Identification of RNA molecules which contain 5 S ribosomal RNA and transfer RNA in an RNAase E-RNAase P- double mutant strain of Escherichia coli

In the analysis of DNAase II digestion of chromatin, as described in the preceding paper, interactions between adjacent nucleosomes play an important part. In order to understand the mechanism of DNAase II cleavage we next investigated the role of histone H1 in these interactions and characterized the nucleoprotein particles arising in the course of DNAase II action.H1-free chromatin prepared by three different procedures, using either 0.6 m-NaCl, transfer RNA or an ion-exchange resin, can be cleaved by DNAase II only at the internucleosomal cleavage site leading to 200-bp² digestion patterns regardless of the ionic conditions. When H1 was added back to the three chromatin preparations the 100-bp cleavage pattern could be restored only with material prepared by the resin method at low concentrations of salt. Addition of polylysine instead of H1 has the same effect, but only with material prepared by that method. A direct correlation between extended and condensed states of chromatin as monitored by electron microscopy and DNAase II cleavage in the 200 and 100-bp modes, respectively, could be established.The continuity of the nucleosome chains in DNAase II-digested chromatin is maintained in spite of intranucleosomal cleavage in the terminal section of the core DNA, even in the absence of H1. Addition of 3 m-urea, however, disrupts the nucleosome chains at the intranucleosomal cleavage sites and leads to the formation of novel nucleoprotein particles as seen in sucrose gradient centrifugations. Those sedimenting between mononucleosomes and dinucleosomes contain, almost exclusively, DNA of 300 bp (mouse) or 315 bp (chicken erythrocyte). They can be formed from particles sedimenting in the absence of urea in the dinucleosome region by either a dissociation process or a massive conformational change.On the basis of the results presented here and in the preceding paper a mechanism for DNAase II cleavage of chromatin in the 200-bp and 100-bp modes is proposed and discussed in the context of structural features of chromatin recognized by DNAase II.     

Two novel RNA binding proteins from Trypanosoma brucei are associated with 5S rRNA.

We have previously reported the identification of two closely related RNA binding proteins from Trypanosoma brucei which we have termed p34 and p37. The predicted primary structures of the two proteins are highly homologous with one major difference, an 18-amino-acid insert in the N-terminal region of p37. These two proteins have been localized to the nucleus based on immunofluorescence microscopy. To gain insight into their function, we have utilized UV crosslinking, coimmunoprecipitation, and sucrose density gradients to identify T. brucei RNA species that associate with p34 and p37. These experiments have demonstrated a specific interaction of both p34 and p37 with the 5S ribosomal RNA and indicate that other RNA species are unlikely to be specifically bound. This suggests a role for p34 and p37 in the import and/or assembly pathway of T. brucei 5S rRNA in ribosome biogenesis.     

Molecular taxonomy of the suborder Bodonina (Order Kinetoplastida), including the important fish parasite,Ichthyobodo necator

Ichthyobodo necator is an important fish ectoparasite with a broad host and ecological range. A novel method, involving the use of an anesthetic, allowed the collection of large numbers of parasites from the skin and gills of hybrid striped bass (Morone saxatilis male x M. chrysops female). Genomic DNA from these samples was used to amplify and clone the 18S rRNA gene. The 18S rRNA gene was similarly cloned from Bodo caudatus, Bodo edax, Bodo saltans, an unidentified Bodo species, and Dimastigella trypaniformis. The resulting sequences were aligned with other representative kinetoplastid species using pileup and similarities in secondary structure. Phylogenetic relationships within the suborder Bodonina and representatives of the suborder Trypanosomatina were determined using maximum-likelihood statistics. The phylogenetic analyses strongly supported the order Kinetoplastida as a monophyletic assemblage consisting of at least two major lineages. One lineage consisted exclusively of L. necator, indicating that it may represent a new suborder. The second lineage consisted of all other kinetoplastid species. This second lineage appeared to contain at least 8 bodonine sublineages, none of which correlated with currently recognized families. For three sublineages, there was a close correspondence between the 18S phylogeny and the classical taxonomy of Dimastigella, Rhynchobodo, and Rhynchomonas. In contrast, Bodo and Cryptobia were polyphyletic, containing species in two or more sublineages that may represent separate genera.     

