Localization of extranuclear genes by investigations of the ultrastructure in Chlamydomonas reinhardii

Summary Investigations of the ultrastructure showed that the localization of the nd gene in the nonmendelian neamin dependent mutant (nd) of Chlamydomonas reinhardii is situated in the chloroplast; the mitochondria of this mutant are neamin resistant. In contrast, the sd gene of the streptomycin dependent mutant (sd) is located in the mitochondria, whereas the chloroplast ist streptomycin resistant.     

Altered chloroplast ribosomal proteins in a yellow mutant of Chlamydomonas reinhardii.

Summary Ribosomes and ribosomal proteins from wild-type and a yellow mutant of Chlamydomonas reinhardii were analysed and compared by two-dimensional gel electrophoresis.Mixothrophycally grown yellow-27 mutant differs from wild-type cells in lowered chlorophyll content and grana fromation of the chloroplast.Analytical ultracentrifuge analyses of cell extracts show a reduced amount of free 70S ribosomes and increased level of 50S subunits in the mutant cells. Similar results were obtained by electronmicroscopical method.Two-dimensional gel electrophoresis shows alterations in protein composition of 70S ribosomes of the mutant. Two proteins of 70S ribosomes have been altered. One of them with high molecular weight is practically absent while there is an additional, intensively stained spot in the mutant.Since the mutation is inherited in a non-Mendelian manner it is possible that the protein alterations in 70S ribosome are localized in the chloroplast DNA.     

Time-sequence of nuclear and chloroplast fusions in the zygote of Chlamydomonas reinhardii

Contradictory data have been published concerning the time-sequence of nuclear and chloroplast fusions in the zygote of Chlamydomonas. In the present study, adjacent ultrathin sections of Chlamydomonas reinhardii zygotes of various ages were examined with the electron microscope. These sections clearly reveal that nuclear fusion precedes chloroplast fusion.     

Ribulose bisphosphate carboxylase from a mutant strain of Chlamydomonas reinhardii deficient in chloroplast ribosomes

The ac-20 mutant strain of the unicellular green alga, Chlamydomonas reinhardii, lacks both chloroplast ribosomes and ribulose bisphosphate carboxylase activity when grown on organic medium. Under these conditions, the cells do not posses pools of either the large or small subunit of this enzyme. When transferred to inorganic medium, the carboxylase activity recovers. During this recovery, de novo synthesis of both subunits occurs. Synthesis of both subunits is inhibited by chloramphenicol even when possible free subunit pools rather than just the subunits incorporated into whole enzyme are examined.Abbreviations RubP ribulose bisphosphate - CAP D-threochloramphenicol - CHI cycloheximide - PPO 2,5-diphenyloxazole - POPOP 1,4-bis[2(5-phenyloxazolyl)]-benzene - SDS sodium dodecyl sulfate     

Isolation of biochemically active chloroplasts from chlamydomonas

Chloroplasts from the cell wall mutant cw-15-2 of Chlamydomonas reinhardii were isolated by disruption of the cells in the Yeda press and fractionation through step gradients of Percoll. The resulting chloroplast fraction contained 80–85% intact chloroplasts. Electron micrographs of thin sections of the chloroplast fraction showed some cytoplasmic impurities, although almost no cytoplasmic ribosomes were detected by analysis of the ribosomal subunits.The isolated chloroplasts are active in photosynthetic O₂-evolution and CO₂-fixation, with the highest rates obtained in the presence of ATP.The chloroplast fraction also showed high rates of light-dependent in organello protein synthesis, with labelling of discrete chloroplast proteins known to be synthesized in the chloroplasts.     

Degradation and compartmentalization of urea in Chlamydomonas reinhardii

Urea is accumulated against a concentration gradient in Chlamydomonas reinhardii. Only energy generated from photosynthesis is used for this accumulation, while degradation of urea utilizes other energy sources. Exogenous supplied urea is distributed between two pools, one large nonmetabolic and one metabolic pool. Ammonia inhibits the transport from the nonmetabolic to the metabolic pool.The nonmetabolic pool is probably located in the chloroplast, and the accumulation of urea is due to an active transport into the chloroplast.Non-Standard Abbreviations N medium medium without nitrogen source - TCA trichloroacetic acid - TCAI trichloroacetic acid insoluble fraction     

Histone - induced changes in the cellular growth of synchronized chlamydomonas reinhardii are due to histone H1

The cellular growth ofChlamydomonas reinhardii is modified by the addition of a total exogenous histone fraction. These modifications may be related to chloroplast DNA replication; they are different according to the different classes of histones. The H₁ subfraction seems to be responsible for the effect of the total histone fraction.     

