Mitochondrial myopathy associated with a novel 5522G>A mutation in the mitochondrial tRNATrp gene

We report a novel pathogenic mutation of the mitochondrial transfer RNA (tRNA) gene for tryptophan in a patient with isolated myopathy and persistently elevated creatine kinase. Muscle studies revealed ragged red fibres and decreased activity of respiratory chain complex I and cytochrome c oxidase (COX). Sequencing of the 22 mitochondrial tRNA genes revealed a mutation m.5522G>A, which alters a conserved base pairing in the D-stem of the tRNA for tryptophan. The mutation was heteroplasmic with a mutational load between 88 and 99% in COX-negative fibres. This case contributes to the genetic heterogeneity of mitochondrial diseases caused by mutations in mitochondrial tRNA genes.     

Functional consequences of mitochondrial tRNATrp and tRNAArg mutations causing combined OXPHOS defects

Combined oxidative phosphorylation (OXPHOS) system deficiencies are a group of mitochondrial disorders that are associated with a range of clinical phenotypes and genetic defects. They occur in approximately 30% of all OXPHOS disorders and around 4% are combined complex I, III and IV deficiencies. In this study we present two mutations in the mitochondrial tRNA^Trp (MT-TW) and tRNA^Arg (MT-TR) genes, m.5556G>A and m.10450A>G, respectively, which were detected in two unrelated patients showing combined OXPHOS complex I, III and IV deficiencies and progressive multisystemic diseases. Both mitochondrial tRNA mutations were almost homoplasmic in fibroblasts and muscle tissue of the two patients and not present in controls. Patient fibroblasts showed a general mitochondrial translation defect. The mutations resulted in lowered steady-state levels and altered conformations of the tRNAs. Cybrid cell lines showed similar tRNA defects and impairment of OXPHOS complex assembly as patient fibroblasts. Our results show that these tRNA^Trp and tRNA^Arg mutations cause the combined OXPHOS deficiencies in the patients, adding to the still expanding group of pathogenic mitochondrial tRNA mutations.     

TGA-Termination codon in the apocytochrome b gene from Oenothera mitochondria

Summary The apocytochrome b gene in the mitochondrial genome of Oenothera contains 1,185 base pairs (bp) in uninterrupted sequence. Comparison with the maize sequence indicates firstly that at the 5 end of the gene an additional triplet, coding for alanine, is inserted immediately behind the initiator ATG. Secondly the carboxy-terminus of the polypeptide is extended by five amino acids. The only in-frame termination codon for the Oenothera gene is a TGA triplet 15 nucleotides downstream from the maize TAG termination, which corresponds to a GAG codon in Oenothera. The plant mitochondria) tryptophan codon CGG and TGG appear to be interchangable although a bias for TGG is observed in this gene (12:1). At position 718–720 in the Oenothera nucleotide sequence TGG is found where a CGG occurs in maize, and nucleotide 361 in Oenothera gene is a T, generating a TGG codon where in maize a CGG codon is found.     

Expression pattern of mitochondrial respiratory chain enzymes in skeletal muscle of patients with mitochondrial myopathy associated with the homoplasmic m.14674T>C variant

Two homoplasmic variants in tRNA^Glu (m.14674T>C/G) are associated with reversible infantile respiratory chain deficiency. This study sought to further characterize the expression of the individual mitochondrial respiratory chain complexes and to describe the natural history of the disease. Seven patients from four families with mitochondrial myopathy associated with the homoplasmic m.14674T>C variant were investigated. All patients underwent skeletal muscle biopsy and mtDNA sequencing. Whole‐genome sequencing was performed in one family. Western blot and immunohistochemical analyses were used to characterize the expression of the individual respiratory chain complexes. Patients presented with hypotonia and feeding difficulties within the first weeks or months of life, except for one patient who first showed symptoms at 4 years of age. Histopathological findings in muscle included lipid accumulation, numerous COX‐deficient fibers, and mitochondrial proliferation. Ultrastructural abnormalities included enlarged mitochondria with concentric cristae and dense mitochondrial matrix. The m.14674T>C variant in MT‐TE was identified in all patients. Immunohistochemistry and immunoblotting demonstrated pronounced deficiency of the complex I subunit NDUFB8. The expression of MTCO1, a complex IV subunit, was also decreased, but not to the same extent as NDUFB8. Longitudinal follow‐up data demonstrated that not all features of the disorder are entirely transient, that the disease may be progressive, and that signs and symptoms of myopathy may develop during childhood. This study sheds new light on the involvement of complex I in reversible infantile respiratory chain deficiency, it shows that the disorder may be progressive, and that myopathy can develop without an infantile episode.     