Evidence for non-coordinated synthesis of 5 S RNA in the thermophilic fungus,Thermomyces lanuginosus

Analysis of ribosomes and the post ribosomal supernatant fraction of actively growing cells ofThermomyces lanuginosus showed the presence of free 5 S RNA in the supernatant fraction. This 5 S RNA was identical to the ribosomal 5 S RNA in its electrophoretic mobility on 10% Polyacrylamide gel and in its base composition. 5 S RNA from both the sources gave evidence for the presence of diphosphate at the 5’ end. Most of the 5 S RNA that appeared in the cytoplasm was that transported from the nucleus during the isolation. This could be prevented by the use of a hexylene glycol-HEPES buffer.     

Azoarcus spp. and their interactions with grass roots

The current knowledge on the divergence within the genus Azoarcus and about interactions with grasses is summarized. Grass-associated members of this genus of diazotrophs have only been isolated from a salt- and flood-tolerant pioneer plant in Pakistan, Kallar grass (Leptochloa fusca (L.) Kunth). Members of these bacteria belong to the beta subclass of the Proteobacteria, most closely related to purple bacteria such as Rhodocyclus purpureus. The isolates from one single host plant showed a surprising divergence, consisting of five groups of Azoarcus distinct at the species level. Molecular diagnostic tests, which are based on 16S ribosomal RNA sequences, allowed preliminary assignment of isolates to Azoarcus by PCR amplification and sequencing of PCR products. Moreover, the moleculer tests enabled us to detect an unculturable strain in Kallar grass roots, stressing that classical cultivation techniques at times fail to detect some groups of the microbial population. Using similar techniques, sequences rooting in the Azoarcus clade were also detected in field-grown rice, indicating that the natural host range might extend to rice. In gnotobiotic laboratory cultures, a member of Azoarcus is able to colonize rice roots endophytically: bacteria invade the roots in the zone of elongation and differentiation, colonize the cortex intra- and inter-cellularly, and penetrate deeply into the vascular system, entering xylem vessels, allowing systemic spreading into the rice shoot. Recently, we detected expression of nitrogenase of Azoarcus cells inside roots of rice seedlings, a result encouraging us to analyze interactions with rice in detail.     

Involvement of lysine and arginine residues in the binding of yeast ribosomal protein YL3 to 5S RNA

The contribution of lysine and arginine residues to the formation of yeast ribonucleoprotein complex 5S RNA. protein YL3 has been investigated by determining the effects on complex formation of modification with chemical reagents specific for either lysine or arginine. Treatment of protein YL3 with acetic anhydride, malefic anhydride or phenylglyoxal is accompanied by loss of its capacity to bind to 5S RNA. This effect is accomplished by modification with phenylglyoxal of only 3 arginine residues per YL3 molecule. In contrast, a large number of protein YL3 amino groups [16] must be modified by acetic anhydride to prevent complex formation.     

Post-maturation of the plastid ribosomal RNA in the plant kingdom

Summary The in vivo fragmentation of the plastid rRNA from plants situated at different places in the evolutionary scale, with the exception ofAlgae, was analysed by electrophoresis using fully denaturing conditions. This fragmentation corresponds to an in vivo post-maturation. It exists only in some bacteria and is not random. Five main groups of fragments with the following real molecular weights (Mr) are found in 23 S:ca 0.9 × 10⁶; 0.7 × 10⁶; 0.45 × 10⁶; 0.35 × 10⁶ and 0.15 × 10⁶. The existence of a large fragment (Mr = 0.9 × 10⁶) corresponds to a primitive type of fragmentation found in some archaic plants. Dicotyledons and several other groups have the same pattern of 23 S fragmentation, often comprising all the fragments mentioned above, whilstGraminaceae (Monocotyledons) constitute a special group with a very predominant 0.35 × 10⁶ dalton fragment and the absence of the 0.45 × 10⁶ dalton fragment. The plastid 16 S rRNA in all plants studied here has aMr of 0.54 × 10⁶ which is smaller than the 16 S ofEscbericbia coli taken as reference (0.56 × 10⁶ dalton).     

