Sequence evolution in and around the mitochondrial control region in birds

By cloning and sequencing 3.4 kilobases of snow goose mtDNA we found that the ND5 gene is followed by the genes for cytochrome b, tRNA(Thr), tRNA(Pro), ND6, tRNA(Glu), the control region, tRNA(Phe), and srRNA. This order is identical to that of chicken, quail, and duck mtDNA but differs from that of mammals and a frog (Xenopus). The mean extent of difference due to base substitution between goose and chicken is generally closer to the same comparison between rat and mouse but less than that between human and cow. For one of the nine regions compared (tRNA(Glu)), the bird differences appear to be anomalous, possibly implicating altered functional constraints. Within the control region, several short sequences common to mammals are also conserved in the birds. Comparison of the goose control region with that of quail and chicken suggests that a sequence element with similarity to CSB-1 duplicated once prior to the divergence of goose and chicken and again on the lineage leading to chicken. Between goose (or duck) and chicken there are four times more transversions at the third positions of fourfold-degenerate codons in mitochondrial than in nuclear genes.     

Complete mitochondrial genome suggests diapsid affinities of turtles

Despite more than a century of debate, the evolutionary position of turtles (Testudines) relative to other amniotes (reptiles, birds, and mammals) remains uncertain. One of the major impediments to resolving this important evolutionary problem is the highly distinctive and enigmatic morphology of turtles that led to their traditional placement apart from diapsid reptiles as sole descendants of presumably primitive anapsid reptiles. To address this question, the complete (16,787-bp) mitochondrial genome sequence of the African side-necked turtle (Pelomedusa subrufa) was determined. This molecule contains several unusual features: a (TA)n microsatellite in the control region, the absence of an origin of replication for the light strand in the WANCY region of five tRNA genes, an unusually long noncoding region separating the ND5 and ND6 genes, an overlap between ATPase 6 and COIII genes, and the existence of extra nucleotides in ND3 and ND4L putative ORFs. Phylogenetic analyses of the complete mitochondrial genome sequences supported the placement of turtles as the sister group of an alligator and chicken (Archosauria) clade. This result clearly rejects the Haematothermia hypothesis (a sister-group relationship between mammals and birds), as well as rejecting the placement of turtles as the most basal living amniotes. Moreover, evidence from both complete mitochondrial rRNA genes supports a sister-group relationship of turtles to Archosauria to the exclusion of Lepidosauria (tuatara, snakes, and lizards). These results challenge the classic view of turtles as the only survivors of primary anapsid reptiles and imply that turtles might have secondarily lost their skull fenestration.     

Evolution of fragmented mitochondrial ribosomal RNA genes in Chlamydomonas

The fragmented mitochondrial ribosomal RNAs (rRNAs) of the green algae Chlamydomonas eugametos and Chlamydomonas reinhardtii are discontinuously encoded in subgenic modules that are scrambled in order and interspersed with protein coding and tRNA genes. The mitochondrial rRNA genes of these two algae differ, however, in both the distribution and organization of rRNA coding information within their respective genomes. The objectives of this study were (1) to examine the phylogenetic relationships between the mitochondrial rRNA gene sequences of C. eugametos and C. reinhardtii and those of the conventional mitochondrial rRNA genes of the green alga, Prototheca wickerhamii, and land plants and (2) to attempt to deduce the evolutionary pathways that gave rise to the unusual mitochondrial rRNA gene structures in the genus Chlamydomonas. Although phylogenetic analysis revealed an affiliation between the mitochondrial rRNA gene sequences of the two Chlamydomonas taxa to the exclusion of all other mitochondrial rRNA gene sequences tested, no specific affiliation was noted between the Chlamydomonas sequences and P. wickerhamii or land plants. Calculations of the minimal number of transpositions required to convert hypothetical ancestral rRNA gene organizations to the arrangements observed for C. eugametos and C. reinhardtii mitochondrial rRNA genes, as well as a limited survey of the size of mitochondrial rRNAs in other members of the genus, lead us to propose that the last common ancestor of Chlamydomonas algae contained fragmented mitochondrial rRNA genes that were nearly co-linear with conventional rRNA genes.     

