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See detailImpact of mutations affecting ND mitochondria-encoded Subunits on the activity and assembly of complex I in chlamydomonas. Implication for the structural organization of the enzyme
Cardol, Pierre ULg; Matagne, René-Fernand ULg; Remacle, Claire ULg

in Journal of Molecular Biology (2002), 319(5), 1211-1221

The mitochondrial rotenone-sensitive NADH:ubiquinone oxidoreductase (complex 1) comprises more than 35 subunits, the majority of which are encoded by the nucleus. In Chlamydomonas reinhardtii, only five ... [more ▼]

The mitochondrial rotenone-sensitive NADH:ubiquinone oxidoreductase (complex 1) comprises more than 35 subunits, the majority of which are encoded by the nucleus. In Chlamydomonas reinhardtii, only five components (ND1, ND2, ND4, ND5 and ND6) are coded for by the mitochondrial genome. Here, we characterize two mitochondrial mutants (dum5 and dum17) showing strong reduction or inactivation of complex I activity: dum5 is a IT deletion in the 3' UTR of nd5 whereas dum17 is a IT deletion in the coding sequence of nd6. The impact of these mutations and of mutations affecting nd1, nd4 and nd4/nd5 genes on the assembly of complex I is investigated. After separation of the respiratory complexes by blue native (BN)-PAGE or sucrose gradient centrifugation, we demonstrate that the absence of intact ND1 or ND6 subunit prevents the assembly of the 850 kDa whole complex, whereas the loss of ND4 or ND4/ND5 leads to the formation of a subcomplex of 650 kDa present in reduced amount. The implications of our findings for the possible role of these ND subunits on the activity of complex I and for the structural organization of the membrane arm of the enzyme are discussed. In mitochondria from all the strains analyzed, we moreover detected a 160210 kDa fragment comprising the hydrophilic 49 kDa and 76 kDa subunits of the complex I peripheral arm and showing NADH dehydrogenase activity. (C) 2002 Elsevier Science Ltd. All rights reserved. [less ▲]

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See detailThe genetics and molecular biology of mitochondria in Chlamydomonas
Matagne, René-Fernand ULg; Remacle, Claire ULg

in Pandalai, S. (Ed.) Recent Research Development in Plant Biology (2002)

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See detailStructure of the Telomeric Ends of Mt DNA, Transcriptional Analysis and Complex I Assembly in the Dum24 Mitochondrial Mutant of Chlamydomonas Reinhardtii
Duby, Franceline ULg; Cardol, Pierre ULg; Matagne, René-Fernand ULg et al

in Molecular Genetics & Genomics (2001), 266(1), 109-14

The dum24 mutant of Chlamydomonas reinhardtii contains four types of altered mitochondrial linear genomes: two types of deleted monomers and two types of dimers resulting from fusions between some ... [more ▼]

The dum24 mutant of Chlamydomonas reinhardtii contains four types of altered mitochondrial linear genomes: two types of deleted monomers and two types of dimers resulting from fusions between some monomers via their deleted ends. All molecules lack at least cob, nd4 and the 3' end of nd5, three adjacent genes located in the left part of the genome. We present evidence showing that in dum24, as in other deletion mutants, the deletions extend to the left telomeric end, and propose that the only replicative forms in the mutants are the dimeric DNA molecules that possess intact telomeric structures at both ends. Two abnormally large transcripts produced from chimeric genes are detected in dum24, which throws some light on the location of potential promoter sequences and processing signals in the mitochondrial genome. Using BN-PAGE analysis and immunological methods to detect complex I, we further show that dum24 mitochondria do not possess the normal multimeric complex I (850 kDa), but produce a smaller, partially assembled, complex (650 kDa), demonstrating a role for ND4 and/or ND5 subunits(s) in complex I assembly. [less ▲]

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See detailMutations Inactivating Mitochondrial Genes in Chlamydomonas Reinhardtii
Remacle, Claire ULg; Duby, Franceline ULg; Cardol, Pierre ULg et al

in Biochemical Society Transactions (2001), 29(Pt 4), 442-6

Chlamydomonas reinhardtii is now becoming a useful model for the study of mitochondrial genetics in a photosynthetic organism. The small (15.8 kb) mitochondrial genome C. reinhardtii has been sequenced ... [more ▼]

Chlamydomonas reinhardtii is now becoming a useful model for the study of mitochondrial genetics in a photosynthetic organism. The small (15.8 kb) mitochondrial genome C. reinhardtii has been sequenced completely and all the genes have been identified. Several mutants inactivated in mitochondrial genes encoding components of the respiratory complexes I, III and IV have been characterized at the molecular level. Assembly of complex I in several mutant strains and mapping of mitochondrial mutations by recombinational analysis are also described. [less ▲]

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See detailMutants of Chlamydomonas reinhardtii deficient in mitochondrial complex I: Characterization of two mutations affecting the nd1 coding sequence
Remacle, Claire ULg; Baurain, Denis ULg; Cardol, Pierre ULg et al

in Genetics (2001), 158(3), 1051-60

The mitochondrial rotenone-sensitive NADH:ubiquinone oxidoreductase (complex I) comprises more than 30 subunits, the majority of which are encoded by the nucleus. In Chlamydomonas reinhardtii, only five ... [more ▼]

