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.

Modification of Chloroplast Gene Transmission in Somatic Fusion Products and Vegetative Zygotes of CHLAMYDOMONAS REINHARDI by 5-Fluorodeoxyuridine.

Sexual crosses and somatic fusions were performed between complementing wall-less arg(-) mutant strains bearing chloroplast markers for resistance to antibiotics. The mode of chloroplast allele transmission was investigated in the diploid colonies developed from both vegetative zygotes and fusion products. Before mating or fusion, one or both of the parental strains were grown for 4 or 8 days on agar containing 5-fluorodeoxyuridine (FUdR, 0.1 to 1.0 mm), which selectively reduces the amount of chloroplast DNA in Chlamydomonas. When one parent was pregrown on FUdR, the frequency of vegetative zygotes transmitting chloroplast alleles of both parents (biparental or BP zygotes) decreased, the reduction being more drastic when the mt(-) parent was treated. Transmission was mainly uniparental maternal (UPm) or paternal (UPp) depending on whether the mt(-) or the mt(+) parent was pregrown for 8 days in the presence of 1.0 mm FUdR. Treatment of both parents led to a strong maternal transmission. In the experiments involving somatic fusion between parent 1 and parent 2 (same or opposite mt), the ratio UP(1)/UP(2), which was approximately equal to 1 in the control, decreased or increased according to whether the cells of parent 1 or 2 were pregrown on FUdR. In parallel, the frequency of BP fusion products always decreased. When both parental strains were treated with FUdR, the frequency of BP fusion products also decreased and the ratio UP(1)/UP(2) was roughly equal to 1. The effect of FUdR can be interpreted in terms of reduction of the input frequencies of parental chloroplast genomes at the time of gametic or somatic cell fusion, the bias in favor of the maternal parent being operational only in sexual crosses.

Isolation and study of mutants lacking a derepressible phosphatase in Chlamydomonas reinhardi.

In the green alga Chlamydomonas reinhardi, removal of inorganic phosphate from the culture medium results in the increase of phosphatase activity (derepression) in the wild-type (WT) strain as well as in a double mutant (P2Pa)) lacking the two main constitutive acid phosphatases. Following treatment of WT and P2Pa with N-methyl-N-nitro-N-nitrosoguanidine (MNNG), mutants were recovered which display very low phosphatase activities when grown in the absence of phosphate; as shown by electrophoresis, they lack one non-migrating phosphatase (PD mutants). This enzyme is active over a wide range of pH with an optimum at pH 7.5. The comparison of elctropherograms form WT and mutants grown on media with or without phosphate allowed us to provide a tentative definition of the pool of derepressible phosphatases in Chlamydomonas: in addition tothe neutral phosphatase lacking in PD mutants, Chlamydomonas produces two electrophoretic forms of alkaline phosphatase showing an optimal activity at pH 9.5.

Acid phosphatase mutants in Chlamydomonas: isolation and characterization by biochemical, electrophoretic and genetic analysis.

In order to isolate acid phosphatase mutants in the green alga Chlamydomonas reinhardi, a staining method for detecting the enzyme activity in colonies has been developed. The occurrence of more than one acid phosphatase brought about some difficulty in the selection of mutants. We have, however, found an original method of selection based on the differential heat sensitivity of the enzymes. After treatment of the wild-type strain with N-methyl-N'-nitro-N-nitrosoguanidine, two types of mutants were recovered, then analyzed by biochemical and electrophoretic methods. In the first class of mutants (P(1), P(2), P(3),...) a heat-stable acid phosphatase bound to cellular debris of the crude extract was missing. The mutant P(a), representing the second class of mutations, was lacking a soluble heat-sensitive enzyme. These mutations were genetically different and exhibited mendelian inheritance.

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

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.

Pleiotropic mutants hypersensitive to heavy metals and to oxidative stress in Chlamydomonas reinhardtii.

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.

Transmission of chloroplast alleles in somatic fusion products obtained from vegetative cells and/or 'Gametes' of Chlamydomonas reinhardi.

