Organization and closing of mitochondrial deoxyribonucleic acid from Paramecium tetraaurelia and Paramecium primaurelia.

Previously we showed that the mitochondrial deoxyribonucleic acid (DNA) from Paramecium aurelia consists of a linear genome and that replication of this genome is initiated at one terminus and proceeds unidirectionally to the other terminus. Analyses of mitochondria from four closely related species (1, 4, 5, and 7) indicated that the species 1, 5, and 7 DNAs are essentially completely homologous but that the species 4 mitochondrial DNA is only 40 to 50% homologous with that from species 1. The major regions of homology are those containing the genes for ribosomal ribonucleic acid (RNA). To understand the replication and organization of the linear mitochondrial genome better, we compared species 1 (Paramecium primaurelia) and 4 (Paramecium tetraaurelia) DNAs with regard to restriction fragment mapping and homology between initiation regions; we also identified the sites of the genes for ribosomal RNA. In general, the structures of the species 1 and 4 mitochondrial genomes were quite similar. Each ribosomal RNA gene was present in one copy per genome, with the large ribosomal RNA gene located near the terminal region of replication and the small ribosomal RNA gene located more centrally. These two genes were separated by about 10 kilobases in the species 1 genome and by about 12 kilobases in the species 4 genome. In contrast to our previous findings, by using nonstringent hybridization conditions we detected homology between the species 1 and 4 DNA fragments containing the initiation regions. We constructed recombinant DNA clones for many fragments, especially those containing the initiation region and the ribosomal RNA genes. We also constructed restriction enzyme maps for six enzymes for both P. primaurelia and P. tetraaurelia.     

Variable arrangement of 5S ribosomal genes within the ribosomal DNA repeats of arthropods.

The polymerase chain reaction and hybridization to genomic blots were used to investigate whether the previously observed inclusion of 5S ribosomal RNA genes in the 28S-18S ribosomal DNA intergenic regions of some crustacean species (copepods) could also be detected in other arthropods. Such an arrangement was found not only in other calanoid copepod species but also in a cirriped, an euphausid, and a spider. It is interesting that species from two different calanoid copepod genera do not have this type of arrangement. We conclude that the inclusion of 5S ribosomal RNA genes within the ribosomal DNA repeats has probably occurred repeatedly during the evolution of arthropod species and that the mechanism(s) responsible for these insertions could also be responsible for their loss.     

Nucleic Acid Homologies Among Species of Saccharomyces

Evolutionary divergence among species of the yeast genus Saccharomyces was estimated from measurements of deoxyribonucleic acid (DNA)/DNA and ribosomal ribonucleic acid (RNA)/DNA homology. Much diversity was found in the DNA base sequences with several species showing little or no homology to the three reference species, S. cerevisiae, S. lactis, and S. fragilis. These three reference species also showed little or no homology to each other. On the other hand the diversity among ribosomal RNA base sequences was small since most species showed a high degree of homology to the reference species. The arrangement of species based on ribosomal RNA homologies agrees in most cases with current taxonomic groupings. A yeast hybrid (S. fragilis x S. lactis) was shown to contain two nonhomologous genomes. A minimum genome size of 9.2 x 10(9) daltons for S. cerevisiae was calculated from the rate of DNA renaturation.     

Electrophoretic Analysis of Ribosomal and Viral Ribonucleic Acids with a Simple Technique for Slicing Low-Concentration Polyacrylamide Gels

The electrophoretic mobilities of ribosomal ribonucleic acids (RNA) from cultured mammalian (HeLa, Vero, MDBK), avian (chick embryo), and bacterial (Escherichia coli) cells, and RNA species extracted from selected viruses (Sindbis, polio, tobacco mosaic, Sendai) were compared, employing a simple, inexpensive technique for slicing low-concentration polyacrylamide gels. The procedure provides for rapid fractionation of gels used for characterization of RNA, incorporating extrusion and serial sectioning of frozen gels. Among 28S ribosomal RNA species, Vero and MDBK were indistinguishable, whereas HeLA RNA had a slightly lower mobility (higher apparent molecular weight) and chick RNA had a higher mobility (lower apparent molecular weight). The 18S ribosomal RNA species of the three mammalian sources were indistinguishable, but chick 18S RNA had a slightly lower apparent molecular weight. The inverse relation between mobility and log-molecular weight among the ribosomal and viral RNA species, though not highly precise, demonstrates the applicability of the technique to the study of molecular weights of viral RNA species.     

