Yoshitaka Suyama

Linear mitochondrial genomes: 30 years down the line

At variance with the earlier belief that mitochondrial genomes are represented by circular DNA molecules, a large number of organisms have been found to carry linear mitochondrial DNA. Studies of linear mitochondrial genomes might provide a novel view on the evolutionary history of organelle genomes and contribute to delineating mechanisms of maintenance and functioning of telomeres. Because linear mitochondrial DNA is present in a number of human pathogens, its replication mechanisms might become a target for drugs that would not interfere with replication of human circular mitochondrial DNA.

Comparative studies on mitochondrial and cytoplasmic ribosomes of Tetrahymena pyriformis

Physico-chemical properties of Tetrahymena mitochondrial and cytoplasmic ribosomes and their components, rRNA† and proteins were investigated. The mitochondrial and cytoplasmic ribosomes were 80 s and indistinguishable in sedimentation analysis at 10−2m-Mg2+. However, these ribosomes exhibit different sensitivity to Mg2+ concentrations. The cytoplasmic ribosomes consist of 60 and 40 s subunits, while the mitochondrial ribosomes produced only one sedimenting species 55 s in low Mg2+- or EDTA-containing buffer. Buoyant densities of mitochondrial and cytoplasmic 80 s ribosomes and respective subunits after formaldehyde fixation showed reproducible differences. Mitochondrial 80 s showed a density 1.46 g/cm3, and two density species, I = 1.52 g/cm3 and II = 1.46 g/cm3 were obtained in a ratio of 1:2 in amount from the 55 s subunit, while the cytoplasmic 80 s ribosomes had a density 1.56 g/cm3 and their subunits 60 and 40 s had densities 1.57 and 1.53 g/cm3, respectively. Electrophoretic differences exist between proteins isolated from mitochondrial and cytoplasmic 80 s ribosomes in polyacrylamide gel columns. Mitochondrial and cytoplasmic rRNAs isolated from their respective 80 s ribosomes were 21 and 14 s, and 26 and 17 s, respectively. 5 and 4 s RNAs were also present. Base compositions of mitochondrial and cytoplasmic rRNAs were different. Furthermore, the 21 and 14 s mitochondrial rRNAs were complementary to 3.8 and 1.9% of the mitochondrial DNA, respectively. No significant complementarity exists between mitochondrial DNA and cytoplasmic rRNAs. These results established that Tetrahymena mitochondria contain unique ribosomes separate from the cytoplasmic ribosomes.

Two dimensional polyacrylamide gel electrophoresis analysis of Tetrahymena mitochondrial tRNA

SummaryTwo dimensional (2D) urea-polyacrylamide gel electrophoresis of tRNA isolated from Tetrahymena mitochondria separated at least 36 spots, while more than 45 major and minor spots were resolved with cytosolic tRNA. Co-electrophoresis of mitochondrial and cytosolic tRNAs revealed that many spots co-migrate. When radioactive mitochondrial tRNA was hybridized to mtDNA under various conditions and tRNA melted from the hybrid was analyzed by 2D gel electrophoresis, only 10 tRNA spots were found. Identified as mtDNA-encoded were 2 spots for tRNAleu, 2 for tRNAmet, and 1 each for tRNAphe, tRNAtrp and tRNAtyr. The remaining three were unidentified. Mitochondrial tRNA spots that correspond to the tRNAs for arg, gly, ile, lys, ser, and val do not hybridize with mtDNA, and in gel positions they correspond to the cytoplasmic tRNA spots for the same respective amino acids. These mitochondrial tRNAs isolated from the gel can be acylated either by the mitochondrial or cytostolic enzymes. Mitochondrial tRNA isolated from a Tetrahymena cell homogenate which was pretreated with RNase A and Micrococcus nuclease exhibited the same 2D gel pattern as a nontreated control. Mitochondrial tRNAs from old and young cells showed generally similar tRNA spots in 2D gels, though more variable spots were seen with old cells. 3H-labeled whole-cell tRNA added to the cell homogenate prior to the mitochondrial isolation procedure did not remain associated with the final mitochondrial tRNA preparation. The present studies also showed mitochondrial tRNAs bound to the mitochondrial 80S monosome and polysome fractions. Radioactive tRNA added to the mitochondrial lysate does not adhere to the ribosomes, suggesting that the ribosome-bound tRNAs are not contaminating cytoplasmic tRNAs. These results are generally in good agreement with our previous data showing that only a small number of tRNAs are coded for by the mitochondrial DNA, while the others are a selected set of imported cytoplasmic tRNAs.

