Localization of Five Antibiotic Resistances at the Subunit Level in Chloroplast Ribosomes of Chlamydomonas

Differences in the distribution of Ava I and BstEII restriction sites in the chloroplast DNA (cpDNA) of Chlamydomonas eugametos and C. moewusii have been used to detect extensive cpDNA recombination in the hybrid progeny of these interfertile algae. In the present study, the inheritance of these restriction-site differences was tested for recombina- MATERIALS AND METHODSStrains. The mt' wild-type strain of C. moewusii (UTEX 97), the male (mt') strain of C. eugametos (UTEX 9), and the mt' strain of C. eugametos carrying the nonmendelian streptomycin resistance mutation sr-2 (5) were provided by C. S. Gowans (University of Missouri-Columbia). The C. moewusii nonmendelian mutant er-nMJ, resistant to erythromycin (400 ,g/ml), was isolated from the mt( wild-type strain after methyl methanesulfonate mutagenesis. The mendelian mutant of C. moewusii requiring nicotinamide at 1 ,ug/ml (nic-J) and the other requiring p-aminobenzoic acid at 1 ,ug/ml (pab-J) were isolated from the mt-wild-type strain (UTEX 96) after UV mutagenesis; both mendelian mutations are unlinked to the Mnt locus. The mt' wild-type strain of C.

Section: Discussionsupporting

confidence: 51%

Chloroplast DNA recombination in interspecific hybrids of Chlamydomonas : Linkage between a nonmendelian locus for streptomycin resistance and restriction fragments coding for 16S rRNA

Lemieux

Turmel

Seligy

et al. 1984

“…Primarily due to a number of advantageous biological characteristics, Chlamydomonas reinhardtia, a unicellular green alga, has been increasingly exploited for fruitful investigations of the cooperation between chloroplast and nuclear genomes in nucleic acid metabolism (1)(2)(3)(4) and protein synthesis (5,6). Genetic studies with a few non-Mendelian mutants have also suggested that there is probably another level of cooperation between the chloroplast and mitochondria genomes in organelle protein synthesis (7)(8)(9).…”

mentioning

confidence: 99%

Isolation and characterization of mitochondrial DNA from Chlamydomonas reinhardtii.

Ryan¹,

Grant²,

Chiang³

et al. 1978

Mitochondrial DNA (mtDNA) has been isolated from a mitochondrial pellet of Chlamydomonas reinhardtii. The mtDNA has a buoyant density of 1.706 g/ml in CsCl, a melting temperature of 87.90 in standard saline citrate, and a nucleoside composition of 47.5% deoxyguanidine plus deoxycytidine with no odd nucleosides. Thermal denaturation and renaturation studies have shown that (i) mtDNA contains no extensive intramolecular heterogeneity nor significant base bias between the complementary polynucleotide chains and (ii) mtDNA renatures as a single homogeneous class with a kinetic complexity of 9.78 X 106 daltons. Although rare ( Primarily due to a number of advantageous biological characteristics, Chlamydomonas reinhardtia, a unicellular green alga, has been increasingly exploited for fruitful investigations of the cooperation between chloroplast and nuclear genomes in nucleic acid metabolism (1-4) and protein synthesis (5, 6). Genetic studies with a few non-Mendelian mutants have also suggested that there is probably another level of cooperation between the chloroplast and mitochondria genomes in organelle protein synthesis (7-9). Dissection of this postulated interorganelle cooperation at the molecular level should prove to be feasible and of interest, because C. reinhardtii is a facultative photosynthetic organism with an abundance of well-characterized nonphotosynthetic mutants (10). The unusually fragile structure of the single large chloroplast in C. reinhardtmi, however, has hampered repeated attempts in several laboratories to isolate contamination-free mitochondria for biochemical characterization.We report in this paper the successful isolation of a DNaseimpermeable mitochondrial preparation from a cell-wall-less mutant of C. reinhardtfil and the characterization of the mitochondrial DNA (mtDNA). We document here the basic physicochemical properties of this mtDNA and its complexity. The unexpected findings of our present study are that (i) the complexity of C. reinhardtii mtDNA is strikingly small in comparison with other plant mtDNAs but very close to that of animal mtDNA, and (ii) the reiterations of mtDNA and chloroplast DNA (cpDNA) molecules are comparable in the vegetative cells of C. reinhardtii. MATERIALS AND METHODSStrain and Culture Conditions. A mating type + subclone of the cell-wall-less mutant CW-15 (11) was used for all studies. Cells were grown in minimal medium supplemented with 0.2% sodium acetate (4). Twelve-or 18-liter cultures were illuminated with six circular 40-W cool-white fluorescent lamps and aerated with 3% CO2 in air.Mitochondria Isolation Procedures. Procedure A. Cells were collected at early stationary phase, washed once in buffer 1 (0.25 M sucrose/0.1 mM EDTA/10 mM Tris-HCl, pH 9), once in buffer 2 (0.25 M sucrose/0.1 mM EDTA/10 mM Tris-HCl, pH 7.4), and suspended in 10 ml of buffer 2 per liter of culture volume (12). This cell suspension was passed once through a chilled French pressure cell at 500 psi (34.5 X 102 kPa), diluted with an equal volume of buffer 2, and, af...

