Sequence of the Small Subunit Ribosomal RNA Gene from the Hypotrichous Ciliate Euplotes aediculatus1

ABSTRACT. We have determined the complete nucleotide sequence of the coding region of the small subunit rRNA gene of the hypotrichous ciliate Euplotes aediculatus. It is 1882 nucleotides long and contains several inserts not present in the small subunit rRNA genes of the hypotrichs Oxytricha nova and Stylonychia pustulata. A comparison of the sequences suggests that E. aediculatus is much less closely related to these other two hypotrichs than they are to each other. Although the gene sequence of E. aediculatus is drifting more rapidly than those of these other two species, its faster evolutionary clock is not enough to account for the degree of difference between them.     

Phylogenetic position of the marine ciliate, Certesia quadrinucleata (Ciliophora; Hypotrichia; Hypotrichida) inferred from the complete small subunit ribosomal RNA gene sequence

The complete small subunit rRNA (SSrRNA) gene sequence of the rare marine hypotrich, Certesia quadrinucleata Fabre-Domergue, 1885, was determined, and found to be 1752 nucleotides long. The phylogenetic position of this species was deduced using distance matrix, maximum parsimony and maximum likelihood methods. Certesia was consistently demonstrated to be a member of the Aspidisca-Euplotes group and clearly exhibits a very close relationship to the well-known genus Euplotes (99% Bay, 99% LS, 99% NJ, 99% MP). The phylogenetic trees further suggest that: (1) Uronychia and Diophrys, traditionally placed in the family Uronychiidae, branch earlier and share a closer relationship to each other than to other hypotrichs; (2) taxa in Gastrocirrhidae, represented by Euplotidium arenarium, might be an "ancestral" group among "traditional" hypotrichs.     

Characterization of Euplotes lynni nov. spec., E. indica nov. spec. and description of E. aediculatus and E. woodruffi (Ciliophora,Euplotidae) using an integrative approach

Four species belonging to the genus Euplotes have been investigated, namely: E. lynni nov. spec., E. indica nov. spec., E. aediculatus, and E. woodruffi. All populations are from India and were investigated using morphological and molecular markers. The phylogenetic relationships were inferred from small subunit ribosomal rRNA gene (SSU rRNA), internal transcribed spacer (ITS) region, and mitochondrial cytochrome c oxidase subunit I (COI) gene. Predicted secondary structure models for two new species using the hypervariable region of the SSU rRNA gene and ITS2 region support the distinctness of both species. Morphological characters were subjected to principal component analysis (PCA) and genetic variations were studied in-depth to analyze the relatedness of the two new species with their congeners. An integrative approach combining morphological features, molecular analysis, and ecological characteristics was carried out to understand the phylogenetic position of the reported species within the different clades of the genus Euplotes.     

Phylogenetic relationships of Blepharisma americanum and Colpoda inflata within the phylum ciliophora inferred from complete small subunit rRNA gene sequences

The complete small subunit rRNA gene sequences of the heterotrich Blepharisma americanum and the colpodid Colpoda inflata were determined to be 1719 and 1786 nucleotides respectively. The phylogeny produced by comparisons with other ciliates indicated that C. inflata is allied more closely with the nassophoreans and oligohymenophoreans than the spirotrichs. This is consistent with the placement of the colpodids in the Class Copodea. Blepharisma americanum was not grouped with the hypotrichs but instead was placed as the earliest branching ciliate. The distinct separation of B. americanum supports the elevation to class status given the heterotrichs based on morphological characters.     

The nucleotide sequence of the 17S ribosomal RNA gene of Tetrahymena thermophila and the identification of point mutations resulting in resistance to the antibiotics paromomycin and hygromycin

We have determined the complete sequence of the nuclear gene encoding the small subunit (17 S) rRNA of the ciliated protozoan Tetrahymena thermophila. The gene encodes an RNA molecule which is 1753 nucleotides in length. The sequence of the Tetrahymena small subunit rRNA is homologous to those of other eukaryotes, and the predicted secondary structure for the molecule includes features which are characteristic of eukaryotic small subunit rRNAs. We have also determined the nature of two different mutations in the Tetrahymena 17 S gene which result in resistance to the aminoglycoside antibiotics paromomycin and hygromycin. In each case we have identified a single base change near the 3' end of the rRNA, within a region that is highly evolutionarily conserved in both sequence and secondary structure. Analysis of the effects of these mutations on rRNA structure, and of the impact of these drugs on translation, should help to elucidate the role of the small subunit ribosomal RNA in ribosome function.     

