Clustering of tRNA genes in Paracentrotus lividus mitochondrial DNA

Summary We have determined the base sequence of the restriction fragment Bam1-2 (3,593) of Paracentrotus lividus (sea urchin) mtDNA. This fragment contains, in addition to genes previously identified (part of the 12S rRNA, ND1 and part of the ND2 mRNA), a cluster of 15 tRNA genes located between the 12S and ND1 genes. Also to be found in the tRNA gene cluster, between the tRNA^Thr and tRNA^Pro genes, is a sequence of 134 bp which constitutes the only non-coding region of this DNA so far identified. The distinctive organization of the tRNA genes and the extreme size reduction of the non-coding region suggest the existence of unique mechanisms for the regulation of gene expression in this organism.     

Mutations in the ND5 subunit of complex I of the mitochondrial DNA are a frequent cause of oxidative phosphorylation disease

Background

Detection of mutations in the mitochondrial DNA (mtDNA) is usually limited to common mutations and the transfer RNA genes. However, mutations in other mtDNA regions can be an important cause of oxidative phosphorylation (OXPHOS) disease as well.

Objective

To investigate whether regions in the mtDNA are preferentially mutated in patients with OXPHOS disease.

Methods

Screening of the mtDNA for heteroplasmic mutations was performed by denaturing high‐performance liquid chromatography analysis of 116 patients with OXPHOS disease but without the common mtDNA mutations.

Results

An mtDNA sequence variant was detected in 15 patients, 5 of which were present in the ND5 gene. One sequence variant was new and three were known, one of which was found twice. The novel sequence variant m.13511A→T occurred in a patient with a Leigh‐like syndrome. The known mutation m.13513G→A, associated with mitochondrial encephalomyopathy lactic acidosis and stroke‐like syndrome (MELAS) and MELAS/Leigh/Leber hereditary optic neuropathy overlap syndrome, was found in a relatively low percentage in two patients from two different families, one with a MELAS/Leigh phenotype and one with a MELAS/chronic progressive external ophthalmoplegia phenotype. The known mutation m.13042G→A, detected previously in a patient with a MELAS/myoclonic epilepsy, ragged red fibres phenotype and in a family with a prevalent ocular phenotype, was now found in a patient with a Leigh‐like phenotype. The sequence variant m.12622G→A was reported once in a control database as a polymorphism, but is reported in this paper as heteroplasmic in three brothers, all with infantile encephalopathy (Leigh syndrome) fatal within the first 15 days of life. Therefore, a causal relationship between the presence of this sequence variant and the onset of mitochondrial disease cannot be entirely excluded at this moment.

Conclusions

Mutation screening of the ND5 gene is advised for routine diagnostics of patients with OXPHOS disease, especially for those with MELAS‐ and Leigh‐like syndrome with a complex I deficiency.Mitochondria are key for many cellular processes. One of the most important mechanisms is oxidative phosphorylation (OXPHOS) resulting in the production of cellular energy in the form of ATP. The OXPHOS system consists of five multiprotein complexes (I–V) and two mobile electron carriers (coenzyme q and cytochrome c) embedded in the lipid bilayer of the mitochondrial inner membrane.^1,2 The mitochondrial genome encodes 13 essential polypeptides of the OXPHOS system and the necessary RNA machinery (two ribosomal RNAs and 22 transfer RNAs (tRNA)). The remaining structural proteins and proteins involved in import, assembly and mitochondrial DNA (mtDNA) replication are encoded by the nucleus and specifically targeted to the mitochondria. OXPHOS disease is characterised by a wide variety of clinical symptoms, in which one or more organs can be involved, and by genetic and clinical heterogeneity.^2,3 With an estimated total number of about 1500 nuclear mitochondrial genes of which 600 have been identified so far,⁴ this complicates the process of identification of the underlying genetic defect. Although mutations in the mtDNA tRNA genes have been reported far more often than other mutations in mtDNA protein‐coding genes,² this figure is highly biased by a preferential screening of these genes.In this study, the complete mtDNA was screened for heteroplasmic mutations using denaturing high‐performance liquid chromatography (DHPLC) analysis in a group of 116 unrelated patients suspected for OXPHOS disease but without the common mutations for mitochondrial encephalomyopathy, lactic acidosis and stroke‐like syndrome (MELAS) m.3243A→G, myoclonic epilepsy, ragged red fibres (MERRF) m.8344A→G, Leigh/neuropathy, ataxia and retinitis pigmentosa m.8993T→G/C or large deletions. For this group of patients, we report that the ND5 gene is a commonly mutated gene.     