“…Wild type Chlamydomonas reinhardi strain 21gr, used in these experiments, was grown on minimal medium (12) in continuous light (15,(0)(1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17),000 lux) at 250. Cultures larger than 100 ml were bubbled with 55% CO2 in air.…”

Section: Methodsmentioning

confidence: 99%

“…The specificity of action of the protein synthesis inhibitors used (spectinomycin, streptomycin, neamine, chloramphenicol, and cleocin), has recently been established in Chlamydomonas by studies of amino acid Abbreviations: EDTA, ethylene diaminetetraacetate; TCA, trichloroacetic acid; n/t, cpm in nuclear fraction divided by cpm in nuclear + chloroplast fractions X 100. * incorporating activity of purified chloroplast and cell sap ribosomes taken from wild-type and from drug-resistant mutant strains, in a poly(U) directed assay system (9). The specificity of action of rifampicin in Chlamydomonas (10) and of rifamycin SV in various systems (11) has been investigated by others.…”

mentioning

confidence: 99%

Regulation of Nuclear DNA Replication by the Chloroplast in Chlamydomonas

Blamire

Flechtner

Sager

1974

Self Cite

The experiments described in this paper implicate chloroplast protein synthesis in the regulation of nuclear DNA replication. The inhibition of nuclear DNA replication in the lower eukaryote, Chlamydomonas reinhardi strain 21gr, was examined after growth of cells with a series of antibiotics (streptomycin, neamine, spectinomycin, cleocin, chloramphenicol, and rifampicin) each of which has a known effect upon Mechanisms for the coordination of nuclear and organelle genetic systems are essential in eukaryotic cells (1). The tight coupling, to the cell cycle, of events in the biogenesis of chloroplasts (2) and mitochondria (3, 4) would seem to require wellsynchronized, genetically controlled programs of biosynthetic activity. Evidence that this control is a two-way process with input from organelles as well as from the nucleus, is beginning to appear (5-8). These reports describe signals of organelle origin which influence nuclear gene activities required for further organelle biogenesis, i.e., feedback loops regulating organelle formation. This paper describes the inhibition of nuclear DNA replication in the lower eukaryote Chlamydomonas, after growth in the presence of antibiotics that block macromolecular synthesis in chloroplasts (transcription of chloroplast RNA and protein synthesis on chloroplast ribosomes). The mechanism of inhibition is a novel one, originating in the chloroplast and regulating cell proliferation. The specificity of action of the protein synthesis inhibitors used (spectinomycin, streptomycin, neamine, chloramphenicol, and cleocin), has recently been established in Chlamydomonas by studies of amino acid Abbreviations: EDTA, ethylene diaminetetraacetate; TCA, trichloroacetic acid; n/t, cpm in nuclear fraction divided by cpm in nuclear + chloroplast fractions X 100. * incorporating activity of purified chloroplast and cell sap ribosomes taken from wild-type and from drug-resistant mutant strains, in a poly(U) directed assay system (9). The specificity of action of rifampicin in Chlamydomonas (10) and of rifamycin SV in various systems (11) has been investigated by others. MATERIALS AND METHODSPreparation of Cells. Wild type Chlamydomonas reinhardi strain 21gr, used in these experiments, was grown on minimal medium (12) in continuous light (15,(0)(1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17),000 lux) at 250. Cultures larger than 100 ml were bubbled with 55% CO2 in air.Uniformity of the cells used as inoculum was found to be essential for reproducibility of radioisotope incorporation rates and antibiotic inhibition levels. Consequently cells were pregrown under the same conditions to be used in each of the experiments, and taken from the mid-log phase of growth. In each experiment, 100 ml cultures were inoculated with 0.5 to 1.0 X 105 cells per ml and grown 24 hr at which time the density was 1 X 106 cells per ml. Then antibiotics, and after 1 hr of incubation, 2 uCi/ml of 2[3H]adenine (New England Nuclear Corp., Boston, Mass.) were added, the flasks were incubated for...