Location,identity, amount and serial entry of chloroplast DNA sequences in crucifer mitochondrial DNAs

Southern blot hybridization techniques were used to examine the chloroplast DNA (cpDNA) sequences present in the mitochondrial DNAs (mtDNAs) of two Brassica species (B. campestris and B. hirta), two closely related species belonging to the same tribe as Brassica (Raphanus sativa, Crambe abyssinica), and two more distantly related species of crucifers (Arabidopsis thaliana, Capsella bursa-pastoris). The two Brassica species and R. sativa contain roughly equal amounts (12–14 kb) of cpDNA sequences integrated within their 208–242 kb mtDNAs. Furthermore, the 11 identified regions of transferred DNA, which include the 5 end of the chloroplast psaA gene and the central segment of rpoB, have the same mtDNA locations in these three species. Crambe abyssinica mtDNA has the same complement of cpDNA sequences, plus an additional major region of cpDNA sequence similarity which includes the 16S rRNA gene. Therefore, except for the more recently arrived 16S rRNA gene, all of these cpDNA sequences appear to have entered the mitochondrial genome in the common ancestor of these three genera. The mitochondrial genomes of A. thaliana and Capsella bursa-pastoris contain significantly less cpDNA (5–7 kb) than the four other mtDNAs. However, certain cpDNA sequences, including the central portion of the rbcL gene and the 3 end of the psaA gene, are shared by all six crucifer mtDNAs and appear to have been transferred in a common ancestor of the crucifer family over 30 million years ago. 1n conclusion, DNA has been transferred sequentially from the chloroplast to the mitochondrion during crucifer evolution and these cpDNA sequences can persist in the mitochondrial genome over long periods of evolutionary time.     

Composition and formation of heterochromatin in Arabidopsis thaliana

The term heterochromatin has been applied to both large-scale, microscopically visible chromocentres and small-scale, silent genes located outside chromocentres. This may cause confusion in the interpretation of epigenetic marks for both features. The model plant Arabidopsis thaliana provides an excellent system to investigate composition and function of chromatin states at different levels of organization. In this review we will discuss recent developments in molecular networks underlying gene silencing and the relationship with visible heterochromatin in Arabidopsis.     

Study of the WFS1 gene and mitochondrial DNA in Spanish Wolfram syndrome families

Wolfram syndrome (WS) is an autosomal recessive neurodegenerative disorder characterized by early onset diabetes mellitus and progressive optic atrophy. Patients with WS frequently develop deafness, diabetes insipidus, renal tract abnormalities, and diverse psychiatric illnesses, among others. A gene responsible for WS was identified on 4p16.1 (WFS1). It encodes a putative 890 amino acid transmembrane protein present in a wide spectrum of tissues. A new locus for WS has been located on 4q22-24, providing evidence for the genetic heterogeneity of this syndrome. Six Spanish families with a total of seven WS patients were screened for mutations in the WFS1-coding region by direct sequencing. We found three previously undescribed mutations c.873C > A, c.1949_50delAT, and c.2206G > C, as well as the duplication c.409_424dup16, formerly published as 425ins16. Several groups had detected deletions in the mitochondrial DNA (mtDNA) of WS patients. For this reason, we also studied the presence of mtDNA rearrangements as well as Leber's hereditary optic neuropathy, mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes, and A1555G point mutations in the WS families. No mtDNA abnormalities were detected.     

Evolution of mitochondrial DNA in yeast: gene order and structural organization of the mitochondrial genome of Saccharomyces uvarum

We have determined the size, the restriction map and the gene order of the mitochondrial genome of the yeast Saccharomyces uvarum. Sequence analysis of the mitochondrial COXII gene confirmed the position of this yeast in the Saccharomyces cerevisiae-like group, near Saccharomyces cerevisiae and Saccharomyces douglasii. Most mitochondrial genes have been positioned on this approximately 57-kb long genome and three regions containing putative replication origins have been identified. The gene order of S. uvarum suggests that the mitochondrial genome of the S.cerevisiae-like yeasts could have evolved from an ancestral molecule, similar to that of S. uvarum, through specific genome rearrangements. Received: 22 April / 2 September 1997     

Yeast mitochondrial Gln-tRNAGln is generated by a GatFAB-mediated transamidation pathway involving Arc1p-controlled subcellular sorting of cytosolic GluRS