Sequence analysis of cDNAs for the human and bovine ATP synthase β subunit: mitochondrial DNA genes sustain seventeen times more mutations

We have cloned and sequenced human and bovine cDNAs for the subunit of the ATP synthase (ATP-synß), a nuclear DNA (nDNA) encoded oxidative phosphorylation (OXPHOS) gene. The two cDNAs were found to share 99% amino acid homology and 94% nucleotide homology. The evolutionary rate of ATPsynß was then compared with that of two mitochondrial DNA (mtDNA) ATP synthase genes (ATPase 6 and 8), seven other mtDNA OXPHOS genes, and a number of nuclear genes. The synonymous substitution rate for ATPsynß proved to be 1.9 × 10^–9 substitutions per site per year (substitutions × site^–1 × year^–1) (SSY). This is less than 1/2 that of the average nDNA gene, 1/12 the rate of ATPase 6 and 8, and 1/17 the rate of the average mtDNA gene. The synonymous and replacement substitution rates were used to calculate a new parameter, the selective constraint ratio. This revealed that even the most variable mtDNA protein was more constrained than the average nDNA protein. Thus, the high substitution mutation rate and strong selective constraints of mammalian mtDNA proteins suggest that mtDNA mutations may result in a disproportionately large number of human hereditary diseases of OXPHOS.     

Analysis of mitochondrial DNA sequences in patients with isolated or combined oxidative phosphorylation system deficiency

Background

Enzyme deficiencies of the oxidative phosphorylation (OXPHOS) system may be caused by mutations in the mitochondrial DNA (mtDNA) or in the nuclear DNA.

Objective

To analyse the sequences of the mtDNA coding region in 25 patients with OXPHOS system deficiency to identify the underlying genetic defect.

Results

Three novel non‐synonymous substitutions in protein‐coding genes, 4681T→C in MT‐ND2, 9891T→C in MT‐CO3 and 14122A→G in MT‐ND5, and one novel substitution in the 12S rRNA gene, 686A→G, were found. The definitely pathogenic mutation 3460G→A was identified in an 18‐year‐old woman who had severe isolated complex I deficiency and progressive myopathy.

Conclusions

Bioinformatic analyses suggest a pathogenic role for the novel 4681T→C substitution found in a boy with Leigh''s disease. These results show that the clinical phenotype caused by the primary Leber''s hereditary optic neuropathy mutation 3460G→A is more variable than has been thought. In the remaining 23 patients, the role of mtDNA mutations as a cause of the OXPHOS system deficiency could be excluded. The deficiency in these children probably originates from mutations in the nuclear genes coding for respiratory enzyme subunits or assembly factors.The oxidative phosphorylation (OXPHOS) system consists of five enzyme complexes composed of >70 subunits encoded by the nuclear genome and 13 subunits encoded by mitochondrial DNA (mtDNA). Both isolated and combined enzyme complex deficiencies have been reported in children with various clinical phenotypes. Defects in the OXPHOS system are common causes of inborn errors in energy metabolism, with an estimated incidence of 1 per 10 000 live births.¹ The inheritance pattern is autosomal recessive in most cases, but autosomal dominant and X‐chromosomal inheritance has also been described. Maternal inheritance points to a mutation in mtDNA as the cause of the disease.²More than 2000 human mtDNA‐coding region sequences have been reported since 2000, and about half of these sequences are from Europeans.^3,4,5,6,7,8 The total number of non‐synonymous mutations leading to an amino acid replacement in mtDNA of European origin has been estimated to be 1081, but as many as 18 100 sequences should be analysed to identify 95% of these substitutions.⁹ Sequencing of the complete mtDNA from patients with an OXPHOS system deficiency will evidently lead to the identification of novel pathogenic mutations. This approach has already yielded several novel mutations in MT‐ND genes so far, and some of them—for example, 10191T→C and 14487T→C—may not be uncommon causes of disease.^10,11

