Small repeated sequences in the chloroplast genome of Chlamydomonas reinhardi

Summary Chloroplast DNA of Chlamydomonas reinhardi contains many inverted repeated sequences. Analysis by hydroxyapatite binding, S1 nuclease digestion, and electron microscopy indicates that these sequences are 0.1–0.3 kilobase pairs in length, are widely distributed in the chloroplast genome, and make up 4–7% of the chloroplast DNA.Abbreviations RNA ribonucleic acid - rRNA ribosomal RNA - RNA complementary RNA - DNA deoxyribonucleic acid - chl DNA chloroplast DNA - HAP hydroxypatite - SSC 0.15 M NaCl, 0.015 M sodium citrate - 0.1xSSC, 2xSSC, 4.67xSSC 0.1, 2, and 4.67 times the concentration of SSC, respectively - TCA trichloroacetic acid - PB NaPO₄ buffer, pH 6.8 - Kb Kilobase - KbP Kilobase pair     

Cell wall organisation in Chlamydomonas reinhardi

Summary Among a series of mutants regulating wall formation, two exhibit both Mendelian and non-Mendelian segregation patterns on crossing with wild type. In addition each, on crossing with an identical mutant, frequently gives wild type and pseudo-wild type forms. Diploid wild types can be generated by fusing two identical haploid mutants. The results of genetic analyses indicated that extra-nuclear information is involved in the regulation of wall formation; this is usually stable in vegetative cells but can be renewed at the diploid spore stage. The degree of autonomy of the extra-nuclear system is considered, and the possibility of its being ultimately based on nuclear information discussed.     

Tunicamycin-sensitive glycoproteins involved in the mating of Chlamydomonas reinhardi

It has been previously shown that mild trypsinization of Chlamydomonas gametes reversibly inhibits steps of the mating process. Gametic agglutination is delayed 30–60 min, while cell wall hydrolysis and zygote formation are delayed 1–3 h. If gametes are pretreated with 5 μg/ml tunicamycin (TM) for 1 h and then trypsinized, the recovery of agglutination is blocked. These results indicate that N-glycosylated glycoproteins are involved in agglutination. Treatment of normal gametes with tunicamycin alone does not have a significant effect on agglutination and mating efficiency, suggesting that there is little or no turnover of the surface receptors before mating. Tunicamycin also interferes with cell growth and prevents the conversion of vegetative cells into gametes.     

Colchicine binding of cell extracts from colchicine-resistant mutants of Chlamydomonas reinhardi

The colchicine-binding activity of a high speed supernatant from fourteen colchicine- and/or vinblastine-resislant mutants of Chlamydomonas reinhardi has been compared to that of wild type. Four of the mutants have reduced binding per unit protein. The low level of binding of one of these mutants is unusually stable. Three other mutants have normal initial binding levels, but show altered kinetics of decay of binding activity. Most of the mutants with altered colchicine-binding activity produce abnormally large cells. Seven other mutants showed only slight or no differences in colchicine binding from wild type.     

RNA levels in colchicine-resistant mutants of Chlamydomonas reinhardi

A colchicine-resistant mutant of Chlamydomonas reinhardi (col⁻_{10, 12}) which appears blocked in the final stages of the cell division cycle is shown to have an RNA: protein ratio over four times that which is observed in wild-type cultures. This does not appear to be simply a consequence of reduced overall growth rate, because a comparable reduction in the overall growth rate of wild type by caffeine inhibition did not produce such a large rise in RNA content. The RNA levels in six other colchicine-resistant mutants, which have various abnormalities in colchicine-binding activity, has also been investigated. Two of them have elevated RNA levels.     

E. coli ribosomal proteins are cross reactive with antibody prepared against Chlamydomonas reinhardi chloroplast ribosomal subunit

Summary Antisera prepared against purified Chlamydomonas reinhardi small chloroplast ribosomal subunit, judged homogenous by sucrose gradient velocity sedimentation and RNA gel electrophoresis was immunologically cross reactive with E. coli ribosomal proteins. The results of three different experimental approaches, namely Ouchterlony double diffusion, sucrose gradient velocity sedimentation and two dimensional crossed immunoelectrophoresis indicate that both E. coli ribosomal subunits and the chloroplast large ribosomal subunit contain proteins which show antigenic similarity to the chloroplast small ribosomal subunit proteins. However, cytoplasmic ribosomal subunits did not contain proteins which were cross reactive with immune antisera.     