It is impossible to predict which pathway, direct glutaminylation of tRNA^Gln or tRNA-dependent transamidation of glutamyl-tRNA^Gln, generates mitochondrial glutaminyl-tRNA^Gln for protein synthesis in a given species. The report that yeast mitochondria import both cytosolic glutaminyl-tRNA synthetase and tRNA^Gln has challenged the widespread use of the transamidation pathway in organelles. Here we demonstrate that yeast mitochondrial glutaminyl-tRNA^Gln is in fact generated by a transamidation pathway involving a novel type of trimeric tRNA-dependent amidotransferase (AdT). More surprising is the fact that cytosolic glutamyl-tRNA synthetase (_cERS) is imported into mitochondria, where it constitutes the mitochondrial nondiscriminating ERS that generates the mitochondrial mischarged glutamyl-tRNA^Gln substrate for the AdT. We show that dual localization of _cERS is controlled by binding to Arc1p, a tRNA nuclear export cofactor that behaves as a cytosolic anchoring platform for _cERS. Expression of Arc1p is down-regulated when yeast cells are switched from fermentation to respiratory metabolism, thus allowing increased import of _cERS to satisfy a higher demand of mitochondrial glutaminyl-tRNA^Gln for mitochondrial protein synthesis. This novel strategy that enables a single protein to be localized in both the cytosol and mitochondria provides a new paradigm for regulation of the dynamic subcellular distribution of proteins between membrane-separated compartments.     

Nucleotide base sequence of the mitochondrial COIII gene of Schizophyllum commune

Summary The cytochrome oxidase subunit III gene (COIII) of the Badidiomycete S. commune has been identified, cloned, and sequenced. The gene contains no introns, is AT-rich (69%) and exhibits a high degree of similarity to sequences from Ascomycetes. While most mitochondrial genes use both TGA and TGG to specify tryptophan, the COIII gene of Schizophyllum uses TGG exclusively. Translation requires no deviation from the universal code.     

The chloroplast trnP-trnW-petG gene cluster in the mitochondrial genomes of Beta vulgaris,B. trigyna and B. webbiana: evolutionary aspects

The chloroplast trnP-trnW-petG gene cluster has been identified in the mitochondrial DNA (mtDNA) of sugar beet (Beta vulgaris). The chloroplast-derived trnW gene is transcribed in the mitochondria; the other two genes, however, do not seem to be transcribed. This gene cluster is also present in the mitochondrial genomes of two wild Beta species, B. trigyna and B. webbiana. Sugar beet and the two wild relatives share 100% sequence identity in the coding regions of both the mitochondrial trnP and trnW genes. On the other hand, the petG genes from the wild Beta mtDNAs were found to be disrupted either by a 5-bp duplication (B. trigyna) or by a deletion of the 5 region (B. webbiana). A data-base search revealed that a conserved sequence of 60 bp is present in the trnP-trnW intergenic region of the mitochondrial genomes of the three Beta species as well as in other higher plants, including wheat and maize, and that the conserved sequence is absent from the chloroplast counterpart. Our results thus favour the hypothesis of a monophyletic origin of the trnP-trnW-petG cluster found in the plant mitochondrial genomes examined.     

Physical map of the COB region in mitochondrial DNA of Saccharomyces cerevisiae

Summary A physical map of the COB region in mtDNA of yeast has been established. This region harbours a split gene coding for apocytochrome b. It includes restriction sites of seven endonucleases (EcoRI, HindII, HindIII, HaeIII, HpaII, AluI and BamHI). Various mtDNA sequences of this region, retained in a series of genetically characterized rho^– clones have been allocated to this map. The combination of this physical map with a genetic map of the rho^– clones revealed that 1) cob^– mutational sites spread over 8,400 bp, 2) mutations in sequences coding for apocytochrome b map in five distinct segments which are separated by intervening sequences with minimum lengths from 350 to 1,900 bp.Abbreviations mtDNA mitochondrial DNA - b bases - bp basepairs     

The mitochondrial genome of the aquatic phycomycete Blastocladiella emersonii

Summary Mitochondrial DNA from the aquatic fungus Blastocladiella emersonii Cantino and Hyatt has been isolated and characterized. By restriction enzyme analysis the size of the mitochondrial genome was found to be 35.5 kb pairs. A restriction site map was constructed using the cleavage data for 6 endonucleases which showed the mitochondrial genome to be circular. The genes for the small and large ribosomal RNA, the ATPase subunits 6 and 9, the cytochrome c oxidase subunits 1, 2, and 3, and the apocytochrome b were located in the mitochonridal genome of B. emersonii by hybridizations with mitochondrial DNA probes from Saccharomyces cerevisiae and Neurospora crassa     

Intergenomic recombination of mitochondrial genomes in a somatic hybrid plant

Summary Restriction site polymorphisms between two parental mitochondrial (mt) genomes were used to characterize the novel mt genome present in a somatic hybrid plant. A cosmid clone containing mtDNA restriction fragments characteristic of both parental plant lines was identified in a library constructed from mtDNA of progeny of a somatic hybrid plant. Restriction mapping revealed the location of several restriction fragments derived from each of the parental mt genomes on the same cloned region of somatic hybrid mtDNA. This result is direct evidence that intergenomic recombination at the molecular level occurs in homologous regions of two parental mt genomes combined in the same plant cell by protoplast fusion.     

