone, however, did not reproduce the expected phylogeny with few exceptions, probably owing to insufficient phylogenetic information in these smaller data sets. This result suggests that further taxonomic sampling and sequencing efforts may clarify limits and intra- and interrelationships of this morphologically and ecologically diverse group of fishes using mitochondrial genomic (mitogenomic) data. Received: August 31, 2000 / Revised: December 20, 2000 / Accepted: January 23, 2001     

Complete nucleotide sequence of the mitochondrial genome of a salamander, Mertensiella luschani

The complete nucleotide sequence (16,650 bp) of the mitochondrial genome of the salamander Mertensiella luschani (Caudata, Amphibia) was determined. This molecule conforms to the consensus vertebrate mitochondrial gene order. However, it is characterized by a long non-coding intervening sequence with two 124-bp repeats between the tRNA(Thr) and tRNA(Pro) genes. The new sequence data were used to reconstruct a phylogeny of jawed vertebrates. Phylogenetic analyses of all mitochondrial protein-coding genes at the amino acid level recovered a robust vertebrate tree in which lungfishes are the closest living relatives of tetrapods, salamanders and frogs are grouped together to the exclusion of caecilians (the Batrachia hypothesis) in a monophyletic amphibian clade, turtles show diapsid affinities and are placed as sister group of crocodiles+birds, and the marsupials are grouped together with monotremes and basal to placental mammals. The deduced phylogeny was used to characterize the molecular evolution of vertebrate mitochondrial proteins. Amino acid frequencies were analyzed across the main lineages of jawed vertebrates, and leucine and cysteine were found to be the most and least abundant amino acids in mitochondrial proteins, respectively. Patterns of amino acid replacements were conserved among vertebrates. Overall, cartilaginous fishes showed the least variation in amino acid frequencies and replacements. Constancy of rates of evolution among the main lineages of jawed vertebrates was rejected.     

Complete mitochondrial genome of Pycnonotus xanthorrhous (Passeriformes,Pycnonotidae) and phylogenetic consideration

The complete mitochondrial genome (mitogenome) of Pycnonotus xanthorrhous was sequenced via next generation sequencing. The full length of the circular genome is 16,952 bp. It consists of 37 typical animal mitochondrial genes including 13 protein-coding genes (PCGs), 22 transfer RNA (tRNA) and 2 ribosomal RNA (rRNA) genes. P. xanthorrhous also contains one control region (CR) and one pseudo-control region, and shares the identical gene arrangements with sequenced Pycnonotus spp. which differs from the typical vertebrates gene order. Phylogenetic analyses indicates that Passerida sensu stricto contains three major clades and the core Sylvioidea form a monophyletic group. Furthermore, we investigated the evolution of control region within this lineage and revealed the multiple independent origins of duplicate control region.     

大壁虎线粒体基因组全序列及其结构(英文)

采用长PCR扩增、克隆和引物步行等方法,测定了大壁虎(Gekkogecko)线粒体基因组全序列。序列全长16435bp,共有13个蛋白质编码基因、2个rRNA基因和22个tRNA基因。基因组的组成、顺序、编码链的选择、tRNA的结构、较低的碱基G含量、对碱基T的偏好以及GC和AT偏斜,都与大部分脊椎动物相同或相近。但有些特征揭示了壁虎类的原始性蛋白质编码基因密码子第3位表现为对碱基A的偏好,更接近两栖类和鱼类而不是羊膜动物;标准终止密码子(TAA)只出现于3个蛋白质编码基因中,比大部分脊椎动物少。tRNA基因核苷酸长度为63～76nt,除了tRNACys和tRNASer(AGY)缺少D臂,其余的二级结构均呈典型的三叶草状。     

Complete mitochondrial genomes from four subspecies of common chaffinch (Fringilla coelebs): New inferences about mitochondrial rate heterogeneity,neutral theory,and phylogenetic relationships within the order Passeriformes

We describe whole mitochondrial genome sequences from four subspecies of the common chaffinch (Fringilla coelebs), and compare them to 31 publicly available mitochondrial genome sequences from other Passeriformes. Rates and patterns of mitochondrial gene evolution are analyzed at different taxonomic levels within this avian order, and evidence is adduced for and against the nearly neutral theory of molecular evolution and the role of positive selection in shaping genetic variation of this small but critical genome. We find evidence of mitochondrial rate heterogeneity in birds as in other vertebrates, likely due to differences in mutational pressure across the genome. Unlike in gadine fish and some of the human mitochondrial work we do not observe strong support for the nearly neutral theory of molecular evolution; instead evidence from molecular clocks, distribution of dN/dS ratios at different levels of the taxonomic hierarchy and in different lineages, McDonald–Kreitman tests within Fringillidae, and site-specific tests of selection within Passeriformes, all point to a role for positive selection, especially for the complex I NADH dehydrogenase genes. The protein-coding mitogenome phylogeny of the order Passeriformes is broadly consistent with previously-reported molecular findings, but provides support for a sister relationship between the superfamilies Muscicapoidea and Passeroidea on a short basal internode of the Passerida where relationships have been difficult to resolve. An unexpected placement of the Paridae (represented by Hume's groundpecker) within the Muscicapoidea was observed. Consistent with other molecular studies the mtDNA phylogeny reveals paraphyly within the Muscicapoidea and a sister relationship of Fringilla with Carduelis rather than Emberiza.     

