The 5S and 5.8S ribosomal RNA sequences of Tetrahymena thermophila and T. pyriformis

The nucleotide sequences of the 5S rRNAs of Tetrahymena thermophila and two strains of T. pyriformis have been determined to be identical. The 5.8S rRNA sequences have also been determined; these sequences correct several errors in an earlier report. The 5.8S rRNAs of the two species differ at a single position. The sequencing results indicate that the species are of recent common ancestry. Molecular evidence that has been interpreted in the past as suggestive of an ancient divergence has been reviewed and found to be consistent with a T. pyriformis complex radiation beginning approximately 30-40 million years ago.     

5S and 5.8S ribosomal RNA evolution in the suborder tetrahymenina (Ciliophora: Hymenostomatida)

Summary The nucleotide sequences of the 5S and 5.8S rRNAs of eight strains of tetrahymenine ciliates have been determined. The sequences indicate a clear distinction betweenTetrahymena paravorax and its suggested conspecificT. vorax, but leave the taxonomic distinction betweenT. vorax andT. leucophrys in doubt. The rRNA sequences of sixTetrahymena species and of three other species of the suborder Tetrahymenina have been used to deduce evolutionary schemes in which ancestral rRNA sequences and changes are proposed. These schemes suggest the predominant acceptance of GA and CT transitions in the 5S rDNA during the evolution of the suborder.     

Comparison of primary and secondary 26S rRNA structures in twoTetrahymena species: Evidence for a strong evolutionary and structural constraint in expansion segments

Summary We have determined the nucleotide sequence of the 26S large subunit (LSU) rRNA genes for twoTetrahymena species,T. thermophila andT. pyriformis. The inferred rRNA sequences are presented in their most probable secondary structures based on compensatory mutations, energy, and conservation criteria. The majority of the nucleotide changes between the twoTetrahymena LSU rRNAs and the positions of a relatively large deletion and of the processing cleavage sites resulting in the generation of the hidden break are all located within the so-called divergent domains or expansion segments. These are regions within the common core of secondary structure where expansions have taken place during the evolution of the rRNA of higher eukaryotes.The dispensable nature of some of the expansion segments has been taken as evidence of their non-functionality. However, our data show that a considerable selective constraint has operated to presesrve the secondary structure of these segments. Especially in the case of the D2 and D8 segments, the presence of a considerable number of compensatory base changes suggests that the secondary structure of these regions is of functional importance. Alternatively, these expansion segments may have maintained characteristic folding patterns because only such structures are being tolerated within otherwise functionally important regions.     

Phylogeny of protozoa deduced from 5S rRNA sequences

Summary The nucleotide sequences of 5S rRNAs from three protozoa,Bresslaua vorax, Euplotes woodruffi andChlamydomonas sp. have been determined and aligned together with the sequences of 12 protozoa species including unicellular green algae already reported by the authors and others. Using this alignment, a phylogenic tree of the 15 species of protozoa has been constructed. The tree suggests that the ancestor for protozoa evolved at an early time of eukaryotic evolution giving two major groups of organisms. One group, which shares a common ancestor with vascular plants, contains a unicellular green flagellate (Chlamydomonas) and unicellular green algae. The other group, which shares a common ancestor with the multicellular animals, includes various flagellated protozoa (includingEuglena), ciliated protozoa and slime molds. Most of these protozoa appear to have separated from one another at a fairly early period of eukaryotic evolution.     

The nucleotide sequences of 5S rRNAs from a rotifer, Brachionus plicatilis, and two nematodes, Rhabditis tokai and Caenorhabditis elegans.

The nucleotide sequences of 5S rRNAs from a rotifer, Brachionus plicatilis, and two nematodes, Rhabditis tokai and Caenorhabditis elegans have been determined. The rotifer has two 5S rRNA species that are composed of 120 and 121 nucleotides, respectively. The sequences of these two 5S rRNAs are the same except that the latter has an additional base at its 3'-terminus. The 5S rRNAs from the two nematode species are both 119 nucleotides long. The sequence similarity percents are 79% (Brachionus/Rhabditis), 80% (Brachionus/Caenorhabditis), and 95% (Rhabditis/Caenorhabditis) among these three species. Brachionus revealed the highest similarity to Lingula (89%), but not to the nematodes (79%).     

