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See detailA mitochondrial half-size ABC transporter is involved in cadmium tolerance in Chlamydomonas reinhardtii
Hanikenne, Marc ULg; Motte, Patrick ULg; Wu, Madeline C.S. et al

in Plant Cell and Environment (2005), 28(7), 863-873

Five cadmium-sensitive insertional mutants, all affected at the CDS1 ('cadmium-sensitive 1') locus, have been previously isolated in the unicellular green alga Chlamydomonas reinhardtii. We here describe ... [more ▼]

Five cadmium-sensitive insertional mutants, all affected at the CDS1 ('cadmium-sensitive 1') locus, have been previously isolated in the unicellular green alga Chlamydomonas reinhardtii. We here describe the cloning of the Cds1 gene (8314 bp with 26 introns) and the corresponding cDNA. The Cds1 gene, strongly induced by cadmium, encodes a putative protein (CrCds1) of 1062 amino acid residues that belongs to the ATM/HMT subfamily of half-size ABC transporters. This subfamily includes both vacuolar HMT-type proteins transporting phytochelatin-cadmium complexes from the cytoplasm to the vacuole and mitochondrial ATM-type proteins involved in the maturation of cytosolic Fe/S proteins. Unlike the Delta sphmt1 cadmium-sensitive mutant of Schizosaccharomyces pombe that lacks a vacuolar HMT-type transporter, the cds1 mutant accumulates a high amount of phytochelatin-cadmium complexes. By epitope tagging, the CrCds1 protein was localized in the mitochondria. Even though mitochondria of cds1 do not accumulate important amounts of 'free' iron, the mutant cells are hypersensitive to high iron concentrations. Our data show for the first time that a mitochondrial ATM-like transporter plays a major role in tolerance to cadmium. [less ▲]

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See detailPleiotropic Mutants Hypersensitive to Heavy Metals and to Oxidative Stress in Chlamydomonas Reinhardtii
Hanikenne, Marc ULg; Matagne, René F.; Loppes, Roland ULg

in FEMS Microbiology Letters (2001), 196(2),

Insertional mutagenesis was used in Chlamydomonas reinhardtii to isolate original mutants hypersensitive to multiple drugs and physical agents. Out of 5200 transformants analyzed, 13 mutants belonging to ... [more ▼]

Insertional mutagenesis was used in Chlamydomonas reinhardtii to isolate original mutants hypersensitive to multiple drugs and physical agents. Out of 5200 transformants analyzed, 13 mutants belonging to seven phenotypic classes were isolated. Five were exclusively sensitive to cadmium and represented two loci. The other mutants were pleiotropic and presented a cross sensitivity to several (2--6) of the following agents: cadmium, copper, lead, paraquat, hydrogen peroxide, UVC and light. In all mutants analyzed, the hypersensitive phenotype was most probably due to a single mutational event. The sensitivity of several pleiotropic mutants to a broad range of physical and chemical agents suggests that the disrupted genes are involved in multiple stress responses. [less ▲]

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See detailGenetic manipulations in Chlamydomonas reinhardtii: Use of the antisense strategy to inhibit the alternative oxidase
Baurain, Denis ULg; Dinant, Monique; Loppes, Roland ULg et al

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 ▲]

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See detailSequence analysis of the ARG7 gene of Schizosaccharomyces pombe coding for argininosuccinate lyase. Expression of the gene in Saccharomyces cerevisiae.
Loppes, Roland ULg; Michels, R; Decroupette, I et al

in Current Genetics (1991), 19(4), 255-60

The complete nucleotide sequence of the ARG7 gene, coding for argininosuccinate lyase (EC 4.3.2.1), in the fission yeast (Schizosaccharomyces pombe) has been determined. It consists of an open reading ... [more ▼]

