Ribosomal RNA Analysis Indicates a Benthic Pennate Diatom Ancestry for the Endosymbionts of the Dinoflagellates Peridinium foliaceum and Peridinium balticum (Pyrrhophyta)

ABSTRACT. The establishment of chloroplasts as cellular organelles in the dinoflagellate, heterokont (stramenopile), haptophyte, and cryptophyte algae is widely accepted to have been the result of secondary endosymbiotic events, that is, the uptake of a photosynthetic eukaryote by a phagotrophic eukaryote. However, the circumstances that promote such associations between two phylogenetically distinct organisms and result in the integration of their genomes to form a single functional photosynthetic cell is unclear. The dinoflagellates Peridinium foliaceum and Peridinium balticum are unusual in that each contains a membrance-bound eukaryotic heterokont endosymbiont. These symbioses have been interpreted, through data derived from ultrastructural and biochemical investigations, to represent an intermediate stage of secondary endosymbiotic chloroplast acquistion. In this study we have examined the phylogenetic origin of the P. foliaceum and P. Balticum heterokont endosymbionts through analaysis of their nuclear small subunit ribosomal RNA genes. Our analyses clearly demonstrate both endosymbionts are pennate diatoms belonging to the family Bacillariaceae. Since members of the Bacillariaceae are usually benthic, living on shallow marine sediments, the manner in which establishment of a symbiosis between a planktonic flagellated dinoflagellate and a botton-dwelling diatom is discussed. In particular, specific environmentally associated life strategy stages of the host and symbiont, coupled with diatom food preferences by the dinoflagellate, may have been vital to the formation of this association.     

Sequence of the Small Subunit Ribosomal RNA Gene of Perkinsus atlanticus-like Isolated from Carpet Shell Clam in Galicia, Spain

Parasites identified as Perkinsus atlanticus have been reported infecting carpet shell clams in Galicia (northwest Spain). We have sequenced the 18S ribosomal RNA gene of in vitro cultured Perkinsus atlanticus-like or hypnospores from diseased clams, and compared it with the same genomic region from P. marinus and Perkinsus sp. We have also compared the sequence of internal transcribed spacer (ITS) 1, ITS 2, and 5.8S rRNA from our isolate with the P. atlanticus GenBank sequence. The phylogenetic analysis of our cultured parasite based on the 18S gene led us to conclude that this isolate is not related to the genus Perkinsus but to the protists Anurofeca, Ichthyophonus, and Psorospermium, located near the animal-fungal divergence. These last two genera have been included, together with Dermocystidium, in the newly described DRIPs (Dermocystidium, rossete agent, Ichthyophonus, and Psorospermium) clade, recently named Mesomycetozoa. Received October 25, 1999; accepted February 11, 2000.     

Genetic Characterization of Clinical Acanthamoeba Isolates from Japan using Nuclear and Mitochondrial Small Subunit Ribosomal RNA

Because of an increased number of Acanthamoeba keratitis (AK) along with associated disease burdens, medical professionals have become more aware of this pathogen in recent years. In this study, by analyzing both the nuclear 18S small subunit ribosomal RNA (18S rRNA) and mitochondrial 16S rRNA gene loci, 27 clinical Acanthamoeba strains that caused AK in Japan were classified into 3 genotypes, T3 (3 strains), T4 (23 strains), and T5 (one strain). Most haplotypes were identical to the reference haplotypes reported from all over the world, and thus no specificity of the haplotype distribution in Japan was found. The T4 sub-genotype analysis using the 16S rRNA gene locus also revealed a clear sub-conformation within the T4 cluster, and lead to the recognition of a new sub-genotype T4i, in addition to the previously reported sub-genotypes T4a-T4h. Furthermore, 9 out of 23 strains in the T4 genotype were identified to a specific haplotype ({"type":"entrez-nucleotide","attrs":{"text":"AF479533","term_id":"28194589","term_text":"AF479533"}}AF479533), which seems to be a causal haplotype of AK. While heterozygous nuclear haplotypes were observed from 2 strains, the mitochondrial haplotypes were homozygous as T4 genotype in the both strains, and suggested a possibility of nuclear hybridization (mating reproduction) between different strains in Acanthamoeba. The nuclear 18S rRNA gene and mitochondrial 16S rRNA gene loci of Acanthamoeba spp. possess different unique characteristics usable for the genotyping analyses, and those specific features could contribute to the establishment of molecular taxonomy for the species complex of Acanthamoeba.     

