Genetic variability of Taenia saginata inferred from mitochondrial DNA sequences

Taenia saginata is an important tapeworm, infecting humans in many parts of the world. The present study was undertaken to identify inter- and intraspecific variation of T. saginata isolated from cattle in different parts of Iran using two mitochondrial CO1 and 12S rRNA genes. Up to 105 bovine specimens of T. saginata were collected from 20 slaughterhouses in three provinces of Iran. DNA were extracted from the metacestode Cysticercus bovis. After PCR amplification, sequencing of CO1 and 12S rRNA genes were carried out and two phylogenetic analyses of the sequence data were generated by Bayesian inference on CO1 and 12S rRNA sequences. Sequence analyses of CO1 and 12S rRNA genes showed 11 and 29 representative profiles respectively. The level of pairwise nucleotide variation between individual haplotypes of CO1 gene was 0.3–2.4 % while the overall nucleotide variation among all 11 haplotypes was 4.6 %. For 12S rRNA sequence data, level of pairwise nucleotide variation was 0.2–2.5 % and the overall nucleotide variation was determined as 5.8 % among 29 haplotypes of 12S rRNA gene. Considerable genetic diversity was found in both mitochondrial genes particularly in 12S rRNA gene. © 2015, Springer-Verlag Berlin Heidelberg

Molecular and morphological characterization of the tapeworm Taenia hydatigena (Pallas, 1766) in sheep from Iran

Although Taenia hydatigena is one of the most prevalent taeniid species of livestock, very little molecular genetic information exists for this parasite. Up to 100 sheep isolates of T. hydatigena were collected from 19 abattoirs located in the provinces of Tehran, Alborz and Kerman. A calibrated microscope was used to measure the larval rostellar hook lengths. Following DNA extraction, fragments of cytochrome c oxidase 1 (CO1) and 12S rRNA genes were amplified by the polymerase chain reaction method and the amplicons were subjected to sequencing. The mean total length of large and small hooks was 203.4 μm and 135.9 μm, respectively. Forty CO1 and 39 12S rRNA sequence haplotypes were obtained in the study. The levels of pairwise nucleotide variation between individual haplotypes of CO1 and 12S rRNA genes were determined to be between 0.3-3.4% and 0.2-2.1%, respectively. The overall nucleotide variation among all the CO1 haplotypes was 9.7%, and for all the 12S rRNA haplotypes it was 10.1%. A significant difference was observed between rostellar hook morphometry and both CO1 and 12S rRNA sequence variability. A significantly high level of genetic variation was observed in the present study. The results showed that the 12S rRNA gene is more variable than CO1. © 2013 Cambridge University Press

Louse (Insecta : Phthiraptera) mitochondrial 12S rRNA secondary structure is highly variable

Lice are ectoparasitic insects hosted by birds and mammals. Mitochondrial 12S rRNA sequences obtained from lice show considerable length variation and are very difficult to align. We show that the louse 12S rRNA domain III secondary structure displays considerable variation compared to other insects, in both the shape and number of stems and loops. Phylogenetic trees constructed from tree edit distances between louse 12S rRNA structures do not closely resemble trees constructed from sequence data, suggesting that at least some of this structural variation has arisen independently in different louse lineages. Taken together with previous work on mitochondrial gene order and elevated rates of substitution in louse mitochondrial sequences, the structural variation in louse 12S rRNA confirms the highly distinctive nature of molecular evolution in these insects

Yeast Rrp8p, a novel methyltransferase responsible for m1A 645 base modification of 25S rRNA