An evaluation of the phylogenetic position of the dinoflagellateCrypthecodinium cohnii based on 5S rRNA characterization

Summary Partial nucleotide sequences for the 5S and 5.8S rRNAs from the dinoflagellateCrypthecodinium cohnii have been determined, using a rapid chemical sequencing method, for the purpose of studying dinoflagellate phylogeny. The 5S RNA sequence shows the most homology (75%) with the 5S sequences of higher animals and the least homology (< 60%) with prokaryotic sequences. In addition, it lacks certain residues which are highly conserved in prokaryotic molecules but are generally missing in eukaryotes. These findings suggest a distant relationship between dinoflagellates and the prokaryotes. Using two different sequence alignments and several different methods for selecting an optimum phylogenetic tree for a collection of 5S sequences including higher plants and animals, fungi, and bacteria in addition to theC. cohnii sequence, the dinoflagellate lineage was joined to the tree at the point of the plant-animal divergence, well above the branching point of the fungi. This result is of interest because it implies that the well-documented absence in dinoflagellates of histones and the typical nucleosomal subunit structure of eukaryotic chromatin is the result of secondary loss. and not anindication of an extremely primitive state, as was previously suggested. Computer simulations of 5S RNA evolution have been carried out in order to demonstrate that the above-mentioned phylogenetic placement is not likely to be the result of random sequence convergence.We have also constructed a phylogeny for 5.8S RNA sequences in which plants, animals, fungi and the dinoflagellates are again represented. While the order of branching on this tree is the same as in the 5S tree for the organisms represented, because it lacks prokaryotes, the 5.8S tree cannot be considered a strong independent confirmation of the 5S result. Moreover, 5.8S RNA appears to have experienced very different rates of evolution in different lineages indicating that it may not be the best indicator of evolutionary relationships.We have also considered the existing biological data regarding dinoflagellate evolution in relation to our molecular phylogenetic evidence.     

Composite structure of the chloroplast 23 S ribosomal RNA genes of Chlamydomonas reinhardii: Evolutionary and functional implications

The chloroplast ribosomal unit of Chlamydomonas reinhardii displays two features which are not shared by other chloroplast ribosomal units. These include the presence of an intron in the 23 S ribosomal RNA gene and of two small genes coding for 3 S and 7 S rRNA in the spacer between the 16 S and 23 S rRNA genes (Rochaix & Malnoë, 1978). Sequencing of the 7 S and 3 S rRNAs as well as their genes and neighbouring regions has shown that: (1) the 7 S and 3 S rRNA genes are 282 and 47 base-pairs long, respectively, and are separated by a 23 base-pair A + T-rich spacer. (2) A sequence microheterogeneity exists within the 3 S RNA genes. (3) The sequences of the 7 S and 3 S rRNAs are homologous to the 5′ termini of prokaryotic and other chloroplast 23 S rRNAs, indicating that the C. reinhardii counterparts of 23 S rRNA have a composite structure. (4) The sequences of the 7 S and 3 S rRNAs are related to that of cytoplasmic 5.8 S rRNA, suggesting that these RNAs may perform similar functions in the ribosome. (5) Partial nucleotide sequence complementarity is observed between the 5′ ends of the 7 S and 3 S RNAs on one hand and the 23 S rRNA sequences which flank the ribosomal intron on the other. These data are compatible with the idea that these small rRNAs may play a role in the processing of the 23 S rRNA precursor.     

