A study of mitochondrial ribosomes from the higher plant Solanum tuberosum L.

Ribosomal subunits are isolated from potato tuber mitochondria devoid of contaminating organelles. The sedimentation constants of the two mitochondrial ribosomal subunits are 33S and 50S respectively. The apparent sizes of the high molecular weight RNAs are 19S and 25S. The proteins of these ribosomes have been analyzed by two-dimensional electrophoresis in SDS polyacrylamide gels to determine their number and molecular weights. The small subunit contains 35 protein species ranging from 8 to 60 kDa. The 50S large subunit contains 33 protein species ranging from 12 to 46 kDa. These preliminary results are the first analysis made on mitochondrial ribosomes from a higher plant.

Electron spectroscopic imaging: parallel energy filtering and microanalysis in the fixed-beam electron microscope.

A new imaging modality in electron microscopy uses energy filtration to produce micrographs with elastically scattered electrons or with electrons that have lost a specific, often characteristic amount of energy in interacting with the specimen. No deleterious effects on microscope performance are encountered. Instead, microanalysis of specimens is made possible with a spatial resolution of 3 to 5 A and a sensitivity of detection of 2 X 10(-21) g corresponding to about 50 atoms of phosphorus. Elements detected range from hydrogen (Z = 1) to uranium (Z = 92). Examples of elemental mapping show membrane structure, DNA within nucleosomes, and RNA within ribosomal particles.

Distinction between Duchenne and other muscular dystrophies by ribosomal protein synthesis.

Ribosome concentration, ribosome distribution on sucrose density gradients, and in-vitro ribosomal amino-acid incorporation (noncollagen and collagen synthesis) were studied in muscle biopsy samples obtained from 30 patients with Duchenne muscular dystrophy, seven patients with Becker muscular dystrophy, and 10 with facioscapulohumeral muscular dystrophy. Ribosome concentration was normal in Duchenne and facioscapulohumeral and decreased in Becker muscular dystrophy. Distribution of ribosomes in sucrose density gradients showed abnormalities (sharp monosomal peak and fewer polyribosomes) only in Duchenne muscular dystrophy and was normal in the other two types. In-vitro amino-acid incorporation of ribosomes in Duchenne muscular dystrophy revealed high collagen and low noncollagen synthesis of the heavy polyribosomes. This abnormality is controlled by an undetermined enzymatic factor belonging to the soluble enzyme fraction. Supplementation of the dystrophic heavy polyribosomes with normal soluble enzymes restored the synthesis of collagen to that of the controls. Heavy polyribosomes extracted from normals or from carriers produce proportionately more collagen in the presence of soluble enzyme fraction from Duchenne muscular dystrophy than in the presence of their homologous enzymes. In Becker muscular dystrophy, both noncollagen and collagen synthesis of the heavy polyribosomes were increased, under the influence of ribosomal factors. The different protein synthesis in Duchenne and Becker muscular dystrophies suggests that these conditions are non-allelic. In facioscapulohumeral muscular dystrophy the changes in protein synthesis occurred only in the early stage of the disease and consisted of increased noncollagen synthesis of the light polyribosomes, while the heavy polyribosomes had normal activity including collagen synthesis. This reaction was controlled by ribosomal factors.

Ribosomal RNA cistron multiplicity and nucleolar organizers in hexaploid wheat.

The ribosomal DNA (DNA complementary to ribosomal RNA) content of twelve different wheat genotypes has been studied. Some of these genotypes are aneuploids with deletions or additions of chromosomes bearing nucleolar organisers. The rDNA contents of these genotypes provide several examples of a clear departure from a correlation between the number of rRNA cistrons and the number of nucleolar organisers. Thus the number of rRNA cistrons per nucleolar organiser is not constant in wheat. Wheat DNA was found to have a mean buoyant density of approximately 1.702 g/cc for all genotypes studied and rRNA hybridized selectively to DNA of buoyant density approximately 1.710 g/cc. The thermal stabilities of all the rRNA/DNA hybrids were essentially identical.

Mitoribosomes from Candida utilis. Morphological, physical, and chemical characterization of the monomer form and of its subunits.