Ribosomal RNA undergoes various modifications to optimize ribosomal structure and expand the topological potential of RNA. The most common nucleotide modifications in ribosomal RNA (rRNA) are pseudouridylations and 2'-O methylations (Nm), performed by H/ACA box snoRNAs and C/D box snoRNAs, respectively. Furthermore, rRNAs of both ribosomal subunits also contain various base modifications, which are catalysed by specific enzymes. These modifications cluster in highly conserved areas of the ribosome. Although most enzymes catalysing 18S rRNA base modifications have been identified, little is known about the 25S rRNA base modifications. The m(1)A modification at position 645 in Helix 25.1 is highly conserved in eukaryotes. Helix formation in this region of the 25S rRNA might be a prerequisite for a correct topological framework for 5.8S rRNA to interact with 25S rRNA. Surprisingly, we have identified ribosomal RNA processing protein 8 (Rrp8), a nucleolar Rossman-fold like methyltransferase, to carry out the m(1)A base modification at position 645, although Rrp8 was previously shown to be involved in A2 cleavage and 40S biogenesis. In addition, we were able to identify specific point mutations in Rrp8, which show that a reduced S-adenosyl-methionine binding influences the quality of the 60S subunit. This highlights the dual functionality of Rrp8 in the biogenesis of both subunits

PCR-Independent Detection of Bacterial Species-Specific 16S rRNA at 10 fM by a Pore-Blockage Sensor.

A PCR-free, optics-free device is used for the detection of Escherichia coli (E. coli) 16S rRNA at 10 fM, which corresponds to ~100-1000 colony forming units/mL (CFU/mL) depending on cellular rRNA levels. The development of a rapid, sensitive, and cost-effective nucleic acid detection platform is sought for the detection of pathogenic microbes in food, water and body fluids. Since 16S rRNA sequences are species specific and are present at high copy number in viable cells, these nucleic acids offer an attractive target for microbial pathogen detection schemes. Here, target 16S rRNA of E. coli at 10 fM concentration was detected against a total RNA background using a conceptually simple approach based on electromechanical signal transduction, whereby a step change reduction in ionic current through a pore indicates blockage by an electrophoretically mobilized bead-peptide nucleic acid probe conjugate hybridized to target nucleic acid. We investigated the concentration detection limit for bacterial species-specific 16S rRNA at 1 pM to 1 fM and found a limit of detection of 10 fM for our device, which is consistent with our previous finding with single-stranded DNA of similar length. In addition, no false positive responses were obtained with control RNA and no false negatives with target 16S rRNA present down to the limit of detection (LOD) of 10 fM. Thus, this detection scheme shows promise for integration into portable, low-cost systems for rapid detection of pathogenic microbes in food, water and body fluids

Exploration of RNA structure spaces

In order to understand the structure of real structure spaces, we are studying the 5S rRNA structure space experimentally. A plasmid containing a synthetic 5S rRNA gene, two rRNA promoters, and transcription terminators has been assembled. Assays are conducted to determine if the foreign 5S rRNA is expressed, and to see whether or not it is incorporated into ribosomes. Evolutionary competition is used to determine the relative fitness of strains containing the foreign 5S rRNA and a control 5S rRNA. By using site directed mutagenesis, a number of mutants can be made in order to study the boundaries of the structure space and how sharply defined they are. By making similar studies in the vicinity of structure space, it will be possible to determine how homogeneous the 5S rRNA structure space is. Useable experimental protocols have been developed, and a number of mutants have already been studied. Initial results suggest an explanation of why single stranded regions of the RNA are less subject to mutation than double stranded regions

The Human Oral Microbiome Database: a web accessible resource for investigating oral microbe taxonomic and genomic information

The human oral microbiome is the most studied human microflora, but 53% of the species have not yet been validly named and 35% remain uncultivated. The uncultivated taxa are known primarily from 16S rRNA sequence information. Sequence information tied solely to obscure isolate or clone numbers, and usually lacking accurate phylogenetic placement, is a major impediment to working with human oral microbiome data. The goal of creating the Human Oral Microbiome Database (HOMD) is to provide the scientific community with a body site-specific comprehensive database for the more than 600 prokaryote species that are present in the human oral cavity based on a curated 16S rRNA gene-based provisional naming scheme. Currently, two primary types of information are provided in HOMD—taxonomic and genomic. Named oral species and taxa identified from 16S rRNA gene sequence analysis of oral isolates and cloning studies were placed into defined 16S rRNA phylotypes and each given unique Human Oral Taxon (HOT) number. The HOT interlinks phenotypic, phylogenetic, genomic, clinical and bibliographic information for each taxon. A BLAST search tool is provided to match user 16S rRNA gene sequences to a curated, full length, 16S rRNA gene reference data set. For genomic analysis, HOMD provides comprehensive set of analysis tools and maintains frequently updated annotations for all the human oral microbial genomes that have been sequenced and publicly released. Oral bacterial genome sequences, determined as part of the Human Microbiome Project, are being added to the HOMD as they become available. We provide HOMD as a conceptual model for the presentation of microbiome data for other human body sites