Linkage of a Known Chloroplast Gene Mutation to the Uniparental Genome of CHLAMYDOMONAS REINHARDII

Data are presented that associate three new markers with the uniparental linkage group in Chlamydomonas reinhardii. One of these, mutant 10-6C, is a genetic marker for the structural gene of the large subunit of ribulose bisphosphate carboxylase. These results provide the first direct link between the uniparental gene map and the physical map of chloroplast DNA. The other two markers, Dr2 (DCMU resistant) and 8-36C (deficient in photosystem II activity), map to a single locus. The data suggest that mixing in zygotic chloroplasts may not be complete so that input genomes do not have equal opportunities to recombine. The data are not compatible with simple linear or circular maps but can be explained on the basis of the known physical structure of chloroplast DNA.     

The location of the Mendelian linkage groups in Chlamydomonas reinhardii

Summary Evidence is presented which contradicts a recent suggestion by Levine and Goodenough that the haploid nucleus of Chlamydomonas reinhardii contains eight chromosomes and that eight of the sixteen known linkage groups may be located within the chloroplast. On the contrary there is direct and indirect evidence that there are sixteen chromosomes located within the nucleus.     

A Petunia hybrida chloroplast DNA region, close to one of the inverted repeats, shows sequence homology with the Euglena gracilis chloroplast DNA region that carries the putative replication origin

Summary Three distinct chloroplast (cp) DNA fragments from Petunia hybrida, which promote autonomous replication in yeast, were mapped on the chloroplast genome. Sequence analysis revealed that these fragments (called ARS A, B and C) have a high AT content, numerous short direct and inverted repeats and at least one yeast ARS consensus sequence 5A/TTTTATPuTTTA/T, essential for yeast ARS activity. ARS A and B also showed the presence of (semi-)conserved sequences, present in all Chlamydomanas reinhardii cpDNA regions that promote autonomous replication in yeast (ARS sequences) or in C. reinhardii (ARC sequences). A 431 bp BamHI/EcoRI fragment, close to one of the inverted repeats and adjacent to the ARS B subfragment contains an AT-rich stretch of about 100 nucleotides that show extensive homology with an Euglena gracilis cpDNA fragment which is part of the replication origin region. This conserved region contains direct and inverted repeats, stem-and-loop structures can be folded and it contains an ARS consensus sequence. In the near vicinity a GC-rich block is present. All these features make this cpDNA region the best candidate for being the origin of replication of P. hybrida cpDNA.     

In-vitro translation of different mRNA-containing fractions of Chlamydomonas chloroplasts

Starting from isolated chloroplasts of the Chlamydomonas reinhardii cw 15 mutant, several mRNA-containing chloroplast subfractions, i.e. thylakoid-bound polysomes, detached polysomes or isolated RNA, were prepared and incubated in homologous and heterologous translation systems. In the reticulocyte lysate these fractions gave rise to strikingly different product patterns. A most prominent difference concerned the in-vivo rapidly labelled 32,000-dalton thylakoid polypeptide. Neither this membrane protein nor its 34,000-dalton precursor was formed when membrane-containing or free polysomes were translated, while the 34,000-dalton precursor was a main product of the RNA isolated from the same membranes. The influence of thylakoid membranes during translation was also observed in homologous translation systems with lysed chloroplasts supplemented with ATP. Membrane and soluble fractions, when translated separately, yielded product patterns which differed from each other, although the RNAs extracted from the respective fractions gave the same product patterns when translated in reticulocyte lysate; the latter included a soluble protein, the large subunit of ribulose-1,5-bisphosphate carboxylase, and a membrane protein, the 34,000-dalton precursor of the 32,000-dalton membrane protein, as major labelled translation products. These results point to a regulatory role of thylakoid membranes in the expression of chloroplast mRNA and argue against compartmentation of the chloroplast mRNAs between the soluble and membrane fractions.Abbreviation SDS sodium dodecyl sulfate     

Untersuchungen an einer p-Aminobenzoesäure-Mangelmutante von Chlamydomonas reinhardii: Chloramphenicol als p-Aminobenzoesäure-Ersatz

Zusammenfassung Die p-Aminobenzoesäure-Mangelmutante pab 1 von Chlamydomonas reinhardii ist in der Lage, Chloramphenicol anstelle von p-Aminobenzoesäure als Supplementstoff zu verwenden. Es konnte jedoch gezeigt werden, daß die Algen selbst nicht imstande sind, Chloramphenicol zu p-Aminobenzoesäure abzubauen. Der Abbau erfolgt vielmehr spontan ohne Mitwirkung der Organismen, allerdings nur in belichteten Versuchsansätzen und nicht in Dunkelheit.Die Entstehung der p-Aminobenzoesäure geht über p-Nitrobenzaldehyd. Sie erfolgt rasch und reichlich, so daß für die Mutante ein Wachstum ermöglicht wird, das dem Wildtyp-Wachstum in Minimalmedium entspricht.