Highly purified mitochondrial ribosomes (mitoribosomes) have been obtained from the yeast Candida utilis. Sedimentation analysis in sucrose gradients made in 5 mM MgCl(2), 1 mM Tris, pH 7.4 and 50 mM KCl clearly distinguishes mitoribosomes (72S) from cytoplasmic ribosomes (cytoribosomes) (78S). Mitoribosomes are completely dissociated into 50S and 36S subunits at 10(-4)M MgCl(2) whereas complete dissociation of cytoribosomes into 61S and 37S subunits occurs only at 10(-6)M MgCl(2) Electron microscopy of negatively stained mitoribosomes (72S peak) shows bipartite profiles, about 265 x 210 x 200 A Characteristic views are interpreted as frontal, dorsal, and lateral projections of the particles, the latter is observed in two enantiomorphic forms Mitoribosome 50S subunits display rounded profiles bearing a conspicuous knoblike projection, reminiscent of the large bacterial subunit. The 36S subunits show a variety of angular profiles. Mitoribosomal subunits are subject to artifactual dimerization at high Mg(2+) concentration Under these conditions, a supplementary 80S peak arises. Electron microscopic observation of the 80S peak reveals closely paired particles of the 50S type Buoyant density determinations after glutaraldehyde fixation show a single peak at rho = 1.48 for mitoribosomes and 1.53 for cytoribosomes In the presence of ethylenediaminetetraacetate (EDTA), two species of RNA, 21S and 16S, are obtained from mitoribosomes, while 25S and 17S RNA are obtained from cytoribosomes It is established that the small and large RNA species are derived from the 36S and 50S subunits, respectively, by extraction of the RNA from each subunit The G + C content of the RNA is lower for mitoribosomes (33%) than for cytoribosomes (50%). Incubation of C utilis mitochondria with leucine-(14)C results in the labeling of 72S mitoribosomes. The leucine-(14)C incorporation is inhibited by chloramphenicol and resistant to cycloheximide Puromycin strips the incorporated radioactivity from the 72S mitoribosomes, which is consistent with the view that leucine-(14)C is incorporated into nascent polypeptide chains at the level of mitoribosomes

The dissociation of Yoshida hepatoma ribosomes into active particles.

1. The sedimentation pattern of Yoshida hepatoma ribosomes shows mainly a high dimer peak and an intermediate peak between those of monomer and dimer. 2. The treatment of the postmitochondrial supernatant with EDTA or potassium chloride leads to the dissociation of ribosomes into subunits, through a decrease ofthe ribosomal Mg(2+)/phosphorus ratio. Provided that the deprivation of endogenous Mg(2+) is not complete, the subunits reassociate into active polyribosomes after addition of magnesium chloride to the medium. If the Mg(2+)/phosphorus ratio is lowered below 0.01 (mumol/mumol), structural changes occur, that become evident by a loss of protein and by a decreased sedimentation rate, which render the subribosomal particles unable to reassociate. 3. In the re-formation of polyribosomes from subunits, during the progressive increase of the concentration of magnesium chloride in the medium, the formation of monomeric ribosomes seems to be intermediate. 4. A dimerization of monomers and subunits occurs at concentrations of magnesium chloride greater than those required for the re-formation of polyribosomes and a preincubation for 1h at 0 degrees C is necessary for the maximum dimerization, whereas the complete reconstitution of polyribosomes is immediate.

Isolation of RNA with properties of messenger RNA from cerebral polyribosomes.

RNA which dissociated from purified cerebral polyribosomes of adult rats in the presence of EDTA was isolated by fractionation in a discontinuous sucrose gradient. The yield was 2% of the total polyribosomal RNA. The base composition resembled the complementary values for rat DNA and was very different from base compositions of ribosomal RNA and transfer RNA. This RNA fraction contained a large proportion of molecules which were rapidly labeled in vivo and hybridized to homologous DNA. The polyribosomal RNA preparation also exhibited high template activity in a cerebral cell-free system which had previously been stripped of the capacity to incorporate amino acids in the absence of added messenger RNA (mRNA). Sedimentation analysis revealed only two peaks, with coefficients of approximately 8 S and 16 S. The data indicate that RNA with the properties of mRNA can be selectively isolated from cerebral polyribosomes under mild conditions which avoid degradation.

The mitochondrial ribosome of Xenopus laevis.

A mitochondrial ribosome with an approximate sedimentation coefficient of 60 S has been isolated from the ovary of the South African clawed toad, Xenopus laevis. This ribosome is active in a submitochondrial protein-synthesizing system. It contains two species of RNA, designated as "21S" and 13S RNA, which have sequences complementary to mitochondrial DNA and share no detectable sequence homology with cytoplasmic ribosomal RNA.