Species Identification and Profiling of Complex Microbial Communities Using Shotgun Illumina Sequencing of 16S rRNA Amplicon Sequences

The high throughput and cost-effectiveness afforded by short-read sequencing technologies, in principle, enable researchers to perform 16S rRNA profiling of complex microbial communities at unprecedented depth and resolution. Existing Illumina sequencing protocols are, however, limited by the fraction of the 16S rRNA gene that is interrogated and therefore limit the resolution and quality of the profiling. To address this, we present the design of a novel protocol for shotgun Illumina sequencing of the bacterial 16S rRNA gene, optimized to capture more than 90% of sequences in the Greengenes database and with nearly twice the resolution of existing protocols. Using several in silico and experimental datasets, we demonstrate that despite the presence of multiple variable and conserved regions, the resulting shotgun sequences can be used to accurately quantify the diversity of complex microbial communities. The reconstruction of a significant fraction of the 16S rRNA gene also enabled high precision (>90%) in species-level identification thereby opening up potential application of this approach for clinical microbial characterization.Comment: 17 pages, 2 tables, 2 figures, supplementary materia

Utilization of tmRNA sequences for bacterial identification

In recent years, molecular approaches based on nucleotide sequences of ribosomal RNA (rRNA) have become widely used tools for identification of bacteria [1-4]. The high degree of evolutionary conservation makes 16S and 23S rRNA molecules very suitable for phylogenetic studies above the species level [3-5]. More than 16,000 sequences of 16S rRNA are presently available in public databases [4,6]. The 16S rRNA sequences are commonly used to design fluorescently labeled oligonucleotide probes. Fluorescence in situ hybridization (FISH) with these probes followed by observation with epifluorescence microscopy allows the identification of a specific microorganism in a mixture with other bacteria [2-4]. By shifting probe target sites from conservative to increasingly variable regions of rRNA, it is possible to adjust the probe specificity from kingdom to species level. Nevertheless, 16S rRNA sequences of closely related strains, subspecies, or even of different species are often identical and therefore can not be used as differentiating markers [3]. Another restriction concerns the accessibility of target sites to the probe in FISH experiments. The presence of secondary structures, or protection of rRNA segments by ribosomal proteins in fixed cells can limit the choice of variable regions as in situ targets for oligonucleotide probes [7,8]. One way to overcome the limitations of in situ identification of bacteria is to use molecules other than rRNA for phylogenetic identification of bacteria, for which nucleotide sequences would be sufficiently divergent to design species specific probes, and which would be more accessible to oligonucleotide probes. For this purpose we investigated the possibility of using tmRNA (also known as 10Sa RNA; [9-11]). This molecule was discovered in E. coli and described as small stable RNA, present at ~1,000 copies per cell [9,11]. The high copy number is an important prerequisite for FISH, which works best with naturally amplified target molecules. In E. coli, tmRNA is encoded by the ssrA gene, is 363 nucleotides long and has properties of tRNA and mRNA [12,13]. tmRNA was shown to be involved in the degradation of truncated proteins: the tmRNA associates with ribosomes stalled on mRNAs lacking stop codons, finally resulting in the addition of a C-terminal peptide tag to the truncated protein. The peptide tag directs the abnormal protein to proteolysis [14,15]. 165 tmRNA sequences have so far (August 2001; The tmRNA Website: http://www.indiana.edu/~tmrna/) been determined [16,17]. The tmRNA is likely to be present in all bacteria and has also been found in algae chloroplasts, the cyanelle of Cyanophora paradoxa and the mitochondrion of the flagellate Reclinomonas americana[10,17,18]