Heterogeneity of the ribosomal DNA episome in strains and species of Entamoeba

Ribosomal DNA sequences in several species of the genus Entamoeba are highly repeated and display restriction fragment-length polymorphism (RFLP), which has been used to identify species and differentiate strains. However, the continuous variability of the non-transcribed repeat sequences in the ribosomal episome hinders an accurate typification. Looking for more reliable markers, we used DNA probes containing conserved sequences in the ribosomal episome — coding regions for the 16S and 5.8S rRNAs and transcribed spacers flanking the rDNA sequences, and the coding region for the 3 end of the 26S rRNA — to analyse hybridization patterns from five cloned pathogenic strains of Entamoeba histoiytica, two strains of the also pathogenic Entamoeba invadens and the non-pathogenic Laredo strain of Entamoeba moshkovskii. Our results provide reliable bases for the differentation of clones, strains and species of Entamoeba and the reconstruction of E. histolytica episomes. Differences in the number and length of rDNA-containing DNA fragments, previously observed by other investigators and confirmed by us, can be better defined by the present analysis.     

Nucleotide sequences of Acanthamoeba castellanii 5S and 5.8S ribosomal ribonucleic acids: phylogenetic and comparative structural analyses.

Sequences of 5S and 5.8S rRNAs of the amoeboid protist Acanthamoeba castellanii have been determined by gel sequencing of terminally-labeled RNAs which were partially degraded with chemical reagents or ribonucleases. The sequence of the 5S rRNA is (formula, see text). This sequence is compared to eukaryotic 5S rRNA sequences previously published and fitted to a secondary structure model which incorporates features of several previously proposed models. All reported eukaryotic 5S rRNAs fit this model. The sequence of the 5.8S rRNA is (formula, see text). This sequence does not fit parts of existing secondary structure models for 5.8S rRNA, and we question the significance of such models.     

The nucleotide sequences of 5S rRNAs from two ribbon worms: Emplectonema gracile contains two 5S rRNA species differing considerably in their sequences.

The nucleotide sequences of 5S rRNAs from two nemerteans (ribbon worms), Lineus geniculatus and Emplectonema gracile have been determined. Emplectonema has two 5S rRNA species that are composed of 119 and 120 nucleotides, respectively. The sequences of these two 5S rRNAs differ at 22 positions. On the other hand, only a single 5S rRNA species was found in Lineus. The sequence similarity percents are 88% (Lineus/Emplectonema longer 5S rRNA), 82% (Emplectonema longer/Emplectonema shorter) and 80% (Lineus/Emplectonema shorter). The comparisons of these sequences with those of other organisms suggest that the phylum Nemertinea is most related to the Mollusca (91%) and the Rotifera (89%), but not to fresh-water planarias (72%).     

Growth Kinetics of Three Species of Tetrahymena On Solid Agar*

A nutrient-agar method without liquid overlay has been developed for cultivation of ciliates. Three species of Tetrahymena-T. pyriformis strain W, T. rostrata strain UNI, and T. vorax strain V₂S, representing the 3 main groups of Tetrahymena species, were used; however the method should apply to other ciliates. Growth on the surface of the agar was facilitated by an optimal surface-to-volume ratio yielding a high density of ciliates (5.8 × 10₅ cells/cm² for T. pyriformis at 25 C) and short generation times (3 h for T. pyriformis at 30 C). At the highest density achieved, the cells became irregularly hexagonal and formed a monolayer “tissue” on the agar. Ciliates grown on agar were like those in liquid culture, typical oral ciliature, food-vacuole formation, and typical cortical patterns being retained. Advantages of this method include high cell density, easy recovery, and optimal O₂ supply. the organisms can also be cultivated on the surface of sterile cellulose-nitrate filters, facilitating in situ fixation and staining as well as transfer into different media by transfer of filters with cells, without prior centrifugation and resuspension.     