16S rRNA序列分析法在医学微生物鉴定中的应用

１６Ｓ ｒＲＮＡ序列分析作为微生物系统分类的主要依据已得到了广泛认同,随着微生物核糖体数据库的日益完善,该技术成为细菌分类和鉴定的一个有力工具。本文概述了 １６５ ｒＲＮＡ序列分析法的技术步骤以及该技术在医学微生物研究中的应用,总结了目前文献报导的各种致病微生物种属特异性 １６５ ｒＲＮＡ引物和探针序列,同时分析了该技术在应用中存在的一些问题。     

Dead-end evolution of the Cnidaria as deduced from 5S ribosomal RNA sequences

Using nucleotide sequences of 5S ribosomal RNAs from 2 hydrozoan jellyfishes, 3 scyphozoan jellyfishes and 2 sea anemones, a phylogenetic tree of Cnidaria has been constructed to elucidate the evolutionary relationships of radial and bilateral symmetries. The 3 classes of Cnidaria examined herein belong to one branch, which does not include other metazoan phyla such as the Platyhelminthes. The Hydrozoa (having radial symmetry without septa) and the Scyphozoa (having radial symmetry with septa) are more closely related to each other than to the Anthozoa (having bilateral symmetry with septa). In classical taxonomy, multicellular animals are considered to have evolved through organisms with radial symmetry (e.g., Cnidaria) to bilateral symmetry. Our results, however, indicate that the emergence of the Bilateria was earlier than that of the Radiata, suggesting (in opposition to Haeckel's view) that the radial symmetry of Cnidaria is an evolutionary dead end.     

Evolution of the 5 S RNA genes in vertebrates

Summary We have built the phylogenetic tree of Vertebrate 5S RNA using the sequence data of thirteen species belonging to six groups. Evolution of the 5S genes has been very slow in Vertebrates since 90 residues are identical in all 5S RNAs which are presently sequenced.In Amphibians and Teleosts different 5S genes are active in oocytes and in somatic cells. This dual gene system has probably been acquired independently by Amphibians and Teleosts. In Amphibians, the oocyte-type 5S genes have evolved much faster than the somatic-type genes. This is not true in all species since the oocyte-type genes of one Teleost (Tinca tinca) have evolved more slowly than the somatic-type genes.There are in all Vertebrate 5S RNAs five complementary regions which can be base-paired. The sequence data are compatible with the three secondary-structure models that have been proposed for 5S RNA.     

Physical mapping of the 5S ribosomal RNA genes on rice chromosome 11

One 5S ribosomal RNA gene (5S rDNA) locus was localized on chromosome 11 of japonica rice by in situ hybridization. The biotinylated DNA probe used was prepared by direct cloning and direct labeling methods, and the locus was localized to the proximal region of the short arm of chromosome 11 (llpl.l) by imaging methods. The distance between the signal site and the centromere is 4.0 arbitrary units, where the total length of the short arm is 43.3 units. The 5S rDNA locus physically identified and mapped in rice was designated as 5SRrn. The position of the 5S rDNA locus reported here differs from that in indica rice; possible reasons for this difference are discussed. DNA sequences of 5S rDNA are also reported.     

