Small Subunit 18S rRNA Research Articles

Use of an rRNA internal transcribed spacer region to distinguish phylogenetically closely related species of the genera Zygosaccharomyces and Torulaspora.

Analyses of the sequences of the small-subunit (18S) rRNA gene and two internal transcribed spacers (ITSs), ITS1 and ITS2, revealed that members of the yeast genera Torulaspora and Zygosaccharomyces are phylogenetically intermixed. Despite some minor differences in 18S rRNA-, ITS1-, and ITS2-derived trees, in general the patterns of the relationships inferred from the three chronometers were in good agreement. The ITS sequences of Torulaspora and Zygosaccharomyces species exhibited far greater interspecies differences than the 18S rRNA sequences and were better than 18S rRNA sequences for measuring close genealogical relationships. Despite the existence of interstrain ITS sequence variation in some species, it is possible to identify conserved regions in both ITSs that are useful in species differentiation.

Phylogenetic diversity of yeast-producing basidiomycetes

Small subunit (18S) rRNA gene sequences from 38 species of fungi (three ascomycetes, 35 basidiomycetes) were analysed using parsimony. The resulting phylogeny supported a hypothesis of three major lineages within the basidiomycetes. The lineages are recognized as classes, the Urediniomycetes, Ustilaginomycetes and Hymenomycetes. Each lineage contained some yeasts or yeast producing taxa. The systematic relationships of basidiomycetous yeasts are discussed with reference to the three class taxonomic system.

The origin of land plants: Phylogenetic relationships among charophytes, bryophytes, and vascular plants inferred from complete small-subunit ribosomal RNA gene sequences

Complete nuclear-encoded small-subunit 18S rRNA (= SSU rRNA) gene sequences were determined for the prasinophyte green alga Mantoniella squamata; the charophycean green algae Chara foetida, Coleochaete scutata, Klebsormidium flaccidum, and Mougeotia scalaris; the bryophytes Marchantia polymorpha, Fossombronia pusilla, and Funaria hygrometrica; and the lycopod Selaginella galleottii to get a better insight into the sequential evolution from green algae to land plants. The sequences were aligned with several previously published SSU rRNA sequences from chlorophytic and charophytic algae as well as from land plants to infer the evolutionary relationships for major evolutionary lineages within the Chlorobionta by distance matrix, maximum parsimony, and maximum likelihood analyses. Phylogenetic trees created by the different methods consistently placed the Charophyceae on the branch leading to the land plants. The Charophyceae were shown to be polyphyletic with the Charales ("charalean" algae) diverging earlier than the Coleochaetales, Klebsormidiales, Chlorokybales, and Zygnematales ("charophycean" algae) which branch from a point closer to the land plants in most analyses. Maximum parsimony and maximum likelihood analyses imply a successive evolution from "charophycean" algae, particularly Coleochaetales, to bryophytes, lycopods, and seed plants. In contrast, distance matrix methods group the bryophytes together with the "charophycean" algae, suggesting a separate evolution of these organisms compared with the club moss and the seed plants.

Development of a PCR-based technique for detection of Helicobacter pylori.

A primer-set was designed for specific detection of genes that encode for 16S rRNA of Helicobacter pylori, using direct polymerase chain reaction (PCR). The primers were selected in the hypervariable regions, derived from a complete small subunit 16S rRNA sequence of the reference strain H. pylori CCUG 17874. The primer-set amplified a 537 base pair (bp) sequence specifically from chromosomal H. pylori DNA. Amplification of purified chromosomal H. pylori DNA was achieved at concentrations as low as 1 femto gram (fg), equivalent to 5 bacteria. Furthermore, as few as 1 lysed H. pylori cell was detected by this PCR technique. The specificity of the primers was 100%, since purified chromosomal DNA was detected from all 32 various H. pylori isolates, whereas no other bacteria species were detected, whether related to Helicobacter or not. The 16S rDNA primers successfully detected H. pylori in antral biopsy specimens collected from infected patients.

Development of a PCR-based technique for detection of Helicobacter pylori

A primer-set was designed for specific detection of genes that encode for 16S rRNA of Helicobacter pylori, using direct polymerase chain reaction (PCR). The primers were selected in the hypervariable regions, derived from a complete small subunit 16S rRNA sequence of the reference strain H. pylori CCUG 17874. The primer-set amplified a 537 base pair (bp) sequence specifically from chromosomal H. pylori DNA. Amplification of purified chromosomal H. pylori DNA was achieved at concentrations as low as 1 femto gram (fg), equivalent to 5 bacteria. Furthermore, as few as 1 lysed H. pylori cell was detected by this PCR technique. The specificity of the primers was 100%, since purified chromosomal DNA was detected from all 32 various H. pylori isolates, whereas no other bacteria species were detected, whether related to Helicobacter or not. The 16S rDNA primers successfully detected H. pylori in antral biopsy specimens collected from infected patients.

The status and composition of the genus Steringophorus Odhner, 1905 (Digenea: Fellodistomidae), based on partial small subunit rRNA sequences

Analysis of partial small subunit (18S) rRNA sequences shows the paraphyly of the genus Steringophorus as currently recognised. In consequence it is necessary to change the conception of the genus by the transfer of the species Steringophorus agnotus and S. sebastodis back to their original genus, Fellodistomum.

Polymerase chain reaction-based detection of dermatophyte DNA with a fungus-specific primer system.