Phylogenetic Relationships of Blepharisma Americanum and Colpoda Inflata Within the Phylum Ciliophora Inferred From Complete Small Subunit Rrna Gene Sequences

ABSTRACT The complete small subunit rRNA gene sequences of the heterotrich Blepharisma americanum and the colpodid Colpoda inflata were determined to be 1719 and 1786 nucleotides respectively. the phylogeny produced by comparisons with other ciliates indicated that C. inflata is allied more closely with the nassophoreans and oligohymenophoreans than the spirotrichs. This is consistent with the placement of the colpodids in the Class Copodea. Blepharisma americanum was not grouped with the hypotrichs but instead was placed as the earliest branching ciliate. the distinct separation of B. americanum supports the elevation to class status given the heterotrichs based on morphological characters.     

Phenotypic effects of targeted mutations in the small subunit rRNA gene of Tetrahymena thermophila.

Tetrahymena thermophila is an ideal organism with which to study functional aspects of the rRNAs in vivo since the somatic rRNA genes of T. thermophila can be totally replaced by cloned copies introduced via microinjection. In this study, we made small insertions into seven sites within the small subunit rRNA gene and observed their phenotypic effects on transformed cells. Two mutated genes coding for rRNA (rDNAs), both of which bear insertions in highly conserved sequences, failed to transform and are therefore believed to produce nonfunctional rRNAs. Three other altered rDNAs produce functional rRNAs that can substitute for most or all of the cellular rRNA. Two of these bear insertions in highly variable regions, and, surprisingly, the other has an insertion in a region that is well conserved for both sequence and secondary structure among eucaryotes. In addition, two other insertions appear to destabilize rRNAs that contain them. Our findings make predictions concerning the positions of some of these sites within the tertiary structure of the small ribosomal subunit and thus serve as an in vivo test of the existing tertiary structure models for the small subunit rRNA. Our results are in good agreement with expectations based on sequence comparison and in vitro work.     

Morphology and molecular phylogeny of two new brackish-water species of Amphisiella (Ciliophora,Hypotrichia), with notes on morphogenesis

Two new species of the taxonomically confused genus Amphisiella were isolated from brackish-water habitats in southern China, and their morphology and morphogenesis were investigated. The genes coding for small subunit (SSU) rRNA were sequenced in each species and included in a phylogenetic analysis with all data available from nominal species of Amphisiella and related hypotrichs. Both new species are diagnosed by an elongate body shape, yellow-brown/grey-brown cell color, one kind of cortical granule, and two macronuclear nodules. The cortical granules in A. pulchra sp. n. are grouped in small clusters which are irregularly distributed both ventrally and dorsally; in A. candida sp. n., they are irregularly and sparsely distributed ventrally and form longitudinal stripes dorsally. The ontogenetic processes of both species show similarities to those of other congeners. Phylogenetic trees based on SSU rRNA gene sequences suggest that the family Amphisiellidae is a paraphyletic assemblage. The results further demonstrate that two isolates identified as Amphisiella annulata (DQ832260 and GU170843) are very likely cryptic species, and a sequence identified as A. milnei (DQ845293) may belong to a genus other than Amphisiella.     

Ribosomal RNA genes of Trypanosoma brucei: mapping the regions specifying the six small ribosomal RNAs