Complete Mitochondrial DNA Sequence of Conger myriaster (Teleostei: Anguilliformes): Novel Gene Order for Vertebrate Mitochondrial Genomes and the Phylogenetic Implications for Anguilliform Families

The complete nucleotide sequence of the mitochondrial genome was determined for a conger eel, Conger myriaster (Elopomorpha: Anguilliformes), using a PCR-based approach that employs a long PCR technique and many fish-versatile primers. Although the genome [18,705 base pairs (bp)] contained the same set of 37 mitochondrial genes [two ribosomal RNA (rRNA), 22 transfer RNA (tRNA), and 13 protein-coding genes] as found in other vertebrates, the gene order differed from that recorded for any other vertebrates. In typical vertebrates, the ND6, tRNA^Glu, and tRNA^Pro genes are located between the ND5 gene and the control region, whereas the former three genes, in C. myriaster, have been translocated to a position between the control region and the tRNA^Phe gene that are contiguously located at the 5′ end of the 12S rRNA gene in typical vertebrates. This gene order is similar to the recently reported gene order in four lineages of birds in that the latter lack the ND6, tRNA^Glu, and tRNA^Pro genes between the ND5 gene and the control region; however, the relative position of the tRNA^Pro to the ND6–tRNA^Glu genes in C. myriaster was different from that in the four birds, which presumably resulted from different patterns of tandem duplication of gene regions followed by gene deletions in two distantly related groups of organisms. Sequencing of the ND5–cyt b region in 11 other anguilliform species, representing 11 families, plus one outgroup species, revealed that the same gene order as C. myriaster was shared by another 4 families, belonging to the suborder Congroidei. Although the novel gene orders of four lineages of birds were indicated to have multiple independent origins, phylogenetic analyses using nucleotide sequences from the mitochondrial 12S rRNA and cyt b genes suggested that the novel gene orders of the five anguilliform families had originated in a single ancestral species. Received: 13 July 2000 / Accepted: 30 November 2000     

Complete sequence and gene organization of the mitochondrial genome of Siamensis Crocodile (<Emphasis Type="Italic">Crocodylus siamensis</Emphasis>)

The complete sequence of mitochondrial genome of Siamensis Crocodile (Crocodylus siamensis) is determined by using PCR amplification, clone and primer-walking sequencing methods. The genome is 16836 bp in size, containing 13 protein-coding, 2 ribosomal and 22 transfer RNA genes. The mtDNA genome of Siamensis Crocodile is similar to most vertebrates in gene component, order, orientation, tRNA structure, low percentage of guanine and high percentage of thymine. The nonstandard stop codes (T) in two protein genes (Cox III and Cyt b) are more than those of most vertebrates. Transfer RNA genes range in length from 61 to 76 nucleotides, and their planar structure present characteristic clover leaf, except for tRNA-Ser (AGY) because of lacking the "DHU" arm.     

Complete mitochondrial genome of the red drum, Sciaenops ocellatus (Perciformes, Sciaenidae): absence of the typical conserved motif in the origin of the light-strand replication

In this study, the complete mitochondrial genome of the red drum Sciaenops ocellatus was determined first. The genome was 16,500?bp in length and contained 13 protein-coding genes, 2 ribosomal RNA genes, 22 transfer RNA genes, and 2 main non-coding regions (the control region and the origin of the light-strand replication); the gene composition and order of which were similar to most other vertebrates. The overall base composition of the heavy strand was T 25.5%, C 30.7%, A 27.5%, and G 16.3%, with a slight AT bias of 53%. Within the control region, the discrete and conserved sequence blocks were identified. Motif 5'-ACCGG-3' rather than 5'-GCCGG-3' was detected in the origin of light-strand replication (O(L)) of red drum, which is rare in the mitogenomes of Sciaenidae species. These results would play an important role in elucidating sequence-function relationships of the O(L).     