Phylogenetic Relationships of Species of the Genus Kluyveromyces van der Walt (Saccharomycetaceae) Deduced from the Partial Sequences of 18S and 26S Ribosomal RNAs

In order to clarify the phylogenetic relationships of the species classified in the genus Kluyveromyces (Saccharomycetaceae), three partial base sequences of 18S and 26S rRNAs of eighteen strains were determined. The regions determined of the strains corresponded to positions 1451 through 1618 (168 bases) of 18S rRNA and to positions 1611 through 1835 (225 bases) and 493 through 622 (130 bases) of a strain (IFO 2376) of Saccharomyces cerevisiae. The analyses of the partial base sequences suggested that the genus Kluyveromyces is phylogenetically heterogeneous, ranging from the strains that are quite close to the strain of S. cerevisiae to the strains that are distinct enough to be classified in genera separate from the genus Saccharomyces. From our sequence data, we concluded that the extent of the genus Kluyveromyces should be restricted to only one species, K. polysporus, the type species of the genus. Kluyveromyces phaffii was also distinct enough to deserve another genus. Kluyveromyces cellobiovorus was not close to any of the strains of Kluyveromyces species examined, and should be excluded from the genus. Most of the strains of the species examined were fairly close to the strain of S. cerevisiae.     

The sequence ofTetrahymena thermophila 5S ribosomal ribonucleic acid

The primary structure ofTetrahymena thermophila 5S rRNA is reported. A secondary structure model is presented which can encompass most published eukaryotic 5S rRNA sequences. Unlike other eukaryotic 5S rRNAs,Tetrahymena is found to contain the sequence-CGAAC- beginning at position 40. The presence of this segment had previously been thought to be an exclusive characteristic of eubacterial 5S rRNAs.     

Tetrahymena australis (Protozoa,Ciliophora): A Well‐Known But “Non‐Existing” Taxon – Consideration of Its Identification,Definition and Systematic Position

A cryptic species of the Tetrahymena pyriformis complex, Tetrahymena australis, has been known for a long time but never properly diagnosed based on taxonomic methods. The species name is thus invalid according to the International Code of Zoological Nomenclature. Recently, a population isolated from a freshwater lake in Wuhan, China was investigated using live observations, silver staining methods and gene sequence data. This organism can be separated from other described species of the T. pyriformis complex by its relatively small body size, the number of somatic kineties and differences in sequences of two genes, namely the small subunit ribosomal RNA (SSU rRNA) and the mitochondrial cytochrome c oxidase subunit I (cox1). We compared the SSU rRNA gene sequences of all available Tetrahymena species to reveal the nucleotide differences within this genus. The sequence of the Wuhan population is identical to two sequences of a previously isolated strain of T. australis (ATCC #30831). Phylogenetic analyses indicate that these three sequences (X56167, M98015, KT334373) cluster with Tetrahymena shanghaiensis (EF070256) in a polytomy. However, sequence divergence of the cox1 gene between the Wuhan population and another strain of T. australis (ATCC #30271) is 1.4%, suggesting that these may represent different subspecies.     

Defining species in Tulasnella by correlating morphology and nrDNA ITS-5.8S sequence data of basidiomata from a tropical Andean forest

The genus Tulasnella comprises important orchid mycobionts. Molecular phylogenetic studies on nrITS-5.8S sequences of Tulasnella species previously isolated from mycorrhizas of epiphytic orchids from a tropical Andean forest showed genomic variability among clones which was difficult to interpret as intra- or interspecific variations or to correlate with described Tulasnella species. To improve this situation, we collected basidiomata of Tulasnella in an Andean forest, studied part of the sequences of fungal ribosomal genes and correlated molecular data with the morphology of the specimens. Within five basidiomata displaying slight morphological variability, we found inter-specimen nrITS1-5.8S-ITS2 variability corresponding to proportional differences of less than 1% except for one clone with 5.1% divergence. Results indicate that the slightly variable basidiomata should be considered as one species, which is morphologically tentatively assigned to the Tulasnella pruinosa complex. However, comparison of nrITS1-5.8S-ITS2 sequences, including sequences of T. pruinosa from other origins, indicate that Tulasnella sp. is only distantly related to the T. pruinosa specimens included in the analyses. Sequences of all morphologically similar and taxonomically well-identified species are required to decide whether the basidiomata analyzed in the present study represent a new species. The new sequences are rather similar to sequences obtained previously from mycorrhizae of epiphytic orchids of the same area indicating mycorrhizal potential of this fungus.     

Unique arrangement of coding sequences for 5 S, 5.8 S, 18 S and 25 S ribosomal RNA in Saccharomyces cerevisiae as determined by R-loop and hybridization analysis.