蓖麻蚕核糖体大亚基RNA基因3‘—端序列分析及进化研究

测定了蓖麻蚕核糖体大亚基ＲＮＡ编码区３’－端ＤＮＡ序列,分析了其二级结构,并与昆虫伊蚊、果蝇;线虫;脊椎动物人、小鼠、爪蟾;低等脊索动物海鞘以及真菌酵母、毛霉相应的保守区段进行了同源比较。邻接法分析表明,昆虫核糖体大亚基ＲＮＡ在进化上与５ＳｒＲＮＡ相似,有加快的趋势。     

Secondary Structure and Molecular Evolution of the Mitochondrial Small Subunit Ribosomal RNA in Agaricales (Euagarics clade,Homobasidiomycota)

The complete sequences and secondary structures of the mitochondrial small subunit (SSU) ribosomal RNAs of both mostly cultivated mushrooms Agaricus bisporus (1930 nt) and Lentinula edodes (2164 nt) were achieved. These secondary structures and that of Schizophyllum commune (1872 nt) were compared to that previously established for Agrocybe aegerita. The four structures are near the model established for Archae, Bacteria, plastids, and mitochondria; particularly the helices 23 and 37, described as specific to bacteria, are present. Within the four Agaricales (Homobasidiomycota), the SSU-rRNA core is conserved in size (966 to 1009 nt) with the exception of an unusual extension of 40 nt in the H17 helix of S. commune. The four core sequences possess 76% of conserved positions and a cluster of C in their 3 end, which could constitute a signal involved in the RNA maturation process. Among the nine putative variable domains, three (V3, V5, V7) do not show significant length variations and possess similar percentages of conserved positions (69%) than the core. The other six variable domains show important length variations, due to independent large size inserted/deleted sequences, and higher rates of nucleotide substitutions than the core (only 31% of conserved positions between the four species). Interestingly, the inserted/deleted sequences are located in few preferential sites (hot spots for insertion/deletion) where they seem to arise or disappear haphazardly during evolution. These sites are located on the surface of the tertiary structure of the 30S ribosomal subunit, at the beginning of hairpin loops; the insertions lead to a lengthening of existing hairpins or to branching loops bearing up to five additional helices.     

Functional Study of the Residue C899 in the 900 Tetraloop of Escherichia coli Small Subunit Ribosomal RNA

A mutant ribosome bearing C899G in the 900 tetraloop of Escherichia coli 16S rRNA, one implicated in a conformational switch in the dynamic movements of the ribosome, showed defects in subunit association and 30S initiation complex formation. Our results explain the basis of the loss of protein synthesis ability caused by a perturbation of the 900 tetraloop.     

芹菜(Apium graveolens L.)叶细胞质5SrRNA的序列测定

应用Gupta等和Tanaka等建立的RNA序列双向直读技术,并辅以部分酶解法、化学法等,测定了芹菜叶细胞质的5SrRNA的全序列:与菠菜和蕃茄细胞质已知5SrRNA序列进行了比较,发现它们之间在序列上有高度的保守性。     

Molecular Identification of Nanoplanktonic Protists Based on Small Subunit Ribosomal RNA Gene Sequences for Ecological Studies1

Nanoplanktonic protists are comprised of a diverse assemblage of species which are responsible for a variety of trophic processes in marine and freshwater ecosystems. Current methods for identifying small protists by electron microscopy do not readily permit both identification and enumeration of nanoplanktonic protists in field samples. Thus, one major goal in the application of molecular approaches in protistan ecology has been the detection and quantification of individual species in natural water samples. Sequences of small subunit ribosomal RNA (SSU rRNA) genes have proven to be useful towards achieving this goal. Comparison of sequences from clone libraries of protistan SSU rRNA genes amplified from natural assemblages of protists by the polymerase chain reaction (PCR) can be used to examine protistan diversity. Furthermore, oligonucleotide probes complementary to short sequence regions unique to species of small protists can be designed by comparative analysis of rRNA gene sequences. These probes may be used to either detect the RNA of particular species of protists in total nucleic acid extracts immobilized on membranes, or the presence of target species in water samples via in situ hybridization of whole cells. Oligonucleotide probes may also serve as primers for the selective amplification of target sequences from total population DNA by PCR. Thus, molecular sequence information is becoming increasingly useful for identifying and enumerating protists, and for studying their spatial and temporal distribution in nature. Knowledge of protistan species composition, abundance and variability in an environment can ultimately be used to relate community structure to various aspects of community function and biogeochemical activity.     