The complete mitochondrial genome of the helmet catfish Cranoglanis bouderius (Siluriformes: Cranoglanididae) and the phylogeny of otophysan fishes

The complete sequence of the 16,539 nucleotide mitochondrial genome from the single species of the catfish family Cranoglanididae, the helmet catfish Cranoglanis bouderius, was determined using the long and accurate polymerase chain reaction (LA PCR) method. The nucleotide sequences of C. bouderius mitochondrial DNA have been compared with those of three other catfish species in the same order. The contents of the C. bouderius mitochondrial genome are 13 protein-coding genes, two ribosomal RNA and 22 transfer RNA genes, and a non-coding control region, the gene order of which is identical to that observed in most other vertebrates. Phylogenetic analyses for 13 otophysan fishes were performed using Bayesian method based on the concatenated mtDNA protein-coding gene sequence and the individual protein-coding gene sequence data set. The competing otophysan topologies were then tested by using the approximately unbiased test, the Kishino-Hasegawa test, and the Shimodaira-Hasegawa test. The results show that the grouping ((((Characiformes, Gymnotiformes), Siluriformes), Cypriniformes), outgroup) is the most likely but there is no significant difference between this one and the other alternative hypotheses. In addition, the phylogenetic placement of the family Cranoglanididae among siluriform families was also discussed.     

Complete mitochondrial genome of Trypauchen vagina (Perciformes, Gobioidei)

The complete mitochondrial genome of Trypauchen vagina was determined first. The genome is 16,686 bp in length and consists of 13 protein-coding genes, 22 tRNA genes, 2 ribosomal RNA genes, and 2 main non-coding regions [the control region (CR) and the origin of the light strand replication], the gene composition and order of which was similar to most other vertebrates. The overall base composition of T. vagina is T 27.6%, C 27.6%, A 29.5%, and G 15.3%, with a slight A+T bias of 57.1%. In addition to the discrete and conserved sequence blocks, an incomplete tandem repeat unit is detected within the CR. This mitogenome sequence data would play an important role in population genetics and phylogenetic analysis of the Gobioidei.     

Complete mitochondrial genome of the rabbitfish Siganus fuscescens (Perciformes, Siganidae).

We determined the complete nucleotide sequence of the mitochondrial genome for the rabbitfish Siganus fuscescens (Perciformes, Siganidae). This mitochondrial genome, consisting of 16,491 base pairs (bp), included 13 protein-coding genes, 2 ribosomal RNAs, 22 transfer RNAs, and a noncoding control region similar those found in other vertebrates; the gene order was identical to that of typical vertebrates. Most of the genes of S. fuscescens were encoded on the H-strand, while the ND6 and eight tRNA (Gln, Ala, Asn, Cys, Tyr, Ser [UCN], Glu, and Pro) genes were encoded on the L-strand. The reading frames of ATPase 8 and 6 and those of ND4L and ND4 overlapped by ten and seven nucleotides, respectively. All mitochondrial protein-coding genes began with an ATG start codon, except for CO1, which started with GTG. Open reading frames of S. fuscescens ended with TAA (ND1, CO1, ATPase 8, ND4L, ND5 and ND6), and the remainder had incomplete stop codons, either TA (ATPase 6 and CO3) or T (ND2, CO2, ND3, ND4, and Cytb). The origin of L-strand replication in S. fuscescens was located in a cluster of five tRNA genes (WANCY) and was 34 nucleotides in length. A major noncoding region between the tRNA-Pro and tRNA-Phe genes (828 bp) was considered to be the control region (D-loop). Within this sequence, we identified a conserved sequence block characteristic of this region. The rabbitfish was grouped with Siganus canaliculatus in most parsimony analyses, which showed 100% bootstrap support for their divergence. These findings are useful for inferring phylogenetic relationships and identification within the suborder Acanthuroidei.     

Complete mitochondrial genome of Oxuderces dentatus (Perciformes, Gobioidei)

The complete mitochondrial genome (mitogenome) of Oxuderces dentatus was determined first. The genome was 17,116?bp in length and consisted of 13 protein-coding genes, 22 tRNA genes, 2 ribosomal RNA genes, and 2 main non-coding regions [the control region (CR) and the origin of the light strand replication], the gene composition and order of which was similar to most other vertebrates. The overall base composition of the heavy strand was T 27.9%, C 26.8%, A 30.2%, and G 15.1%, with a slight A+T bias of 58.1%. In addition to the discrete and conserved sequence blocks, unusual long tandem repeat unit (three 150-bp tandem repeat units and an incomplete copy of 146?bp) was also detected within CR. This mitogenome sequence data would play an important role in population genetics and phylogenetic analysis of the Gobioidei.