Triallelic complementation and hybrid enzyme formation in Chlamydomonas reinhardi

Summary In Chlamydomonas, the arg-7 cistron (linkage group I) is the structural gene for the multimeric (probably pentameric) enzyme argininosuccinate lyase. Most of the alleles of the cistron were previously shown to complement in some pair combinations, giving rise to phenotypically wild-type diploids.By crossing diploid (mt^-) and haploid (mt⁺) cells bearing different markers of auxotrophy, seven different presumptive triploid strains, phenotypically wild-type, were isolated. Each strain had 3 different arg-7 alleles or 2 mutant alleles associated with a wild one.The isolates were cytologically and biochemically analyzed: it could be concluded that they were triploid or ar least trisomic for the linkage group I.The specific activity and the thermosensitivity of the lyase were compared in the different triploids and in the diploids bearing two of the three corresponding arg-7 alleles. In most cases, the enzyme formed by triallelic complementation was more active and more heat resistant than the enzyme formed by diallelic complementation. These results can be interpreted by assuming that hybrid enzyme is formed by interaction between the products of the three different alleles. They provide a molecular basis for explaining the increased vigor often found in polyploids.     

The polypeptides of stacked and unstacked Chlamydomonas reinhardi chloroplast membranes and their relation to photosystem II activity

When cells of the ac-5 mutant strain of Chlamydomonas reinhardi are cultured mixotrophically, their chloroplast membranes are unstacked and they lack a group of membrane polypeptides that have been reported to be associated with a membrane fraction enriched for Photosystem II activity. On the other hand, the chloroplast membranes of cells grown phototrophically are stacked and they possess the membrane polypeptides. Since the unstacked membranes possess Photosystem II activity, we suggest that the polypeptides must be present in the chloroplast membrane if stacking is to occur.     

Genes involved in the regulation of the neutral phosphatase in Chlamydomonas reinhardi

Summary In Chlamydomonas reinhardi, mutations in either of two unlinked genes (PD₂ and PD₃) abolish the activity of the derepressible neutral phosphatase. The question arose whether these genes (or one of them) specify the structure of the enzyme or whether they have a regulatory function.Three mutants producing an active phosphatase at 25°C but not at 35°C were isolated and investigated. One of these mutants (PD ₁₁ ^ts ) was allelic with PD₂, another one (PD ₁₂ ^ts ) was linked to PD₃ and the third one (PD ₁₃ ^ts ) was linked to PD₂.PD ₁₁ ^ts and PD ₁₃ ^ts affected the formation of the neutral phosphatase only whereas PD ₁₂ ^ts interfered with the formation of both neutral and alkaline phosphatases at 35°C. The neutral phosphatase produced by the three mutants at low temperature was not more thermosensitive in vitro than the wild enzyme. Moreover, quite similar K_m values were found in WT, PD ₁₁ ^ts and PD ₁₂ ^ts using naphthyl phosphate as a substrate.On the other hand, revertants of PD ₂ ^- and PD ₃ ^- were isolated: their neutral phosphatases could not be distinguished from the wild enzyme on the basis of their thermosensitivities and K_m values for naphthyl phosphate.These results are consistent with the idea that PD₂ and PD₃ are regulatory genes. Other possible regulatory genes were revealed through PD ₁₂ ^ts and PD ₁₃ ^ts mutations.Chercheur qualifié du Fonds National Belge de la Recherche Scientifique     

Low molecular weight sulphydryl compounds and the expression of a cell division mutant of Chlamydomonas reinhardi

Cultures of the cytokinesis-deficient mutant of Chlamydomonas reinhardi, cyt₁ normally contain a mixture of uninucleate, binucleate and multinucleate cells. Inclusion of ethanol, diamide or mercaptoethanol in the culture medium increases the proportions of binucleate and multinucleate cells. A similar effect has previously been found with both cobalt and benzimidazole. Ethanol, cobalt and benzimidazole all raise internal cysteine or cystine levels very markedly in both mutant and wild-type cells. Changes in free cysteine or cystine levels and proportions of multinucleate cells in the mutant are induced over similar concentrations of ethanol and over similar time periods in a fixed concentration of ethanol. Diamide, thought to be a specific glutathione oxidising agent, and mercaptoethanol do not raise internal cysteine/ cystine levels. Agents which induce changes in levels of sulphydryl compounds or their oxidation-reduction status therefore seem to alter the expression of the mutant. Since similar changes in sulphydryl compounds only produce much lesser effects in cell division in wild type cells, it is proposed that cells of the mutant cyt₁ are in some way hypersensitive to such changes.     