Key points

• Enzyme deficiencies of the oxidative phosphorylation (OXPHOS) system may be caused by mutations in the mitochondrial DNA (mtDNA) or in the nuclear DNA. The sequence of mtDNA‐coding region was analysed in 25 patients with OXPHOS system deficiency to identify the underlying genetic defect.
• 4681T→C, a novel substitution in MT‐ND2, was found in a patient with Leigh''s disease. Further analyses suggested a pathogenic role for this substitution.
• 3460G→A, one of the mutations causing Leber''s hereditary optic neuropathy, was identified in a patient with progressive myopathy. The finding suggests that the clinical phenotype caused by this mutation is more variable than what has been known.

There is a growing need to analyse complete mtDNA sequences with a high throughput and in a cost‐efficient manner. We analysed the entire coding region of mtDNA in 28 patients (consisting of children and young adults) with OXPHOS system deficiency using a protocol consisting of conformation‐sensitive gel electrophoresis (CSGE) of amplified mtDNA fragments and subsequent sequencing of those fragments that differed in mobility in CSGE. Obtained sequences were compared with previously reported mtDNA sequences to identify haplotype‐specific or novel variants, and to detect possible sequencing errors.¹² The quality of the sequences was confirmed by comparison of the sequences obtained using the CSGE protocol with those obtained using direct mtDNA sequencing, and by correct identification of three samples with a known pathogenic mutation. Three novel non‐synonymous substitutions and one novel rRNA substitution were detected, and their pathogenic potential was estimated on several criteria.     

Characterization of mitochondrial DNA in chloramphenicol-resistant interspecific hybrids and a cybrid

We have examined the restriction endonuclease cleavage patterns exhibited by the mitochondrial DNAs (mtDNA) of four chloramphenicolresistant (CAP^R)human × mouse hybrids and one CAP^R cybrid derived from CAP^R HeLa cells and CAP^S mouse RAG cells. Restriction fragments of mtDNAs were separated by electrophoresis and transferred by the Southern technique to diazobenzyloxymethyl paper. The covalently bound DNA fragments were hybridized initially with ³² P-labeled complementary RNA (cRNA) prepared from human mtDNA and, after removal of the human probe, hybridized with mouse [³²P]cRNA prepared from mouse mtDNA. Three hybrids which preferentially segregated human chromosomes and the cybrid exhibited mtDNA fragments indistinguishable from mouse cells. One hybrid, ROH8A, which exhibited reverse chromosome segregation, contained only human mtDNA. The pattern of chromosome and mtDNA segregation observed in these hybrids and the cybrid support the hypothesis that a complete set of human chromosomes must be retained if a human mouse hybrid is to retain human mitochondrial DNA.     

Deletions and rearrangements in Kluyveromyces lactis mitochondrial DNA

Summary Three classes of respiratory deficient mutants have been isolated from a fusant between Kluyveromyces lactis and Saccharomyces cerevisiae that contains only K. lactis mtDNA. One class (15 isolates), resemble ⁰ mutants of S. cerevisiae as they lack detectable mtDNA. A second class (16 isolates), resemble point mutations (mit ^–) or nuclear lesions (pet ^–) of S. cerevisiae as no detectable change is found in their mtDNA. The third class (five isolates), with deletions and rearrangements in their mtDNA are comparable to S. cerevisiae petite (^–) mutants. Surprisingly, three of the five deletion mutants have lost the same 8.0 kb sector of the mtDNA that encompasses the entire cytochrome oxidase subunit 2 gene and the majority of the adjacent cytochrome oxidase subunit 1 gene. In the other strains, deletions are accompanied by complex rearrangements together with substoiciometric bands and in one instance an amplified sector of 800 bp. By contrast to G+C rich short direct repeats forming deletion sites in S. cerevisiae mtDNA, excision of the 8.0 kb sector in K. lactis mtDNA occurs at an 11 bp A+T rich direct repeat CTAATATATAT. The recovery of three strains manifesting this deletion suggests there are limited sites for intramolecular recombination leading to excision in K. lactis mtDNA.     