The Small Subunit Ribosomal RNA Gene Sequence of Pleistophora anguillarum and The Use of PCR Primers for Diagnostic Detection of the Parasite

Using the polymerase chain reaction (PCR) and two primers for conserved regions of the small subunit ribosomal RNA (SSU-rRNA.) of Microsporidia, a DNA segment about 1,195 base pairs long was amplified from a DNA template prepared from purified spores of the microsporidian species Pleistophora anguillarum. These spores had been isolated from adult eels ( Anguilla japonica ) with "Beko Disease." A comparison of sequence data from other microsporidian species showed P. anguillarum SSU-rRNA to be most similar to Vavraia oncoperae. When juvenile eels were artificially infected with P. anguillarum , enzyme-linked immunosorbent assay could detect a positive infection only 12 days post-infection. However, when suitable PCR primers were used, a DNA fragment of about 0.8 kb was detected from these juvenile eels after only 3 days post-infection. No PCR product was obtained with templates prepared from clinically healthy control animals.     

Characterization of Two Perkinsus spp. from the Softshell Clam, Mya arenaria Using the Small Subunit Ribosomal RNA Gene

Sequence analysis and riboprinting of the small subunit ribosomal RNA genes were used to characterize two morphologically different Perkinsus species isolates from the gill (G117) and the hemolymph (H49) of the softshell clam, Mya arenaria. Sequence data of the polymerase chain reaction amplified ribosomal RNA loci of G117 and H49 indicated that these genes are 1803 and 1806 base-pair long, respectively. A sequence similarity of > 98.9% was calculated among ribosomal RNA sequences of the two isolates of this study and the published sequences of Perkinsus marinus from the American eastern oyster, Crassostrea virginica, and Perkinsus sp. from the blood cockle of the Australian mollusc, Anadara trapezia. From a phylogenetic tree obtained from Jukes-Cantor distances of the aligned ribosomal RNA gene sequences of 13 eukaryotic taxa using the Neighbor-Joining method, we showed that G117 and H49 clustered within the genus Perkinsus. Guided by the sequence data of Perkinsus marinus (accession # X75762) and Perkinsus sp. (accession # L07375), restriction endonucleases were selected for restriction fragment analysis of polymerase chain reaction products of the small subunit ribosomal RNA genes (riboprinting). Riboprinting was used to distinguish the four members of the genus Perkinsus from each other.     

The Large Ribosomal Subunit Protein L9 Enables the Growth of EF-P Deficient Cells and Enhances Small Subunit Maturation

The loss of the large ribosomal protein L9 causes a reduction in translation fidelity by an unknown mechanism. To identify pathways affected by L9, we identified mutants of E. coli that require L9 for fitness. In a prior study, we characterized L9-dependent mutations in the essential GTPase Der (EngA). Here, we describe a second class of L9-dependent mutations that either compromise or inactivate elongation factor P (EF-P, eIF5A in eukaryotes). Without L9, Δefp cells are practically inviable. Cell fractionation studies revealed that, in both the Der and EF-P mutant cases, L9''s activity reduces immature 16S rRNA in 30S particles and partially restores the abundance of monosomes. Inspired by these findings, we discovered that L9 also enhances 16S maturation in wild-type cells. Surprisingly, although the amount of immature 16S in 30S particles was found to be elevated in ΔrplI cells, the amount in polysomes was low and inversely correlated with the immature 16S abundance. These findings provide an explanation for the observed fitness increases afforded by L9 in these mutants and reveal particular physiological conditions in which L9 becomes critical. Additionally, L9 may affect the partitioning of small subunits containing immature 16S rRNA.     