Native and imported transfer rna in mitochondria

Tetrahymena mitochondrial and cytoplasmic tRNAs for valine, lysine, arginine and phenylalanine produced identical or different isoacceptor patterns on reversed-phase chromatography. Positive bindings of individual isoacceptors to Escherichia coli ribosomes were demonstrated in response to trinucleoside diphosphates and polynucleotides. It was shown that different isoacceptors exhibited different but degenerate coding recognition patterns. One of two phenylalanyl-tRNA species in mitochondria, however, showed no recognition toward either phenylalanine codon triplets or poly(U). Under suitable annealing conditions, two phenylalanyl-tRNA species could hybridize with mitochondrial DNA. The other mitochondrial tRNAs for valine, lysine and arginine showed no hybridization with mitochondrial DNA but produced significant binding to nuclear DNA-immobilized filters. From these and other coding data presented here, it is postulated that there are two classes of tRNA in Tetrahymena pyriformis mitochondria; the one, “native” tRNA, is mitochondrial DNA-transcript whose coding specificity is unique to mitochondrial tRNA. The other, termed “imported” tRNA, is transcribed from the nuclear DNA and probably transported into mitochondria. This class of tRNA exhibits the same or similar coding specificity as the corresponding cytoplasmic tRNA.

A nuclear tRNA gene cluster in the protozoan Leishmania tarentolae and differential distribution of nuclear-encoded tRNAs between the cytosol and mitochondria.

All mitochondrial tRNAs in the protozoan Leishmania are believed to be encoded in the nuclear genome and imported selectively into the mitochondria by an as yet unknown mechanism. Previously, we reported that two tRNAs whose genes are tightly linked were imported by mitochondria. In contrast, a tRNA encoded by a lone tRNA gene was not detectable in mitochondria. The lone tRNA gene had flanking sequences that were different from the linked genes. These studies implied a possible correlation between tRNA gene organization and gene flanking sequence, and selective tRNA import into mitochondria. Here, we report the identification of a cluster of 10 tRNA genes and show the distribution of the corresponding tRNAs in cytosolic and mitochondrial fractions. tRNA(leu)(CAG) and tRNA2(arg)(TCG) are abundant in the cytosol, but relatively scarce in mitochondria. Conversely, tRNA(ile)(TAT) and tRNA1(lys)(TTT) are abundant in mitochondria, but relatively scarce in the cytosol. tRNA(val)(TAC) and tRNA2(thr)(TGT) are barely detectable in either cellular compartment, while tRNA(gln)(TTG), tRNA1(arg)(ACG), tRNA(gly)(TCC), and tRNA(trp)(CCA) are detected in approximately equal levels in both compartments. Sequencing of the 2600 bp that comprise the tRNA gene cluster also encoding the genes for 5S RNA and URNAB RNA indicates that nucleotide composition, length, and location of genes within the cluster do not clearly correlate with import characteristics. The unexpected presence of the tRNA(trp)(CCA)-gene transcript in mitochondria is also reported. Evidence suggests that this tRNA may have unidentified base modifications at the anticodon triplet.

Restriction map of the mitochondrial DNA of the true slime mould, Physarum polycephalum : linear form and long tandem duplication

SummaryThe mitochondrial DNA (mtDNA) of the true slime mould, Physarum polycephalum strain CH934xCH938, was isolated and characterized by restriction mapping. Cloned fragments of the mtDNA were assembled and used to construct the restriction map. This map showed that the mtDNA was a linear molecule of 86.0 kb with a tandem duplication of 19.6 kb. The terminal fragments were identified by sensitivity to Bal31 exonuclease. One of the duplications was located at the right end and the other was located 5 kb from the left end. Each duplicated segment contained 26 restriction sites for ten enzymes and these restriction sites were completely conserved in each duplication. Genes for the large and small rRNAs were mapped to positions about 30 kb from the right end of the mtDNA by hybridization with its own rRNAs. With the exception of a probe for the gene for the large rRNA in Tetrahymena pyriformis mtDNA, various probes from the mtDNAs of Saccharomyces cerevisiae and T. pyriformis showed no significant hybridization to any of the restriction fragments of the mtDNA from P. polycephalum.

Protein synthesis in vitro with Tetrahymena mitochondrial ribosomes

Abstract Phenylalanine incorporation studies in vitro have established that 80-S ribosomes from Tetrahymena mitochondria function in protein synthesis. Rate of incorporation of [ 14 C]phenylalanine in the presence of poly(U) was approx. 126 pmoles/h per mg ribosomal protein. Incorporation was sensitive to chloramphenicol but not to cycloheximide. Mitochondrial ribosomes were found to be functionally distinct from cytoplasmic ribosomes and capable of synthesizing high-molecular-weight proteins in the absence of poly(U).