The 5S ribosomal RNA sequences of a red algal rhodoplast and a gymnosperm chloroplast. Implications for the evolution of plastids and cyanobacteria

Summary The 5S ribosomal RNA sequences have been determined for the rhodoplast of the red algaPorphyra umbilicalis and the chloroplast of the coniferJuniperus media. The 5S RNA sequence of theVicia faba chloroplast is corrected with respect to a previous report. A survey of the known sequences and secondary structures of 5S RNAs from plastids and cyanobacteria shows a close structural similarity between all 5S RNAs from land plant chloroplasts. The algal plastid 5S RNAs on the other hand show much more structural diversity and have certain structural features in common with bacterial 5S RNAs. A dendrogram constructed from the aligned sequences by a clustering algorithm points to a common ancestor for the present-living cyanobacteria and the land plant plastids. However, the algal plastids branch off at an early stage within the plastid-cyanobacteria cluster, before the divergence between cyanobacteria and land plant chloroplasts. This evolutionary picture points to the occurrence of multiple endosymbiotic events, with the ancestors of the present algal plastids already established as photosynthetic endosymbionts at a time when the ancestors of the present land plant chloroplasts were still free-living cells.     

Tandem 5S ribosomal RNA genes and the spacer region sequences of three Japanese Laminaria species

The organization of 5S ribosomal RNA genes (rDNA) was examined for threeJapanese Laminaria species, L. japonica, L.religiosa and L. ochotensis. The linkage of 5SrDNA with the 18S-5.8S-25S rDNAs unit known in the brown algaScytosiphon lomentaria could not be detected inLaminaria. Instead, the tandem repeats of 5S rDNA were notassociated with the 18S-5.8S-25S rDNAs unit. The nucleotide sequence of 5S rDNAwas completely identical among these three species and its length was 118bp. However, a difference of nucleotide arrangement was detectedinthe spacer region of the tandemly repeated 5S rDNAs. Several nucleotideinsertion / deletions and substitutions were confirmed between differentindividuals of L. japonica, which were collected from notonly disjunct localities, but also the same locality. The lengths of the spacerregion of L. japonica, L. religiosaand L. ochotensis were 247–252 bp, 232bp and 252 bp, respectively.     

Evolution of green plants and their relationship with other photosynthetic eukaryotes as deduced from 5S ribosomal RNA sequences

The nucleotide sequence of cytoplasmic 5S ribosomal RNAs from three gymnosperms,Pinus contorta, Taxus baccata andJuniperus media and from one fern,Pteridium aquilinum, have been determined. These sequences were aligned with all hitherto known cytoplasmic 5S ribosomal RNA sequences of photosynthetic eukaryotes. A dendrogram based on that set of sequences was constructed by a distance matrix method and the resulting tree compared with established views concerning plant and algal evolution. The following monophyletic groups of photosynthetic eukaryotes are recognizable: theRhodophyta, a group consisting ofPhaeophyta, Bacillariophyta andChrysophyta, and the green plants, the latter comprising green algae,Bryophyta, Pteridophyta andSpermatophyta. According to our 5S ribosomal RNA tree, green plants may have originated from some type of a green flagellated organism such asChlamydomonas. The land plants seem to have originated from some form of charophyte such asNitella. 5S ribosomal RNA seems to be less appropriate to estimate dissimilarities between species which have diverged relatively recently, like the angiosperms. Therefore, a precise evolutionary process is difficult to reconstruct for members of this group.     

基于18S rRNA和线粒体16S rRNA基因序列的柳蚕进化分析

为探讨柳蚕Actias selene Hübner与鳞翅目昆虫的系统发育关系,本研究利用PCR扩增获得了柳蚕核糖体18S rRNA和线粒体16S rRNA基因的部分序列,长度分别为391bp和428bp。并采用邻近距离法(NJ)、最大简约法(MP)、类平均聚类法(UPGMA)构建系统进化树。结果表明,柳蚕线粒体16SrRNA基因序列与大蚕蛾科昆虫的16SrRNA基因序列均表现出偏好于碱基AT的倾向。柳蚕与所研究的其它蚕类的遗传距离介于0.016至0.140之间,其中与温带柞蚕Antheraea roylii的遗传距离最小,与野桑蚕Bombyx mandarina的遗传距离最大。而基于鳞翅目昆虫18S rRNA基因部分序列的进化分析显示,柳蚕与柞蚕Antheraea pernyi之间的遗传距离最小(0.010),与蓖麻蚕Samia ricini的遗传距离最大(0.017)。     

