Structure of the Telomeric Ends of Mt DNA, Transcriptional Analysis and Complex I Assembly in the Dum24 Mitochondrial Mutant of Chlamydomonas Reinhardtii

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 ▲]

Mutants of Chlamydomonas reinhardtii deficient in mitochondrial complex I: Characterization of two mutations affecting the nd1 coding sequence

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 ▲]

Characterization of two genes encoding the mitochondrial alternative oxidase in Chlamydomonas reinhardtii

in Current Genetics (2001), 39(2), 101-108

Two cDNA clones (AOX1 and AOX2) and the corresponding genes encoding the alternative oxidases (AOXs) from Chlamydomonas reinhardtii were isolated and sequenced. The cDNAs, AOX1 and AOX2, contained open ... [more ▼]

Two cDNA clones (AOX1 and AOX2) and the corresponding genes encoding the alternative oxidases (AOXs) from Chlamydomonas reinhardtii were isolated and sequenced. The cDNAs, AOX1 and AOX2, contained open reading frames (ORFs) encoding putative proteins of 360 amino acids and 347 amino acids, respectively. For each of the ORFs, a potential mitochondrial-targeting sequence was found in the 5'-end regions. In comparison to AOX enzymes from plants and fungi, the predicted amino acid sequences of the ORFs showed their highest degree of identity with proteins from Aspergillus niger (38.1% and 37.2%) and Ajellomyces capsulatus (37% and 34.9%). Several residues supposed either to be Fe ligands or to be involved in the ubiquinol-binding site were fully conserved in both C. reinhardtii putative AOX proteins. In contrast, a cysteine residue conserved in the sequences of all higher plants and probably involved in the regulation of the enzyme activity was missing both from the AOX1 and AOX2 amino acid sequences and from protein sequences from various other microorganisms. The transcriptional expression of the AOX1 and AOX2 genes in wild-type cells and in mutant cells deficient in mitochondrial complex III activity was also investigated. [less ▲]

Pleiotropic drug sensitive mutants induced by insertional mutagenesis in Chlamydomonas

Poster (2000, May 05)

Isolation and characterization of respiratory NADH:ubiquinone oxidoreductase (complex I) mutants in Chlamydomonas reinhardtii

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

Genetic manipulations in Chlamydomonas reinhardtii: Use of the antisense strategy to inhibit the alternative oxidase

Poster (1998, May)

Besides the cytochrome pathway (CP), mitochondria of higher plants and many microeukaryotes possess a second electron pathway, the alternative pathway (AP), that bypasses complexes III and IV and ... [more ▼]

Besides the cytochrome pathway (CP), mitochondria of higher plants and many microeukaryotes possess a second electron pathway, the alternative pathway (AP), that bypasses complexes III and IV and catalyzes the oxidation of the ubiquinol pool by molecular oxygen. Electrons transferred by this way are not coupled to ATP production and free energy is lost as heat. The alternative oxidase (AOX) is the sole enzyme implicated in this process. Despite a great research effort during these last years, the exact role of AP is poorly understood, except in Araceae (such as Sauromatum guttatum, the ‘voodoo lily’) in which it is implicated in the thermogenesis of the inflorescence. In order to investigate the physiological role of AOX in the unicellular green alga Chlamydomonas reinhardtii, two different plasmids derived from pSP105 (Stevens et al., 1996) were constructed by placing in antisense orientation an AOX cDNA from this species under the control of the regulatory regions of the Chlamydomonas RBCS2 gene. The first plasmid possessed the full promoter, while the second one had a truncated promoter, supposed to be more efficient (Purton, personal communication). Cell wall-deficient arginine-requiring strains were co-transformed by the glass beads method with linearized pASL plasmid (complementing the arginine requirement) and linearized first or second construction. More than 1100 transformants able to grow on an arginine-free medium were screened for AOX alteration by growth tests in the presence of the two complex III inhibitors antimycin A and myxothiazol. Out of seventeen clones selected for their sensitivity to these inhibitors, only one (A1 clone) was totally unable to grow in the presence of antimycin A and myxothiazol. A PCR analysis showed that the seventeen selected clones had integrated the antisense construction. The A1 cell line was further investigated for its mitochondrial respiration activity. The cells grown under mixotrophic conditions (light + acetate as a carbon source), showed a 70% drop of the AP capacity. Moreover, the total respiration rate represented only 60% of the wild-type rate. Interestingly, despite these alterations, the A1 transformant displayed a 25% faster growth rate than the wild-type cultivated under the same mixotrophic conditions. This work was supported by grants from the Belgian FRFC (2.4527.97). Actions de recherches concertées (ARC 93-98/170) and Fonds spéciaux pour la recherche dans les universités. Reference STEVENS D.R., ROCHAIX J.D., and PURTON S. (1996), Mol. Gen. Genet. 251, 23-30 [less ▲]