Mapping of chloroplast genes involved in chloroplast ribosome biogenesis in Chlamydomonas reinhardtii

Summary Chloroplast gene mutations which confer antibiotic resistance on chloroplast ribosomes of the green alga Chlamydomonas reinhardtii have been tested for allelism and mapped by recombination analysis of progeny from biparental zygote clones. Thirty-one independently isolated streptomycin resistant mutants have chloroplast ribosomes which are resistant to this drug in an assay based on misreading of isoleucine in response to a poly U template, and comprise one nuclear and four chloroplast gene loci. Four mutants resistant to spectinomycin, and three mutants resistant to neamine and kanamycin, which have chloroplast ribosomes resistant to their respective antibiotics in poly U directed phenylalanine incorporation, appear to map in a single chloroplast gene locus. Representative alleles of this nr/spr locus, the four streptomycin resistance loci, and two chloroplast gene loci for erythromycin resistance, have been analyzed in a series of parallel crosses to establish the following map order for these seven genes in the chloroplast genome: er-u-la-er-u-37-nr-u-2-1/spr-u-1-H-4-sr-u-2-23-sr-u-2-60-sr-u-sm3-sr-u-sm2. These seven genes may constitute a ribosomal region within the chloroplast genome of Chlamydomonas comparable to the ribosomal gene clusters in bacteria.     

The methyl viologen-catalyzed mehler reaction and catalase activity in blue-green algae and Chlamydomonas reinhardi

1. The methyl viologen-catalyzed Mehler reaction was investigated in intact cells of five species of blue-green algae and Chlamydomonas reinhardi.

2. In the presence of methyl viologen, all the blue-green algae except Anabaena flos-aquae show a light-dependent O₂ consumption as well as a post-illumination O₂ evolution. The rate of O₂ consumption is stimulated by 1 mM KCN, an inhibitor of catalase, but the dark O₂ evolution becomes suppressed.

3. A. flos-aquae shows a light-dependent methyl viologen-catalyzed O₂ uptake which is not affected by 1 mM KCN. Furthermore, there is no release of O₂ in the dark following illumination.

4. With C. reinhardi, the cells do not show any net O₂ exchange during or after illumination. Addition of 1 mM KCN, however, results in an immediate O₂ uptake in the light.

5. Based on the mechanism postulated for the Mehler reaction in isolated chloroplasts, it was deduced that the differences in the kinetics of the O₂ exchange catalyzed by methyl viologen reflect differences in the endogenous catalase activity in these algae. Cells of A. flos-aquae are deficient in catalase activity whereas those of the other blue-green algae possess catalase, although at low activity. C. reinhardi, on the other hand, has high catalase activity in vivo.

6. These findings are corroborated by results obtained from O₂ electrode measurements of catalase activity in cell-free extracts of these algae.

7. The possible roles of catalase in algae and the implications of these results are also discussed.     

The fate of chloroplast DNA during cell fusion, zygote maturation and zygote germination in Chlamydomonas reinhardi as revealed by DAPI staining

Chlamydomonas reinhardi, a haploid isogamous green alga, presents a classic case of uniparental inheritance of chloroplast genes. Since the molecular basis of this phenomenon is poorly understood, an examination of the cytology of the C. reinhardi plastid DNA was made in gametes, newly formed zygotes, maturing zygotes, and at zygote germination.The single plastid per cell of Chlamydomonas contains a small number of DNA aggregates (‘nucleoids’) which can be seen after staining with DNA-binding fluorochromes. In zygotes formed by pre-stained gametes, the fluorescing nucleoids disappear from the plastid of mating type minus (male) gamete plastids but not from the plastid of mating type plus (female) gamete plastids about 1 h after zygote formation. Subsequently, nucleoids aggregate slowly to a final average of two or three in the single plastid of the mature zygote.Quantitative microspectrofluorimetry indicates that gametes of both mating types have equal amounts of plastid DNA, and that zoospores arising from zygotes have 3.5 × as much as gametes. Assuming degradation of male plastid DNA, there must be a very major synthesis of plastid DNA between zygote formation and zoospore release when zygotes produce the typical 8–16 zoospores. That synthesis appears to occur at germination, where there is a massive increase in plastid DNA and nucleoid number beginning just prior to meiosis. The results support the theory that uniparental inheritance results from degradation of plastid DNA entering the zygote via the male gamete and suggest further studies, using mutants and altered conditions, which might explain how male plastid DNA sometimes survives.     