Novel non‐neutral mitochondrial DNA mutations found in childhood acute lymphoblastic leukemia

Mitochondria produce adenosine triphosphate (ATP) for energy requirements via the mitochondrial oxidative phosphorylation (OXPHOS) system. One of the hallmarks of cancer is the energy shift toward glycolysis. Low OXPHOS activity and increased glycolysis are associated with aggressive types of cancer. Mitochondria have their own genome (mitochondrial DNA [mtDNA]) encoding for 13 essential subunits of the OXPHOS enzyme complexes. We studied mtDNA in childhood acute lymphoblastic leukemia (ALL) to detect potential pathogenic mutations in OXPHOS complexes. The whole mtDNA from blood and bone marrow samples at diagnosis and follow‐up from 36 ALL patients were analyzed. Novel or previously described pathogenic mtDNA mutations were identified in 8 out of 36 patients. Six out of these 8 patients had died from ALL. Five out of 36 patients had an identified poor prognosis genetic marker, and 4 of these patients had mtDNA mutations. Missense or nonsense mtDNA mutations were detected in the genes encoding subunits of OXPHOS complexes, as follows: MT‐ND1, MT‐ND2, MT‐ND4L and MT‐ND6 of complex I; MT‐CO3 of complex IV; and MT‐ATP6 and MT‐ATP8 of complex V. We discovered mtDNA mutations in childhood ALL supporting the hypothesis that non‐neutral variants in mtDNA affecting the OXPHOS function may be related to leukemic clones.     

Codon usage,genetic code and phylogeny of Dictyostelium discoideum mitochondrial DNA as deduced from a 7.3-kb region

We have sequenced a region (7 376-bp) of the mitochondrial (mt) DNA (54 kb) of the cellular slime mold, Dictyostelium discoideum. From the DNA and amino-acid sequence comparisons with known sequences, genes for ATPase subunit 9 (ATP), cytochrome b (CYTB), NADH dehydrogenase subunits 1, 3 and 6 (ND1, ND3 and ND6), small subunit rRNA (SSU rRNA) and seven tRNAs (Arg, Asn, Cys, Lys, f-Met, Met and Pro) have been identified. The sequenced region of the mtDNA has a high average A+T-content (70.8%). The A+T-content of protein-genes (73.6%) is considerably higher than that of RNA genes (61.3%). Even with the strong AT-bias, the genetic code employed is most probably the universal one. All seven tRNAs are able to form typical clover leaf structures. The molecular phylogenetic trees of CYTB and SSU rRNA suggest that D. discoideum is closer to green plants than to animals and fungi.     

Influence of a 24 h fast on high intensity cycle exercise performance in man

Summary The influence of a 24 h fast on endurance performance and the metabolic response to maximal cycle exercise was investigated in 6 healthy men (mean±SD: age = 21±7 years; weight = 73±10 kg; = 46±10 ml·kg^–1·min^–1). Subjects performed in randomised order two exercise bouts to exhaustion separated by one week. Test rides were performed in fasted (F) and post-absorptive (normal-diet, ND) conditions on an electrically braked cycle ergometer at a workload equivalent to 100% of . Acid-base status and selected metabolites were measured on arterialised venous blood at rest prior to exercise and at intervals for 15 mins following exercise. Exercise time to exhaustion was shorter after F compared with ND (p<0.01). Pre-exercise blood bicarbonate (HCO₃ ^–) concentration, and base excess (BE) were lower after F compared with ND (p<0.05). Prior to exercise, circulating concentrations of free fatty acids (FFA), gb-hydroxybutyrate (B-HB) and glycerol were higher after F compared with ND (p<0.01) but blood glucose and lactate concentration were not different. On the F treatment, after exercise, blood pH, HCO₃ ^–, and BE were all significantly higher (p<0.01) than on ND; blood lactate concentration was significantly lower for the whole of the post-exercise period after F compared with ND (p<0.01). Circulating levels of FFA and B-HB after exercise on the F treatment fell but levels of these substrates were not altered by exercise after ND. Blood glucose and glycerol concentrations increased following exercise on both treatments. The present study provides evidence that a 24 h fast is detrimental to high-intensity exercise performance and possibly influences the metabolic response following maximal cycle exercise. These changes may be related to the altered pre-exercise acid-base status and/or a change in the pattern of substrate utilisation.     