Heterogeneity of the conserved ribosomal ribonucleic acid sequences of Bacillus subtilis

Doi, Roy H. (University of California, Davis), and Richard T. Igarashi. Heterogeneity of the conserved ribosomal ribonucleic acid sequences of Bacillus subtilis. J. Bacteriol. 92:88-96. 1966.-Hybrid formation was demonstrated between Bacillus subtilis ribosomal ribonucleic acid (RNA) and deoxyribonucleic acid (DNA) from various bacterial species. The high degree of complementarity between B. subtilis ribosomal RNA and the DNA from B. cereus and B. stearothermophilus suggested a method to test whether the same RNA sequences were hybridizing with the DNA from these two species. Saturation studies with 16S and 23S RNA preparations from B. subtilis showed that a definite number of complementary sites was present in each DNA. Base composition analyses of the RNA in the hybrid demonstrated that ribosomal RNA sequences were involved. Hybrid competition studies revealed that B. stearothermophilus ribosomal RNA could compete totally against B. subtilis ribosomal RNA for B. stearothermophilus DNA, although it could compete only partially against the B. subtilis ribosomal RNA hybridizing with B. cereus DNA. These observations were made independently with both 16S and 23S ribosomal RNA preparations. These results revealed that different nucleotide sequences of B. subtilis ribosomal RNA were hybridizing with the DNA from B. cereus and B. stearothermophilus. Two possible interpretations of these results are: (i) different nucleotide sequences from a homogeneous ribosomal RNA population are hybridizing with heterologous DNA preparations, and (ii) ribosomal RNA cistrons are heterogeneous.     

Unusual pattern of ribonucleic acid components in the ribosome of Crithidia fasciculata, a trypanosomatid protozoan.

In a previous study from this laboratory, presumptive ribosomal ribonucleic acid (RNA) species were identified in the total cellular RNA directly extracted from intact cells of the trypanosomatid protozoan Crithidia fasciculata (M. W. Gray, Can. J. Biochem. 57:914-926, 1979). The results suggested that the C. fasciculata ribosome might be unusual in containing three novel, low-molecular-weight ribosomal RNA components, designated e, f, and g (apparent chain lengths 240, 195, and 135 nucleotides, respectively), in addition to analogs of eucaryotic 5S (species h) and 5.8S (species i) ribosomal RNAs. In the present study, all of the presumptive ribosomal RNAs were indeed found to be associated with purified C. fasciculata ribosomes, and their localization was investigated in subunits produced under different conditions of ribosome dissociation. When ribosomes were dissociated in a high-potassium (880 mM K+, 12.5 mM Mg2+) medium, species e to i were all found in the large ribosomal subunit, which also contained an additional, transfer RNA-sized component (species j). However, when subunits were prepared in a low-magnesium (60 mM K+, 0.1 mM Mg2+) medium, two of the novel species (e and g) did not remain with the large subunit, but were released, apparently as free RNAs. Control experiments have eliminated the possibility that the small RNAs are generated by quantitative and highly specific (albeit artifactual) ribonuclease cleavage of large ribosomal RNAs during isolation. In terms of RNA composition and dissociation properties, therefore, the ribosome of C. fasciculata is the most "atypical" eucaryotic ribosome yet described. These observations raise interesting questions about the function and evolutionary origin of C. fasciculata ribosomes and about the organization and expression of ribosomal RNA genes in this organism.     

The course of the assembly of ribosomal subunits in yeast

The course of the assembly of the various ribosomal proteins of yeast into ribosomal particles has been studied by following the incorporation of radioactive individual protein species in cytoplasmic ribosomal particles after pulse-labelling of yeast protoplasts with tritiated amino acids. The pool of ribosomal proteins is small relative to the rate of ribosomal protein synthesis, and, therefore, does not affect essentially the appearance of labelled ribosomal proteins on the ribosomal particles. From the labelling kinetics of individual protein species it can be concluded that a number of ribosomal proteins of the 60 S subunit (L6, L7, L8, L9, L11, L15, L16, L23, L24, L30, L32, L36, L40, L41, L42, L44 and L45) associate with the ribonucleoprotein particles at a relatively late stage of the ribosomal maturation process. The same was found to be true for a number of proteins of the 40 S ribosomal subunit (S10, S27, S31, S32, S33 and S34). Several members (L7, L9, L24 and L30) of the late associating group of 60-S subunit proteins were found to be absent from a nuclear 66 S precursor ribosomal fraction. These results indicate that incorporation of these proteins into the ribosomal particles takes place in the cytoplasm at a late stage of the ribosomal maturation process.     

The relationship between ribosomal repeat length and genome size in Vicia

The organization of the ribosomal RNA genes was examined in several species of Vicia in an attempt to determine whether a relationship exists between genome size and ribosomal repeat length. Species within this genus exhibit a sevenfold variation in haploid DNA content. Our data suggest that species with an intermediate genome size maintain one predominant Eco RI class of ribosomal repeat of about 9 kilobases (kb). In contrast, the smallest and largest genomes of Vicia possess one major and several minor classes. The possible relationship between repeat classes among species is discussed. We examined the species with the smallest (V. villosa) and largest (V. faba) genomes in closer detail by R-loop analysis of a satellite DNA from Hoechst 33258 dye-CsCl gradients. Heterogeneity was found in the length of the ribosomal repeat for both species, but no appreciable difference was observed in the distribution of these lengths, which averaged 11–12 kb. This heterogeneity is associated with the nontranscribed spacer region. Intervening sequences were not found in either the 25S or 18S coding regions of the ribosomal repeat of either of these two plants. A putative ribosomal RNA precursor of 7 kb was identified for both species.     