The complete nucleotide sequence of the ARG7 gene, coding for argininosuccinate lyase (EC 4.3.2.1), in the fission yeast (Schizosaccharomyces pombe) has been determined. It consists of an open reading frame of 461 codons. The deduced protein has a molecular weight of 51,200 Da. The gene is devoid of introns which is confirmed by the fact that it is expressed in Escherichia coli after spontaneous insertion of a bacterial sequence probably bearing a prokaryotic promoter. A perfect "TATA" box is found at -72 and the major transcription initiation site in Saccharomyces cerevisiae is located at -11 as shown by primer extension experiments. Comparison of the S. pombe lyase with related proteins from other organisms reveals an important degree of conservation except in the carboxyterminal part of the polypeptide. Additionally, a deletion removing 66 amino acids of the carboxy terminus yields an enzyme exhibiting some biological activity. A unique 1,500 b transcript was found in S. cerevisiae when the intact gene was present, but the deleted version of the gene gave rise to at least three transcripts of 1,800, 2,800 and 3,900 b. [less ▲]

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See detailThree-dimensional electron microscopy of ribosomal chromatin in two higher plants: a cytochemical, immunocytochemical, and in situ hybridization approach.
Motte, Patrick ULg; Loppes, Roland ULg; Menager, M. et al

in Journal of Histochemistry and Cytochemistry : Official Journal of the Histochemistry Society (1991), 39(11), 1495-506

We report the 3-D arrangement of DNA within the nucleolar subcomponents from two evolutionary distant higher plants, Zea mays and Sinapis alba. These species are particularly convenient to study the ... [more ▼]

We report the 3-D arrangement of DNA within the nucleolar subcomponents from two evolutionary distant higher plants, Zea mays and Sinapis alba. These species are particularly convenient to study the spatial organization of plant intranucleolar DNA, since their nucleoli have been previously reconstructed in 3-D from serial ultra-thin sections. We used the osmium ammine-B complex (a specific DNA stain) on thick sections of Lowicryl-embedded root fragments. Immunocytochemical techniques using anti-DNA antibodies and rDNA/rDNA in situ hybridization were also applied on ultra-thin sections. We showed on tilted images that the OA-B stains DNA throughout the whole thickness of the section. In addition, very low quantities of cytoplasmic DNA were stained by this complex, which is now the best DNA stain used in electron microscopy. Within the nucleoli the DNA was localized in the fibrillar centers, where large clumps of dense chromatin were also visible. In the two plant species intranucleolar chromatin forms a complex network with strands partially linked to chromosomal nucleolar-organizing regions identified by in situ hybridization. This study describes for the first time the spatial arrangement of the intranucleolar chromatin in nucleoli of higher plants using high-resolution techniques. [less ▲]

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See detailMitochondrial Genome Transmission in Chlamydomonas Diploids Obtained by Sexual Crosses and Artificial Fusions: Role of the Mating Type and of a 1 Kb Intron
Remacle, Claire ULg; Bovie, C.; Michel-Wolwertz, M. R. et al

in Molecular & General Genetics [=MGG] (1990), 223(2), 180-4

The linear mitochondrial DNAs of the two infertile algal species Chlamydomonas smithii and C. reinhardtii are co-linear with the exception of a 1 kb intron (alpha intron) located in the cytochrome b gene ... [more ▼]

The linear mitochondrial DNAs of the two infertile algal species Chlamydomonas smithii and C. reinhardtii are co-linear with the exception of a 1 kb intron (alpha intron) located in the cytochrome b gene of C. smithii. C. smithii also possesses an additional HpaI restriction site (H marker) located in the COXI gene, about 5 kb from the intron. In reciprocal crosses, C. smithii (H+ alpha +) x C. reinhardtii (H- alpha -), the alpha intron is transmitted to all diploid progeny, whereas the H marker is frequently transmitted either biparentally or paternally depending on whether the C. smithii parent is maternal (mt+) or paternal (mt-). In diploids resulting from artificial fusion between vegetative cells, the absolute transmission of alpha is accompanied by the frequent transmission of the H+ marker, irrespective of the mating type of the parental strains. Finally, in reciprocal crosses between C. smithii (H+ alpha +) and recombinant H- alpha + clones, the transmission of the H marker is predominantly paternal or biparental. These results allow us to conclude that (1) the alpha intron behaves as a group I intron whose unidirectional conversion influences the transmission of the H marker; and (2) the mt- paternal mitochondrial genome is transmitted more often than the mt+. The mating type has no effect in diploids obtained by artificial fusion. [less ▲]

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