Is the woodmouse (Apodemus sylvaticus) of sicily a distinct species?

[No abstract available

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

peer reviewedTwo 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

A mutation in the GTPase domain of the large subunit rRNA is involved in the suppression of a -1T frameshift mutation affecting a mitochondrial gene in Chlamydomonas reinhardtii

The dum19 mutation isolated in Chlamydomonas reinhardtii is due to the deletion of one T at codon 152 of the mitochondrial cox1 gene sequence. Phenotypically, the dum19 mutant is characterized by a lack of cytochrome c oxidase activity and is unable to grow under heterotrophic conditions. A spontaneous pseudo-revertant that grows slowly in the dark was isolated from the dum19 mutant strain. A genetic and molecular analysis allowed us to demonstrate that the revertant phenotype is the consequence of two additional mutations that together act as a frameshift suppressor: an m mutation affecting a mitochondrial gene other than cox1 and an n mutation affecting a nuclear gene. On its own the n mutation does not act as a suppressor, whereas the m mutation very slightly compensates for the effect of the -1T mutation. Sequencing analysis showed that the m mutation affects the GTPase-associated domain of the large subunit (LSU) ofmitochondrial rRNA. Surprisingly, two substitutions, A1090 to G and A1098 to C, were found in the LSU rRNA of the revertant, the latter one being already present in the dum19 mutant strain itself. The A1090 to G substitution is thus involved in the suppression of the frameshift mutation, but it is not clear whether the change at position 1098 is also required for the expression of the suppressed phenotype. To our knowledge, this is the first example of a mutation in the GTPase-associated domain acting as a suppressor of a frameshift mutation

Transmission, Recombination and Conversion of Mitochondrial Markers in Relation to the Mobility of a Group I Intron in Chlamydomonas

Mitochondrial DNA transmission has been analyzed in diploids produced from sexual crosses or artificial fusions between Chlamydomonas strains which differ by several genetic markers: a group I intron (Cs cob. 1 or alpha intron), three restriction sites (Nh, Nc and H markers) located 0.5-5 kb from the insertion site of the intron, and a MUD2 point mutation (27 bp from the insertion site) conferring resistance to myxothiazol. Recombination between mitochondrial markers is a general property of all crosses and fusions analyzed. In crosses between two intron-containing (alpha+) strains or two intron-less (alpha-) strains, the transmission is preferentially paternal (mt-), with a preponderance depending on the nature of the parental genomes. In crosses between alpha+ and alpha- strains, the conversion of intron-less molecules intron+ is frequent when the alpha+ parent is maternal (mt+) and nearly absolute when the alpha+ parent is paternal (mt-). In 94% of cases, the conversion is accompanied by the co-conversion of the MUD2 marker. In both crosses and artificial fusions, the conversion of alpha- into alpha+ also influences the transmission of the more distant Nh, Nc and H markers. It is hypothesized that the more frequent transmission of the genome containing the intron results from the elimination of alpha- molecules, as a result of a double-strand cut which is induced by an endonuclease encoded by the intron

Suppression 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

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

Transcriptional regulation of the Chlamydomonas alternative oxidase (Aox1) gene studied with a reporter gene construct

Beside the cytochrome pathway of respiration, mitochondria of higher plants and many microeukaryotes possess an alternative pathway that bypasses complexes III and IV to directly catalyze the oxidation of the ubiquinol pool by molecular oxygen. No proton motive force is generated during this reaction and free energy is thus lost as heat. The alternative oxidase (AOX), which is the sole enzyme implicated in this wasteful process, is generally subject to both transcriptional and post-translational regulation. — Recently, we have cloned and characterized two cDNAs and the corresponding gene sequences (AOX1 and AOX2) encoding the alternative oxidase in the green alga Chlamydomonas reinhardtii (1). In order to investigate the transcriptional regulation of the AOX1 gene (which is more strongly expressed than AOX2), we fused the AOX1 promoter to the arylsulfatase-encoding ARS reporter gene. Several transformants expressing the ARS protein were selected and characterized at the molecular level. While AOX transcription is induced or at least enhanced by cytochrome pathway inhibitors in fungi and higher plants, our results indicate that the expression of the AOX1:ARS construct remains unchanged following inhibitor addition in Chlamydomonas. Moreover, its expression was not affected by light, acetate as a carbon source and osmotic stress. A five-to-ten-fold stimulation was however achieved by using nitrate instead of ammonium as a nitrogen source. Effects of toxic compounds (oxidative stress agents and heavy metals) are currently investigated. This work was supported by grants from the Belgian FRFC (2.4527.97) and Fonds Spéciaux pour la Recherche dans les Universités. D. Baurain is a FNRS Research Fellow. (1) Dinant, M., Baurain, D., Coosemans, N., Joris, B. and Matagne, R. F. (2001) Characterization of two genes encoding the mitochondrial alternative oxidase in Chlamydomonas reinhardtii. Curr. Genet. (in press

Characterization of a cDNA encoding the mitochondrial alternative oxidase (AOX) in Chlamydomonas reinhardtii and assays of AOX inactivation by the antisense strategy

peer reviewe

The alternative oxidase genes and their expression in Chlamydomonas

Mitochondria of Chlamydomonas possess a terminal cyanide-resistant alternative oxidase (AOX) that reduces oxygen to water with electrons derived from the ubiquinone pool. Mutants inactivated in the cytochrome pathway of respiration are still able to consume oxygen via the alternative oxidase pathway. – By combining PCR, RT-PCR and screening of a Chlamydomonas cDNA library, two genes, AOX1 and AOX2, were identified. The cDNAs from AOX1 and AOX2 contain ORFs encoding predicted proteins of 360 and 347 amino acids, respectively. In both cases, a potential targeting presequence was identified in the protein sequence. The two AOX proteins have only 57.6% identity. Compared to AOX proteins from plants and fungi, the highest degree of identity (35-38%) was found with proteins from Aspergillus niger and Ajellomyces capsulatus. – The expression of AOX1 and AOX2 was analyzed at transcriptional level in wild-type and in dum19 mutant cells deprived of cytochrome c oxidase activity. Very low amounts of the 1.9-kb AOX2 transcript were present compared to the 2.3-kb AOX1 transcript. The expression of AOX1 was 2-6 times higher in the mutant than in wild-type, indicating that the absence of the cytochrome pathway of respiration enhances the transcriptional activity of the gene. Supported by FRFC grant 2.4527.97