There are six small ribosomal RNAs in trypanosome ribosomes. sRNA3 and sRNA5 of Trypanosoma brucei brucei have been partially sequenced. Sequence homologies indicate that sRNA3 is 5.8S RNA and sRNA5 is 5S RNA of T. b. brucei. The regions specifying these two, and the remaining four small RNAs, have been identified within clones of rRNA genes and in the genome. Five of the small RNAs, 1, 2, 3, 4 and 6, hybridise exclusively within the major rRNA gene repeat. A map of the regions specifying these small RNAs is presented. sRNA3 (5.8S RNA) hybridises to a region corresponding to the transcribed spacer of other eukaryotes. sRNA1 hybridises to a region between sequences specifying the two large subunit RNA molecules of 2.3 kb and 1.8 kb. Sequences specifying sRNAs 2 and 4 are present near the sequence specifying sRNA1, while sRNA6 appears to be specified 3' to the sequence specifying the 1.8-kb RNA sequence. In addition regions of secondary hybridisation for small RNAs 2, 3, 4 and 6 have also been identified. Though sRNA5 (5S RNA) hybridises within the major rRNA repeat, a separate 5S RNA gene repeat with unit size of 760 bp is also present. It is 10 to 20 times more abundant than the major rRNA gene repeat.     

Paramecium mitochondrial genes. I. Small subunit rRNA gene sequence and microevolution

The sequences of the small subunit mitochondrial rRNA genes from two divergent species of Paramecium (primaurelia and tetraurelia) were determined. The gene lies near the center of the linear mitochondrial genome, on the same strand as are all other currently identified genes. The sequences generally resemble their counterparts found in cytoplasmic, procaryotic, and other mitochondrial sources. The rDNA gene boundaries were located by nuclease S1 protection. Small subunit rDNA spans about 1680 nucleotides, including an extraneous 83-base pair sequence very near the 3' end which is unique to Paramecium mitochondria. This "insert" occurs at the apex of the highly variable in length penultimate helix, according to proposed models for small subunit rRNA secondary structure. A discontinuity occurs in isolated rRNA near the start of the insert, resulting in a stable 13 S RNA species and a small segment containing the remaining 3' portion of the gene. The overall rRNA gene sequence was 94% conserved between the two species, and the nucleotide differences consisted of 53% transitions, 37% transversions, and 9% insertions plus deletions. These substitutions were somewhat clustered, and the two most divergent regions coincided with the gene boundaries. The sequence was aligned with Escherichia coli 16 S rRNA for direct comparison of sequence and structure.     

The gene coding for small ribosomal subunit RNA in the basidiomycete Ustilago maydis contains a group I intron.

The nucleotide sequence of the gene coding for small ribosomal subunit RNA in the basidiomycete Ustilago maydis was determined. It revealed the presence of a group I intron with a length of 411 nucleotides. This is the third occurrence of such an intron discovered in a small subunit rRNA gene encoded by a eukaryotic nuclear genome. The other two occurrences are in Pneumocystis carinii, a fungus of uncertain taxonomic status, and Ankistrodesmus stipitatus, a green alga. The nucleotides of the conserved core structure of 101 group I intron sequences present in different genes and genome types were aligned and their evolutionary relatedness was examined. This revealed a cluster including all group I introns hitherto found in eukaryotic nuclear genes coding for small and large subunit rRNAs. A secondary structure model was designed for the area of the Ustilago maydis small ribosomal subunit RNA precursor where the intron is situated. It shows that the internal guide sequence pairing with the intron boundaries fits between two helices of the small subunit rRNA, and that minimal rearrangement of base pairs suffices to achieve the definitive secondary structure of the 18S rRNA upon splicing.     

The Small Ribosomal Subunit RNA Isoforms in Plasmodium Cynomolgi

We report the isolation, characterization and analysis of the small subunit rRNA genes in Plasmodium cynomolgi (Ceylon). As in other Plasmodium species, these genes are present in low copy number, are unlinked and form two types that are distinct in sequence and are expressed stage specifically. The asexually expressed (type A) genes are present in four copies in the Ceylon(-) and in five copies in the Berok(-) strain. Surprisingly, the sexually expressed (type B) gene is present in a single copy. The vast majority of the differences between gene types is confined to the variable regions. The pattern of divergence is different from that observed in Plasmodium berghei or in Plasmodium falciparum. Analysis of the small subunit rRNA sequences of P. cynomolgi, P. berghei and P. falciparum, indicates that the two gene types do not evolve independently but rather interact (through gene conversion or some form of recombination) to such an extent as to erase whatever stage-specific sequence signatures they may have had in the last common ancestor.     

