Induction and characterization of mitochondrial DNA mutants in Chlamydomonas reinhardtii.

In addition to lethal minute colony mutations which correspond to loss of mitochondrial DNA, acriflavin induces in Chlamydomonas reinhardtii a low percentage of cells that grow in the light but do not divide under heterotrophic conditions. Two such obligate photoautotrophic mutants were shown to lack the cyanide-sensitive cytochrome pathway of the respiration and to have a reduced cytochrome c oxidase activity. In crosses to wild type, the mutations are transmitted almost exclusively from the mating type minus parent. A same pattern of inheritance is seen for the mitochondrial DNA in crosses between the two interfertile species C. reinhardtii and Chlamydomonas smithii. Both mutants have a deletion in the region of the mitochondrial DNA containing the apocytochrome b gene and possibly the unidentified URFx gene.

Estimation of protochlorophyll(ide) contents in plant extracts; re-evaluation of the molar absorption coefficient of protochlorophyll(ide).

In an attempt to solve the controversy about the evaluation of the molar absorption coefficient of PChl(ide), this coeffecient is estimated in this work by using an original experimental approach. The calculated molar absorption coefficient of PChl(ide) is 30.4.10(3) l mole(-1) cm(-1) at 626 nm in acetone 80%; it is close to that derived from the specific absorption coefficient of Koski and Smith when assurning that the pigment extracted by these authors was the esterified pigment: PChl. Sets of equations for the quantification of Chl(ide) a, Chl b and PChl(ide) in 80% acetone extracts are derived.

Estimation of protochlorophyll(ide) contents in plant extracts; re-evaluation of the molar absorption coefficient of protochlorophyll(ide).

In an attempt to solve the controversy about the evaluation of the molar absorption coefficient of PChl(ide), this coefficient is estimated in this work by using an original experimental approach. The calculated molar absorption coefficient of PChl(ide) is 30.4.10(3) 1 mole(-1) cm(-1) at 626 nm in acetone 80%; it is close to that derived from the specific absorption coefficient of Koski and Smith when assuming that the pigment extracted by these authors was the esterified pigment: PChl. Sets of equations for the quantification of Chl(ide) a, Chl b and PChl(ide) in 80% acetone extracts are derived.

Some properties of purified fractions from bean etioplast membranes.

Distribution of proteins, protochlorophyllide and photoactivity was studied in fractions obtained from bean etioplast membranes purified by linear or discontinuous sucrose density gradient centrifugation. The polypeptide pattern of two selected membrane fractions was examined by means of polyacrylamide gel electrophoresis. Both fractions contained the doublet of molecular weights 34.000-35.000 characteristic of NADPH-Protochlorophyllide oxido-reductase but differed by accompanying proteins. When both fractions were illuminated, protochlorophyllide was photoreduced in chlorophyllide fluorescing at 688 nm. During a subsequent 30 min. dark period, the emission peak shifted rapidly up to 678 nm in one of the fractions while it moved slowly to 683 nm in the other one. The possible occurrence of two phototransformable protochlorophyllide-complexes is discussed. Delta-aminolevulinic acid pretreatment of etiolated leaves led to an increase in the amount of proteins in the etioplast membranes.

Mitochondrial genome transmission in Chlamydomonas diploids obtained by sexual crosses and artificial fusions: role of the mating type and of a 1 kb intron.

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.

The apocytochrome b gene of Chlamydomonas smithii contains a mobile intron related to both Saccharomyces and Neurospora introns.

The mitochondrial DNA of the two interfertile algal species Chlamydomonas smithii and Chlamydomonas reinhardtii are co-linear with the exception of ca. 1 kb insertion (the alpha insert) present in C. smithii DNA only. In vegetative diploids resulting from interspecific crosses, mitochondrial genomes are transmitted biparentally except for the alpha insert which is transmitted to all C. reinhardtii molecules in a manner reminiscent of the intron-mediated conversion event that occurs at the omega locus in yeast mitochondria, under the action of the I-SceI endonuclease. Here we report that the alpha insert corresponds to a typical group I intron of 1075 bp, inserted within the gene for apocytochrome b and containing a 237 codon open reading frame (ORF). We also report the complete sequence of the apocytochrome b gene of C. smithii. Comparison with the sequence of the same gene in C. reinhardtii reveals the precise intron insertion site. These data, together with the previous genetic data provide the first example of intron mobility in mitochondria of the plant kingdom. The product of the intronic ORF shows 36% amino acid identity with the I-SceI endonuclease whereas the intron ribozyme shows a 60% identity at the nucleotide level with the Neurospora crassa cob.1 intron. The possibility of a recent horizontal transfer of introns between fungi and algae is discussed.

Biochemical, genetic and molecular characterization of new respiratory-deficient mutants in Chlamydomonas reinhardtii.

Eight respiratory-deficient mutants of Chlamydomonas reinhardtii have been isolated after mutagenic treatment with acriflavine or ethidium bromide. They are characterized by their inability to grow or their very reduced growth under heterotrophic conditions. One mutation (Class III) is of nuclear origin whereas the seven remaining mutants (Classes I and II) display a predominantly paternal mt- inheritance, typical of mutations residing in the mitochondrial DNA. Biochemical analysis has shown that all mutants are deficient in the cyanide-sensitive cytochrome pathway of the respiration whereas the alternative pathway is still functional. Measurements of complexes II + III (antimycin-sensitive succinate-cytochrome c oxido-reductase) and complex IV (cytochrome c oxidase) activities allowed to conclude that six mutations have to be localized in the mitochondrial apocytochrome b (COB) gene, one in the mitochondrial cytochrome oxidase subunit I (COI) gene and one in a nuclear gene encoding a component of the cytochrome oxidase complex. By using specific probes, we have moreover demonstrated that five mutants (Class II mutants) contain mitochondrial DNA molecules deleted in the terminal end containing the COB gene and the telomeric region; they also possess dimeric molecules resulting from end-to-end junctions of deleted monomers. The two other mitochondrial mutants (Class I) have no detectable gross alteration. Class I and Class II mutants can also be distinguished by the pattern of transmission of the mutation in crosses. An in vivo staining test has been developed to identify rapidly the mutants impaired in cyanide-sensitive respiration.