16S-23S and 23S-5S intergenic spacer regions of lactobacilli: nucleotide sequence, secondary structure and comparative analysis

Lactobacilli have been used as industrial starters for a long time, but in several cases their identification was, and still is, neither easy nor reliable. The aim of the present work was to examine whether the intergenic spacer regions could be of value in the identification of Lactobacillus species. For that purpose, the polymerase chain reaction (PCR) was used to amplify 16S-23S and 23S-5S spacer regions of Lactobacillus (L.) acidophilus, L. delbrueckii subsp. bulgaricus, L. casei, L. helveticus and L. curvatus. The PCR products were directly sequenced, and two forms of ribosomal RNA (rrn) operons were identified in each species studied: one with tandem tRNA(Ile)/tRNA(Ala) genes and the other one without tRNA genes. Our study revealed that the rrn operons of Lactobacillus species studied comprise the genes of 16S, 23S and 5S rRNA, in that order. Only the tRNA genes and the rRNA processing stems are highly conserved in spacer regions of lactobacilli. The divergence between the lactobacilli spacer region sequences arises from insertions and deletions of short sequences. These sequences could be interesting candidates for the development of species-specific probes. Theoretical RNA/RNA secondary structure models of the interaction between the two spacer region sequences were constructed. In conclusion, the two spacer region sequences may prove to be a useful alternative to 16S and 23S rDNA sequencing for designing species-specific probes and for establishing phylogenetic relationships between closely related species such as L. curvatus and L. casei or L. acidophilus and L. helveticus.     

Gene rearrangements in snake mitochondrial genomes: highly concerted evolution of control-region-like sequences duplicated and inserted into a tRNA gene cluster

Mitochondrial DNA (mtDNA) regions corresponding to two major tRNA gene clusters were amplified and sequenced for the Japanese pit viper, himehabu. In one of these clusters, which in most vertebrates characterized to date contains three tightly connected genes for tRNA(Ile), and tRNA(Gln), and tRNA(Met), a sequence of approximately 1.3 kb was found to be inserted between the genes for tRNA(Ile) and tRNA(Gln). The insert consists of a control-region-like sequence possessing some conserved sequence blocks, and short flanking sequences which may be folded into tRNA(Pro), tRNA(Phe), and tRNA(Leu) genes. Several other snakes belonging to different families were also found to possess a control-region-like sequence and tRNA(Leu) gene between the tRNA(Ile)and tRNA(Gln) genes. We also sequenced a region surrounded by genes for cytochrome b and 12S rRNA, where the control region and genes for tRNA(Pro) and tRNA(Phe) are normally located in the mtDNAs of most vertebrates. In this region of three examined snakes, a control-region-like sequence exists that is almost completely identical to the one found between the tRNA(Ile) and tRNA(Gln) genes. The mtDNAs of these snakes thus possess two nearly identical control-region-like sequences which are otherwise divergent to a large extent between the species. These results suggest that the duplicate state of the control-region-like sequences has long persisted in snake mtDNAs, possibly since the original insertion of the control-region-like sequence and tRNA(Leu) gene into the tRNA gene cluster, which occurred in the early stage of the divergence of snakes. It is also suggested that the duplicated control-region-like sequences at two distant locations of mtDNA have evolved concertedly by a mechanism such as frequent gene conversion. The secondary structures of the determined tRNA genes point to the operation of simplification pressure on the T psi C arm of snake mitochondrial tRNAs.     