Chlamydomonas reinhardtii proteomics

Proteomics, based on the expanding genomic resources, has begun to reveal new details of Chlamydomonas reinhardtii biology. In particular, analyses focusing on subproteomes have already provided new insight into the dynamics and composition of the photosynthetic apparatus, the chloroplast ribosome, the oxidative phosphorylation machinery of the mitochondria, and the flagellum. It assisted to discovered putative new components of the circadian clockwork as well as shed a light on thioredoxin protein-protein interactions. In the future, quantitative techniques may allow large scale comparison of protein expression levels. Advances in software algorithms will likely improve the use of genomic databases for mass spectrometry (MS) based protein identification and validation of gene models that have been predicted from the genomic DNA sequences. Although proteomics has only been recently applied for exploring C. reinhardtii biology, it will likely be utilized extensively in the near future due to the already existing genetic, genomic, and biochemical tools.     

Temperature-sensitive yellow mutants of Chlamydomonas reinhardtii

Summary We have isolated and genetically characterized 10 mutants of Chlamydomonas reinhardtii carrying single, mendelian, temperature-sensitive yellow mutations. The mutants have a yellow phenotype at the restrictive temperature (33°C), but have a wildtype phenotype at the permissive temperature (25°C). Based on complementation and recombination tests, the ten mutations include alleles of two previously described yellow loci (y-1 and y-6) and three new yellow loci (y-8, y-9, and y-10). At the restrictive temperature, y-8, y-9, and y-10 are physiologically similar to other yellow mutants. They accumulate small amounts of protochlorophyllide when grown under dim light, but synthesize normal amounts of chlorophyll when grown in the light. Linkage tests indicate that the three new mutations are not linked to each other. y-8 is linked to y-7 on linkage group III, and y-10 is linked to y-5 and y-6 on linkage group I. y-9 is located on linkage group II. We conclude that the control of light-independent protochlorophyllide reduction is complex, involving several genetic loci which are scattered in the genome and which code for gene products able to complement in trans. Temperature-sensitive alleles at several of the yellow loci suggest that the gene products made by these loci are proteins.     

Ribosomal subunits affected by antibiotic resistance mutations at seven chloroplast loci in Chlamydomonas reinhardtii

Summary Mutations at seven recombinationally distinct chloroplast loci confer antibiotic resistance on chloroplast ribosomes of the green alga Chlamydomonas reinhardtii. Assays of polynucleotide-directed amino acid incorporation by ribosomes reconstituted from mutant and wild type subunits demonstrate that streptomycin, neamine/kanamycin and spectinomycin resistance mutations specifically affect the small ribosomal subunit, whereas mutations to erythromycin resistance affect the large subunit. Although in each case the subunit site of antibiotic resistance is the same as that observed in analogous mutations in Escherichia coli, the number of loci conferring resistance to a given antibiotic differs in the two organisms. We have previously shown that streptomycin resistance mutations in Chlamydomonas map at five discrete loci (one nuclear and four chloroplast), and that mutations to neamine/kanamycin and spectinomycin resistance appear to define a single chloroplast locus. Results presented here confirm our previous report that all chloroplast erythromycin resistance mutations isolated to date fall into two recombinationally distinct loci, and indicate that mutants at one of these loci may be further divided on the basis of their level of cross resistance to other macrolide antibiotics.     

Isolation of a cDNA fragment coding for Chlamydomonas reinhardtii ferredoxin and expression of the recombinant protein in Escherichia coli

A cDNA clone coding for mature C. reinhardtii ferredoxin has been isolated from a cDNA library using PCR and two oligonucleotide primers based on the N- and C-termini of the protein's amino acid sequence. The nucleotidic sequence of the PCR fragment (299 bp) agreed well with the amino acid sequence since a single conservative substitution (Thr-7 to Ser) could be deduced. The PCR fragment was inserted into the expression vector pTrc 99A, using the incorporated NcoI and BamHI restriction sites and the construction used to transform E. coli (DH5α F′). After subsequent large scale expression and purification of the recombinant protein, biochemical and biophysical analysis have indicated that the product isolated from E. coli is homologous to native ferredoxin isolated from green algae.