Somatic fusion between cells of Chlamydomonas containing complementing cell wall and auxotrophic mutations and bearing chloroplast markers for resistance to antibiotics (streptomycin or spectinomycin) have been performed to analyze the mode of chloroplast gene transmission in the fusion products. Prototrophic colonies developed from mitotic divisions of 'diploid' fusion products were isolated on minimal medium and analyzed for their resistance to antibiotics. Fusion was performed between vegetative or nitrogen-starved cells (non-flagellated gametes) of the same or of opposite mating type. In all cases, about one third of the fusion products (mt (+)/mt (+), mt (-)/mt (-), or mt (+)/mt (-)) transmitted chloroplast markers from both parents (= biparental fusion products). The rest of the population was equally distributed between fusion products transmitting the chloroplast marker of one parent or the other exclusively (uniparental, or UP fusion products). The results indicate that the preferential elimination of paternal chloroplast alleles (i.e. maternal inheritance) observed after sexual fusion does not occur following artificially induced cell fusion, and that heterozygosity at the mt locus is not sufficient to ensure a directionality in uniparental chloroplast gene transmission.When somatic fusions were made between vegetative cells and nitrogen-starved cells, preferential transmission of the chloroplast alleles of the vegetative parent was observed, independently of the mating type of the parent. The data can be interpreted in terms of differences in the input frequencies of parental chloroplast genomes at the time of cell fusion. The possible importance of flagellar contact between opposite mating types in determining patterns of chloroplast gene transmission is also discussed.

Fine structure of the arg-7 ciston in chlamydomonas reinhardi. Complementation between arg-7 mutants defective in argininosuccinate lyase.

In Chlamydomonas reinhardi, the arg-7 cistron is the structural gene for the enzyme argininosuccinate lyase which catalyzes the last reaction in the biosynthesis of arginine. Fourteen mutants (nine previously analyzed and five new mutants) defective in the lyase have been investigated so far: they all map within a cistron (length: 1.0--1.6 recombination units) of the linkage group I and fall within six groups of complementation. The enzyme activity found in the diploids formed by intragenic complementation was always lower than in wild-type haploid or diploid strains. The study of the denaturation curves obtained by heat treatment of the lyase indicates that in some diploids, several enzyme varieties can be present. These results and those previously obtained with diploids formed by intragenic and intergenic complementation (Matagne and Loppes, 1972; Matagne, 1976) are discussed in relation to the recent data showing that the argininosuccinate lyase is a multimeric enzyme probably composed of five identical polypeptide chains (Matagne and Schlösser, 1977).

Arg-7 mutant X wild-type crosses in Chlamydomonas reinhardi: study of the enzyme produced in diploid strains.

The arg-7 locus is the structural gene for the argininosuccinate lyase (ASL). Interallelic complementation was previously found to occur between several mutants of the locus: this is indicative for the homomultimeric nature of ASL. Two complementing (arg-7-5 and arg-7-7) and two non-complementing (arg-7-1 and arg-7-6) mutants of the arg-7 locus were crossed to the pab-2 strain (which is wild-type for the arg-7 locus). In each cross, heterozygote phenotypically wild-type strains were isolated; their diploid pattern was demonstrated by various criteria: mating type, cell volume, nuclear size. The four heterozygotes were compared to the haploid wild-type and in some experiments, to the diploid strain arg-1 X pab-2 homozygous for the arg-7 locus. No difference was found in growth rate and in the Michaelis constant values for ASL. The specific activity of the enzyme produced in the heterozygotes was about 50 percent of the activity found in haploid or diploid wild-type. The heat sensitivity of ASL was also investigated in the different strains: two (containing the complementing mutations arg-7-5 and arg-7-7) of the four heterozygotes produce ASL varieties different from the wild-type enzyme as far as the thermolability is concerned. These results suggest that hybrid ASL can be formed by interaction between the products of wild-type and mutant genes. A clear dominance of the wild-type allele is expected only when the mutant allele has no product of the gene: this could be the case for arg-7-1 and arg-7-6.

Alteration of dark respiration and reduction of phototrophic growth in a mitochondrial DNA deletion mutant of Chlamydomonas lacking cob, nd4, and the 3' end of nd5.

We describe here a new type of mitochondrial mutation (dum24; for dark uniparental minus inheritance) of the unicellular photosynthetic alga Chlamydomonas reinhardtii. The mutant fails to grow under heterotrophic conditions and displays reduced growth under both photoautotrophic and mixotrophic conditions. In reciprocal crosses between mutant and wild-type cells, the meiotic progeny only inherit the phenotype of the mating-type minus parent, indicating that the dum24 mutation exclusively affects the mitochondrial genome. Digestion with various restriction enzymes followed by DNA gel blot hybridizations with specific probes demonstrated that dum24 cells contain four types of altered mitochondrial genomes: deleted monomers lacking cob, nd4, and the 3' end of the nd5 gene; deleted monomers deprived of cob, nd4, nd5, and the 5' end of the cox1 coding sequence; and two types of dimers produced by end-to-end fusions between monomers similarly or differently deleted. Due to these mitochondrial DNA alterations, complex I activity, the cytochrome pathway of respiration, and presumably, the three phosphorylation sites associated with these enzyme activities are lacking in the mutant. The low respiratory rate of the dum24 cells results from the activities of rotenone-resistant NADH dehydrogenase, complex II, and alternative oxidase, with none of these enzymes being coupled to ATP production. To our knowledge, this type of mitochondrial mutation has never been described for photosynthetic organisms or more generally for obligate aerobes.