Strategies for Amplification by Polymerase Chain Reaction of the Complete Sequence of the Gene Encoding Nuclear Large Subunit Ribosomal RNA in Corals

The nearly complete nuclear large subunit ribosomal RNA (LSU rRNA) gene in corals was amplified by primers designed from polymerase chain reaction (PCR) strategies. The motif of the putative 3′-terminus of the LSU rRNA gene was sequenced and identified from intergenic spacer (IGS) clones obtained by PCR using universal primers designed for corals. The 3′-end primer was constructed in tandem with the universal 5′-end primer for the LSU rRNA gene. PCR fragments of 3500 bp were amplified for octocorals and non-Acropora scleractinian corals. More than 80% of the Acropora LSU rRNA gene (3000 bp) was successfully amplified by modification of the 5′-end of the IGS primer. Analysis of the 5′-end of LSU rDNA sequences, including the D1 and D2 divergent domains, indicates that the evolutionary rate of the LSU rDNA differs among these taxonomic groups of corals. The genus Acropora showed the highest divergence pattern in the LSU rRNA gene, and the presence of a long branch of the Acropora clade from the other scleractinian corals in the phylogenetic tree indicates that the evolutionary rate of Acropora LSU rDNA might have accelerated after divergence from the common ancestor of scleractinian corals. Received February 17, 2000; accepted June 12, 2000.     

Partial Sequence of a Sponge Mitochondrial Genome Reveals Sequence Similarity to Cnidaria in Cytochrome Oxidase Subunit II and the Large Ribosomal RNA Subunit

A 2550-bp portion of the mitochondrial genome of a Demosponge, genus Tetilla, was amplified from whole genomic DNA extract and sequenced. The sequence was found to code for the 3′ end of the 16S rRNA gene, cytochrome c oxidase subunit II, a lysine tRNA, ATPase subunit 8, and a 5′ portion of ATPase subunit 6. The Porifera cluster distinctly within the eumetazoan radiation, as a sister group to the Cnidaria. Also, the mitochondrial genetic code of this sponge is likely identical to that found in the Cnidaria. Both the full COII DNA and protein sequences and a portion of the 16S rRNA gene were found to possess a striking similarity to published Cnidarian mtDNA sequences, allying the Porifera more closely to the Cnidaria than to any other metazoan phylum. The gene arrangement, COII—tRNA^Lys—ATP8—ATP6, is observed in many Eumetazoan phyla and is apparently ancestral in the metazoa. Received: 24 November 1997 / Accepted: 14 September 1998     

Interaction between Ribosome Assembly Factors Krr1 and Faf1 Is Essential for Formation of Small Ribosomal Subunit in Yeast

Ribosome formation in Saccharomyces cerevisiae requires a large number of transiently associated assembly factors that coordinate processing and folding of pre-rRNA and binding of ribosomal proteins. Krr1 and Faf1 are two interacting proteins present in early 90 S precursor particles of the small ribosomal subunit. Here, we determined a co-crystal structure of the core domain of Krr1 bound to a 19-residue fragment of Faf1 at 2.8 Å resolution. The structure reveals that Krr1 consists of two packed K homology (KH) domains, KH1 and KH2, and resembles archaeal Dim2-like proteins. We show that KH1 is a divergent KH domain that lacks the RNA-binding GXXG motif and is involved in binding another assembly factor, Kri1. KH2 contains a canonical RNA-binding surface and additionally associates with an α-helix of Faf1. Specific disruption of the Krr1-Faf1 interaction impaired early 18 S rRNA processing at sites A0, A1, and A2 and caused cell lethality, but it did not prevent incorporation of the two proteins into pre-ribosomes. The Krr1-Faf1 interaction likely maintains a critical conformation of 90 S pre-ribosomes required for pre-rRNA processing. Our results illustrate the versatility of KH domains in protein interaction and provide insight into the role of Krr1-Faf1 interaction in ribosome biogenesis.     

Phylogeny of the Rumen Ciliates Entodinium, Epidinium and Polyplastron (Litostomatea: Entodiniomorphida) Inferred from Small Subunit Ribosomal RNA Sequences

ABSTRACT. Three complete 18S ribosomal RNA gene sequences from the rumen ciliates, Entodinium coudatum (1,639 bp), Epidinium caudarum (1,638 bp), and Polyplastron multivesiculatum (1,640 bp) were determined and confrimed in the opposite direction. Trees produced using maximum parsimony and distance-matrix methods (lest squares and neighbour-joining). with strong bootstrap support, depict the rumen ciliates as a monophyletic group, Entodinium caudatum is the earliest branching rumen ciliate. However, Entodiniwn simplex does not pair with En. caudatum , but rather with Polyplastron multivesiculatum. Signature sequences for these rumen ciliates reveal that the published SSrRNA gene sequence from En. simplex is in fact a Polyoplastron species. The free-living haptorian ciliates, Loxophyllum, Homalozoon and Spathidium (Subclass Hoptoria), are monophyletic and are the sister group to the rumen cilates. The litostomes (class Litostomatea), consisting of the haptorians and the rumen ciliates, are also a monophyletic group.     