There is significant clinical interest in primers which are specific for fungi and do not hybridize to DNA of other eukaryotes or prokaryotes. Such primers would allow specific amplification of fungal DNA from human tissue samples containing fungi. Fungal identification to the species level could follow by direct sequencing or restriction analysis. Several previously described primer systems cross-react with DNA of plants and animals. We have designed a primer system that amplifies a fragment of the gene coding for the small ribosomal subunit 18S rRNA. Database searches and sequence analyses were performed using the HUSAR (Heidelberg Unix Sequence Analysis Resources) computer system at the German Cancer Research Centre, Heidelberg, Germany. Primers TR1 (5'-GTTTCTAGGACCGCCGTA) and TR2 (5'-CTCAAACTTCCATCGACTTG) bind to sequences which are homologous within the fungi, but differ from corresponding DNA fragments of plants and animals. The amplified fragment is 581 base pairs in length and contains variable, and therefore species-specific, regions. The DNA of Trichophyton rubrum, Trichophyton mentagrophytes, Trichophyton verrucosum, Trichophyton terrestre, Microsporum canis, Microsporum gypseum and Epidermophyton floccosum and of several yeast species was amplified by the primers, but not the DNA from 42 normal human skin samples. Furthermore, other DNA preparations from plants and animals, including those from radish, cabbage, wheat and mouse, did not show amplification reactions.

Rapid presumptive identification of medically relevant yeasts to the species level by polymerase chain reaction and restriction enzyme analysis.

A method for the rapid presumptive differentiation of a panel of 12 clinically relevant yeasts to the species level was developed on the basis of evaluation by the polymerase chain reaction (PCR) of the gene coding for the small ribosomal subunit 18S-rRNA. The method involved restriction enzyme analysis of PCR products obtained with primers common to all fungi. Using six restriction enzymes, AluI, BanI, BbsI, DraII, Eco147I and NheI, characteristic PCR-restriction enzyme patterns were obtained for Candida albicans, Candida tropicalis, Candida krusei, Candida kefyr, Candida lusitaniae, Candida guilliermondii, Candida glabrata and Saccharomyces cerevisiae, as well as for the pairs Candida parapsilosis/Candida viswanathii and Trichosporon beigelii/Cryptococcus neoformans. The procedure does not involve hybridization steps or the use of radioactivity and can be completed within one working day.

Partial small subunit (18S) rRNA gene sequences from fish parasites of the families Lepocreadiidae and Fellodistomidae (Digenea) and their use in phylogenetic analyses

Digeneans of the families Lepocreadiidae and Fellodistomidae are common parasites of shallow and deep-water marine fishes. Worms recovered from these contrasting environments may be indistinguishable or very similar morphologically and little is known concerning their inter-relationships. To investigate the systematic status of these forms, partial sequences from a small subunit 18S rRNA gene were generated from four species of lepocreadiids (Lepidapedon elongatum, L. gaevskayae, L. rachion and Opechona bacillaris) and two fellodistomids (Fellodistomum fellis and Steringophorus agnotus). Approximately 1,637 bases were sequenced for L. elongatum, L. rachion and F. fellis. A comparison of these sequences identified regions which varied between the two families. As a result, two regions were identified as containing potential phylogenetically informative sites and sequenced from the remaining species. Analyses of these regions by both maximum parsimony and distance methods showed that nucleic acid sequence data can be used to resolve the two families. The results indicate that 18S rRNA sequences are likely to be more satisfactory at resolving higher levels of phylogeny, i.e. at the family level, than at the generic level due to the similarity shown between the sequences of congeners.

Phylogenetic relationships in the Gracilariales (Rhodophyta) as determined by 18S rDNA sequences

Nucleotide (nt) sequences of the nuclear-encoded small-subunit (18S) rRNA genes are compared in representatives of the non-parasitic genera of the marine red algal order Gracilariales. Sequence div...

Sequence and organization of a 7.2 kb region of wheat mitochondrial DNA containing the large subunit (26S) rRNA gene.

We report the sequence of a 7.2 kilobase pair DNA fragment containing a copy of the wheat mitochondrial gene (rrn26) that encodes the mitochondrial large-subunit ribosomal RNA (26S rRNA). The mature 26S rRNA was determined by direct RNA sequencing to be 3467 nucleotides long, and to share a 5'-terminal pentanucleotide (5'-AUCAU), thought to be important in post-transcriptional processing, with the wheat mitochondrial small-subunit (18S) rRNA. Two other prominent features of the sequence were noted. First, upstream of rrn26 are located two tandem copies of a 70 base pair element containing a putative mitochondrial promoter motif (TCGTATAAAAA). Second, downstream of rrn26 is a sequence element that, if transcribed, would produce an RNA with a secondary structure resembling that of tRNAs but differing sufficiently from the latter structure to preclude any transcript from functioning normally in translation. These upstream and downstream sequence elements may play a role in the expression of rrn26 in wheat mitochondria.

Ribosomal RNA Sequence Divergence Among Sibling Species of Yeasts

The genetic and implicit taxonomic resolution provided by ribosomal RNA sequence divergence was estimated from comparisons of sibling yeast species. Two regions of the large subunit (25S) rRNA and four regions of the small subunit (18S) rRNA comprising a total of 855 nucleotides were examined in all strains. Within these regions, no nucleotide differences were detected between strains of the same species. The sibling species pairs Pichia mississippiensis/P. amylophila, P. americana/P. bimundalis, and Issatchenkia scutulata and its variety exigua could be separated from each other by differences in a ca. 200-base region of the large subunit rRNA. Sequence differences within the sibling species complex composed of Saccharomyces bayanus, S. pastorianius (S. carlsbergensis), and S. cerevisiae were consistent with the prior proposal that S. pastorianus is a partial amphidiploid formed by a rare mating between the other two Saccharomyces species. From these data, the highly variable region identified in the 25S subunit rRNA appears suitable for rapid separation of nearly all species by oligonucleotide probe technology.