Automated chemical synthesis of biologically active tRNA having a sequence corresponding to Ascaris suum mitochondrial tRNA(Met) toward NMR measurements

RNA samples corresponding to Ascaris suum mitochondrial tRNA(Met) were chemically and automatically synthesized in amounts sufficient for NMR measurement. Conventional and rapid deprotection methods gave tRNA samples with the same amino acid-accepting activity as those prepared by other method; enzymatic synthesis, and enzymatic ligation of chemically synthesized fragments. The synthetic tRNA showed the same 1H-NMR spectrum in the iminoproton region as the ligated tRNA. This rapid and reliable preparation method thus provides biologically active tRNA for NMR measurement, and further, it is applicable for synthesis of other large synthetic RNAs, by combining the site-specific isotopic labeling method.     

Ascidian mitochondrial tRNA(Met) possessing unique structural characteristics

Methionine tRNA was purified from muscle mitochondria of the ascidian Halocynthia roretzi and its RNA sequence was determined. Analysis of the nucleotide sequence revealed that unlike most metazoan mitochondrial tRNAs(Met), which have a highly conserved cytidine (C) or C-derivative at the wobble position, the H. roretzi mitochondrial tRNA(Met) possesses 5-carboxymethylaminomethyluridine (cmnm5U) at the first position of the anticodon. This is the first report of a single mitochondrial tRNA(Met) species having uridine (U) or a U-derivative at the wobble position.     

Effects of scatter and attenuation correction on quantitative assessment of regional cerebral blood flow with SPECT

The pyruvate dehydrogenase complex (PDC) plays a key role in the anaerobic mitochondrial metabolism of the parasitic nematode Ascaris suum. A cDNA coding for an A. suum pyruvate dehydrogenase kinase (APDK) has been cloned and sequenced from poly(A)+ RNA isolated from adult A. suum muscle.2 APDK exhibited significant sequence identity to mammalian PDKs. Nucleotide sequence analysis of the APDK cDNA revealed a 22-nucleotide spliced leader, characteristic of many nematode mRNAs, a 5'-UTR of 6 nucleotides, an open reading frame of 1197 nucleotides, and a 3'-UTR of 101 nucleotides that included a putative polyadenylation signal. The open reading frame predicted a protein of 399 amino acids with a molecular weight of 45,402 that included a putative 18-aminoacid leader peptide. Recombinant APDK (rAPDK) was functionally expressed in Escherichia coli with a his tag at its N-terminus and purified to apparent homogeneity on Ni-NTA-agarose. Recombinant APDK was a dimer and was not autophosphorylated and its activity was stimulated in the presence of APDK-deficient adult A. suum muscle PDC presumably by the binding of APDK to the dihydrolipoyl transacetylase (E2) core of the complex. After binding to the core, rAPDK activity was stimulated by elevated NADH/NAD+ and acetyl CoA/CoA ratios within the same ranges as observed for the native APDK. Immunoblotting suggested that native APDK focused as a series of 43-kDa spots (pI 6.1-6.8) on two-dimensional gels of the purified adult A. suum muscle PDC.     

5-formylcytidine (f5C) found at the wobble position of the anticodon of squid mitochondrial tRNA(Met)CAU

In squid (Loligo breekeri) mitochondria, AUA codons are translated as methionine instead of the universal isoleucine. Here, we present the nucleotide sequence of squid mitochondrial tRNA(Met)CAU. This tRNA(Met)CAU has 5-formylcytidine (f5C) at the wobble position of the anticodon, though it is partially modified. This result indicates the common feature with bovine and nematoda mitochondrial systems in that f5C at the wobble position of the anticodon is very likely involved in translation of AUA codons as methionine in squid mitochondria.     

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 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.     

Expression of bovine mitochondrial tRNASer GCU derivatives in Escherichia coli

By replacing a stretch of five A-U base pairs in the acceptor stem with G-C pairs, mitochondrial tRNA-SerGCU lacking a D arm could be expressed in Escherichia coli cells in considerable amounts. The expressed tRNA with no modified nucleoside was serylated in vitro with the mitochondrial enzyme. The tRNASerGCU derivatives carrying identity elements for alanine tRNA and the related anticodons were expressed. However, this expression event did not affect cell growth, probably because the expression started from the late log phase, which suggests that these mitochondrial tRNA derivatives are not involved in E.coli gene expression systems. Although there are some restrictions in the secondary structure of tRNAs that can be expressed by this method, it could prove useful for preparing large amounts of heterologous tRNAs in vivo.     