猕猴(Macaca mulatta)肝5S核糖核蛋白体RNA的分离提纯

应用酚-去污剂和1摩尔/升NaCl抽提低分子量RNA,通过DEAE-葡聚糖A-50离子交换层析提纯后,经含有7摩尔/升尿素的10%聚丙烯酰胺凝胶垂直板电泳制备纯化猕猴肝5S核糖核蛋白体RNA是简易而行之有效的方法。制纯的5SrRNA经聚丙烯酰胺凝胶电泳鉴定呈现单一的一条区带。与大肠杆菌5SrRNA标准品具有相同的电泳迁移率。     

Single-chain ribosome inactivating proteins from plants depurinate Escherichia coli 23S ribosomal RNA.

The rRNA N-glycosidase activities of the catalytically active A chains of the heterodimeric ribosome inactivating proteins (RIPs) ricin and abrin, the single-chain RIPs dianthin 30, dianthin 32, and the leaf and seed forms of pokeweed antiviral protein (PAP) were assayed on E. coli ribosomes. All of the single-chain RIPs were active on E. coli ribosomes as judged by the release of a 243 nucleotide fragment from the 3′ end of 23S rRNA following aniline treatment of the RNA. In contrast, E. coli ribosomes were refractory to the A chains of ricin and abrin. The position of the modification of 23S rRNA by dianthin 32 was determined by primer extension and found to be A₂₆₆₀, which lies in a sequence that is highly conserved in all species.     

Ribosomal RNA genes inQuercus spp. (Fagaceae)

The taxonomy of the genusQuercus is still unclear. In order to elucidate the taxonomy of Mediterranean oaks we have analyzed ribosomal RNA genes ofQuercus cerris, Q. coccifera, Q. trojana, Q. ilex, Q. suber, andQ. macrolepis by means of Southern blot hybridization. Oak nuclear DNA was extracted from root tips of 300 acorns and from catkins of single plants. EcoRI and BamHI restriction endonucleases were used. DNA electrophoresis and rRNA/DNA hybridization were performed usingVicia faba rRNA 18 S and 25 S as probes. The rRNA genes of all the species studied have an identical restriction mapping in the 18 S and 25 S regions, while differences in length are present in the intergenic regions.Q. cerris possesses at least four types of genes of 12.1, 11.5, 8.5, and 8.3 kb;Q. coccifera at least three types of 12.4, 10.4, and 10.1 kb;Q. trojana possesses the same rRNA genes asQ. cerris plus another gene type 12.0 kb long, with EcoRI and BamHI restriction sites in the intergenic spacer;Q. ilex at least three types of 12.4, 10.85, and 9.5 kb;Q. suber at least five types of 11.5, 11.0, 8.6, 8.5, and 8.3 kb;Q. macrolepis, finally, at least seven types of 11.5, 11.0, 10.2, 8.6, 8.5, 8.3, and 8.15 kb.Q. coccifera andQ. ilex rDNA appears quite different respect to other species examined, while high similarity seems to exist betweenQ. cerris, Q. trojana, Q. suber, andQ. macrolepis. These results are in agreement with the taxonomic model proposed bySchwarz for the genusQuercus.     

Preparative-scale isolation and purification of procaryotic and eucaryotic ribosomal 5 S RNA: Bacillus subtilis, Neurospora crassa, and wheat germ

Ribosomal 5 S RNA from three different organisms has been isolated in high yield and purity. Without prior isolation of ribosomes, a presoak in buffer followed by phenol extraction, DE-32 ion-exchange chromatography, and Sephadex G-75 gel-permeation chromatography yields at least 5-10 mg of electrophoretically homogeneous 5 S RNA from 100 g of cells. Ribonuclease activity is eliminated by various combinations of low temperature, sodium dodecyl sulfate, phenol, and bentonite. High-molecular-weight contaminants are suppressed by either 65 degrees C heat treatment or lowered sodium dodecyl sulfate concentration. For the eucaryotes, 5.8 S RNA contamination is reduced either by low temperature in the initial solubilization or by postponing 65 degrees C heat treatment until after the phenol extraction step.