The cytochrome oxidase subunit I gene of Tetrahymena: a 57 amino acid NH2-terminal extension and a 108 amino acid insert

SummaryThe gene sequence for cytochrome oxidase subunit I (COI) in the ciliate Tetrahymena mitochondrial DNA has been determined and shown to be coded by the same strand as codes the genes (in order) for 14S rRNA, tRNAtrp tRNAglu 21S rRNA, tRNAleu and tRNAmet. The predicted protein has 698 amino acids, including an NH2-terminal 57 amino acid extension and a 108 amino acid insert originally found in Paramecium COI. These extension and insert segments are not highly hydrophobic but are relatively rich in lysine, arginine and serine. In analogy with the presequence of nuclear-encoded mitochondrial proteins, they might function as a transmembrane signal. The remaining poly-peptide segments show a hydrophobicity characteristic of membrane spanning proteins. TCOI shows a 64% amino acid identity with Paramecium COI but less than a 38% amino acid conservation with human COI. The Tetrahymena mitochondrial code is analogous with the mammalian mitochondrial code; but differs from the Tetrahymena nuclear genetic code; TGA is exclusively translated as tryptophan; ATA is used as an initiation codon probably for methionine, and TAA as a stop codon; the arginine codons (CGN) are not used. The use of the leucine codon TTA in TCOI is contradictory to the codon recognition pattern previously obtained from the isolated tRNAleu isoacceptors recognizing only the CUN codons, but consistent with the tRNAleu (anticodon UAA) gene encoded in the genome. The reason for this inconsistency has not been resolved.

Three isoaccepting forms of leucyl transfer RNA in mitochondria

Abstract This paper presents the evidence that Tetrahymena pyriformis mitochondria contain three isoaccepting leucyl tRNA species differing in (1) charging responses to the mitochondrial and cytoplasmic leucyl tRNA synthetases, (2) melting temperatures of hybrids between tRNA Leu species and mitochondrial DNA and (3) codon recognition patterns. Reversed-phase column chromatography of whole-cell tRNA from Tetrahymena resolves at least six isoaccepting leucyl tRNA species. Out of these, three species (II, V and VI) are found in mitochondria and preferentially charged by the mitochondrial leucyl tRNA synthetase. Species II and VI are not acylatable with the cytoplasmic enzyme, while species V can be charged with this enzyme. Three individual tRNA Leu species form stable hybrids with mitochondrial DNA, whose melting temperatures ( T m ) are: 44 °C (II), 52 °C (V) and 51·5 °C (VI) in 50% formamide, 0·30 m -NaCl, 0·03 m -sodium citrate, 0·05 m -ammonium acetate. Competitive hybridization between these tRNAs and DBAE-cellulose chromatography of RNase T 1 digests of three tRNA Leu species indicate the existence of considerable sequence homology at their —CCA terminal regions. Binding studies of individual tRNA Leu species to six leucine triplets and polynucleotides, poly(U), poly(U 2 C) and poly(U 7 G), showed that three mitochondrial and two cytoplasmic tRNA Leu species all responded to different leucine codons. The significance of these findings in relation to the function and transcriptional origin of mitochondrial tRNA is discussed.

Regulated tRNA import in Leishmania mitochondria

The genes for three new tRNA and a 5S RNA were identified from a genomic DNA clone of 917 nucleotide pairs from the protozoon Leishmania tarentolae. They were encoded in the following order. The transcriptional directions and anticodons are in parentheses: tRNA(Val) (CAC-->)-5SRNA (-->)-tRNA(His) (<--GUG)-tRNA(Phe) (GAA-->). The tRNA(His) and tRNA(Phe) sequences have not been reported previously in trypanosomatid organisms. By northern analysis, tRNA(Val) and tRNA(Phe) were equally distributed between the cytosol and mitochondria, while tRNA(His) was less abundant in mitochondria than in the cytosol. Accordingly, the latter tRNA is classified as Import restricted (Impr). As shown before, 5S RNA was not imported. Recently, Mahapatra and Adhya [S. Mahapatra, T. Ghosh, S. Adhya, Nucl. Acids Res. 22 (1994) 3381-3386; S. Mahapatra, S. Adhya, J. Biol. Chem. 271 (1996) 20432-20437] have developed an in vitro import system in Leishmania and suggested that the D-loop sequence could serve as the import determinant. We examined all available tRNA gene sequences in trypanosomatids but found no apparent consensus within the D-loop that might account for tRNA-import regulation.