Mitochondrial gene rearrangement in the sea cucumber genus Cucumaria

A novel mitochondrial tRNA gene arrangement is described for two species of sea cucumber. The mitochondrial tRNA gene cluster common to sea stars, sea urchins, and the sea cucumber Parastichopus californicus has been significantly modified in the genus Cucumaria as a result of dispersal of the tRNA genes into two separate areas of the genome. The tRNA genes in the novel clusters are interspersed with short unassigned sequences (UASs). Alignment of the two separated novel clusters indicates that the rearrangement was most likely the result of a tandem duplication of approximately 7 kb, encompassing the putative control region, the tRNA cluster, NADH dehydrogenase subunits 1 and 2, the large ribosomal RNA (lrRNA), cytochrome oxidase subunit I, and tRNAArg. Subsequently, deletion of the duplicated lrRNA and protein-coding genes occurred. In addition, the degeneration of one of each of the duplicated tRNA gene pairs has resulted in the interspersed UAS segments observed in each cluster. In contrast, the second copy of the putative control region has been maintained with a very high degree of sequence conservation, suggesting either some functional constraint or concerted evolution for the duplicated element. Analysis of gene organization in other sea cucumber species may provide (1) important insights into the mechanism of mitochondrial gene rearrangements and (2) an informative character set for deep-level phylogenetic analysis of this echinoderm class.     

Comparison between the complete mtDNA sequences of the blue and the fin whale, two species that can hybridize in nature

The sequence of the mitochondrial DNA (mtDNA) molecule of the blue whale (Balaenoptera musculus) was determined. The molecule is 16,402 bp long and its organization conforms with that of other eutherian mammals. The molecule was compared with the mtDNA of the congeneric fin whale (B. physalus). It was recently documented that the two species can hybridize and that male offspring are infertile whereas female offspring may be fertile. The present comparison made it possible to determine the degree of mtDNA difference that occurs between two species that are not completely separated by hybridization incompatibility. The difference between the complete mtDNA sequences was 7.4%. Lengths of peptide coding genes were the same in both species. Except for a small portion of the control region, disruption in alignment was usually limited to insertion/deletion of a single nucleotide. Nucleotide differences between peptide coding genes ranged from 7.1 to 10.5%, and difference at the inferred amino acid level was 0.0-7.9%. In the rRNA genes the mean transition difference was 3.8%. This figure is similar in degree to the difference (3.4%) between the 12S rRNA gene of humans and the chimpanzee. The mtDNA differences between the two whale species, involving both peptide coding and rRNA genes, suggest an evolutionary separation of > or = 5 million years. Although hybridization between more distantly related mammalian species may not be excluded, it is probable that the blue and fin whales are nearly as different in their mtDNA sequences as hybridizing mammal species may be.     

Mitochondrial gene order is not conserved in arthropods: prostriate and metastriate tick mitochondrial genomes

The entire mitochondrial genome was sequenced in a prostriate tick, Ixodes hexagonus, and a metastriate tick, Rhipicephalus sanguineus. Both genomes encode 22 tRNAs, 13 proteins, and two ribosomal RNAs. Prostriate ticks are basal members of Ixodidae and have the same gene order as Limulus polyphemus. In contrast, in R. sanguineus, a block of genes encoding NADH dehydrogenase subunit 1 (ND1), tRNA(Leu)(UUR), tRNA(Leu)(CUN), 16S rDNA, tRNA(Val), 12S rDNA, the control region, and the tRNA(Ile) and tRNA(Gln) have translocated to a position between the tRNA(Glu) and tRNA(Phe) genes. The tRNA(Cys) gene has translocated between the control region and the tRNA(Met) gene, and the tRNA(Leu)(CUN) gene has translocated between the tRNA(Ser)(UCN) gene and the control region. Furthermore, the control region is duplicated, and both copies undergo concerted evolution. Primers that flank these rearrangements confirm that this gene order is conserved in all metastriate ticks examined. Correspondence analysis of amino acid and codon use in the two ticks and in nine other arthropod mitochondrial genomes indicate a strong bias in R. sanguineus towards amino acids encoded by AT-rich codons.     

