Restriction endonuclease map of the chloroplast DNA of Chlamydomonas reinhardii

The chloroplast DNA of Chlamydomonas reinhardii has been examined by restriction endonuclease analysis. EcoRI, BamHI and BglII produce 30, 17 and 12 fragments, respectively, whose sites have been determined by electron microscopy and by comparative gel electrophoresis. These fragments have been ordered into a circular map which corresponds to a genome size of M_r = 126 × 10⁶. The map was established by comparing the double digests of individual restriction fragments and by hybridizing purified labelled fragments to restriction enzyme digests of chloroplast DNA. The restriction fragments were isolated by molecular cloning or by preparative agarose gel electrophoresis.The two sets of chloroplast ribosomal RNA genes are contained within two inverted repeats of 13 × 10⁶ molecular weight, which are located nearly at opposite sides of the map. In addition, the mapping studies have revealed the presence of short repeated base sequences which are interspersed throughout the chloroplast genome.     

Comparative investigation of the cytoplasmic ribosomal proteins of Chlamydomonas reinhardii and Polytoma mirum

The cytoplasmic and ribosomal proteins of Chlamydomonas reinhardii and Polytoma mirum were compared by 2-D gel electrophoresis. 56 proteins with molecular weights from 12 500 to 46 000 could be detected in Chlamydomonas and 53 proteins with molecular weights from 10 000 to 50 000 in Polytoma. 15 ribosomal proteins from Chlamydomonas were identical with those of Polytoma, and it could be concluded that 25 were corresponding to each other.     

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.     

DNA polymerases from Chlamydomonas reinhardii. Purification and properties.

Three DNA polymerase activities, A, B and C, were identified in extracts of exponentially growing synchronous cultures of Chlamydomonas reinhardii, and DNA polymerases A and B were characterized in detail. Both enzymes have the same binding affinity for DEAE-cellulose at pH 7.8, but can be distinguished from each other by their behaviour on phosphocellulose and DNA-agarose. 'Activated' calf thymus DNA was used as template, and the pH, K+ and bivalent-cation optima were measured. DNA polymerase A sediments at 5.3 S in glycerol gradients, with an apparent mol.wt. of 90000-100000. Polymerase B sediments between 8S and 10S in 100mM-KCl, the predominant species having an apparent mol.wt. of 200000. In 200mM-KCl, polymerase B dissociates to a single species, which sediments at 5.8S. A 3S species was found in aged preparations of both enzymes. The activity of polymerase B from cells harvested during nuclear DNA synthesis is twice that found in Chlamydomonas at other times during the cell cycle.     

Urate oxidase of Chlamydomonas reinhardii

Urate oxidase (EC 1.7.3.3) of Chlamydomonas reinhardii cells grown on purines and purine derivatives has been partially characterized. Crude enzyme preparations have a pH optimum of 9.0, require O₂ for activity, have an apparent K_m of 12 μ M for urate, and are inhibited by high concentrations of this substrate. Enzyme activity was particularly sensitive to metal ion chelating agents like cyanide, cupferron, diethyldithiocarbamate and o -phenanthroline, and to structural analogues of urate like hypoxanthine and xanthine. Chlamydomonas cells grow phototrophically on adenine, guanine, hypoxanthine, xanthine, urate, allantoin or allantoate as sole nitrogen source, indicating that in this alga the standard pathway of aerobic degradation of purines of higher plants, animals and many microorganisms operates. As deduced from experiments in vivo , urate oxidase from Chlamydomonas is repressed in the presence of ammonia or nitrate.     

Electron microscopic localization of the chloroplast DNA replicative origins in Chlamydomonas reinhardii.

Chloroplast DNA, isolated from a synchronized culture of Chlamydomonas reinhardii, was digested with restriction endonucleases and examined in the electron microscope. Restriction fragments containing displacement loops (D-loop) were photographed and measured to determine the position of replicated sequences in relation to the restriction enzyme sites. D-loops were located at two positions on the physical map of chloroplast DNA. One replication origin was mapped at about 10 kb upstream of the 5' end of a 16s rRNA gene. The second origin was spaced 6. 5kb apart from the first origin and was about 16.5 kb upstream of the same 16s rRNA. Initiations at those two sites were not always synchronized. Replication initiated with the formation of a D-loop resulting from the synthesis of one daughter strand. After a short initial lag phase, corresponding to the synthesis of 350 +/- 130 bp of one daughter strand, DNA synthesis then proceeded in both directions. Both D-loop regions were preferred binding sites of undetermined protein complexes.     

Biosynthesis of ferredoxin in Chlamydomonas reinhardii

The selective action of the antibiotics chloramphenicol and cycloheximide on the synthesis of ferredoxin in liquid cultures of Chlamydomonas reinhardii was studied. Highly specific antibodies raised against Chlamydomonas ferredoxin were used to determine the in vivo synthesis of apoferredoxin and conversion into native protein. The results indicate that 80S ribosomes are involved in the synthesis. Chlamydomonas cells growing in the absence of iron did not synthesize immunologically detectable amounts of ferredoxin. We suggest that this is based upon feed-back inhibition of apoferredoxin synthesis at the translational level.Abbreviations CAP chloramphenicol - CHI cycloheximide - IgG Immunoglobulin G - PBS 140.4 mM NaCl. 9 mM Na₂HPO₄, 1.3 mM NaH₂PO₄ (pH 74) - SDS sodium dodecvl sulphate - Fd Ferredoxin - apoFd Apoferredoxin - CM-Fd Scarboxymethylated Fd - TCA-Fd Fd treated with trichloroacetic acid     

Comparison of the nuclear ribosomal units of five Chlamydomonas species

The organization of the nuclear ribosomal units of five different species of Chlamydomonas has been examined by hybridization of their nuclear DNA fragments produced by several restriction endonucleases with a radioactively labelled probe consisting of the two cloned BamHI ribosomal fragments of C. reinhardii. The results indicate that a) the ribosomal units of these five species are structurally related, b) changes in the non transcribed spacer occur in C. eugametos and in C. globosa, c) the rDNA unit of C. intermedia contains either an enlarged internal transcribed spacer or a ribosomal intervening sequence, d) the rDNA units of C. reinhardii and C. callosa are indistinguishable.     

Sequence organization of repetitive elements in the flanking regions of the chloroplast ribosomal unit of Chlamydomonas reinhardii.

The flanking regions and the end of the chloroplast ribosomal unit of Chlamydomonas reinhardii have been sequenced. The upstream region of the ribosomal unit contains three open reading frames coding for 111, 117 and 124 amino acids, respectively. The latter polypeptide is partially related to the ribosomal protein L16 of E. coli. Two of the open reading frames overlap each other and are oriented in opposite direction. The region between these open reading frames and the 5' end of the 16S rRNA gene contains numerous short direct and inverted repeats which can be folded into large stem-loop structures. Sequence elements that resemble prokaryotic promoters are found in the same region. Several of the repeated elements are distributed throughout the non-coding regions of the chloroplast inverted repeat. Sequence comparison between the 5S rRNA and its gene does not reveal any significant sequence heterogeneity between the chloroplast 5S rRNA genes.