Identification and phylogenetic relationships of Vahlkampfia spp. (free-living amoebae) by riboprinting

Abstract The riboprinting technique (restriction fragment length polymorphism analysis of polymerase chain reaction amplified ribosomal DNA) was applied to 8 strains representing 7 species of amoebae from the Vahlkampfia genus (Family Vahlkampfiidae, Class Heterolobosea). The length of the 18S ribosomal gene was found to vary significantly between species. Restriction fragment length polymorphism analysis following digestion of the amplified ribosomal DNA with 18 restriction enzymes confirmed the separate identity of each species, originally based on morphological characteristics, and generated a phylogenetic tree. Two strains assigned to the same species yielded identical riboprints.     

Divergence of primate ribosomal RNA genes as assayed by restriction enzyme analysis

Primate ribosomal RNA (rRNA) genes have been compared by restriction endonuclease mapping. In all species examined, the restriction map of the reiterated ribosomal DNA is simple (within the limits of detection by hybridization with rRNA) and is consistent with a high degree of homogeneity among the repeats. Within a species, all members have similar rDNA restriction patterns. However, different species of primates have distinctly different rDNA restriction maps; even chimpanzee and man can be discerned by their rDNA restriction patterns. Possible mechanisms for maintenance of homogeneity of the rDNA repeats within a species, while allowing divergence among closely related species, are discussed.     

Genetic Variation of Nuclear Ribosomal DNA in Cockroaches (Order Blattaria): Phylogenetic Analysis of Restriction Fragment Length Polymorphism

Species-specific characteristics of nuclear ribosomal DNA (rDNA) have been determined for the first time for six insect species of order Blattaria (Insecta, Dictyoptera) with the use of restriction analysis and Southern blotting. Probes containing highly conserved fragments of 18S- and 28S-like genes of the ribosomal RNA (rRNA) of Tetrahymena pyriformis were tested in this study. The genetic similarity tree constructed for the species studied agrees with evidence from classical taxonomy based on morphological, ecological, anatomical, and physiological characters.     

Ribosomes and ribonucleic acids of Coxiella burneti

This report describes the direct isolation and characterization of rickettsial ribosomes. Ribosomes from the rickettsia Coxiella burneti were isolated and partially characterized. The ribosomes had a sedimentation constant of about 70S and could be dissociated into 50 and 30S subunits. Electron microscopy revealed ribosomal particles with dimensions similar to those reported for other procaryotic organisms. Ribonucleic acid (RNA) species (23 and 16S) were isolated from the ribosomal particles. The nucleotide compositions of the ribosomal RNAs were found to be similar to those reported for bacterial ribosomal RNA. In addition to the high-molecular-weight ribosomal RNA, 5S RNA was also extracted from the organism.     

Comparison of ribosomal and isozymic phylogenies of tetrahymenine ciliates

A recent analysis of sequence variations in ribosomal RNA's from 31 species of tetrahymenine ciliates groups them into 9 sets referred to as "ribosets." These species associations are not well correlated with the distributions of distinctive morphological characteristics. The phylogenetic structure suggests that modern "pyriform" tetrahymenines may be paraphyletic survivors of primitive design and that the morphologically distinctive forms may include examples of convergent evolution of derived forms. Alternatively, the common ancestor may have been a polymorphic species that has lost its plasticity in some derived lineages. In an attempt to test the ribosomal phylogeny, we here compare it with a phylogeny based on isozymic variation. The main features of the ribosomal and isozymic phylogenies are similar. The carnivorous (macrostome-forming) species are widely scattered in both, as are the bacteriophagous pyriform species. Isozymic and ribosomal analyses are optimally useful, however, in different contexts. Isozymic variations can distinguish species that are ribosomally identical. Ribosomal variations provide more secure evaluations of distant relationships.     

Comparison of Ribosomal and Isozymic Phylogenies of Tetrahymenine Ciliates

A recent analysis of sequence variations in ribosomal RNA's from 31 species of tetrahymenine ciliates groups them into 9 sets referred to as "ribosets." These species associations are not well correlated with the distributions of distinctive morphological characteristics. The phylogenetic structure suggests that modem "pyriform" tetrahymenines may be paraphyletic survivors of primitive design and that the morphologically distinctive forms may include examples of convergent evolution of derived forms. Alternatively, the common ancestor may have been a polymorphic species that has lost its plasticity in some derived lineages. In an attempt to test the ribosomal phylogeny, we here compare it with a phytogeny based on isozymic variation. The main features of the ribosomal and isozymic phylogenies are similar. The carnivorous (macrostome-forming) species are widely scattered in both, as are the bacteriophagous pyriform species. Isozymic and ribosomal analyses are optimally useful, however, in different contexts. Isozymic variations can distinguish species that are ribosomally identical. Ribosomal variations provide more secure evaluations of distant relationships.     