Molecular evidence of intraspecific variability in different habitat-related populations of Triatoma dimidiata (Hemiptera: Reduviidae) from Costa Rica

Intraspecific genetic variation among Triatoma dimidiata (Hemiptera: Reduviidae) from seven Costa Rican populations and from different domestic, peridomestic, and sylvatic ecotopes were analyzed. The complete nucleotide sequence of the nuclear ribosomal DNA internal transcribed spacer (ITS-2) and partial sequences of the cytochrome B (Cyt b) gene and the large ribosomal subunit RNA (16S) of mitochondrial DNA (mtDNA) were analyzed and compared. All ITS-2 sequences analyzed were identical and correspond to the haplotype T.dim-H1, the most common haplotype in Central American populations. Sequences of mtDNA revealed a 10.17% of polymorphism in Cyt b and 2.39% in 16S, suggesting that the Cyt b fragment is a useful marker to describe the genetic structure of populations, even at habitat-related level. The analyses of the 18 new combined T. dimidiata haplotypes (Cytb/16S/ITS-2) showed that the two main geographical locations and populations studied are genetically structured showing different haplotype profiling. Only one combined haplotype was shared in the studied areas (Cytb.d/16S.a). Seven haplotypes exclusive for domestic/peridomestic populations, five for sylvatic, and six shared haplotypes for both habitat-related ecotopes are described. Although the relationship between the habitat and the haplotype profiling is less clear, there are different patterns of haplotype distribution in each geographic area between the two habitat-related ecotopes studied (domestic/peridomestic and sylvatic), some of them reflected in the phylogenetic relationships analyzed. The intraspecific variability detected may underlie the known plasticity of T. dimidiata, an important vector for Chagas disease transmission, suggesting that this species must be continuously monitored.     

Neutron flow exposure as a test for survival of Artemia Salina spores

Live and heat-inactivated Artemia salina spores (samples with the same mass and filling density) were exposed to a flow of thermal neutrons from a ²⁵²Cf radioactive source at an equivalent dose power of about 1 µSv/h. Irradiation led to a 4-fold acceleration of nauplius development and to modification of the element profiles of live spores. The difference between absorption/diffusion of thermal neutrons by live and dead spores was revealed.Translated from Byulleten Eksperimentalnoi Biologii i Meditsiny, Vol. 138, No. 11, pp. 530–534, November, 2004     

Repair properties in yeast mitochondrial DNA mutators

Summary After ethy1methanesulfonate mutagenesis of the strain Saccharomyces cerevisiae D273-1013, out of 100,000 survivors, 1,000 were selected for their high production of petite mutants at 36 °C. Among these 1,000 mutators, 5 also showed an increased frequency of spontaneous point mutations measured at 25 °C. Further analysis revealed that in all mutators, except 2, petite accumulation proceeded at 25 °C as well as 36 °C. In these 2 mutants, the production of petite mutants was much higher at 36 °C than at 25 °C. In one of them, however, the mutator and the thermosensitive petite phenotypes were due to mutations in two unlinked nuclear genes. In the other mutants, both traits were the result of a mutation in a single nuclear gene. The mutators fell into three complementation groups (tpm1, tpm2, mup1). No complementation was observed between tpm1 mutants and the gam4 mutant previously described by Foury and Goffeau (1979). From the latter and the present works, only four complementation groups (gam1, gam2, gam4 or tpml, mupl) have been identified and it is likely that the number of genes controlling specifically the spontaneous mutability of the mtDNA is low. The mutators exhibited a variety of responses to damaging agents such as UV light and ethidium bromide; especially in a representative mutant from the complementation group tpm1, the induction of ^– mutants was sensitive to UV light and resistant to ethidium bromide. In addition, we found that in the mutants from this complementation group, the synthesis of mtDNA in isolated mitochondria was low; however their mitochondrial DNA polymerase activity was similar to that of the wild type strain. A relationship might exist between the mutator phenotype and the low mtDNA synthesis in the tpm1 mutants.     