Amber suppression in Escherichia coli by unusual mitochondria-like transfer RNAs

The "cloverleaf" base-pairing pattern was established as the structural paradigm of active tRNA species some 30 years ago. Nevertheless, this pattern does not accommodate the folding of certain mitochondrial tRNAs. For these recalcitrant tRNAs, we have proposed structures having from 5 to 10 base pairs in the anticodon stem rather than the canonical 6. The absence of these types of tRNAs in cytoplasmic translation systems, however, raises the possibility that they may not be bona fide alternate folding patterns for active tRNA molecules. For this reason, we have designed new tRNA genes based on our model of unusual mitochondrial tRNAs, having 7, 8, 9, and 10 base pairs in the anticodon stem with other modifications to the D-stem and connector regions. We show here that these synthetic genes produce tRNAs that actively suppress amber codons in vivo.     

Evolution of a mitochondrial tRNA PHE gene in A. thaliana: import of cytosolic tRNA PHE into mitochondria

Previously we have described a putative tRNATyr in Arabidopsis thaliana mitochondria, the sequence of which is different from that of other plant mitochondrial tRNATyr genes. We show here that this tRNATyr gene sequence is present in several copies in the mitochondrial genome of A. thaliana. One copy of these tRNATyr gene sequences, termed here tRNATyr-1, could encode a functional tRNA. Expression analysis has shown that the tRNATyr-1 gene is cotranscribed with the downstream tRNAGlu gene, and that the corresponding mature-sized tRNA is present in mitochondria. We also show that the native tRNATyr gene, similar to the mitochondrial tRNATyr genes found in plants, is present in the A. thaliana mitochondrial genome and expressed. The tRNATyr-1 gene has been previously suggested to be derived from a tRNAPhe gene sequence. We show here that, as a consequence, there is no tRNAPhe gene in the mitochondrial genome of A. thaliana and that a cytosolic tRNAPhe is imported in A. thaliana mitochondria.     

A mitochondrial tRNA(Val) gene mutation (G1642A) in a patient with mitochondrial myopathy, lactic acidosis, and stroke-like episodes

We studied a patient with the diagnosis of mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS) for mitochondrial DNA mutations in muscle. Established MELAS mutations were excluded. Mitochondrial DNA was further analyzed for mutations in the 22 tRNA genes by single-strand conformation polymorphism (SSCP) analysis; a tRNA(Val) mutation (G1642A) was found. The structure of the altered tRNA, the heteroplasmy, and the absence of the mutation in the mother and in 100 control subjects suggests that the tRNA(Val) mutation is associated with the MELAS syndrome.     

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.     

Stochastic context-free grammars for tRNA modeling

Stochastic context-free grammars (SCFGs) are applied to the problems of folding, aligning and modeling families of tRNA sequences. SCFGs capture the sequences' common primary and secondary structure and generalize the hidden Markov models (HMMs) used in related work on protein and DNA. Results show that after having been trained on as few as 20 tRNA sequences from only two tRNA subfamilies (mitochondrial and cytoplasmic), the model can discern general tRNA from similar-length RNA sequences of other kinds, can find secondary structure of new tRNA sequences, and can produce multiple alignments of large sets of tRNA sequences. Our results suggest potential improvements in the alignments of the D- and T-domains in some mitochondrial tRNAs that cannot be fit into the canonical secondary structure.     

In vivo import of unspliced tRNATyr containing synthetic introns of variable length into mitochondria of Leishmania tarentolae

The mitochondrial genomes of trypanosomatids lack tRNA genes. Instead, mitochondrial tRNAs are encoded and synthesized in the nucleus and are then imported into mitochondria. This also applies for tRNATyr, which in trypanosomatids contains an 11 nt intron. Previous work has defined an exon mutation which leads to accumulation of unspliced precursor tRNATyr. In this study we have used the splicing-deficient tRNATyr as a vehicle to introduce foreign sequences into the mitochondrion of Leishmania tarentolae. The naturally occurring intron was replaced by synthetic sequences of increasing length and the resulting tRNATyr precursors were expressed in transgenic cell lines. Whereas stable expression of precursor tRNAsTyr was obtained for introns up to a length of 76 nt, only precursors having introns up to 38 nt were imported into mitochondria. These results demonstrate that splicing-deficient tRNATyr can be used to introduce short synthetic sequences into mitochondria in vivo. In addition, our results show that one factor which limits the efficiency of import is the length of the molecule.     

A correlation between N2-dimethylguanosine presence and alternate tRNA conformers

Even though the evolutionary conservation of the cloverleaf model is strongly suggestive of powerful constraints on the secondary structure of functional tRNAs, some mitochondrial tRNAs cannot be folded into this form. From the optimal base pairing pattern of these recalcitrant tRNAs, structural correlations between the length of the anticodon stem and the lengths of connector regions between the two helical domains, formed by the coaxial stacking of the anticodon and D-stems and the acceptor and T-stems, have been derived and used to scan the tRNA and tRNA gene database. We show here that some cytosolic tRNA gene sequences that are compatible with the cloverleaf model can also be folded into patterns proposed for the unusual mitochondrial tRNAs. Furthermore, the ability to be folded into these atypical structures correlates in the mature RNA sequences with the presence of dimethylguanosine, whose role may be to prevent the unusual mitochondrial tRNA pattern folding.     

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.