Universal Primers for Amplification of Mitochondrial Small Subunit Ribosomal RNA-Encoding Gene in Scleractinian Corals

We describe the construction of polymerase chain reaction primers designed to amplify a portion of the mitochondrial (mt) small subunit ribosomal (SSU) RNA-encoding genes in scleractinian corals. Combinations of cloning and sequencing show that the amplified fragments are between 694 and 896 bp in length. Alignment of the amplified DNA sequences to the published mt SSU rRNA genes of Metridium senile and Sarcophyton glaucum indicates several conserved regions among actiniarian, corallimorpharian, octocorallian, and scleractinians, suggesting this primer set can successfully amplify over 80% of the mt SSU rDNA region of scleractinian corals. Surveys of sequence variation and estimation of the rate of evolution show an extremely slow divergence of the SSU rRNA gene in the family Acroporidae. Received June 11, 1999; accepted October 4, 1999.     

Phylogeny of Six Genera of the Subclass Haptoria (Ciliophora,Litostomatea) Inferred from Sequences of the Gene Coding for Small Subunit Ribosomal RNA

ABSTRACT. The small subunit ribosomal RNA genes of nine species belonging to six genera of litostome ciliates, namely Amphileptus aeschtae, Chaenea teres, Chaenea vorax, Lacrymaria marina, Litonotus paracygnus, Loxophyllum sp.‐GD‐070419, Loxophyllum jini, Loxophyllum rostratum, and Phialina salinarum, were sequenced for the first time. Phylogenetic trees were constructed using different methods to assess the inter‐ and intra‐generic relationships of haptorians, of which Chaenea, Lacrymaria, Litonotus, and Phialina were analyzed for the first time based on molecular data. Monophyly of the order Pleurostomatida was strongly confirmed, and the two existing families of pleurostomatids, created on the basis of morphology, were confirmed by molecular evidence. Within the Pleurostomatida, Siroloxophyllum utriculariae occupied a well‐supported position basal to the Loxophyllum clade, supporting the separation of these genera from one another. Both the subclass Haptoria and the order Haptorida were partially unresolved, possibly paraphyletic assemblages of taxa in all analyses, creating doubts about the traditional placement of some haptorid taxa. The existing sequence of L. rostratum in GenBank (DQ411864) was conspicuously different from that of the isolate from Qingdao, China sequenced in the present work, indicating that they are different species. The isolate from Qingdao was verified as L. rostratum by morphological analysis, and the published morphology of existing GenBank record of L. rostratum is different from it. Based on both morphological and molecular evidence, the latter may be congeneric with an undescribed species of Loxophyllum from Guangdong Province, China.     

Nucleotide structure of the Scytalidium hyalinum and Scytalidium dimidiatum 18S subunit ribosomal RNA gene: evidence for the insertion of a group IE intron in the rDNA gene of S. dimidiatum

The molds Scytalidium dimidiatum (Nattrassia mangiferae synanamorph) and Scytalidium hyalinum are responsible for dermatomycosis in humans. We sequenced their 18S subunit ribosomal RNA gene to identify these species with molecular biology-based methods. The coding sequences differed by a single polymorphism (A in S. dimidiatum, G in S. hyalinum). Moreover, we found an insert at position 1199 in the 18S rRNA gene sequence of S. dimidiatum. Its potential secondary structure was characteristic of a group IE intron. Bioinformatic and phylogenic group IE intron analyses generated four main homogeneous clusters. The S. dimidiatum intron is original and not related with other known IE group introns.     

A Mutation in the Large Subunit Ribosomal RNA Gene of Tetrahymena Confers Anisomycin Resistance and Cold Sensitivity

Anisomycin, an antibiotic that specifically inhibits the peptidyl transfer function of eukaryotic ribosomes, has been used to select resistant mutants in Tetrahymena thermophila. A mutation conferring anisomycin resistance (an-r) has been localized to a 1.2-kb fragment of the large subunit ribosomal RNA (rRNA) gene by transformation via microinjection. A single base pair change was detected within this region. Nine independently isolated an-r mutants had the same base pair change. T. thermophila strains that are homozygous for this mutation are cold sensitive, unable to mate and grossly abnormal in cell morphology.