Control of final seed and organ size by the DA1 gene family in Arabidopsis thaliana

Although the size of an organism is a defining feature, little is known about the mechanisms that set the final size of organs and whole organisms. Here we describe Arabidopsis DA1, encoding a predicted ubiquitin receptor, which sets final seed and organ size by restricting the period of cell proliferation. The mutant protein encoded by the da1-1 allele has a negative activity toward DA1 and a DA1-related (DAR) protein, and overexpression of a da1-1 cDNA dramatically increases seed and organ size of wild-type plants, identifying this small gene family as important regulators of seed and organ size in plants.     

The mitochondrial genome of the aquatic phycomycete Allomyces macrogynus. Physical mapping and mitochondrial DNA instability

Summary A physical map of the mitochondrial genome of the aquatic phycomycete Allomyces macrogynus strain Burma 3–35 (35°C) has previously been published (Borkhardt and Delius 1983). This map has been extended in this study by locating 37 additional recognition sites for five new restriction enzymes in the mitochondrial genome. Homologous regions for the genes coding for cytochrome oxidase subunits 1, 2, and 3, apocytochrome b, ATPase subunits 6 and 9, the small and large ribosomal RNA, URF1, URF5, and perhaps urfa, a presumptive gene hitherto found only in the mitochondrial genome of the fission yeast Schizosaccharomyces pombe, were located in the mitochondrial genome of A. macrogynus by heterologous hybridizations with specific, mitochondria) gene probes from Saccharomyces cerevisiae, Aspergillus nidulans, Neurospora crassa, and S. pombe. The mitochondrial gene order in A. macrogynus was found to be identical to that of A. arbuscula; a gene order hitherto found only among members of the family Blastocladiaceae. Spontaneous insertion mutations have been found to occur quite frequently in the mitochondrial genome of A. macrogynus. In all mutated mitochondrial genomes so far studied, insertions have been located in a specific region located between the genes coding for the ATPase subunit 9 and the large ribosomal RNA. In two of the mutated mitochondrial genomes the insertional event(s) resulted in the presence of mitochondrial DNA molecules differing in size by multiples of approximately 70 base pairs.     

Characterization of the nuclear gene encoding chloroplast ribosomal protein S13 from Arabidopsis thaliana

We have characterised a cDNA clone and a nuclear gene encoding the chloroplast 30 s ribosomal protein S13 from Arabidopsis thaliana. The identification is based on the high similarity of the predicted amino-acid sequence with eubacterial S13 protein sequences, and immunodetection of a 14.5-kDa chloroplast ribosomal polypeptide using antibodies raised against the polypeptide produced from part of the cDNA expressed in bacteria. The predicted amino-acid sequence contains an N-terminal extension which has several features characteristic of chloroplast transit peptides. Experiments suggest there is a single copy of this gene in A. thaliana and multiple copies in Brassica species. The origin of the mitochondrial S13 polypeptide in crucifers is also discussed.     

Chimeric organization of two genes for the soybean mitochondrial ATPase subunit 6

Summary There are two copies of the ATPase subunit 6 (atp6) gene in the soybean mitochondrial genome which differ in their gene organization but share extensive homology with the maize atp6 gene except at their 5 ends. The two soybean genes are chimeric, containing regions with homology to other known mitochondrial genes at their 5 ends. Sequences homologous to the cytochrome oxidase subunit II (coxII) are located in one copy and sequences homologous to the ATPase subunit 9 (atp9) gene are located in the other copy, both of which contain methionine (ATG) codons that are in-frame with the remainder of the atp6 open reading frame. At least the copy of atp6 that contains the coxII sequence at its 5 end is abundantly transcribed to give an RNA of approximately 1,200 nucleotides.     

A tRNASer(UCN) gene in Artemia salina mitochondrial DNA: a case of mistaken identity

We have sequenced a segment of mitochondrial DNA (mtDNA) of a crustacean, the brine shrimp, Artemia salina, that includes 3 end-proximal regions of the genes for subunit 1 of the NADH dehydrogenase complex (ND1) and cytochrome b (Cyt b). From our data we conclude that in this mtDNA, as in the mtDNAs of Drosophila species, a tRNA^Ser(UCN) gene separates the ND1 and Cyt b genes. This is contrary to an earlier report that the A. salina ND1 and Cyt b genes are immediately adjacent to each other.