Complete sequence of the amphioxus (Branchiostoma lanceolatum) mitochondrial genome: relations to vertebrates

The complete nucleotide sequence of the mitochondrial DNA of the amphioxus Branchiostoma lanceolatum has been determined. This mitochondrial genome is small (15 076 bp) because of the short size of the two rRNA genes and the tRNA genes. In addition, this genome contains a very short non-coding region (57 bp) with no sequence reminiscent of a control region. The organisation of the coding genes, as well as of the two rRNA genes, is identical to that of the sea lamprey. Some differences in the repartition of the tRNA genes occur when compared to the lamprey. The mitochondrial codon usage of the amphioxus is reminiscent of that of urochordates since the AGA codon is read as a glycine and not as a stop codon as in vertebrates. Moreover, the base composition at the wobble positions of the codon is strongly biased toward guanine. Altogether, these data clearly emphasise the close relationships between amphioxus and vertebrates, and reinforce the notion that prochordates may be viewed as the brother group of vertebrates.     

A review of orthodontic face-bow injuries and safety equipment

A nucleotide sequence for the tRNA(phe) gene of Carp mitochondria was determined. Sequence data comparisons made among the whale, human, Xenopus laevis, bovine, mouse, chicken and carp, showed that a novel conservative structure was found in the D. stem (dihydrouridine stem), which was known had variant nucleotides in any other vertebrate mitochondrial tRNA and cytoplasmic tRNA genes. This conservative structures contains 13 bp. When we compared the front 7 bp of the conservative structure with the eukaryotic RNA Pol III recognitive A domain, we found these two kinds of different species had partly homologue. As the mitochondrial tRNA(phe) gene is located between the displacement loop and mitochondrial rRNA gene, we inferred that the novel conservative structure might have some extra interesting functions.     

ART-CH: a VL30 in chickens?

The complete sequence of ART-CH, a recently found chicken retrotransposon (A. V. Gudkov, E. A. Komarova, M. A. Nikiforov, and T. E. Zaitsevskaya, J. Virol. 66:1726-1736, 1992), was characterized. ART-CH has the structure of a 3,300-bp-long provirus, including two 388-bp long terminal repeats (LTRs) (U3, 245 bp; R region, 17 bp; and U5, 126 bp), a tRNA(Trp)-binding site, and a polypurine tract, similar to avian leukosis viruses. At least some of the approximately 50 genomic copies of ART-CH are transcribed into polyadenylated RNA, which is initiated and terminated at the expected sites within the LTRs. In contrast to the regulatory sequences involved in proviral expression and replication, the internal regions of ART-CH seem to be completely defective. Several short regions of homology with avian leukosis virus genes, most of which encode gag-related sequences, were found among different reading frames of ART-CH, which are not organized like regular retroviral genes. Both sequence analysis and restriction fragment length polymorphism analysis revealed a high degree of sequence (97% homology) and structural similarity among members of the ART-CH family, indicating their common origin and recent penetration into chicken DNA. ART-CH sequences were detected in mouse cells infected with Rous sarcoma virus produced by an ART-CH-expressing Rous sarcoma. These data are consistent with the hypothesis that ART-CH belongs to a class of defective retrotransposons whose replication strategy requires the use of helper viruses. They might originate from an avian leukosis virus-related retrovirus which completely lost its coding capacities as a result of multiple mutations and deletions. These features apparently group ART-CH with the VL30 retrotransposons of rodents.     

A conserved sequence in tRNA and rRNA promoters of Lactococcus lactis

A tRNA operon (trnA) from Lactococcus lactis consisting of seven tRNA genes and a 5S rRNA gene was cloned and sequenced. Promoter-fusion of the trnA promoter to a promoter-less beta-galactosidase gene of Leuconostoc mesenteroides resulted in high levels of beta-galactosidase activity in L. lactis. Searching for sequences with similarity to the sequence of the promoter region revealed a consensus sequence of promoters preceeding rRNA operons and tRNA operons from Lactococcus species including a not previously described conserved sequence (AGTT).     