The structure of the yeast ribosomal RNA genes. I. The complete nucleotide sequence of the 18S ribosomal RNA gene from Saccharomyces cerevisiae.

The cloned 18 S ribosomal RNA gene from Saccharomyces cerevisiae have been sequenced, using the Maxam-Gilbert procedure. From this data the complete sequence of 1789 nucleotides of the 18 S RNA was deduced. Extensive homology with many eucaryotic as well as E. coli ribosomal small subunit rRNA (S-rRNA) has been observed in the 3'-end region of the rRNA molecule. Comparison of the yeast 18 S rRNA sequences with partial sequence data, available for rRNAs of the other eucaryotes provides strong evidence that a substantial portion of the 18 S RNA sequence has been conserved in evolution.     

Analysis of the Primary Sequence and Secondary Structure of the Unusually Long SSU rRNA of the Soil Bug, Armadillidium vulgare

The complete nucleotide sequence of the SSU rRNA gene from the soil bug, Armadillidium vulgare (Crustacea, Isopoda), was determined. It is 3214 bp long, with a GC content of 56.3%. It is not only the longest SSU rRNA gene among Crustacea but also longer than any other SSU rRNA gene except that of the strepsipteran insect, Xenos vesparum (3316 bp). The unusually long sequence of this species is explained by the long sequences of variable regions V4 and V7, which make up more than half of the total length. RT-PCR analysis of these two regions showed that the long sequences also exist in the mature rRNA and sequence simplicity analysis revealed the presence of slippage motifs in these two regions. The putative secondary structure of the rRNA is typical for eukaryotes except for the length and shape variations of the V2, V4, V7, and V9 regions. Each of the V2, V4, and V7 regions was elongated, while the V9 region was shortened. In V2, two bulges, located between helix 8 and helix 9 and between helix 9 and helix 10, were elongated. In V4, stem E23-3 was dramatically expanded, with several small branched stems. In V7, stem 43 was branched and expanded. Comparisons with the unusually long SSU rRNAs of other organisms imply that the increase in total length of SSU rRNA is due mainly to expansion in the V4 and V7 regions. Received: 2 March 1999 / Accepted: 22 July 1999     

Streptomycin-resistance of Euglena gracilis chloroplasts: identification of a point mutation in the 16S rRNA gene in an invariant position.

We sequenced the chloroplast 16S rRNA gene of two Euglena gracilis mutants which contain streptomycin-resistant chloroplasts (Smr 139.12/4 and Smr 139.20/2). These mutants are known to contain a single intact rrn operon per circular chloroplast genome. Nucleotide sequence comparison between a 16S rRNA gene of wild type Euglena gracilis, strain Z, with streptomycin-sensitive chloroplasts, and the 16S rRNA gene of both Smr-strains reveals a single base change (C to T) at position 876. This position is equivalent to the invariant position 912 of the E. coli 16S rRNA gene. The analogous position is also conserved in all chloroplast small subunit RNA genes from lower and higher plants sequenced so far. Light dependent protein synthesis with purified chloroplasts from streptomycin-resistant cells is not inhibited by streptomycin. Based on the results reported here we postulate linkage between the observed point mutation on the 16S rRNA gene and streptomycin-resistance of chloroplast 70S ribosomes.     

Phylogenetic relationships and altered genome structures among Tetrahymena mitochondrial DNAs.

Tetrahymena thermophila mitochondrial DNA is a linear molecule with two tRNAs, large subunit beta (LSU beta) rRNA (21S rRNA) and LSU alpha rRNA (5.8S-like RNA) encoded near each terminus. The DNA sequence of approximately 550 bp of this region was determined in six species of Tetrahymena. In three species the LSU beta rRNA and tRNA(leu) genes were not present on one end of the DNA, demonstrating a mitochondrial genome organization different from that of T. thermophila. The DNA sequence of the LSU alpha rRNA was used to construct a mitochondrial phylogenetic tree, which was found to be topologically equivalent to a phylogenetic tree based on nuclear small subunit rRNA sequences (Sogin et al. (1986) EMBO J. 5, 3625-3630). The mitochondrial rRNA gene was found to accumulate base-pair substitutions considerably faster than the nuclear rRNA gene, the rate difference being similar to that observed for mammals.