Restricted immunoglobulin constant heavy G chain genes in primary immunodeficiencies

Some primary immunodeficiencies (PIDs) express low serum levels of antibodies. The constant heavy G chain (IGHG) genes, also representing Fc domains of γ3, γ1 and γ2 on chromosome 14q32.3, genotyped by the alternative IgG subclass allotypes, found in four fixed IGHG haplotypes, designating four B cell variants, were identified by a competitive ELISA and double immunodiffusion. IGHG genes were hypothesized to contribute to the development of PIDs. From 235 Caucasian patients, the homozygous IGHGbf-n/bf-n diplotype (B^bf-n/B^bf-n cells) dominated significantly in 43 IgG2 deficiency (OR 6.0), 32 common variable immunodeficiency (OR 4.6) and 22 Ataxia telangiectasia (OR 3.0) and the IGHGga-n/ga-n diplotype (B^ga-n/B^ga-n cells) dominated in 53 IgG3 deficiency (OR 10.6) and 21 Wiscott–Aldrich syndrome (OR 4.1). 62 IgA deficiency patients were dominated by both diplotypes (OR 2.3 and OR 2.8 respectively). Restricted IGHG genes, restricted IgG allotypes (Fc domains) and restricted B cells are significant in PIDs for diagnosis, treatment and pathogenetic mechanisms.     

Depletion of mitochondrial DNA in leucocytes harbouring the 3243A->G mtDNA mutation

Background

The 3243A→G MTTL1 mutation is the most common heteroplasmic mitochondrial DNA (mtDNA) mutation associated with disease. Previous studies have shown that the percentage of mutated mtDNA decreases in blood as patients get older, but the mechanisms behind this remain unclear.

Objectives and method

To understand the dynamics of the process and the underlying mechanisms, an accurate fluorescent assay was established for 3243A→G heteroplasmy and the amount of mtDNA in blood with real‐time polymerase chain reaction was determined. The amount of mutated and wild‐type mtDNA was measured at two time points in 11 subjects.

Results

The percentage of mutated mtDNA decreases exponentially during life, and peripheral blood leucocytes in patients harbouring 3243A→G are profoundly depleted of mtDNA.

Conclusions

A similar decrease in mtDNA has been seen in other mitochondrial disorders, and in 3243A→G cell lines in culture, indicating that depletion of mtDNA may be a common secondary phenomenon in several mitochondrial diseases. Depletion of mtDNA is not always due to mutation of a nuclear gene involved in mtDNA maintenance.The 3243A→G MTTL1 gene mutation of mitochondrial DNA (mtDNA) is the most common heteroplasmic pathogenic mtDNA mutation and is found in approximately 1 in 6000 of the general population.¹ Although first described in mitochondrial encephalomyopathy with lactic acidosis and stroke‐like episodes (MELAS), the phenotypic spectrum is extremely diverse, including isolated diabetes and deafness, hypertrophic cardiomyopathy and retinitis pigmentosa.² The clinical variability can be explained partly by tissue‐specific differences in the percentage of mutated mtDNA.^3,4Intriguingly, the percentage of mutated mtDNA is consistently lower in peripheral blood than in post‐mitotic tissues such as skeletal muscle and brain.^3,5 Serial measurements in the same subject have shown that the percentage of the 3243A→G mutation in blood decreases over time,^6,7 but the reasons for this are not clear. One possibility is that vegetative segregation in rapidly proliferating leucocyte precursors leads to high percentages of mutated mtDNA in some cells. This causes a biochemical defect of the respiratory chain, which either impairs the further proliferation of that cell lineage or leads to cell death.⁷ This would ultimately lead to a decrease in the percentage of mutated mtDNA in the daughter cells present in the peripheral blood. However, it is currently not known whether the biochemical defect is primarily because of high amounts of mutated mtDNA,⁸ low amounts of wild‐type mtDNA⁹ or a combination of both.To advance our understanding of this process, we developed and validated a highly sensitive fluorescent assay to measure the changes in heteroplasmy over time, and also measured the absolute amount of mutated and wild‐type mtDNA in 11 subjects known to harbour 3243A→G.     