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Investigations on a p-Aminobenzoic auxotrophic mutant of Chlamydomonas reinhardii: Chloramphenicol as a substitute for p-aminobenzoic-acid

Summary The p-aminobenzoic-acid mutant pab 1 of Chlamydomonas reinhardii is able to use chloramphenicol as a supplement instead of p-aminobenzoic-acid. Nevertheless, it could be shown, that the algae are not in the position to reduce chloramphenicol to p-aminobenzoic-acid. On the contrary, the decomposition occurs spontaneously without the participation of the organisms, but only in experimental arrangements, exposed to light.The formation of the p-aminobenzoic-acid works rapidly and a high yield via p-nitrobenzaldehyde. Therefore the growth of the mutant is possible, corresponding to the growth of the wildtype cells in minimal-medium.

     

Transfer RNA gene mapping studies on chloroplast DNA from Chlamydomonas reinhardii

Total tRNA of Chlamydomonas reinhardii was fractionated by 2-dimensional gel electrophoresis. Sixteen tRNAs specific for eleven amino acids could be identified by aminoacylation with Escherichia coli tRNA synthetases. Hybridization of these tRNAs with chloroplast restriction fragments allowed for the localization of the genes of tRNA^Tyr, tRNA^Pro, tRNA^Phe (2 genes), tRNA^Ile (2 genes) and tRNA^His (2 genes) on the chloroplast genome of C. reinhardii. The genes for tRNA^Ala (2 genes), tRNA^Asn and tRNA^Leu were mapped by using individual chloroplast tRNAs from higher plants as probes.     

Chlamydomonas reinhardii gene for the 32 000 mol. wt. protein of photosystem II contains four large introns and is located entirely within the chloroplast inverted repeat

The chloroplast psbA gene from the green unicellular alga Chlamydomonas reinhardii has been localized, cloned and sequenced. This gene codes for the rapidly-labeled 32-kd protein of photosystem II, also identified as as herbicide-binding protein. Unlike psbA in higher plants which is found in the large single copy region of the chloroplast genome and is uninterrupted, psbA in C. reinhardii is located entirely within the inverted repeat, hence present in two identical copies per circular chloroplast genome, and contains four large introns. These introns range from 1.1 to 1.8 kb in size and fall into the category of Group I introns. Two of the introns contain open reading frames which are in-frame with the preceding exon sequences. We present the nucleotide sequence for the C. reinhardii psbA 5'-and 3' -flanking sequences, the coding region contained in five exons and the deduced amino acid sequence. The algal gene codes for a protein of 352 amino acid residues which is 95% homologous, excluding the last eight amino acid residues, with the higher plant protein.     

Construction of a Chlamydomonas reinhardtii mutant with an intronless psbA gene

Efficient chloroplast transformation systems now available allow the manipulation of the evolutionarily highly conserved psbA gene in the eucaryotic organism Chlamydomonas reinhardtii. Two copies of this gene in the inverted repeat region of the chloroplast genome contain four large group I introns. To analyse possible functions of these introns and to generate a mutant for simplified psbA gene manipulations, a psbA cDNA fragment was introduced into a psbA deletion mutant using the biolistic transformation method. A transformant with no introns in the psbA gene has been obtained and represents the first example of the removal of a complete set of introns from a chloroplast gene. The newly generated strain is photosynthetically competent and contains no detectable recipient genome copies. The loss of all four introns appears to be phenotypically silent.     