The complete mitochondrial DNA (mtDNA) of the donkey and mtDNA comparisons among four closely related mammalian species-pairs

The nucleotide sequence of the complete mitochondrial genome of the donkey, Equus asinus, was determined. The length of the molecule is 16,670 bp. The length, however, is not absolute due to pronounced heteroplasmy caused by variable numbers of two types of repetitive motifs in the control region. The sequence of the repeats is (a) 5'-CACACCCA and (b) 5'-TGCGCGCA, respectively. The order of (a) and (b) can be expressed as {n[2(a)+(b)]+m(a)}. In 32 different clones analyzed the number of n and m ranged from 0 to 9 and 1 to 7. The two rRNA genes, the 13 peptide-coding genes, and the 22 tRNA genes of the donkey and the horse, Equus caballus, were compared in detail. Total nucleotide difference outside the control region was 6.9%. Nucleotide difference between peptide-coding genes ranged from 6.4% to 9.4% with a mean of 8.0%. In the inferred protein sequences of the 13 peptide-coding genes the amino acid difference was 0.2-8.8%, and the mean for the 13 concatenated amino acid sequences was 1.9%. In the 22 tRNA genes, the mean difference was 3.5%, and that in the two rRNA genes was 4.1%. The mtDNA differences between the donkey and the horse suggest that the evolutionary separation of the two species occurred approximately 9 million years ago. Analyses of differences among the mtDNAs of three other species-pairs, harbor seal/grey seal, fin whale/blue whale, and Homo/common chimpanzee, showed that the relative evolutionary rate of individual peptide-coding genes varies among different species-pairs and modes of comparison. The findings show that the superimposition of sequence data of one lineage for resolving and dating evolutionary divergences of other lineages should be performed with caution unless based on comprehensive data.     

Mitochondrial import of only one of three nuclear-encoded glutamine tRNAs in Tetrahymena thermophila

The mitochondrial genome of Tetrahymena does not appear to encode enough tRNAs to perform mitochondrial protein synthesis. It has therefore been proposed that nuclear-encoded tRNAs are imported into the mitochondria. T.thermophila has three major glutamine tRNAs: tRNA(Gln)(UUG), tRNA(Gln)(UUA) and tRNA(Gln)(CUA). Each of these tRNAs functions in cytosolic translation. However, due to differences between the Tetrahymena nuclear and mitochondrial genetic codes, only tRNA(Gln)(UUG) has the capacity to function in mitochondrial translation as well. Here we show that approximately 10-20% of the cellular complement of tRNA(Gln)(UUG) is present in mitochondrial RNA fractions, compared with 1% or less for the other two glutamine tRNAs. Furthermore, this glutamine tRNA is encoded only by a family of nuclear genes, the sequences of several of which are presented. Finally, when marked versions of tRNA(Gln)(UUG) and tRNA(Gln)(UUA) flanked by identical sequences are expressed in the macronucleus, only the former undergoes mitochondrial import; thus sequences within tRNA(Gln)(UUG) direct import. Because tRNA(Gln)(UUG) is a constituent of mitochondrial RNA fractions and is encoded only by nuclear genes, and because ectopically expressed tRNA(Gln)(UUG) fractionates with mitochondria like its endogenous counterpart, we conclude that it is an imported tRNA in T.thermophila.     

23S rRNA positions essential for tRNA binding in ribosomal functional sites

rRNA plays an important role in function of peptidyl transferase, the catalytic center of the ribosome responsible for the peptide bond formation. Proper placement of the peptidyl transferase substrates, peptidyl-tRNA and aminoacyl-tRNA, is essential for catalysis of the transpeptidation reaction and protein synthesis. In this report, we define a small set of rRNA nucleotides that are most likely directly involved in binding of tRNA in the functional sites of the large ribosomal subunit. By binding biotinylated tRNA substrates to randomly modified large ribosomal subunits from Escherichia coli and capturing resulting complexes on the avidin resin, we identified four nucleotides in the large ribosomal subunit rRNA (positions G2252, A2451, U2506, and U2585) whose modifications prevent binding of a peptidyl-tRNA analog in the P site and one residue (U2555) whose modification interferes with transfer of peptidyl moiety to puromycin. These nucleotides represent a subset of positions protected by tRNA analogs from chemical modification and significantly narrow the number of 23S rRNA nucleotides that may be directly involved in tRNA binding in the ribosomal functional sites.