Plastid DNA from Pyrenomonas salina (Cryptophyceae): physical map,genes, and evolutionary implications

Summary Cryptomonads are thought to have arisen from a symbiotic association between a eukaryotic flagellated host and a eukaryotic algal symbiont, presumably related to red algae. As organellar DNAs have proven to be useful tools in elucidating phylogenetic relationships, the plastid (pt) DNA of the cryptomonad alga Pyrenomonas salina has been characterized in some detail. A restriction map of the circular 127 kb ptDNA from Pyrenomonas salina was established. An inverted repeat (IR) region of about 5 kb separates two single-copy regions of 15 and 102 kb, respectively. It contains the genes for the small and large subunit of rRNA. Ten protein genes, coding for the large subunit of ribulose-1,5-bisphosphate carboxylase, the 47 kDa, 43 kDa and 32 kDa proteins of photosystem II, the ribosomal proteins L2, S7 and S11, the elongation factor Tu, as well as the - and -subunits of ATP synthase, have been localized on the restriction map either by hybridization of heterologous gene probes or by sequence homologies. The gene for the plastidal small subunit (SSUr) RNA has been sequenced and compared to homologous SSU regions from the cyanobacterium Anacystis nidulans and plastids from rhodophytes, chromophytes, euglenoids, chlorophytes, and land plants. A phylogenetic tree constructed with the neighborliness method and indicating a relationship of cryptomonad plastids with those of red algae is presented.     

Organisation of the mitochondrial genome of Trichophyton rubrum III. DNA sequence analysis of the NADH dehydrogenase subunits 1, 2, 3, 4, 5 and the cytochrome b gene

In this paper, we present the nucleotide sequence of a 9761 nt-long segment of the mitochondrial genome of the dermatophyte Trichophyton rubrum that bridges the gap between two previously published segments, making a unique contig that represents approximately 80% of the molecule. The location of all genes on the map is determined except for some tRNA genes expected to flank the LSU rRNA gene not yet sequenced. Starting from the 5′ end of the present sequence, we recognized the ND5 and ND2 genes, the cytochrome b gene, an unusually long intergenic spacer of unknown function, as well as the ND3, ND1 and ND4 genes. This sequence extends and confirms the similarity with the mitochondrial genome of Aspergillus nidulans. Interestingly, two cases of partial overlaps between the terminator and initiator codons of successive genes (ND4–ND5 and ND5–ND2) are encountered. Received: 9 July / 10 November 1998     

Investigation of tRNALys/Leu and ATPase 6/8 gene mutations in Iranian ataxia telangiectasia patients

Introduction

Ataxia telangiectasia (AT) is a rare human neurodegenerative autosomal recessive multisystem disease. AT is the result of mutations in the AT-mutated (ATM) gene. ATM protein is required for radiation-induced apoptosis and acts before mitochondrial collapse. The tRNA genes are considered one of the hot spots for mutations causing mitochondrial disorders. Due to the important role of ATM in apoptosis and its effect on the cell cycle it might be possible that it has a central role in mtDNA mutations. On the other hand, the tRNA^Lys/Leu gene and also ATPase6 and ATPase8 genes are important for many mitochondrial diseases and many causative mutations have been reported from these genes.

Material and methods

In the present research, we performed mutation screening for these genes in 20 patients who were diagnosed with ataxia telangiectasia by a PCR sequencing method.

Results

The results showed a significant level of mtDNA variations in AT patients. Among 20 patients in this study, 12 patients (60%) were detected with point mutations, among which 8 mutations (40%) belonged to the MT-ATP6 gene. There was probably a second effect of mtDNA mutations in AT disease and mtDNA plays a main role in establishment of AT.

Conclusions

MtDNA mutations might be responsible for the decline of mitochondrial function in AT patients. Mitochondrial investigation can help to understand the mechanism of damage in AT disease.     

Functional Consequences of Mitochondrial DNA Deletions in Human Skin Fibroblasts : Increased Contractile Strength in Collagen Lattices Is Due to Oxidative Stress-Induced Lysyl Oxidase Activity