The mitochondrial rotenone-sensitive NADH:ubiquinone oxidoreductase (complex I) comprises more than 30 subunits, the majority of which are encoded by the nucleus. In Chlamydomonas reinhardtii, only five components of complex I are coded for by mitochondrial genes. Three mutants deprived of complex I activity and displaying slow growth in the dark were isolated after mutagenic treatment with acriflavine. A genetical analysis demonstrated that two mutations (dum20 and dum25) affect the mitochondrial genome whereas the third mutation (dn26) is of nuclear origin. Recombinational analyses showed that dum20 and dum25 are closely linked on the genetic map of the mitochondrial genome and could affect the nd1 gene. A sequencing analysis confirmed this conclusion: dum20 is a deletion of one T at codon 243 of nd1; dum25 corresponds to a 6-bp deletion that eliminates two amino acids located in a very conserved hydrophilic segment of the protein. [less ▲]

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See detailIsolation and characterization of respiratory NADH:ubiquinone oxidoreductase (complex I) mutants in Chlamydomonas reinhardtii
Remacle, Claire ULg; Baurain, Denis ULg; Colin, Martine et al

in Archives of Physiology & Biochemistry (2000), 108(Supplement 1), 10

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See detailSuppression of a +1 T Mutation by a Nearby Substitution in the Mitochondrial Cox1 Gene of Chlamydomonas Reinhardtii: A New Type of Frameshift Suppression in an Organelle Genome
Remacle, Claire ULg; Colin, Martine ULg; Matagne, René-Fernand ULg

in Molecular & General Genetics [=MGG] (1998), 259(3), 294-8

In Chlamydomonas reinhardtii, mutants defective in the cytochrome pathway of respiration lack the capacity to grow under heterotrophic conditions (in darkness on acetate). In the dark- strain duM18, a + 1 ... [more ▼]

In Chlamydomonas reinhardtii, mutants defective in the cytochrome pathway of respiration lack the capacity to grow under heterotrophic conditions (in darkness on acetate). In the dark- strain duM18, a + 1 T addition in a run of four Ts, located at codon 145 of the mitochondrial cox1 gene encoding subunit I of cytochrome c oxidase, is responsible for the mutant phenotype. A leaky revertant (su11) that grows heterotrophically at a lower rate than wild-type cells was isolated from dum18. Its respiration sensitivity to cyanide was low and its cytochrome c oxidase activity was only 4% of that of the wild-type enzyme. Meiotic progeny obtained from crosses between revertant and wild-type cells inherited the phenotype of the mt- parent, showing that the suppressor mutation, like dum18 itself, is located in the mitochondrial genome. In order to map the su11 mutation relative to dum18, a recombinational analysis was performed on the diploid progeny. It demonstrated that su11 was very closely linked to the dum18 mutation less than 20-30 bp away. The cox1 gene of the su11 revertant was then sequenced. In addition to the + 1 T frameshift mutation still present at codon 145, an A-->C substitution was found at codon 146, leading to the replacement of a glutamic acid by an alanine in the polypeptide chain. No other mutations were detected in the cox1 coding sequence. As the new GCG codon (Ala) created at position 146 is very seldom used in the mitochondrial genome of C. reinhardtii, we suggest that the partial frameshift suppression by the nearby substitution is due to an occasional abnormal translocation of the ribosome (+ 1 base shift) facilitated both by the run of Ts and the low level of weak interaction of alanyl-tRNA. [less ▲]

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See detailA new type of frameshift mutation in a mitochondrial genome
Remacle, Claire ULg

Poster (1998, June)

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See detailMitochondrial genetics
Remacle, Claire ULg; Matagne, René-Fernand ULg

in Rochaix, Jean-David; Goldschmidt-Clermont, Michel; Merchant, Sabeeha (Eds.) The Molecular Biology of Chloroplasts and Mitochondria in Chlamydomonas (1998)

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See detailPoint mutations of mitochondrial genes of Chlamydomonas reinhardtii
Remacle, Claire ULg

Poster (1996, May)

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See detailA promoter element active in run-off transcription controls the expression of two cistrons of nad and rps genes in Nicotiana sylvestris mitochondria
Lelandais, C.; Gutierres, S.; Mathieu, C. et al

in Nucleic Acids Research (1996), 24(23), 4798-4804

The expression of two mitochondrial gene clusters (orf87-nad3-nad1/A and orf87-nad3-rps 12) was studied in Nicotiana sylvestris. 5' and 3' termini of transcripts were mapped by primer extension and ... [more ▼]

The expression of two mitochondrial gene clusters (orf87-nad3-nad1/A and orf87-nad3-rps 12) was studied in Nicotiana sylvestris. 5' and 3' termini of transcripts were mapped by primer extension and nuclease S1 protection. Processing and transcription initiation sites were differentiated by in vitro phosphorylation and capping experiments. A transcription initiation site, present in both gene clusters, was found 213 nucleotides upstream of orf87. This promoter element matches the consensus motif for dicotyledonous mitochondrial promoters and initiates run-off transcription in a pea mitochondrial purified protein fraction, Processing sites were identified 5' of nad3, nad1/A and rps12 respectively. These results suggest that (i) the expression of the two cistrons is only controlled by one duplicated promoter element, and (ii) multiple processing events are required to produce monocistronic nad3, nad1/A and rps12 transcripts. [less ▲]

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