Genetic analysis of nitrate reductase-deficient mutants in Chlamydomonas reinhardii.

Six mutants (305, 301, 203, 307, 104 and 102) of Chlamydomonas reinhardii, all defective in nitrate reductase (NR) activity, have been genetically analyzed. All except 102 carry single Mendelian mutations.Mutant 305, defective in diaphorase activity and mutant 301, defective in terminal enzyme activity, did not give rise to wild-type recombinants when crossed to each other or with the nit-1 mutant isolated from strain 137c (which is actually a double mutant nit-1 nit-2). Nit-1 was shown to lack both diaphorase and terminal activities. Whether the mutated sites in 305 and 301 are located in a unique cistron (nit-1) or in two adjacent cistrons (nit-1a and nit-1b) coding for a diaphorase subunit and a terminal subunit of NR is discussed in the light of previous biochemical findings.The 203 mutation affecting a regulatory gene did not recombine with nit-2, the other mutated locus present in strain 137c.Mutants 307, 104 and 102, all lacking molybdenum cofactor for both NR and xanthine dehydrogenase, where shown to be affected in different loci. The genes mutated in 307 and 104 have been designated nit-3 and nit-4, respectively. The 102 strain is mutated in two non-linked loci, nit-5 and nit-6, with both mutations required to confer the mutant phenotype. One of these cryptic mutations is present in the "wild" strain 21gr.The results indicate that at least six or seven loci are involved in the production of an active NR enzyme: one (nit-1) or two (nit-1a and nit-1b) cistrons to produce the NR apoproteins responsible for the partial activities diaphorase and terminal, one locus (nit-2) for the regulation of NR synthesis, and four loci (nit-3, nit-4, nit-5 and nit-6) to produce the molybdenum cofactor. The loci nit-1a and nit-2 seem to correspond to the nit-A and nit-B loci described by Nichols and Syrett (J Gen Microbiol 108:71-77, 1978).

In vivo complementation analysis of nitrate reductase-deficient mutants in Chlamydomonas reinhardtii.

In vivo complementation between different wild and mutant strains defective for nitrate assimilation has been performed by isolating diploid strains from the appropriate crosses. Twenty-two diploids homozygous or heterozygous with respect to nitrate reduction and able to grow on nitrate medium were obtained and their diploid character demonstrated from analyses of mating type, cell volume, nuclear size and progeny of crosses with haploid wild-type. All diploids were assayed for overall- and terminal-nitrate reductase (NR) activity and for the occurrence of the NR-diaphorase subunit. Data on NR activities in heterozygotes carrying mutation(s) in structural gene(s) (nit-1 or nit-1a, nit-1b) agree with the heteromultimeric nature of the enzyme complex previously described (Franco et al. (1984) EMBO J 3: 1403-1407), and indicate that subunits are exchangeable to form hybrid enzymes. In addition, in vitro complementation tests with mutant nit-1 of C. reinhardtii indicate that this mutant has defective NR-diaphorase subunits but intact terminal subunits. Super-repression caused by the mutant allele nit-2 is suppressed by the wild allele in heterozygotes, which suggests a positive control by the nit-2 product on structural gene(s) transcription. Mutant alleles of genes for the biosynthesis of molybdenum-containing cofactor, either nit-4 or nit-5 and nit-6, were recessive in diploids carrying them. The mutant allele of nit-3, from strain 307, was codominant in all heterozygotes suggesting that nit-3 codes for a protein whose activity is limiting for the molybdenum-cofactor biosynthetic pathway.

Isolation and genetic analysis of Chlamydomonas reinhardtii strains resistant to cadmium.

In Chlamydomonas reinhardtii, cadmium induces reduction of growth, reduction of chlorophyll content, and lethality. The toxicity was higher in a cell wall-deficient strain than in the wild type. By growing the cells on agar medium containing cadmium at concentrations inducing high lethality, stable resistant clones were isolated. The resistance was due to a nuclear mutation (cadAR) which probably preexisted in the wild-type cell population, as suggested by the fluctuation test. A double mutant (cadAR cadBR) was selected on media containing higher concentrations of cadmium. The cadBR mutation, which is unlinked to cadAR, determines a resistance intermediate between the CadAR mutant and the wild-type strain. Both cadAR and cadBR mutations are partially dominant.

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.

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 of chloroplast genes in triploid and tetraploid zygospores of Chlamydomonas reinhardtii: Roles of mating-type gene dosage and gametic chloroplast DNA content.