Deletions within the mitochondrial DNA (mtDNA) are thought to contribute to extrinsic skin aging. To study the translation of mtDNA deletions into functional and structural changes in the skin, we seeded human skin fibroblasts into collagen gels to generate dermal equivalents. These cells were either derived from Kearns-Sayre syndrome (KSS) patients, who constitutively carry large amounts of the UV-inducible mitochondrial common deletion, or normal human volunteers. We found that KSS fibroblasts, in comparison with normal human fibroblasts, contracted the gels faster and more strongly, an effect that was dependent on reactive oxygen species. Gene expression and Western blot analysis revealed significant upregulation of lysyl oxidase (LOX) in KSS fibroblasts. Treatment with the specific LOX inhibitor β-aminopropionitrile decreased the contraction difference between KSS and normal human fibroblast equivalents. Also, addition of the antioxidant N-tert-butyl-α-phenylnitrone reduced the contraction difference by inhibiting collagen gel contraction in KSS fibroblasts, and both β-aminopropionitrile and N-tert-butyl-α-phenylnitrone diminished LOX activity. These data suggest a causal relationship between mtDNA deletions, reactive oxygen species production, and increased LOX activity that leads to increased contraction of collagen gels. Accordingly, increased LOX expression was also observed in vivo in photoaged human and mouse skin. Therefore, mtDNA deletions in human fibroblasts may lead to functional and structural alterations of the skin.Oxidative stress can damage biological macromolecules including lipids, proteins, and DNA.^1,2 In this regard, mitochondrial DNA (mtDNA), a circular molecule comprising 16,569 bp in human cells, is particularly vulnerable, due to its close proximity to the mitochondrial electron transport chain as the major intracellular source of reactive oxygen species (ROS), its lack of histones, and a limited repertoire of DNA repair capacity.^3,4 Mitochondrial DNA encodes for 13 essential components of the electron transport chain, 22 tRNAs and 2 rRNAs involved in their translation. As a consequence, mutations of mtDNA including point mutations and large scale deletions interfere with mitochondrial physiology and result in cellular dysfunction. So far, more than 100 point mutations associated with a heterogeneous spectrum of pathological abnormalities have been reported.⁵ Large scale deletions such as the 4977 bp-containing common deletion are found in a number of mitochondrial disorders that can occur sporadically or can be inherited maternally. The best known mitochondrial disease associated with the common deletion is Kearns-Sayre syndrome (KSS). KSS appears to be a sporadical disease that is clinically characterized by skeletal muscle weakness, progressive ptosis, external ophthalmoplegia, retinopathy, cardiac conduction defects, and brain damage.⁶Mutations of mtDNA are not only found in mitochondrial diseases but are also frequently detected in aged tissues with high energy demands such as skeletal muscle, heart, and neurons,^7,8,9,10 and it has therefore been proposed that mtDNA mutations are causally related to the aging process. At least for point mutations, this assumption has recently been supported by a number of elegant studies using mtDNA mutator mice.^11,12 The precise molecular mechanisms, however, through which mtDNA mutations in general and mtDNA deletions in particular contribute to the aging process of a given tissue, are not yet understood.In this regard recent studies suggest a role for large scale deletions of mtDNA in premature (=extrinsic) aging of human skin.^13,14,15 Among all environmental factors, solar ultraviolet (UV) radiation is the most important in extrinsic skin aging, a process accordingly also termed photoaging.¹⁶ In photoaged skin, the amount of large scale deletions of mtDNA such as the common deletion is increased up to tenfold, as compared with sun-protected skin of the same individuals.¹⁷ Also, chronic exposure to UV radiation induces large scale deletions of mtDNA in human skin fibroblasts in vitro as well as in vivo,^10,15 and UV-induced mtDNA mutagenesis is associated with a decline of mitochondrial functions.¹⁸ In human skin, UV-induced deletions were found to persist for years and their levels increased after cessation of UV irradiation even in the absence of further exposures.¹⁵In the present study, we have addressed the question how the presence of mtDNA deletions in human skin fibroblasts translates into structural and functional alterations in human skin by using dermal equivalents. Dermal equivalents can be generated by seeding human skin fibroblasts into a collagen gel, which is then remodeled and contracted by these cells within several days. After finishing the contraction process, the dermal equivalents can be kept in culture for several weeks. This organotypic model system has been shown to closely resemble the dermal compartment of living human skin¹⁹ and has extensively been used in cutaneous biological research.^20,21,22,23 To exclude that any of the observed changes result from UV radiation-induced effects that are independent of the generation of large scale mtDNA deletions, either unirradiated normal healthy human skin fibroblasts (NHFs) or unirradiated human skin fibroblasts from KSS patients, which constitutively carry the UV-inducible common deletion, were used to generate dermal equivalents. Here, we report on differences between NHFs and KSS fibroblasts that occur within the initiation phase of the dermal equivalents, ie, the first 4 days of culture.