The arrangement of the coding sequences for the 5 S, 5.8 S, 18 S and 25 S ribosomal RNA from Saccharomyces cerevisiae was analyzed in λ-yeast hybrids containing repeating units of the ribosomal DNA. After mapping of restriction sites, the positions of the coding sequences were determined by hybridization of purified rRNAs to restriction fragments, by R-loop analysis in the electron microscope, and by electrophoresis of S₁ nuclease-treated rRNA/rDNA hybrids in alkaline agarose gels. The R-loop method was improved with respect to the length calibration of RNA/DNA duplexes and to the spreading conditions resulting in fully extended 18 S and 25 S rRNA R-loops. The qualitative results are: (1) the 5 S rRNA genes, unlike those in higher eukaryotes, alternate with the genes of the precursor for the 5.8 S, 18 S and 25 S rRNA; (2) the coding sequence for 5.8 S rRNA maps, as in higher eukaryotes, between the 18 S and 25 S rRNA coding sequences. The quantitative results are: (1) the tandemly repeating rDNA units have a constant length of 9060 ± 100 nucleotide pairs with one SstI, two HindIII and, dependent on the strain, six or seven EcoRI sites; (2) the 18 S and 25 S rRNA coding regions consist of 1710 ± 80 and 3360 ± 80 nucleotide pairs, respectively; (3) an 18 S rRNA coding region is separated by a 780 ± 70 nucleotide pairs transcribed spacer from a 25 S rRNA coding region. This is then followed by a 3210 ± 100 nucleotide pairs mainly non-transcribed spacer which contains a 5 S rRNA gene.     

The nucleotide sequences of 5S rRNAs from two Annelida species, Perinereis brevicirris and Sabellastarte japonica, and an Echiura species, Urechis unicinctus.

The nucleotide sequences of 5S rRNAs from two Annelida species, Perinereis brevicirris and Sabellastarte japonica, and an Echiura species, Urechis unicinctus have been determined. Their sequences are all 120 nucleotides long. The sequence similarity percents are 88% (Perinereis/Sabellastarte), 90% (Sabellastarte/Urechis) and 92% (Perinereis/Urechis), indicating that the Echiura is indistinguishable from the Annelida by their 5S rRNAs. The 5S rRNA sequences from the Annelida/Echiura are most related to those from the Nemertinea (87%), the Mollusca (87%) and the Rotifera (88%).     

Cytological localization of the genes for the four classes of ribosomal RNA (25S, 18S, 5.8S and 5S) in polytene chromosomes of Phaseolus coccineus

Homologous tritiated 25S, 18S and 5.8S rRNAs were used separately for in situ hybridization to the polytene chromosomes of the embryo suspensor cells of Phaseolus coccineus. Hybridization occurred at the same chromosomal sites which were labeled in previous in situ hybridization experiments with 25+18S rRNAs in the same material (Avanzi et al., 1972), namely: nucleolus organizing system (satellite, nucleolar constriction and organizer) of chromosome pairs I (S₁) and V (S₂), proximal heterochromatic segment of the long arm of chromosome pair I, and terminal heterochromatic segment of chromosome pair II. Competition hybridization experiments confirmed for P. coccineus the high sequence homology between 25S and 18S rRNA already known for other plants.Homologous ¹²⁵I-5S rRNA was found to hybridize to three sites in the polytene chromosomes of P. cocdneus: the proximal heterochromatic segment in the long arm of chromosome pair I (which also bears the sequences complementary to 25S, 18S and 5.8S RNAs), most of the proximal heterochromatic segment plus a small portion of adjoining euchromatin in the long arm of chromosome pair VI and the large intercalary heterochromatic segment in the same chromosome pair. Simultaneous labeling of the two 5S RNA sites in chromosome VI was quite rare (3%), the rule being labelling of one site to the exclusion of the other, with a labeling frequency of 43.7% and 53.3% for sites no. 1 and no. 2 respectively. These results are interpreted as being due to differential hybridizability of chromosomal sites such as described in other materials.