A mitochondrial glycolate: Cytochrome C reductase in Chlamydomonas reinhardii

Summary A glycolate: cytochrome c reductase is reported in a thin-walled mutant of Chlamydomonas reinhardii. The reductase is of mitochondrial origin and is sensitive to certain mitochondrial electron transport system inhibitors.Abbreviations ETS electron transport system - DCIP 2.6 dichlorophenol indophenol - PVP polyvinyl pyrrolidone     

Universal genetic code evidenced in mitochondria ofChlamydomonas reinhardii

Summary With the ultimate intent to establish a transformation system for eukaryotic organelles, the structure and organization of mitochondrial genes from the unicellular algaChlamydomonas reinhardii has been investigated. Using DNA hybridisation and DNA sequencing techniques, 3.9 kb of DNA, comprising about 25% of the mitochondrial genome, have been analysed in detail. By comparing the primary structure of homologous genes from other eukaryotic systems, we were able to identify the continuous genes coding for cytochrome oxidase subunit I (COI) and a NADH dehydrogenase subunit (ND5). The two genes are coded by opposite DNA strands and are not overlapping. The COI and the ND5 genes code for 505 and 567 amino acids, respectively. Interestingly, the comparative analysis with homologous genes from other eukaryotes shows that the universal genetic code is used in mitochondria ofC. reinhardii. This situation is different from all other mitochondrial systems studied so far. The results provide evidence thatC. reinhardii would be the appropriate organism for development of a transformation and expression system, where foreign genes, translated via the universal genetic code, are introduced into mitochondria.     

CO2 exchange characteristics during dark-light transitions in wild-type and mutant Chlamydomonas reinhardii cells

A burst of net CO₂ uptake was observed during the first 3–4 min after the onset of illumination in both wild-type Chlamydomonas reinhardii in which carbonic anhydrase was chemically inhibited with ethoxyzolamide and in a mutant of C. reinhardii (ca-1-12-1C) deficient in carbonic anhydrase activity. The burst was followed by a rapid decrease in the CO₂ uptake rate so that net evolution often occurred. After a 2–3 min period of CO₂ evolution, net CO₂ uptake again increased and ultimately reached a steady-state, positive rate. From [¹⁴CO₂]-tracer studies it was determined that CO₂ fixation proceeded at a nearly linear rate throughout the period of illumination. Thus, prior to reaching a steady state, there was a rapid accumulation of inorganic carbon inside the cells which apparently reached a supercritical concentration and the excess was excreted, causing a subsequent efflux of CO₂. A post illumination burst of net CO₂ efflux was also observed in ethoxyzolamide-inhibited wild type and ca-1 mutant cells, but not in the unihibited wild type. [¹⁴CO₂]-tracer experiments revealed that this burst was the result of a collapse of a large internal inorganic carbon pool at the onset of darkness rather than a photorespiratory post-illumination burst. These results indicate that upon illumination, chemical or genetic inhibition of carbonic anhydrase initially causes an accumulation of excess inroganic carbon in C. reinhardii cells, and that unknown regulatory mechanisms correct for this imbalance by first excreting the excess inorganic carbon and then, after several dampened oscillations, achieving an equilibrium between bicarbonate uptake, bicarbonate dehydration, and CO₂ fixation.     

Characterization of photosystem II mutants of Chlamydomonas reinhardii lacking the psbA gene

Summary We have examined 78 chloroplast mutants of Chlamydomonas reinhardii lacking photosystem II activity. Most of them are unable to synthesize the 32 Kdalton protein. Analysis of 22 of these mutants reveals that they have deleted both copies of the psbA gene (which codes for the 32 Kdalton protein) in their chloroplast genome. Although these mutants are able to synthesize and to integrate the other photosystem II polypeptides in the thylakoid membranes, they are unable to assemble a stable functional photosystem II complex. The 32 Kprotein appears therefore to play an important role not only in photosystem II function, but also in stabilizing this complex.     

Calcium lonophore-induced stimulation of secretory activity in Chlamydomonas reinhardii

The cell-wall lysin in gametes from Chlamydomonas reinhardii which under normal mating conditions is activated by flagellar cell contact was found to be susceptible to stimulation by the antibiotic ionophore A 23187 provided that CA²⁺ was included in the medium. Ionophore-induced release of the cell-wall lysin did not deend on the mating type or the gametic state of the cells. Vegetative cells which normally do not exhibit any mating capacity reacted with cell-wall lysis like gametes stimulated by cell contact.Ionophore-dependent Ca²⁺-transfer across the cell membranes generated a signal for cell-wall lysis only in cells with intact flagella. Deflagellated cells did not respond to A 23187 before regeneration of the amputated organelles. Another indication for a possible role of flagella in Ca²⁺-mediated cell-wall lysis was obtained from a conditional flagellar-assembly mutant of C. reinhardii which had been isolated and described by Huang et al. (1977). Upon shift-up the mutant strain immediately became unresponsive to A 23187 and Ca²⁺ but regained susceptibility soon after being retransferred to permissive conditions (20°C).