The Relationship between Satellite Deoxyribonucleic Acid, Ribosomal Ribonucleic Acid Gene Redundancy, and Genome Size in Plants

The buoyant density of ribosomal DNA is similar in species with or without satellite DNA, and in all species examined was distinguishable from that of the satellite DNA. In melon tissues (Cucumus melo) the percentage satellite DNA is not correlated with the percentage hybridization to ribosomal RNA. Satellite DNA sequences do not appear to be dispersed between those coding for ribosomal RNA. There is no correlation between the presence of satellite DNA and high ribosomal RNA gene redundancy, but there is a correlation between satellite DNA and small genome size, which results in a correlation between satellite DNA and a high percentage hybridization to ribosomal RNA. Satellite DNAs are defined as minor components after CsCI centrifugation.     

Isolation and Characterization of Bacterial Ribosomal RNA Cistrons

The DNA sequences which code for ribosomal DNA have been isolated and purified. The technique used has general application for RNA:DNA hybridization studies and enables the isolation of any gene for which sufficient gene product can be obtained. Experiments with isolated ribosomal RNA cistrons demonstrated that (a) the majority of the ribosomal cistrons are similar to one another; (b) the cistrons which are similar to one another are virtually identical to one another; (c) ribosomal cistrons of different bacterial species are closely related to one another.     

Extraction of proteins from Saccharomyces cerevisiae ribosomes under nondenaturing conditions

The differential sensitivity of ribosomal proteins to removal by salts has been studied. Proteins were extracted from the large and small subunits of cytoplasmic ribosomes from Saccharomyces cerevisiae by washing the individual subunits with a series of solutions containing increasing concentrations of NH4Cl (0.74-3.56 M) for a defined time (20 min) at 0 degrees C. The molar ratio of magnesium to ammonium ions of 1:40 was maintained to protect the ribosomal subparticles from complete disassembly. Proteins extracted under each salt condition were analyzed for composition by two-dimensional polyacrylamide gel electrophoresis. The relative quantity of each protein was determined. Most proteins were not removed from the ribosomal particle completely by any one condition, but were preferentially enriched in a single fraction. Whereas most proteins could be solubilized, several proteins remained predominantly or exclusively with the final core particle. The kinetics of protein release from both subunits at a single NH4Cl concentration (0.74 M) were also studied. Release of protein was time dependent, i.e., longer extraction generally removed more of the same proteins. However, prolonged treatment (240 min) of subunits, even at the same salt concentration, resulted in removal of additional species of proteins in varying amounts. Among the ribosomal RNA species, only the 5 S RNA species was released from the ribosomal particles upon treatment.     

Characterization of the ribosomal DNA units in two related Prunus species (P. Cerasifera and P. Spinosa)

Summary The genetic relationships between two Prunus species, involved in rootstock breeding, were examined at the level of the ribosomal RNA genes. Twenty clones of P. cerasifera, a diploid species, and 12 clones of P. spinosa, a tetraploid wild species, were studied. The use of three heterologous ribosomal DNA probes covering different regions of the ribosomal tandem repeats enabled us to construct restriction maps for EcoRI and BamHI. We identified two unit types (unit I and unit II) in P. cerasifera. In P. spinosa, P. cerasifera units were present in addition to a third ribosomal unit type (unit III). These results appeared to confirm previous cytological studies (Salesses 1973) indicating that one of the genomes in P.spinosa has homology with the one from P. cerasifera.     

Conservation and variation of ribosomal proteins in several species of the cellular slime molds Dictyostelium and Polysphondylium

Genus- and species-specific composition of ribosomal proteins was investigated in four species of the genus Dictyostelium (D. discoideum, D. purpureum, D. murcoroides and D. giganteum) and two species of the genus Polysphondylium (P. pallidum and P. violaceum). Ribosomal proteins were resolved by a high-resolution, two-dimensional gel method. In general, the numbers and distributions for the majority of ribosomal proteins were similar within the species of each genus, although some differences were detected. More differences were observed between Dictyostelium and Polysphondylium than among the individual species within each genus. Stage-specific ribosomal proteins previously demonstrated in D. discoideum were found to be developmentally regulated in other Dictyostelium species, and in both Polysphondylium species. The study shows that ribosomal proteins may be a potentially useful new biochemical parameter for the molecular taxonomy of the cellular slime molds.