Diploid clones homozygous (mt(+)/mt(+) or mt(-)/mt(-)) or heterozygous (mt(+)/mt(-); phenotypically mt(-)) for the mating-type locus and homoplasmic for a chloroplast marker conferring resistance to an antibiotic were isolated by artificially induced cell fusion or sexual mating. These diploids were crossed with haploid or diploid strains of opposite mating type and carrying another chloroplast marker. The transmission of the chloroplast genes was analyzed in the triploid and tetraploid zygospores in comparison with diploid zygospores used as controls. The transmission was almost exclusively maternal (mt(+)) (>94%) in the crosses mt(+) x mt(-), mt(+)/mt(+) x mt(-), and mt(+)/mt(+) x mt(-)/mt(-). The transmission was preferentially maternal (>76%) in the crosses mt(+) x mt(-)/mt(-) whereas in the crosses mt(+) x mt(+)/mt(-), <50% of the zygospores transmitted the chloroplast allele of maternal (mt(+)) origin. The zygospores produced in crosses mt(+)/mt(+) x mt(+)/mt(-) transmitted the alleles from both parents in >60% of cases. The results show that (i) the presence of one mt(+) allele in the mt(+)/mt(-) (phenotypically mt(-)) diploid gametes and (ii) the higher amount of chloroplast DNA molecules (input) present in the diploid gametes versus the haploid ones favor the transmission of the chloroplast allele contributed by these gametes. Moreover, because the zygospores issued from crosses mt(+)/mt(+) x mt(-) and mt(+) x mt(+)/mt(-) were genotypically identical mt(+)/mt(+)/mt(-)) but behaved very differently in their chloroplast gene transmission, it was concluded that the molecular events leading to preferential elimination of paternal DNA copies must occur before the fusion of nuclei or chloroplasts in the newly formed zygotes.

Perturbation of chloroplast gene transmission in diploid and triploid zygotes of Chlamydomonas reinhardi by 5-fluorodeoxyuridine.

Haploid cells or diploid cells homozygous (mt(+)/mt(+) or mt(-)/mt(-)) or heterozygous (mt(+)/mt(-) phenotypically mt(-)) for the mating-type locus and homoplasmic for a chloroplast marker conferring resistance to an antibiotic were crossed with haploid cells of opposite mating-type and carrying another chloroplast marker. Before mating, one or both of the parental strains were grown for 8 days on agar containing 1 mM 5-fluorodeoxyuridine (FUdR), which selectively reduces the amount of chloroplast DNA in Chlamydomonas. In all cases, the chloroplast allele of the treated parent was less frequently transmitted to the meiotic progeny of the zygote than in the corresponding control cross. The effect of FUdR was more pronounced on haploid cells than on diploid cells which initially contained a two-fold higher amount of chloroplast DNA.The results are discussed in relation to current models for uniparental inheritance of non-Mendelian genes.

Chloroplast gene inheritance studied by somatic fusion in Chlamydomonas reinhardtii.

Somatic fusion between strains of Chlamydomonas containing complementing cell-wall and auxotrophic mutations, having the same mating-type (mt) and bearing chloroplast markers, have been performed to study the mode of chloroplast gene inheritance in the fusion products. About one third of the fusion products (mt (+)/mt (+) or mt (-)/mt (-)) transmitted chloroplast markers from both parents (= biparental fusion products). The rest of the population was equally distributed between fusion products transmitting the chloroplast marker of one parent or the other (uniparental fusion products) exclusively. Incubation of the fusion products in the dark for 48 hours, immediately after the fusion, decreases the frequency of biparental fusion products. The results indicate that the general process of elimination of chloroplast alleles is independent of the presence of both mt (+) and mt (-) alleles in the cell. In contrast, directional elimination (i.e. preferential elimination of paternal chloroplast alleles) does appear to depend upon heterozygosity at the mt locus. These results are discussed in relation to the models which have been proposed to explain the maternal inheritance of chloroplast genes in Chlamydomonas.

Purification and subunit structure of argininosuccinate lyase from Chlamydomonas reinhardi.

Argininosuccinate lyase (EC 4.3.2.1) was purified by (NH4)2SO4 fractionation, chromatography on DEAE-cellulose and gel filtration on Sephadex G-200. The final enzyme preparation was purified 46-fold compared with the crude extract. Electrophoresis of this preparation revealed three bands, the major one having the enzyme activity. Analysis of the enzyme by gel filtration and by disc electrophoresis (in two different concentrations of acrylamide) gave mol.wts. of 200000 (+/- 15000) and 190000 (+/- 20000) respectively. Treatment with sodium dodecyl sulphate and mercaptoethanol dissociated the enzyme into subunits of mol.wt. 39000 (+/-2000). The results are indicative of the multimeric structure of the enzyme, which is composed of five (perhaps four or six) identical subunits.