Mitochondrial genetics

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

Biogeography and taxonomy of Apodemus sylvaticus (the woodmouse) in the Tyrrhenian region: Enzymatic variations and mitochondrial DNA restriction pattern analysis

in Heredity (1996), 76(Part 3), 267-277

In the western Mediterranean area, the taxonomic status of the various forms of Apodemus sylvaticus is quite unclear. Moreover, though anthropogenic, the origins of the island populations remain unknown ... [more ▼]

In the western Mediterranean area, the taxonomic status of the various forms of Apodemus sylvaticus is quite unclear. Moreover, though anthropogenic, the origins of the island populations remain unknown in geographical terms. In order to examine the level of genetic relatedness of insular and continental woodmice, 258 animals were caught in 24 localities distributed in Belgium, France, mainland Italy, Sardinia, Corsica and Elba. Electrophoresis of 33 allozymes and mtDNA restriction fragments were performed and a UPGMA dendrogram built from the indices of genetic divergence. The dendrogram based on restriction patterns shows two main groups: 'Tyrrhenian', comprising all the Italian and Corsican animals and 'North-western', corresponding to all the other mice trapped from the Pyrenees to Belgium. Since all the Tyrrhenian mice are similar and well isolated from their relatives living on the western edge of the Alpine chain, they must share a common origin. The insular populations are consequently derived from peninsular Italian ones. From a taxonomic point of view and taking the priority rules into account, we have to invalidate A. s. clanceyi Harrison, 1948 and to consider the Tyrrhenian woodmice as belonging to A. s. milleri de Beaux, 1926, whereas the North-western ones must be referred to as the nominal subspecies. As far as the Elban woodmouse is concerned, at the moment we prefer to keep its present subspecific status because we only studied one animal. [less ▲]

Genetic Mapping of Mitochondrial Markers by Recombinational Analysis in Chlamydomonas Reinhardtii

in Molecular & General Genetics [=MGG] (1995), 249(2), 185-90

The mitochondrial genome of Chlamydomonas reinhardtii is a 15.8 kb linear DNA molecule present in multiple copies. In crosses, the meiotic products only inherit the mitochondrial genome of the mating type ... [more ▼]

The mitochondrial genome of Chlamydomonas reinhardtii is a 15.8 kb linear DNA molecule present in multiple copies. In crosses, the meiotic products only inherit the mitochondrial genome of the mating type minus (paternal) parent. In contrast mitotic zygotes transmit maternal and paternal mitochondrial DNA copies to their diploid progeny and recombinational events between molecules of both origins frequently occur. Six mitochondrial mutants unable to grow in the dark (dk- mutants) were crossed in various combinations and the percentages of wild-type dk+ recombinants were determined in mitotic zygotes when all progeny cells had become homoplasmic for the mitochondrial genome. In crosses between strains mutated in the COB (apocytochrome b) gene and strains mutated in the COX1 (subunit 1 of cytochrome oxidase) gene, the frequency of recombination was 13.7% (+/- 3.2%). The corresponding physical distance between the mutation sites was 4.3 kb. In crosses between strains carrying mutations separated by about 20 bp, a recombinational frequency of 0.04% (+/- 0.02%) was found. Two other mutants not yet characterized at the molecular level were also used for recombinational studies. From these data, a linear genetic map of the mitochondrial genome could be drawn. This map is consistent with the positions of the mutation sites on the mitochondrial DNA molecule and thereby validates the method used to generate the map. The frequency of recombination per physical distance unit (3.2% +/- 0.7% per kilobase) is compared with those obtained for other organellar genomes in yeasts and Chlamydomonas. [less ▲]

The Fate of Mitochondrial Dnas of Mt+ and Mt- Origin in Gametes and Zygotes of Chlamydomonas

in Current Genetics (1991), 20(3), 239-43

In order to study the mechanism responsible for the uniparental transmission of the mitochondrial genome in crosses between Chlamydomonas reinhardtii and C. smithii, we have analyzed the fate of ... [more ▼]

In order to study the mechanism responsible for the uniparental transmission of the mitochondrial genome in crosses between Chlamydomonas reinhardtii and C. smithii, we have analyzed the fate of mitochondrial DNA during gametogenesis, zygospore differentiation and sporulation by hybridization experiments. Both mt+ and mt- gametes contain the same amount of mitochondrial DNA and the two parental genomes persist for several days in the zygotes. The DNA of mt+ origin is slowly eliminated during the period of zygote maturation. Light is required for total elimination of mt+ mitochondrial DNA in the zygospores. Using appropriate restriction enzymes, we have been unable to detect methylation of the mitochondrial DNA during gametogenesis or zygospore formation. The possibility that the mt+ mitochondria themselves are specifically eliminated in the course of zygote maturation is discussed. [less ▲]